Author P

roof

Review

© Future Drugs Ltd. All rights reserved. ISSN 1473-7159 89

CONTENTS

Traditional leishmaniasis diagnostic approaches

Diagnosis of visceral leishmaniasis

Diagnosis of tegumentary leishmaniasis

Molecular diagnosis methods

DNA-based methods

Molecular research: the search for new leishmania diagnostic molecules

Leishmania genome approaches

DNA microarrays

Protein microarrays

Expert opinion

Five-year view

Key issues

Information resources

References

Affiliations

www.future-drugs.com

Molecular diagnosis of leishmaniasisCarlos Alberto P Tavares†, Ana Paula Fernandes and Maria Norma Melo

This review describes the worldwide situation of visceral and tegumentary leishmaniasis with an emphasis on diagnosis, including methods for the detection of antibodies, antigens, parasite DNA and of skin testing. The advantages and problems of each method are discussed and the need for a rapid, sensitive and low-cost diagnostic method for use in field conditions is highlighted. Recent advances in leishmania genome sequencing, the use of DNA microarrays and protein microarray methodologies and their potential use for leishmaniasis diagnosis are presented.

Expert Rev. Mol. Diagn. 3(5), (2003)

†Author for correspondenceInstituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Caixa Postal 486 - 30161970-Belo Horizonte, Minas Gerais, BrazilTel.: +55 313 499 2501Fax: +55 313 499 2525capt@icb.ufmg.br

KEYWORDS:DNA microarrays, Leishmania genome, molecular diagnosis, serological diagnosis, tegumentary leishmaniasis, visceral leishmaniasis

Leishmaniasis comprises a spectrum of humandiseases widely distributed in 88 countries, infive continents. These diseases are transmittedby the bite of an infected sand fly and arecaused by over 20 species of Leishmania [1],which are obligatory intracellular parasitessurviving within phagolysosomes of host mac-rophages. There are three different clinicalforms of leishmaniasis:

• Visceral

• Cutaneous

• Mucocutaneous

Worldwide, leishmaniasis affects approxi-mately 12 million people. Every year, 1–1.5 mil-lion cases of the cutaneous form and 500,000visceral leishmaniasis (VL) occur, whichcaused 59,000 deaths in 2001 [2]. VL, alsoknown as kala-azar, is caused by Leishmaniadonovani in India, Pakistan, Oriental China,Bangladesh, Nepal, Sudan and Kenya. Inthese regions, humans act as the reservoir ofparasites and VL has an anthroponotic profile.L. infantum is the etiologic agent of zoonoticVL in central and southeast Asia, northeastChina, north Africa and the Europe–Mediter-ranean basin, and L. chagasi in Argentina,Bolivia, Brazil, Colombia, El Salvador, Guate-mala, Honduras, Martinique, Mexico, Para-guay and Venezuela. Patients with VL presentprolonged fever, splenomegaly, hepatomegaly,

leucopenia (mainly neutropenia), anemia,hypergammaglobulinemia, cough, abdominalpain, diarrhea, weight loss and cachexia. It isthe more severe form of leishmaniasis and, ifuntreated, is usually fatal [3]. Post-kala-azardermal leishmaniasis (PKDL) is a knowncomplication of VL in the Old World, in par-ticular in Sudan [4], where it develops duringor in the short interval after treatment of VLand a clinical diagnosis can be made. In Indiaand Bangladesh, it develops 1–5 years afterthe apparent cure of VL and treatment willoften be necessary. Diagnosis confirmation,by demonstration of the parasite in a smear orbiopsy, is recommended. The spread of VL iscaused by population displacement as a resultof war, drought, famine or economic develop-ment, including widespread urbanization,deforestation and development of new settle-ments, besides migration from rural to urbanareas. These factors were responsible for anepidemic in Sudan, with mortality rates of upto 36% [5], and are contributing to the resur-gence of the disease in India [6,7] and its urbanspread in Brazil [8].

Several risk factors for clinical VL includemalnutrition, immunosuppressive drugs andespecially HIV coinfection. The number ofcoinfections are increasing in India and Brazil,where the urban HIV epidemic and the rural VLepidemic are coming into contact [9]. In Africaand in southern Europe, Leishmania–HIV

Author P

roof

Tavares, Fernandes & Melo

90 Expert Rev. Mol. Diagn. 3(5), (2003)

coinfection is regarded as an emerging disease. In Europe,25–70% of adults with VL also have AIDS and leishmaniasisbehaves as an opportunistic infection [10]. In HIV-infectedpatients, the Leishmania parasite is found in the peripheral blood,outside the reticuloendothelial system, making these patients areservoir and source of infection for the vectors. The parasite loadin peripheral blood is generally so high that transmission amongintravenous drug users, by use of shared syringes, has been dem-onstrated. In addition, coinfected patients may be difficult todiagnose, respond poorly to treatment and relapse repeatedly [11].

Tegumentary (cutaneous and mucocutaneous) leishmaniasisresults from multiplication of Leishmania in the phagocytes ofthe skin or mucosal membranes. In the USA, it occurs from thesouthern states to northern Argentina and is caused by 14 spe-cies belonging to two subgenus: Viannia and Leishmania.Members of the subgenus Viannia are: L. braziliensis, L. pana-mensis, L. guyanensis, L. peruviana, L. lainsoni, L. shawi,L. naiffi, L. colombiensis and L. lindenbergi, which are zoonoticand cause cutaneous or mucocutaneous lesions. Those of thesubgenus Leishmania are L. mexicana, L. amazonensis, L. vene-zuelensis, L. pifanoi and L. garnhami, which are zoonotic andcause localized lesions or diffuse cutaneous leishmaniasis. In theOld World, L. tropica (anthroponotic), L. major, L. aethiopicaand L. killicki (zoonotic) cause cutaneous leishmaniasis.

The clinical characteristics of cutaneous leishmaniasis varydepending on the infecting Leishmania species and theimmune response of the host. The typical lesion first appearsas an erythematous papule or small nodule at the site wherepromastigotes were inoculated, which may itch but are usu-ally painless. It increases slowly in size, progressing to ulcersthat persist for months and in some cases years. The lesionremains painless and its chronic evolution leads most often tospontaneous cure, but leaves flat, hypopigmented, atrophicscars. L. braziliensis infection may be accompanied by regionallymphadenopathy, fever and constitutional symptoms eitherbefore or after the skin lesion becomes apparent [11,12]. Amas-tigotes probably disseminate to distant mucosal sites duringthe early phase of infection.

In one study in Guatemala, 22 of 25 lesions (85%) due toL. mexicana infection healed in a median observation time of14 weeks [14]. In contrast, only 22% of L. braziliensis lesionshealed during a similar median time of 13 weeks [15]. Theseresults suggest that, at least in the New World, patients mayexhibit lesions as early as a few weeks after exposure. Nodularlesions and lymphadenopathy as well as the more typical ulcersshould be evaluated for the presence of Leishmania. A smallpercentage of individuals infected with L. braziliensis in LatinAmerica develop mucosal lesions, due to metastasis of the pri-mary lesion. While L. braziliensis is the organism usually associ-ated with this complication, such infections have also beenreported from the Middle East and Africa, often due to conti-guity of the cutaneous lesion. The mucous involvement tendsto vary from one endemic area to another [16]. Althoughmucosal leishmaniasis is defined as infection of the mucosalmembranes of the nose and mouth, infection begins with nasal

inflammation and stuffiness and then may involve the orophar-ynx and larynx. The period of time between the appearance ofthe cutaneous lesion and the subsequent appearance of mucosalinvolvement is extremely variable, extending from several weeksto many years. In 14–28% of the cases, mucous involvementcan even be concomitant to an active primary lesion [16].Mucosal disease characteristically does not heal spontaneouslyand evolves slowly (with a mean time of 3 years), before beingbrought to medical attention.

Traditional leishmaniasis diagnostic approachesCurrently, routine diagnosis of leishmaniasis is usually based onclinical and epidemiological parameters but definitive diagnosisstill relies on the parasitological observation of the parasite indirect smear, culture or animal inoculation. Parasitological con-firmation is important because the current drugs of choice,mostly antimonials, have high cost and significant toxicity andtherefore should not be given without justification. In addition,treatment has significant impact on the control of anthro-ponotic leishmaniasis, thus early diagnosis is required for avoid-ing a reservoir effect. Furthermore, accurate identification ofthe infecting Leishmania species permits better orientation ofthe medical follow-up, since clinical manifestations may vary,depending largely on the immune response of the host and thespecies to which the parasite belongs. Detection of the particu-lar Leishmania species involved is also essential for epidemiolog-ical studies. Although major advances have been made in thediagnosis of leishmaniasis during the last decade, there is nospecific method that can be adopted as the gold standard fordetection and diagnosis of Leishmania infections. The chosenmethod often depends upon the application.

Several methods are used for diagnosis of leishmaniasis, all ofwhich have important limitations. As illustrated by the schemepresented in FIGURE 1, the main tests used are:

• Demonstration of parasites in tissues of relevance by microscopic examination

• Detection of antileishmanial antibodies in patient sera

• Detection of parasite antigens in tissue, blood or urine samples

• Detection of parasite DNA in tissue samples

• Detection of specific cell mediated immunity

Diagnosis of VLThe clinical diagnosis of VL is complex because other com-monly occurring diseases, such as trypanosomiasis, malaria,typhoid, schistosomiasis and tuberculosis, share the same clinicalfeatures. Many of these diseases can be present along with VL(in cases of coinfection) and sequestration of the parasite in thespleen, bone marrow or lymph nodes further complicates thisissue [3]. The best laboratory test for diagnosing visceral leishma-niasis is parasite demonstration in tissue smears, with spleenaspirates being more sensitive than bone marrow or lymph nodeaspirates. In spite of this, there are contraindications, precautionsare necessary and complications, though rare, may be serious [9].

Author P

roof

Diagnosis of leishmaniasis

www.future-drugs.com 91

The material obtained is used for culture, cytology or exami-nation for the presence of parasites. Examination of peripheralblood smear may be used, particularly in patients coinfectedwith HIV.

Although demonstration of even a single amastigote uponmicroscopic examination of tissue smears is considered sufficientfor positive diagnosis of the disease, the sensitivity of the tissueexamination, except in the case of spleen aspirate, is low. Besides,identification of amastigotes requires considerable expertise andtraining and is subject to the ability of the observer [3]. Even withthe described problems, spleen aspirates are used for routinediagnosis in the field, for example in Kenya and Sudan [9].

In endemic areas, the recognition of Leishmania species is notusually performed. However, identification of an organism tothe species level is helpful epidemiologically and is importantfor the treatment of, and prognosis determination for, globaltravelers and soldiers returning from war in endemic areas,which are not immune to the parasite and tend to developunusual manifestations of the disease [17].

To avoid the problems described for parasite identification intissue smears, several noninvasive diagnostic tests have beendeveloped. The most employed are immunodiagnostic tests forantibody detection. Serodiagnosis is particularly useful for VLpatients, since they present hypergammaglobulinemia. Currentserologic tests, such as direct agglutination test (DAT), immun-ofluorescence antibody test (IFAT) and enzyme-linked immu-nosorbent assay (ELISA), use crude antigen preparations and arelimited in terms of both specificity and assay reproducibility [18].

Over the last 15 years, DAT has proven to be a very importantserodiagnostic tool, combining high levels of intrinsic validityand ease of performance. The test has so far been semiquantitative,where test readings are given in end-point titers. The reading of

end-point titers is bound to involve subjec-tive errors, thus presenting a problem inthe reproducibility of the test. Nevertheless,the test has been in field use in manyendemic areas. In Sudan, especially in fieldlaboratories and because of the epidemicnature of the disease, DAT is used for diag-nosis of VL. Patients with high titersreceive treatment and a confirmatory para-sitic diagnosis is performed in those withlow titers [19]. In India, several laboratoriesreported satisfactory sensitivity and specifi-city levels for this test [3]. Although DATshowed a high degree of repeatabilitywithin centers, its reproducibility acrosscenters was quite weak [20]. Like most ofthe antibody-based tests, DAT may yieldpositive results for a long time followingcomplete cure and thus has not proved tobe of much prognostic value [3].

Recently, a modified version of DAT,the fast agglutination screening test(FAST) was described, which maintains

the high levels of intrinsic validity described for DAT [21]. Thistest basically employs the DAT in a system designed to use asingle serum dilution and in which test results are availablewithin 3 h. The test was developed especially for application inepidemic situations and for screening of large populations.

IFAT uses an antigen promastigotes of Leishmania. It haslow specificity, demands highly trained personnel, is timeconsuming and expensive, thus is not adaptable to large-scaleepidemiological studies.

ELISA is the most commonly used test for immunodiagnosisof Leishmania. The antigens used are traditionally derivedfrom promastigotes cultivated in vitro and consist of a reper-toire of at least 30 somatic antigens and several surface com-ponents. As a result, to date, most immunodiagnostic methodshave been hampered by the problem of cross-reactivity ofspecies within a family as well as phylogenetically distantmicro-organisms [18]. The problem is further complicated inareas where different forms of leishmaniasis and trypanosomalinfections occur simultaneously.

Thus, emphasis has been placed on the characterization ofLeishmania antigenic components as a tool for obtaining spe-cific diagnosis. Several antigens with different molecular masseshave been identified and a specificity of 100% has beenachieved, however, sensitivity fell as low as 37% [22].

An antigenic glycoprotein complex, the fructose–mannose lig-and from promastigotes of L. donovani, was tested for diagnosisand prognosis of human kala-azar and canine VL. The majorcomponent of this antigen is a 36-kDa glycoprotein designatedgp36, which produces 100% sensitivity and 96% specificity inELISA [23]. Recently, an antigen prepared from Leishmaniamajor-like promastigotes resulted in 100% specificity and 92%sensitivity for diagnosis of human VL, without cross-reactivity

Clinical and epidemiological parameters

Parasitological evaluation

Immunological methods

DNA-based methods

Needle aspirationSuperficial scrapingSlit smearImpression smear

CultureAnimal inoculation

Skin testIFATELISADATImmunoblotFASTMonoclonal antibodies DIFMA

Nucleic acidHibridizationPCR

Histology

Figure 1. Diagnosis of leishmaniasis.DAT: Direct agglutination test; DIFMA: Direct immunofluorescence antibody test using Leishmania genus-specific monoclonal antibody; ELISA: Enzyme-linked immunosorbent assay; FAST: Fast agglutination screening test; IFAT: Immunofluorescence antibody test.

Author P

roof

Tavares, Fernandes & Melo

92 Expert Rev. Mol. Diagn. 3(5), (2003)

with sera from several other diseases [24]. The use of antigensderived from promastigotes cultivated in a protein-freemedium showed a sensitivity of 95% when tested with ELISA[25]. Thus, tests using purified proteins may show the highsensitivity and increased specificity when compared withthose using crude extracts or promastigotes of Leishmania asantigens. However, their preparation is time consuming andmay require sophisticated methods of purification.

On the other hand, recombinant antigens, once identified,can be more easily obtained. The recombinant antigen A2,expressed in amastigotes as a family of proteins that display avariable number of repeats of a unit of ten amino acids, wasreactive in ELISA with 60 and 82% of sera from patients withkala-azar from India and Sudan, respectively [26]. In Brazil,antiA2 antibodies were detected by ELISA in 77% of patient’ssera with symptomatic VL, and in 87% of sera from dogs thattested positive with IFAT for Leishmania or in parasitologicalevaluation [27].

The best-studied and tested Leishmania recombinant antigenis a 39-amino acid repeat from a kinesin-like protein that is pre-dominant in L. chagasi amastigotes [28]. The recombinant pro-tein rK39 has been reported to be 100% sensitive and 100%specific in the diagnosis of VL and PKDL by ELISA [29].Important features of this antigen are that it could be used inHIV-positive patients and antibody levels against rK39 declinerapidly after successful treatment [28,30].

As there is a need for a simple and accurate test for use infield conditions without the requirement of any specific exper-tise, a rapid immunochromatographic dipstick test has beendeveloped which is based on the rK39 antigen. Several studiesin India reported the test to be 100% sensitive [31,32]. However,when evaluated in Sudan, the sensitivity of the test was only67% [33]. When tested in southern Europe, the rK39 stripresults were positive in only 71.4% of VL cases [34]. In Brazil,when applied to the sera of 1798 dogs from an endemic area, itpresented a sensitivity of 92.1% and specificity of 99.5%.However, the test was not able to detect active infection inanimals with low IFAT titers, in the range of 1:40 to 1:320 [35].

Despite the large number of serological tests available, thereis no gold standard diagnostic test for VL. The main reason isthat none of these tests are 100% sensitive and specific [36].

Antigen detectionAntibody-based diagnostics present inherent limitations, suchas weak responses in some patients, persistence of antibodiesafter cure, presence of antibodies in some healthy individualsand particularly in immunocompromised patients [36]. An anti-gen detection test would, in principle, provide better means fordiagnosis, since antigen levels are expected to broadly correlatewith the parasite load. Antigen detection in urine has beentested for the diagnosis of several parasitic infections.

Detection of Leishmania antigens in urine of VL patients wasfirst performed by Kohanteb [37]. Two polypeptide fractions of72–75 kDa and 123kDa were detected in urine of kala-azarpatients. The sensitivity of the 72–75-kDa fraction was 96%

and the specificity was 100%. These antigens were not detectedwithin 3 weeks of antikala-azar treatment, suggesting that thetest has a good prognostic value. A new latex agglutination test(so-called Katex) for detecting Leishmania antigen in urine ofpatients with VL has showed sensitivities between 68 and100% and a specificity of 100%. The antigen is detected quiteearly during the infection and the results of animal experimentssuggest that the amount of detectable antigen tends to declinerapidly following chemotherapy. The test performed betterthan any of the serological tests when compared with micros-copy. Field trials are underway to evaluate its utility for thediagnosis and prognosis of VL [38].

Diagnosis of tegumentary (cutaneous and mucocutaneous) leishmaniasisAlthough major advances have been made in the diagnosis oftegumentary leishmaniasis during the last decade, as for VL,there is no specific method which can be adopted as a goldstandard for detection and diagnosis of these infections.

Current diagnosis is usually made on a clinical basis. Prob-lems in the clinical diagnosis are due to the diverse clinicalmanifestations of the disease. In the cutaneous forms, the clini-cal diagnosis is usually suspected on examination of compatiblelesions on an individual living in or having traveled to anendemic area. Definitive diagnosis requires confirmation of tis-sue amastigotes in cutaneous, mucosal or lymphoid tissue(smears and histology) or the isolation of parasites from lesionsin culture. The sensitivity of these methods depends on theinfecting species and whether or not their pathological featureshave been described [39]. Combination of direct examinationwith culture increases sensitivity for the diagnosis of tegumen-tary leishmaniasis. The paucity of parasites in mucous lesionsmakes biopsy difficult to justify [13]. However, the presumptiveclinical diagnosis cannot often be confirmed by parasitologicaldiagnosis because these methods, although appropriate forreferral centers and investigations, are not widely available tothe health services in endemic areas.

In endemic areas, a presumptive diagnosis is often made on thebasis of clinical presentation and the Montenegro skin test is usedto support clinical suspicion. The skin test is negative in earlycases of cutaneous leishmaniasis and in patients with diffuse cuta-neous leishmaniasis, however, it is a decisive method for the diag-nosis of older leishmanial lesions and mucosal lesions in whichthe number of parasites is low and therefore difficult to detect.

Unfortunately, for American cutaneous leishmaniasis, theoptimal diagnostic methods (culture or inoculation of biopsymaterial in golden hamsters), although highly specific, are only70–75% sensitive at best. The sensitivity is reduced in mucosal,chronic and recurrent lesions.

Skin testingThe Montenegro skin test is a delayed-type hypersensitivitytest used to diagnose leishmaniasis. In this method, 0.5 ml ofphenol-killed whole promastigotes (5 × 107 promastigotes) isinjected on the forearm of the patient. After 48–72 h, the size

Author P

roof

Diagnosis of leishmaniasis

www.future-drugs.com 93

of induration is measured and compared with the size of indu-ration produced by injection of a phenol-saline control in theother forearm. Presently, there is no standardized antigen forthis test but there are a few preparations which are financed andtested by the World Health Organization (WHO)/Special Pro-gramme for Research & Training in Tropical Diseases (TDR)and produced under Good Manufacturing Practices inpremises licensed by their respective governments to producebiologicals for humans: the Pasteur Institute (Tehran, Iran),Istituto Superiore di Sanità (Rome, Italy), Instituto de Biome-dicina (Caracas Venezuela) and BIOBRAS–Montes (Claros,Brazil). The size of induration considered to represent a positivereaction depends on the purpose of the test and the antigenconcentration. The test is negative in active VL due to absenceof appropriate T-cell responses, although in most cases willbecome positive after treatment. The skin test cannot distin-guish between current and past infections in tegumentary leish-maniasis since T-cell responses usually develop in tegumentarypatients shortly after infection [2].

Serological diagnosisSerodiagnosis is an alternative to parasite detection in biopsysamples, either by staining or culturing the parasites. The mostcommonly used serodiagnostic methods for tegumentary leish-maniasis include the same tests described for VL. Sensitivityand specificity of such methods depend on the type, source andpurity of antigens used. It must be pointed out that in patientswith cutaneous leishmaniasis, the antibody titers are usuallylower than those found in patients with VL. IFAT is a sensitivetest and is group-specific. It is the most widely used test and hasan important role in evaluating the effectiveness of chemother-apy [18]. In Old World cutaneous leishmaniasis, sensitivity ofIFAT was 56–81% in parasitological-proven cases and the titersdeclined in most cases after recovery. ELISA using whole para-sites as antigens has a higher sensitivity. In the New World, thistest has been used for diagnosis and/or subsequent control-of-cure of cutaneous leishmaniasis [13,40]. The specificity of IFATis lower than culture and histopathology combined. Antibodytiters are higher in patients with mucosal or multiple lesionsthan those with single lesions. In areas which L. braziliensis isthe prevalent parasite, although both tests are valuable, ELISAhad higher sensitivity when compared with IFAT.

Molecular diagnosis methodsNowadays, there is increasing information on new techniquesfor diagnosis of leishmaniasis using monoclonal antibodies(mAbs), DNA probes and PCR. MAbs and isoenzyme electro-phoresis are undoubtedly the most precise methods of Leishma-nia identification but these techniques are not applicable to theroutine diagnosis of leishmaniasis. MAbs have the advantage ofallowing the characterization of the parasite species usingELISA, IFAT or immunohistochemistry. In recent years,hundreds of mAbs against various species and molecules ofLeishmania have been produced (sponsored by WHO/TDR),not only for diagnosis but also as a research tool.

A direct immunofluorescence antibody test using a Leishma-nia genus-specific monoclonal antibody was significantly supe-rior to standard diagnostic methods in samples taken fromactive lesions of cutaneous leishmaniasis in Ecuador, with noloss of sensitivity for chronic lesions [41].

DNA-based methodsMany studies on DNA-based techniques have been per-formed in order to develop tools for detection and identifica-tion of Leishmania parasites. A number of species-specificprobes are now available for identification of parasites inmany different samples, including biopsies, touch prepara-tions, aspirates from lymph nodes, spleen or bone marrowand blood spots [42]. For maximum sensitivity, the probesused to investigate the sample often target repetitive elements,such as kinetoplast minicircle DNA (kDNA; due to it’s highcopy number, kDNA provides multiple targets for DNAprobes and contains a conserved region of at least 120 basepairs that can provide evidence in every molecule), ribosomalRNA genes, miniexon-derived RNA genes or genomic repeats[43]. The major problem with nucleic acid probes, even thosedirected against repetitive targets, is their lack of sensitivity inreal samples. Due to the low sensitivity, probes are not adaptableon a wide-scale in individual diagnosis.

The advent of the PCR technique has provided a powerfulapproach for the application of molecular biology techniquesfor use in parasite identification, especially in a diagnostic con-text. PCR offers greater sensitivity compared with DNAprobes. The technique has several advantages over other detec-tion methods, especially in the field situation. PCR advantagesinclude: the ability to work with very small amounts of targetmaterial; the fast detection of Leishmania in all materials thatare used for diagnosis, including biopsies, touch preparations,aspirates from lymph nodes, bone marrow or spleen, the buffycoat of patients with VL and in blood spots collected on filterpaper; it permits follow-up of treatment through analysis ofaspirates of healing lesions; it allows assessement of the successof VL treatment; and it can be used for the simultaneousdetection and typing of the parasite.

Several detection systems based on PCR methodology havebeen developed and are available for Leishmania [43]. The besttargets for PCR, as for DNA probes, are still repetitivesequences. They are either based on kDNA minicircle con-served region or complete minicircle amplification [45–47],miniexon-derived RNA genes [48], small subunit ribosomalgenes [49] or gp63 [51].

Many centers have evaluated the use of PCR for diagnosis ofVL, especially on peripheral-blood (PB) samples [50]. WhenPCR is performed on blood, the sensitivity for the detection ofLeishmania DNA ranges from 80.8% [49] to 92.5% [44]. The useof PCR in the diagnosis of VL using a PB sample is particularlypromising because lymph node, bone marrow or spleen aspira-tion is painful and can be dangerous for the patient. Thismethod could be used as an alternative, noninvasive method ofscreening individuals suspected of having VL, including HIV

Author P

roof

Tavares, Fernandes & Melo

94 Expert Rev. Mol. Diagn. 3(5), (2003)

coinfected patients, or as a tool for monitoring the efficacy oftreatment and the appearance of disease relapses in thesepatients [45,52]. PCR seems to be one of the most sensitivemeans of detecting VL among HIV-infected patients. The pres-ence of a positive result by PCR in PB is always associated withclinical disease [52]. It must be emphasized that if PCR on bloodis negative, a PCR on lymph node and/or bone marrow materialshould be performed [43].

In diagnosis of PKDL, PCR of either lymph node aspiratesor biopsies is more sensitive than microscopy, probably due tothe low concentration of the parasites. When using slit skinsmears, 83% of the samples were PCR positive compared withonly 30% positive samples in microscopy [50].

Several PCR protocols have been reported for simultaneousdetection and typing of Leishmania species that cause tegu-mentary leishmaniasis [47,48,54,55]. In Sudan, the detection ofL. major using PCR showed sensitivity of 86% comparedwith 55% of slit smears and 48% of impression smears [45]. Inother studies, PCR showed detection rates ranging from80.8% [54] to more than 90% [55] with samples from differentendemic areas. PCR employing oligonucleotides directedagainst conserved regions of kDNA, with subsequent hybridi-zation with species-specific cloned minicircle molecules [46],proved superior (94%) to conventional touch preparation.This study also showed that PCR is a useful tool for the diag-nosis of mucosal disease (ML) directly from biopsy samples,since conventional methods were positive in only 17% of thepatients with ML, whereas PCR coupled with hybridizationwas positive for 71%. In addition, discrimination could bemade between L. braziliensis and L. mexicana complexes thathave important implications for therapy.

A new PCR-based assay, real-time PCR, has been describedfor detection, quantification and identification of Leishmaniaspecies in clinical samples [57,58]. Advantages attributed tothis technique are when the parasites are difficult to detectdue to their sparseness in the sample, as in some types ofleishmaniasis, and the elimination of the need to employDNA amplification in order to detect and confirm the iden-tity of the PCR product. However, the application of real-time PCR in diagnosis of Leishmania is just beginning and isstill very expensive.

It can be concluded that PCR is a reliable tool for the detec-tion of Leishmania in a variety of samples and for all clinicalmanifestations of the disease. However, despite its undoubtedpotential, this technique is more routinely used in studiesrelated to epidemiological aspects rather than in clinical tests.

Presently, no test is suitable for use under the field condi-tions that usually prevail in the medical facilities in areaswhere the disease is endemic. The ideal test would be a sim-plified, low-cost production that can be easily interpretedwith high sensitivity and specificity. However, most of thenew and complex methods are not available for routine use inthe health services in developing countries because of theirelevated costs, infrastructural requirements and lack oftrained personnel.

Molecular research: the search for new Leishmania diagnostic moleculesEfforts are now being applied to the identification of new Leish-mania genes and proteins through the use of high-throughputtechnologies, such as DNA and protein microarrays. As forother pathologies, these strategies embody the promise of iden-tification of new molecules, which may contribute to develop-ment of more sensitive and specific diagnostic methods as wellas new therapeutic and vaccine strategies.

Leishmania genome approachesThe task of performing genome sequencing of parasites wasfirst envisaged by the United Nations Development Pro-gramme/World Bank/WHO/TDR. TDR has played an impor-tant role in the generation of knowledge on parasite genomes.Starting in 1994, TDR has helped to establish five internationalparasite genome networks and opened the door for scientistsfrom disease-endemic countries (DECs) to participate andcollaborate in genome and postgenome projects. One of themost successful networks was the Leishmania Genome Network(LGN). The aim of the LGN was to obtain a detailed high-resolution map of the reference strain L. major Friedlin(MHOM/IL/81/Friedlin), which is approximately 33.6 Mb insize and distributed among 36 chromosomes. This is now beingachieved by the application of a number of complementaryapproaches: determination of a pulsed field gel (PFG) chromo-somal karyotype, shuttle cosmid clone fingerprinting to gener-ate overlapping contigs, sequencing and mapping of expressedsequence tags to PFG-separated chromosomes, and the generationof DNA sequences from entire chromosomes.

The pace of sequence generation in the Leishmania genomeproject has increased substantially over the past year. The com-plete genome sequence of L. major will soon be available.Sequences of chromosomes 1, 3, 4, 5, 15, 24, 25 and 31 havebeen completed and sequencing of several other chromosomesare currently underway.

There is an urgent need to make the link between these DNAsequences and the functional proteins of the parasite whichthey encode. With the aim of deciphering and understandingthe information in parasite genomes, TDR is enhancing DECcapacity to use the parasite genome data and is supportingdevelopments in applied genomics and bioinformatics. Bioin-formatics may contribute to the identification of new targetsfor drugs, vaccines and diagnostics, and to the understanding ofthe basis of drug resistance, antigenic diversity, infectivity andpathology [59].

DNA microarraysSince sequencing of various Leishmania chromosomes and mostgenes has been concluded, the challenge now is the interpreta-tion of gene sequence information regarding biological struc-ture and placing gene expression in the context of the life cycleof the parasite [60]. This can be achieved at the level of RNAtranscription, protein expression and metabolic pathways. Thelife cycle of Leishmania involves adaptations to a variety of

Author P

roof

Diagnosis of leishmaniasis

www.future-drugs.com 95

conditions in the mammalian macrophages and sand fly vector.There are successive changes in morphology, biochemistry andplasma membrane proteins [61,62]. Many of these changes arelikely to be directed by changes in mRNA abundance andtranslation. The complete sequencing of several genomes,including the human, has signaled the beginning of a new erain which scientists are becoming increasingly interested in func-tional genomics [63]. Functional genomics represents the globalgenetic approaches for elucidating the function of the novelgenes revealed by complete genome sequences. In recent years,microarray technology has become a major tool for the investi-gation of transcriptional variations in gene expression. Already,many studies are underway in several laboratories to implementthe next stage of high-throughput genome-wide analysis, suchas DNA microarrays and proteomics. To date, based on tradi-tional molecular biological methods which work on one-geneexperiment basis, the biological functions of only a fraction ofthe Leishmania genes that were identified in genome projectsare known. The application of microarray technology will per-mit the analysis of many genes at once and determine whichones are differentially expressed in the promastigote and amas-tigote stages, in the host-parasite interactions or in a particularcell type [64,65].

Thus, the expression of thousands of Leishmania genes isbeing analyzed at the same time by DNA microarrays andnumerous protein-coding genes have been identified whichshow significant levels of differential expression between vari-ous life cycle stages. In the near future, DNA microarrays mayreveal antigen coding genes suitable for diagnostic purposes,such as A2, K39 and ORFF [66], three of the most promisingdefined antigens identified to date. Curiously, A2 and K39 areboth more abundantly expressed in the amastigote stage and arecomposed of repetitive units of amino acids which representamplified targets for serological diagnosis. These characteristicsmay offer important guidelines for the search for new antigencoding genes. Epitope mapping is another possibility once newantigen coding genes are identified.

Protein microarraysVery few proteins and even fewer cloned molecules have beendescribed which can be used in the diagnosis of leishmaniasis.Therefore, there is a need to identify new proteins that couldimprove the diagnosis of leishmaniasis. After the sequencing ofthe human genome and that of numerous pathogens, there wasan interest in studying the proteins expressed by the genes. Theterm proteome was then coined and defined as the proteincomplement of the genome and the process of studying theproteome became know as proteomics.

Array-based methods are becoming prevalent within pro-teomics research due to the desire to analyze proteins in ananalogous format to that of the DNA microarray [67]. Thesetechnologies are emerging for basic biological research, molecu-lar diagnostics and therapeutic development, with the potentialof providing parallel functional analysis of hundreds or perhapshundreds of thousands of proteins simultaneously.

The study of a proteome, or complete complement of pro-teins, their interactions and functions within a cell or organism,has employed protein-profiling techniques for protein discoveryand analysis. Using these methods, the totality of protein com-ponents within a cell or organism can be viewed on a specialized2D gel, where proteins are separated on a polyacrylamidematrix. Each protein species is identified using mass spectrome-try [67]. This is called an open high-throughput platform forprotein expression analysis. These techniques are best suited forfirst-pass comparisons of proteomes to identify a few, typicallynovel, proteins that exhibit the greatest differences in abundance[68]. El-Fakhry and coworkers are using these methodologies tohighlight and identify proteins that are differentially expressedin the intracellular stage of L. donovani infantum [69].

Closed architecture proteomic platforms will be best suitedfor precise analysis of quantitative differences in abundanceamong known protein families and pathways. Both open andclosed architecture expression proteomic methods are likely toflourish. For example, upon identification of differentiallyexpressed proteins in an open system, further analyses, suchas time- and dose-response, can be performed in a closedarchitecture system [68].

Depending of the type of biological material, such as pro-teins, peptides or antibodies, different microarray formats canbe applied to proteomic analysis [70–72]. Microarrays of captureantibodies have been used to measure protein levels, such ascytokines [72], a mixture of analytes [73], and microarraysprinted with antigens have been used for detection of circulatingantibodies in clinical specimens [74].

Proteome alterations in disease may occur in many differentways that are not predictable from genomic analysis and it isclear that a better understanding of these alterations will have asubstantial impact in medicine. A useful repertoire of pro-teomic technologies is currently available for disease-relatedapplications, although further technological innovations wouldalso be beneficial to increase sensitivity, reduce sample require-ment, increase throughput and more effectively uncover varioustypes of protein alterations such as post-translational modifica-tions. The use of these technologies will likely expand substan-tially, particularly to meet the need for better diagnostics and toshorten the path for developing effective therapy [75].

The complete sequencing of a number of microbial genomeshas provided a framework for identifying proteins encoded bythese genomes. The genome sequencing of the malaria parasitePlasmodium falciparum has provided the basis for conductingcomparative proteomic studies of this pathogen, leading to theidentification of new potential drug and vaccine targets [76,77].Aside from comprehensive identification of microbial proteins,proteomics is relevant to numerous aspects of microbial diseasepathogenesis and treatment [78,79].

The study of leishmaniasis proteomics could approach thefollowing targets:

• Comparative analysis of different strains. Analysis of secretedproteins, surface proteins or proteins responsible for crucial

Author P

roof

Tavares, Fernandes & Melo

96 Expert Rev. Mol. Diagn. 3(5), (2003)

metabolic pathways can reveal those that could be importantfor diagnosis, evaluation of the mechanism of action of drugsor vaccination studies

• Comparative analysis of different parasitic stages [80]. Theidentification of amastigote-expressed genes that could beused in high-throughput DNA vaccine screens is being usedto identify new vaccine candidates [59]

• Identification of proteins related to pathogenicity

• Identification of proteins involved in host–pathogen interaction

A number of questions remain unanswered. Why L. bra-ziliensis causes cutaneous or mucocutaneous disease andL. amazonensis causes localized or diffuse cutaneous leishmaniasis?In localized leishmaniasis, why lesions induced by L. mexicanaheal rapidly and those induced by L. braziliensis do not? Theseand other aspects are probably determined by proteins involvedin the host–pathogen interaction and could be answered bycomparative proteomic studies.

In achieving these targets, the main contribution of proteom-ics to leishmaniasis would be:

• The development of a trustworthy diagnosis test, as there isno gold standard in use

• The discovery of new targets for drugs, as there is a need fordeveloping new therapies due to the increasing resistance ofthe parasites to the commonly used drugs

• The discovery of new immunogens to be used in vaccinationstudies

Expert opinionA huge amount of effort and knowledge is now being applied tothe identification of new Leishmania genes and proteins throughthe use of high-throughput technologies. However, as men-tioned previously, there is a gap between the scientific advancesand the diagnostics and management of Leishmania infectionsin the field. Thus, the real challenge for scientists and health andgovernmental organizations in the coming years will be toshorten the distance between all the refinements gathered fromthe scientific effort and the practice of health services in manyendemic areas around the world. Rapid, sensitive and low-costdiagnostic methods will certainly have an impact in the incidence,morbidity and mortality of the different forms of leishmaniasis.

Five-year viewDespite the introduction of novel techniques and the better per-formance of the existing ones, there is a need for the develop-ment of more precise and sensitive methods for the diagnosis ofleishmaniasis. The efforts of leishmaniasis diagnosis shouldfocus upon the development of rapid, inexpensive, sensitive andspecific diagnostic tests which can be used in field conditions inendemic areas.

Special attention should be paid to the development ofimmunological tests (mainly serological) due to their simplic-ity, noninvasiveness and low cost. As a result of genomic andproteomic approaches, the identification of new parasite proteins

will certainly allow the identification of diagnostic antigens orepitopes to be tested either as recombinant or synthetic pep-tides in ELISA or rapid diagnostic tests. Moreover, the availa-bility of genomic or proteomic data for different species willallow, by comparison, the identification of species-specificmolecules for differential diagnosis, especially for tegumentaryleishmaniasis infection.

Information resources• WHO/TDR Genome Projects, including the

Leishmania Genome Projectwww.who.int/tdr/index.html(Viewed August 2003). In the section ‘Genomes on the web’, all data are stored indatabases. This information provides the building blocks forfunctional and structural analyses, which can be performedby researchers all over the world.

• Leishmania major GeneDBwww.genedb.org/genedb/leish/index.jsp(Viewed August 2003).The genome of L. major Friedlin, the reference strain, isbeing sequenced as part of a multicenter collaboration.Extrapolating from the currently available data, it isexpected that the approximate 8000 genes will be identifiedin a 33.6-Mb haploid genome spread over 36 chromo-somes. As both sequencing and annotation are in progress,the data are continually updated.

• The Leishmania major Friedlin Genome Projectwww.sanger.ac.uk/Projects/L_major(Viewed August 2003).Information on the Leishmania Genome Network (LGN) canalso be obtained in the Sanger Institute Genome SequencingSite, one of the institutes responsible for the LGN.

Key issues

• Current leishmaniasis diagnosis is usually made on a clinical basis. There is a need for simplified laboratory diagnostic tests which can be easily interpreted with high sensitivity and specificity and with low-cost production and can be used in the prevalent field conditions.

• Purified leishmania proteins and recombinant antigens are improving immunological methods based on detection of antibodies in patient sera. However, few proteins were described which can be used in the diagnosis of leishmaniasis and even fewer cloned molecules have been used.

• Diagnostic tests based on antigen detection in urine of patients showed better performance than serological tests.

• The PCR technique has several advantages including the ability to work with small amounts of target material, fast detection of Leishmania and the follow-up of treatment as well as the assessement of the success of visceral leishmaniasis.

Author P

roof

Diagnosis of leishmaniasis

www.future-drugs.com 97

ReferencesPapers of special note have been highlighted as:• of interest•• of considerable interest

1 Desjeux P. Human leishmaniasis: epidemiology and public health aspects. World Health Stat. Q45, 267–275 (1992).

2 Davies CR, Kaye P, Croft S, Sundar S. Leihmaniasis: new approaches to disease control. Br. Med. J. 326, 377–382 (2003).

• A concise clinical review on diagnosis, treatment, vector and reservoir control of leishmaniasis.

3 Sundar S, Rai M. Laboratory diagnosis of visceral leishmaniasis. Clin. Diagn. Lab. Immunol. 9(5), 951–958 (2002).

•• A comprehensive review on diagnosis of visceral leishmaniasis.

4 Zijlstra EE, Musa AM, Khalil EA,el-Hassam IM, el-Hassan AM. Post-kalar dermal leishmaniasis. Lancet Infect. Dis. 3(2), 87–98 (2003).

5 Seaman J, Mercer AJ, Sondorp E. The epidemic of visceral leishmaniasis in western upper Nile, southern Sudan: course and impact from 1984 to 1994. Int. J. Epidemiol. 25, 862–871 (1996).

6 Thakur CP. Socio-economics of visceral leishmaniasis in Bihar (India). Trans. R. Soc. Trop. Med. Hyg. 94, 156–157 (2000).

7 Hermaldt BL. Leishmaniasis. Lancet 354, 1191–1199 (1999).

8 Arias JR, Monteiro PS, Zicker F. The re-emergence of visceral leishmaniasis in Brazil. Emerg. Infect. Dis 2, 146–146 (1996).

9 Guerin PJ, Olliaro P, Sundar S et al. Visceral leishmaniasis: current status of control, diagnosis and treatment and a proposed research and development agenda. Lancet Infect. Dis. 2, 494–501 (2002).

•• A review of the current situation and needs of vector control, prevention of disease, diagnosis, treatment and burden of visceral leishmaniasis

10 Desjeux P. The increase in risk factors for leishmaniasis worldwide. Trans. R. Soc. Trop. Med. Hyg. 95, 239–243 (2001).

11 Pintado V, Lopez-Velez R. HIV-associated visceral leishmaniasis. Clin. Microbiol. Infect. 7, 291–300 (2001).

12 Barral A, Guerreiro J, Bonfim G et al. Lymphadenopathy as the first sign of human cutaneous infection by Leishmania braziliensis. Am. J. Trop. Med. Hyg. 53, 256–259 (1995).

13 Carvalho MLR, Fontes CJF, Hueb M et al. Tegumentary leishmaniasis in the State of

Mato Grosso (Brazil): clinical, laboratory and therapeutic studies. An. Bras. Dermatol. 77, 45–56 (2002).

14 Herwaldt BL, Arana BA, Navin TR. The natural history of cutaneous leishmaniasis in Guatemala. J. Infect. Dis. 165, 518–527 (1992).

15 Berman JD. Human leishmaniasis: clinical, diagnostic and chemotherapeutic developments in the last 10 years. Clin. Infect. Dis. 24, 684–703 (1997).

16 Marsden PD. Mucosal leishmaniasis(‘espundia’ Escomel 1911). Trans. R. Soc. Trop. Med. Hyg. 80, 859–876 (1993).

17 Magill AJ, Grogl M, Gasser A et al. Viscerotropic leishmaniasis caused by Leishmania tropica in soldiers returning from Operation Desert Storm. N. Engl. J. Med. 328, 1383–1387 (1993).

18 Kar K. Serodiagnosis of leishmaniasis. Crit. Ver. Microb. 21, 123–152 (1995).

19 Boelaert M, Lynen P, Desjeux P, Van der Stuyft P. Cost-effectiveness of competing diagnostic-therapeutic strategies for visceral leishmaniasis. Bull. World Health Organ. 77, 667–674 (1999).

20 Boelaert M, El Safi S, Mousa H et al. Multicenter evaluation of repeatability and reproducibility of the direct agglutination test for visceral leishmaniasis. Trop. Med. Int. Health 4, 31–37 (1999).

21 Schoone GJ, Hailu A, Kroon CM et al. A fast agglutination screening test (FAST) for the detection of anti-leishmania antibodies. Trans. R. Soc. Trop. Med. Hyg. 95, 400–401 (2001).

22 Vinayak VK, Mahajan D, Sobti RC et al. Anti-66 kDa anti-leishmanial antibodies as specific immunodiagnostic probe for visceral leishmaniasis. Indian J. Med. Res. 99, 109–114 (1994).

23 Palatnik de Sousa CB, Gomes EM, Paraguai de Souza et al. Leishmania donovani: titration of antibodies to the fucose mannose ligand as an aid in the diagnosis and prognosis of visceral leishmaniasis. Trans. R. Soc. Trop. Med. Hyg. 89, 390–393 (1995).

24 Barbosa de Deus R, Mares Guia ML, Nunes AZ et al. Leishmania major-like antigen for specific and sensitive serodiagnosis of human and canine visceral leishmaniasis. Clin. Diagn. Lab. Immunol. 9, 1361–1366 (2002).

25 Rajasekariah GH, Rykan JR, Hillier SR et al. Optimization of an ELISA for the serodiagnosis of visceral leishmaniasis using in vitro derived promastigote antigens. J. Immunol. Methods 252, 105–119 (2001).

26 Ghedin E, Zhang WW, Charest H et al. Antibody response against a Leishmania donovani amastigote-stage-specific protein in patients with visceral leishmaniasis. Clin. Diagn. Lab. Immunol. 4, 530–535 (1997).

27 Carvalho FA, Charest H, Tavares CAP et al. Diagnosis of American visceral leishmaniasis in human and dogs using recombinant Leishmania donovani A2 antigen. Diagn. Microbiol. Infect. Dis. 43, 289–295 (2002).

28 Burns JM, Shreffler WG, Benson DR et al. Molecular characterization of a kinesin-related antigen of Leishmania chagasi that detects specific antibody in African and American visceral leishmaniasis. Proc. Natl Acad. Sci. 15, 775–779 (1993).

29 Kumar R, Pai K, Pathak K, Sundar S. Enzyme -linked immunosorbent assay for recombinat K39 antigen in diagnosis and prognosis of Indian visceral leishmaniasis. Clin. Diagn. Lab. Immunol. 8, 1220–1224 (2001).

30 Hougton RL, Petrescu M, Benson DR et al. A cloned antigen (recombinant K39) of Leishmania chagasi diagnostic for visceral leishmaniasis in human immunodeficiency virus Type 1 patients and a prognostic indicator for monitoring patients undergoing drug therapy. J. Infect. Dis. 177, 1339–1344 (1998).

31 Bern C, Jha SN, Joshi AB et al. Use of the recombinant K39 dipstick test and the direct agglutination testing in a setting endemic for visceral leishmaniasis in Nepal. Am. J. Trop. Med. Hyg. 63, 153–157 (2000).

32 Sundar S, Pai K, Sahu M et al. Immunochromatographic strip test detection of anti K39 antibody in Indian visceral leishmaniasis. Ann. Trop. Med. Parasitol. 96, 19–23 (2202).

33 Zijlstra E, Nur EY, Desjeux P et al. Diagnosing visceral leishmaniasis with recombinant K39 strip test: experience form the Sudan. Trop. Med. Int. Health 6, 108–113 (2001).

34 Jelinek T, Eichenlaub S, Loscher T. Sensitivity and specificity of a rapid immunochromatographic test for diagnosis of visceral leishmaniasis. Eur. J. Clin. Microbiol. Infect. Dis. 18, 669–670 (1999).

35 Genaro O, Costa RT, França Silva JC et al. Evaluation of an immunocromatographic asssay for the diagnosis of dogs experimentally and naturally infected with Leishmania chagasi in Brazil. Acta Parasitologica Turcica 21(1), (1997).

36 World Health Organization. Leishmania/HIV Co-infection in Southwestern Europe 1990–1998. Retrospective Analysis of 965 Cases. WHO/LEISH/2000/42. WHO, Geneva, (2000).

Author P

roof

Tavares, Fernandes & Melo

98 Expert Rev. Mol. Diagn. 3(5), (2003)

37 Kohanteb J, Ardehali SM, Rezai HR. Detection of Leishmania donovani soluble antigen and antibody in the urine of visceral leishmaniasis patients. Trans. R. Soc. Trop. Med. Hyg. 81, 578–580 (1987).

38 Attar ZJ, Chance L, el-Safi S et al. Latex agglutination test for the detection of urinary antigen in visceral leishmaniasis. Acta Trop. 78, 11–16 (2001).

39 Romero G, Guerra MVF, Paes MG, Macedo VO. Comparison of cutaneous leishmaniasis due to Leishmania (Viannia) braziliensis and L. (V.) guyanensis in Brazil: clinical findings and diagnostic approach. Clin. Infect. Dis. 32, 1304–1312 (2001).

40 Silveira TG, Arraes SM, Bertolini DA et al. The laboratory diagnosis and epidemiology of cutaneous leishmaniasis in Parana State, southern Brazil. Rev. Soc. Bras. Med. Trop. 32, 413–423 (1999).

41 Chico ME, Guderian RH, Coopre P et al. Evaluation of a direct immunofluorescent antibody (DIFAMA) test using Leishmania genus-specific monoclonal antibody in the routine diagnosis of cutaneous leishmaniasis. Rev. Soc. Bras. Med. Trop. 28, 99–103 (1995).

42 Wilson BSM. DNA-based methods in the detection of Leishmania parasites: field applications and practicalities. Ann. Trop. Med. Parasitol. 89(Suppl. 1), 95–100 (1995).

43 Schallig HDFH, Oskan L. Molecular biological applications in the diagnosis and control of leishmaniasis and parasite identification. Trop. Med. Int. Health 7, 641–651 (2002).

44 Andresen K, Gasim S, Elhassan AM et al. Diagnosis of visceral leishmaniasis by polymerase chain reaction using blood, bone marrow and lymph node samples from patients from the Sudan. Trop. Med. Int. Health 2, 440–444 (1997).

45 Osman OF, Oskam L, Zijlstra EE et al. Use of the polymerase chain reaction to assess the success of visceral leishmaniasis treatment. Trans. R. Soc. Trop. Med. Hyg. 92, 397–400 (1998).

46 Pirmez C, Trajano VS, Neto MPO et al. Use of PCR in diagnosis of human american tegumentary leishmaniasis in Rio de Janeiro, Brazil. J. Clin. Microbiol. 37, 1819–1823 (1999).

47 Harris E, Kroop G, Belli A et al. Single step multiplex PCR assay for characterization of New World Leishmania complexes. J. Clin. Microbiol. 36, 1989–1995 (1998).

48 Uliana SR, Ishikuwa E, Stempliuk VA et al. Geographical distribution of neotropical Leishmania of the subgenus Leishmania

analysed by ribosomal oligonucleotide probes. Trans. R. Soc. Trop. Med. Hyg. 94, 261–264 (2000).

49 Lechaud L, Chabbert E, Dubessay P et al. Comparison of various sample preparation methods for PCR diagnosis of visceral leishmaniasis using peripheral blood. J. Clin. Microbiol. 39, 613–617 (2001).

50 Salotra P, Screenivas G, Pouge GP et al. Development of a species-specific PCR assay for detection of Leishmania donovani, in clinical sample from patients with kala-azar and pos-kala-azar dermal leishmaniasis. J. Clin. Microbiol. 39, 849–854 (2001).

51 Dujardin JC, Victoir K, De Doncker S et al. Molecular epidemiology and diagnosis of Leishmania: what have we learnt from genome structure, dynamics and function. Trans. R Soc. Trop. Med. Hyg. 96(Suppl. 1), S81–S86 (2002).

52 Pizzuto M, Piazza M, Senese D et al. Role of PCR in diagnosis and prognosis of visceral leishmaniasis in patients coinfected with human immunodeficiency virus Type I. J. Clin. Microbiol. 39, 357–361 (2001).

53 Ramirez JR, Agudelo S, Muskus C et al. Diagnosis of cutaneous leishmaniasis in Colombia: the sampling site with lesions influences the sensitivity of parasitologic diagnosis. J. Clin. Microbiol. 38, 3768–3773 (2000).

•• Reports on how to take samples of cutaneous lesions for enhancing the sensitivity of microscopic examination. It is very useful in local health services in developing countries.

54 Rodrigues EH, Felinto de Brito ME, Mendonça MG et al. Evaluation of PCR for diagnosis of American cutaneous leishmaniasis in an area of endemicity in northeastern Brazil. J. Clin. Microbiol. 40, 3572–3576 (2002).

55 Belli A, Rodriguez B, Avilles H, Harris E. Simplified polymerase chain reaction detection of New World Leishmania in clinical specimens of cutaneous leishmaniasis. Am. J. Trop. Med. Hyg. 58, 102–199 (1998).

56 Romero GAS, Guerra MVF, Paes MG et al. Sensitivity of the polymerase chain rection for the diagnosis of cutaneous leishmaniasis due to Leishmania (Viannia) guyanensis. Acta Trop. 79, 225–229, (2001).

57 Nicolas L, Prina E, Lang T, Milon G. Real-time PCR for detection and quantification of Leishmania in mouse tissues. Clin. Microbiol. 40, 1666–1669 (2002).

58 Schulz A, Mellenthin K, Schonian G et al. Detection, differentiation and

quantification of pathogenic Leishmania organisms by a fluorescence resonance energy transfer-based in real-time PCR assay. J. Clin. Microbiol. 41, 1529–1535 (2003).

59 Almeida R, Norrish A, Levick M et al. From genomes to vaccines: Leishmania as a model. Philos. Trans. R. Soc. Lond. B. Biol. Sci. 357, 5–11 (2002).

60 Beverley SM, Akopyants NS, Goyard S et al. Putting the Leishmania genome to work: functional genomics by transposon trapping and expression profiling. Philos. Trans. R. Soc. Lond. B. Biol. Sci . 357, 47–53 (2002).

61 Butcher BA, Turco SJ, Hilty BA et al. Deficiency in β1,3- galactosyltransferase of a Leishmania major lipophosphoglycan mutant adversely influences in the Leishmania–sand fly interaction. J. Biol. Chem. 271, 20573–20579 (1996).

62 Sacks DL, Modi G, Rowton E et al. The role of phosphoglicans in Leishmania–sand fly interactions. Proc. Natl Acad. Sci. USA 97, 406–411 (2000).

63 Mutch DM, Berger A, Mansourian R et al. The limit fold change model: a practical approach for selecting differentially expressed genes from microarray data. BMC Bioinformatics 3, 17 (2002).

64 Myler PJ, Sisk E, McDonagh PD et al. Genomic organization and gene function in Leishmania. Biochem. Soc. Trans. 28, 527–531 (2000).

65 Buates S, Matlashewski G. General suppression of macrophage gene expression during Leishmania donovani infection. J. Immunol. 166, 3416–3422 (2001).

66 Raj VS, Ghosh A, Dole VS et al. Serodiagnosis of leishmaniasis with recombinant ORFF antigen. Am. J. Trop. Med. Hyg. 61, 482–487 (1999).

67 Albala JS. Array-based proteomics: the latest chip challenge. Exp. Rev. Mol. Diagn. 1, 145–152 (2001).

•• Describes array-based protein technologies with emphasis on array-based proteomics, protein production for generation of protein arrays, high-throughput antibody production and applications of array-based proteomics.

68 Schweitzer B, Kingsmore SF. Measuring proteins on microarrays. Curr. Opin. Biotechnol. 13, 14–19 (2002).

•• A clear explanation on how to measure proteins on microarrays, including microarray immunoassays, microarrays for diagnostics and protein profiling on microarrays.

Author P

roof

Diagnosis of leishmaniasis

www.future-drugs.com 99

69 El Fakhry Y, Ouellete M, Papadopoulou B. A proteomic approach to identify developmentally regulated proteins in Leishmania infantum. Proteomics 2(8), 1007–1017 (2002).

70 Cahill D. Protein and antibody arrays and their medical applications. J. Immunol. Methods 250, 81–91 (2001).

•• Focuses on current strategies used to generate protein and antibody arrays and their applications in biological research, medicine and diagnostics.

71 Zhu H, Synder M. Protein arrays and microarrays. Curr. Opin. Chem. Biol. 5, 40–45 (2001).

72 Moody MD, Van Arsdell SW, Murphy KP, Orencole SF, Burns C. Array-based ELISAs for high-throughput analysis of human cytokines. Biotechniques 31, 186–194 (2001).

73 Wiese R, Belosludtsev Y, Powdrill T, Thompson P, Hogan M. Simultaneous multianalyte ELISA performed on a microarray platform. Clin. Chem. 47, 1451–1457 (2001).

74 Haab BB, Dunham MJ, Brown PO. Protein microarrays for highly parallel detection and quantitation of specific proteins and antibodies in complex solutions. Genome Biol. 2, 4–9 (2001).

75 Hanash S. Disease proteomics. Nature 422, 226–232 (2003).

•• Describes the application of proteomics to foster a better understanding of disease process, develop new biomarkers for diagnosis and early detection of disease and accelerate drug development.

76 Lasonde E, Ishihama Y, Andersen JS et al. Analysis of the Plasmodium falciparum proteome by high-accuracy mass spectrometry. Nature 419, 537–542 (2002).

77 Florens L, Washburn MP, Raine JD et al. A proteomic view of the Plasmodium falciparum life cycle. Nature 419, 520–526 (2002).

78 Nilsson CL. Bacterial proteomics and vaccine development. Am. J. Pharmacogenomics 2, 59–65 (2002).

79 Haas G, Karaali G, Ebermayer K et al. Immunoproteomics of Helicobacter pylori infection and relation to gastric disease. Proteomics 2, 313–324 (2002).

80 Thiel M, Bruchhaus I. Comparative proteome analysis of Leishmania donovani at different stages of transformation from promastigotes to amastigotes. Med. Microbiol. Immunol. 190, 33–36 (2001).

Affiliations• Carlos Alberto P Tavares, PhD

Departamento de Bioquímica e Imunologia and Director Instituto de Ciências Biológicas,Universidade Federal de Minas Gerais,Caixa Postal 486 – 30161970-Belo Horizonte,Minas Gerais, BrazilTel.: +55 313 499 2501Fax: +55 313 499 2525capt@icb.ufmg.br

• Ana Paula Fernandes, PhDAdjunct ProfesssorDepartamento de Análises Clínicas e Toxicológicas,Faculdade de Farmácia da Universidade Federal de Minas Gerais,Av. Olegário Maciel,2360 – 30 180-112- Belo Horizonte,Minas Gerais, BrazilTel.: +55 313 339 7649Fax: +55 313 339 7644anav@uai.com.br

• Maria Norma Melo, PhD

Full ProfessorDepartamento de Parasitologia,Instituto de Ciências Biológicas,Universidade Federal de Minas Gerais,30161970- Belo Horizonte,Minas Gerais, BrazilTel.: +55 313 499 2850Fax: +55 313 499 2525normamello@aol.com