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Keywords:

  • Leishmania;
  • cutaneous;
  • visceral;
  • leishmaniasis;
  • molecular biology;
  • diagnosis;
  • control

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Diagnosis
  5. Control
  6. Identification
  7. New developments
  8. Concluding remarks
  9. References

Molecular biology is increasingly relevant to the diagnosis and control of infectious diseases. Information on DNA sequences has been extensively exploited for the development of polymerase chain reaction-based assays for the diagnosis of leishmaniasis and the identification of parasite species. It has also led to the use of cloned antigen for serodiagnosis. It is expected that the sequencing of the Leishmania major genome and the genomes of other Leishmania species will enable important progress in further improving diagnosis and control. The ability to use genome data to clone and sequence genes, which, when expressed, provide antigens for vaccine development, will increase the possibilities for rational vaccine development. Moreover, DNA on its own will provide the basis for the development of DNA vaccines that may overcome some of the problems encountered with protein-based vaccines. One of the greatest threats to parasite control is the development of drug resistance in parasites. Knowing the molecular basis of drug resistance and the ability to monitor its development with sensitive and specific DNA-based assays for `resistance alleles' may aid maintaining the effectiveness of available anti-Leishmania drugs. Finally, techniques such as microarrays and nucleic acid sequence-based amplification will eventually allow rapid screening for specific parasite genotypes and assist in diagnostic and epidemiological studies.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Diagnosis
  5. Control
  6. Identification
  7. New developments
  8. Concluding remarks
  9. References

The World Health Organization (WHO) considers leishmaniasis to be one of the most important parasitic diseases with approximately 350 million people at risk of contracting the disease. Leishmaniasis has a worldwide distribution, it is endemic in at least 88 countries, and the disease occurs on all continents except Antarctica and Australia where no suitable vectors are present. There are three major clinical forms of the disease: (1) Visceral leishmaniasis (VL), affecting an estimated 500 000 people each year, is a deadly disease if left untreated. The parasites colonize the internal organs, in particular the spleen, liver, bone marrow and lymph nodes; (2) cutaneous and (3) muco-cutaneous leishmaniasis (CL and MCL, respectively) affecting approximately 1.5 million people per year. In the case of CL, the parasite is confined to the skin where it causes one or more slow-healing sores. In the case of MCL, the infection spreads to the mucosal membranes, especially those of mouth and nose, where it may cause extensive damage. More than 20 recognized Leishmania species are responsible for these diseases, each species having distinct epidemiological patterns.

Research in Leishmania has gained momentum in the last decade because of the application of molecular biology and is accelerated by the Leishmania genome project. This paper reviews some of the latest developments in the application of molecular tools in the diagnosis and control of leishmaniasis and parasite identification.

Diagnosis

  1. Top of page
  2. Abstract
  3. Introduction
  4. Diagnosis
  5. Control
  6. Identification
  7. New developments
  8. Concluding remarks
  9. References

The early diagnosis of leishmaniasis is important in order to avoid severe damage or even death of the patient. The routine diagnosis of leishmaniasis relies on either the microscopical demonstration of Leishmania amastigotes in aspirates from lymphoid tissue or liver, in slit skin smears or in peripheral blood or culturing. However, the retrieval of samples is uncomfortable for the patient and the isolation of parasites by culturing is time-consuming, difficult and expensive. Because of these limitations, a number of indirect immunological methods, such as enzyme-linked immunosorbent assay (ELISA), dipsticks and direct agglutination test (DAT), have been developed. Despite the large number of serological tests that are available, there is still no gold standard diagnostic test. This is in part because of the fact that none of the tests is 100% sensitive and specific. Moreover, the spread of Leishmania/HIV coinfection complicates the use of the serological techniques as a result of low or lack of antibody responses of these patients (WHO 2000).

Identification of parasite antigens for serodiagnosis

Western blotting techniques have been extensively used to identify antigens for the serodiagnosis of (visceral) leishmaniasis. The most promising candidate so far is a 39 amino-acid repeat from a kinesin-like protein that is predominant in Leishmania chagasi tissue amastigotes (Burns et al. 1993). The recombinant protein rK39 was initially used in the ELISA, after which it was used for the development of a rapid immunochromatographic dipstick test (Jelinek et al. 1999). Both the ELISA and the dipstick test were tested for the diagnosis of visceral leishmaniasis. Initial reports were very promising: rK39-based tests were very sensitive and specific for visceral leishmaniasis, could be used in HIV-positive patients, and antibody levels against rK39 declined rapidly after successful treatment (Burns et al. 1993; Qu et al. 1994; Houghton et al. 1998). However, more recent reports show that the dipstick test lacks sensitivity (Jelinek et al. 1999; Zijlstra et al. 2001) and specificity (Veeken 2001). Currently, there are no other widely available diagnostic tests that are based on defined, cloned Leishmania antigen(s). The DAT remains the serological test of choice, in particular, in many developing countries (Schallig et al. 2001).

Diagnosis by polymerase chain reaction (PCR)

Over the years, a number of different PCR assays has been developed for the detection of Leishmania DNA in a variety of clinical samples such as skin biopsies and smears, bone marrow and lymph node aspirates and peripheral blood. Several target sequences have been used for the PCR. Maximum sensitivity can be achieved by using multicopy sequences as the PCR target (Lachaud et al. 2002). Examples of such targets are ribosomal RNA genes, kinetoplast DNA, mini-exon-derived RNA genes and genomic repeats (Osman 1998). The specificity of the PCR can be adapted to specific needs by targeting conserved or variable regions. In this way it is possible to characterize the parasite to the level of the genus complex, species or even the individual isolate.

For diagnosis of VL, bone marrow and lymph node aspirates as well as blood samples have been evaluated. Bone marrow aspirates from parasitologically confirmed VL patients were always PCR positive in several studies (Mathis & Deplazes 1995; Andresen et al. 1997; Osman et al. 1997a). In another study, comparison of PCR on bone marrow aspirates with microscopic examination and culture for diagnosis of VL in immune-compromised patients showed that PCR exhibited a higher sensitivity (82%) than microscopy (55%) and culture (55%) (Piarroux et al. 1994). In human lymph node aspirates Leishmania DNA was detected by PCR in all six samples in one study (Andresen et al. 1997) and in 33/38 samples in another (Osman et al. 1997a). In general, PCR is a more sensitive method for the detection of Leishmania in lymph node and especially bone marrow aspirates of VL patients than microscopy and is especially useful for the confirmation of cases of suspected VL.

Because lymph node, bone marrow and splenic aspiration is painful and can even be dangerous for the patient, peripheral blood, which is easy to obtain, may be used for the initial PCR screening of people suspected of having VL. The sensitivity of PCR for the detection of Leishmania DNA in blood samples ranges from around 70% (Adhya et al. 1995; Osman et al. 1997a) to 90% (Nuzum et al. 1995) and higher (Andresen et al. 1997; Salotra et al. 2001). It must be emphasized that if PCR on blood is negative, a PCR on lymph node and/or bone marrow material should be performed, because PCR on these materials is more often positive (Osman et al. 1997a).

PCR may also be useful for the confirmation of the diagnosis in HIV/Leishmania coinfected patients. Pizzuto et al. (2001) showed that all 76 HIV/Leishmania coinfected patients were parasitaemic by PCR on peripheral blood before therapy. In another study, 15 of 20 (75%) patients were PCR positive (Campino et al. 2000).

Patients with cutaneous or muco-cutaneous leishmaniasis (CL or MCL) often have low or no Leishmania antibodies, because of the localized character of the disease, and thus serological tests are mostly negative. Therefore, PCR is an important tool for the diagnosis of CL and MCL. Six of 10 skin biopsies from patients with CL were positive in PCR (Mathis & Deplazes 1995) and Andresen et al. (1996) found that 24 of 28 (86%) samples taken from CL patients were PCR positive, whereas microscopy detected parasites in only 55% of the samples that for reasons of epidemiology, clinical aspect, LST-status and pathology were considered to be CL. Later studies essentially confirmed these early findings with PCR being positive in more than 90% of CL cases (Aviles et al. 1999; Pirmez et al. 1999). In MCL, PCR was capable of detecting parasites in 17 of 24 (71%) patients, whereas diagnosis by conventional techniques could only confirm the disease in four of 24 (17%) patients (Pirmez et al. 1999).

Also in post-kala-azar dermal leishmaniasis (PKDL) PCR has proved its value: when using slit skin smears from PKDL patients, 19 of 23 (83%) samples were PCR positive compared with only seven of 23 (30%) positive samples in microscopy (Osman et al. 1998a). In another study, 45 of 48 PKDL patients were PCR positive (Salotra et al. 2001).

Finally, PCR detection of parasite DNA in either lymph node aspirates or peripheral blood can be used as a prognostic marker for the development of relapse or PKDL after apparently successful treatment. Parasite DNA could still be detected by PCR in 80 and 40%, respectively, of the lymph node aspirates that were obtained from Sudanese VL patients either immediately after treatment (Osman et al. 1998) or at least 3 months after treatment (Osman et al. 1997b). While patients who were PCR negative remained free of signs and symptoms and were apparently cured, 36% of the patients with a positive PCR after treatment developed PKDL and 23% showed recurrence of VL symptoms with microscopic reappearance of parasites in the aspirates (Osman et al. 1998). In VL patients that relapsed after treatment, positive PCR results on peripheral blood almost always appeared before the clinical onset of disease (Lachaud et al. 2000; Pizzuto et al. 2001). Persistent infection in apparently healed scars has been reported for MCL (Delgado et al. 1996; Schubach et al. 1998). Guevara et al. (1994) reported the presence of Leishmania braziliensis in patients cured by immunotherapy or at different stages of treatment. They also found the parasite in subjects who had never suffered from leishmaniasis but who had lived in endemic areas and migrated to non-endemic regions many years earlier.

So far, only one report was published on the use of real-time PCR, namely for the quantification of Leishmania parasites in mouse liver (Bretagne et al. 2001). In the near future, this technique may play an increasingly important role in the quantification of promastigotes or amastigotes present in clinical samples and in the (simultaneous) identification of the infecting species or strain.

It can be concluded that PCR is capable of detecting the Leishmania parasite in a variety of clinical samples and for all clinical manifestations of the disease. PCR has caused a revolution in the diagnosis of leishmaniasis. However, one has to keep in mind that the execution of this very sensitive technique requires precautions:

• the risk of contamination necessitates dedicated laboratory areas for mix preparation, sample preparation and amplification/detection;

• false positive results can be further prevented by using the uracil nucleotide glycosylase/dUridine tri-phosphate (UNG/dUTP system) (Longo et al. 1990);

• appropriate and sufficient positive and negative controls need to be included in each experiment to confirm the sensitivity and specificity of the technique.

Another important issue that, so far, has hardly been addressed is the standardization of PCR technology for the diagnosis of leishmaniasis. Most laboratories use `in house' PCR methods that are based on different primer pairs and DNA targets; the Leishmania PCR does not exist (Lachaud et al. 2002). There are only few comparative studies available in the literature (Meredith et al. 1993; Reithinger et al. 2000; Lachaud et al. 2002) and it would be very valuable to establish a universal PCR for the diagnosis of leishmaniasis.

Nucleic acid sequence-based amplification (NASBA)

Although PCR certainly has proved its merit in detecting Leishmania parasites and the diagnosis of leishmaniasis, a disadvantage of this technique is the fact that it is based on the detection of parasite DNA, which may be present a long time after the parasite has been cleared. NASBA technology, for the amplification of specific RNA sequences, has proven to be a very sensitive and specific assay in diagnostic microbiology (Compton 1991). Tests have been developed for HIV, human papillomavirus, mycobacteria and Plasmodium falciparum. The technique has not yet been developed for leishmaniasis. NASBA has several advantages over PCR: it detects RNA in a background of DNA and may thus serve to measure viable parasites; the NASBA reaction is isothermal (a thermo-cycler is not required) and quick (90 min); it is specific and sensitive, as little as 10–100 target molecules in a sample can be amplified. Moreover, NASBA can be used for the accurate quantification of RNA levels, which allows the accurate quantification (i.e. determining the actual number) of the infectious agent (Schoone et al. 2000). Quantitative analysis of RNA levels after drug treatment could be a useful method to assess the efficacy of anti-Leishmania treatment.

Despite these advantages, NASBA is not widely used. This not only holds true for leishmaniasis, but with the exception of HIV/AIDS, for most other diseases where its value was proved (such as malaria, tuberculosis and leprosy) as well. This is probably because of the fact that PCR and RT-PCR already fill the niche where NASBA could be of value.

Control

  1. Top of page
  2. Abstract
  3. Introduction
  4. Diagnosis
  5. Control
  6. Identification
  7. New developments
  8. Concluding remarks
  9. References

Vaccination is considered to be the best option to develop an effective strategy in controlling leishmaniasis (Tesh 1995; Dye 1996). In the past, numerous antigens have been tested with variable success for protection against CL in in vitro and mouse models (Handman 2001), but there are currently no (commercially) available effective vaccines against Leishmania in routine use anywhere in the world. However, the general opinion prevails that a vaccine against leishmaniasis is feasible (Handman 2001). Furthermore, vaccines have the additional advantage of avoiding the problems encountered with drug resistance. The latter is an increasing problem in the control of leishmaniasis. The mechanisms underlying resistance are being elucidated and may result in the development of molecular tools for monitoring the development of drug resistance.

Vaccine development

Leishmaniasis control would greatly benefit from the development of an effective vaccine. Traditionally, vaccine development has mainly been focused on cutaneous leishmaniasis, as cured individuals are protected from further disease. However, despite many efforts, as recently extensively reviewed by Handman (2001), to date there are no effective vaccines against any form of leishmaniasis available. The development of vaccines has been hampered by several factors: the complex life cycle of Leishmania involving at least two hosts; the antigenic diversity of the parasite makes it difficult to identify potential protective antigens; experimental animal models used to evaluate potential vaccines are not representative for disease in humans and the lack of sufficient financial support to develop a vaccine for a relatively poor part of the world population. Notwithstanding these difficulties, several antigens have been identified that may be potential vaccine candidates and molecular biological techniques have made them available as recombinant antigens for second generation vaccines.

Recombinant antigens

It is outside the scope of this review to extensively present all potential protective recombinant antigens. However, recently the UNDP/WORLD BANK/WHO Special Programme for Research and Training in Tropical Diseases (TDR) has organized a comparative study of several leading recombinant antigens as part of an effort to systematically test candidate antigens as potential vaccine candidates. A report (TDR/PRD/LEISH/VAC/01.1) on this study can be downloaded from: www.who.int/tdr/publications/publications/leishmaniasis-vaccines.htm.

The study comprised different recombinant L. major, L. braziliensis and L. mexicana antigens expressed in Escherichia coli either as a single recombinant protein or as a mixture. The evaluation consisted of: (1) testing the antigens for human peripheral blood mononuclear cell (PBMC) proliferation and (2) challenge experiments in immunized mice. Mice were immunized with two doses of 25 μg of recombinant antigen plus either recombinant interleukin 12 (rIL-12) or monophospholipid A as adjuvant.

Peripheral blood mononuclear cell stimulation experiments showed that most antigens induced some proliferative responses and interferon-gamma production, suggesting that the selected antigens have some degree of immunogenicity in humans. In contrast, none of the antigens produced significant protection in the mouse challenge experiments. These antigens were either used as a single recombinant protein, i.e. LACK (Leishmania homologueue of receptors for activated C kinase) and 4H6 (a L. major antigen), or as a mixture containing several antigens like heat shock proteins of L. braziliensis, L. major leishmanolysin (GP63) and LACK. Some of the tested antigens, irrespective of which type of adjuvant was used, showed some protection against L. major challenge in BALB/c mice in one laboratory, but not in another. Because of these conflicting results it is difficult to draw definitive conclusions from these experiments and the usefulness of these antigens as a vaccine in humans remains to be elucidated.

DNA vaccination

DNA vaccination may be a new approach for preventing infectious diseases for which conventional vaccines have failed (Ulmer et al. 1996; Wahren 1996). In DNA vaccination plasmid DNA containing the gene encoding the vaccine candidate and necessary regulatory elements to express it is introduced into the tissue via intramuscular injection or particle bombardment. Once the DNA reaches the tissue, it is taken up by cells and translocated to the nucleus, where it is transcribed into RNA and subsequently translated in the cytoplasm. Although the uptake and expression in DNA must be very low, the antigen is expressed in enough quantity to induce a potent and specific immune response and to confer protection against further infections (Hassan et al. 1999). The effectiveness of DNA vaccines against infectious agents has been demonstrated in numerous animal models, including a murine model for leishmaniasis in which the effectiveness of a GP63-based DNA vaccine was evaluated (Walker et al. 1998). In addition, vaccination with DNA encoding LACK afforded protection to mice against infection with L. major (Gurunathan et al. 1997). The amino acid sequence of the Leishmania LACK antigen is highly conserved, but the efficacy of this vaccine antigen in preventing disease caused by strains other than L. major could not be demonstrated. DNA vaccination studies with a L. donovani LACK DNA vaccine, although inducing a robust parasite-specific Th1 immune response (IFN-gamma but not IL-4 production) and priming for an in vivo T-cell response to inoculated parasites, did not induce protection against cutaneous or systemic L. donovani challenge in mice (Melby et al. 2001).

DNA vaccination certainly holds great promise for the future of vaccine development against infectious diseases, especially in developing countries. Such vaccines are attractive because they generate appropriate immune responses, ensure correct folding of the antigen, do not require adjuvants, are simple to produce, which may make them affordable and stable and do not require a strict cold chain for distribution (Handman 2001). Furthermore, DNA vaccines are most likely to induce long-term cellular immunity (Gurunathan et al. 2000). However, transition of DNA vaccines from the laboratory to clinical studies in humans raises important safety issues, such as the integration of the vaccine DNA in the human genome and the induction of auto-immune disease, which must be appropriately addressed before any DNA vaccine becomes reality (Klinman et al. 2000; Mor & Eliza 2001).

Drug resistance

Because of the fact that an effective vaccine against leishmaniasis is not available, chemotherapy is the only effective way to treat all forms of disease. Throughout the world, pentavalent antimony containing drugs have been the first-line treatment of choice for more than 50 years. However, the use of these drugs is becoming limited by the emergence of drug resistance. Primary resistance or unresponsiveness to antimonials is found in approximately 1% of all previously untreated patients (Ben Salah et al. 2000); and in some cases relapse after initial response occurs (secondary resistance or unresponsiveness). The occurrence of drug resistant Leishmania strains is increasing, which is most evident in India, where in hyperendemic districts of north Bihar, 50–65% of the patients fail to respond to treatment with pentavalent antimonial compounds (Sundar et al. 2000). Important reasons for this increase are rampant use of subtherapeutic doses, incomplete duration of treatment and substandard drugs (Sundar 2001).

Understanding drug resistance is crucial for the future control of leishmaniasis. In contrast to the malaria situation, where for example point mutations in two genes (dihydrofolate reductase and dihydropteroate synthetase) confer the occurrence of resistance to the antimalaria drug sulfadoxine-pyrimethamine (Fansidar), the mechanisms underlying drug resistance in leishmaniasis are just beginning to be understood (Croft 2001; Ouellette 2001). This hampers the development of molecular probes or PCR-based diagnostics to monitor the development and spread of antileishmanial drug resistance.

Recent insights suggest that drug resistance comprises at least the following features. Firstly, the active form of pentavalent antimony is the trivalent form of the metal. The reduction of the pentavalent compound to a trivalent metal may take place either in the host macrophage (Sereno et al. 1998) or in the parasite (Shaked-Mishan et al. 2000). If the latter is the case, reduction or loss of reductase activity may lead to drug resistance. This hypothesis is supported by the observation that pentavalent antimony resistant L. donovani amastigotes lost their reductase activity (Shaked-Mishan et al. 2000). Secondly, increased levels of trypanothione (TSH), the major reduced thiol of Leishmania cells, are found in cells selected for resistance to trivalent antimony (Haimeur et al. 2000). This increase is caused by amplification of the γ-glutamylcysteine synthase (GHS1) gene (Grondin et al. 1997) and overexpression of the ornithine decarboxylase (ODC) gene (Haimeur et al. 1999), which are both involved in TSH biosynthesis. It is possible to reverse antimonial drug resistance by using TSH biosynthesis inhibitors. On the other hand, however, increasing TSH biosynthesis in wild type cells by transfecting GSH1 or ODC does not lead to resistance, suggesting that high TSH levels are essential but not sufficient for resistance (Grondin et al. 1997; Haimeur et al. 1999). Thirdly, transport experiments indicate that a high level of drug resistance is mediated by an active extrusion system, an ATP-dependent transport system in membrane vesicles of L. tarentolae (Dey et al. 1996). Independently of this, the Leishmania ATP-binding cassette (ABC) transporter p-glycoprotein A (PGPA) is also involved in metal resistance, although the exact mechanism by which PGPA confers resistance to antimonials is not fully known (Ouellette et al. 1990). Co-transfection experiments, transport assays, and the use of inhibitors suggest that PGPA recognizes metals conjugated to glutathione or TSH. Localization studies indicate that PGPA is localized in membranes that are close to the flagellar pocket, the site of endocytosis and exocytosis in this parasite (Legare et al. 2001). Transport experiments using radioactive conjugates clearly showed that PGPA recognizes and actively transports thiol–metal conjugates. These results suggest that PGPA confers resistance by sequestrating the metal–thiol conjugates (Legare et al. 2001).

Most of the molecular and biochemical mechanisms underlying drug resistance elucidated so far are determined in vitro using promastigotes. It remains to be established whether comparable mechanisms are present in the amastigote stage of the parasite. There is a long way to go to identify a suitable in vivo amastigote marker for drug resistance.

Identification

  1. Top of page
  2. Abstract
  3. Introduction
  4. Diagnosis
  5. Control
  6. Identification
  7. New developments
  8. Concluding remarks
  9. References

Leishmania transmission cycle and epidemiology depend on the particular infecting (sub)species. Therefore, exact identification of the parasite species causing the disease is important in order to design the correct control strategy and to make decisions regarding treatment strategies. Leishmania parasites have been divided into different species primarily on the basis of clinical, biological, geographical and epidemiological criteria. During the past two decades, intrinsic characteristics, such as biochemical and molecular data, have been used for the classification of Leishmania isolates. At present, isoenzyme analysis by electrophoresis is widely used for the identification of Leishmania (sub)species and up to now it still represents the reference technique for Leishmania identification. The parasites can be identified by their enzymatic profile and be grouped in taxonomic units termed zymodemes. However, isoenzyme analysis is time consuming and laborious, requires culturing and obtaining the profile of 10–20 different enzymes. Molecular methods, such as Southern hybridization and PCR-based methods have become available that are less laborious and more powerful to study variability between Leishmania species (Morales et al. 2001).

Southern blotting using DNA probes

One of the first molecular characterization methods in use was Southern blotting using DNA probes. This method is tedious as it requires cultivation of promastigotes, DNA extraction, gel electrophoresis, Southern blotting and hybridization (Van Eys et al. 1989). DNA probes for identifying Leishmania spp. generally target kinetoplast DNA (kDNA), because the kDNA minicircle molecules are present at 10 000 copies and have a variable region that differs from minicircle classes in the same network (Barker 1989). kDNA minicircle sequence probes have been developed for L. major (Smith et al. 1989), dermatropic and viscerotropic strains of L. infantum (Gramiccia et al. 1992) and L. aethiopica (Laskay et al. 1991). Recently, a new minicircle class exclusive to Leishmania (Viannia) guyanensis was identified (Rodriguez et al. 2000). This allowed the development of a specific probe, which hybridized strongly only to L. (V.) guyanensis kDNA after medium stringency washing.

Probes derived from different sequences of the nuclear DNA have also been described. A cDNA probe containing multiple copies of a 60-bp repetitive degenerate sequence isolated from L. donovani specifically hybridized only with isolates of the L. donovani complex (Howard et al. 1991). Van Eys et al. (1989, 1991) described two probes which could be used for the differentiation of Leishmania species. One probe, pDK10, can be used to distinguish the Old World CL causing species from L. donovani complex and the other, pDK20, to differentiate between all Old World Leishmania species.

PCR-based techniques for identification

Many PCR-based techniques have been developed for the identification of Leishmania species.

Species specific primers: the specificity of any given PCR assay can be adapted to specific needs by targeting conserved or variable regions in parasite DNA. In this way it is possible to characterize Leishmania to the level of the genus complex, species or even the individual isolate. For example, PCR amplification of mini-exon gene repeating units showed size and sequence variation amongst many Leishmania species (Ramos et al. 1996), especially in the non-transcribed spacer region, which is distinct in length and in sequence among different Leishmania species (Katakura et al. 1998).

Random amplified polymorphic DNA (RAPD) uses a single primer of arbitrary sequence at a low annealing temperature to produce a PCR fingerprint-banding pattern after gel electrophoresis. This method does not depend upon previous knowledge or availability of the nucleotide sequence of the target nor does it require DNA hybridization (Welsh & McCleland 1990). However, the RAPD method has one major drawback: it can only be used on cultured parasites free of contaminating host DNA, which would mask the signal from the parasite DNA (Noyes et al. 1996). A correlation between RAPD pattern and isoenzyme analysis results was observed among different Leishmania isolates using six different arbitrary primers (Tibayrenc et al. 1993). Four different, closely related Leishmania spp. from the Viannia group were used to evaluate 28 different RAPD primers. Thirteen of these primers yielded reproducible multiple banding patterns of taxonomic value (Noyes et al. 1996).

Amplification of genomic DNA with primers annealing to mini- and microsatellite DNA sequences yielded distinctive and reproducible sets of amplified DNA fragments for all Leishmania isolates tested. The number and size of amplification products were found to be characteristic for a given species. By comparing PCR patterns of unidentified Leishmania isolates with those obtained from reference strains it is possible to identify these isolates at the species level (Schönian et al. 1996).

Restriction fragment length polymorphism (RFLP): with RFLP a PCR amplicon is digested with a set of different restriction enzymes and resulting fragments are separated according to molecular size using gel electrophoresis. For example, by digesting the amplified small subunit ribosomal RNA PCR product with the restriction enzyme RsaI, it is possible to distinguish L. donovani from L. tropica and L. major (Van Eys et al. 1992).

The GP63 locus has been successfully used for the genetic characterization of a large number of natural isolates belonging to four species of the subgenus Viannia, namely L. (V.) braziliensis, L. (V.) peruviana, L. (V.) guyanensis and L. (V.) lainsoni (Victoir et al. 1998). Cupolillo et al. (1995) has exploited the variability of the transcribed non-coding regions between the small and large subunit rRNA genes to examine relationships in the Viannia subgenus. In a method termed intergenic region typing (IRT), PCR amplification products were obtained for the rapidly evolving 1–1.2-kb internal transcribed spacers (ITS) between the SSU and LSU rRNAs, from 50 parasites isolated from different hosts and geographical areas. Amplified DNAs were cut with different restriction enzymes, and fragment patterns compared after acrylamide gel electrophoresis. High levels of intra- and interspecific variation were observed, and quantitative similarity comparisons were used to associate different lineages. A complex evolutionary tree was obtained. Some species formed tight clusters (L. equatorensis, L. panamensis, L. guyanensis, L. shawi), while L. braziliensis was highly polymorphic and L. naiffi showed intraspecific distances comparable to the largest obtained within all Viannia. L. colombiensis, L. equatorensis and L. lainsoni clearly represent distinct lineages. Good agreement was obtained with molecular trees based upon isoenzyme or mini-exon repeat sequence comparisons.

Single strand conformation polymorphism (SSCP) analysis is based on the differences in the secondary structure of single-strand DNA molecules differing in a single nucleotide, which also is frequently reflected in an alteration of their electrophoretic mobility in non-denaturing gel electrophoresis. SSCP analysis was able to detect genetic diversity at the level of a single nucleotide in a set of Sudanese L. donovani isolates (El Tai et al. 2001) and in 29 L. tropica strains (Schönian et al. 2001).

Molecular techniques and taxonomy

Molecular techniques have provided new or improved views with regard to a number of taxonomic matters:

So far no differences were found between L. infantum and L. chagasi, supporting the idea that L. chagasi and L. infantum are identical and that this species was introduced in the New World in the recent past (Mauricio et al. 2000), maybe as part of the Spanish and Portuguese conquest of Latin America.

In another matter, the question whether Leishmania is a sexually or asexually reproducing organism, the current consensus seems to be that Leishmania has a basically clonal population structure with occasional bouts of genetic exchange or hybridization (Tibayrenc & Ayala 1999).

RFLP data suggest that the Old World Leishmanias comprise a monophyletic lineage, with species associated with cutaneous disease exhibiting the greatest level of divergence (Pogue et al. 1996).

However, a number of taxonomic questions with regard to Leishmania remain open to debate. These are related to the Viannia subgenus and the taxonomic status of a number of New World complexes and have partly been addressed by Cupolillo et al. (2001).

New developments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Diagnosis
  5. Control
  6. Identification
  7. New developments
  8. Concluding remarks
  9. References

Genome sequencing project

The Leishmania genome is a relatively small eukaryotic genome with an estimated size of 33.6 Mbp with a karyotype of 36 chromosomes, ranging in size from 0.3 to 2.5 Mbp (Sunkin et al. 2000). In imitation of other genome sequencing projects, the Leishmania Genome Network (LGN) was established in Rio de Janeiro (Brazil) in 1994 and aimed at the genomic sequencing of the reference strain Leishmania major Friedlin (Ivens & Blackwell 1999). The LGN is chaired by Professor J. M. Blackwell (Cambridge, UK) and 10 laboratories from seven countries are working for this project. The LGN is a programme supported by the UNDP/WORLD BANK/WHO – TDR and its website www.ebi.ac.uk/parasites/leish.html is a important source for Leishmania sequence information, general information on parasites and their genomes and allows access to valuable bioinformatic tools via its links.

The aim of the LGN was to obtain a detailed high-resolution map of the reference strain L. major Friedlin (MHOM/IL/81/Friedlin). This has been achieved by the application of a number of complementary approaches: the determination of a pulsed field gel (PFG) chromosomal `karyotype', shuttle cosmid clone fingerprinting to generate overlapping contigs, sequencing and mapping of expressed sequence tags (ESTs) to PFG-separated chromosomes and the generation of DNA sequence from entire chromosomes. Sequencing of the whole genome is in progress. A first-generation cosmid contig map of L. major Friedlin genome has been constructed and sequencing of the genome is well under way with chromosome 1 (Chr1) and Chr2 completed and Chr4 virtually complete (Myler & Stuart 2000). Furthermore, active sequencing of Chr19, 23 and 35 is underway (LGN web site 2001).

More than 600 completely sequenced genes have been identified, an estimated 8% of the genome of approximately 8600 genes of Leishmania. Up to date, a large portion of the newly identified genes (> 60%) remains unclassified, with 40% of these being potentially Leishmania specific.

Chr1, the smallest chromosome of L. major Friedlin, consists of three homologues which differ in size by approximately 29 kb. The complete sequence of Chr1 predicts that this chromosome has 79 protein coding sequences. The first 29 of these genes are encoded in tandem on one strand of DNA, and the remaining 50 genes are encoded on the other (McDonagh et al. 2000). No RNA polymerase promoters, centromeric sequences of origins of DNA replication have been identified in the DNA sequence. Statistical analysis of Chr1 showed that the divergent junction region between the two polycistronic gene clusters may be a candidate for an origin of DNA replication (Myler et al. 2000).

In the coming years, the LGN will shift into the next phase: functional genomics and proteomics. This emerging area of research in the `post-genomic era' deals with the global analysis of gene expression using a battery of techniques to resolve (by 2D PAGE), identify (by peptide sequencing, mass spectrometry, immunoblotting, etc.), quantify and characterize proteins, as well as to store and interlink protein and DNA sequence and mapping information from the genome projects. This will aid identification of the molecular determinants of virulence in Leishmania and new strategies that can be exploited in the search for new therapies and vaccines.

Microarrays

The biological functions of only a fraction of the genes that are identified in genome projects are known. Traditional molecular biological methods work on one gene – one experiment basis and because of the limited throughput of such methods, the whole picture of gene function is difficult to obtain. In contrast, the recently developed microarray technology, in which DNA molecules representing many genes are placed in thousands of discrete spots on a microscope slide, allows scientists to look at many genes at once and to determine which ones are expressed in a particular cell type. Total mRNA from cells in two different conditions is extracted and labelled with two different fluorescent labels: for example, a green dye for cells at condition 1 (e.g. the Leishmania promastigote stage) and a red dye for cells at condition 2 (amastigote stage). Both extracts are washed over the microarray. Labelled gene products from the extracts hybridize to their complementary sequences in the spots because of base-pairing. The fluorescent dyes enable measuring the amount of sample. If the RNA from the promastigote is in abundance, the spot will be green, if the RNA from the amastigote, it will be red. If both are equal, the spot will be yellow, while if neither is present the spot will appear black. Thus, from the fluorescence intensities and colours for each spot, the relative translation levels of the genes in both samples can be estimated. More background information on DNA array technology can be found in Anthony et al. (2001), including several references to relevant websites.

It is expected that the application of microarray technology in combination with the Leishmania genome project will allow the identification of many new drug and vaccine targets. Recently, Professor J.M. Blackwell reported at the Fourth TDR/IDRI meeting on second generation vaccines against leishmaniasis on the use of microarrays to identify potential new vaccine targets. DNA microarrays were used to simultaneously monitor the expression profiles for 2183 unique Leishmania genes as the parasite undergoes developmental transition from the logarithmic promastigote to the metacyclic form, and in the host-derived amastigote form. From this analysis, more than 100 previously unknown genes were identified that are up-regulated in expression in amastigotes. These are now being tested as new vaccine candidates; some cocktails of them appear to be effective as DNA vaccines in mice.

Concluding remarks

  1. Top of page
  2. Abstract
  3. Introduction
  4. Diagnosis
  5. Control
  6. Identification
  7. New developments
  8. Concluding remarks
  9. References

The field of molecular biology is developing rapidly and genome projects hold great promise for the future development of tools that can be used for the diagnosis and control of infectious diseases. These developments must be exploited in full in Leishmania research as we are still facing major problems, e.g. lack of an effective vaccine and the development of drug resistance, in controlling leishmaniasis. In addition, new technology may become available for the diagnosis of the disease in particular for HIV/Leishmania coinfections.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Diagnosis
  5. Control
  6. Identification
  7. New developments
  8. Concluding remarks
  9. References
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