Molecular diagnosis of. leishmaniasis. Carlos Alberto P Tavares, Ana Paula Fernandes and Maria Norma Melo. Author Proof

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1 Review Molecular diagnosis of leishmaniasis Carlos Alberto P Tavares, Ana Paula Fernandes and Maria Norma Melo 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 Author for correspondence Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Caixa Postal Belo Horizonte, Minas Gerais, Brazil Tel.: Fax: capt@icb.ufmg.br KEYWORDS: DNA microarrays, Leishmania genome, molecular diagnosis, serological diagnosis, tegumentary leishmaniasis, visceral leishmaniasis 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) Leishmaniasis comprises a spectrum of human diseases widely distributed in 88 countries, in five continents. These diseases are transmitted by the bite of an infected sand fly and are caused by over 20 species of Leishmania [1], which are obligatory intracellular parasites surviving within phagolysosomes of host macrophages. There are three different clinical forms of leishmaniasis: Visceral Cutaneous Mucocutaneous Worldwide, leishmaniasis affects approximately 12 million people. Every year, million cases of the cutaneous form and 500,000 visceral leishmaniasis (VL) occur, which caused 59,000 deaths in 2001 [2]. VL, also known as kala-azar, is caused by Leishmania donovani in India, Pakistan, Oriental China, Bangladesh, Nepal, Sudan and Kenya. In these regions, humans act as the reservoir of parasites and VL has an anthroponotic profile. L. infantum is the etiologic agent of zoonotic VL in central and southeast Asia, northeast China, north Africa and the Europe Mediterranean basin, and L. chagasi in Argentina, Bolivia, Brazil, Colombia, El Salvador, Guatemala, Honduras, Martinique, Mexico, Paraguay and Venezuela. Patients with VL present prolonged fever, splenomegaly, hepatomegaly, leucopenia (mainly neutropenia), anemia, hypergammaglobulinemia, cough, abdominal pain, diarrhea, weight loss and cachexia. It is the more severe form of leishmaniasis and, if untreated, is usually fatal [3]. Post-kala-azar dermal leishmaniasis (PKDL) is a known complication of VL in the Old World, in particular in Sudan [4], where it develops during or in the short interval after treatment of VL and a clinical diagnosis can be made. In India and Bangladesh, it develops 1 5 years after the apparent cure of VL and treatment will often be necessary. Diagnosis confirmation, by demonstration of the parasite in a smear or biopsy, is recommended. The spread of VL is caused by population displacement as a result of war, drought, famine or economic development, including widespread urbanization, deforestation and development of new settlements, besides migration from rural to urban areas. These factors were responsible for an epidemic in Sudan, with mortality rates of up to 36% [5], and are contributing to the resurgence of the disease in India [6,7] and its urban spread in Brazil [8]. Several risk factors for clinical VL include malnutrition, immunosuppressive drugs and especially HIV coinfection. The number of coinfections are increasing in India and Brazil, where the urban HIV epidemic and the rural VL epidemic are coming into contact [9]. In Africa and in southern Europe, Leishmania HIV Future Drugs Ltd. All rights reserved. ISSN

2 Tavares, Fernandes & Melo coinfection is regarded as an emerging disease. In Europe, 25 70% of adults with VL also have AIDS and leishmaniasis behaves as an opportunistic infection [10]. In HIV-infected patients, the Leishmania parasite is found in the peripheral blood, outside the reticuloendothelial system, making these patients a reservoir and source of infection for the vectors. The parasite load in peripheral blood is generally so high that transmission among intravenous drug users, by use of shared syringes, has been demonstrated. In addition, coinfected patients may be difficult to diagnose, respond poorly to treatment and relapse repeatedly [11]. Tegumentary (cutaneous and mucocutaneous) leishmaniasis results from multiplication of Leishmania in the phagocytes of the skin or mucosal membranes. In the USA, it occurs from the southern states to northern Argentina and is caused by 14 species belonging to two subgenus: Viannia and Leishmania. Members of the subgenus Viannia are: L. braziliensis, L. panamensis, L. guyanensis, L. peruviana, L. lainsoni, L. shawi, L. naiffi, L. colombiensis and L. lindenbergi, which are zoonotic and cause cutaneous or mucocutaneous lesions. Those of the subgenus Leishmania are L. mexicana, L. amazonensis, L. venezuelensis, L. pifanoi and L. garnhami, which are zoonotic and cause localized lesions or diffuse cutaneous leishmaniasis. In the Old World, L. tropica (anthroponotic), L. major, L. aethiopica and L. killicki (zoonotic) cause cutaneous leishmaniasis. The clinical characteristics of cutaneous leishmaniasis vary depending on the infecting Leishmania species and the immune response of the host. The typical lesion first appears as an erythematous papule or small nodule at the site where promastigotes were inoculated, which may itch but are usually painless. It increases slowly in size, progressing to ulcers that persist for months and in some cases years. The lesion remains painless and its chronic evolution leads most often to spontaneous cure, but leaves flat, hypopigmented, atrophic scars. L. braziliensis infection may be accompanied by regional lymphadenopathy, fever and constitutional symptoms either before or after the skin lesion becomes apparent [11,12]. Amastigotes probably disseminate to distant mucosal sites during the early phase of infection. In one study in Guatemala, 22 of 25 lesions (85%) due to L. mexicana infection healed in a median observation time of 14 weeks [14]. In contrast, only 22% of L. braziliensis lesions healed during a similar median time of 13 weeks [15]. These results suggest that, at least in the New World, patients may exhibit lesions as early as a few weeks after exposure. Nodular lesions and lymphadenopathy as well as the more typical ulcers should be evaluated for the presence of Leishmania. A small percentage of individuals infected with L. braziliensis in Latin America develop mucosal lesions, due to metastasis of the primary lesion. While L. braziliensis is the organism usually associated with this complication, such infections have also been reported from the Middle East and Africa, often due to contiguity of the cutaneous lesion. The mucous involvement tends to vary from one endemic area to another [16]. Although mucosal leishmaniasis is defined as infection of the mucosal membranes of the nose and mouth, infection begins with nasal inflammation and stuffiness and then may involve the oropharynx and larynx. The period of time between the appearance of the cutaneous lesion and the subsequent appearance of mucosal involvement is extremely variable, extending from several weeks to many years. In 14 28% of the cases, mucous involvement can even be concomitant to an active primary lesion [16]. Mucosal disease characteristically does not heal spontaneously and evolves slowly (with a mean time of 3 years), before being brought to medical attention. Traditional leishmaniasis diagnostic approaches Currently, routine diagnosis of leishmaniasis is usually based on clinical and epidemiological parameters but definitive diagnosis still relies on the parasitological observation of the parasite in direct smear, culture or animal inoculation. Parasitological confirmation is important because the current drugs of choice, mostly antimonials, have high cost and significant toxicity and therefore should not be given without justification. In addition, treatment has significant impact on the control of anthroponotic leishmaniasis, thus early diagnosis is required for avoiding a reservoir effect. Furthermore, accurate identification of the infecting Leishmania species permits better orientation of the medical follow-up, since clinical manifestations may vary, depending largely on the immune response of the host and the species to which the parasite belongs. Detection of the particular Leishmania species involved is also essential for epidemiological studies. Although major advances have been made in the diagnosis of leishmaniasis during the last decade, there is no specific method that can be adopted as the gold standard for detection and diagnosis of Leishmania infections. The chosen method often depends upon the application. Several methods are used for diagnosis of leishmaniasis, all of which have important limitations. As illustrated by the scheme presented 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 VL The clinical diagnosis of VL is complex because other commonly occurring diseases, such as trypanosomiasis, malaria, typhoid, schistosomiasis and tuberculosis, share the same clinical features. Many of these diseases can be present along with VL (in cases of coinfection) and sequestration of the parasite in the spleen, bone marrow or lymph nodes further complicates this issue [3]. The best laboratory test for diagnosing visceral leishmaniasis is parasite demonstration in tissue smears, with spleen aspirates being more sensitive than bone marrow or lymph node aspirates. In spite of this, there are contraindications, precautions are necessary and complications, though rare, may be serious [9]. 90 Expert Rev. Mol. Diagn. 3(5), (2003)

3 Diagnosis of leishmaniasis Needle aspiration Superficial scraping Slit smear Impression smear Parasitological evaluation Histology Clinical and epidemiological parameters Culture Animal inoculation Immunological methods Skin test IFAT ELISA DAT Immunoblot FAST Monoclonal antibodies DIFMA The material obtained is used for culture, cytology or examination for the presence of parasites. Examination of peripheral blood smear may be used, particularly in patients coinfected with HIV. Although demonstration of even a single amastigote upon microscopic examination of tissue smears is considered sufficient for positive diagnosis of the disease, the sensitivity of the tissue examination, except in the case of spleen aspirate, is low. Besides, identification of amastigotes requires considerable expertise and training and is subject to the ability of the observer [3]. Even with the described problems, spleen aspirates are used for routine diagnosis in the field, for example in Kenya and Sudan [9]. In endemic areas, the recognition of Leishmania species is not usually performed. However, identification of an organism to the species level is helpful epidemiologically and is important for the treatment of, and prognosis determination for, global travelers and soldiers returning from war in endemic areas, which are not immune to the parasite and tend to develop unusual manifestations of the disease [17]. To avoid the problems described for parasite identification in tissue smears, several noninvasive diagnostic tests have been developed. The most employed are immunodiagnostic tests for antibody detection. Serodiagnosis is particularly useful for VL patients, since they present hypergammaglobulinemia. Current serologic tests, such as direct agglutination test (DAT), immunofluorescence antibody test (IFAT) and enzyme-linked immunosorbent assay (ELISA), use crude antigen preparations and are limited in terms of both specificity and assay reproducibility [18]. Over the last 15 years, DAT has proven to be a very important serodiagnostic tool, combining high levels of intrinsic validity and 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 subjective errors, thus presenting a problem in the reproducibility of the test. Nevertheless, the test has been in field use in many endemic areas. In Sudan, especially in field laboratories and because of the epidemic nature of the disease, DAT is used for diagnosis of VL. Patients with high titers receive treatment and a confirmatory parasitic diagnosis is performed in those with low titers [19]. In India, several laboratories reported satisfactory sensitivity and specificity levels for this test [3]. Although DAT showed a high degree of repeatability within centers, its reproducibility across centers was quite weak [20]. Like most of the antibody-based tests, DAT may yield positive results for a long time following complete cure and thus has not proved to be 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]. This test basically employs the DAT in a system designed to use a single serum dilution and in which test results are available within 3 h. The test was developed especially for application in epidemic situations and for screening of large populations. IFAT uses an antigen promastigotes of Leishmania. It has low specificity, demands highly trained personnel, is time consuming and expensive, thus is not adaptable to large-scale epidemiological studies. ELISA is the most commonly used test for immunodiagnosis of Leishmania. The antigens used are traditionally derived from promastigotes cultivated in vitro and consist of a repertoire of at least 30 somatic antigens and several surface components. As a result, to date, most immunodiagnostic methods have been hampered by the problem of cross-reactivity of species within a family as well as phylogenetically distant micro-organisms [18]. The problem is further complicated in areas where different forms of leishmaniasis and trypanosomal DNA-based methods Nucleic acid Hibridization PCR 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. infections occur simultaneously. Thus, emphasis has been placed on the characterization of Leishmania antigenic components as a tool for obtaining specific diagnosis. Several antigens with different molecular masses have been identified and a specificity of 100% has been achieved, however, sensitivity fell as low as 37% [22]. An antigenic glycoprotein complex, the fructose mannose ligand from promastigotes of L. donovani, was tested for diagnosis and prognosis of human kala-azar and canine VL. The major component of this antigen is a 36-kDa glycoprotein designated gp36, which produces 100% sensitivity and 96% specificity in ELISA [23]. Recently, an antigen prepared from Leishmania major-like promastigotes resulted in 100% specificity and 92% sensitivity for diagnosis of human VL, without cross-reactivity 91

4 Tavares, Fernandes & Melo with sera from several other diseases [24]. The use of antigens derived from promastigotes cultivated in a protein-free medium showed a sensitivity of 95% when tested with ELISA [25]. Thus, tests using purified proteins may show the high sensitivity and increased specificity when compared with those using crude extracts or promastigotes of Leishmania as antigens. However, their preparation is time consuming and may 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 a variable number of repeats of a unit of ten amino acids, was reactive in ELISA with 60 and 82% of sera from patients with kala-azar from India and Sudan, respectively [26]. In Brazil, antia2 antibodies were detected by ELISA in 77% of patient s sera with symptomatic VL, and in 87% of sera from dogs that tested positive with IFAT for Leishmania or in parasitological evaluation [27]. The best-studied and tested Leishmania recombinant antigen is a 39-amino acid repeat from a kinesin-like protein that is predominant in L. chagasi amastigotes [28]. The recombinant protein 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 in HIV-positive patients and antibody levels against rk39 decline rapidly after successful treatment [28,30]. As there is a need for a simple and accurate test for use in field conditions without the requirement of any specific expertise, a rapid immunochromatographic dipstick test has been developed which is based on the rk39 antigen. Several studies in India reported the test to be 100% sensitive [31,32]. However, when evaluated in Sudan, the sensitivity of the test was only 67% [33]. When tested in southern Europe, the rk39 strip results were positive in only 71.4% of VL cases [34]. In Brazil, when applied to the sera of 1798 dogs from an endemic area, it presented a sensitivity of 92.1% and specificity of 99.5%. However, the test was not able to detect active infection in animals with low IFAT titers, in the range of 1:40 to 1:320 [35]. Despite the large number of serological tests available, there is no gold standard diagnostic test for VL. The main reason is that none of these tests are 100% sensitive and specific [36]. Antigen detection Antibody-based diagnostics present inherent limitations, such as weak responses in some patients, persistence of antibodies after cure, presence of antibodies in some healthy individuals and particularly in immunocompromised patients [36]. An antigen detection test would, in principle, provide better means for diagnosis, since antigen levels are expected to broadly correlate with the parasite load. Antigen detection in urine has been tested for the diagnosis of several parasitic infections. Detection of Leishmania antigens in urine of VL patients was first performed by Kohanteb [37]. Two polypeptide fractions of kda and 123kDa were detected in urine of kala-azar patients. The sensitivity of the kDa fraction was 96% and the specificity was 100%. These antigens were not detected within 3 weeks of antikala-azar treatment, suggesting that the test has a good prognostic value. A new latex agglutination test (so-called Katex) for detecting Leishmania antigen in urine of patients with VL has showed sensitivities between 68 and 100% and a specificity of 100%. The antigen is detected quite early during the infection and the results of animal experiments suggest that the amount of detectable antigen tends to decline rapidly following chemotherapy. The test performed better than any of the serological tests when compared with microscopy. Field trials are underway to evaluate its utility for the diagnosis and prognosis of VL [38]. Diagnosis of tegumentary (cutaneous and mucocutaneous) leishmaniasis Although major advances have been made in the diagnosis of tegumentary leishmaniasis during the last decade, as for VL, there is no specific method which can be adopted as a gold standard for detection and diagnosis of these infections. Current diagnosis is usually made on a clinical basis. Problems in the clinical diagnosis are due to the diverse clinical manifestations of the disease. In the cutaneous forms, the clinical diagnosis is usually suspected on examination of compatible lesions on an individual living in or having traveled to an endemic area. Definitive diagnosis requires confirmation of tissue amastigotes in cutaneous, mucosal or lymphoid tissue (smears and histology) or the isolation of parasites from lesions in culture. The sensitivity of these methods depends on the infecting species and whether or not their pathological features have been described [39]. Combination of direct examination with culture increases sensitivity for the diagnosis of tegumentary leishmaniasis. The paucity of parasites in mucous lesions makes biopsy difficult to justify [13]. However, the presumptive clinical diagnosis cannot often be confirmed by parasitological diagnosis because these methods, although appropriate for referral centers and investigations, are not widely available to the health services in endemic areas. In endemic areas, a presumptive diagnosis is often made on the basis of clinical presentation and the Montenegro skin test is used to support clinical suspicion. The skin test is negative in early cases of cutaneous leishmaniasis and in patients with diffuse cutaneous leishmaniasis, however, it is a decisive method for the diagnosis of older leishmanial lesions and mucosal lesions in which the number of parasites is low and therefore difficult to detect. Unfortunately, for American cutaneous leishmaniasis, the optimal diagnostic methods (culture or inoculation of biopsy material in golden hamsters), although highly specific, are only 70 75% sensitive at best. The sensitivity is reduced in mucosal, chronic and recurrent lesions. Skin testing The Montenegro skin test is a delayed-type hypersensitivity test used to diagnose leishmaniasis. In this method, 0.5 ml of phenol-killed whole promastigotes ( promastigotes) is injected on the forearm of the patient. After h, the size 92 Expert Rev. Mol. Diagn. 3(5), (2003)

5 Diagnosis of leishmaniasis of induration is measured and compared with the size of induration produced by injection of a phenol-saline control in the other forearm. Presently, there is no standardized antigen for this test but there are a few preparations which are financed and tested by the World Health Organization (WHO)/Special Programme for Research & Training in Tropical Diseases (TDR) and produced under Good Manufacturing Practices in premises licensed by their respective governments to produce biologicals for humans: the Pasteur Institute (Tehran, Iran), Istituto Superiore di Sanità (Rome, Italy), Instituto de Biomedicina (Caracas Venezuela) and BIOBRAS Montes (Claros, Brazil). The size of induration considered to represent a positive reaction depends on the purpose of the test and the antigen concentration. The test is negative in active VL due to absence of appropriate T-cell responses, although in most cases will become positive after treatment. The skin test cannot distinguish between current and past infections in tegumentary leishmaniasis since T-cell responses usually develop in tegumentary patients shortly after infection [2]. Serological diagnosis Serodiagnosis is an alternative to parasite detection in biopsy samples, either by staining or culturing the parasites. The most commonly used serodiagnostic methods for tegumentary leishmaniasis include the same tests described for VL. Sensitivity and specificity of such methods depend on the type, source and purity of antigens used. It must be pointed out that in patients with cutaneous leishmaniasis, the antibody titers are usually lower than those found in patients with VL. IFAT is a sensitive test and is group-specific. It is the most widely used test and has an important role in evaluating the effectiveness of chemotherapy [18]. In Old World cutaneous leishmaniasis, sensitivity of IFAT was 56 81% in parasitological-proven cases and the titers declined in most cases after recovery. ELISA using whole parasites as antigens has a higher sensitivity. In the New World, this test has been used for diagnosis and/or subsequent control-ofcure of cutaneous leishmaniasis [13,40]. The specificity of IFAT is lower than culture and histopathology combined. Antibody titers are higher in patients with mucosal or multiple lesions than those with single lesions. In areas which L. braziliensis is the prevalent parasite, although both tests are valuable, ELISA had higher sensitivity when compared with IFAT. Molecular diagnosis methods Nowadays, there is increasing information on new techniques for diagnosis of leishmaniasis using monoclonal antibodies (mabs), DNA probes and PCR. MAbs and isoenzyme electrophoresis are undoubtedly the most precise methods of Leishmania identification but these techniques are not applicable to the routine diagnosis of leishmaniasis. MAbs have the advantage of allowing the characterization of the parasite species using ELISA, IFAT or immunohistochemistry. In recent years, hundreds of mabs against various species and molecules of Leishmania have been produced (sponsored by WHO/TDR), not only for diagnosis but also as a research tool. A direct immunofluorescence antibody test using a Leishmania genus-specific monoclonal antibody was significantly superior to standard diagnostic methods in samples taken from active lesions of cutaneous leishmaniasis in Ecuador, with no loss of sensitivity for chronic lesions [41]. DNA-based methods Many studies on DNA-based techniques have been performed in order to develop tools for detection and identification of Leishmania parasites. A number of species-specific probes are now available for identification of parasites in many different samples, including biopsies, touch preparations, aspirates from lymph nodes, spleen or bone marrow and blood spots [42]. For maximum sensitivity, the probes used to investigate the sample often target repetitive elements, such as kinetoplast minicircle DNA (kdna; due to it s high copy number, kdna provides multiple targets for DNA probes and contains a conserved region of at least 120 base pairs that can provide evidence in every molecule), ribosomal RNA genes, miniexon-derived RNA genes or genomic repeats [43]. The major problem with nucleic acid probes, even those directed against repetitive targets, is their lack of sensitivity in real samples. Due to the low sensitivity, probes are not adaptable on a wide-scale in individual diagnosis. The advent of the PCR technique has provided a powerful approach for the application of molecular biology techniques for use in parasite identification, especially in a diagnostic context. PCR offers greater sensitivity compared with DNA probes. The technique has several advantages over other detection methods, especially in the field situation. PCR advantages include: the ability to work with very small amounts of target material; the fast detection of Leishmania in all materials that are used for diagnosis, including biopsies, touch preparations, aspirates from lymph nodes, bone marrow or spleen, the buffy coat of patients with VL and in blood spots collected on filter paper; it permits follow-up of treatment through analysis of aspirates of healing lesions; it allows assessement of the success of VL treatment; and it can be used for the simultaneous detection and typing of the parasite. Several detection systems based on PCR methodology have been developed and are available for Leishmania [43]. The best targets for PCR, as for DNA probes, are still repetitive sequences. They are either based on kdna minicircle conserved region or complete minicircle amplification [45 47], miniexon-derived RNA genes [48], small subunit ribosomal genes [49] or gp63 [51]. Many centers have evaluated the use of PCR for diagnosis of VL, especially on peripheral-blood (PB) samples [50]. When PCR is performed on blood, the sensitivity for the detection of Leishmania DNA ranges from 80.8% [49] to 92.5% [44]. The use of PCR in the diagnosis of VL using a PB sample is particularly promising because lymph node, bone marrow or spleen aspiration is painful and can be dangerous for the patient. This method could be used as an alternative, noninvasive method of screening individuals suspected of having VL, including HIV 93

6 Tavares, Fernandes & Melo coinfected patients, or as a tool for monitoring the efficacy of treatment and the appearance of disease relapses in these patients [45,52]. PCR seems to be one of the most sensitive means of detecting VL among HIV-infected patients. The presence of a positive result by PCR in PB is always associated with clinical disease [52]. It must be emphasized that if PCR on blood is negative, a PCR on lymph node and/or bone marrow material should be performed [43]. In diagnosis of PKDL, PCR of either lymph node aspirates or biopsies is more sensitive than microscopy, probably due to the low concentration of the parasites. When using slit skin smears, 83% of the samples were PCR positive compared with only 30% positive samples in microscopy [50]. Several PCR protocols have been reported for simultaneous detection and typing of Leishmania species that cause tegumentary leishmaniasis [47,48,54,55]. In Sudan, the detection of L. major using PCR showed sensitivity of 86% compared with 55% of slit smears and 48% of impression smears [45]. In other studies, PCR showed detection rates ranging from 80.8% [54] to more than 90% [55] with samples from different endemic areas. PCR employing oligonucleotides directed against conserved regions of kdna, with subsequent hybridization 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 diagnosis of mucosal disease (ML) directly from biopsy samples, since conventional methods were positive in only 17% of the patients with ML, whereas PCR coupled with hybridization was positive for 71%. In addition, discrimination could be made between L. braziliensis and L. mexicana complexes that have important implications for therapy. A new PCR-based assay, real-time PCR, has been described for detection, quantification and identification of Leishmania species in clinical samples [57,58]. Advantages attributed to this technique are when the parasites are difficult to detect due to their sparseness in the sample, as in some types of leishmaniasis, and the elimination of the need to employ DNA amplification in order to detect and confirm the identity of the PCR product. However, the application of realtime PCR in diagnosis of Leishmania is just beginning and is still very expensive. It can be concluded that PCR is a reliable tool for the detection of Leishmania in a variety of samples and for all clinical manifestations of the disease. However, despite its undoubted potential, this technique is more routinely used in studies related to epidemiological aspects rather than in clinical tests. Presently, no test is suitable for use under the field conditions that usually prevail in the medical facilities in areas where the disease is endemic. The ideal test would be a simplified, low-cost production that can be easily interpreted with high sensitivity and specificity. However, most of the new and complex methods are not available for routine use in the health services in developing countries because of their elevated costs, infrastructural requirements and lack of trained personnel. Molecular research: the search for new Leishmania diagnostic molecules Efforts are now being applied to the identification of new Leishmania genes and proteins through the use of high-throughput technologies, such as DNA and protein microarrays. As for other pathologies, these strategies embody the promise of identification of new molecules, which may contribute to development of more sensitive and specific diagnostic methods as well as new therapeutic and vaccine strategies. Leishmania genome approaches The task of performing genome sequencing of parasites was first envisaged by the United Nations Development Programme/World Bank/WHO/TDR. TDR has played an important role in the generation of knowledge on parasite genomes. Starting in 1994, TDR has helped to establish five international parasite genome networks and opened the door for scientists from disease-endemic countries (DECs) to participate and collaborate in genome and postgenome projects. One of the most successful networks was the Leishmania Genome Network (LGN). The aim of the LGN was to obtain a detailed highresolution map of the reference strain L. major Friedlin (MHOM/IL/81/Friedlin), which is approximately 33.6 Mb in size and distributed among 36 chromosomes. This is now being achieved by the application of a number of complementary approaches: determination of a pulsed field gel (PFG) chromosomal karyotype, shuttle cosmid clone fingerprinting to generate overlapping contigs, sequencing and mapping of expressed sequence tags to PFG-separated chromosomes, and the generation of DNA sequences from entire chromosomes. The pace of sequence generation in the Leishmania genome project has increased substantially over the past year. The complete genome sequence of L. major will soon be available. Sequences of chromosomes 1, 3, 4, 5, 15, 24, 25 and 31 have been completed and sequencing of several other chromosomes are currently underway. There is an urgent need to make the link between these DNA sequences and the functional proteins of the parasite which they encode. With the aim of deciphering and understanding the information in parasite genomes, TDR is enhancing DEC capacity to use the parasite genome data and is supporting developments in applied genomics and bioinformatics. Bioinformatics may contribute to the identification of new targets for drugs, vaccines and diagnostics, and to the understanding of the basis of drug resistance, antigenic diversity, infectivity and pathology [59]. DNA microarrays Since sequencing of various Leishmania chromosomes and most genes has been concluded, the challenge now is the interpretation of gene sequence information regarding biological structure and placing gene expression in the context of the life cycle of the parasite [60]. This can be achieved at the level of RNA transcription, protein expression and metabolic pathways. The life cycle of Leishmania involves adaptations to a variety of 94 Expert Rev. Mol. Diagn. 3(5), (2003)

7 Diagnosis of leishmaniasis conditions in the mammalian macrophages and sand fly vector. There are successive changes in morphology, biochemistry and plasma membrane proteins [61,62]. Many of these changes are likely to be directed by changes in mrna abundance and translation. The complete sequencing of several genomes, including the human, has signaled the beginning of a new era in which scientists are becoming increasingly interested in functional genomics [63]. Functional genomics represents the global genetic approaches for elucidating the function of the novel genes revealed by complete genome sequences. In recent years, microarray technology has become a major tool for the investigation of transcriptional variations in gene expression. Already, many studies are underway in several laboratories to implement the next stage of high-throughput genome-wide analysis, such as DNA microarrays and proteomics. To date, based on traditional molecular biological methods which work on one-gene experiment basis, the biological functions of only a fraction of the Leishmania genes that were identified in genome projects are known. The application of microarray technology will permit the analysis of many genes at once and determine which ones are differentially expressed in the promastigote and amastigote stages, in the host-parasite interactions or in a particular cell type [64,65]. Thus, the expression of thousands of Leishmania genes is being analyzed at the same time by DNA microarrays and numerous protein-coding genes have been identified which show significant levels of differential expression between various life cycle stages. In the near future, DNA microarrays may reveal antigen coding genes suitable for diagnostic purposes, such as A2, K39 and ORFF [66], three of the most promising defined antigens identified to date. Curiously, A2 and K39 are both more abundantly expressed in the amastigote stage and are composed of repetitive units of amino acids which represent amplified targets for serological diagnosis. These characteristics may offer important guidelines for the search for new antigen coding genes. Epitope mapping is another possibility once new antigen coding genes are identified. Protein microarrays Very few proteins and even fewer cloned molecules have been described which can be used in the diagnosis of leishmaniasis. Therefore, there is a need to identify new proteins that could improve the diagnosis of leishmaniasis. After the sequencing of the human genome and that of numerous pathogens, there was an interest in studying the proteins expressed by the genes. The term proteome was then coined and defined as the protein complement of the genome and the process of studying the proteome became know as proteomics. Array-based methods are becoming prevalent within proteomics research due to the desire to analyze proteins in an analogous format to that of the DNA microarray [67]. These technologies are emerging for basic biological research, molecular diagnostics and therapeutic development, with the potential of providing parallel functional analysis of hundreds or perhaps hundreds of thousands of proteins simultaneously. The study of a proteome, or complete complement of proteins, their interactions and functions within a cell or organism, has employed protein-profiling techniques for protein discovery and analysis. Using these methods, the totality of protein components within a cell or organism can be viewed on a specialized 2D gel, where proteins are separated on a polyacrylamide matrix. Each protein species is identified using mass spectrometry [67]. This is called an open high-throughput platform for protein expression analysis. These techniques are best suited for first-pass comparisons of proteomes to identify a few, typically novel, proteins that exhibit the greatest differences in abundance [68]. El-Fakhry and coworkers are using these methodologies to highlight and identify proteins that are differentially expressed in the intracellular stage of L. donovani infantum [69]. Closed architecture proteomic platforms will be best suited for precise analysis of quantitative differences in abundance among known protein families and pathways. Both open and closed architecture expression proteomic methods are likely to flourish. For example, upon identification of differentially expressed proteins in an open system, further analyses, such as time- and dose-response, can be performed in a closed architecture system [68]. Depending of the type of biological material, such as proteins, peptides or antibodies, different microarray formats can be applied to proteomic analysis [70 72]. Microarrays of capture antibodies have been used to measure protein levels, such as cytokines [72], a mixture of analytes [73], and microarrays printed with antigens have been used for detection of circulating antibodies in clinical specimens [74]. Proteome alterations in disease may occur in many different ways that are not predictable from genomic analysis and it is clear that a better understanding of these alterations will have a substantial impact in medicine. A useful repertoire of proteomic technologies is currently available for disease-related applications, although further technological innovations would also be beneficial to increase sensitivity, reduce sample requirement, increase throughput and more effectively uncover various types of protein alterations such as post-translational modifications. The use of these technologies will likely expand substantially, particularly to meet the need for better diagnostics and to shorten the path for developing effective therapy [75]. The complete sequencing of a number of microbial genomes has provided a framework for identifying proteins encoded by these genomes. The genome sequencing of the malaria parasite Plasmodium falciparum has provided the basis for conducting comparative proteomic studies of this pathogen, leading to the identification of new potential drug and vaccine targets [76,77]. Aside from comprehensive identification of microbial proteins, proteomics is relevant to numerous aspects of microbial disease pathogenesis and treatment [78,79]. The study of leishmaniasis proteomics could approach the following targets: Comparative analysis of different strains. Analysis of secreted proteins, surface proteins or proteins responsible for crucial 95

8 Tavares, Fernandes & Melo metabolic pathways can reveal those that could be important for diagnosis, evaluation of the mechanism of action of drugs or vaccination studies Comparative analysis of different parasitic stages [80]. The identification of amastigote-expressed genes that could be used in high-throughput DNA vaccine screens is being used to 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. braziliensis causes cutaneous or mucocutaneous disease and L. amazonensis causes localized or diffuse cutaneous leishmaniasis? In localized leishmaniasis, why lesions induced by L. mexicana heal rapidly and those induced by L. braziliensis do not? These and other aspects are probably determined by proteins involved in the host pathogen interaction and could be answered by comparative proteomic studies. In achieving these targets, the main contribution of proteomics to leishmaniasis would be: The development of a trustworthy diagnosis test, as there is no gold standard in use The discovery of new targets for drugs, as there is a need for developing new therapies due to the increasing resistance of the parasites to the commonly used drugs The discovery of new immunogens to be used in vaccination studies Expert opinion A huge amount of effort and knowledge is now being applied to the identification of new Leishmania genes and proteins through the use of high-throughput technologies. However, as mentioned previously, there is a gap between the scientific advances and the diagnostics and management of Leishmania infections in the field. Thus, the real challenge for scientists and health and governmental organizations in the coming years will be to shorten the distance between all the refinements gathered from the scientific effort and the practice of health services in many endemic areas around the world. Rapid, sensitive and low-cost diagnostic methods will certainly have an impact in the incidence, morbidity and mortality of the different forms of leishmaniasis. Five-year view Despite the introduction of novel techniques and the better performance of the existing ones, there is a need for the development of more precise and sensitive methods for the diagnosis of leishmaniasis. The efforts of leishmaniasis diagnosis should focus upon the development of rapid, inexpensive, sensitive and specific diagnostic tests which can be used in field conditions in endemic areas. Special attention should be paid to the development of immunological tests (mainly serological) due to their simplicity, noninvasiveness and low cost. As a result of genomic and proteomic approaches, the identification of new parasite proteins will certainly allow the identification of diagnostic antigens or epitopes to be tested either as recombinant or synthetic peptides in ELISA or rapid diagnostic tests. Moreover, the availability of genomic or proteomic data for different species will allow, by comparison, the identification of species-specific molecules for differential diagnosis, especially for tegumentary leishmaniasis infection. Information resources WHO/TDR Genome Projects, including the Leishmania Genome Project (Viewed August 2003). In the section Genomes on the web, all data are stored in databases. This information provides the building blocks for functional and structural analyses, which can be performed by researchers all over the world. Leishmania major GeneDB (Viewed August 2003). The genome of L. major Friedlin, the reference strain, is being sequenced as part of a multicenter collaboration. Extrapolating from the currently available data, it is expected that the approximate 8000 genes will be identified in a 33.6-Mb haploid genome spread over 36 chromosomes. As both sequencing and annotation are in progress, the data are continually updated. The Leishmania major Friedlin Genome Project (Viewed August 2003). Information on the Leishmania Genome Network (LGN) can also be obtained in the Sanger Institute Genome Sequencing Site, 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. 96 Expert Rev. Mol. Diagn. 3(5), (2003)

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