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Review Mb

Keywords: tuberculosis, hiV-AiDS, iFn-gamma, immunodiagnosis, iGRA, cD4+ t cells

Introductiontuberculosis (tB) remains the leading cause of death among hiV-infected persons, and the risk of active tB is still increased even among those who receive highly active antiretroviral therapy (hAARt) (20, 32). Alarmingly, fewer than half of tB cases in hiV-infected patients are diagnosed before death. Despite this enormous and increasing global burden, there is still no reliable test to confirm active M. tuberculosis (MtB) infection in culture-negative cases, or to detect latent infection in asymptomatic individuals (17).

Although not official policy in many countries, the tuberculin skin test (tSt), which provides evidence of MtB infection, is widely used to lend support to clinical and radiological findings in the assessment of individuals with suspected tB. however, the tSt has limited sensitivity and specificity (2, 17). In addition, the sensitivity of the TST is reduced in hiV positive patients (24). the lack of a reliable and rapid test for smear-negative tB in hiV-positive patients

means that the correct diagnosis is often missed and/or patients are erroneously started on anti-tuberculosis therapy on the basis of clinical presentation alone. therefore, what is needed is an accurate test for rapid diagnosis of latent tB infection (ltBi); that is of assistance with the diagnosis of active tB; and remains effective in hiV co-infected individuals (24).

The identification of MTB specific antigens such as ESAT-6 and cFP-10 has, over recent years, led to the development of a new generation of MTB-specific diagnostic tests (2). ESAT-6 and cFP-10 are encoded by regions of the M. tuberculosis genome which are absent from M. bovis BcG, M. avium and most of the non-tuberculous mycobacteria. these antigens have been shown in studies to elicit strong IFN-γ responses from the t cells of persons infected with M. tuberculosis but not from the t cells of those at low risk of infection or those solely BcG-vaccinated (1, 2, 4). two commercial iFn-gamma release assays (IGRAs) utilizing these specific antigens are available in europe: the whole blood-based enzyme-linked immunoassay (quantiFeRon®-tB Gold in-tube, cellestis ltd., Australia; qFt) and the enzyme-linked immunospot assay (t-SPot.tB™, oxford immunotec, UK; SPot) (23,

USEFULNESS OF INTERFERON-GAMMA RELEASE ASSAYS IN THE DIAGNOSIS OF TUBERCULOSIS INFECTION IN HIV-INFECTED PATIENTS IN BULGARIA

R. Markova1, Y. todorova1, R. Drenska1, i. elenkov2, M. Yankova2, D. Stefanova3

National Center of Infectious and Parasitic Diseases, Department of Immunology and Allergology, Sofia, Bulgaria1

Infectious Diseases Hospital “Prof. Ivan Kirov”, AIDS Clinic, Sofia, Bulgaria2

University Hospital for Lung Diseases “St. Sofia”, TB Clinic, Sofia, Bulgaria3

corespondence to: Roumiana Markovae-mail: markova@ncipd.org

ABSTRACTWe aimed to evaluate the usefulness of incorporating the result from an IFN-gamma release assay (IGRA) in the algorithm for initial diagnosis of tuberculosis (TB) in prospectively enrolled HIV-infected subjects. Ninety HIV-infected subjects with clinical and/or radiological features consistent with a possible diagnosis of active-TB were tested with QuantiFERON®-TB Gold In-Tube (QFT) and T-SPOT.TB™ (SPOT). All information, including IGRA results, was available to clinicians for making their diagnosis. Those diagnosed with TB were given anti-TB treatment and samples were collected for microbiological culture. Overall, 35/90 patients were QFT positive and 30/90 by SPOT. QFT was indeterminate for 5/90 subjects (5.5%) and SPOT for 11/90 (12.2%; p=0.0313). Active-TB was microbiologically confirmed in 13 subjects and twelve (92%) were positive by QFT and one was negative. For SPOT, 8/12 (62%; p=0.125) were positive, one- negative, and 4 were indeterminate. Of the 77 HIV-positive subjects without microbiologically confirmed active-TB, 23 (30%) were positive, 49 (64%) negative, and 5 (6%) indeterminate by QFT, compared with 22 (29%), 48 (62%) and 7 (9%) by SPOT. Those IGRA positive, and two who were IGRA indeterminate, had a final presumptive diagnosis of active-TB, based upon initial clinical symptoms and improvement with anti-TB treatment. All IGRA negative subjects had a diagnosis other than tuberculosis and none have since developed active TB over a period of more than 12 month.In conclusion, both IGRAs provided useful additional information for assisting the diagnosis and early treatment of Mycobacterium tuberculosis infection in immunocompromised HIV/AIDS patients with negative smear microscopy. A positive IGRA result was a significant factor in making initial diagnoses of TB, diagnoses that were later supported by clinical outcome following anti-TB therapy, and diagnoses other than TB in those IGRA negative. The QFT and SPOT tests had similar high sensitivity, with QFT having fewer indeterminate results, in our HIV-infected immunocompromised population.

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30). Both IGRAs measure IFN-γ responses to peptides of eSAt-6 and cFP-10. Further, the qFt test also incorporates an additional peptide from the M. tuberculosis-complex specific antigen tB7.7 (p4) for maximal sensitivity (31).

While the tests have been designed for the diagnosis of latent tB infection, both iGRAs have been reported to have high sensitivity (>85%) for the detection of active-tB and also to have very high specificity (97-100%) in low TB risk populations, independent of BcG vaccination status (5, 8, 19, 26, 29). While there are a number of studies that have investigated the performance of these tests in hiV-positive individuals (6, 10, 13, 21, 28), there is a paucity of data on the performance of the tests in hiV-positives with concurrent active-tB.

Although Bulgaria has a low prevalence of hiV infection, with a cumulative number of 815 hiV positive individuals as of end of December 2007 (Public health Statistics, 2007, national center of health informatics, Republic of Bulgaria), the chance of hiV/tB co-infection is raised due to the intermediate incidence of tB (37.1/100 000 population; Public health Statistics, 2007, national center of health informatics, Republic of Bulgaria). Due in part to their suppressed immune response, but also to the testing methods currently available, diagnosis of both active and latent MtB infection remains a difficult issue in HIV/AIDS patients.

the aim of this study was to estimate the usefulness of the two iGRAs as additional tools for assisting in the

initial diagnosis and management of active tB infection in prospectively enrolled hiV-infected patients in Bulgaria, a country where BcG-vaccination is mandatory at childbirth.

Materials and MethodsStudy populationhiV-1-infected Bulgarian patients attending the AiDS clinic of the Infectious Diseases Hospital in Sofia were recruited prospectively into the study. individuals of more than 18 years age, with a documented hiV-1 infection and clinical and/or radiological features compatible with possible tB were eligible. Patients were evaluated during their routine quarterly check-up which comprised clinical appraisal, measurement of hiV-viral load, cD4+ t-cell count, chest x-ray result and evaluation of compliance with treatment. clinical information such as age, sex, year of hiV diagnosis, previous treatment for active-tB, was recorded.

From January 2006 to December 2007, 90 hiV-1-positive patients (59 male; 66%) meeting the study criteria were recruited and tested for MtB infection with both iGRAs. All subjects provided informed consent.

QuantiFERON-TB Gold In-Tube assaythe qFt-tB Gold in-tube assay was performed as per the manufacturer’s instructions (cellestis limited, carnegie, Australia). Samples with IFN-γ ≥ 0.35 IU/mL following stimulation with MTB-specific antigens (after subtracting the

TABLE 1clinical characteristics and baseline data for all hiV+ Bulgarian patients (n=90) at the time of iGRA testing

Variable Other than TB TB clinical TB confirmednumber 52 25 13Age:

MeanRange

37(21-66)

34(18-60)

43(29 – 63)

Gender:MaleFemale

3319

187

85

BcG Vaccinated 52 25 13M. tuberculosis culture positive 0 0 13t-cell count (cells / cD4+ml)

MeanRange

248(36 - 785)

284(58 - 646)

195(15 - 450)

Duration of hiV-1 diagnosis (years) MeanRange

7(3 - 17)

5.9(3 - 17)

4(2 – 21)

quantiFeRon-tB Gold in-tubePositivenegativeindeterminate

0 (0%)49 (94%)3 (6%)

23 (92%)0 (0%)2 (8%)

12 (92%)1 (8%)0 (0%)

t-SPot.tBPositivenegativeindeterminate

0 (0%)48 (92%)4 (8%)

22 (88%)0 (0%)3 (12%)

8 (62%)1 (8%)4 (31%)

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value for the negative control) were considered positive. the qFt test result was considered indeterminate if negative to the MTB-antigens and production of IFN-γ after stimulation with PhA was < 0.5 iU/ml above the negative control.

T-SPOT.TB assay the SPot test was performed according to manufacturer’s instructions (oxford immunotec, Abingdon, UK). Responses were scored as positive if either or both of the eSAt-6 or CFP-10 test wells contained ≥ 6 IFN-γ-SFCs more than the negative control when it was 0 - 5 spots, or ≥ 2x the negative control when it was ≥ 6 spots. The SPOT test was deemed indeterminate if the PhA positive control had less than 20 spots more than the respective negative control.

Statistical analysis Data were analyzed by using Mann-Whitney non-parametric t-test, exact Mcnemar test and Spearman rank order correlation test (GraphPad Prism software, Version 4.0; San Diego, cA, USA).

Results and Discussionof the 90 subjects enrolled in the study, 35 (39%) subjects were qFt positive and 30 (33%) were positive by the SPot assay (Table 1). Five (6%) and 11 (12%) patients were indeterminate by the qFt and SPot tests respectively, with the 5 indeterminate by qFt also being SPot indeterminate. there was significant correlation between the magnitude of response for the two tests (r=0.6972; p < 0.0001, Fig. 1). Disregarding indeterminate results, there was complete agreement between the two iGRAs (Table 2).

the graph shows the correlation between the iFn-g response results obtained for each hiV+ patient (n=90). each dot represents the combination of iGRA data for one study subject. on the abscissa are reported the SPot results as Spot Forming Units/106 PBMcs (SFUs) and on the ordinate the qFt results as iFn-gamma international units/ml (iFn-g iU/ml). Dashed lines indicate the test threshold as reported in the Methods section. the overall correlation observed was r=0.6972; p < 0.0001.Fig. 1. correlation between quantiFeRon-tB Gold in-tube and t-SPot.tB assays.

By design, results for both iGRAs were available to clinicians, along with the usual clinical, historical and

radiological data, to assist in making their final diagnoses. Based on this information, an initial diagnosis active tB was made for 38 of the 90 hiV+ patients and all 38 were treated with standard anti-tB chemotherapy. only 10 (26%) of the tB patients were positive for acid-fast bacilli by sputum smear microscopy. Respiratory and other samples were collected from all 38 tB patients and sent for microbiological culture, with 13 (34%) having active TB subsequently confirmed by culture of MtB. All ten of those with positive sputum smear microscopy results were also MtB culture positive and the other 3 patients with positive culture had MtB meningitis. Diagnosis of active-tB for the other 25 patients (66%) was not confirmed by culture; however, consistent with the diagnoses, all resolved their clinical symptoms of tB with chemotherapy.

TABLE 2comparison of iGRA results from hiV+ patients

qFt-tB Gold in-tubetotal

Positive negative indet^

t-SPot.tBPositive 30 0 0 30negative 0 49 0 49

indeterminate 5 1 5 11total 35 50 5 90

^ indeterminate

there were 52 hiV-positive individuals who received initial diagnoses other than tB and who did not receive anti-tB medication. Most were iGRA-negative (49 for qFt, 48 for SPot) or indeterminate (3 for qFt, 4 for SPot), suggestive that the IGRA result played a significant role in their initial diagnosis. The final diagnoses for all 52 remained other than tB and are shown in Table 3. none have subsequently developed active-tB, 12 months after the end of recruitment.

TABLE 3Diagnosed infection/condition of hiV+ patients

infection/condition number of patientsTB (culture confirmed) 13Presumptive tB (clinical diagnosis) 25toxoplasmosis 6Non-specific pneumonia 10cryptococcal menengitis 4Non-defined bacterial menengitis 3hcV infection 4non-hodgkin lymphoma 4Pyelonephritis (e.coli) 1chronic pancreatitis 1Syphilis of the cnS 2Rheumatoid arthritis 1Systemic candidiasis 16totAl 90

1106 Biotechnol. & Biotechnol. eq. 23/2009/1

characteristics of the study subjects are shown in Table 1. the median age of the 52 patients without tB (33 male; 63.46%) was 37 years (range 21 to 66), with a mean cD4+ t cell count of 248 cells/µl (range 36 to 785). For the 13 patients with confirmed TB, 8 (62%) were male, their mean age was 43 years (range 29 to 63) and their mean cD4+ t cell count was 195 cells/µl (range 15 to 450). eighteen (72%) of those with clinically diagnosed active-tB were male with a mean age of 34 years (range 18 to 60) and a mean cD4+ count of 284 cells/µl (range 58 to 646). current hAARt use was reported in 57 (63%) patients. All subjects tested had received BCG vaccination at birth, which was confirmed by examination for a scar.

For the 13 hiV-positive patients with microbiologically confirmed TB, 12 (92%) were QFT positive and 8 (62%) were positive by the SPot test, however the difference in sensitivity did not reach statistical significance (Exact McNemar test p=0.125). one tB patient was negative by both tests and the other 4, who were qFt-positive, were indeterminate by the SPot test due to low responses to the Mitogen positive control. the patient negative to both tests had a cD4+ t cell count of 138/µl, and the four indeterminate by SPot had a mean count of 246/µl (range 62 to 443), compared with 195/µl (range 15 to 372, p<0.05) for those that were positive by both iGRAs.

qFt was positive for 23 (92%) of the 25 patients in the clinically diagnosed (and treated) tB group and the SPot test for 22 (88%; ns). two of this group of patients were indeterminate by both iGRAs and another patient by SPot alone. the cD4+ t-cell counts for the two iGRA indeterminate subjects were 63 and 210 cells/µl. Both converted to a positive qFt result when re-tested after 2 to 3 months of therapy, at which time their condition had improved, both factors supportive of the original diagnosis. Similarly, all patients with clinically diagnosed tB improved and resolved their symptoms with anti-tB therapy.

For all 38 patients diagnosed with active tB (culture-confirmed or clinically diagnosed) 35 (92.1%) were QFT positive and 30 (78.9%) positive by SPot (p=0.0625). one patient was negative to both tests, two were indeterminate

to both iGRAs, and 5 were SPot indeterminate, but qFt positive.

overall, 5 subjects were both qFt and SPot indeterminate and, two of them were diagnosed and treated for active-tB based on their clinical symptoms and history of exposure and the other 3 had a final diagnosis other than TB (HCV; HCV/hBV; and cryptococcal meningitis). there were further 6 subjects who had indeterminate responses by the SPot test alone. Four of these were culture-positive for MtB, one was diagnosed with cryptococcal meningitis and one with non-hodgkin lymphoma.

We analysed the possible influence of CD4 T-cell counts in the performance of the two iGRA tests (Table 4). there were no significant differences between the rates of positive results obtained by either IGRA when stratified by CD4+ T-cell count, even for the group of hiV patients with counts less than 50/µl. the number of indeterminate results, as evidenced by responses to the mitogen control less than the respective test cut-offs, was significantly greater for the SPOT test (11/90; 12%) as compared to qFt (5/90: 6%; exact Mcnemar test p=0.0313). For qFt, 4 of the 5 patients (80%) with indeterminate results had cD4+ t-cell counts less than 200/µl, and for the SPot test, 7 of the 11 patients (64%). However, there was no significant association between the level of mitogen-stimulated IFN-γ production and cD4+ t cell counts for either of the iGRAs.

We evaluated the utility of two iGRAs, in conjunction with other clinical parameters, to assist in the initial diagnosis of active-tB in 90 hiV-infected subjects with clinical suspicion of tB. By study design, clinicians were made aware of iGRA results and were free to use these results, along with traditional diagnostic criteria, in making their diagnostic decisions. overall, 38/90 (42%) of the hiV-infected cohort were diagnosed with tB and treated accordingly. it is likely that the IGRA results had significant influence on the diagnostic decisions of active-tB, as 35 of these patients were iGRA positive. consistent with poor sensitivity of MtB culture in HIV-infected, culture confirmation was only obtainable for 13/38 (34%) of these tB patients.

For the 13 patients with confirmed MTB, the sensitivity of qFt was high (92%; 12/13), but lower for SPot (62%; 8/13),

TABLE 4Positive, negative and indeterminate QFT and SPOT results in HIV+ patients stratified by CD4+ T-cell count

cD4+ counts (cells/ml)

no. subjectsquantiFeRon-tB Gold in tube t-SPot.tB

Positive negative indet* Positive negative indet*0 – 50 13 5 6 2 4 6 3

51 – 100 15 6 8 1 5 8 2101 – 200 24 8 15 1 7 15 2201 -350 13 6 7 0 6 6 1

>350 25 10 14 1 8 14 3

totAl 90 35 (38.8%)50

(55.6%)5

(5.6%)30

(33.3%)49

(54.4%)11

(12.2%)* indet = indeterminate

1107Biotechnol. & Biotechnol. eq. 23/2009/1

primarily due to 4 of the 13 patients having indeterminate SPot results. however, were the iGRA results useful in assisting timely diagnosis (prior to culture results being available) and treatment of tB in these 13 patients? ten of the 13 patients were sputum-smear positive at the time of initial diagnosis and undoubtedly this finding, along with their clinical picture, would have led to anti-tB treatment independent of their iGRA result. the other 3 culture positive patients all had tB meningitis and were qFt positive. in these cases, the iGRA result, in conjunction with clinical symptoms and knowledge of the difficulty of definitive diagnosis of TB meningitis provided us with enhanced confidence of our decision to treat for TB infection rather than other potential causes of meningitis.

The main benefit we anticipated from the availability of iGRA results was assistance in the diagnosis of smear-negative (and therefore likely culture negative) active-tB in our hiV infected population. there were 25 smear negative patients clinically diagnosed and treated for active tB without subsequent culture confirmation. In this group a positive IGRA result was viewed as highly suggestive for MtB infection and 23 of these 25 patients had a positive qFt (22 SPot positive and one indeterminate), with the remaining two diagnosed on clinical grounds only (both iGRA indeterminate). conversely, none of the 52 subjects with an initial diagnosis other than tB were iGRA positive. it is therefore highly probable that clinician’s knowledge of IGRA results influenced their diagnoses for the majority of subjects. Was this influence of assistance in making the correct diagnoses? Our finding that all 25 clinically diagnosed tB patients improved with anti-tB treatment and resolved their symptoms, supports the initial diagnosis of tB. Similarly, all of the patients who were iGRA-negative had an alternative diagnosis made, with none having developed symptoms compatible with tB progression up until the time of writing (>12 months). together, these results suggest that the iGRA tests provided valuable information to clinicians to aid correct initial diagnoses in this hiV-infected cohort with clinically suspected tB.

Although our data suggest that iGRAs assist diagnosis of tB in hiV-infected, they do not provide a “rule in” or “rule out” result and should be interpreted in light of other diagnostic criteria and methods. two patients from our cohort were diagnosed with tB purely on conventional grounds. A limitation of iGRAs for the diagnosis of active-tB is that they do not discriminate between active and latent tB infection.

the Who reported incidence of active tB for 2006 in Bulgaria was 39/100 000, which is relatively high compared with most european countries and suggests that the rate of ltBi infection should also be similarly high (32). Given that iGRAs have high sensitivity for MtB infection (25), it is possible that a number of our patients with clinically diagnosed tB had ltBi rather than active tB and that an infection/condition other than tB, which responded to the tB therapy, was the cause of their symptoms; although we have no evidence this was the case. limitations of our study were that the design did not provide a control group for whom iGRA results were blinded to the

clinicians, and that we did not determine the ltBi background rate (as measured by the iGRAs) in those without symptoms of active tB. these data would assist in further determining the value of iGRAs in aiding initial diagnosis of active tB in hiV positive populations.

comparison of results of the two iGRAs suggested that qFt was more sensitive (92%) than SPot (62%) in the 13 patients with confirmed HIV/TB co-infection, however, this difference did not reach statistical significance (p=0.125) and was due to indeterminate SPot results. the observed trend in sensitivity was in contrast to that reported in a meta-analysis by Menzies and colleagues, which suggested a higher sensitivity, albeit, lower specificity, for the SPOT test (25). However, we used the “in tube” version of the qFt test rather than the earlier liquid-antigen version that was used for the majority of studies cited in the meta-analysis. the in tube qFt test has recently been shown to be significantly more sensitive than the earlier liquid antigen version for detection of active-tB (15). to our knowledge, there are four currently published studies directly comparing the “in tube” qFt format with SPot in patients with active-tB (9, 11, 12, 14). one study shows a higher sensitivity for active tB for the SPot test (9), with the other three studies finding no significant difference and thus, compatible with our findings.

We found significantly more indeterminate results for the SPot test (12%; 11 of 90) than for qFt (6% 5 of 90; p=0.0313). our rate of SPot indeterminate results was commensurate with the 9.4% found in hiV infected patients in Switzerland (16), however, four other published studies of SPot in hiV positive populations have found lower indeterminate rates of 1.4%, 3.4%, 4.7% and 4.8% (10, 13, 22, 28). the qFt indeterminate rate in our study is in agreement with a number of other studies that have been conducted in developed countries in hiV-infected subjects using the in tube version of qFt (3.4%, 4.8% and 5.1%) (6, 18, 21). in contrast, two qFt studies in Africa in hiV/AiDS patients found higher rates of indeterminate results of approximately 25% and 17% (3, 27). These differing rates may reflect the immune status of the subjects in the different studies, as all 5 qFt studies found an association between low cD4+ t-cell counts and indeterminate results. the reasons for the higher rate of indeterminate results for SPot in our study are unclear, but may be related to the ease of use of the “in tube” qFt as compared with more technically demanding SPot test. As suggested by hoffman and colleagues, “differentiation between an anergic t-Spot result and technical failures is difficult” (16), which is further supported by a study of immunocompetent people in the USA, where 12.7% (14 of 110) failed SPot tests due to low response to the Mitogen control or technical difficulties (7).

ConclusionsBoth iGRAs appear useful for aiding the diagnosis of active MtB infection in hiV-co-infected patients. their use in conjunction with conventional diagnostic methods provided additional confidence in our early diagnosis of those who

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were negative by smear microscopy but later had infection confirmed by culture of MTB. Similarly, the IGRA results were an important component of the diagnosis and early treatment of those hiV-positive subjects with clinical suspicion of active-tB but negative by smear microscopy. the value of iGRAs for aiding active tB diagnosis seems clear in our hiV positive population, but may have low specificity, and thus limited benefit, in populations where the rate of LTBI is high. The qFt test was more convenient than and at least as sensitive as the SPot test, with fewer indeterminate results, in our hiV-infected immunocompromised population.

Acknowledgementsthis study was supported by Grant l-1503/05 of the national Fund for Scientific Research at the Ministry of Education and Science, Republic of Bulgaria. the authors are grateful to all patients and nursing staff who took part in this study. the authors thank Prof. h. taskov and his team for providing data about cD4+ t-cell counts.

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