CURRENT LITERATURE

INTERNATIONAL JOURNAL OF LEPROSY^

Volume 63, Number IPrinted in the U.S.A.

CURRENT LITERATURE

This departntent carnes selected abstracts of articles published in current med-icai journals dealing with leprosy and other mi'cobacterial diseases.

General and Historical

Jacob, M. S., Amar, D., Christopher, A. andKeystone, J. S. Transmission of healthinformation on leprosy from children totheir families in an urban centre. Lepr.Rev. 65 (1994) 272-278.

A health education study utilizing ahomework assignment was carried out in aprivate secondary school in Bangalore, SouthIndia, to determine whether health infor-mation about leprosy would be transferredfrom children to their families. After a pre-test questionnaire on knowledge and atti-tude about leprosy was administered to threeStandard VII classes and their family mem-bers, a different comprehensive health ed-ucation session was given to each class: (i)leprosy plus a homework assignment; (ii)leprosy alone; and (iii) tuberculosis alone.A post-test questionnaire was administeredto ali participants 1 month later.

Of the 118 children and 229 family mem-bers who entered the study, almost 80% ofthe participants completed it. The childrenin the leprosy-educated groups showed sig-nificant improvement in knowledge com-pared with controls, but no change in theirattitude toward leprosy. Although post-testresponses of household members showedmodest improvement in knowledge aboutleprosy, attitudes remained the same orworsened. The homework assignment didnot appear to improve the transmission ofhealth information to household members.This study showed that the knowledge leveiof family members in South India could beimproved modestly by educating their chil-dren about leprosy. However, attitudes to-ward leprosy were unaffected or wors-ened. — Authors' Summary

Murray, C. J. L. Quantifying the burden ofdisease: the technical basis for disability-adjusted life years. Bull. WHO 72 (1994)429-445.

Detailed assumptions used in construct-ing a new indicator of the burden of disease,the disability adjusted life year (DALY), arepresented. Four key social choices in anyindicator of the burden of disease are care-fully reviewed. First, the advantages anddisadvantages of various methods of cal-culating the duration of life lost due to adeath at each age are discussed. DALYs usea standard expected-life lost based on modellife-table West Level 26. Second, the valueof time lived at different ages is captured inDALYs using an exponential function whichrefiects the dependence of the young and theelderly on adults. Third, the time lived witha disability is made comparable with thetime lost due to premature mortality by de-fining six classes of disability severity. As-signed to each class is a severity weight be-tween O and 1. Finally, a 3% discount rateis used in the calculation of DALYs. Theformula for calculating DALYs based onthese assumptions is provided. — Authors'Abstract

Murray, C. J. L. and Lopez, A. D. Globaland regional cause-of-death patterns in1990. Bull. WHO 72 (1994) 447-480.

Demographic estimation techniques sug-gest that worldwide about 50 million deathsoccur each year, of which about 39 millionare in the developing countries. In countrieswith adequate registration of vital statistics,the age at death and the cause can be reliablydetermined. Only about 30%-35% of alldeaths are captured by vital registration (ex-cluding sample registration schemes); for theremainder, cause-of-death estimation pro-cedures are required. Indirect methodswhich model the cause-of-death structure asa function of the levei of mortality can pro-vide reasonable estimates for broad cause-of-death groups. Such methods are gener-ally unreliable for more specific causes. In

133

134^ International Journal of Leprosy^ 1995

this case, estimares can be constructed fromcommunity-level mortality surveillancesystems or from epidemiological evidenteon specific diseases. Some check on theplausibility of the estimates is possible inview of the hierarchical structure of cause-of-death Iists and the well-known age-spe-cific patterns of diseases and injuries.

The results of applying these methods toestimate the causes of death for over 120diseases or injuries, by age, sex and region,are described. The estimates have been de-rived in order to calculate the years of lifelost due to premature death, one of the twocomponents of overall disability-adjustedlife years (DALYs) calculated for the 1993World development report. Previous at-tempts at cause-of-death estimation havebeen limited to a few diseases only, withlittle age-specific detail. The estimates re-ported in detail here should serve as a usefulreference for further public health researchto support the determination of health sec-tor priorities.—Authors' Abstract

Murray, C. J. L. and Lopez, A. D. Quan-tifying disability: data, methods and re-sults. Bull. WHO 72 (1994) 481-494.

Conventional methods for collecting, an-alyzing and disseminating data and infor-mation on disability in populations haverelied on cross-sectional censuses and sur-veys which measure prevalence in a givenperiod. While this may be relevant for de-fining the extent and demographic patternof disabilities in a population, and thus in-dicating the need for rehabilitative services,prevention requires detailed information onthe underlying diseases and injuries thatcause disabilities. The Global Burden ofDisease methodology described in this pa-per provides a mechanism for quantifyingthe health consequences of the years of lifelived with disabilities by first estimating theage-sex-specific incidence rates of underly-ing conditions, and then mapping these toa single disability index which collectivelyreflects the probability of progressing to adisability, the duration of life lived with thedisability, and the approximate severity ofthe disability in terms ofactivity restriction.Detailed estimates of the number of dis-ability-adjusted life years (DALYs) lived areprovided in this paper, for eight geograph-

ical regions. The results should be useful tothose concerned with planning health ser-vices for the disabled and, more particular-ly, with determining policies to prevent theunderlying conditions which give riso to se-rious disabling sequelae.—Authors' Ab-stract

Murray, C. J. L., Lopez, A. D. and Jamison,D. T. The global burden ofdisease in 1990:summary results, sensitivity analysis andfuture directions. Bull. WHO 72 (1994)495-509.

A basic requirement for evaluating thecost-effectiveness of health interventions isa com prehensive assessment of the amountofill health (premature death and disability)attributable to specific diseases and injuries.A new indicator, the number of disability-adjusted life years (DALYs), was developedto assess the burden of disease and injuryin 1990 for over 100 causes by age, sex andregion. The DALY concept provides an in-tegrative, comprehensive methodology tocapture the entire amount ofill health whichwill, on average, be incurred during one'slifetime because of new cases of disease andinjury in 1990. It differs in many respectsfrom previous attempts at global and re-gional health situation assessment whichhave typically been much less comprehen-sive in scope, less detailed, and limited toa handful of causes.

This paper summarizes the DALY esti-mates for 1990 by cause, age, sex and region.For the first time, those responsible for de-ciding priorities in the health sector haveaccess to a disaggregated set of estimateswhich, in addition to facilitating cost-effec-tiveness analysis, can be used to monitorglobal and regional health progress for overa hundred conditions. The paper also showshow the estimates depend on particular vai-ues of the parameters involved in the cal-culation. — Authors' Abstract

Talhari, S. [Hanseniasis: actual situation.]An. Bras. Dermatol. 69 (1994) 209-215.(in Portuguese)This is a review of the main aspects re-

lated to the etiology, transmission, classi-fication and clinical aspects of leprosy andan analysis of the effectiveness and side ef-

63, 1^ Current Literature^ 135

fects of multidrug therapy and its opera-tional problcros. The world distribution ofthis disease and the goal of its eliminationby the year 2000 are discussed.—Author'sEnglish Summary

Turk, J. I,. Sir James Simpson: leprosy andsyphilis. J. R. Soc. Med. 87 (1994) 549-551.

Sir James Simpson (1811-1870) had along-standing intcrest in antiquarian stud-ies. In 1841 he published .Intiquarian No-tices o/ Leprosy and Leprosy Hospitais inScotland and England. Scotland was se-verely affected by leprosy, and the diseaselingered therc long after it had almost van-ished from England. King Robcrt the Brucccontracted leprosy in 1339. Simpson liststhe Scottish leprosy hospitais and recordsthe history of thcir foundation and organi-zation. He mentioned the role of the Knightsof St. Lazarus who supervised a number ofleprosy hospitais in England and Scotland.Simpson points out that leprosy was knownin France as early as the 8th century and in

Britain from the early 10th century, thusbefore the Crusades. During the 17th and18th centuries it was believed that leprosywas introduced finto northern Europe fromthe East by those returning from the Cru-sades.—Froco the Article

Walter, C. S. Changing role of voluntaryorganizations in the context of elimina-tion of leprosy. Indian J. Lepr. 66 (1994)37-43.

Elimination of leprosy as a disease stillleaves hundreds and thousands of affectedwho requirc carc and assistance. Hence de-cades of opportunities and challenges arethcre and the voluntary organizations willcontinue to play a major role in cooperationwith the government of India and WHOuntil the disease is eliminated and finallyeradicated. The main opportunities lie inthe arcas of expansion of MDT coverage,rehabilitation ofcured patients, and spread-ing the knowledge and awareness about lep-rosy to general public.—Author's Conclu-sion

Chemotherapy

Altagracia, M., Monroy Noyola, A., Oso-riorico, L., Kravzov, J., Alvarado Calvillo,R., Manjarrez Marmolejo, J. and Rios, C.Dapsone attenuates kainic acid-inducedseizures in rats. Neurosci. Lett. 176 (1994)52-54.

We tested the ability of dapsone (4,4'-diamino-diphenyl sulfone) to attenuatekainic acid-induced seizures. We observedthat 9.375 and 12.5 mg/kg doses of dapsoneadministered 30 min before a single kainicacid (10 mg/kg) i.p. injection were able todecrease the time of electroencephalograph-ic seizures by 52% and 82%, respectively,as compared with rats administered 10 mg/kg kainic acid only. The 12.5 mg/kg doseof dapsone was also able to diminish bothkainic acid-evoked body and head shakes(58%) and kainic acid-induced mortality(75%). These results suggest that dapsonecould be used in clinical trials as an anti-convulsant.—Authors' Abstract

Bwire, R. and Kav uma, H. J. S. Type 1reactions in leprosy, neuritis and steroidtherapy: the impact of the human im-munodeficiency virus. Trans. R. Soc.Trop. Med. Hyg. 88 (1994) 315-316.The incidence of type 1 reactions and

neuritis among HIV seronegative and HIVseropositive leprosy patients was investi-gated. HIV seropositivity was associatedwith an increased incidence of type 1 re-actions among multibacillary (MB) pa-tients, which were observed in 9 of 12 se-ropositive MB patients and in 8 of 40 HIVseronegative MB patients (p < 0.0005).Similarly, the incidence of neuritis was sig-nificantly increased among the HIV sero-positive MB patients, of whom 8 developedacute neuritis compared to 3 of the HIVseronegative patients (p < 0.0005). Therewas no significant difference betwecn thenumbers of paucibacillary HIV seropositiveand HIV seronegative patients who devel-


oped these complications. Both the HIV se-ronegative and HIV seropositive patientsshowed a similar response to steroid ther-apy for the management ofacutc neuritis.-Authors' Abstract

Dietrich, M., Gaus, W., Kern, P. and Mey-ers, W. M. An international randomizcdstudy with long-term follow-up of singleversus combination chemotherapy ofmultibacillary leprosy. Antimicrob.Agents Chemother. 38 (1994) 2249-2257.

A total of 307 patients with lepromatousleprosy and borderline lepromatous leprosywere randomizcd to dapsone monotherapyor to one oftwo types ofdrug combinations.A 3-year treatment phase was followed bya 5-year observation phase. The evaluationincluded 233 patients for whom togetherthere were 1404 years of observation. A to-tal of 1956 blinded histopathological spec-imens were processed centrally. When en-tering the trial isolates from 13 patients(5.6%) showed dapsone resistance in themouse foot pad test, and these patients wereevaluated separately. Dapsonc monother-apy (68 patients) had the same frequency ofcure as the combination of dapsone and rif-ampin (77 patients) or the four-drug regi-men consisting of dapsone, rifampin, iso-niazid, and prothionamide (75 patients). Wedid not find a significant difference in theclearance of bacteria either between themonotherapy and the two-drug combina-tion or the monotherapy and the four-drugcombination. Six months after the initiationof treatment, disease in 15% of the patientswho received dapsone monotherapy butnone of the patients who received combinedtreatment werc clinically progressive: Afteranother 1 to 9 months of treatment the dis-case in ali patients was stable or regressive.There was no difference in the type or fre-quency of reactions. Only after the end ofthe scheduled observation phase were threerelapses reported. All three treatment regi-mens were well tolerated. Dapsone mon-otherapy is highly effective in the treatmentof multibacillary leprosy under the condi-tions of well-controlled treatment. Combi-nation regimens seem only to accelerate theregression of active disease when they arecompared with monotherapy with dapsone.The mouse foot pad test does not reflect the

clinical resistance and cannot be recom-mended for use in making therapeutic de-cisions. — Authors' Abstract

dos Santos, S. N. M. B., Araujo, M. G.,Patrus, O. A. and Samaha, D. [Variationin the concentration of hemoglobin in thedosage of sulfone used to treat hansen-iasis.] An. Bras. Dermatol. 69 (1994) 281-284. (in Portuguese)

Hemolysis has been obscrved frequentlyas one of sulfone's efFects. This seems tohappen also in doses applied to the treat-ment of leprosy paticnts. One-hundred-fourleprosy patients using sulfones were studicdand 33 werc statistically analyzed. There wasno significant statistical correlation with he-moglobin (hb) variation and the initial leveiof hb, interval between laboratory tests, ageand weight in the male. In the female therewas moderate correlation with weight.Eighty-four percent of patients had a fali inhb concentration of 1 g/dl or more, confirm-ing reports in the literature. It is very im-portant to observe this fact carefully. Thismay have serious clinicai implications, es-pecially in endemic arcas where, owing tonutrition, malaria, and intestinal parasit-ism, the hb concentration is aiready com-promised.—Authors' English Summary

Herbert, D., Paramasivan, C. N., Prabhak-ar, R. and Swaminathan, G. In vitro ex-periments with Centella asiatica: inves-tigation to elucidate the effect of an in-digenously prepared powder of this planton the acid-fastness and variability of Al.tuberculosis. Indian J. Lepr. 66 (1994) 65-68.

The herb Centella asiatica (Linn.), foundthroughout India, is acclaimed to have me-dicinal properties and has been used in lep-rosy patients from very early times. It isconsidered that the active compound of thisherb, called asiaticoside, probably acts onthe waxy covering ofMi'cobacterium leprae.The in vitro effect of an indigenously pro-duced dry powder of Centella asiatica (CA)on the acid-fastness and viability of M. tu-berculosis was investigated in the presentstudy. The results indicate that CA may nothave any direct action on the acid-fastnessor viability of AI. tuberculosis H37Rv invitro. Further studies using purified asiati-

63, 1^ Current L iterat ure^ 137

coside of the plant or in vivo studies arerequired.—Authors' Abstract

Matz, J., Borish, L. C., Routes, J. M. andRosenwasser, L. J. Oral desensitizationto rifampin and ethambutol in mycobac-terial disease. Am. J. Respir. Crit. CareMed. 149 (1994) 815-817.

The incidente of disease caused by Mv-cobacteriunn tuberculosas (including drug-re-sistant strains) and 111. aviam complex(MAC) is increasing. Hypersensitivity re-actions to antimycobacterial agents are rel-atively uncommon, but when they occurthey may result in cessation of therapeuticmedications. We report our experiente withrapid oral desensitization to ethambutol andrifampin in a group of 10 patients with my-cobacterial disease who had cxperienced eu-tancous hypersensitivity reactions to thesedrugs. An adaptation of the rapid oral de-sensitization protocol for penicillin was used,with the dosing intervals increased to ac-count for the different kinetics of these drugs.Adverse reactions were few and easily treat-ed without necessitating cessation of ther-apy. We conclude that oral desensitizationto rifampin and ethambutol by our protocolis safe and effective, allowing these patientsto proceed with an optimal antimycobac-terial regimen.—Authors' Abstract

Nau, R., Kinzig, M., Dreyhaupt, T., Kolen-da, H., Sorgel, F. and Frange, H. W. Ki-netics of ofloxacin and its metabolites incerebrospinal fluid after a single intra-venous infusion of 300 milligrams of of-loxacin. Antimicrob. Agents Chemother.38 (1994) 1849-1853.

Ofloxacin has been reported to diffusereadily into the cerebrospinal fluid (CSF) insubjects with both infiamed and uninfiamedmeninges. However, with moderately sus-ceptible bacteria, ofloxacin concentrationsin CSF may be subtherapeutic after admin-istration of an intravenous (i.v.) dose of 200mg. For this reason, the kinetics of a higherdose of ofloxacin in CSF was studied withhumans. Six patients with occlusive hydro-cephalus caused by cerebrovascular diseaseswho had undergone external ventriculos-tomy received 400 mg of ofloxacin i.v. over30 min. Serum and CSF samples were drawnrepeatedly. Serum from 12 healthy volun-

teers was sampled repeatedly after they hadreceived 400 mg of ofloxacin i.v. over 60min. Ofloxacin, ofloxacin-N-oxide, andN-desmethyl-ofloxacin concentrations weredetermined by high-pressure liquid chro-matography with fluorescente detection.The maximum ofloxacin concentrations inthe serum of the patients ranged from 7.36to 11.6 mg/liter (mean, 9.55 mg/liter), theapparent volume of distribution/bodyweight was 0.96 to 1.19 liters/kg (mean, 1.11liters/kg), and the total body clearance was115 to 280 ml/min (mean 192 ml/min). Inhealthy volunteers, the volume of distri-bution/body weight and the total bodyclearance were higher and amounted to 1.27± 0.18 liters/kg and 217 ± 43 ml/min(means ± standard deviations), respective-ly. These differences were attributed to theolder ages of the patients than the volun-teers. In the CSF ofpatients, maximum con-centrations of 1.00 to 2.85 mg/liter (mean2.04 mg/liter) were observed 0.5 to 4 hrfollowing the completion of the ofloxacininfusion. Ofloxacin elimination from CSFwas slightly slower than that from serum(half-lives, 4.33 to 10.02 versus 4.27 to 9.14hr). The overall penetration ofofloxacin fintoCSF, as expressed by the ratios of the arcasunder the concentration-time curves,amounted to 0.59 to 0.81 (mean 0.65). Themore hydrophilic metabolites ofloxacin-N-oxide and N-desmethyl-ofloxacin passed lessreadily than ofloxacin into the CSF. In con-clusion, the concentrations of CSF attainedafter a single i.v. infusion of 400 mg of of-loxacin in the absence of meningeal inflam-mation appear to be high enough to inhibitthe growth of most staphylococci and mem-bers of the family Enterobacteriaceae, whichare often involved in CSF shunt infections.Yet, in view of pharmacodynamic studiessuggesting a peak concentration of CSF ofat least 10-fold the MIC, the use ofofloxacinfor central nervous system infections is op-timal only with highly susceptible patho-gens (MIC _< 0.12 mg/liter).—Authors' Ab-stract

O'Reilly, J. R., Corrigan, O. I. and O'Dris-coll, C. M. The effect of mixed micellarsystems, bile salt/fatty acids, on the sol-ubility and intestinal absorption of clo-fazimine (B663) in the anaesthetised rat.Int. J. Pharmaceut. 109 (1994) 147-154.


Clofazimine (B663) is a highly lipophilicdrug used in the treatment of leprosy. Thesolubility and gastrointestinal membrancpermeability (P-app) of B663 in mixed mi-cellar systems were examined. Membranepermeability was determined using a rat gutperfusion model and, in addition, thesestudies incorporated the hydrophilic mark-er PEG 4000. The mixed micellar systemsstudied contained the bile salt, sodium cho-late (NaC), in association with difTerent fattyacids including caprylic acid, olcic acid andlinolcic acid. At a set concentration of NaC(40 mM) the solubility of 13663 increasedwith increasing concentration of each fattyacid. Relative to NaC, the maximunm en-hancement in solubility (16-fold) was ob-tained with the NaC/linoleic acid (40:40mM) system. An optimum bile salt/fattyacid ration of 1:1 existed for maxinium sol-ubility enhancement. All mixed micellarsystems enhanced the absorption of I3663relative to the simple micelle. The P-apptended to increase with increasing fatty acidconcentration, maximunm enhancement be-ing obtained with the NaC/linoleic acid 40:40 mM system. With each mixed micellarsystem a higher P-app was obtained withlower drug loading. The efTects of the mixedmicellar systems on the absorption of PEG4000 varied with fatty acid loading. Theseresults have shown that mixed micelles canenhance the absorption of B663 to a greaterextent relative to non-micellar and simplemicellar systems. Maximum enhancement(> 800-fold) in the rate of B663 absorptionwas obtained with the NaC/linolcic acid 40:40 mM system. Thesc results offer a pos-sible explanation for the reported enhance-ment in gastrointestinal absorption of B663when co-administered with fatty materi-als.—Authors' Abstract

Pertel, P. and Hirschtick, R. Adverse re-actions to dapsone in persons infectedwith human immunodeficiency virus.Clin. Infect. Dis. 18 (1994) 630-632.

Dapsone is used in prophylaxis for andtreatment of Pneumnoét'stis carinii pneu-monia. We present a case of Stevens-John-son syndrome that was likely induced byadministration of dapsone. A review ofcharts at the HIV Treatment Center ofNorthwestern University (Chicago, Illinois,

U.S.A.) revealed that 40.3% of patientstreated with trimethoprim-sulfamethoxa-zole could not tolerate the medication, while25.2% of those treated with dapsone wereintolerant of the drug. We also found a high-er rate of adverse reactions to dapsoneamong patients with prior intolerance to tri-methoprim-suflamethoxazole than amongpatients without such a history; however,the difference was not significant.—Au-thors' Summary

Rao, P. S., Ramachandran, A., Sekar, B.,Ravi, S. and Subramanian, M. Ofloxacin-containing combined drug regimens in thetreatment of lepromatous leprosy. Lepr.Rev. 65 (1994) 181-189.

A total of 26 clinically diagnosed adultpatients, with active untreated lepromatousleprosy, with a bacterial index of4+ or more,were admitted to the hospital of the CentralLeprosy Teaching and Research Instituto,Chengalpattu, India, between 1989 and1991. After prescribed investigations, thepatients were randomly allocated in groupsof 3 to three treatment regimens, namely:1)clofazimine 50 mg daily and 300 mg oncein 4 weeks + dapsone 100 mg daily (AA);2) (AA)+ofloxacin 400 mg daily (BB); and3) (AA)+ofioxacin 800 mg daily (CC). Thedrugs were administered for 56 days con-tinuously under supervision. Sequential bi-opsy results on days 0, 7, 14, 28 and 56 innormal mouse foot pad revealed no growthby days 28 and 56 in ali patients treatedwith CC and BB regimens, respectively.Calculation of the proportion of viable Alt'-cobacteriuni leprae through analysis of me-dian infectious dose (ID 50) showed signifi-cant differences on day 7 in the percentageofkill between the ofioxacin-containing reg-imens and the other. Moderate-to-markedclinical improvement has been observed ina significantly higher proportion of patientstreated with ofioxacin-containing regimens.All three regimens were well tolerated. Nosevere complications or side effects to thedrugs were noticed with any of the regimensthat required any suspension of treatmentor the administration of steroids. Additionof ofioxacin to the standard WHO recom-mended MDT regímen for multibacillarypatients may reduce the present duration oftherapy. Ofloxacin may also be considered


as an alternative drug in rifampin-resistantcases or where rifampin is contraindicat-ed. —Authors' Summary

Reepmeyer, J. C., Rhodes, M. O., Cox, D.C. and Silverton, J. V. Characterizationand crystal structure of two polymorphicforms of racemic thalidom ide. J. Chem.Soc. Perkin 2 (1994) 2063-2067.There are two polymorphic forms of ra-

cemic thalidomide. The alpha-polymorphwas formed by crystallization from2-ethoxycthanol, methanol or dichlorome-thane, while the beta-polymorph was formedby crystallization from a supersaturated so-lution in refluxing 2-ethoxycthanol. The twopolymorphs were characterized by IR spec-tra, differential scanning calorimetry (DSC),mclting points, powder X-ray diffractionpatterns, and X-ray crystallography. Theywere easily differentiated by IR (KBr) inwhich thc alpha-polymorph absorbed at3196, 3098 and 859 cm - ' and the beta at3276 and 755 cm - '. DSC scans of the alphaand beta forms showed endothermic pcaksat 272.3°C and 275.7°C, respectively. Dur-ing this measurement the alpha form waspartially or fully converted finto the beta formif the rate of temperature change was slowor if the alpha form contained some betawhich provided seed crystals for the inter-conversion. Upon remelting both forms gaveone pcak corresponding to the beta-poly-morph. The powder X-ray diffraction pat-terns for thc two forms differ significantly.The crystal structurcs of the two poly-morphs differ primarily in their mode ofhydrogen bonding. In the alpha-polymorphthe molecules forro dimers, while in the betathe dimers form infinite linear strings linkedby bifurcated hydrogen bonds along theb-axis.—Authors' Abstract

Rege, V. L., Shukla, P. and Mascarenhas,M. F. Dapsone syndrome in Goa. IndianJ. Lepr. 66 (1994) 59-64.

Dapsone syndrome was noted within 6weeks ofstarting treatment in 1.3% of about700 leprosy patients on MDT reporting tothe skin department of Goa Medical Col-lege. Skin rash, photosensitivity, fever,

lymphadenopathy, sore throat, hepato-splenomegaly, abnormal liver function testsand raised reticulocyte counts were consis-tent features in all the patients. Other drugs,infectious mononucleosis and virai exan-themata were considered in a differentialdiagnosis. Withdrawal of dapsone and ad-ministration of prednisolone controlled thecondition within 3 to 4 weeks in majorityof the patients. One patient died ofischemicheart disease unrelated to dapsone syn-drome. —Authors' Summary

Vage, C. and Svensson, C. K. Evidence thatthe biotransformation of dapsone andmonoacetyldapsone to their respectivehydroxylamine metabolites in rat livermicrosomes is mediated by cytochromeP450 2C6/2C11 and 3A1. Drug Metab.Dispos. 22 (1994) 572-577.

The formation of dapsone hydroxyla-mine (DDS-NOH) and monoacetyldapsonehydroxylamine (MADDS-NOH) was foundto be greater in male vs. female rat livermicrosomes, suggesting a role for eitherCYP2C1 1 or CYP3A2. Preincubation withcimetidine (selective for inhibition ofCYP2C 1 1), but not trolcandomycin (selec-tive for inhibition of CYP3A 1 /2), inhibitedmetabolite formation. Furthermore, incu-bation with monoclonal antibodies (Mabs)to CYP2C6/2C11 reduced metabolite for-mation to below the levei of detection. To-gether, these data indicate that N-hydrox-ylation of DDS and MADDS in rat livermicrosomes from untrcated male rats is cat-alyzed by CYP2C6/2C11. Interestingly,dexamethasone pretreatment increased thehydroxylation of both metabolites. Prein-cubation with cimetidine or Mabs toCYP2C6/2C11 (at an antibody:protein ra-tio of 26:1) in microsomes from dexameth-asone pretreated animals did not reduce theN-hydroxylation of DDS; whereas prein-cubation with troleandomycin reduced me-tabolite formation by < 50%. Collectively,these data indicate that the constitutive en-zymes CYP2C6 and/or CYP2C11, as wellas CYP3A1 (nonconstitutive), are capableof catalyzing thc hydroxylation of DDS andMADDS.—Authors' Abstract


Clinicai Sciences

Breen, G. A., Brocavich, J. M., Etzel, J. V.,Shah, V., Schaefer, P. and Forlenza, S.Evaluation of elfects of gastric pH on ab-sorption of dapsone in healthy volun-teers. Antimicrob. Agents Chemother. 38(1994) 2227-2229.

A prospective, randomized, crossoverstudy was performed with seven hcalthyvolunteers to address the e(fect of increasedgastric pH on dapsonc absorption. Subjectswcre randomized to receive a single 100-mg dose of dapsonc ora single 100-mg doseof dapsone in addition to 30 ml of a highpotcncy antacid 1 hr before dapsone ad-ministration and hourly thcreafter for a to-tal of 10 doses. Dapsone concentrations inserum were measured periodically for 48 hr.No statistical differences between the tworegimens were noted when mean dapsonemaximal initial concentrations, times topeak, and arcas under the curve were com-pared. These data suggest that an increasein gastric pH has little or no effect on theabsorption of dapsone in healthy sub-jects.—Authors' Abstract

Courtright, P., Hu, L. F., Li, H. Y. and Le-wallen, S. Multidrug therapy and eye dis-case in lcprosy; a cross-sectional study inthe Peoplc's Republic of China. Int. J.Epidemiol. 23 (1994) 835-842.

Factors associated with lcprosy-related eyedisease in a multidrug therapy (MDT) treat-ed population in China were assessed to de-termine if status prior to inclusion in theMDT program (newly diagnosed leprosypatient or leprosy patient on prior dapsonemonotherapy) contributed to the preva-lence of ocular pathology. Trained leprosyparamedical workers in Sichuan Provinceexamined 974 leprosy patients in a stan-dardized fashion. Univariate analyses andmultiple logistic regression were used to as-sess the contribution of demographic andclinical parameters to leprosy-related eyedisease. In both groups (prior dapsone andnew MDT) leprosy-related eye disease wasassociated with a longer distance to leprosyhealth worker or health center. Among pa-tients with a history of prior dapsone mon-otherapy, age and duration on dapsone

monotherapy were also associated with lep-rosy-related ocular morbidity. Among new-ly diagnosed leprosy patients the prevalenceof ocular morbidity remained between 8%and 11% regardless of when the patientstarted MDT. Our findings suggest that, evenwhen case detection is good, ocular pathol-ogy will still occur in MDT-treatcd leprosypatients. There remains an important rolefor health workers in the prevention of oc-ular morbidity. Our data also demonstratedthat pooling of results from all paticnts(newly diagnosed and on prior dapsoncmonotherapy) in a leprosy control programwill likely give riso to inadequate estimatesof risk of ocular disease due to variable clin-icai disease histories in these groups.—Au-thors' Abstract

Daniel, A. E., Arunthathi, S., Bhat, L. andRao, P. S. S. Intraocular pressure in lep-rosy patients without clinically apparentanterior segment pathology. Indian J.Lepr. 66 (1994) 165-172.

A widely prevalent notion is that intra-ocular pressures are generally lower in lep-rosy patients than in normal individuals.Applanation intraocular pressures were rc-corded in 166 lcprosy paticnts who had noclinically visible anterior segment pathol-ogy and in 1 1 1 healthy controls. Mcan (S.D.)intraocular pressures in leprosy patients(13.6 (3.0) mm Hg) did not differ signifi-cantly from that of controls (13.1 (2.7) mmHg). Eyes of only 1.5% of the leprosy pa-tients had pressures of 7 mm Hg or less.Correlation coefficients (r) between age, sexand intraocular pressures were not statisti-cally significant both in lcprosy patients andin controls. No statistically significant dif-ferences in mean intraocular pressures werenoted when leprosy patients were groupedaccording to the Ridley and Jopling classi-fication. Duration of disease also did notaffect the intraocular pressures. Neither didsmear positivity or differing bacterial indi-ces. This study questions the widely heldbelief that low intraocular pressures are acommon feature in leprosy and contendsthat in the era of MDT where ocular com-plications associated with low intraocular


pressures are thought to be less, the occur-rence of low intraocular pressure may notbe as common a phenomenon as it is be-lieved to be.—Authors' Abstract

Frommel, D., Tekle Haimanot, R., Verdier,M., Negesse, Y., Bulto, R. and Denis, F.HIV infection and leprosy: a four-yearsurvey in Ethiopia. Lancet 344 (1994)165-166.

Between 1988 and 1992 three cross-sec-tional surveys for antibodies to HIV weredone in 644 Ethiopian patients with histo-logically proven leprosy. Whereas the fre-quency of HIV-I infection gradually in-creased from 3.2% to 6.5%, the clinical pre-sentation, number of new patients, and fre-quency of relapse did not dilfer betweenHIV-negative and -positive patients. Thus,HIV appears unlikely to have a significantimpact on the incidence of leprosy in sub-Sahelian populations.—Authors' Abstract

Glaser, J. B., Levis, W. R., Gruber, T., Ca-brera, A. and Poiesz, B. J. Prevalence ofhuman T cell lymphotrophic virus(HTLV) types I and II and human im-munodeficiency virus type 1 infectionsamong persons with Hansen's diseasc inNew York City. J. Infect. Dis. 170 (1994)1007-1009.

One-hundred-seven consecutive patientsattending a New York Hansen's diseaseclinic from November 1990 through June1991 were tested for retroviruscs. This co-hort included 58 patients diagnosed withHansen's disease after the onset of the AIDSepidemic, 54 of whom immigrated to theUnited States before diagnosis of Hansen'sdisease (median, 7 years). The overall rate(1.9%) of human T-cell lymphotrophic vi-rus (HTLV) type I infection was low. Two(3.6%) of 55 Caribbean-born patients hadpolymerase chain reaction (PCR)-docu-mented HTLV-I infection, but this inci-dence was not higher than expected in per-sons without Hansen's disease. No patienthad PCR-documented evidence of eitherHTLV-II or human immunodeficiency vi-rus (HIV) type 1 infection. The low rate ofHIV-1 among those studied was likely re-lated to an absence of classic HIV risk be-havior because about halfofthe cohort could

have incubated Mvcobacteriuni leprae for aprolonged period while infected with HIV-1.—Authors' Abstract

Hiran, S., Pande, T. K., Pani, S. and Vish-w anathan, K. A. Dapsone agranulocytosisin a leprosy patient. Lepr. Rev. 65 (1994)279-281.

Dapsone-induced agranulocytosis is a rareadverse effect. There are various reports ofagranulocytosis in patients treated withdapsone for malaria prophylaxis and otherdermatological diseases. However, this ad-verse reaction in leprosy is not often en-countered. We describe agranulocytosis ina young patient who was taking dapsone(100 mg) for borderline tuberculoid leprosyin a rural environment.—Authors' Sum-mary

Lora Robles, M. and Gonzalez, K. [Dimor-phous leprosy with a lesion of the radialnervo; a case report.] Rev. Domin. Re-publ. 20 (1993) 55-57. (in Spanish)

The authors study a case of dimorphousleprosy with damage to the radial nervc ina 9-year-old male paticnt.—Authors' En-glish Summary

Lubbers, W. J., Schipper, A., Hogeweg, M.and de Soldenhofl, R. Eye disease in new-ly diagnosed leprosy patients in easternNepal. Lepr. Rev. 65 (1994) 231-238.

To determine the magnitude of eye le-sions in newly diagnosed leprosy patientswe examined their eyes. The Eastern Lep-rosy Control Project was supported by TheNetherlands Leprosy ReliefAssociation; weused the regional clinic in Biratnagar and 5mobile clinics in surrounding districts as oursurvey arca. All patients who presented atthe clinics over 10 weeks, diagnosed as hav-ing untreated leprosy were included. Of the260 examined patients 97 (37.3%, 95% con-fidence interval 28.3-40.3%) had an eye le-sion; 12/260 patients (4.6%, 95% confi-dence interval 2.0-7.2%) had sight-threat-ening lesions (lagophthalmos, iris involve-ment, corneal anesthesia), directly relatedto leprosy; 46 (17.7%) patients were diag-nosed as having some degree of cataract; 2patients were aphakic; 3 patients (1.2%) wereblind according to the WHO definition.


In this series ofnew and untreatcd leprosypatients many eye lesions found are not rel-evant or leprosy reeated. There were 9 newpatients with lagophthalmos, some toolongstanding to treat with steroids. We found3 patients with iris involvement. The figureswe found for eye lesion, sight-threateninglesions and blindness are low when com-pared to other studies. The number of pa-tients with any grade ofcataract is high. Thcaverage total of leprosy patients who wereblind can be compared with the average to-tal who are blind in the general popula-tion.—Authors' Summary

Mahajan, P. M., Jadhav, V. H., Patki, A.H., Jogaikar, D. G. and Mehta, J. M.Oral zinc therapy in recurrent erythemanodosum leprosum: a clinical study. In-dian J. Lepr. 66 (1994) 51-57.

The effect of oral zinc as an immuno-modulator was studied clinically in patientswith recurrent erythema nodosum lepro-sum (ENL) over a period of 1 year. In thisstudy, 40 leprosy patients with chronic ENL,requiring more than 30-40 mg of predni-solone/day for the control of thcir reactions,were given oral zinc sulfate for a period of4 months, and marked improvement in thefrequency, duration and severity of reac-dons was observed after zinc therapy. Alsoevident was marked reduction in the steroidrequirement after oral zinc therapy. It ap-pears that zinc may be a good substitute forthe present day antireaction treatment whichis not free from disadvantages. Further in-vestigations to know the precise action ofzinc on the immune system may help tounderstand the role of zinc therapy and itsoptimum duration. —Authors' Abstract

Pavithran, K. Palatal palsy in a case of lep-romatous leprosy. Lepr. Rev. 65 (1994)248-252.

A male patient with lepromatous leprosydeveloped nasal regurgitation of food dueto palatal palsy during type 2 reaction. Earlyhigh-dose administration of corticosteroidachieved a prompt therapeutic response andhe completely recovered from palatal palsy.The associated lagophthalmos, foot drop andulnar paralysis persisted.—Author's Sum-mary

Saran, B. R., Maguire, A. NI., Nichols, C.,Frank, I., Hertle, R. W., Brucker, A. J.,Goldman, S., Brown, M. and Vanuitert,B. Hypopyon uveitis in patients with ac-quired immunodcficiency syndrometreated for systemic Mvcobacteriunr ar-me complex infection with rifabutin.Arch. Ophthalmol. 112 (1994) 1159—1 165.

Iridocyclitis has been identified as a dos-age-dependent sido effect in patients withthe acquired immunodcficiency syndrome(AIDS) who are treated for AIycobactcrirmnarium complex (MAC) infection with sys-temic rifabutin. We reviewed cases of acutehypopyon uveitis occurring in patients withAIDS to establish whether there was an as-sociation. The cases carne from an outpa-tient clinic and inpatient hospital-basedophthalmology referral practice and infec-tious disease specialty service. Seven pa-tients with AIDS were studied, aged 10 to40 years, presenting with acute unilateralhypopyon mimicking infectious endo-phthalmitis. At the time of presentation, all7 patients were receiving treatment for MACinfection with rifabutin (dosage range 300to 600 mg/d) and clarithromycin. Resultsof microbiological investigations in 5 pa-tients were negative. Iridocyclitis becamebilateral in all 7 patients, and hypopyon de-veloped in the contralateral eye in 5 of 7patients. Hypopyon resolved rapidly withintensive topical corticosteroid therapy. Re-sidual inflammation responded to topicalcorticosteroids with or without reduction ofthe rifabutin dosage. Concomitant use ofrifabutin, clarithromycin, and fluconazolemay precipitate hypopyon uveitis in pa-tients with AIDS being treated for MACinfection.—Authors' Abstract

Schipper, A., Lubbers, W. J., Hogeweg, M.and de Soldenhoff, R. Disabilitics ofhands, feet and eyes in newly diagnosedleprosy patients in eastern Nepal. Lepr.Rev. 65 (1994) 239-247.

The objective of the study was to deter-mine the magnitude of hand/feet/eye dis-abilities in newly diagnosed leprosy patientsby examining all newly diagnosed leprosypatients who presented at the Eastern Lep-rosy Control Project (supported by TheNetherlands Leprosy Relief Association),


made up of a regional clinic in Biratnagarand 5 mobile clinics in surrounding dis-tricts. The study comprised of ali new andpreviously untreated patients who present-ed at the clinics over a 10-week period whowere diagnosed as leprosy sufferers. Of the260 leprosy patients examined 12 (4.6%)had sight-threatening lesions (lagophthal-mos, iris involvement, corneal anesthesia);3 patients were blind due to cataract; 96/260 patients (37.0%, 95% confidence inter-val 35.0-43.0%) had 1 or more disabilitiesof their hands and/or feet. The most fre-quently found disabilities were sensory lossof the hands and feet, claw hand and plantarulccrs. According to the WHO disabilitygrading 60% had no disabilities, 19% hadgrade 1, and 21% had grade 2 disability.Disability assessment is very important notonly to evaluate the effectiveness of the con-trol program but also for the patient, whosemost important worry is the stigmatizingdeformities leprosy patients suffer. The ear-lier detection of sensory loss might reducethese secondary deformities.—Authors'Summary

Sekar, B., Sharma, R. N., Anandan, D.,Vasanthi, B. and Jayasheela, M. Inde-terminate leprosy: a seroimmunologicaland histochemical evaluation. Lepr. Rev.65 (1994) 167-174.

An effort was made to differentiate in-determinate (IND) leprosy from other typesof the paucibacillary (PB) group of leprosyand to identify among indeterminate lep-rosy cases those which may evolve to mul-tibacillary (MB) leprosy, using serological,immunological and histochemical param-eters. A total of 92 untreated, histologicallyclassified (TT-19, BT-30, IND-32) patients,including 11 cases diagnosed as nonspecificdermatitis (NSD), which were clinicallystrongly suspected to be leprotic, werescreened for antibodies against PGL-I, 35-kDa and LAM antigens. Lepromin tests andantigen demonstration in tissue by indirectimmunoperoxidase staining were also car-ried out. Though a qualitative analysis didnot differentiate, a quantitative analysis interms of a cumulative index (CI) showed ahigher antibody levei among the indeter-minate group of patients than the othergroups included in PB leprosy. Also, the

lepromin-negative indeterminate group pa-tients showed a higher CI than the lepro-min-positive cases, indicating that perhapsthese may be the cases which may developinto MB leprosy. Thus, the semiquantifi-cation of antibody leveis in the form of aCI may be a useful parameter to predict thepossible evolution of a given case of inde-terminate leprosy. Interestingly 64% of NSDcases had either antigen or antibody, whichindicated that they were probably cases ofleprosy.—Authors' Summary

Soni, N. K. Eustachian tubc functions inlepromatous leprosy: a tympanometricstudy. Indian J. Lepr. 66 (1994) 45-49.

Tympanometry was performed in 20 pa-tients with lepramatous leprosy. About 30%showed tympanogram B type, indicatingmiddle-ear pathology which was shown tobe related to the stage of lepramatous rhi-nitis. The pathogenesis of middle-ear mal-function in lepromatous leprosy is dis-cussed. —Author's Abstract

Subramaniam, K., Nah, S. H. and Marks,S. C. A longitudinal study of alveolar boneloss around maxillary central incisors inpatient with leprosy in Malaysia. Lepr.Rev. 65 (1994) 137-142.

The loss of alveolar bone supporting themaxillary central incisors and the generalperiodontal conditions were evaluated after14 years in the 12 patients remaining froman original group of 47 under treatment inMalaysia. Alveolar bone loss was minimalduring this period even in the presence ofperiodontal infiammation. These data sug-gest that treatment protects patients withleprosy from alveolar bone loss and suggeststhat other skeletal deformities might re-spond similarly.—Authors' Summary

Turkof, E., Tambwekar, S., Mansukhani,K., Millesi, H. and Mayr, N. Intraoper-ative spinal root stimulation to detectmost proximal site of leprous ulnar neu-ritis. Lancet 343 (1994) 1604-1605.

In 10 borderline leprosy patients with lep-rous ulnar neuritis, we investigated the mostproximal site oflesion in the affected nerves.Spinal roots C8 and Ti were stimulated in-traoperatively to evoke efferent mixed com-

144^ International Journal of Leproso^ 1995

pound nerve action potentials which wererecorded from the exposed ulnar ncrves. Thesite at which amplitudes reached a maxi-mum was considered the most proximal siteoflesion. Nerve damage was found far prox-imally from the thickened segments in oth-erwise inconspicuous sections. The mostproximal site of lesion was finally localizedat the proximal third of the upper arm in 9cases and further centrally in 1 case. Epi-neuriotomy within thesc apparently unaf-fected segments revcaled fibrosis of the in-terfascicular epineurium in 9 patients, whichis an indication for microsurgical interfas-cicular neurolysis.—From the report

van Brakel, VV. H. and Khawas, I. B. Nervedamage in leprosy: an epidemiological andclinical study of 396 patients in west Ne-pal—Part I. Definitions, methods andfrequencies. Lepr. Rev. 65 (1994) 204-221.

An historie cohort study was performedto determine the prevalence and incidencerates of nerve function impairment (NFI)as demonstrated by sensory testing with anylon monofilament and standard tests ofmotor function. The records of 396 new lep-rosy patients registering at Green PasturesHospital, Pokhara, West Nepal, betweenJanuary 1988 and January 1992 were ana-lyzed. The mean follow-up period was 21months.

In all, 36% (141/396) of patients had ei-ther sensory or motor function impairmentat their initial examination. For each nervethe prevalence of sensory and motor im-pairment is reported separately. The pos-terior tibial nerve was the most frequentlyaffected (sensory) nerve (21 %). Sensory im-pairment of the ulnar nerve was found in17% of the patients; 8.8% had sensory im-pairment of the median nerve. The overallincidence rate of motor function impair-ment was 7.5 (5.4-10) per 100 person yearsat risk (PYAR). Sensory impairment had asignificantly higher rate of 13 (10-17)/ 100PYAR (rate ratio [ 1.8 (1.2-2.7), p = 0.0076].BL patients had a significantly higher inci-dence rate of nerve function impairmentthan BT patients (rate ratio 2.3 (1.4-3.7), p= 0.006). Altogether 152/396 (39%) of thepatients required corticosteroid treatmentfor "recent" or "acquired" impairment, and

78 of the patients (20%) developed severenerve function impairment during or afterantileprosy treatment. Analysis of potentialrisk factors for nerve function impairmcntshowed a significant association with theextent of clinical disease expressed as thenumber of body arcas (out of 9) with pri-mary or secondary signs of leprosy (rate ra-tio 5.0 (1.5-17), p = 0.0091).

It was concluded that nerve function im-pairment is a serious problem, often occur-ring during or after multidrug therapy. Theextent ofclinical disease expressed as a countof body arcas involved, or of skin or nervelesions, may identify patients who are atincreased risk of nerve damage.—Authors'Summary

van Brakel, W. H., Khawas, I. B. and Lucas,S. B. Reactions in leprosy: an epidemi-ological study of 386 patients in west Ne-pal. Lepr. Rev. 65 (1994) 190-203.

This paper presents epidemiological dataon reversal reaction (RR) and erythema no-dosum leprosum reaction (ENL) from a ret-rospective study of 386 leprosy patientsnewly registered at Green Pastures Hospi-tal, Pokhara, West Nepal. The average fol-low-up time was 21 months. The prevalenceof RR at first examination was 28% (23-32), and the prevalence ofENL reaction was5.7% (2.3-9.2). The overall incidence ratesamong the 335 patients that were availablefor follow up were 8.7 (6.5-12)/ 100 personyears at risk (PYAR) for RR and 3.2 (1.5-6.7)/100 PYAR for ENL. Reiapse of RRwas common (1.4/patient). In all, 52% ofRR were complicated by new nerve func-tion impairmcnt, against 59% of ENL re-actions. The finding of other investigatorsthat most RRs occur during the first year oftreatment was confirmed by this study. Themost significant risk factor for RR was ex-tent of clinical disease measured by a countof body arcas with clinical signs of leprosy.The risk of developing a RR for patientswith "extensive disease" (3 or more out of9 body arcas involved) was 10 times that ofpatients with limited disease (rate ratio 10(1.3-76), p = 0.026).

The study indicated that the followingcategorias of patients in Nepal are at highor increascd risk of developing a RR: 1)

63, 1^ Curreni Literatura^ 145

borderline paticnts during their first year of clinicai disease as described above.—Au-MDT; and 2) patients with more extensive thors' Summary

lmmuno-Pathology

Adams, E., Britton, W., Morgan, A., Ser-geantson, S. and Basten, A. Individuaisfrom different populations identify mul-tiple and diverse T-cell determinants onmycobacterial HSP70. Scand. J. Immu-nol. 39 (1994) 588-596.

The 70-kDa heat-shock protein (HSP) ofMvcobacieriurn leprae stimulates both cel-lular and antibody responses in leprosy pa-tients and subclinically infected individuaisdespite partial homology with host HSP70.Furthermore, mycobacterial HSP70 can actas a carrier protein in unprimed mico, sug-gesting the presence of widely shared T-celldeterminants on this protein. In order toclucidate the frcquency and genetic restric-tion of these T-cell epitopes, we have un-dertaken a systematic analysis of the pro-liferative responses to 20mer peptides en-compassing the whole protein in differentpopulations. Caucasian BCG vaccinees whorespondcd to recombinant M. leprae HSP70identified multiple scattered T-cell deter-minants, four of which were recognized by60% ofsubjects in association with a varictyof HLA-DR haplotypes. When a group ofNepali leprosy and tuberculosis patientswere tested, significant differences in thepattern of peptide recognition were ob-served. The dominant peptides recognizedby Caucasian subjects were infrequently re-active and other peptides were stimulatory,again in association with a variety of HLA-DR phenotypes. The C-terminal 70 resi-dues of the M. leprae HSP70 are specific toM. leprae and sera from lepromatous lep-rosy patients bind to this region. However,few T-cell determinants were identified inthese residues, indicating that this region isunhelpful as a diagnostic tool for detectingM. /eprae-specific T-cell responses. Whencompared with the equivalent regions of thehuman HSP70, the commonly recognizedpeptides showed significant differences inamino-acid sequence. When taken in con-junction with the failure of human HSP70

to stimulate M. leprae HSP70-reactive T-cellclones (E. Adams et al., unpublished ob-servations), this finding indicates that thehuman T-cell response to this protein islargely directed at mycobacterial-specificdeterminants. The presence of multipleT-cell epitopes on AI. leprae HSP70 withvaried patterns of HLA-DR associationsuggests that the whole protein is requiredfor stimulating effective T-cell responses ingenetically diverse populations.—Authors'Abstract

Anand, P., Pandya, S., Ladiwala, U., Singh-al, B., Sinicropi, D. V. and WilliamsChestnut, R. E. Depletion of nerve growthfactor in leprosy. (Letter) Lancet 344(1994) 129-130.

Recent advances have led to the identi-fication of neurotrophic factors that rescuenerve fibers after injury and disease in an-imal models of neuropathy and help nerveregeneration. The best established of these,nerve growth factor (NGF), is normally pro-duced in skin and taken up by nerve fibersvia receptors on nerve terminals. We reportthat NGF leveis are depleted in skin andnerve from patients with leprosy.—Froco theletter

Appelberg, R., Castro, A. G., Pedrosa, J.and Minoprio, P. Role of interlcukin-6 inthe infection of protective T cells duringmycobacterial infections in mice. Im-munology 82 (1994) 361-364.

Interleukin-6 (IL-6) has been shown toregulate numerous functions of the immunesystem, including the differentiation ofT-ccllsubpopulations. Here we examined the in-volvement of this cytokine in the in vivogeneration of a population ofT cells able toprotect mice against mycobacterial infec-tions. BALB/c mice were infected intrave-nously with AI. anan i 2447 and anti-IL-6monoclonal antibodies were administered


intraperitoneally throughout the course ofthe infection. Control mice were able tocontrol the mycobacterial proliferation 1month after inoculation; whereas micewhosc IL-6 had been blocked showed pro-gressive bacterial growth. To distinguish arole for IL-6 associated to the induction orexpression of immunity mediated by T cells,we immunized mice with AI. bons bacillusCalmette-Guérin (BCG) Pasteur and chal-lenged them 2 months later with AI. ariuni.One group of mice received anti-IL-6 dur-ing the BCG vaccination and another duringthe Al. aviunl challenge. When AI. ariumproliferation was assessed at day 30 of thechallenge, it was found that the administra-tion of anti-IL-6 during vaccination re-duced the protection afforded by BCG com-pared to administration of the isotype con-trol antibody. No difference in bacterialproliferation was observed at day 30 ofchal-lenge when antibodies were administeredduring Al. anui! challenge. Our results showthat protective T cells arise during AI. ariunlinfections in mice after differentiating in thepresence of IL-6.—Authors' Abstract

Barbosa, A. D., Silva, T. C., Patel, B. N.,Santos, M. I. R., Wakamatsu, A. andAlves, V. A. F. Demonstration of myco-bacterial antigens in skin biopsies fromsuspected leprosy cases in the absence ofbacilli. Pathol. Res. Prac. 190 (1994) 782-785.

Skin-biopsies from 56 patients suspectedof early leprosy from Bahia State, Brazil,were examined histopathologically. TheFite-Faraco staining failed to demonstrateacid-fast bacilli in this material. The prom-inent features of the lesions were inflam-mation of the neurovascular bundles andsometimes inflammation of the skin ap-pendages. The nonspecific infiltrate waspredominantly composed of histiocytes andlymphocytes. In 41 cases (73.2%) epidermalatrophy was also present. The avidin-biotinperoxidase technique was used with pri-mar antibodies to detect bacillary antigens(anti-BCG serum) and nerve branches (anti-S-100 protein serum). Immunohistochem-ical detection of bacillary antigens using theanti-BCG serum was positive in 28 cases(50%). A positive staining for S-100 proteinwas observed in 40 cases (71.4%) in den-

dritic antigen-presenting cells of the skin.The detection of bacillary antigens, togetherwith the clear demonstration of nerve bun-dles enhanced our capacity to fulfill mor-phologic criteria for the diagnosis of earlyleprosy. Our observations indicate that theuse of immunohistochemical methods rep-resent a useful tool for the carly diagnosisof leprosy.—Authors' Abstract

Bharadwaj, V. P. The present status of se-rological techniques and future perspec-tives in the study of epidemiology of lep-rosy. Jpn. J. Lepr. 62 Suppl. (1993) 152-172.

Severa! seroassays are now available fordetection of Myeobacteriunl lepr•ae infec-tion. It appears that at present FLA-ABS isthe most sensitive test for detection of Al.leprae infection in the community and, be-sides estimating the prevalence of infectionit is likely to be quite useful for monitoringthe transmission of the disease. Great stridesin the serological techniques and their ap-plication to epidemiology of leprosy havebeen made. Traditionally, the presence ofantigcn/antibody lias been investigated insera/leprous tissues but it might bc worth-while to investigate other body fluids/cx-cretory fluids as well. The field of serologyof leprosy is still evolving. It appears thatin coming years there would be more in-formation available on the combined ap-plication of various serological assays (whichmay be further modified by the newer ad-vances about antigens/epitopes) and molec-ular techniques for better understanding ofthe epidemiology of the disease.—From theArtic!e

Brown, D. H. and Zwilling, B. S. Activationof the hypothalamic-pituitary-adrenal axisdifferentially affects the antimycobacter-ial activity of macrophages from BCG-resistant and -susceptible mice. J. Neu-roimmunol. 53 (1994) 181-187.

The effect of hypothalamic-pituitary-ad-renal (HPA) axis activation and exogenousglucocorticoids on the ability of splenicmacrophages to control the growth of My-cobacteriunl ariunl was evaluated. We foundthat activation of the HPA axis by restraintstress or the addition of corticosterone in-


creased the susceptibility of macrophagesfrom mice that are innately susceptible tothe in vivo growth of Al. aviam. In contrast,the ability of macrophages from innatelyresistant, congenic mice to control thegrowth ofM. aviam was not affected by HPAactivation or the addition of corticosterone.The effect of restraint and of corticosteroneon macrophage function was abrogated byeither trcating mice with the glucocorticoidreceptor antagonist RU486 or the additionof the drug to cultures of macrophages. Ac-tivation of the HPA axis as well as the ad-dition of corticosterone to cultures of mac-rophages resulted in a suppression of theproduction of tumor necrosis factor (TNF)-alpha and of reactive nitrogen intermediatesby macrophages from both strains of mice.The lack of effect of HPA activation and ofcorticosterone on the mycobacterial resis-tance of macrophages from BCG-resistantmice, while at the same time suppressingthe production of reactive nitrogen inter-mediates, appears to rule out a role for thisantimicrobial pathway in innate resistanceto mycobacterial growth.—Authors' Ab-stract

Castells Rodellas, A., Garcia-Patos Briones,V., Repiso 1Iontero, T. and Terencio delas Aguas, J. [The immunology of leprosy1993.] Rev. Leprol. Fontilles 19 (1994)477-532. (in Spanish)

The macrophage immune response, hu-moral and ccllular immunity against .11r-cobacteriuin leprae and its different antigensare revicwed (270 references). Total andpartial defects of the ccllular immunity existwith disturbed secretion pattcrns of cyto-kines while the humoral immunity is un-altered. The immunodeficiency is not sat-isfactorily explained.—Authors' EnglishSummary

Chui, D. H., Tabira, T., Izumi, S., Koya, G.and Ogata, J. Decreased beta-amyloid andincreased abnormal tau deposition in thebrain of aged patients with leprosy. Am.J. Pathol. 145 (1994) 771-775.

We examined the brains of 37 leprosypatients (mean age 76.3 ± 7.8 years), 5 pa-tients with Alzheimer-type dementia (meanage 79.0 ± 9.5 years), and 23 age-matched

nondementia controls (mean age 77.6 ± 5.4years). The frequency of beta-amyloid(Abeta)-positive cases was lower (27.0%) inleprosy patients (N = 37) than in controls(47.8%; p = 0.05, Z = 1.49). When senileplaque subtypes were examined, type III(classical) plaques were significantly fewer(p < 0.05) in leprosy subjects compared withcontrols. Interestingly, neurofibrillary tan-gles in the temporal cortei were much morefrequent in leprosy patients than in controls(p < 0.05). Howevcr hippocampal CA3 py-ramidal neurons in leprosy patients werewell preserved. These data indicate that: 1)leprosy patients have a low risk of Abetadeposition but a high risk of abnormal taudeposition, 2) abnormal tau deposition isunrelated to AP deposition in leprosy, and3) neuronal loss is unrelated to abnormaltau deposition. It is not clear at presentwhether the results is related to the diseaseprocess itsclf, antileprosy treatmcnt, envi-ronmental fictors, or the genetic back-ground in leprosy patients.—Authors' Ab-stract

Denis, M. Intcrlcukin-12 (IL-12) augmentscytolytic activity of natural killcr cell to-ward Mrcobacterium tuberculosis - infcct-ed human monocytes. Ccll. Immunol. 156(1994) 529-536.

We tested the impact of interleukin-12(IL-12) and of cytotoxic leukocytes [partic-ularly natural killer (NK) cclls] from normaland HIV-1-infected subjects on lysis of hu-man monocytes infected with Mycobacte-riunm tuberculosis. Nylon -wool -nonadher-ent cells stimulated with interleukin-2 (IL-2) or with IL-12 developed significant kill-ing activity against infected monocytes, withIL-12 being a superior stimulant on a molarbasis. Cells of the CD16+ phenotypc me-diated most of the cytotoxicity against in-fected monocytes, and this lytic activity wasassociated with a significant decrease in my-cobacterial numbers. When peripheral bloodmononuclear cells (PBMC) froni HIV-1-in-fected subjects were examined, it was alsofound that IL-12 very significantly in-creased lytic activity against JI. tubercalo-sis-infected cells. Moreover, purified NKcells from normal voluntecrs or from HIV-1-infected subjects were shown to have el-evated lytic activity against AI. tuberculosis-


infected monocytes after IL-2 or IL-12stimulation. These data suggest an impor-tant involvement of NK cens and their ac-tivating stimuli (particularly IL-12) in hostresistance to tuberculosis.—Authors' Ab-stract

Desikan, P., Parkash, O. and Narang, P.The role of antiperipheral nerve antibod-ics in nerve damage in leprosy. Lepr. Rev.65 (1994) 222-230.

The objective of this study was to deter-mine the role of antineural antibodies inleprosy. Indirect ELISA using antigen pre-pared from normal human peripheral nerveswas carried out on the Bera from 100 leprosypatients and 18 normal controls. In total,9% of the patients had demonstrable levelsof IgG antineural antibodies and 1 1 % haddemonstrable levels of IgM antibodies.There was no correlation with the type ofleprosy, bacteriological index, treatmenttaken, the presence of a reactional state, thepresence of enlarged nerves or active neu-ritis.—Authors' Summary

Dumarey, C. H., Labrousse, V., Rastogi, N.,Vargaftig, B. B. and Bachelet, M. Sclec-tive Mycobacteriunt avium-induced pro-duction of nitric oxide by human mono-cyte-derived macrophages. J. Leuk. Biol.56 (1994) 36-40.

Infection with a virulent strain of Mv-cobacteritnn ariunt, but not with virulentM. tuberculosis or avirulent M. smegmatis,induced the formation of nitric oxide byhuman monocyte-derived macrophages.This process was not affected by lipopoly-saccharide or cytokines such as interferon-gamma or tumor necrosis factor alpha. Af.ati•itn-induced nitric oxide production wassignificantly decreased by N-G-monome-thyl-L-arginine, a potent inhibitor of nitricoxide synthase activity, without any signif-icant enhancement of intramacrophagicmycobacterial growth. Infection with all thethree mycobacterial species induced a sig-nificant activation of phospholipase A(2)activity of macrophages as evidenced by theincreased release of thromboxane A(2). Fi-nally, nitric oxide production by humanmonocyte-derived macrophages requiredinfection with live M. arium, as neither

gamma-irradiated M. anuiu nor the sub-cellular fractions of this microorganism (cellwall, cytosol) were able to trigger nitric ox-ide synthesis.—Authors' Abstract

Flesch, I. E. A., Hess, J. H., Oswald, I. P.and Kaufmann, S. H. E. Growth inhibi-tion of Atycobacterium bons by IFN-gamma stimulated macrophages— regu-lation by endogenous tumor necrosis fac-tor-alpha and by IL-10. Int. Immunol. 6(1994) 693-700.

Murine bone marrow-derived macro-phages (BMM) are able to inhibit the intra-cellular growth of Mycobacterium bons andM. tuberculosis H37Rv after activation withrecombinant (r) IFN and growth inhibitionis mediated by reactive nitrogen interme-diates (RNI) derived from L-arginine. Wenow demonstrate that tumor necrosis factor(TNF)-alpha acts as an endogenous cofactorin the induction of mycobacterial growthinhibition. TNF-alpha was produced byBMM stimulated with rIFN-gamma and in-fected with mycobacteria, and a specifie an-tiserum to TNF-alpha inhibited rIFN-gam-ma-induced production of RNI as well asgrowth inhibition of M. bons. IL-10, a cy-tokine which suppresses antimycobactcrialmacrophage functions, was also producedby BMM activated with rIFN-gamma andinfected with A7. bons. IFN-gamma-in-duced production of TNF-alpha and of re-active nitrogen intermediates as well as my-cobacterial growth inhibition were inhibit-ed by exogenous IL-10, but only when givenprior to IFN-gamma stimulation. We con-clude that the outcome of mycobacterial in-fection is regulated by a coordinate inter-play between IFN-gamma, TNF-alpha andIL-10. —Authors' Abstract

Gelber, R. H., Mehra, V., Bloom, B. R.,Murray, L. P., Siu, P., Tsang, M. andBrennan, P. J. Vaccination with pure My-cobacterium leprae proteins inhibits M.leprae multiplication in mouse foot pads.Infect. Immun. 62 (1994) 4250-4255.

In this study, we evaluated vaccinationwith a number of purified, as well as recom-binant, Mycobacterium leprae proteins forprotective efficacy in mice. BALB/c micewere immunized intradermally with vari-

63, 1^ C rrent Literature^ 149

ous nativo somatic (purified) or recombi-nant AI. leprae proteins and their syntheticpolypeptides emulsified in Frcund's incom-plete adjuvant. The protective efficacy ofthese preparations was assessed by enu-meration of bacilli in thc foot pads of micechallenged with viable AI. leprae 1 to 2months following immunization. Protec-tion was afforded by the purified and re-combinant 10-kDa M. leprae cytoplasmicheat shock protein, the recombinant cellwall-associated 65-kDa AI. leprae heat shockprotcin and, to a lesser extent, the purified28-kDa AI. leprae cytoplasmic protcin (su-peroxide dismutase). Vaccination with ei-ther the purified or recombinant 35-kDa A7.leprae cell membrane protein, the synthetic27-amino-acid N-terminal peptide of the 10-kDa protein, the recombinant 18-kDa Al.leprae protein, or the purified 22-kDa cellmembrane protein was ineffective. Whenthe interval between immunization andchallenge was increased to 6 months, thepurified 10-kDa M. leprae protein and therecombinant 65-kDa AI. leprae protein lostvaccine efficacy, while a sodium dodecylsulfate-soluble protein fraction of the AI.leprae cell wall (solublc proteins), as hadbeen found previously, continued to pro-tect, suggesting that multiple Al. leprae pro-tein epitopes are criticai for solid vaccincprotection.—Authors' Abstract

Gheorghiu, M., Lagranderie, M. R. R., Gic-quel, B. M. E. and LeClerc, C. D. My-cobacterium bons BCG priming inducesa strong potentiation of the antibody re-sponse induced by recombinant BCG ex-pressing a foreign antigen. Infect. Immun.62 (1994) 4287-4295.

Several recent studies have dcmonstratedthat strong cellular or humoral immune re-sponses can be induced against foreign an-tigens expressed by recombinant Mycobac-terium bons BCG. It has therefore been sug-gested that BCG could represent one of thebest candidate vectors for live recombinantvaccines. However, a large percentage of thehuman population has been immunized byBCG, and this priming could modify theimmune response to future recombinantBCG vaccines. In the present study, we havetherefore compared the immune responsesinduced in naive and BCG-primed mice by

two recombinant BCG vaccines expressingeither beta-galactosidase or human immu-nodeficiency virus type 1 Nef antigens. Ourresults demonstrated that BCG priminglimits thc growth of recombinant BCG inmouse splcen or lymph nodes. This reduc-tion in BCG growth was associated with de-creased prolifcrative responses against Nefor beta-galactosidase antigens. This sup-pression, however, never exceeded 50%. In-terestingly, in contrast to these reducedT-cell responses, BCG-primed mice devei-oped high leveis of anti-beta-galactosidaseantibodies after immunization with recom-binant BCG expressing this antigen. Thisstimulation of antibody responses was notdue to polyclonal stimulation or to a non-specific adjuvant ef ect of BCG. The isotyp-ic patterns of anti-beta-galactosidase anti-body responses induced by the recombinantBCG were similar in naive and BCG-primedmice. These results indicate that primingwith BCG will not be a limitation for theuse of recombinant BCG vaccines in hu-mano.—Authors' Abstract

Haftel, H. NI., Chang, Y., Hinderer, R.,Hanash, S. M. and Holoshitz, J. Induc-tion of the autoantigen proliferating cellnuclear antigen in T lymphocytes by amycobacterial antigen. J. Clin. Invest. 94(1994) 1365-1372.

Mycobacteria have been implicated in thepathogenesis of autoimmunity. To deter-mine the potential efrect of mycobacterialantigens on peripheral blood mononuclearcells (PBMC), we analyzed PBMC incubat-ed with the acetone-precipitable fraction ofMycobacterium tuberculosis (AP-MT) forchanges in cellular induction of a 36-kDapolypeptide identified as proliferating cellnuclear antigen (PCNA), a known autoan-tigen, after incubation with AP-MT. PCNAplays a role in cell proliferation and is ex-pressed as a late growth-regulated factor.However, its synthesis in response to AP-MT was induced as an early event. The earlyinduction of PCNA was regulated at a post-transcriptional levei and was restricted to Tcells. Trcatment of PBMC with known T-cellmitogens, namely PHA, anti-CD3 antibod-ies, and staphylococcal superantigens, failedto induce an early PCNA increase. The dis-tinct characteristics of the AP-MT el%et on


PCNA expression suggest a separate mech-anism of induction in response to AP-MT,compared with the late incrcase observedin response to mitogens. The induction ofPCNA in response to mycobactcrial anti-gens may represcnt a pathogenically rele-vant mechanism in autoimmunity.—Au-thors' Abstract

Hetland, G. and Wiker, H. G. Antigen 85Con Alvcobacterium bonis, BCG and ,11. tu-berculosis promotes monocyte-CR3-me-diated uptake of microbeads coated withmycobacterial products. Immunology 82(1994) 445-449.The uptake in monocytes of monodis-

persed latex microbeads precoated withwhole bacillus Calmctte-Guérin (BCG) cells,Mvcobacteriunt tuberculosis sonicatc or cul-ture fluid, or antigen (Ag) from the culturefluid was examined by microscopy. Therewas a significantly higher ccll association ofbcads coated with wholc BCG edis, the se-creted 85C component of the Ag85 complexor M. nrberculosis sonicate than of phos-phate-buffcred salino (PI3S)-trcated controlbeads. Antibodies (Ab) to Ag85 inhibitedthe uptake of BCG- and Ag85C-trcatedbeads. A monoclonal antibody (mAb) tocomplement receptor type 3 (CR3), but notmAb to CRI, inhibited the uptake ofAg85C-coated beads, indicating that the mycobac-terial Ag-dependent uptake of particles wasmediated via CR3 on the monocytes. Thispoints to the existence of a ligand on Ag85Cwhich may promote monocyte uptake ofM.bons, BCG and M. tuberculosis. —Authors'Abstract

Ilangumaran, S., Narayan, N. P. S., Ramu,G. and Muthukkaruppan, V. Ceilular andhumoral immune responses to recombi-nant 65-kD antigen of Rlvcobacterirnnleprae in leprosy patients and hcalthycontrols. Clin. Exp. Immunol. 96 (1994)79-85.

Cellular and humoral immune responsesto recombinant 65-kDa antigen of Myco-bacterium leprae (rML65) were studied inleprosy paticnts and hcalthy contacts froma leprosy-endemic population. Peripheralblood mononuclear edis from a corisider-able proportion of tuberculoid leprosy pa-tients, hcalthy contacts and noncontacts

showed proliferative response to rML65 irtnitro. A strong positive correlation was ob-served between the responses to rML65 andbacille Calmctte-Guérin (BCG) or leprosinA. Addition ofrecombinant IL-2 (rIL-2) en-hanced the proportion of responders torML65 considerably in ali groups of leprosypatients, hcalthy contacts and noncontacts.Among lepromatous patients this enhance-ment was more pronounced in the bacterialindex (BI)-negative group. These results in-dicate that the 65-kDa antigen of .11. lepraeis a dominant T-cell immunogen in ourstudy population. Though lepromatous pa-tients showed poor lymphoproliferative re-sponse to rML65, their IgG antibody levclsto the same antigen were markedly high.Most of the BI-positive lepromatous pa-tients with elevated anti-rML65 IgG levclsdid not show T-ceil reactivity evcn with theaddition of rIL-2. On the other hand, tu-berculoid leprosy patients, hcalthy contactsand noncontacts showed good T-cell reac-tivity but low levcls of IgG antibodics torML65, thus indicating the presence of aninverso relationship between cell-mediatedand humoral immune responses to a definedprotein antigen of A1. leprae in humans. Asignificant proportion of individuais amongtubcrculoid leprosy patients, hcalthy con-tacts and noncontacts showed neither T-ceilreactivity nor elevated levcls of IgG anti-body to rML65. However, in most of thesesubjects, a T-cell response to rML65 wasdemonstrable with the addition of rIL-2.These results are discussed with referenceto the immunoregulatory mechanisms oc-curring during RI. leprae infection on thebasis of differential activation of Th 1 andTh2 subsets.—Authors' Abstract

Kawamura, I., Yang, J. F., Takaesu, Y., Fu-jita, M., Nomoto, K. and Mitsuyama, M.Antigen provoking gamma interferonproduction in response to ,llvcobacteriumbons BCG and functional difference inT-cell responses to this antigcn betweenviable and killed BCG-immunized mico.Infect. Immun. 62 (1994) 4396-4403.

It has been shown that gamma interferon(IFN-y)-producing CD4+ T cells, which aregenerated only by immunization with via-ble bacteria, exert a significant role in pro-tective immunity against mycobacteria in


mice. In this study, we have tried to deter-mine the antigen recognized by the T edisin scarch of a possible protective antigen. Tcells from viable Mvcobacterium buris BCG-immunized mice were stimulated with sev-eral antigens, and IFN-y production wasmeasured. Purified protein derivative andviable and killed BCG lysates caused sig-nificant IFN-y production, and almost thesame levei of IFN-y activity was detectedin both groups stimulated with viable endkilled BCG lysates. However, heat shockprotein (HSP) 65 and HSP 70 were not amajor antigen for IFN-y production. Theantigen provoking IFN-y production is lo-calized mainly in the membranc fraction ofBCG cens, and the approximate molecularsize was 18 kDa. On the other hand, T cellsfrom killed BCG-immunized mico never re-sponded to this antigen for IFN--y produc-tion; whereas they could mount a delayed-type hypersensitivity response. These re-sults showed that the antigen provokingIFN-y production was present in killed aswell as viable BCG. In addition to the an-tigen presentation by antigen-presentingcens, some kinds of diffcrentiation factor(such as monokines) that are produced onlyby stimulation with viable cens scemed tobe necessary for the development of IFN-y-producing T cens.—Authors' Abstract

Launois, P., Deleys, R., Niang, M. N., Dro-wart, A., Andrien, M., Dierckx, P., Car-tel, J.-L., Sarthou, J. L., Van Vooren, J.P. and Huygen, K. T-cell-epitope map-ping of the major secreted mycobacterialantigen Ag85A in tuberculosis and lep-rosy. Infect. Immun. 62 (1994) 3679-3687.

Lymphoproliferation and gamma inter-feron (IFN-y) secretion in response to 28overlapping 20-mer synthetic peptides cov-ering the complete sequence of the mature(295-amino-acid) 85A component of themajor secreted, fibronectin-binding antigen85 complex from Mvcobacterii nn tubercu-losis and M. bons BCG (MTAg85A) wasexamined by using peripheral blood mono-nuclear cell (PBMC) cultures from healthytuberculin- and lepromin-positive volun-tecrs and from patients with tuberculosisand leprosy. Peptide recognition was largelypromiscuous, with a variety of human leu-

kocyte antigen haplotypes reacting to thesame peptides. PBMC from ali tuberculin-positive subjects rcacted to Ag85, and themajority proliferated in response to peptide6 (amino acids 51 to 70), peptides 13, 14,and 15 (amino acids 121 to 160), or peptides20 and 21 (amino acids 191 to 220). PBMCfrom tuberculosis patients demonstrated avariable reactivity to Ag85 and its peptides,and the strongest proliferation was observedagainst peptide 7 (amino acids 61 to 80).MTAg85A peptides were also recognized byPBMC from healthy lepromin-positive vol-unteers and paucibacillary leprosy patients(again in a promiscuous manner), but de-spite a 90% homology between the 85A pro-teins of M. leprae and Aí. tuberculosis, thepeptides recognized were different. PBMCfrom lepromin-positive healthy contacts re-acted against peptide 2 (amino acids 11 to30), peptide 5 (amino acids 41 to 60), andpeptides 25 and 26 (amino acids 241 to 270).PBMC from paucibacillary patients reactedpreferentially against peptide 1 (amino ac-ids 1 to 20) and peptide 5. Multibacillarypatients were not reactive to Ag85 or theMT85A peptides. IFN-y production wasgenerally detected simultaneously with pos-itive lymphoproliferative responses, al-though peptide 1 mostly stimulated prolif-eration and peptides 27 and 28 mostly elic-ited an IFN-y response. In conclusion,regions 41 to 80 and 241 to 295 demon-strated powerful and promiscuous T-cell-stimulatory properties, resulting in prolif-erative responses and IFN-y secretion, re-spectively, in the majority of reactive sub-jects tested in this study. These results couldbe of value in the development of a subunitvaccine for tuberculosis and leprosy.—Au-thors' Abstract

Launois, P., Ndiaye, M. N., Sarthou, J. L.,Drowart, A., Van Vooren, J. P., Cartel,J. L. and Huygen, K. T-cell reactivityagainst antigen-85 but not against the 18-kD and 65-kD heat shock proteins in theearly stages of acquired immunity againstMycobacterium leprae. Clin. Exp. Im-munol. 96 (1994) 86-90.

T cell proliferation and interferon-gam-ma (IFN-y) production of peripheral bloodmononuclear cells (PBMC) from 20 house-hold contacts were tested against the 18-


and 65-kDa heat shock proteins from My-cobactcriuni leprae (ML18 and ML65, re-spectively) and antigen 85 from ,11. bonsbacille Calmette-Guérin (BCG) (Ag 85) dur-ing a 12-months follow-up study. Amongthe eight contacts that became positive, eightshowed positive reactivity against Ag 85,5/8 against ML65 and 4/8 against ML18 atthe end of the study. Of the 16 contacts whowere lepromin-positive either at first or sec-ond testing, ali responded to Ag 85, 11 toML65, but only eight reacted to ML18 an-tigen. Contacts who were lepromin-positiveat first testing developed responses to M L 18only at second testing. In contrast, amongthe four contacts that remained lepromin-negative during the follow up, three prolif-erated to Ag 85 either at first or second test-ing, but only one produced IFN-y againstAg 85 at the end of the study. Thesc resultsdemonstrated that T-cell reactivity and par-ticularly IFN-y secretion against Ag 85, butnot against ML18 and ML65, might be apredominant mechanism in the early stagesof acquired protective immunity against M./eprac.— Authors' Abstract

Modlin, R. L. Thl-Th2 paradigm: insightsfrom leprosy. J. Invest. Dermatol. 102(1994) 828-832.

The mechanism by which T cell and cy-tokines regulate immune processes in skincan be investigated by studying patients withleprosy. The diseasc, caused by the obligateintraceilular bacterium M1'cobacteritmt lep-rae, forms a spectrum. At one polo, patientswith tuberculoid leprosy are able to restrictthe growth of the pathogen and their skinlesions are characterized by a predominanceof CD4+ T ceils and type 1 cytokines in-cluding interleukin 2 and interferon gam-ma. At the opposite pole, patients with lep-romatous leprosy are unable to contain theinfection and their skin lesions are charac-terized by a predominance of CD8 + T censand type 2 cytokines including interlcukins4 and 10. A key determinant of the T-cellcytokine response may be interleukin 12,which selectively favors expansion of CD4+T cens producing interferon gamma. By un-derstanding the factors that regulate T-celland cytokine responses in leprosy, it shouldbe possible to devise specific immunologicinterventions in diseases ofskin.—Authors'Abstract

Nogueira, A. C., Neuhert, R., Helge, H. andNeuhert, D. Thalidomide and the im-mune system. 3. Simultaneous up- anddown-regulation of different integrin re-ceptors on human white blood edis. LifeSci. 55 (1994) 77-92.

Time-dependcnt changes in the surfaccreceptor expression of various maturationaland integrin receptors on peripheral bloodcell were studied in two healthy human vol-unteers following oral applications of tha-lidomide (Thd). In cach measurement thereceptor density was quantified by prior cal-ibration of the flow cytometer with latexbeads bearing a determined number of flu-orescence molecules. The effects observedin the course of the Thd-treatment werepractically identical or at least very similarin both of the volunteers during four dif-ferent trials, and were in accord with pre-vious results obtained in large-scale studies(68 trealed animais) with nonhuman pri-nmates. It should be stressed that no clear-cut changes were observed in the percentageor absolute numbers ofprimary lymphocytesubsets suei as CD3, CD4 and CD20. Afterthe first two doses of 7 mg Thd/kg body wtthe CD 18 (the common beta-chain of thebeta 2-integrins) marker already decreasedin surface density or was no longer detect-able on granulocytes, monocytes and lym-phocytes. This effect persisted throughoutthe treatment period and slowly subsidedafter discontinuation of treatment. With afew days lag phase, the surface density ofCD54 (ICAM- I) on granulocytes increasedand many cens previously not bearing thisreceptor newly acquired such surface mark-ers. On monocytes, however, the CD54 re-ceptor was lost on many cens. Within thelymphocyte fraction a loss of the CD54marker could be noted on CD4 cens but noton CD8 cens, where an increase of the re-ceptor expression could be observed. Othermarkers, such as the alpha chains of the beta1 integrins CD49b (VLA alpha 2) andCD49d (VLA alpha 4) showed contrastingreactions to the Thd-treatment. Whereas apronounced loss of the receptor density ofCD49d was observed and only few cells withhigh epitope density were left in the bloodat the end of the complete dosing schedule,no such effect was observable on cens bear-ing the CD49b epitope. A distinct reductionof the number of rcceptors was also notice-

63, 1^ Current Literalure^ 153

able on L-selectin (Leu8) bearing cens. OnCD4 positive lymphocytes, the majority ofthe described effects on the integrin and ad-hesion receptors was seen on edis bearingthe CD45RO maturational epitope. Thisfunctional receptor is strongly downregu-lated and the pathway of CD45RA toCD45RO maturation is apparently alteredby Thd-treatment. These multiple changeswe observed may explain the large varietyof therapeutic effects experienced in thetreatment with Thd.—Authors' Abstract

Ohmen, J. D., Barnes, P. F., Grisso, C. L.,Bloom, B. R. and Modlin, R. L. Evidenceof a superantigen in human tuberculosis.Immunity 1 (1994) 35-43.

T cens are not only required for resistanceto tuberculosis, but they likely contribute tothe tissue damage characteristic of the dis-case. To define better the T-cell populationsthat contribute to the immunopathogenesisof human tuberculosis, we investigated theT-cell receptor (TCR) beta chain repertoireexpressed in patients with tuberculous pleu-ritis. Analysis by polymerase chain reactionand flow cytometry indicated an expansionof V beta 8(+) T cens at the site of diseasein some donors, suggesting the possibilitythat Mvcobacteriunz tuberculosis contains asuperantigen. M. tuberculosis induced strongT-cell proliferative responses in tuberculin-negative healthy donors in ri!ro, with pref-erential expansion of V beta 8(+) T cens,independent of the CDR3 region. T-cellstimulation was MHC class II-dependentand did not require antigen processing bythe antigen-presenting cens. These findingsare consistent with the presence of a super-antigen in M. tuberculosis, aspects of whichmay contribute to the immunopathology oftuberculosis and to the adjuvant propertiesof Al. tuberculosis. —Authors' Abstract

Ortega, V. V., Diaz, F. M., Pacheco, G. O.and Rubiales, F. C. Ultrastructural studyacross the leprosy spectrum. Ultrastruct.Pathol. 18 (1994) 423-432.

We have carried out a systematic ultra-structural study of the bacilli, the cell-me-diated response in the host, and the dermalmicrovasculature in lepromatous (LL), bor-derline lepromatous (BL), and borderlinetuberculoid (BT) types of active leprosy

(eight cases). In the types of least resistance(LL and BL), macrophages with large cy-toplasmic processes were observed; in ad-dition, numerous peripheral vacuoles werefound in BL. Mast cens were abundant andvascular alterations constant. BT macro-phages showed more regular outlines andmultivacuolated cytoplasms with plentifulrough endoplasmic reticulum. Giant censwere scarce. Bacilli, both isolated and inglobi, were contained within the vacuolesand appeared constantly in macrophages andendothelial and Schwann edis in LL andBL. Conversely, in BT they were found sin-gly, infrequently in the endothelial cens, andnot at all in Schwann cens. Forms in theprocess of destruction or degradation weremore common than intact forms, in whichthe symmetric outline of the membranecould be seen clearly.—Authors' Abstract

Peetermans, W. E., Raats, C. J. I., Langer-mans, J. A. M. and Van Furth, R. My-cobacterial heat-shock protein 65 inducesproinflammatory cytokines but does notactivate human mononuclear phagocytes.Scand. J. Immunol. 39 (1994) 613-617.

The 65-kDa heat-shock protein (Hsp65),a well-conserved and immunodominant an-tigen which elicits a cellular and humoralimmune response, may play a role in hostdefense against invading microorganismsand autoimmune disorders. The aim of thepresent study was to assess the effects ofHsp65 on the functional activities of humanmononuclear phagocytes in the absence oflymphocytes. Incubation with Hsp65 re-sulted in an enhanced release of TNF-alphaand IL-1 beta by human monocytes andmonocyte-derived macrophages (MDM).The amount of cytokines released by theseeens in response to Hsp65 was similar tothat released in response to IFN-gamma to-gether with LPS. Incubation with ovalbu-min did not stimulate the release of thesecytokines. In ritro stimulation of monocyteswith Hsp65 enhanced the membrane ex-pression of complement receptor III but didnot influente either the expression of Fcgamma-receptor I and HLA class-II anti-gens or the release of reactive oxygen inter-mediates. Therefore, Hsp65-stimulatedmonocytes cannot be considered to be ac-tivated according to classical criteria. Therelease of the proinflammatory cytokines


TNF-alpha and IL-1 beta by human mono-nuclear phagocytes in response to Hsp65indicates that this protein can contributo toboth host defense and tissue damage in in-flammatory lesions characterized by anabundant expression of Hsp65.—Authors'Summary

Roche, P. W., Peake, P. W., Davenport, M.P. and Britton, W. J. Identification of aAlvcobacteriunr leprae-specific T-cell ep-itope on the 70 kDa heat shock protein.Immunol. Ccll Biol. 72 (1994) 215-221.

A major antigen of the leprosy bacillus,Mvcobacterium leprae, is the 70-kDa heat-shock protein (Hsp70), which has signifi-cant sequence homology with Hsp70 fromother mycobacterial spccics as well as Hsp70from eukaryotes. A unique region of 70amino acids at the C-terminus of the AI.leprae Hsp70 has been previously identi-fied. This study investigated whether miceimmunized with the C-terminal fragmentof AI. leprae Hsp70 recognize T-cell epi-topes in this spccics-specific portion of themolecule. Murine lymphoproliferative re-sponses to overlapping peptides spanningthe C-terminal 70 amino acids were re-stricted to mice of an H-2 (b) haplotype andidentified the presence of a determinant insequence 567-591. Lymph-node cells frommice immunized with this peptide recog-nized both the C-terminal fragment and thewhole Hsp70 molecule. Moreover, miceimmunized with the same peptide respond-ed to the whole Hsp70 molecule in a de-layed-type hypersensitivity reaction. Thesignificance of Aí. leprae-specific T-cell ep-itopes in the host response to mycobacterialinfection is discussed.—Authors' Abstract

Santos, D. O., Suffys, P. N., Moreira, A. L.,Bonifacio, K., Salgado, J. L., Esquenazi,D., Bertho, A. L. and Sarno, E. N. Eval-uation of chemiluminescence, procoagu-lant activity and antigen presentation bymonocytes from lepromatous leprosy pa-tients with or without reactional episodes.Lepr. Rev. 65 (1994) 88-99.

In this study, we evaluated the activity ofperipheral blood mononuclear cells (PBMC),isolated from treated and untreated lepro-matous leprosy patients, from lepromatous

leprosy patients during and after reactionalepisodes [erythema nodosum leprosum(ENL) and reversal reaction (RR)], and fromnormal healthy individuais. We determinedreactive oxygen intermediate (ROI) pro-duction, procoagulant activity (PCA) andH LA-DR antigen expression of monocytes,besides lymphoproliferation, both in thepresence and absence ofvarious stimulatoryagents. Phorbol myristate acetato (PMA)stimulated ROI production by monocytesfrom ali the groups studied, with patientsduring reactional episodes (ENL and RR)showing a significantly highcr response (p< 0.009 and p < 0.00001). Irradiated Alv-cobacteriunr leprae, although having littleeffect when added alone, strongly sup-pressed PMA-stimulated ROI production.Muramyl dipeptide (MDP) had no influenceon cither basal or on PMA-induccd ROIproduction. Basal monocyte PCA, as wellas AI. leprae or concanavalin A (ConA)-in-duced monocyte PCA, was comparable inmonocytes from ali the groups studied.ConA was able to induce mitogcnic activityin mononuclear cells isolatcd from ali thegroups studied. AI. leprae, although stimu-latory for normal individuais, did not in-ducc lymphoproliferation in lepromatousleprosy patients, except for cells from pa-tients during RR, which responded equallyto AI. leprae and to ConA. The absence ofM. leprae-induccd lymphoproliferation inlepromatous leprosy patients is not causedby the lack of basal HLA-DR expression,as PBMC from ali individuais studicdshowed the same levei of this antigen. Ourresults suggest an increase of spontaneousor PMA-induced monocyte activity, as de-tected by ROI production, during the reac-tional episode; addition of M. leprae sup-pressed this response. The increase inmonocyte activity could be correlated withthe increase of lymphoproliferation re-sponse to AI. leprae during RR, but not dur-ing ENL. The importance of a possible im-mune suppressive aation of AI. leprae is dis-cussed. —Authors' Summary

Schoel, B., Sprenger, S. and Kaufmann, S.H. E. Phosphate is essencial for stimu-lation of V gamma 9V delta 2 T lympho-cytes by mycobacterial low molecularweight ligand. Eur. J. Immunol. 24 (1994)1886-1892.


T lymphocytes are divided into two sub-sets which express different T-cell receptorheterodimers. In the peripheral blood ofhealthy individuais, the majority of T censexpress the alpha/beta T-cell receptor (>90%) while a minority have the gamma/delta T-cell receptor (< 10%). The gamma/delta T cells of adults use preferentially theV gamma 9V delta 2 chain combination.Although the stimulation requirements forgamma/delta T lymphocytes are still un-determined, it has been reported that gam-ma/delta T cens are not only stimulated,like alpha/beta T edis, by conventional pro-tein antigens and superantigens, but also byunusual ligands. Mycobacteria selectivelystimulate V gamma 9V delta 2 T cens, anda nonproteinacious low molecular weightfraction of 1-3 kDa lias been identified asthe tentative active component. Here, weconfirm the nonproteinacious nature of thisligand, and show that it is comprised of un-usual carbohydrate and phosphate. Impor-tantly, cleavage of the terminal phosphateby alkaline phosphatase completely abro-gates the stimulatory activity of the low mo-lecular weight ligand for V gamma 9V delta2 T cells. Even mycobacterial whole lysateloses its stimulatory activity, for this T-cellsubset, after dephosphorylation with alka-line phosphatase. These findings identifyphosphocarbohydrates as a novel molecularentity with selective stimulatory activity fora defined T-cell subset.—Authors' Abstract

Shetty, V. P., Uplekar, NI. W. and Antia,N. H. Immunohistological localization ofmycobacterial antigens within the periph-eral nerves oftreated leprosy patients andtheir significance to nerve damage in lep-rosy. Acta Neuropathol. 88 (1994) 300-306.

The presence and distribution of Mvco-bacteriurn leprae-specific and crossreactingantigens within the peripheral nerves ofmultidrug-treated patients with leprosy wereinvestigated to gain a better understandingof the mechanism of nerve damage and theeffect of multidrug therapy (MDT) on it.There was no specific qualitative differencein the type of antigens in the leprosy spec-trum. However, our results indicate thatthere may be differential handling of anti-gens by the macrophages as compared to

Schwann edis. This could play a key role inthe pathogenesis of the disease. Multidrugtreatment is effective in arresting the pro-gression of the disease process as well as inreducing the viable bacterial ioad, both inborderline lepromatous and lepromatous(MB) and borderline tuberculoid and tu-berculoid (PB) cases. However, the presenceof AI. leprae antigens in ali the multidrug-treated MB nerve lesions and 87% of PBnerve lesions suggest that the antigens per-sist for a prolonged period. Hence, the riskof immunological reaction and antigen-as-sociated silent nerve damage may continueeven after the majority of AI. leprae werekilled. The findings give further support tothe view that most of the nerve damage isdue to bacterial antigens.—Authors' Ab-stract

Torres, M., Mendez Sampeiro, P., JimenezZamudio, L., Teran, L., Camarena, A.,Quezada, R., Ramos, E. and Sada, E.Comparison of the immune responseagainst Alrcobacteritnn tuberculosis an-tigens between a group of patients withactive pulmonary tuberculosis and healthyhousehold contacts. Clin. Exp. Immunol.96 (1994) 75-78.

The mycobacterial antigens and the fac-tors related to protection for the develop-ment of active tuberculosis are not known.In a natural model of tuberculosis, we stud-ied 10 patients with active pulmonary tu-berculosis (nonprotcctive immune re-sponse) and 38 healthy household contacts(protective immune response). We tested thelymphocyte proliferati ve response by T-cellWestern blotting to eight different antigenfractions and to two purified mycobacterialantigens of 30 and 64 kDa. Patients withactive tuberculosis recognized fractions withmolecular weights of 80-114, 60-80, 28-41 and 14-19 kDa. Household contacts rec-ognized the same fractions except the 14-19 kDa. The response to the 64-kDa antigenwas not significantly different betweengroups. In contrast, 10% of the patients withactive tuberculosis and 73% of the house-hold contacts responded to the 30-kDa an-tigen. The humoral response against the 30-kDa antigen by ELISA showed a signifi-cantly higher production of antibodies intuberculosis patients compared with house-


hold contacts. We conclude that patientswith active pulmonary tuberculosis developan immune response characterized by poorproliferative response to the 30-kDa antigenwith a strong humoral response; whereas theopposite occurs in healthy subjects infectedby AÍvcobacte ritnn tuberculosis.— Authors'Abstract

Wadee, A. A., Sussman, G., Kuschke, R. H.and Reddy, S. G. Suppression of cytokineproduction by supernatants from CD8+lymphocytes activated by mycobacterialfractions: the role of interleukins 4 and 6.13iotherapy 7 (1994) 125-136.

Supernatants derived from CD8+ lym-phocytes treated with mycobacterial com-ponents, or the partially purified carbohy-drates from these supernatants, increasedthe production of IL-4 and IL-6 by mono-nuclear cells. Thc addition of anti-IL4 oranti-IL6 antibodies to LPS-stimulated MNcells incubated with supernatants fromCD8+ lymphocytes or carbohydrates re-sulted in the restoration of other cytokineproduction by these MN cells. Recombi-nant IL-4 and IL-6 on their own suppressedthe production ofIL-1 beta, TNF-alpha, IL-2, and IFN-gamma by mononuclear cells.Such suppression could be reversed with an-tibodies to IL-4 and IL-6. The addition ofrIL-4 and rIL-6 did not increase the sup-pression of cytokinc production induced bysuppressor supernatants or carbohydrates.IL-4 decreased the production of IL-6 byMN edis; IL-6 suppressed IL-4 productionin a dose-dependent manner. Both effectscould be reversed with the appropriate an-tisera. Our results suggest that mycobacteriacould evade host immunity by inducing theproduction of IL-4 and I L-6 by host mono-nuclear cells. These cytokines, in turn, wouldsuppress the production of other cytokinesnecessary for effective cellular immunity.-Authors' Abstract

Wheeler, P. R., Raynes, J. G. and McAdam,K. P. W. J. Autoantibodies to cerebrosidesulphate (sulphatide) in leprosy. Clin. Exp.Immunol. 98 (1994) 145-150.

Sera from 40 leprosy patients werescreened for autoantibodies to cerebrosidesulfate (sulfatide). Anti-sulfatide IgM ingroups of patients with lepromatous (LL)

and borderline (BL + BB + BT), but notwith tuberculoid (TT) disease, were signif-icantly elevated above the levels found inendemic control subjects. Eight-six percent(18 out of 21; mean 1.59 OD units) of LL,33% (four out of 12; mean 1.59 OD units)of borderline and 13% (one out of eight;mean 0.69 OD units) oftuberculoid patientshad anti-sulfatide IgM in their sera abovea cut-oW value of 2 S.D. above the meanvalue (0.66 OD units) for control sera. El-evated anti-sulfatide IgG was detected inonly one patient's serum, an individual withLL disease. Thc levei of anti-sulfatide IgMwas strongly correlated to expression of theTH3 idiotype, an idiotype previously de-fined by a human MoAb that bound A/v-cobacterium leprae phenolic glycolipid,K/ebsiella capsular polysaccharide, poly-nucleotides and human tissues. The puri-fied, TH3 MoAb was found in this study tobind sulfatide, but not cholesterol-3-sulfateor cerebroside. It is suggested that antisul-fatide IgM is elevated in leprosy, in relationto the bacterial load. Anti-sulfatide IgM fellat the onset oferythema nodosom leprosum(ENL) reaction, consistent with the depo-sition of serum antibodies, and thus mayplay a part in pathology during periods ofinflammation, particularly in multibacillarypatients. — Authors' Abstract

Xu, S. M., Cooper, A., Sturgill Koszycki, S.,Van Heyningen, T., Chatterjee, D., Orme,I., Allen, P. and Russell, D. G. Intracel-lular tralficking in Atvcobacteriurn tuber-culosis and Alvcobacteriunn ariuin-infect-ed macrophages. J. Immunol. 153 (1994)2568-2578.

Despite the potential role of the macro-phage in the eradication of invading mi-crobes, Mvcobacterium species have evolvedmechanisms to ensure their survival andreplication Inside the macrophage. Particlesphagocytosed by macrophages normally willbe delivered into acidic lysosomal com-partments for degradation. Mycobacteriamust, in some way, avoid this fate by mod-ulation of their phagosome. Immunoelec-tron microscopy of macrophages infectedwith Mvcobacteriunz arünn or M. tubercu-losis indicates that the vacuolar membranesurrounding the bacilli possesses the late en-dosomal/lysosomal marker, LAMP-1 (ly-sosomal-associated membrane protein-1),


but lacks the vesicular proton-ATPase.Analysis of the intersection of the bacteria-containing vacuoles with the endocytic net-work of the macrophage supports previousstudies indicating that these bacilli restrictthe fusion capability of their intracellularcompartments. The occurrence of vesiclescontaining lipoarabinomannan, discretefrom those containing mycobacteria, indi-cate that material does traffic out from themycobacterial vacuole. To compensate forthis loss of membrane, the vacuole mustremain dynamic and fuse with LAMP-1-containing vesicles to maintain the densityof this marker.—Authors' Abstract

Yassin, R. J. and Hamblin, A. S. Alteredexpression of CD 1 1 /CD 18 on the periph-eral blood phagocytes of patients with tu-berculosis. Clin. Exp. Immunol. 97 (1994)120-125.

Tuberculosis (TB), caused by Mvcobac-teriu n i tuberculosis, is characterized bygranulomatous lesions made up of epithe-lioid cells, giant cells and mononuclear leu-kocytes. Cell-cell adhesion is important ingranuloma formation and in the leukocytemigration which accompanies it. We have

recently shown increased expression of theadhesion molecules CD 1 1 /CD 18 (Leu-CAMs, beta (2) integrins) on peripheralblood leukocytes from patients with sar-coidosis (Shakoor and Hamblin, 1992). Herewe have studied the expression of CD! 1 /CD18 and CD29 (VLA beta (1) integrin) onthe peripheral blood leukocytes of 10 TBpatients by flow cytometry. The density (ex-pressed as mean fluorescence intensity) ofCD! l b on monocytes and polymorphs wasincreased (p < 0.005), as was CD! 1 c (p <0.005) and CD18 (p < 0.05) on poly-morphs. CD1 la expression was significant-ly reduced on polymorphs (p < 0.05). Nodifferences were found in the expression ofCD29, the percentages of cells expressingany molecule and, in contrast to sarcoidosis,the density of any molecule on lympho-cytes. Although the cytokine tumor necrosisfactor (TNF) has been implicated in the pro-cess of up-regulation, an ELISA for TNFfailed to detect significant leveis in plasma.The results suggest increased peripheralphagocyte CD1 I/CD18 expression is a fea-ture of TB, which may contribute to thepathological processes involved.—Authors'Abstract

Microbiology

Baelden, M. C., Bouckaert, A. E., Gregoire,D., Vandepoele, M. and Coene, M. Com-parison of thermostable macromolecularantigens from leprosy-associated bacte-ria. Can. J. Microbiol. 40 (1994) 508-512.

Three types of bacteria are associated withleprosy: MVcobacteriunn leprae, leprosy -de-rived corynebacteria (LDC), and armadillo-derived mycobacteria (ADM). The immu-nological relationships between these threetypes of bacteria and AI. bons BCG, usedas a reference, were determined by cross-immunoelectrophoresis. When comparedwith the reference, crossreactions were ob-served with a variable number of antigens:2 in the case of strain LDC 15, 4 with M.leprae, and from 1 to 10 in the case of theADM, depending on their subgroup. Next,thermostable macromolecular antigens(TMAs), the major crossreactive antigens of

leprosy-associated bacteria, were comparedby anti-TMA antibody ELISA tests. TheLDC TMAs displayed high crossreactivitybetween the subgroups and lower crossre-activity with the TMAs of :II. bovis BCG.Evidence for the presence of a species-spe-cific moiety in TMA of the different LDCwas obtained by using depleted anti-TMAantisera. Western blot analysis revealed thepresence of many proteins in the TMAs ofLDC and Al. bovis BCG, some of them beingspecies-specific and other crossreactive.-Authors' Abstract

Bhaduri, T. and Nagaraja, V. DNA topo-isomerase I from AIvcobacteriunt smeg-nzatis. Indian J. Biochem. Biophys. 31(1994) 339-343.

DNA topoisomerase I has been purifiedfrom AI}'cobacteriunt snlegnlatis to near ho-

158^ International Journal of Leprosr^ 1995

mogeneity using diflcrent column chro-matographic techniques. The enzyme activ-ity relaxes form I DNA into form iV DNA,requiring Mg2+, but not ATP or any othercolactors for its activity. Several propertiesof the enzyme were found to be similar tothat of the prototype enzyme, Escherichiacoli topoisomerase I.—Authors' Abstract

Bhatia, V. N. Repeated isolation of der-matophilus-Iike organism from leprosymaterial. Indian J. Lepr. 66 (1994) 149-156.

An organism having actinomyatoid-typecolonies has been grown repeatedly in pureculture from leprosy material using a solidmedium. The isolates obtained from diffcr-en! biopsies, skin scrapes and mouse foot-pad harvests were found to be similar toeach other on the routine taxonomical testsand had many characters common withDern/o/ophilus congolensis. Similar growthcould be obtained from a strain of Mrco-bacterium leprae from armadillo and alsofrom a DOPA-positive mycobacterium iso-lated previously from the blood of a leprosypatient.—Author's Abstract

Cirillo, J. D., Weisbrod, T. R., Banerjee, A.,Blocou, B. R. and Jacobs, W. R., Jr. Ge-netic determination of the mcso-diami-nopimelate biosynthetic pathway of my-cobacteria. J. Bacteriol. 176 (1994) 4424-4429.

The increasing incidence of multiple-drug-resistant mycobacterial infections indicatesthat the development of new methods fortreatment of mycobacterial diseases shouldbe a high priority. coeso-Diaminopimelicacid (DAP), a key component of a highlyimmunogenic subunit of the mycobacterialpeptidoglycan layer, has been implicated asa potential virulence factor. The mycobac-terial DAP biosynthetic pathway could serveas a target for design of new antimycobac-terial agents as well as the construction ofin vivo selection systems. We have isolatedthe asd, dap.-1, dapB, dapD, and dapE genesinvolved in the DAP biosynthetic pathwayof Mvcobacteriunl bovis BCG. These geneswere isolated by complementation of Esch-erichia coli mutations with an expressionlibrary of BCG DNA. Our analysis of these

genes suggests that BCG may use more thanone pathway for biosynthesis of DAP. Thenucleotide sequence of the I3CG dapB genewas determined. The activity of the productof this gene in E. coli provided evidencethat the gene may encode a novel bi func-tional dihydrodipicolinate reductase andDAP dehydrogenase.—Authors' Abstract

Colston, M. J. The molecular biology ofMrcobactericnn leprae. (Editorial) Lepr.Rev. 64 (1993) 289-294.

This editorial is a modified version of apresentation made to the Royal Society ofTropical Medicine and Hygiene at a mect-ing to mark the 30th anniversary of the Rid-ley-Jopling classification for leprosy. The fullpaper was to be published in the Transac-tions of the Roval.Societ r o/ Tropical Med-icine and IIygiene. Topics covered here in-clude the taxonomic position of .11rcobac-terium lepra(' based on nucleic acid analysis,cloning and expression of M. leprae genes,molecular methods for rapid detection andidentification of Al. leprae, the A1. lepraegenome map, and the molecular basis ofrifampin resistance and rapid detection ofresistant isolates.—C. A. Brown (Trop. Dis.Bull.)

de Wit, NI. Y. L. and Klatser, P. R. Mrco-bacteriunl leprae isolates from differentsources have identical sequences of thespacer region between the 16S and 23Sribosomal RNA genes. Microbiology 140(1994) 1982-1987.

To test for genotypic variations betweendifferent isolates of Alrcobacteriunt leprae,the causative agent of leprosy, the 282-bpspacer region between the 16S and 23SrRNA genes was amplified using PCR, andsubmitted to single-strand conformationpolymorphism (SSCP) anlaysis. The pro-cedure was optimized using four modifiedspacer fragments, containing mutations atone, three, four and six positions, respec-tively. Seventy-fìve Al. leprae isolates fromdifferent sources, including isolates fromleprosy patients, healthy individuais, ar-madillos and mouse foot pads, were iden-tical in the SSCP analysis. DNA sequencingand restriction enzyme analysis performedon four and 40 samples, respectively, con-


fìrmed the results obtained with SSCP anal-ysis. — Authors' Abstract

Griggs, D. J., Hall, M. C., Jin, Y. F. andPiddock, L. J. V. Quinolone resistance inveterinary isolates of salmonella. J. An-timicrob. Chemother. 33 (1994) 1173—1 189.

Twenty-seven nalidixic acid-resistant(MIC ? 256 mg/L) isolates of salmonellafrom veterinary sources were also less sus-ceptible to fluoroquinolones (range of MICsof ciprofloxacin, 0.12-2 mg/L). Six isolateswere crossresistanl to one or more chemi-cally unrelated antibacterial agents. Theconcentrations of enrofloxacin that inhib-ited DNA synthesis by 50% were similar tothe MIC values for 23 of 27 isolates, sug-gesting a mutation in gi'r.•l. Insertion ofpNJR3-2 (gt'rA) in 9 of 20 isolates increasedsusceptibility to quinolones, suggesting thatresistance was due to mutation in gt'r; I. Fiveof 27 isolates had reduced leveis of accu-mulation of enrofloxacin. Two of the Eivealso had increased susceptibility to quino-lones when pNJR3-2 was introduced. Noneof the outer membrane protein profiles ofthe resistam isolates differed from those ofsensitive control strains. Three of 27 iso-lates had differences in lipopolysaccharideprofiles compared to control strains. Al-though the MIC of ciprofloxacin was lessthan the recommended U.K. break pointconcentrations for most isolates, the in-creasing incidence of quinolone-resistancein salmonella from veterinary sources is amatter of concern.—Authors' Abstract

Ji, Y., Kempsell, K. E., Colston, NI. J. andCox, R. A. Nucleotide sequences of thespacer-1, spacer-2 and trailer regions ofthe rrn operons and secondary structuresof precursor 23S rRNAs and precursor 5SrRNAs of slow-growing mycobacteria.Microbiology 140 (1994) 1763-1773.The single ribosomal RNA (rrn) operons

of slow-growing mycobacteria comprise thegenes for 16S, 23S and 5S rRNA, in thatorder. PCR methodology was used to am-plify parts of the rrn operons, namely thespacer-1 region separating the 16S rRNAand 23S rRNA genes and the spacer-2 re-gion separating the 23S rRNA and 5S rRNA

genes of Mi'cobacterium ai'ium, M. intra-cellulare, "AL Iuf r"and AI. simiae. The am-plified DNA was sequenced. The spacer-2region, the 5S rRNA gene, the trailer regionand the downstream region of the rrn op-erou of M. tuberculosis were cloned and se-quenced. These data, together with thoseobtained previously for Al. Ieprac, were usedto identify putative antitermination signalsand RNasc III processing sites within thespacer-1 region. Notablc features includetwo adjacent potencial Box B elements anda Box A element. The latter is located withina sequence of 46 nucleotides which is veryhighly conserved among the slow-growerswhich were examined. The conserved se-quence has the capacity to interact throughbase-pairing with part of the spacer-2 re-gion. Secondary structures for mycobacter-ial precursor 23S rRNA and for precursor5S rRNA were devised, based on sequencehomologies and homologous nucleotidesubstitutions. All the slow-growers, includ-ing Al. Ieprae, conform to the same schemeof secondary structure. A putative motif forthe intrinsic termination oftranscription wasidentified approximately 33 bp downstreamfrom the 3'-end of the 5S rRNA gene. Thespacer-1 and spacer-2 sequences may provea useful supplement to 16S rRNA sequencesin establishing phylogenetic relationshipsbetween very closely related species.—Au-thors' Abstract

Linton, C. J., Jalal, H., Leeming, J. P. andMillar, NI. R. Rapid discrimination ofAIi'cobacteriuni tuberculosis strains byrandom amplified polymorphic DNAanalysis. J. Clin. Microbiol. 32 (1994)2169-2174.

Investigations of the epidemiology of tu-berculosis have been hampered by the lackof strain-specific markers that can be usedto differentiate isolates of Alt'cobacteriumtuberculosis. We report the development ofa rapid protocol for random amplified poly-morphic DNA analysis which included theuse of a commercially available DNA ex-traction kit (GeneReleaser). This was ap-plied to 14 strains of AI. tuberculosis, in-cluding strains associated with temporal andgeographical clusters of tuberculosis in theUnited Kingdom and those from India, Áf-rica, and Saudia Arabia. Strains of Al. tu-

160^ International Journal of Lcprosy^ 1995

herculosis could be discriminated in about8 hr by this method, which is, therefore, arapid and simple alternativa to restrictionfragment length polymorphism analysis.-Authors' Abstract

Lopez Marin, L. M., Quesada, 1)., Lakh-darghazal, F., Tocanne, J. F. and La-neelle, G. Interactions of mycobacterialglycopeptidolipids with membranes: in-fluence of carbohvdrate on induced alter-ations. 13iochemistry 33 (1994) 7056-7061.

Glycopeptidolipids (GPLs) are specifìcconstituents of mytohactcria known as op-portunistic pathogens. The influence of thecarbohvdrate moiety on GPL-induccdmembrane alterations was examined withGPLs bearing 1-5 sugar residues (GPL-1 toGPL-5) and a sulfated GPL (S-GPL-2).GPLs decreased the ADP/O ratio and in-creased controlled respiration of isolatedmitochondria. The more polar GPLs werethe less active, with the following order ofefficiency: GPL-1 > GPL-2 > S-GPL-2 =GPL-3 = GPL-5. GPL-1 and GPL-2 in-creased passive permcability of liposomesto carboxyfluorescein (GPL-1 > GPL-2),while GPL-3 and GPL-5 were inactivo.GPL-2 and GPL-3 decreased the trans-membrane electrical potential (Delta Psi) inisolated mitochondria (GPL-2 > GPL-3).These results suggest that GPLs uncoupleoxidative phosphorylation by increasing thepassive permcability of the mitochondrialmembrane to protons. Compression iso-therms of GPL-2 monolayers showed that,at low surface pressure, the arca per GPL-2molecule was about 5 times that of an acylchain: it is likely that the peptide moietywas at the air/water interface. With an in-crease in the surface pressure, its arca de-creased, down to that of a tightly packedacyl chain. It is postulated that the glyco-peptidic moiety can be either at the interfaceor dipping finto the water. GPL-2 insertionin liposomes rendered the acyl-chain partof the bilayer more accessiblc to ions, lincea fluorescent probe located deep in the bi-layer was much more quenched by Cu2+ions in liposomes containing GPL-2 thanin control liposomes, suggesting a distur-bance of the bilayer interface. A modal isproposed to explain the influence of the po-

larity of GPLs on their activity towardmembrane properties.—Authors' Abstract

Pessolani, M. C. V., Smith, I). R., Rivoire,B., NIcCormick, .I., IIefta, S. A., Cole, S.T. and Brcnnan, P. J. Purifìcation, char-acterization, gene sequence, and signifi-cance of a bacterioferritin from .Ilt co-bacierium leprae. J. Exp. Med. 180 (1994)319-327.

The study of tissue-derived Mycobactc-riunr lcprac providos insights to the im-munopathology of leprosy and hclps iden-tify broad molecular features necessary formycobactcrial parasitism. A major mem-brane protein (MMP-II) of in viro-derived.11. lepra(' previously recognized (Hunter, S.W., 13. Rivoire, V. Mehra, B. R. Bloom, andP. J. Brcnnan. 1990. J. Biol. Chem. 265:14065) was purifìed from extracts of the or-ganism and partia! amino acid sequence ob-tained. This information allowed recogni-tion, within one of the cosmids that encom-pass the entire A1. lcprac genome, of a com-plete gene, bfr, encoding a protein ofsubunitsize 18.2 kD. The amino acid sequence de-duced from the major membrane protein II(MMP-II) gene revealed considerable ho-mology to severa! bacterioferritins. Analy-sis of the native protein demonstrated theiron content, absorption spectrum, and largcnative molecular mass (380 kD) of severa!known bacterioferritins. The ferroxidaso-center residues typical of ferritins were con-served in the Al. lepra' product. Oligonu-cleotides derived from the amino acid se-quence of A1. lepra(' bacterioferritin enabledampliUcation of much of the MMP-II geneand the detection of homologous sequencesin R7. paratuberculosis, M. avium, Al. tu-berculosis, A. intraccllulare, and M. scro-fulaceum. The role of this iron-rich proteinin the virulence ofM. lepra(' is discussed.-Authors' Abstract

Rastogi, N., Goh, K. S., Wright, E. L. andBarrow, W. W. Potencial drug targets forMi'cobactcriuni aviunt defined by radio-metric drug-inhibitor combination tech-niques. Antimicrob. Agents Chemother.38 (1994) 2287-2295.

Previously established radiometric tech-niques werc used to assess the efTectiveness


of combined antimicrobial drug-inhibitorydrug (drug-inhibitor) trcatment on two clin-ical isolates of the Alycobacteriunl aviunlcomplex representing threc colony variants:smooth opaque (dome) (SmO), smoothtransparent (SmT), and rough (Rg). All var-iants were identified as members of the M.arllun complex; however, only the SmT col-ony type of strain 373 possessed character-istic serovar-specific glycopeptidolipid(GPL) antigens. MICs, determined radio-metrically, of drugs with the potential toinhibit the biosynthesis of GPL antigens orother cell-envelope constituents wcre sim-ilar for ali strains. These drugs included cer-ulenin, N-carbamyl-DL-phenylalanine,N-carbamyl-L-isoleucinc, trans-cinnamicacid, ethambutol, 1-fluoro- l -deoxy-beta-D-glucose, 2-deoxy-D-glucose, and m-fluoro-phenylalanine. The MICs of the antimicro-bial drugs amikacin, sparfloxacin, and clar-ithromycin varied, but overall the MiCs forthe SmO variant were the lowest. Radio-metric assessment of drug-inhibitor com-binations by using established x/y deter-minations revealed enhanced activity wheneither ethambutol or cerulenin were used incombination with ali antimicrobial agentsfor ali variants except the Rg variant of strain424, for which ethambutol was not effec-tive. Enhanced activity with amino acid an-alogs was obscrved with the Rg colony var-iants of strains 373 and 424. Two potentialsites for drug targeting were identified: fattyacid synthesis, for ali strains assayed, andpeptide biosynthesis, particularly for Rgcolony variants that possess previouslyidentified phenylalanine-containing lipo-peptides as potential targets for future drugdevelopment.—Authors' Abstract

Revel, V., Cambau, E., Jarlier, V. and Sou-gakoff, W. Characterization of mutationsin Alt'cobacterium smegnlatis involved inresistance to fluoroquinolones. Antimi-crob. Agents Chemother. 38 (1994) 1991-1996.

Fluoroquinolone-resistant mutants ofMvcobacteriunl smegmatis have been ob-tained ili nitro by using ofloxacin as a se-lecting agent. Two types of mutants wercidentified according to their quinolone re-sistance patterns. Type 1 showed a low leveiof resistance to ofloxacin (MIC of 8 pg/m1);

whereas a high levei of resistance to thisdrug (MICs of 32 to 64 µg/ml) characterizedtype 2. By using two oligonucleotide prim-ers homologous to DNA sequences flankingthe quinolone resistance-determining re-gion (QRDR) in the gvr,l gene of Esche-richia coli and Staphilococcus aurcus, a 150-bp DNA fragment was obtained by PCRamplification from total DNA of two wild-type and five mutant strains of AI. snmg-nratis. The nucleotide sequences of the am-plifìed fragmenta were determined. The de-duced amino acid sequente from the wild-type strains showed ca. 79% similarity withthe QRDR in the gyrase A subunit fromothergram-positive and gram-negativo bac-teria. The DNA sequences obtained fromthe fluoroquinolone-resistant mutants of.1/.snlcgnlatis exhibited nucleotide modifica-tions compared with the wild-typc QRDR.The QRDR from type 1 mutants had a C-Tor an A-G transition leading to a changefrom Ala-83 to Val or Asp-87 to Gly, re-spectively. The QRDR from type 2 mutantshad a Vai-83 mutation or both Vai-83 andGly-87 mutations detected in the type 1 mu-tants. These results suggest that point mu-tations in the QRDR of the mycobacterialghrrl gene are responsibie for acquired quin-olone resistance in AI. snlegnlatis. —Au-thors' Abstract

Shannon, E. J., Frommel, D., Guebre-Xab-ier, M. and Haile-Mariam, H. S. Titra-tion of numbers of human-derived A'Ir-cobacteriunt leprae required to progres-sively oxidize "C palmitic acid and re-lease 14 CO2. Lepr. Rev. 65 (1994) 100-105.

Aíycobacteriuni leprae was isolated fromskin-punch biopsies of two untreated lep-romatous leprosy patients. The bacteria wcreenumerated, diluted 10-fold and cultured inMiddlebrook 7H9 medium supplementedwith albumin, dextrose, catalase and 14 C-palmitic acid. The cultures were incubatedat 33°C in a modified Buddemeyer radi-orespiratory detection vessel. Those cul-tures containing at least 10 7 mycobacteriademonstrated a progressive evolution of"CO,.—Authors' Summary

Takano, M., Ohara, N., Mizuno, A. and Ya-mada, T. Cloning, sequencing and ex-


pression in F_schcrichia coli of the genefor alpha antigen from Mycobactcriuniscrolulaccuni. Scand. J. Immunol. 40(1994) 165-170.

The gene for the extracellular a antigenof ,1Ivcobacteriin scrolulaccun1 (S-a) wascloned by using the a antigcn gene frag-mente of AI. bons BCG as probes. The com-plete nucleotide sequence was determined.The gene was expressed in Escherichia coli.The gene encodes 330 amino acids, includ-ing 40 amino acids for the signal peptide,followed by 290 amino acids for the matureprotcin. The deduced amino acid sequenceswere highly homologous to the a antigen ofthe species of other mycobacteria. Interest-ingly, the antigens of MAIS complex (i1/.aviam-AI. intraccllularc-M. scrolulaccinn)were closely homologous even at the C-ter-minal regions which were variable amongthose of AI. bons BCG, Al. lepras and JI.kansasii. —Authors' Abstract

'Irias, J. and Benz, R. Permeability of thecell wall of Afvcobactcrium sntcgnlatis.Mol. Microbiol. 14 (1994) 283-290.

The cell wall of Mncobacterium smcg-nmtis me 2 155 was shown to be an effectivepermeability barrier to hydrophilic com-pounds. Permcability coefficients to 0-lac-tams ranged from 10 x 10 - ' to 0.5 x10 -7cm sec - I. Cell-wall proteins were sol-ubilized with EDTA and Genapol and weretested for channel-forming activity by re-constitution into lipid bilayers. Proteins wereable to induce a voltage-gated cation-selec-tive channel. The mycobacterial porinchannel appeared to be water-filled since thesingle-channel conductance followed themobility sequence of hydrated ions in theaqueous phase. On the basis of the Renkinequation and the single-channel conduc-tance, the channel diameter was estimatedto be around 3 nm. Model calculationsshowed that cation selectivity may be causedby four negative point-charges at the chan-nel mouth. The permeability properties ofthe cell wall of intact cells were in goodagreement with those of the reconstitutedchannel. Negatively charged cephalospo-rins, cefamandole and cephalothin, diffusedat a 10- to 20-fold lower rate than the zwit-terionic cephaloridine. The mycobacterial

porin represents a major hydrophilic path-way of the cell wall of AI. sntcginatis. —Au-thors' Summary

van der Vliet, G. M. E., Schepers, P., Schuk-kink, R. A. F., van Gemen, B. and Klatser,P. R. Assessment of mycobacterial via-bility by RNA amplification. Antimi-crob. Agents Chemother. 38 (1994) 1959-1965.

We investigated whether the presence ofintact RNA is a valuable indicator of via-bility of mycobacteria with ,lfrcobactcriumsmegmatis. Al. smcgmatis was exposed tovarious concentrations of rifampin and of-loxacin suspended in broth for diflerent pe-riods of time. The nucleic acid-sequencebased amplification (NASBA) nucleic acidamplification system was used because ofits rapid, sensitive, and specific detection of16S rRNA. During drug exposurc, the vi-ability of the mycobacteria, expressed bythe number of cfu, was compared with thepresence of 16S rRNA as determined byNASBA and with the presence of DNA cod-ing for 16S rRNA as determined by PCR.Both NASBA and PCR were shown to havea detection limit of approximately 5 x 10 2

cfu/ml. The intensity of the NASBA signalcorresponded well with the number of cfu,and the lack of NASBA signal coincidedwith a loss of viability, which was reachedafter 3 days of exposurc to bactericida) con-centrations of both drugs. The presence ofmycobacterial DNA, as determined by theintensity of the PCR signal, and the viabilityof AI. snu'gnratis were not related, but anincrease in the number ofcelis and intensityof PCR signal correlated well. Bacterial vi-ability may thus be assessed by a rapid, sen-sitive, and specific, and semiquantitativetechnique by using NASBA. This system ofviability testing provides the potential forrapid evaluation of drug susceptibility test-ing. — Authors' Abstract

Verma, A., Rattan, A. and Tyagi, J. S. De-velopment of a 23S rRNA-based PCR as-say for the detection of mycobacteria. In-dian J. Biochem. Biophys. 31 (1994) 288-294.

The partial nucleotide sequence of a re-combinant plasmid containing the 23S


rRNA gene of A71'cobacterium tuberculosiswas determined and an assay was developedfor amplifying 23S rRNA gene sequences ofmycobacteria. The PCR-based non-radio-active test enabled us to distinguish AIt'-cobacteriunt from other closcly related gen-era and was sensitive enough to detect twobacterial genome equivalents. The assay wasextended to the detection of mycobactcrialDNA in uncultured clinica] specimens; 23SrRNA sequences were detected in 34 of 48(70.8%) sputum and cerebrospinal fluid(CSF) specimens by the PCR assay; whercasdirect smear examination and culture meth-ods dcmonstrated a positivity rate of 29.2%and 16.7%, respectively, for the same spec-imens. A RNA-based PCR assay with a de-tection limit of 1 genome equivalent wasalso developed. These PCR assays shouldprove useful for carly and rapid detectionof mycobactcrial infection in unculturedclinicai specimens.—Authors' Abstract

Walker, G. T., Nadeau, J. G., Spears, 1'. A.,Schram, J. L., Nycz, C. NI. and Shank,D. D. Multiplex strand displacement am-plifications (SDA) and detection of DNAsequences from Mycobacterium tubercu-losis and other mycobacteria. Nucl. AcidsRes. 22 (1994) 2670-2677.

Strand Displacement Amplification(SDA) is an isothermal, in nitro method ofamplifying a DNA target sequente prior todetection [Walker, et ai. (1992) Nucleic Ac-ids Res., 20, 1691-1693]. Here we describea multiplex forro of SDA that allows twotarget sequences and an internai amplifi-cation control to be co-amplified b,, , a singlepair of primers after common priming se-quences are spontaneously appended to theends of target fragments. Multiplex SDAoperates at a single temperature, under thesame simple protocol previously developedfor single-target SDA. We applied multiplexSDA to co-amplification of a target se-quence (IS6110) that is specific to membersof the Mycobacteriuin tuberculosis-complexand a target (16S ribosomal gene) that iscommon to most clinically relevant speciesof mycobacteria. Both targets are amplified10 8 -fold during a 2 hr, single temperatureincubation. The relativo sensitivity of thesystem was evaluated across a number ofclinically relevant mycobacteria and checked

for crossreactivity against organisms that areclosely related to mycobacteria.—Authors'Abstract

Wayne, L. G. and Sramek, H. A. Metro-nidazole is bactericidal to dormant cellsof Mvcobacteritnn tuberculosis. Antimi-crob. Agents Chemother. 38 (1994) 2054-2058.

Very abrupt exposure to anaerobic con-ditions has a lethal cffect on actively grow-ing cultures of Alycobactcrium tuberculosis.However, incubation under conditions inwhich oxygen is depleted gradually causesM. tuberculosis to shift down from activereplication to dormancy. Thc dormant ba-cilli are resistant to the bactericidal effectsof anaerobiosis and also exhibit partial orcomplete resistance to the bactericidal ef-fects of isoniazid and rifampin. On the otherhand, metronidazole, a drug specific for an-aerobes, kills dormant tubercle bacilli underanaerobic conditions, but it lias no cffect onactively growing aerobic cultures. The lethaleffect of metronidazole under anaerobicconditions is enhanced by rifampin. Thepossible implications of these findings onthe phenomenon of latcncy in tuberculosisare discussed.—Authors' Abstract

Williams, D. L., Waguespack, C., Eisenach,K., Crawford, J. T., Portaels, F., Salfin-ger, 1%I., Nolan, C. M., Abe, C., Sticht-Groh, V. and Gillis, T. P. Characteriza-tion of rifampin resistance in pathogenicmycobacteria. Antimicrob. AgentsChemother. 38 (1994) 1380-1386.

The emergence of rifampin-resistantstrains of pathogenic mycobacteria hasthreatened the usefulness of this drug intreating mycobacterial diseases. Criticai tothe trcatment of individuais infected withresistant strains is the rapid identificationof these strains directly from clinicai spec-imens. It has been shown that resistance torifampin in Mrcobacterium tuberculosis and11. leprae apparently involves mutations inthe rpoB gene encoding the ,6-subunit of theRNA polymerases of these species. DNAsequences were obtained froni a 305-bpfragment of the rpoB gene from 110 rif-ampin-resistant and 10 rifampin-suscepti-ble strains of M. tuberculosis from diverse

164^ International Journal of Lcprosy^ 1995

geographical regions throughout the world.in 102 of 110 rifampin-resistant strains 16mutations affecting 13 amino acids were ob-served. No mutations werc observed in rif-ampin-susceptible strains. No associationwas found between particular mutations inthe 17)013 gene and drug susceptibility pat-terns of multidrug-resistant ,i I. tuberculosisstrains. Drug-resistant M. tuberculosisstrains from the same outbreak and cxhib-iting the same iS6110 DNA fìngerprint anddrug susceptibility patíern containcd thesame mutation in the rpo13 gene. However,mutations are not correlatcd with IS6110profiling outside of epidemics. The evolu-tion ofrifampin resistance as a consequenceof mutations in the rpoB gene was doeu-mented in a patient who developed rifam-pin resistance during the course of treat-ment. Rifimpin-resistant strains of M. lep-rae, M. ariuni, and AI. a%ricanum containedmutations in the rpol3 gene similar to thatdocumented for Al. tuberculosis. This in-formation served as the basis for developinga rapid DNA diagnostic assay (PCR-het-eroduplex formation) for the detection ofrifampin susceptibility ofM. tuherculos/s. —Authors' Abstract

Zwadyk, P., Jr., Down, J. A., Myers, N. andDey, M. S. Rendering of mycobacteriasafe for molecular diagnostic studies and

development of a lysis method for stranddisplacemcnt amplification and PCR. J.Clin. Microbiol. 32 (1994) 2140-2146.

Two criteria must bc met before myco-bacterial specimens can be tested by DNAamplification methods: (i) the samples mustbe rendered noninfectious, and (ii) the or-ganisms must be lysed to frec the DNA.Previous publications reporting DNA am-plification of mycobacteria have concen-trated on lysis and amplification proceduresand have not addressed the issue of samplesafety. We have shown that heating of sam-ples below 100°C may not consistently killmycobacteria; however, heating at 100°C ina boiling-water bati] ora forced-air oven fora minimum of 5 min kills mycobacteria,including Mt'cobacteriuni therntoresistibile.Furthermore, heating at 100°C for 30 minconsistently lyses mycobacteria to produceshort fragments of DNA that are suitablefor amplification by PCR and strand dis-placemcnt amplification. This procedurcworks with clinicai samplcs digcsted by then-acetyl cysteine-NaOH method as well aswith suspensionsoforganisms in phosphatcbuficr. This paper also demonstrates thefeasibility of using strand displacemcnt ofamplification with clinicai specimens.-Authors' Abstract

Experimental Infections

De Blaquiere, G. E., Santamaria, L., Curtis,J., Terenghi, G., Polak, J. M. and Turk,J. L. A morphological and functional as-sessment of Mvcobacteriunz lepras-in-duced nerve damage in a guinea-pig mod-el ofleprous neuritis. Neuropathol. Appl.Neurobiol. 20 (1994) 261-271.

Nerve damage, resembling that caused byMycobacteriunt leprae in man, was createdby the injection of cobalt-irradiated M. lep-rae organisms into the tibial nerve ofguineapigs. Assessment of nerve damage was madeby clinicai, electrophysiological and mor-phometric means that intervals up to 13weeks after injection. Quantitative immu-nohistochemical analysis of neuropeptide-containing fibers in the skin of the foot was

also carried out. Significant nerve damageoccurred 3 weeks after injection ofAí. lepraeorganisms. Motor and sensory functionalloss peaked at 5 weeks after injection, andthere was a significam decrease of peptide-immunoreactive nerves in ali skin com-partments. The nerve damage was self-lim-iting and functional recovery had occurredby 13 weeks. The model shows that manyof the features found in the nerve damageof treated leprosy patients.—Authors' Ab-stract

Gelber, R. H., Hunter, S. W., Murray, L.P., Siu, P., Tsang, i\I. and Brennan, P. J.Effective vaccination of mice against Mr-cobacterium leprae with density-gradient


subfractions of soluble Aí. leprae pro-teins: clues to effective protein epitopes.Lepr. Rev. 65 (1994) 175-180.

It had previously been discovered thatintradermal mousc vaccination with a pro-tein fraction ofA lt'cobacteriun1 /cprae (calledsoluble proteins) in Freund's incompleteadjuvam (FIA) resulted in consistent andlong-lived protection against AI. /eprae mul-tiplication from subsequent viablc foot padchallenges. In this study certain density-gra-dient subfractions of this soluble protein,but not others, in FIA afforded vaccine pro-tection. The results of this study suggestwhich AI. Ieprac proteins may be involvedin protective immunity, particularly 1-3 kD,10 kD, 65 kD, and those ofhigher molecularwcight.—Authors' Summary

Ramakrishnan, L. and Falkow, S. Afvco-bacteriu111 111arinu 11 persists in culturedmammalian cells in a temperature-re-

stricted fashion. Infect. Immun. 61 (1994)3222-3229.

We have explored the relatively rapidlygrowing animal and human pathogen Aly-cobacterium marinunl as an experimentalmodel for mycobacterial pathogenesis. Aí.marinunl, which has a lower temperaturefor optimal growth than does AI. tubercu-losis, lias a much shorter generation timeand can be safely studied in ordinary lab-oratory facilities and examined in multipleanimal infection models. We have estab-lishcd an in nitro essay for its interactionwith eukaryotic cells and shown that it per-sists in these cells in a temperature-specificfashion that correlates with its ability tocause disease in vivo at lower temperatures.Additionally, preliminary evidence that AI.marinunl causes a chronic disease with somefeatures resembling tuberculosis in frogs ofthe species Rana pipiens is presented.—Au-thors' Summary

Epidemiology and Prevention

Gupte, M. D. Elimination of leprosy: fore-casts and projections. Indian J. Lepr. 66(1994) 19-35.

It is possible to diagrammatically repre-sem the pattern of prevalence and incidenceunder dapsone and MDT interventions ob-served in various endemic regions. A sub-stantial decline in prevalence following dap-sone was observed in several regions.Though a substantial proportion of this de-cline was attributable to cure on account ofdapsone, it was also on account of someother factors such as patients found to benot in need of treatment, migration anddeaths. Prevalence of leprosy was gettingstabilized after the initial 10 to 15 years ofthe dapsone-based program. Interventionswith the MDT campaigns have produced adramatic decline in leprosy prevalence in avery short period, again followed by stabi-lization at a much lower levei. Decline inprevalence was again attributable partly totherapy and partly to other factors. Newcase detection did not show a downwardtrend that could be attributed to dapsone or

MDT per se. New case detection rates inchildren have not shown signs of reduction.It is also important to note that despite theoccurrence of dapsone resistance or irreg-ular intake of dapsone or possible short-comings in the program, no recrudescenceof leprosy on a large scale was ever recorded.There is a definite perceptible change in theprofile ofnew cases of leprosy for the better.The essential effect of MDT campaigns hasbeen a phenomenal decline in leprosy prev-alence, providing opportunities to deliverdisability care services to leprosy patientsand to expand coverage of the program.There is a definite need to continue vigor-ously the MDT implementation and pro-vide curative services and ensure availabil-ity of those services to all leprosy patientsin the country. Available data do not in-dicate any immediate effect of MDT ontransmission. It is quite likely that personsalready infected will keep on developingleprosy lesions for some years. But then onewould expect an increase in the proportionof cases occurring in adults as compared tochildren. This is not observed. There will


be several questions like this and more.Monitoring leprosy trends may providesome clues and development of sentinelcenters would be an essential requirementto generate the necessary data. It is expectcdthat MDT implementation would becomeoperationally easier over a period of timeafter devising shorter and cffective drug reg-imens. Thcre is a distinct need to improvethe operational performance in terms of casedetection. The need for diagnosing single-lesion evanescent forms of leprosy is beingdoubted, at least in some quarters. Empha-sis on diagnosing leprosy patients mainlyresponsible for transmission or those at riskof deformities cannot be qucstioned. Per-haps there is a need to optimize the programby adopting some of there kinds of sugges-tions. Will there modifications lead to re-crudescence? Based on earlier experienceswith dapsone therapy program such a sce-nario is an unlikely event. It is thus possibleto adopt some of there reforms while keep-ing a Glose watch on the situation throughthe sentinel centers. These centers wouldprovide the kind of data that can be effec-tively used for predictions of trends, cor-rections of strategies and developing ration-al programs.—Authors' Conclusions

Isa Isa, R. A. and Castellazzi, Z. [Scope ofthe Action Plan for the Elimination ofLeprosy in the Dominican Republic.] Rev.Dominica Republ. 30 (1993) 19-22. (inSpanish)

The principal features of the Action Planfor the Elimination of Leprosy as a PublicHealth Problem in the Dominican Republicare presented. The essential components ofthe epidemiological situation, projections,intervention strategy, support and epide-miological observation are reviewed. Anoutline of their projection roto the near fu-ture is presented.—Authors' English Sum-mary

Krishnamurthy, P., Rao, P. S., Subraman-ian, M. and Inderparkash. The influenteof operational factors in the profile ofmonolesional leprosy cases in South In-dia. Lepr. Rev. 65 (1994) 130-136.

A comparison of the profile of monole-sional cases among new PB cases detectedin a Governmental Leprosy Control Unit(GLCU) and the field arca of a Central Lep-

rosy Teaching and Research Institute(CLTRI), both Iocated in South India, dcm-onstrates that the proportion of monole-sional cases among new cases detected be-tween 1987 and 1991 was higher in childrenthan adults, higher in females than males(only in the CLTRI)—over 95% were thetuberculoid type. A significantly incrcasingtrend in this proportion could be seen in theGLCU but not in the CLTRI; an explana-tion of this is based on the difference inoperational aspects in case detection meth-odology adopted by the two arcas—e.g., in-tersurvey interval and mode of case detec-tion. Such studies, focusing on single skinlesions, hclp us in understanding the role ofvarious possible operational factors in in-fluencing the behavior of the disease.—Au-thors' Summary

Sansarricq, H. Some points on the elimi-nation of leprosy. (Editorial) Lepr. Rev.65 (1994) 81-87.

Is the elimination strategy an improve-ment compared to the control strategy? Itwould appear that, ifcompared to the con-trol strategy, the elimination strategy is like-ly to result in increased case detection, in-creased MDT coverage and better moni-toring of progress. The reasons for whichthe elimination strategy should be more ef-fective than the control strategy are its spe-cific components: a "time limit" and a "de-fincd target."

What is the importance of the drawbacksof the elimination strategy? (a) The need foradditional resources does not appear a se-rious constraint. (b) These will be importantoperational difficulties: (1) related to activ-ities (case detection, MDT delivery, treat-ment compliance, etc.); and (2) related tomonitoring of progress as we do not havean adequate indicator for measuring prog-ress in low prevalence/incidence situations.

Is it timely to engage in an eliminationstrategy? It seems that if the eliminationstrategy is implemented as a further stepand natural expansion of the control strat-egy, there can be only advantages, such as:(a) It can be built on the infrastructure andfacilities already established for the controlstrategy, and can take advantage of the ex-periente gained by its implementation. (b)In terms of caseload, a better operationaland possibly epidemiological (concerning


incidence) impact than with the controlstrategy; in any case, the greatest impactwhich could be obtained from an MDT-based strategy.

It is important to note that these advan-tages will become effective only if the twofollowing conditions are met. (a) Opera-tional methods are developed to overcomeoperational difficulties. This should be fea-sible, although unconventional solutions tosome problems should be accepted. (b)Methods for monitoring progress in lowprevalence/incidence situations will have tobe developed. This may cause real difficul-ties. However, full use should be made ofthe ratio prevalence/case detection rate. Thisindicator may prove to have a greater sig-nificance than expected.

Altogether, it seems that the advantagesof implementing the elimination strategy insequence with the control strategy, and as anatural expansion of the latter, outweighsthe disadvantages. In contrast, if imple-mcnting the elimination strategy was de-layed, there is no certainty that the methodsrequired to facilitate case-detection and case-holding, and to monitor progress in lowprevalence/incidence situations, would bedeveloped without the commitment of na-tional authorities to eliminate leprosy as apublic health problem.—Froco the Editorial

Suite, M., Edinborough, N. B., Lewis, M.and Tollefson, J. A survey to determinethe prevalence ofleprosy in a communityin East Trinidad. Lepr. Rev. 65 (1994)122-129.

A house-to-house survey was conductedin a community in East Trinidad, where aclustering of cases has been observed. Therewere 1355 residents, of whom 73.5% had acomplete visual skin examination. No newcases of leprosy were found but a variety ofskin disorders were diagnosed. The mostcommon disorder was pityriasis versicolor,which is one of the differential diagnoses ofhypopigmented skin lesions. This has seri-ous implications for the delayed diagnosisofleprosy. In all, 5 of the 9 old cases residingin the survey arca suffered from pauciba-cillary disease, and had a history of contactwith a lepromatous case. They were not list-ed initially as contacts of this index case.Contact lists should therefore include non-familial persons having frequent contactwith an index case. The definition of "fre-quent" should be determined by each pro-gram. It may also be necessary to review theduration of surveillance of contacts. Thesurvey was estimated to have cosi about US$2500 and was not considered to be cost-effective.—Authors' Summary

Rehabilitation

Birke, J. A., Foto, J. G., Deepak, S. andWatson, J. Measurement of pressurewalking in footwear used in leprosy. Lepr.Rev. 65 (1994) 262-271.

Pressure measurements were madc on 10leprosy patients while walking barefoot andwhile using 6 sample shoes. The sampleshoes, which represented footwear currentlyused worldwide in leprosy programs, in-cluded: 1. a U.S.A. extradepth shoe withoutinsole; 2. a U.S.A. extradepth shoe with in-sole; 3. a Chinese tennis shoe; 4. a Mozam-bique sandal; 5. a Bombay sandal; 6. a Bom-bay sandal with rigid sole; and 7. the pa-tients prescribed footwear. Peak pressurewas significantly lower while walking in allfootwear, except with the extradepth shoewithout an insole, when compared to bare-

foot walking. Pcak pressure was signifi-cantly lower walking in the Bombay san-dals, the Chinese tennis shoe, the extradepthshoe with an insert and the patient's pre-scribed shoe when compared to the extra-depth shoe without an insert. Regressionanalysis showed a significant inverse rela-tionship between pressure and insole thick-ness.—Authors' Summary

Kuipers, M. and Schreuders, T. The pre-dictive value of sensation testing in thedevelopment ofneuropathic ulceration onthe hands of leprosy patients. Lepr. Rev.65 (1994) 253-261.

The early detection of the loss of protec-tive sensation in leprosy patients is vital ifneuropathic ulceration and subsequent dis-


abilities are to be avoided. The aim of thisstudy was to find the protective value ofsensory thresholds in the hands of leprosypatients. Thresholds for touch-pressure, vi-bration and temperature were assessed inarcas on leprosy-alrected hands near ulcersor ulcer scars (LU-group), in arcas withoutlesions (LN-group), and in controls (N-group). Semmes-Weinstein monolìlaments(SWF) were used for testing the touch-pres-sure threshold (PST), a biothesiometer forthe vibration threshold (VST) and a Ther-mo Sensation Tester for thc temperaturethreshold (TST). The distribution of ulcerswas about equal on both palmar and dorsalaspects of thc hands. In the LU-group thcrcwas a negativo response to SWF of 2.0 g inall patients, while 74% could feel the 2.0 gin LN-areas and in N-areas 100% could de-tect thc 2.0 g SWF. In thc LU-group about11% felt 8 V VST, in thc LN-group about60% and in the N-group 89%. Testing tem-perature sensation was given up prema-turely because the results in controls wereunsatisfactory. Both palmar and dorsal sidosof the hands should be tested for sensation.The thresholds for protective sensation are2.0 g SWF and 8 V for vibration sense. Itis recommended that Semmes-Weinsteinmonofilaments should always be used forearly detection of loss of protective sensa-tion.—Authors' Summary

Malaviya, G. N., lHusain, S., Girdhar, A.and Girdhar, B. K. Sensory functions inlimbs of normal persons and leprosy pa-tients with peripheral trunk damage. In-dian J. Lepr. 66 (1994) 157-164.

The threshold to touch was tested in thehands and feet of normal persons usingSemmes-Weinstein graded monofilamentnylons. The minimum stimulus to whichresponse could be elicited was nylon num-ber 3.61 in palms and 4.31 in soles. 'fhesenumbers relate to the logarithm of the forceapplied, 3.61 corresponding to 0.217 gmforce and 4.31 to 2.35 gm force, respec-tively. The arca of pain insensitivity com-plained by the patient more or less corre-sponds to that revealed by objective testing.It was interesting to observe that loss of painsensitivity was confined to a smallcr arcacompared to touch and thermal insensibil-

ity in the part innervated by the same nervetrunk.—Authors' Abstract

Terencio de Ias Aguas, J. [Pathology of thefeet in leprosy.] Rev. Leprol. Fontilles 19(1994) 543-555. (in Spanish)

The a(linity of leprosy for the skin andperiphcral nervous system leads to situa-tions that frequently affect the foot regard-ing skin and nerve lesions. The different le-sions are explained, the most important, be-ing ncurotrophic and plantar ulcers, disso-ciated tarsus, and bone and joint lesions; allare causes of disabilities and need hospi-taliiation, and are an important cause ofdifliculty for social reinsertion.—Authors'English Summary

van Brakel, W. 1-1., Shute, J., Dixon, J. A.and Arzet, II. Evaluation of sensibility ofleprosy—comparison of various clinicalmethods. Lepr. Rev. 65 (1994) 106-121.

In order to determine whether varioussensibility tests, not in common use at ourhospital, are appropriate for the neurolog-ical screening of leprosy patients, an ex-tended nervo function assessment (NFA)was done on 50 in- and outpaticnts who hadbeen diagnosed as sulTering from leprosy(100 hands and feet). The nerve functionassessment battcry consisted of Semmes-Weinstein monofilament testing (SWMT),moving 2-point discrimination (M2PD),pinprick (PP), position sense (PS), vibrationsense (VS) and voluntary muscle testing(VMT). In addition the SWMT was per-formed on 637 hands and 634 feet of "fieldpatients" in order to get a betler indicationof the prevalence of sensory impairment asmeasured with the SWMT. The SWMT hasbeen shown to be a sensitive test of periph-eral nerve function; therefore the other testswere compared with the SWMT. Resultswcre reported separately for the ulnar, me-dian and posterior tibial nerve. Test siteswere the pulp of the distal phalanx of theindex finger, the little finger and the big toe.Correlation betwecn the SWMT and eachof the other tests proved statistically signif-icant; the closest correlations were betweenthe SWMT, M2PD and PP for both ulnarand median nerves (r > 0.7, F test > 100,p < 0.0001). It is argued that the first tests


to show nerve function impairment (NFI)are the M2PD and the SWMT. VS and PSwere also absent in a significant proportionof patients. Arguments are presentcd that

this may indicate advanced nerve functionimpairment. Results are compared withother data currently available in the litera-ture.—Authors' Summary

Other Mycobacterial Diseases and Related Entities

Adal, K. A., Anglim, A. M., Palumbo, C. L.,Titus, 1\I. G., Coyner, B. J. and Farr, B.M. The use of high-efriciency particulateair-filter respirators to protect hospitalworkers from tuberculosis—a cost-effec-tiveness analysis. N. Engl. J. Med. 331(1994) 169-173.

After outbreaks ofmultidrug-resistant tu-berculosis (TB), the Centers for DiseaseControl and Prevention proposed the use ofrespirators with high-efficiency particulateair filters (HEPA respirators) as part of iso-lation precautions against TB, along with arespiratory-protection program for healthcare workers that includes medicai evalua-tion, training, and tests of the fit of the res-pirators. Each HEPA respirator costs be-tween $7.51 and $9.08, about 10 times thecost of respirators currently used.

We conducted a cost-efrectiveness anal-ysis using data from the University of Vir-ginia Hospital on the exposure to patientswith TB and rates at which the purified-protein-derivativa (PPD) skin test becamepositive in hospital workers. The costs of arespiratory-protection program were basedon those of an existing program for workersdealing with hazardous substances.

During 1992, 11 patients with docu-mented TB were admitted to our hospital.Eight of 3852 workers (0.2%) had PPD teststhat became positive. Five of these conver-sions were believed to be due to the boosterphenomenon; one followed unprotected ex-posure to a patient not yet in isolation; theother two occurred in workers who had nev-er entered a TB isolation room. These datasuggest that it will take more than 1 yearfor the use of HEPA respirators to preventa single conversion of the PPD test. Assum-ing that one conversion is prevented peryear, however, it would take 41 years at ourhospital to prevent one case of occupation-

ally acquired TB, at a cost of $1.3 millionto $18.5 million.

Given the efrectiveness of currently rec-ommended measures to prevent nosocom-ial transmission of TB, the addition of HEPArespirators would offer negligible protectiveefficacy at great cost.—Authors' Abstract

Adams, J. L. and Czuprynski, C. J. My-cobacterial cell wall components inducethe production of TNF-alpha, IL-1, andIL-6 by bovine monocytes and the mu-rine macrophage cell line RAW 264.7.Microb. Pathogen. 16 (1994) 401-411.

Johne's disease is characterized by achronic enteritis that results in granulo-matous inflammation, cachexia, and even-tual death of cattle infected with Alrcobac-teriam paratuberculosis. The cytokines tu-mor necrosis factor-alpha (TNF-a),interleukin-1 (IL-1), and interleukin-6 (IL-6) have been associated with granuloma for-mation and wasting in other disease syn-dromes. The potential role of these cyto-kines in the development and progressionofJohne's disease lias not been investigated.Freshly isolated bovine peripheral bloodmonocytes and the murine macrophage cellline RAW 264.7 were examined for theirability to release inflammatory cytokines inresponse to mycobacterial cell wall com-ponents. Bovino monocytes and RAW 264.7cells incubated with .lf. paratuberculosis li-poarabinomannan (LAM), muramyl dipep-lide (MDP), or lipopolysaccharide (LPS) re-leased INF-a, IL-1 a, and IL-6 as detectedby appropriate bioassays. Using the RAW264.7 cells, cytokine mRNA levels were el-evated after in nitro incubation with live AI.paratuberculosis or LPS as determined us-ing a reverse-transcriptase polymerase chainreaction procedure.—Authors' Abstract


Altamirano, M., Marostenmaki, J., Wong,A., Fitzgerald, M., Black, W. A. andSmith, J. A. Mutations in the catalase-peroxidase gene from isoniazid-resistant,1Iycobacterium tuberculosis isolates. J.Infect. Dis. 169 (1994) 1162-1165.

Isoniazid resistant in Mvcobacteriuni tu-berculosis has been associated with total de-letion of the katG gene, which codes forcatalase-peroxidase production. To deter-mine whether this is a common mechanismof drug resistance, 9 isolates of isoniazid-resistant and 1 of isoniazid-sensitive Al. tu-berculosis were analyzed by polymerasechain reaction amplification of a 237-bp se-quence of the katG gene. Amplification wasobserved in the isoniazid-sensitive isolateand in 8 resistant isolates; in only 1 isoni-azid-resistant isolate was there no amplifi-cation of the expected band, suggesting genedeletion. DNA sequencing showed that 8 ofthe 9 isolates had point mutations, dele-tions, or insertions of 1-3 bases. Evidencecorroborating the presence of mutations inthe katG gene was obtained by single-strandconformation polymorphism analysis inthese 8 isolates. Thus, mutations as well asinsertions and deletions in the katG genecan account for inactive catalase peroxi-dase, leading to isoniazid resistance; genedeletion occurs only infrequently, similar to11% of cases.—Authors' Abstract

Appelberg, R., Castro, A. G., Pedrosa, J.,Silva, R. A., Orme, I. M. and Minoprio,P. Role of gamma interferon and tumornecrosis factor alpha during T-cell-inde-pendent and -dependent phases of AIt'-cobacteriuln aviam infection. Infect. Im-mun. 62 (1994) 3962-3971.

To design an effective immunotherapy forMycobacteriulzz aviam infections, the pro-tective host response to the infection mustbe known. Here were analyzed the role ofgamma interferon (IFN-y) and tumor ne-crosis factor alpha (TNF-a) in the innateand acquired responses to Aí. aviam infec-tions in mice. T-cell depletion studiesshowed that CD4+ T cells were requiredfor control of the infection. CD4+-dèpletedmice showed enhanced bacterial prolifera-tion and at the same time showed a reduc-tion in the level ofexpression ofboth IFN-y

and TNF-a mRNAs in spleen cens. In con-trast, AI. bovis BCG immunization restrict-ed M. aviam proliferation and at the sametime promoted expression of the mRNAsfor the two cytokines. hz vivo depletion stud-ies using specific monoclonal antibodiesshowed that both IFN-y and TNF-a are in-volved in an early protection possibly in-volving NK eells, and furthermore, IFN-yis involved in the later T-cell-protective re-sponse to infection. In vivo neutralizationof IFN-y during M. aviam infection alsoblocked the priming for enhanced TNF-asecretion triggered by endotoxin. Both cy-tokines were found to be involved in theresistance expressed in BCG-immunizedanimais and exhibited additive bacterio-static effects in vitro on bone marrow-de-rived macrophages infected with differentstrains of Al. aviam. These data suggest thatboth cytokines act in an additive or syner-gistic fashion in the induction of bacterio-stasis and that IFN-y is also involved inpriming TNF-a secretion.—Authors' Ab-stract

Bermudez, L. E., Kolonoski, P., Young, L.S. and Inderlied, C. B. Activity of KRM1648 alone or in combination withethambutol or clarithromycin againstMvcobacteriunz aviam in beige mousemodel ofdisseminated infection. Antimi-crob. Agents Chemother. 38 (1994) 1844-1848.

Rifamycins are active against slowlygrowing mycobacteria, such as Alt'cobacte-riam tuberculosis and Al. kansasii, but themajority of rifamycins thus far investigatedboth in vitro and in vivo are inactive or haveonly modest activity against the Al. aviamcomplex (MAC). We investigated the activ-ity of three doses of semisynthetic benzox-azinorifamycin KRM 1648, alone or incombination with ethambutol or clarith-romycin, in beige coice challenged with theMAC strain 101. Our results show the fol-lowing: (i) KRM 1648 was significantly ef-fective against MAC infection as deter-mined by the reduction of the number ofbacteria in the blood, liver, and spleen whenadministered at doses of 20 and 40 mg/kgof body weight per day but not at 10 mg/kg/day, compared with untreated controls.

63, 1^

Current Literature^ 171

(ii) KRM 1648 (40 mg/kg/day) adminis-tered in combination with ethambutol (100mg/kg/day) resulted in significant reductionin bacteremia compared with values for un-treated controls (p < 0.001), KRM 1648alone (p = 0.019), and ethambutol alone (p= 0.003). Furthermore, the combination ofKRM 1648 and ethambutol was associatedwith a significant decrease in the number ofbacteria in the spleen and the liver com-pared with values for both untreated con-trols and each drug alone (p < 0.001 for alicomparisons). (iii) KRM 1648 (40 mg/kg/day) administcred in combination withclarithromycin (200 mg/kg/day) resulted ina significant decrease in the number of bac-teria in the blood and the spleen comparedwith the number for untreated controls (p< 0.001 for ali comparisons). In our ex-perience, using MAC 101 as the challengingorganism, KRM 1648 is the first rifamycinwith significant activity in vivo against MACinfection in beigc mice.—Authors' Abstract

Bila, V. and Kren, V. Evidence for terato-genicity ofthalidomidc using congcnic andrecombinant inbred rat strains. Folia Biol.40 (1994) 161-171.

Teratogenic properties of thalidomidewere tested in two systems of laboratory ratstrains carrying the mutant lx alicie that de-termines the polydactyly-luxate syndrome.In agreement with our previous experi-menta, we have confirmed that the responseof fetuses basically depends on their geno-type. Fetuses of LEW/BN, +/+ genotyperemained unaffected following 500 or 3 x500 mg/kg thalidomide doses (43 and 56fetuses, respectively). In LEW/BN, +/lx fe-tuses these doses elicited 24% and 87% hindfeet polydactyly (14/59 and 53/61 fetuses,respectively), which was highly significantwhen compared with 84 vehiculum-treatedand 235 untreated controls (p < 0.001).However, in 48 SHR/RI 2, lx/lx fetuses bothpairs of limbs were affected in an oppositeway after the 500 mg/kg thalidomide dose:hind feet oligodactyly (94/96 limbs) and in-creased front feet polydactyly occurred (incomparison with 70 controls, p < 0.001).The mutant lx allele as well as modifyinggenes are involved in the response to tha-lidomide.—Authors' Abstract

Cambau, E., Sougakoff, W., Besson, M.,Truffot-Pernot, C., Grosset, J. and Jar-lier, V. Selection of a gyrA mutant of My-cobacteritun tuberculosis resistant tofiuoroquinolones during treatment withofioxacin. J. Infect. Dis. 170 (1994) 479-483.

A strain of 1tlycobacterium tuberculosisresistant to ofloxacin was selected in a pa-tient with a long history of multidrug-resis-tant tuberculosis eventually treated by of-ioxacin combined with other second-linedrugs. A mutation in the gyrA gene was hy-pothesized to be the mechanism ofacquiredresistance to ofloxacin in this strain. Chro-mosomal DNA of strains MTB1, isolatedbefore treatment and susceptible to ofiox-acin (MIC, 1 µg/mL), and MTB2, isolatedduring treatment and resistant to ofioxacin(MIC, 32 µg/mL), was amplified by poly-merase chain reaction (PCR) using two oli-gonucleotide primers highly homologous toDNA sequences fianking the quinolone re-sistance-determining region in gyrA of my-cobacteria. Comparison of the nucleotidesequences of the 150-bp fragments obtainedby PCR revealed a point mutation in MTB2leading to the substitution of histidine foraspartic acid at a position corresponding toresidues involved in quinolone resistance inEscherichia coli (Asp87), Staphylococcusaureus (G1u88), and Campylobacter jejuni(Asp90).—Authors' Abstract

Cauthen, C. M., Snider, D. E. and Onorato,I. M. Boosting of tuberculin sensitivityamong Southeast Asian refugees. Am. J.Respir. Crit. Care Med. 149 (1994) 1597-1600.Following an initial negative Mantoux tu-

berculin skin test, a second test, given assoon as 1 wk later, has been shown to elicitmarkedly larger reactions (boosting) in 20%to 40% of refugees tested in the UnitedStates. We conducted a study to determinethe explanation for this phenomenon. Usingthe Mantoux method of intradermal skintesting, 2469 refugees from Southeast Asiawere initially tested with tuberculin fol-lowed by sequential retesting 7 and/or 90 dlater. They were also tested initially withnontubuculous mycobacterial antigens. Ahigh proportion (35.5%) of Southeast Asian


refugees had rcactions ? 10-mm indurationto an initial tuberculin test, and 30.9% ofthe nonreactors exhibited boosting on asubscquent tuberculin test. Boosting, unlikereactivity to the initial tuberculin test, wasnot associated with exposure to a personwith tuberculosis. However, boosting wasassociated with reactivity to nontubercu-lous mycobacterial antigens and a historyof bacille Calmette-Guérin (BCG) vacci-nation. Boosting in this population is there-fore attributable to environmental exposureto nontuberculous mycobacteria that areendemic in Southeast Asia or to BCG vac-cination, rather than to remote infection withMvcobacteriutn tuberculosis. Sequential tu-berculin screening and preventive therapyof persons with boosted reactions is not rec-ommended as a tuberculosis preventionstrategy in this population.—Authors' Ab-stract

Chin, D. P., Reingold, A. L., Stone, E. N.,Vittinghoff, E., Horsburgh, C. R., Simon,E. M., Yajko, D. M., Hadley, W. K., Os-troff, S. M. and Hopewell, P. C. The im-pact of Alvcobacterium aviou? complexbacteremia and its treatment on survivalof AIDS patients—a prospective study. J.Infect. Dis. 170 (1994) 578-584.

It is currently recommended that patientswith AIDS and Mvcobacteriunt aviara com-plex (MAC) bacteremia receive antimyco-bacterial treatment. However, no study hasprospectively evaluated the impact of thisinfection and its treatment on survival. Thisstudy prospectively followed a cohort of 367AIDS patients with50 CD4+ cells/µL andfound that MAC bacteremia was indepen-dently associated with an increased risk ofdeath (relative hazard [RH] = 1.8, 95% con-fidente interval [CI] = 1.3-2.4, p < 0.001).Patients with MAC bacteremia who weretreated had a longer median survival thanthose who were not (263 vs 139 days, p <0.001); treatment was independently asso-ciated with a lower risk ofdeath (RH = 0.45,95% CI = 0.23-0.89, p , 0.001). However,23% of patients with bacteremia died within28 days of that diagnosis; few were treated.MAC bacteremia contributes to the deathof patients with AIDS, and treatment in-creases survival. However, many patientswill not survive long enough to receive

treatment. These results underscore the im-portancc of early diagnosis and chemopro-phylaxis for MAC bacteremia.—Authors'Abstract

Curcic, R., Dhandayuthapani, S. and Der-etic, V. Gene expression in mycobacteria:transcriptional fusions based on xyIE andanalysis of the promoter region of the re-sponse regulator nttr.4 from Alvcobacte-riunt tuberculosis. Mol. Microbiol. 13(1994) 1057-1064.

Understanding promoter regulation andsignal-transduction systems in pathogenicmycobacteria is criticai for uncovering theprocesses that govern interactions of thesebacteria with the human host. In order todevelop additional genetic tools for analysisof mycobacterial promoters, the xylE genefrom Pseudotnonas was tested as a tran-scriptional fusion reporter in fast- and slow-growing mycobacteria. Initially, its utilitywas demonstrated by expression behind thehsp60 promoter in Afvcobacteriunt smeg-ntatis and A1. bons BCG. The presente ofan active promoter in front of the promo-terless xyIE cassette on a plasmid was scoredby development of a bright yellow colorupon spraying of mycobacterial colonies onplates with a solution of catechol. The geneproduct of xyIE, catechol 2,3 dioxygenase,was measurable in sonic extracts and wholecells, permitting quantitative determinationof promoter activity in both fast- and slow-growing mycobacteria. The xy/E-based my-cobacterial transcriptional fusion plasmidpRCX3 was constructed and used to assesspromoter activity within the sequentes lo-cated upstream of the newly characterizedM. tuberculosis H37Rv response regulatorttttrA, a member of the superfamily of bac-terial signal-transduction systems. — Au-thors' Summary

Darling, T. N., Sidhumalik, N., Corey, G.R., Allen, N. B., Kamino, H. and Murray,J. C. Treatment of Alycobacteriuttt hae-tnophilunt infection with a antibiotic reg-imen including clarithromycin. Br. J.Dermatol. 131 (1994) 376-379.

A patient with rheumatoid arthritis de-veloped ulcerated nodules predominantlyon his legs. Skin biopsy and culture dem-


onstrated rheumatoid vasculitis and infec-tion with Mycobacterium haeniophilumi.Improvement was not seen until clarithro-mycin was added to his treatment regi-men.—Authors' Abstract

Denis, M. Tat protein from HIV-1 binds toMycobacterium aviam via a bacterial in-tegrin—effects on extracellular and intra-cellular growth. J. Immunol. 153 (1994)2072-2081.

We examined the interaction betweenHIV-1 Tat protein and the opportunisticpathogen Mycobacterium aviam. AIDS -as-sociated strains of Al. aviam were shown tobind Tat protein quite avidly in an attach-ment assay. The attachment of Al. aviam toTat was shown to occur via the integrin al-pha(5) beta(1) present on the mycobacterialcell surface. AI. aviam strains ere shown tobind to virai Tat protein with high afrinityin a specific fashion (600 binding sites witha Kd of 1 to 5 nM). Al. aviam coated withTat protein were shown to be more infectivefor human alveolar macrophages than un-treated AI. aviam. Other HIV-1 Ags had nosuch effects (e.g., p24, p17). Examination ofthe cytokine profile of infected macrophagesshowed that Al. aviam -Tat complexes in-duced higher levels of TGF beta-1 [TGFbeta(1)] than M. aviam alone or Al. aviamthat had been in contact with other viralproteins. Conditioned media from HIV-1-infected H9 cens released a factor that en-hanced M. aviam intramacrophage growth,and was partially neutralized by an anti-TatAb. Finally, Tat protein (purified or presentin conditioned media from infected cells)moderately enhanced the growth of Al. ar-iam strains in extracellular media, and ex-posure of M. aviam to Tat protein in thepresence of IL-6 enhanced the growth ofAIDS-associated strains. These data arguefor an interaction between the Tat viraiproduct an the opportunistic pathogen Al.aviam which may contribute to the exqui-site susceptibility of AIDS subjects to thispathogen.—Authors' Abstract

de Wit, M. Y. L., Falou, M. and Content,J. Nucleotide sequence of the 85B-pro-tein gene of Mycobacterium bovis BCGand Mycobacterium tuberculosis. DNASeq. 4 (1994) 267-270.

We have cloned and sequenced the genescoding for the 85B antigen from Al. bonsBCG and AÍ. tuberculosis. Within this gene,the only difference in sequence between M.bons BCG and Al. tuberculosis corresponds,respectively, to a C —* T yielding a Leu —>Phe replacement at position 100 of the ma-ture 85B protein. Therefore, as we describedpreviously for the 85A gene, there is alsovery little variation between there two spe-cies within the 85B gene.—Authors' Ab-stract

Elsaatari, F. A. K., Engstrand, L., Hachem,C. Y., Graham, D. Y. and Naser, S. A.Identification and characterization ofMycobacterium paratuberculosis recom-binant proteins expressed in E. coli. Curr.Microbiol. 29 (1994) 177-184.

Mycobacterium paratuberculosis is thecausative agent ofJohne's disease, a chronicenteritis in ruminants, and it has also beenisolated and identified from patients withCrohn's disease, an inflammatory boweldisease. The control ofJohne's disease hasbeen hampered by the lack of a reliable di-agnostic test because of the large degree ofantigenic crossreactivity between mycobac-terial and nonmycobacterial species. To helpidentify specific antigen(s) or epitope(s), anAl. paratuberculosis expression library wasscreened with antibodies and DNA probes.In total, 54 clones were randomly picked,purified, and characterized by DNA probesand monoclonal antibodies with knownspecificity to individual mycobacterial an-tigens. Four clones carrying the heat shockprotein 65K-, two representing the secretedprotein 32K-, three representing the 21 K-,and 20 clones representing the specific in-sertion element of M. paratuberculosis(IS900)-encoding genes and their gene prod-ucts were identified and characterized. Well-defined recombinant antigens and/or epi-topes representing M. paratuberculosis mayfacilitate the development of specific diag-nostic tests and the investigation of theirrole in there chronic diseases.—Authors'Abstract

Gevaudan, M. J., Bollet, C. and Demicco,P. In nitro evaluation of clarithromycinalone and in combination against Myco-


bacterium chelonae complex. Pathol. Biol.42 (1994) 412-418.

The in vitro activity of clarithromycinalone and in combination with amikacin,ethambutol and rifabutin was tested against12 strains of Alt'cobacterium chelonae ab-scessos and eight strains of AI. chelonae che-lonae isolated from patients. Extracellularactivity of clarithromycin was assessed bydetcrmining MICs using the 1% proportionmethod in Middlebrook 7H11 agar mediacompared to the radiometric methodologyin 7H12 broth at two pHs, 6.8 and 7.4. TheMICs obtained at pH 7.4 were 2 to 4 moredilutions lower than thore obtained at pH6.8. By both methods, clarithromycin ap-pcared more active against isolates of Al.chelonae chelonae than against isolates ofM. chelonae abscessus. Clarithromycin-amikacin combination demonstrated themost important additive effect. The use ofthree drugs in association resulted in a syn-ergistic effect. Studies of intracellular bac-teria showed that the most effective bacte-ricidal combination was clarithromycin,amikacin and ethambutol together.—Au-thors' Abstract

Ghossein, R. A., Ross, D. G., Salomon, R.N. and Rabson, A. R. A search for my-cobacterial DNA in sarcoidosis using thepolymerase chain reaction. Am. J. Clin.Pathol. 101 (1994) 733-737.

The etiology of sarcoidosis is unknown,but mycobacteria have been considered asa possible etiologic agent. The authors usedthe polymerase chain reaction (PCR) tosearch for mycobacterial DNA in paraffin-embedded granulomatous tissues from pa-tients with sarcoidosis. The target sequenceused for PCR amplification is a 383-basepair segment of the gene encoding the 65-kDa mycobacterial surface antigen. This as-say can detect A1t'cobactcrium tuberculosisand atypical mycobacteria in archival ma-terial. Its sensitivity, which is superior toZiehl-Nielsen staining for acid-fast bacilli,is 1 bacterium per 2500 cells. Ten sarcoid-osis blocks and 10 normal controls werenegative with mycobacterial PCR but pos-itive with beta-actin PCR, indicating thepresence of amplifiable DNA. Mycobactcr-ial PCR gave positive results for six acid-

fast bacilli stain/culture-positive blocks frompatients with tuberculosis. These results in-dicate that sarcoidosis probably does notrepresent an active mycobacterial infection.These data also suggest that mycobacterialPCR is helpful in differcntiating tubercu-losis and sarcoidosis.—Authors' Abstract

Harris, D. P., Vordermeier, H.-M., Brett,S. J., Pasvol, G., Moreno, C. and Ivanvi,J. Epitope spccificity and isoforms of themycobacterial 19-kilodalton antigen. In-fect. Immun. 62 (1994) 2963-2972.

The topography and specificity of B- andT-cell stimulatory epitopes from the 19-kDaprotein of A71'cobacteriuni tuberculosis wereinvestigated by using overlapping syntheticpeptides. Murine antisera identified twocryptic epitopes (residues 11 to 30 and 61to 80) and one species-specific immuno-dominant epitope (residues 140 to 159). Im-munoglobulins G1 and G2a antibody iso-types varied for the respective peptide im-munogens but without relationship to theT-ceil cytokine profiles which were char-acterized by high gamma-interferon and lowinterleukin 5 leveis. Antisera to recombi-nant Al. tuberculosis 19-kDa protein (rGST-19) crossreacted with homologous proteinsof similar size from organisms of the AI.ariunt - intracellulare complex. Two -dimen-sional gel electrophorcsis revealed differ-ences in the number, relative mobility, andcharge of isoforms of the 19-kDa protein,possibly reflecting post-translational mod-ifications. The immunodominant T-cell ep-itope from the M. tuberculosis 19-kDa pro-tein (residues 61 to 80) and the correspond-ing peptide sequence from Al. aritmi subsp.intracellulare (residues 64 to 83), differingat five residues, werc both recognized in agenetically permissive manner. Peptides 61-80 and 64-83 stimulated crossreactive re-sponses in BALB/c (H-2d) mice, while inthe C57BL/10 (H-2e) strain, responses topeptide 61-80 were species specific. In pu-rified protein derivative-positive healthyindividuais, the AI. arium subsp. intracel-lulare peptide stimulated strongcr responsesthan did the Al. tuberculosis peptide; where-as patients with active tuberculosis had en-hanced in nitro T-cell responses to both pep-tides. — Authors' Abstract


Harth, G., Clemens, D. L. and Horwitz, M.A. Glutamine synthetase of Altcobactc-riunr tuberculosis: extraccllular release andcharacterization of its enzymatic activity.Proc. Natl. Acad. Sci. U.S.A 91 (1994)9342-9346.

We have investigated the activity and ex-tracellular release of glutamine synthetase[L-glutamate:ammonia ligase (ADP-form-ing), EC 6.3.1.2] of Altcobacteriunr tuber-culosis. The purified, homogeneous M. tu-berculosis glutamine synthetase appears toconsist of 12 most likely identical subunitsof M(r) 58,000, arranged in two superim-posed hexagons. In the catalysis of L-glu-tamine, the enzyme has an apparent Km forL-glutamate of approximately 3 mM at thepH optimum of 7.5. M. tuberculosis releasesa large proportion (approximately 30%) ofits total measurable enzyme activity into theculture medium, a feature that is highly spe-cific for pathogenic mycobacteria. Immu-nogold electron-microscopy revealed thatAI. tuberculosis also releases the enzyme intoits phagosome in infected human mono-cytes. Two potentially important roles forglutamine synthetase in the pathogenesis ofA1. tuberculosis infection are: (i) the synthe-sis of L-glutamine, a major component ofthe cell wall of pathogenic but not nonpath-ogenic mycobactcria, and (ii) the modula-tion of the ammonia levei in the M. tuber-culosis phagosome, which may in turn in-fluence phagosomal pH and phagosome-ly-sosome fusion.—Authors' Abstract

Harth, M., Ralph, E. D. and Faraawi, R.Septic arthritis due to Mtcobacteriunrnrarinu n r. J. Rheumatol. 21 (1994) 957-960.

We describe a patient with septic arthritisand osteomyelitis of the ring finger due toMicobactcriunr marinurn. A review of theliterature shows fewer than 40 reported casesof joint infection with this organism. Mostof the patients reported had been previouslyin good health, and had been in contact withfish or otherwise involved in aquatic activ-ities. The arthritis affects mainly the handsor wrists and is insidious in onset. Delay indiagnosis, and initial inappropriale treat-ment with intraarticular steroids are fre-quent. Thcrapy with antibiotics and/or sur-

gical debridement is usually successful.-Authors' Abstract

Heger, W., Schmahl, H. J., Klug, S., Felies,A., Nau, H., Merker, H. J. and Neubert,D. Embryotoxic effects of thalidomidederivatives in the non-human primateCallithrix jacchus. 4. Teratogcnicity of µg/kg doses of the EM 12 enantiomers. Te-ratogen. Carcinogen. Mutagen. 14 (1994)115-122.

The dose-response of the teratogenic po-tency of the thalidomide (Thd) derivativeEM 12 was evaluated in the common mar-moset (Callithrix jacchus). The smallestdaily dose found to be effective was 30 µgEM 12/kg body wt. This is the lowest doseof a Thd derivative ever reported to inducesevere skeletal abnormalities. Ten micro-grams EM12/kg body wt may be consid-ered the no-observed-adverse-effect-level(NOAEL) under the experimental condi-tions chosen. The teratogenic potencies ofthe two EM 12 enantiomers were tested at100 pg/kg body wt, the dose which just in-duces an almost 100% effect in the case ofthe racemate. The S(—)-EM 12 was found toinduce typical severe limb abnormalitiessuch as amelia, phocomelia, and radiusaplasia, and nono of the exposed fetuses weredevoid of skeletal defects. In contrast, onlyfew and minor skeletal defects were ob-served after application of the R(+) enan-tiomer. Although a pronounced teratogenicpotency of the R(+)-EM12 can now largelybe excluded, these low-dose studies are notsufficient to completely rule out any tera-togenic potential of this enantiomer, sinceracemization to small amounts of the S(—)form may occur in vivo. Further studies withThd derivatives which are unable to racem-ize are necessary to prove the assumed com-plete ineffectiveness of the R(+) enantio-mers.—Authors' Abstract

Henriques, B., Hoffner, S. E., Petrini, B.,Juhlin, I., Wahlen, P. and Kallenius, G.Infection with Mvcobacterium mal-moense in Sweden; report of 221 cases.Clin. Infect. Dis. 18 (1994) 546-600.

Mycobacteriunr malnroense was first de-scribed in 1977 and today is second only tothe M. arir n n complex as a cause of atypical


mycobacterial infection in Sweden. We ret-rospectivcly studied the records of 221 pa-tients from whom AI. mah noense was iso-lated during 1968-1989. AI. malinoense wasrecovered from the respiratory tract of 1 71patients (170 adults and one child) and fromcervical lymph nodes of 36 patients (35 chil-dren and one adult). In addition, the organ-ism was isolated from the urine of six pa-tients, one of whom had disseminated dis-case and two of whom had abscessos causedby AI. malnzoense. A majority of the pa-tients with pulmonary infection as well asthe patient with disseminated disease hadothcr underlying diseases.—Authors' Ab-stract

Heym, B., Honore, N., Truffot-Pernot, C.,Banerjee, A., Schurra, C., Jacobs, W. R.,van Embden, J. D., Grosset, J.-H. andCole, S. T. Implications of multidrug re-sistance for the future of short-course che-motherapy of tuberculosis: a molecularstudy. Lancei. 344 (1994) 293-298.

Tuberculosis-control programs are com-promised by the increased frequency ofmultidrug-resistant strains of Alrcobacteri-um tuberculosis. We used the polymerasechain reaction (PCR) and single-strand con-formation polymorphism (SSCP) analysistechniques to establish the molecular basisof resistance in 37 drug-resistant isolates ofM. tuberculosis, and correlated these find-ings with clinical and antibiotic-sensitivitydata. Resistance to isoniazid was found in36 strains, 16 of which were also resistantto ethionamide. Of the 36 isoniazid-resis-tant strains, 23 had mutations in the katGgene, and 5 of these also had mutations inthe inhA gene. A further 5 strains had al-terations in the inhA locus without the katGgene being mutated. Rifampin resistance wasless frequent (13 strains) and usually asso-ciated with isoniazid resistance (11 of 13strains). Mutations in the rpoB gene weredetected for all these rifampin-resistant iso-lates. Mutations in the rpsL and rrs genes,associated with streptomycin resistance,were found in 13 of 25 and 2 of 25 strep-tomycin-resistant strains, respectively. Thesame chromosomal mutations, or combi-nations of mutations, were found in strainsdisplaying single or multidrug resistance,from cases of both primary and secondary

resistance, and from patients infected withhuman immunodcficiency virus. Thus,multidrug resistance is not due to a novelmechanism and tuberculosis chemotherapyis not subject to a new threat.—Authors'Abstract

Hill, A. R., Mateo, F. and Hudak, A. Tran-sient exacerbation of tuberculous lym-phadenitis during chemotherapy in pa-tients with AIDS. Clin. Infect. Dis. 19(1994) 774-776.

We describe thrce men with disseminat-ed, drug-sensitive tuberculosis and ad-vanced human immunodcficiency virusdisease (CD4+ lymphocyte count, <50/mm 3 ) who had (lares of tuberculous lym-phadenitis with suppuration during the ini-tial weeks of successful chemotherapy. Bac-tericidal drugs may kindle these transientexacerbations, which involve neutrophilsbut apparently do not require normal helperT-ceil function. In patients with AIDS, asin immunocompetent individuais, treat-ment-related fiares of lymphadenitis areusually not an adverse sign, provided thatdrug resistance and nonadherence have beenexcluded. —Authors' Abstract

Hoffner, S. E., Klintz, L., Olsson-Liljequist,B. and Bolstrom, A. Evaluation of Etestfor rapid susceptibility testing of Myco-bacteriunt chelonae and AI. fortuituni. J.Clin. Microbiol. 32 (1994) 1946-1949.

Etest is a new concept for MIC determi-nations for antimicrobial agents that is basedon a predefined antibiotic gradient on aplastic strip calibrated with a continuouslogarithmic MIC scale covering 15 twofolddilutions. Etest was compared with a ref-erence agar dilution method for suscepti-bility testing of clinicai isolates of Alnco-bacleriuni chelonae and M. fortuitunt. Re-sults read after 3 days showed good agree-ment between MICs obtained with Etest andthose obtained with the reference methodwithin ±2 dilutions for 90% of ali test com-binations. All but one of the strains wereinhibited by low concentrations of cipro-floxacin or amikacin. Susceptibility to clar-ithromycin, erythromycin, imipenem, rif-ampin, doxycycline, and fusidic acid wasvariable, and ali strains were resistant to


ceftazidime and trimethoprim. The resultssuggest that Etest is well suited for studiesof drug resistance in rapidly growing my-cobacteria. —Authors' Abstract

Horsburgh, C. R., Chin, D. P., Yajko, D.M., Hopewell, P. C., Nassos, P. S., Elkin,E. P., Hadley, W. K., Simon, E. M., Gon-zalez, P., Ostroff, S. and Reingold, A. L.Environmental risk factors for acquisi-tion ofMvcobacteriunr aviam complex inpersons with human immunodeficiencyvirus infection. J. Infect. Dis. 170 (1994)362-367.

A case-control study was done to deter-mine risk factors for Mvcobactcriunr aviamcomplex (MAC) disease in persons infectedwith human immunodeficiency virus (HIV)with <50 CD4+ cells/mm 3 . In univariateanalysis, cases (N = 83) had lower CD4+cell counts than controls (N = 177) (median,10 vs 17/mm 3 ; p < 0.001) and were morelikely to have consumed hard cheese (oddsratio [OR], 5.44; 95% confidence interval[CI], 1.61-18.4) but were less likely to havetaken daily showers (OR, 0.55; 95% CI,0.33-0.94). In multivariate analysis, CD4+cell count < 25/mm 3 (OR, 3.58; 95% CI,1.71-7.49) and consumption of hard cheese(OR, 5.63; 95% CI, 1.58-20.1) remainedassociated with disease, while daily show-ering (OR, 0.58; 95% CI, 0.28-0.88) re-mained protective. Increased risk for MACdisease in persons with HIV infection andlow CD4+ cell counts is not associated withexposure to water or a variety of other en-vironmental sources but may be associatedwith the consumption of hard chccsc.—Au-thors' Abstract

Horsburgh, C. R., Metchock, B., Gordon,S. M., Havlik, J. A., McGowan, J. R. andThompson, S. E. Predictors of survival inpatients with AIDS and disseminatedMycobacteriinn aviam complex disease.J. Infect. Dis. 170 (1994) 573-577.

Patients with AIDS and disseminatedMycobacteriunt aviam complex disease(DMAC), as defined by the presence of apositive blood culture for MAC, were stud-ied retrospectively to define the natural his-tory of DMAC. All patients had fevers, se-vere anemia (hematocrit <26%), or both.

Eighty-seven (76%) had signs, symptoms,or laboratory findings related to the gastro-intestinal tract, but no distinct syndromewas identified. Sixty-nine patients receivedantimycobacterial therapy; assignment totherapy was not randomized. In a propor-tional hazards analysis, shorter survival wasassociated with higher initial level of my-cobacteremia (relative risk [RR], 1.86; 95%confidence interval [CI], 1.49-2.31; p <0.001), while administration of antimyco-bacterial chemotherapy (RR, 0.42; 95% CI,0.26-0.70; p < 0.001) and antiretroviraltherapy (RR, 0.40; 95% CI, 0.22-0.73; p <0.01) had protective effects. Thus, the initiallevei of mycobacteremia of patients withDMAC may have prognostic value, and ad-ministration of antimycobacterial and an-tiretroviral agents may be associated withprolonged survival. — Authors' Abstract

Hunt, J. M., Roberts, G. D., Stockman, L.,Felmlee, T. A. and Persing, D. 1-1. Detec-tion of a genetic locus encoding resistanceto rifampin in mycobacterial cultures inclinicai specimens. Diagn. Microbiol. In-fect. Dis 18 (1994) 219-227.

The polymerase chain reaction (PCR) andautomated DNA sequencing were used todetect a genetic locus, rpoB, associated withrifampin resistance in ,tlrcobacterium tu-berculosis (TB) in clinical isolates and di-rectly in clinical specimens. Primers de-rived from the sequence of a TB rpoB genefragment were used to amplify DNA frombacterial and mycobacterial isolates. AnrpoB-specific PCR product was obtained forfive of five TB, sevcn of eight other myco-bacterial specics, Nocardia sp., Corvnebac-teriam sp., Streptomyces sp., Actinomycessp., and Rhodococcus sp., but not for 15isolates (eight genera) representing usualbacterial flora. Sequence comparison of theamplified rpoB region revealed the occur-rence ofTB-specific "signature nucleotides"at three positions. PCR yielded amplifica-tion products for seven of 16 clinical spec-imens. Five of the seven contained TB-spe-cific DNA, as well as sequences that pre-dicted rifampin susceptibility in accord withagar dilution results. None of ten specimensthat were culture negative for TB yieldedTB-specific PCR products. These resultswith a limited number of clinical specimens


demonstrate the feasibility of direct detec-tion by PCR of rifampin-resistant TB inclinicai specimens. Such testing may serveas a rapid surrogate test for multidrug-re-sistant TB in laboratories with PCR andautomated sequencing capability.—Au-thors' Abstract

Inderlied, C. B., Barbara-Burnham, L., Wu,M., Young, L. S. and Bermudez, L. E. M.Activities of the benzoxazinorifamycinKRM 1648 and ethambutol against My-cobac7eriunl aviam complcx in Nitro andin macrophages. Antimicrob. AgentsChemother. 38 (1994) 1838-1843.

KRM 1648 is a 4-aminobenzoxazine de-rivative of rifamycin S with potent in nitroactivity against the Mt'cobacteril un aviamcomplex (MAC); MIC for 90% of 24 MACisolates from AIDS paticnts was 0.25 pg/mlas determined by a radiometric broth ma-crodilution assay. KRM 1648 was bacteri-cidal for MAC isolates in Middlebrook 7H9broth, with a reduction in viability of 1 to4 orders of magnitude over 72 hr. In humanmacrophages, KRM 1648 also was bacte-ricidal, with a reduction of 3 to 4 orders ofmagnitude in CFU per ml of macrophagelysate at a concentration of 1 pg/ml; how-ever, the bactericidal activity varied ap-proximately 10-fold among the three MACserovars tested. In growth medium, etham-butol potentiated the effect of KRM 1648,but this potentiation was modest when test-ed against MAC in macrophages and alsovaried between MAC strains. KRM 1648has potential as an antimycobactcrial agentfor MAC disease, perhaps in combinationwith other agents so that the use of lowerdosages of KRM 1648 than are needed withother rifamycins may be possible.—Au-thors' Abstract

Ji, B., Lounis, N., Truffot-Pernot, C. andGrosset, J. Effectiveness of various anti-microbial agents against Mvcobacteriumaviam complex in the beige mouse model.Antimicrob. Agents Chemother. 38 (1994)2521-2529.

The results of five chemotherapeu,tic ex-periments in beige mice infected with or-ganisms of the Mycobacterium aviam com-plex are presented. After monotherapy withvarious antimicrobial agents for 4 weeks,

only clarithromycin, amikacin, and etham-butol displaycd definite bactericida) effects;sparfloxacin and clofazimine showcd mod-est bacteriostatic effects; and rifampin andrifabutin were totally inactive against theisolate tested. After treatment for 4 weeks,the large quantities of clofazimine that hadaccumulated in the organs of mice scriouslyinterfered with the enumeration of the CFUand assessment of the efficacy of the treat-ment. The in vitro synergistic effects ofdrugcombinations against ;1I. aviam complexwere not confirmed in beige mice. In com-bination with clarithromycin, amikacincould prevent the selection of clarithro-mycin-resistant mutants, whereas minocyc-line could not.—Authors' Abstract

Kemper, C. A., I-Iavlir, D., I-Iaghighat, D.,Dube, M., Bartok, A. E., Sison, J. P., Yao,Y. Z., Yagco, B., Leedom, J. M., Tilles,J. G., McCutchan, J. A. and Deresinski,S. C. Thc individual microbiologic effectof three antimycobacterial agents, clofa-zimine, ethambutol and rifampin, on MY-cobacterilhnb aviam complex bacteremiain patients with AIDS. J. Infect. Dis. 170(1994) 157-164.

The individual antibacterial activities ofclofazimine, ethambutol, and rifampin inthe treatment of ,14vcobac1eriunl aviamcomplex bacteremia in patients with AIDSwere determined. Sixty human immuno-deficiency virus 1-infected patients who hadat least one blood culture positive for ,11.aviam complex were randomized to receiveeither clofazimine (200 mg), ethambutol (15mg/kg), or rifampin (600 mg) once daily for4 weeks. Only ethambutol resulted in a sta-tistically significant reduction in the levei ofmycobacteremia. The median change in in-dividual baseline colony counts was —0.60log o cfu/ml after 4 weeks of ethambutol (p= 0.046). In contrast, median changes inindividual baseline colony counts were —0.21og, 0 cfu/ml and +0.2 log, o cfu/ml for clo-fazimine and rifampin, respectively (both,p > 0.4). Ethambutol had greater antibac-terial activity, as determined by changes inthe levei of mycobacteremia, than either rif-ampin or clofaziminc, supporting its con-tinued use in combination with other agentsin the treatment of aviam infection.-Authors' Abstract


Kennedy, N., Gillespie, S. H., Saruni, A. O.S., Kisyombe, G., McNerney, R., Ngowi,F. I. and Wilson, S. Polymerase chain re-action for assessing treatment response inpatients with pulmonary tuberculosis. J.Infect. Dis. 170 (1994) 713-716.The use of the polymerase chain reaction

(PCR) for assessing treatment response intuberculosis was investigated. Serial spu-tum samples were analyzed from 10 Tan-zanian patients treated for smear-positivepulmonary tuberculosis, including 4 whorclapsed after initially successful trcatment.A one-tube nestcd PCR with a colorimetricdetection system was compared with mi-croscopy and culture. Samples were foundto be negative by microscopy before theywere by PCR or culture, often remainingpositive 1-2 months longer by PCR thanby culture. For the 76 samples available forboth culture and PCR, there was a 76% (58/76) agreement between the methods. Ninesamples were negative by culture but posi-tive by PCR; 7 were either negative (5) orequivocai (2) by PCR despite being positiveby culture. Two of the 4 relapse cases weredetected earlier by PCR than by culture.These results demonstrate that PCR is apromising method for assessing treatmcntresponse in pulmonary tuberculosis.—Au-thors' Abstract

Kenney, T. J. and Churchward, G. Cloningand sequence analysis of the rpsL and rpsGgenes of Mrcobacterium smegmatis andcharacterization of mutations causing re-sistance to streptomycin. J. I3acteriol. 176(1994) 6153-6156.

The Mi^cobacteriimr smegnratis rpsL andrpsG genes, encoding the ribosomal proteinsS12 and S7, were cloned, and their DNAsequence was determined. The third nucle-otide of the S12 termination codon over-lapped the first nucleotide of the S7 trans-lation initiation codon. A collection of 28spontaneous streptomycin-resistant mu-tants of ALI. smegmatis was isolated. All hadsingle-base-pair substitutions in the rpsLgene which were changed to a streptomycin-sensitive phenotype by complementationwith a low-copy-number plasmid carryingthe wild-type M. sntegmatis rpsL gene. Atotal ofeight different mutations were foundin two specific regions of the rpsL gene. Fif-

ty-seven percent (16 of 28) altered the Lyscodon at position 43. Forty-six percent ofthe mutations (13 of 28) were due to a tran-sition changing an AAG Lys codon to anAGG Arg codon, with eight changes at co-don 43 and five at codon 88.—Authors' Ab-stract

Kikuchi, K., Fukumoto, M., and Takahashi,H. Iron storage in Mycobacterium smeg-matis grown under iron-sufficient andiron-overload conditions. Biosci. Bio-technol. Biochem. 58 (1994) 885-888.

Two kinds ofiron-containing proteins themolecular masses of which were about 10kDa and 24 kDa were isolated from cyto-plasmic fractions of Mi'cobacteriunt smeg-matis grown under iron-sufficient (50 µMFe) and iron-overload (500 µM Fe) condi-tions. Based on the elution profiles in twochromatographic systems, spectrophoto-metric analysis, and ESR spectrum mea-surement, the protein of 10 kDa met thecriteria for classification as a ferredoxin.Another protein of 24 kDa showed no en-zymatic activity, though its detailed struc-ture was unknown. The ferredoxin and theprotein of 24 kDa contained about 30% and50% of the total cellular iron, respectively,when cells were grown under the above con-ditions. The synthesis of the protein of 24kDa was, however, completely repressed incells grown under iron-deficient (0.5 µM Fe)conditions, although the ferredoxin was stillsynthesized to some extent even in iron-deficient cells. These results suggested thatboth ferredoxin and the protein of 24 kDacould be synergistically involved in ironstorage in this organism.—Authors' Ab-stract

Klemens, S. P. and Cynamon, M. H. Inter-mittent azithromycin for treatment ofi%lrcobacterium avium infection in beigemice. Antimicrob. Agents Chemother. 38(1994) 1721-1725.The activity of azithromycin (AZI) was

evaluated in the beige mouse model of dis-seminated %Ii'cobacterium avium infection.Mice were infected intravenously with ap-proximately 10' viable M. avium ATCC49601. AZI at 50, 100, or 200 mg/kg ofbody weight or clarithromycin (CLA) at 200mg/kg was given by gavage 5 days per week

180^ International Journal of Leprosv^ 1995

for 4 weeks. Groups of treated mice werecompared with untreated control animais.A dose-related reduction in ccll counts inorgans was observed with AZI treatment.AZI at 200 mg/kg was more active thanCLA at 200 mg/kg against organisms inspleens. The activities of these two agentsat 200 mg/kg were comparable against or-ganisms in lungs. In a second study, AZI at200 mg/kg was given daily for 5 days; thiswas followed by intermittent AZI treatmentfor the next 3 weeks. The activitics of AZIgiven on a three-times- and five-times-per-week basis in the continuation phase werecomparable. AZI given on a once-weeklybasis was less active. The regimen of AZIgiven in combination with rifapentine on aonce-weekly basis for 8 weeks showedpromising activity. Clinical evaluation ofAZI and rifapentine will help to define theroles of these agents in the treatment of dis-seminated Al. ariunr complex infection.-Authors' Abstract

Klemens, S. P., Grossi, M. A. and Cynamon,M. H. Activity of KRM- 1 648, a new ben-zoxazinorifamycin, against Al'cobacteri-um tuberculosis in a murine model. An-timicrob. Agents Chemother. 38 (1994)2245-2248.

The activity of KRM-1648 was evaluatedin a murine model of tuberculosis. Approx-imately 10' viable Aíycobacteriunn tuber-culosis ATCC 35801 organisms were givenintravenously to 4-week-old female outbredmice. Treatment was started 1 week post-infection and given by gavage for 4 weeks.Viable-cell counts were determined fromhomogenates ofspleen and lung tissues. Theactivity of KRM-1648 was compared withthose of rifampin and rifabutin at 20 mg/kg of body weight. KRM-1648 was moreactive than either rifampin or rifabutinagainst organisms in spleens and lungs.KRM-1648 alone and in combination witheither isoniazid, ethambutol, pyrazinamide,or levofloxacin was evaluated. Other treat-ment groups received isoniazid, ethambu-tol, pyrazinamide, or levofloxacin as singleagents. KRM-1648 was the most active sin-gle agent evaluated. KRM-1648-pyrazin-amide and KRM-1648-isoniazid were themost active combinations. These combi-nations were more active than KRM-1648

alone. The promising activity of KRM-1648in Al. tuberculosis-infected mice suggests thatit is a good candidate for clinical develop-ment as a new antituberculosis agent.—Au-thors' Abstract

Klopman, G., Li, J.-Y., Wang, S., Pearson,A. J., Chang, K., Jacobs, M. R., Bajak-souzian, S. and Ellner, J. J. In nitro anti-Mvcobacterium anum activities of quin-olones: predicted active structures andmechanistic considerations. Antimicrob.Agents Chemother. 38 (1994) 1794-1802.

The relationship between the structuresof quinolones and their anti-Mvcobacteri-uni arium activities has been previously de-rived by using the Multiple Computer-Au-tomated Structure Evaluation program. Anumber of substructural constraints re-quired to overcome the resistance of mostof the strains have been identified. Nineteennew quinolones which qualify under thesesubstructural requirements were identifiedby the program and subsequently tested. Theresults show that the substructural attri-butes identificd by the program produced asuccessful a priori prediction of the anti-A1.anuiu activities of the new quinolones. All19 quinolones were found to be active, and4 of them are as active or better than cip-rofloxacin. With these new quinolones, theupdated multiple computer-automatedstructure evaluation program structure-ac-tivity relationship analysis has helped to un-cover additional information about the na-ture of the substituents at the C5 and C7positions needed for optimal inhibitory ac-tivity. A possible explanation of drug resis-tance based on the observation of suicideinactivation of bacterial cytochrome P-450by the cyclopropylamine moiety has alsobeen proposed and is discussed in this re-port. Furthermore, we confirm the view thatthe amount of the uncharged form presentin a neutral pH solution plays a crucial rolein the drug's penetration ability.—Authors'Abstract

Mabilat, C., Desvareene, S., Panteix, G.,Machabert, N., Bernillon, M.-H., Guar-diola, G. and Cros, P. Routine identifi-cation of Alycobacteriu,n tuberculosis


complex isolated by automated hybrid-ization. J. Clin. Microbiol. 32 (1994)2702-2705.

Methodologies for biochemical identifi-cation of mycobacteria isolated from clin-ical samples are still cumbersome, takingskilled technicians 3 to 6 weeks. We de-scribe here a 2-hr identification system formycobacterial isolates belonging to Mvco-bacteriunt tuberculosis complex using aDNA probe. After 30 min of hand-off sam-ple preparation, the I.5-hr hybridization testis totally automated in the newly developedVIDAS system (bioMérieux, Marcyl'Etoile,France), which performs solid-phase spe-cific hybridization of 16S rRNA at 37°C.The strain collection of actinomycetes test-ed was composed of 662 isolates from 27species: 461 members of the M. tuberculosiscomplex (443 M. tuberculosis, 10 Af. bons,and 8 AI. bons BCG isolates) and 201 iso-lates of other species, including 55 AI. ar-iunt - intracellulare isolates). They wereidentified by traditional methods: growthrate, colonial morphology, pigmentation,and biochemical profiles. The automatedprobe assay displayed an excellent correla-tion with the reference results. The fourmembers of the Nocardia and Rhodococcusgenera tested did not cross-hybridize. Thisflexible random-access and automated tech-nology was shown to suit the routine contextof the laboratory by rapidly delivering theresults.—Authors' Abstract

Majumder, S. and Gupta, R. Effect ofinterleukin-1 alpha on the growth of Alv-cobacteriuni microti within J774A.1 cells.FEMS Microbiol. Lett. 120 (1994) 329-334.

The effect of mouse recombinantinterleukin-1 alpha (IL-1a) on the intracel-lular growth of Afvcobacteriuni microti in amurine macrophage cell line J774A.1 wasinvestigated. IL- l a added after infection tothe M. microti-infected macrophage mono-layers enhanced the growth of M. microti ina concentration-dependent manner and thisgrowth enhancement was abrogated by neu-tralization of IL-1a with anti-IL- l a anti-body. Cyclic adenosine monophosphatelevei in J774A.1 cells was increased by theaddition of IL- l a. Addition of dibutyryl cy-

clic adenosine monophosphate to infectedJ774A.1 cells increased the number of in-tracellular bacteria in a concentration-de-pendent manner. There results suggest thatIL-la acts as a growth enhancer for intra-cellular M. microti and the growth-enhanc-ing effect of IL- l a may be due to enhancedcellular cyclic adenosine monophosphatelevei. —Authors' Abstract

McCormick, P. A., Scott, F., Epstein, O.,Burroughs, A. K., Scheuer, P. J. andMcIntyre, N. Thalidomide as therapy forprimary biliary cirrhosis: a double-blindplacebo controlled pilot study. J. Hepatol.21 (1994) 496-499.

Thalidomide has been reported to be ef-fective in treating graft-versus-host disease,a condition with many clinicai and patho-logical similarities to primary biliary cir-rhosis. We performed a double-blind, pla-cebo-controlled pilot study to assess the ef-ficacy of thalidomide in 18 patients withbiopsy-proven primary biliary cirrhosis (10thalidomide, 8 placebo). Each patient wastreated for 6 months and had a Tiver biopsybefore and after treatment. Side effects, par-ticularly sedation and fatigue, were morecommon on thalidomide and two patientswere withdrawn from this group. There wereno improvements in Tiver function tests orin liver histology, assessed morphometri-cally. A number of patients treated with tha-lidomide reported an improvement in pru-ritus. This study suggests that thalidomideis unlikely to be effective in altering the nat-ural history of primary biliary cirrhosis.-Authors' Abstract

Mills, J. A., McNeil, M. R., Belisle, J. T.,Jacobs, W. R., Jr. and Brennan, P. J. LociofAlvcobacterium aviam ser2 gene clusterand their functions. J. Bacteriol. 176(1994) 4803-4808.The highly antigenic glycopeptidolipids

present on the surface of members of theAlrcobacterium avium complex serve todistinguish these bacteria from ali othersand to define the various serovars that com-pose this complex. Previously, the genesresponsible for the biosynthesis of the dis-accharide hapten [2,3-di-O-methyl-a-L-fu-copyranosyl-(1—>3)-a-L-rhamnopyranose] of


serovar 2 of the M. avilun complex wereisolated, localized to a contiguous 22- to 27-kb fragment of the Al. arillnl genome, anddesignated the ser2 gene cluster (J. T. Be-lisle, L. Pascopella, J. M. Inamine, P. J.Brennan, and W. R. Jacobs, Jr., J. Bacteriol.173:6991-6997, 1991). In the present study,transposon saturation mutagenesis was usedto map the specific genetic loci within theser2 gene cluster required for expression ofthis disaccharide. Four essential loci, termedser2/1,-I3,-C, and -D, constituting a total of5.7 kb within the ser2 gene cluster, weredefined. The se121J and ser2D loci encodethe methyltransferases required to methyl-ate the fucose at the 3 and 2 positions, re-spectively. The rhamnosyl-transferase wasencoded by ser2,1; whereas either ser2C orser2D encoded the fucosyltransferase. Thescr2C and ser2D loci are also apparentlyinvolved in the de novo synthesis of fucose.Isolation of the truncated versions of thehapten induced by the transposon inser-tions provides genetic evidence that the gly-copeptidolipids of AI. arillnl serovar 2 aresynthesized by an initial transfer of therhamnose unit of the peptide core followedby fucose and finally O methylation of thefucosyl unit.—Authors' Abstract

Montano, L. F., Masso, F., Paez, A., San-doval, S., Vazquez, L., Sanchez, L., Four-net, B. and Zenteno, E. Isolation of a 32kDa Mycobacteriunl tuberculosis proteinby lectin affinity chromatography. Com-par. Biochem. Physiol. [B] 108 (1994)265-272.

A 32-kDa antigen from delipidated My-cobacteriu,n tuberculosas H37Rv culture fil-trate protein extract (CFPE) was purified byaffinity chromatography on immobilizedLens culinaris lectin and electroelution. Thisantigen represents 0.4% of the total CFPEcarbohydrate content and possesses galac-tose, xylose, mannose and G1cNAc (5:2:3:1mol. ratio). A monoclonal antibody againstthe purified antigen reacted with the 32-kDaas well as a 30-kDa antigen in H37Rv CFPE,thus suggesting that both antigens representclosely related allelomorphic forms'of thesame antigen.—Authors' Abstract

Newman, G. W., Guarnaccia, J. R., Vance,E. A., Wu, J. Y., Remold, H. G. and Ka-zanjian, P. H. Interleukin-12 enhances

antigen-specific proliferation of periph-eral blood mononuclear cens from HIV-positive and -negative donors in respdnseto Mvcobactcr/u,n arium. AIDS 8 (1994)1413-1419.

Objective: To determine if the additionof recombinant (r) human interleukin (IL)-12 enhances in vitro proliferative responsesto A1t'cobacterh nn arillnl ofperipheral bloodmononuclear cens (PBMC) from HIV-pos-itive donors with CD4 cell counts <100 x10 6 /1.

Design and methods: PBMC proliferativeresponses to virulent and avirulent serovarsof Al. avim)/ in the presence and absence ofexogenously added IL-12 were determincdin 24 HIV-positive and 11 HIV-negativedonors by H-3-thymidine uptake assay.Changes in CD4 and CD8 cell populationsafter IL-12 treatment and AI arilnn stim-ulation were analyzed by FACS.

Results: I L-12 significantly enhancedproliferation of PBMC to both virulent andavirulent AI. arilnn from all 24 HIV-posi-tive donors (p = 0.0001) although the mag-nitude varied for each donor. In contrast,addition of IL-12 to PBMC from HIV-neg-ative donors only increased the proliferativeresponses to the virulent A1. anu iu serovar4 (p = 0.0044). PBMC from HIV-positivedonors in the presence of IL-12 respondedbetter to the avirulent serovar of AI. arilnnthan the virulent serovar 4. Proliferative re-sponses of HIV-positive donors to M. av-iunl alone, however, were significantly less(p = 0.0013) than that of HIV-negative do-nors. Increased proliferative responses ofHIV-positive donors were independent ofCD4 counts. No significant changes in theratio of CD4+ to CD8+ T cens occurredin either HIV-positive or negative donorsunder any culture conditions.

Conclusion: In Atro proliferative re-sponses of PBMC from HIV-positive do-nors to M. avrlull were significantly en-hanced by the addition of human rIL-12,which was not dependent on their CD4 cellcounts. The use of IL-12 as an enhancer ofcell-mediated immunity in AIDS patientsagainst M. aviunl infections deserves fur-ther study.—Authors' Abstract

Nibbering, P. H., Yoshida, S. I., Van denBarselaar, M. T. and Van Furth, R. Bac-teriostatic activity of BCG/PPD-activat-


ed macrophages against Mycobacteriunifortuiturn does not involve reactive nitro-gen or oxygen intermediates. Scand. J.Immunol. 40 (1994) 187-194.

Mycobacteria preferentially reside in res-ident macrophages whereas activated mac-rophages are presumed to eliminate the bac-teria effectively. The aim of the present studywas to determine the antibacterial activitiesof resident and activated murine peritonealmacrophages against Mvcobacteriunt for-tuition and the intracellular mechanisms in-volved. After phagocytosis M. fortuitunlcould not be killed by either BCG/PPD-activated and IFN-gamma-activated mac-rophages and resident macrophages. Themycobacteria did not multiply in BCG/PPD-activated macrophages and the rate ofproliferation of AI. fortuitlun in IFN-gam-ma-activated macrophages was only slightlyinhibited compared to that in resident mac-rophages. Experiments with selective inhib-itors of the production of reactive nitrogenintermediates (RNI) and reactive oxygcn in-termediates (ROI) demonstrated that thesefactors are not essential for the mycobac-tcriostatic activity of BCG/PPD-activatedmacrophages. After phagocytosis of AI. for-tuitunl, BCG/PPD-activated and IFN-gam-ma-activated macrophages produced sub-stantial amounts of both RNI and ROI. Nocorrelation was found between the levels ofthese intermediates and the proliferation ofLI. for/ui/uni in the macrophages. In con-clusion, BCG/PPD-activated macrophagesare bacteriostatic, but not bacteriocidal forAI. fortuitum and the former does not in-volve reactive nitrogen and oxygen inter-mediates. —Authors' Abstract

Perez, M. H., Fomukong, N. G., Hellyer,T., Brown, I. N. and Dale, J. W. Char-acterization of IS 1 1 10 a highly mobile ge-netic element from Afi cobacteriurn ar-iunr. Mol. Microbiol. 12 (1994) 717-724.

A highly mobile insertion sequence des-ignated IS 1 1 10 was detected in Aíj'cobac-teriunl ariunr strain LR541 following an ob-served increase in size of the plasmid pLR20.Genomic libraries of AI. ariunr strains car-rying either parental pLR20 or the modifiedplasmid (pLR20') were constructed and thesequence of the relevam clones was deter-mined to characterize the insertion se-

quence and the target region. IS! 1 10 is a1457-bp element lacking terminal invertedrepeats, and is related to IS900 (from Al.paratuberculosis), IS901 and IS902 (fromM. ariunr) and to IS 116 (from Streptonlvicesclavuligerus). LR541 carries several copiesof IS1110. Individual colonies from thesame plate show differences in Southern blotpatterns when tested with an IS 1 1 10-de-rived probe; the ability to detect transpo-sition events in random colonies, withoutany selection pressure, indicates an excep-tionally high degree of mobility, which willbe invaluable for transposon mutagenesis.Analyses ofAI. ariunr isolates from human,vetcrinary, and environmental sourcesshowed that IS1110-hybridizing sequencesare present in some AI. ariunr isolates butthey were not detected in strains of othermycobacterial species. The polymorphismexhibited in AI. ariurn isolates suggests thatthis element may be useful for molecularepidemiological studies of M. ariunr infec-tions.—Authors' Summary

Petty, R. E., Hunt, D. W. C., Mathers, D.M., McCormick, A. Q., Barker, H.,Southwood, T. R. and Corson, L. Exper-imental arthritis and uveitis in rats as-sociated with Ahvcobacterium butrricunl.J. Rheumatol. 21 (1994) 1491-1496.

The objective was to determine if the an-terior uveitis associated with adjuvant ar-thritis (AA) in the rat can be passively trans-ferred with arthritis to syngeneic recipienteusing spleen cells or T-cell lines preparedfrom animais given complete Freund's ad-juvant (CFA) and Alvcobacteriunl butt'ri-cum (AI. butt°ricunl) in incomplete Freund'sadjuvant (IFA). Splcen cells from Lewis orLewis SsN rats given IFA, CFA, type I col-lagen in IFA (CI-IFA), or type II collagenin IFA (CII-IFA) were administered to na-ive rats or rats treated with pertussis toxinor bacterial endotoxin. Three CD4+ T-celllines, propagated from CFA injected ratsand maintained in litro with AI. bulvricunl(M-1), bovine proteoglycan (PR-1) or an ex-tract of ABI. but}'ricunl (MBE-1) were ad-ministered to naive or immunosuppressedrats. The arthritogenic and uveitogenicproperties of these cell preparations and in-tradermal MBE-IFA, CII-IFA and intra-peritoneal (i.p.) AI. but}'ricunz without ad-juvant were evaluated. Uveitis was ob-


served in 15/69 (22%) arthritic rats givenCFA. Spleen cells prepared from CFA-in-jected rats caused arthritis in 55 (85%) anduveitis in 2 (3%) of 67 cell recipients. Uve-itis occurred in 2/6 cell recipients pretreatedwith bacterial endotoxin. Neither uveitis norarthritis was obscrvcd in rats given IFA (0/6) or spleen cells prepared from rats givenIFA (0/27), CI-IFA (0/6), or CII-IFA (0/28).CII-IFA produced polyarthritis in 5/6 rats,but no uveitis. CII-IFA induced arthritis as-sociated uveitis in 1/15 animais receivingspleen cells from rats givcn CII-IFA, but notthose givcn CI-IFA (0/3) or IFA (0/13).Uveitis was obscrvcd in one recipient of theM-1 T-cell line and in two recipients of thePR-1 T-ccll line. Immunization with 400 µgof MBE-IFA induced uveitis but not ar-thritis in 3/11 animais. The MBE-spccificT-cell line was neither arthritogenic noruveitogenic. A high frequency (5/6) of uve-itis accompanied arthritis in male Lewis ratsgivcn i.p. M. butt'ricum. Arthritis occurredin 4/10 female Lewis rats given i.p. AI. hu-ti'ricu,n and 2 arthritic animais also devel-oped uveitis. Uveitis occurs infrequently inarthritic rats given spleen cells from CFA-injected animais. Thc i.p. administration ofM. butvricum constitutes a novel diseasemodel in which the immunopathological re-lationships between arthritis and uveitis maybc more reliably studied.—Authors' Ab-stract

Popper, H. H., Winter, E. and Hotler, G.DNA of Mycobacterium tuberculosis informalin-fixed, parallìn-cmbedded tissuein tuberculosis and sarcoidosis detectedby polymerase chain reaction. Am. J. Clin.Pathol. 101 (1994) 738-741.

Infection with Mycobacterium tubercu-losis is a major cause of death worldwide.Identification of mycobacteria in tissue sec-tions is usually easily achieved by acid-faststains, but this method sometimes gives un-satisfactory results. The authors thereforecompared conventional staining techniquesand polymcrasc chain reaction (PCR) formycobacterial DNA sequences in 24 se-lected tissue samples from patients with tu-berculosis. In ali samples, either positive ornegative with acid-fast stain, mycobacterialDNA fragments were detected. In addition,tissue samples from patients with clinically

proven sarcoidosis were included as con-trols. Surprisingly, strong signals for my-cobacterial DNA were found in 2 of 15 cases.PCR is a useful technique in the demon-stration of mycobacterial DNA fragmentsin patients with clinically suspected tuber-culosis who have acid-fast stain-negativehistology. An epitheiioid granulomatous re-action in the lung, negative by acid-fast stainand positive for mycobacterial DNA byPCR, however, does not permit the diag-noses of tuberculosis, because a positive re-sult can also be obtained in cases of sar-coidosis. In some cases of sarcoidosis, thecausal agent might be either cell-wall defec-tive mycobacteria or persistent intracellularDNA from mycobacteria.—Authors' Sum-mary

Sanchez Navarro, A., Martinez Cabarga, M.and Dominguez-Gil Hurle, A. Oral ab-sorption of ofioxacin administered to-gether with aluminum. Antimicrob.Agents Chcmother. 38 (1994) 2510-2512.

A clinicai study was carried out to estab-lish the influence of aluminum on the oralabsorption of ofloxacin. Ten healthy vol-unteers were included in a crossover studybased on a Latin square design. Thc treat-ments that ali volunteers received were A(consisting of 400 mg of ofloxacin) and B(consisting of 400 mg of ofloxacin plus 11g of colloidal aluminum phosphate). Theabsorption constant and other ofloxacin pa-rameters were calculated from data on lev-els in plasma by using modei-independentcalculation methods. There were no statis-tically significant differences between themean values of the arcas under the curvecorresponding to the administration of of-loxacin alone and those of ofloxacin withaluminum. Regarding the other pharma-cokinetic parameters, a significant differ-ence between the absorption constants wasfound. The presence of aluminum reducesthe absorption rate of this quinolone butdoes not modify the percentage of the ab-sorbed dose.—Authors' Abstract

Sathe, S. S., Sarai, A., Tsigler, D. and Ne-dunchezian, D. Pentoxifylline aggravatesimpairment in tumor necrosis factor-al-pha secretion and increases mycobacter-ial load in macrophages from AIDS pa-


tients with disseminated Mycobacteriumanum - intracellulare complex infection.J. Infect. Dis. 170 (1994) 484-487.

Pentoxifylline, which inhibits tumor ne-crosis factor-alpha (TNF-a), decreases hu-man immunodeficiency virus replication inperipheral blood mononuclear cells. How-ever, TNF-a is important in cellular defenseagainst Aivcobacterium avium - intracellu-lare complex (MAC), a common infectionin advanced AIDS. The eWect of pentoxi-fylline on mycobacterial colony counts inmacrophages with in vivo MAC infectionwas evaluated, and differences in lipopoly-saccharide (LPS)-induced TNF release ininfected and uninfected macrophages weredetermined. Macrophages with in vivo MACinfection released much less TNF-a in re-sponse to LPS (p = 0.01). The response waspartially restored after antimycobactcrialtherapy. Pentoxifylline, in a concentrationthat inhibited LPS-induced TNF-a by52.4%, incrcased MAC counts by 2.5- to50.0-fold. Thus, macrophages from AIDSpatients with disseminated MAC infectionare deficient in their ability to release TNF-aand further inhibition by pentoxifylline maybe detrimental.—Authors' Abstract

Schluger, N. W., Condos, R., Lewis, S. andRom, W. N. Amplification of DNA ofMycobacterium tuberculosis from periph-eral blood of patients with pulmonary tu-berculosis. Lancet 344 (1994) 232-233.

Sputum examination for rapid diagnosisof pulmonary tuberculosis is not always sat-isfactory. We examined peripheral bloodwith the polymerase chain reaction (PCR).Blood samples were collected from 8 con-secutive patients with suspected pulmonarytuberculosis and from 18 healthy controls,half of whom were tuberculin skin-test pos-itive. All 8 patients had evidence of circu-lating Mycobacteriuni tuberculosis DNA inthe lymphocyte fraction of peripheral blood,and positive sputum cultures indicating ac-tive pulmonary tuberculosis. None of thehealthy controls had positive PCR results.This PCR technique may prove useful forthe rapid diagnosis of tuberculosis.— Au-thor's Abstract

Sekosan, M., Cleto, M., Senseng, C., Far-olan, M. and Sekosan, J. Spindle ccll

pscudotumors in the lungs due to My-cobacterium tuberculosis in a transplantpatient. Am. J. Surg. Pathol. 18 (1994)1065-1068.

A rare spindle-cell pseudotumor in theskin, lymph nodes, and bons marrow hasbeen previously reported in immunosup-pressed transplant patients and patients withacquired immunodeficiency syndrome. Allreported cases were caused by Mycobacte-rium avium - intracellulare or other nontu-berculous mycobacteria. We are reportingspindle-cell pseudotumors in the lungscaused by M. tuberculosis. The patient hadinsulin-dependent diabetes mellitus and wasstatus post-cadaveric renal and pancreatictransplants. His hospital course was com-plicated by pulmonary tuberculosis due toAI. tuberculosis. At autopsy, the lungsshowed numerous, bilateral gray nodulesranging from 0.2 to 2.5 cm. Microscopicexamination uncovered a cellular prolifer-ation composed of spindle cells arranged infascicles. There were no granulomata. Anacid-fast stain showed numerous acid-fastbacilli within the spindle edis. To ourknowledge, this is the first case of spindle-ccll pseudotumor caused by M. tuberculosisof the lungs. Awareness of this unusualmanifestation of mycobacterial infection inimmunosuppressed patients underscores thenecd for acid-fast staining of biopsies withspindle-cell proliferation even in the ab-sence of overt granulomatous lesions in or-der to prevent misdiagnosis.—Authors' Ab-stract

Sepulveda, R. L., Heiba, I. M., King, A.,Gonzalez, B., Elston, R. C. and Sorensen,R. U. Evaluation of tuberculin reactivityin BCG-immunized siblings. Am. J. Res-pir. Crit. Care Med. 149 (1994) 620-624.

The purpose of the present study was todetermine the BCG-immunized sibships<_ 14 yr of age whether the correlations ofintensity of tuberculin reactivity support agenetic regulation of the response to BCGimmunization. The study population con-sisted of 659 healthy children living in 265households exposed to an adult with tuber-culosis: 38 children did not have a BCGscar, 327 children had one BCG scar, and294 had two BCG scars from vaccinationsat birth and at 6 yr of age. There were 603


full-siblings, 16 half-siblings, and 40 unre-lated children. Tuberculin testing was per-formed by one trained nurse. Sibling cor-relations of the intensity of the tuberculinresponse were calculated after adjusting forvarious nongenetic covariates that could beimportant in predicting it. The sibling cor-relations were significant at the 1% signifi-cance levei. There was no significant cor-relation of tuberculin reactivity among un-related children in the same household.These results are consistent with genetic reg-ulation of the development and persistenceof tuberculin reactivity after BCG immu-nization.—Authors' Abstract

Silva, S. R. B., Viana, P. C. F., Lugon, N.V., I-loette, M., Ruzany, F. and Lugon, J.R. Thalidomide for the treatment ofuremic pruritus: a crossover randomizeddouble-blind trial. Nephron 67 (1994)270-273.

Our observation that thalidomide ad-ministration to a dialysis patient with lep-rosy alleviated his pruritus led us to conductthis short-terra study to assess the efficacyof the drug in this regard. From 210 he-modialysis patients, 29 cases of refractoryuremic pruritus were entered finto the study.Patients were instructed to score theirsymptoms from O to 3, three times a day,and assigned to reccive thalidomide or pla-cebo at bedtime for 7 days. After a washoutperiod of 7 days, drugs were crossed over.Response was defined as a reduction of atlcast 50% in the pruritus scoring. Eighteenpatients finished the study. In the first phase,55% of patients responded showing a meanreduction in the pruritus scoring of 78% (p< 0.05 vs placebo); no response to placebowas observed. A similar proportion of pa-tients responded to thalidomide in the sec-ond phase with a mean reduction in theirpruritus scoring of 81%. In conclusion, tha-lidomide can be a precious tool in the han-dling of uremic pruritus unresponsive toavailable therapy. —Authors' Abstract

Skuce, R. A., Brittain, D., Hughes, M. S.,Beck, L.-A. and Neill, S. D. Genomicfingerprinting of Mvcobacterium bovisfrom cattle by restriction fragment lengthpolymorphism analysis. J. Clin. Micro-biol. 32 (1994) 2387-2392.

Two insertion sequences, IS6110 andIS1081, specific to the tuberculosis complexmycobacteria and a highly reiterated DNAelement (pTBN 12) cloned from MMrcobac-tcriunl tuberculosis were systematically usedto identify restriction fragment lcngth poly-morphism (RFLP) types among bovine iso-lates of AI. bons in Northern Ireland. In asample of 109 isolates, probes IS61 10,IS1081, and pTBN12 identified 10, 2, and12 distinct patterns, respectively. By com-bining the patterns generated by the threeprobes it was possible to identify 28 distinctRFLP types. The standard protocol advo-cated for RFLP analysis of M. tuberculosiswas used and would facilitate computer-based gel documentation and image anal-ysis to establish a database of AI. bons typesfor large-scale epidemiological studies.Thcse procedures will facilitate interlabor-atory comparisons of M. bons isolates andwill help to elucidate the precise epidemi-ology of bovine tuberculosis in differentcountries.—Authors' Abstract

Spies, II. S. C. and Steenkamp, D. .I. Thiolsof intracellular pathogens—identificationofovothiol A in Lcish/naltia donovani andstructural analysis of a novel thiol fromAlt'cobactcrium bovis. Eur. J. Biochem.224 (1994) 203-213.

Leishl/lalna donovani, the causative agentof visceral leishmaniases, is an intracellularpathogen which proliferares within the hostmacrophages. Analysis of the thiol com-position of L. donovani by means of thethiol-specific reagent, 7-diethylamino-3-(4'-maleimidylphenyl)-4 indicated that this or-ganism produces substancial amounts ofovothiol A. This observation was furthersubstantiated by HPLC of extracts of L.donovani after derivatization with bromo-bimane. L. donovani extracts contained athiol, the bimane derivative of which hadidentical retention time and fluorescencequenching to a thiol from Crithidia fasci-culata, which had previously been identifiedas ovothiol A. By comparison, the intra-cellular bacterial pathogen, A7rcobactcriurnbovis, contained only one major low-mo-lecular-mass thiol, which was assigned atrivial name mycothiol. The structure ofbi-mane derivative of mycothiol was solvedby a combination of one- and two-dimen-


sional H-1 and C-13 NMR spectroscopy.Spatial relationships in the molecule werefurther refined by NOE experiments and al-lowed identification of mycothiol as 1-D-myo-inosito1-2-(N-acetyl-L-cysteinyl)am i-no-2-deoxy-alpha-D-glucopyranoside. Thisassignment was confirmed by positive-ionfast-atom-bombardment mass spectrome-try which gave m/z = 677.6 Da and a so-diated species at 699.6 Da. Analysis of thedansylated hydrolysis products of perform-ic-acid-oxidized mycothiol indicated thepresence of 0.85 moi glucosamine and 1.02moi cysteic acid/moi sulfhydryl groups.Crude extracts ofAM. bons contained an en-zyme which catalysed the NAD(P)H,-de-pendent reduction of mycothiol disulfide tothe free thiol. Analysis of perchloric acidextracts ofAi. tuberculosis H37Rv indicatedthe presence of a thiol which comigratedwith mycothiol, both as the free thiol andas the 7-diethylamino-3-(4'-maleimidyl-phenyl)-4 and bimane derivates, on reverse-phase HPLC. The significance of these find-ings in tcrms of the evasion of the host de-fense mechanisms by leishmania parasitesand mycobacteria is considered.—Authors'Abstract

Tomiyama, T., Asano, S., Suwa, Y., Morita,T., Kataoka, K., Mori, H. and Endo, N.Rifampicin prevents the aggregation ofneurotoxicity of amyloid beta protein innitro. Biochem. Biophys. Res. Comm. 204(1994) 76-83.

The aggregation and cerebral depositionof amyloid beta protein (AP), which is amajor component of senile plaques in Al-zhcimer's disease (AD) brains, is believedto be involved in the pathogenesis of AD.Inhibition ofA beta aggregation would seemto be a promising strategy for the treatmentof AD. Here, we show that rifampin, whichis an antibiotic widely used in the treatmentof tuberculosis and leprosy, inhibited theaggregation and fabril formation ofsyntheticA beta 1-40 peptide in a dose-dependentmanner at reasonable concentrations. Fur-thermore, rifampin was found to prevent Abeta 1-40-induced neurotoxicity on ratpheochromocytoma PC12 cens. Rifampinmay have therapeutic potential as an agentfor inhibiting the initial step of amyloid for-mation in AD.—Authors' Abstract

Valero, G., Moreno, F. and Graybill, J. R.Activities of clarithromycin, ofioxacin,and clarithromycin plus ethambutolagainst Mvrobacterium sitniae in murinemodel of disseminated infection. Anti-microb. Agents Chemother. 38 (1994)2676-2677.After 2 weeks of intravenous challenge

with Mi'cobacteriunt simiae, ICR outbredmice were treated with clarithromycin, of-loxacin, or clarithromycin plus ethambutolfor 4 weeks. All three therapy groups dem-onstrated a decrease in the levei of infectionin both the lungs and the spleen. There wereno significam differences among the threetreated groups in decreasing mycobacterialcounts in the lungs; however, both ofloxacinand clarithromycin plus ethambutol weresuperior to clarithromycin alone in reducingthe levei of infection in the spleen. Resultsof the study suggest a potential role for theseagents in the treatment of human Al. sitniaeinfection.—Authors' Abstract

Valway, S. E., Richards, S. B., Kovacovich,J., Greifinger, R. B, Crawford, J. T. andDooley, S. W. Outbreak of multi-drug-resistant tuberculosis in a New York stateprison, 1991. Am. J. Epidemiol. 140(1994) 113-122.

In the summer of 1991, four inmates fromprison A in Upstate New York [U.S.A.] diedof multidrug-resistant tuberculosis. To de-termine the extent of resistant tuberculosisat prison A and transmission patterns, theauthors interviewed staff and reviewedmedicai records and inmate movement his-tories. Contact investigation results wereexamined to determine tuberculin skin-testconversions and to estimate risk ofinfectionand disease for inmates who were seropos-itive for human immunodeficiency virus(HIV). Eight HIV-positive inmates and oneHIV-negative guard, who was immuno-compromised with cancer, had multidrug-resistant tuberculosis. Eight died a medianof 28 days after the first culture-positivespecimen was collected. All isolates hadidentical seven-drug resistance and DNAfingerprint patterns. Ofexposed inmates, 92out of 306 (30%) had skin-test conversions.HIV infection was not associated with be-coming infected with drug-resistant tuber-


culosis (active disease or skin-test conver-sion), but once infected, HIV-positive in-mates were significantly more likely to de-velop disease than were HIV-negativeinmates (p < 0.001). The source case trans-ferred to prison A in February 1991, was illwith undiagnosed multidrug-resistant tu-berculosis, refused medicai care, and livedin the general prison population where hetransmitted the disease to other inmates.Lapsos in infection control and laboratorydelays contributed to this outbreak. Prisonsshould fully implement infcction controlguidelines to prevent tuberculosis trans-mission.—Authors' Abstract

van Rensburg, C. E. J., Anderson, R., Myer,IVl. S., Joone, G. K. and O'Sullivan, J. F.The riminophenazine agents clofazimineand B669 reverse acquircd multidrug re-sistance in a human lung cancer cell line.Cancer Lett. 85 (1994) 59-63.

The potential of the riminophcnazineagents clofazimine and 13669, at therapeu-tically relevant concentrations, to reverseP-glycoprotein-mediated multidrug-resis-tance (MDR) in a human lung cancer cellline (H69/LX4) has been investigated innitro. Cyclosporin A, a well-documentedMDR-modifying agent, was included forcomparison. Clofazimine, B669 and cy-closporin A at minimally cytotoxic concen-trations of 1, 0.5 and 5 pg/ml, respectively,were equally effective in restoring sensitiv-ity to vinblastine, doxorubicin, daunorub-icin and mitomycin C in the H69-LX4 cellline. All three chcmosensitizing agents alsoincreased the accumulation of [C-14]vinblastine by H69/LX4 cells. Rimino-phenazines, which are relatively nontoxic,noncarcinogenic and non-myelosuppres-sive agents, are promising contenders forevaluation in experimental and clinicai on-cology as modulators of acquircd MDR.-Authors' Abstract

van Soolingen, D., de Haas, P. E. W.,Haagsma, J., Eger, T., Hermans, P. W.M., Ritacco, V., Alito, A. and van Emb-den, J. D. A. Use of various genetic mark-ers in differentiation of Rlvcobacteriumbovis strains from animais and humansand for studying epidemiology of bovine

tuberculosis. J. Clin. Microbiol. 32 (1994)2425-2433.

One-hundred-fifty-three Mycobacteriunrbons strains from cattle, various animalspccies from zoos and wild parks, and hu-mans were analyzed for three different ge-netic markers for use in the epidemiologyof bovine tuberculosis. llf. bons strains iso-lated from cattle were found to carry a singleIS6110 element; whereas the majority ofstrains from other animais, such as ante-lopes, monkeys, and seals, harbored mui-tiple IS6110 elements, suggesting that thereservoirs in cattle and wild animais are sep-arated. Because the single IS6110 elementin cattle strains is located at the same chro-mosomal position, strain differentiation byinsertion sequente fingerprinting was ham-pered. Therefore, we investigated the use-fulness of the direct repeat and polymorphicGC-rich repeat elements for strain differ-entiation. Both markers allowed sufficientstrain discrimination for epidemiologicalpurposes. Evidente is presented that in Ar-gentina, most human AI. bons infections aredue to transmission from cattle; whereas Al.bons infections among humans in the Neth-erlands are mainly contracted from animaisother than cattle. Various outbreaks of AI.bovis among animais and humans are de-scribed, including a small one which likelyinvolved transmission from human to hu-man.—Authors' Abstract

Vila, J., Ruiz, J., Marco, F., Barcelo, A.,Goni, P., Giralt, E. and Jimenez de Anta,T. Association between double mutationin gt'rA gene of ciprofloxacin-resistantclinicai isolates of Escherichia coli andMICs. Antimicrob. Agents Chemother.38 (1994) 2477-2479.

The mutations in the quinolone resis-tance-determining region of the gi rAA andgvrB genes from 27 clinicai isolates of Esch-enchia coli with a range of MICs of cipro-floxacin from 0.007 to 128 pg/mi and ofnalidixic acid from 2 to >2000 pg/mi weredetermined by DNA sequencing. All 15 iso-lates with ciprofìoxacin MICs of ? 1 pg/m1showed a change in Ser-83 to Leu of GyrAprotein; whereas in clinicai isolates with aMIC of ? 8 pg/mi (11 strains), a doublechange in Ser-83 and Asp-87 was found. All


isolates with a MIC ofnalidixic acid of :̂128µg/ml showed a mutation at amino acid co-don Ser-83. Only 1 of the 27 clinicai isolatesof E. coli analyzed showed a change in Lys-447 of the B subunit of DNA gyrase. Achange in Ser-83 is sufficient to generale ahigh levei of resistance to nalidixic acid;whereas a second mutation at Asp-87 in theA subunit of DNA gyrase may play a com-plementary role in developing the strain'shigh leveis ofciprofioxacin resistance.—Au-thors' Abstract

Vilagut, L., Vila, J., Vinas, O., Pares, A.,Gines, A., Deanta, M. T. J. and Rodes,J. Cross-reactivity of anti-Mhcobacieri-uni gordonae antibodies with the majormitochondrial autoantigens in primarybiliary cirrhosis. J. Hepatol. 21 (1994)673-677.

Primary biliary cirrhosis is a chronic cho-lestatic liver disease associated with auto-immune disorders. Antimitochondrial au-toantibodies and granulomatous portal le-sions are characteristic in primary biliarycirrhosis. Since granuloma may be inducedby mycobacteria, and there is evidence im-plicating mycobacteria as infectious agentscapable of initiating autoimmunity, a studywas performed to determine the presence ofantibodies against 10 atypical mycobacteriain 19 patients with primary biliary cirrhosis,and in 35 controls (25 patients with otherchronic liver diseases and 10 healthy sub-jects). All primary biliary cirrhosis sera andnone of the controls reacted with the extractfrom Mirobacterium gordonae, showingidentical recognition profiles with two poly-peptides of 70-65 and 55 kDa. No otherreaction was found in primary biliary cir-rhosis patients and in controls with the ex-tracts from the other nine atypical myco-bacteria tested. Eluted immunoglobulinswhich reacted with the 70-65 and 55 kDapolypeptides from AI. gordonae, bound tothe mitochondrial antigens PDH-E2 andBCKDH-E2. Furthermore, when the ex-tract from M. gordonae was tested with elut-ed immunoglobulins from recognized PDH-E2 and BCKDH-E2 by primary biliary cir-rhosis patients, cross observed both 70-65and 55 kDa polypeptides. These data in-dicate that antibodies to Al. gordonae, found

in ali primary biliary cirrhosis patients,crossreact with the major mitochondrialtargets of the disease. We suggest that M.gordonae may play a potential pathogenicrole in primary biliary cirrhosis.—Authors'Abstract

Villar, I., Hernandez, E., Cozzi, J., Paletta,C. and Mathurin, S. [Glomerulonephritisdue to immune complexes associated withpulmonary tuberculosis.] Medicina (B.Aires) 54 (1994) 237-240. (in Spanish)

A 32-year-old man was admitted for dys-pnea, hemoptysis, macroscopic hematuria,hypertension (140/100), peripheral edemaand hemodynamic decompensation. LungX-rays revealed pulmonary edema and acavity in the left apex. Laboratory deter-minations revealed an altered renal func-tion with increased creatinine and urea lev-els and nephrotic syndrome. There was leu-cocyturia, hematuria and cylindruria. Thesputum showed a large number of acid-fastbacilli. The patient began anti-tuberculosistreatment with three drugs (isoniazid, rif-ampin, pyrazinamide). On ultrasonogra-phy, both kidneys revealed ecogenic lesionswith size, shape and cortico-medular rela-tionship preserved. The patient persistedwith altered renal function, steady leveis ofurea nitrogen, creatinine and potassium,preserved diuresis and hypertension. Bidi-mensional echocardiogram: LVDD 55 mm,hypoquinetic septum, pericardic effusion,thickened pericardium, pleural effusion,shortening fraction decreased. He receivedtreatment for this congestive cardiac failureand hypertension with enalapril, nifedipineand furosemide. A percutaneous renal bi-opsy was performed with anatomopathol-ogic diagnosis of diffuse encocapillar pro-liferative glomerulonephritis with crescents(15%) and total glomerular sclerosis (33%).Immunofiuorescence: positive, immune-complexes with IgM and C3. The patientgradually recovered his normal renal func-tion, improved his pleural effusions andnormalized his cardiac function. He wasdischarged in good clinical condition on the69th day of anti-tuberculosis treatment. Anassociation between pulmonary tuberculo-sis and glomerulonephritis is discussed. Itis proposed that renal lesions might be the


consequence of the tuberculosis due to thesedimentation of circulating immune com-plcxcs.—Authors' English Abstract

Wallace, R. J., Brown, B. A., Griffith, D.E., Girard, W. M., Murphy, D. T., Onyi,G. O., Steingrube, V. A. and Mazurek, G.H. Initial clarithromycin monotherapy forMycobacteriunl ar'nun - intracellularecomplex lung disease. Am. J. Respir. Crit.Care Med. 149 (1994) 1335-1341.

Sputum conversion rates in illvcobacte-riunl aviuln - intracellulare (MAI) complexlung disease have ranged from only 50% to80% despite the use of three to five anti-tuberculosis agents. We initiated a prospec-tive, open, noncomparative trial of initialclarithromycin monotherapy at 500 mgtwice a day for 4 months in HIV-negativepatients with MAI lung disease. The pri-mary study end point was microbiologic im-provement. Of 30 patients enrolled, 20completed therapy. This latter group waspredominantly male (60%), smokcrs (70%),older than 45 yr of age (90%), infected withAI. intracellulare (70%) and with bilateraldisease (85%). Of 19 patients with pretreat-ment minimum inhibitory concentrations(MIC) for clarithromycin <16 pg/ml, 58%became sputum-negative, and 21% showedsignificant reductions in sputum positivity.Heavily positive sputum cultures (>200colonies) were reduced from 30 to 47 sam-ples pretherapy (64%) to three of 54 (6%)post-therapy (p < 0.0001); 18 of 19 patients(95%) showed an improvement in sputumcultures, chest radiographs, or both. Onlytwo patients (7%) discontinued the drug be-cause of adverse events. Only three (16%)of 19 isolates developed clarithromycin re-sistance (MIC > 32 µg/ml). Clarithromy-cin-susceptible and -resistant MAI isolatesfrom the same paticnt had identical DNAlarge-restriction fragment patterns. Clarith-romycin is the first single agent to be shownefficacious in the treatment of MAI lung dis-ease.—Authors' Abstract

Wormser, G. P., Horowitz, H. and Dworkin,B. Low-dose dexamethasone as adjunc-tive therapy for disseminated Alvcobac-terium avium complex infections in AIDSpatients. Antimicrob. Agents Chemother.38 (1994) 2215-2217.

Five, human immunodeficiency virus-in-fected patients with disseminated A1vco-bacterium avium complex infection hadprogressive weight loss and persistent feverdespitc multidrug antimycobacterial ther-apy. These patients were given daily low-dose oral dexamethasone (typically 2 mg/day) as adjunctive therapy. All had sub-stantial and sustained weight gain (12% to50% of presteroid treatment body weight [p< 0.03]), reduction in fever, and an im-proved sense of well-being. The serum al-bumin levei increased during dexametha-sone therapy (from 3.06 ± 0.59 g/dl [mean± standard deviation] to 3.9 ± 0.22 g/dl [p< 0.01]), while the serum alkaline phos-phatase levei fell (from 368 ± 247 U/literto 128 ± 43.6 U/liter [p < 0.04]). Furtherstudies of the potential role for corticoste-roids in the management of disseminatedAI. avium complex infections in human im-munodeficiency virus-infccted patients arewarranted. —Authors' Abstract

Yew, W. W., Piddock, L. J. V., Li, M. S.K., Lyon, D., Chan, C. Y. and Cheng, A.F. B. In - nitro activity of quinolones andmacrolides against mycobacteria. J. An-timicrob. Chcmothcr. 34 (1994) 343-351.The activities of eight quinolones (cip-

rofloxacin, clinafloxacin, levofloxacin, of-loxacin, A-80556, sparfloxacin, tcmafloxa-cin and tosufloxacin) and three macrolides(azithromycin, clarithromycin, and eryth-romycin) against 98 clinical isolates of Aly-cobacteriln tuberculosis and 120 isolates offive different atypical mycobacterial speciesincluding 20 M. kansasii, 25 Al. scrofilla-ceunl, 25 Al. aviunl/iniracellulare, 25 M.chelonae and 25 AI. foriuitunl were deter-mined with the Middlebrook 7H9 brothmacro-dilution method. Sparfloxacin, cli-nafloxacin, levofloxacin, ciprofloxacin andofloxacin were active against AI. tubercu-losis (MIC90 0.06-0.5 mg/L; MBC, O 0.125-2.0 mg/L). However, higher MIC 90 s andMBC90 s of theee quinolones were obtainedfor strains of multidrug-resistant AI. tuber-culosis. The macrolides tested had poor ac-tivity against AI. tuberculosis isolates (MIC90> 8.0 mg/L). Furthermore, high MIC 9o s ofthe quinolones and macrolides (2.0 to 8.0mg/L) were obtained for clinical isolates ofatypical mycobacteria, with the exception


ofclarithromycin against AI. kansasii (MIC 90

= 1.0 mg/L) and sparfloxacin against M.scrofirlaceum (MIC90 = 1.0 mg/L).—Au-thors' Abstract

Yuan, S. N., Tan, P. L. J. and Skinner, M.A. The effect of prostaglandin E(2) andindomcthacin on the cytotoxic responseto mycobacterial antigens. Int. J. Im-munopharmacol. 16 (1994) 525-531.

The effect of prostaglandin E(2) and in-dometbacin on the generation of cytotoxicT-Iymphocytes in response to Alycobactc-riunr tuberculosis (MTB) antigens was com-pared between health controls and nceu-matoid arthritis (RA) patients. Peripheralblood mononuclear cells (PBMC) from 16healthy individuais and 15 RA patients werestimulated for 7 days with an irradiated,sonicated preparation of MTB in the pres-ence or absence of PGE(2) or indomethacinand assayed for cytotoxic activity on au-tologous target cells prepulsed with MTB.The mean cytotoxic activity generated waslower in patients than in controls. Exoge-nous PGE(2) suppressed the cytotoxicity di-rected against MTB-pulsed targets in 12 of16 controls, but in only 1 of 11 patients.Indomethacin enhanced this cytotoxicity inonly 2 of 16 controls but in 6 of 10 RApatients. When effector cells were derivedfrom the synovial fluid, PGE(2) again hadno effect and indomcthacin enhanced thecytotoxicity. Our data suggest that the de-pressed cytotoxic response of RA patientsto MTB may be due to the production ofendogenous PGE(2). Cyclooxygenase inhib-itors commonly used in the treatment of RAmay influente MTB-induced cytotoxicity inpatients. In addition to their antiinflam-matory effects within the joint, nonsteroidalantiinflammatory drugs may potentially en-hance cytotoxic reactions which are inducedby antigens, such as MTB crossreactive heat-shock proteins.—Authors' Abstract

Zhang, Y. and Young, D. Molecular geneticsof drug resistance in Mycobacteriwn tu-berculosis. (Review) J. Antimicrob.Chemother. 34 (1994) 313-319.

Tuberculosis (TB) is the single largest kill-er among infectious diseases. The recent re-surgence of TB together with outbreaks of

multidrug-resistant tuberculosis has fo-cused attention on understanding the mech-anisms of such drug resistance. Because ofthe relativo neglect of TB research in thepast and late arrival of mycobacterial ge-netic tools, the molecular mechanisms ofdrug resistance in TB remained iargely un-known until very recently. In this paper wereview recent progress on the mechanismsof resistance to three major anti-TB drugs:isoniazid, rifampin and streptomycin. Whilein resistance mechanisms for rifampin andstreptomycin are similar to those found inother bacteria, isoniazid susceptibility andresistance is unique to Mycobacterium tu-berculosis. So far, mutations in two chro-mosomal loci, katG and inhtf have beenfound to be involved in isoniazid resistancein TB. Identification and characterizationof mutations responsible for resistance opensup new possibilities for rapid detection ofdrug-resistant strains. Molecular under-standing of drug resistance and drug actionin AI. tuberculosis may eventually lead torational design of new anti-TB drugs.—Au-thors' Abstract

Zolg, J. W. and Philippi-Schulz, S. The su-peroxide dismutase gene, a target for de-tection and identification of mycobacte-ria by PCR. J. Clin. Microbiol. 32 (1994)2801-2812.

The superoxide dismutase gene has beenidentified as a target in screening for thepresence of mycobacteria on the genus leveiand differentiating relevant mycobacterialspecies from one another by PCR. Consen-sus primers deduced from known super-oxide dismutase gene sequences allowed theamplification of DNAs from a variety ofbacteria, fungi, and protozoa. Selected am-plicons from Actinontyces viscosas, Cor.t'-nebacteriuni diphtheriae, Cor.i'nebacteriumpsetrdodiphtheriticurn, Mycobacteriuni ar-iam, AI. Fortuitam, M. gordonae, AI. intra-cellu lare, AI. kansasü, Al. scrof rlaceum, AI.sirniae, M. tuberculosis, M. xenopi, and No-cardia asteroides were subsequently clonedand sequenced. The alignment of those se-quences facilitated the selection of primerstargeting conserved regions present in my-cobacterial species but absent in nonmy-cobacterial species and thus allowed the ge-nus-specific amplification of all 28 different

192^ International Journal ofLeprosy^ 1995

mycobacterial species tested. A pool of ge-nus-specific probes recognized 23 of the 28mycobacterial species and did not crossre-act with any of the 96 nonmycobacterialspecies tested. In addition, probes recog-nizing species-specific variable regionswithin the superoxide dismutase genes ofAl. aviam, M..fortuittnn, Al. gordonae, Al.intracellulare, Al. kansasü, AI. scrofieia-ceuna, AI. simiae, the AI. taberculosis com-plex, and M. xenopi were identified. Aliprobes recognized only the species from

which they were derived and did not cross-react with any other mycobacterial speciesor with any of the nonmycobacterial speciestested. We conclude that the superoxide dis-mutase gene is a suitable target for ampli-fying mycobacteria by PCR on the genuslevei, confirming correct amplification bygenus-specific probes, and differentiatingrelcvant species from one another by a setof species-specific probes.—Authors' Ab-stract

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