Applications of molecular methods for Leishmania control

Review

10.1586/14737159.5.2.251 © 2005 Future Drugs Ltd ISSN 1473-7159 251www.future-drugs.com

Applications of molecular methods for Leishmania controlSarman Singh†, Ayan Dey and Ramu Sivakumar

†Author for correspondenceAll India Institute of Medical Sciences, Division of Clinical Microbiology, New Delhi-110029, IndiaTel.: +91 112 658 8484Fax: +91 112 658 [email protected]

KEYWORDS: drug targeting, gene expression profiling, gene sequencing, Leishmania, microarrays, PCR, recombinant antigens, vaccine

This article reviews the recent advances made in the field of human leishmaniasis. Special emphasis is placed upon the application of various molecular tools for accurate and rapid diagnosis, understanding the mechanisms of drug resistance and identification of vaccine candidates. The focus will be on the major role played by recombinant antigens in the immunoserodiagnosis and progress of the Leishmania genome project, which has enabled researchers to design better PCR primers and molecular probes for microarrays. A special interest is placed on the recombinant antigen (rK39) cloned from the Leishmania chagasi kinesin gene and a very recently cloned recombinant antigen (KE16) from the Old World Leishmania donovani species with high sensitivity and specificity. Advances made in the specific PCR primer designed to diagnose and differentiate various species and strains of Leishmania causing visceral and post-kala-azar-dermal leishmaniasis have been covered. Molecular methods (e.g., DNA and protein microarrays) applied to understanding the pathobiology of the parasite, mechanism of host invasion, drug interaction and drug resistance to develop effective therapeutic molecules, gene expression profiling studies that have opened doors to understand many host–parasite relations, effective therapy and vaccine candidates are extensively covered in this review.

Expert Rev. Mol. Diagn. 5(2), 251–265 (2005)

Leishmaniasis is a parasitic disease caused by ahemoflagellate Leishmania. More than 21 speciescause human infection. The disease manifestsmainly in three predominant forms; visceral,cutaneous and mucocutaneous leishmaniasis(MVL) [1,2]. Approximately 50 species of thefemale sandflies of genus Phlebotomous and sixsubgenera in the Old World [3] and genus Lut-zomiya in the New World [4] are suspected orproven vectors that transmit parasites from ani-mal to animal, animal to man and man to man.On rare occasions, transmissions also occur con-genitally or as a result of blood transfusions. Atotal of approximately 21 Leishmania specieshave been identified to be pathogenic to human.Leishmania are one of several genera within thefamily Trypanosomatidae and are characterizedby the possession of kinetoplast (k)DNA, aunique form of mitochondrial DNA [3,4].

The disease is endemic in 88 countries onfive continents with a total of 350 million peo-ple at risk. In fact, according to the WorldHealth Organization (WHO), 12 million cases

of leishmaniasis occur worldwide. Diseases inhumans occur in three predominant forms witha broad range of clinical manifestations [1–3].Though all forms can have devastating conse-quences, visceral leishmaniasis (VL), also knownas kala-azar (KA), is the most severe form of thedisease, which if untreated, has a mortality rateof almost 100%. KA was first described in Indiain 1903 and the manifestations of this diseasehad been known by various other names in the19th century [5]. In the 1950s, KA disappearedfrom almost all parts of India due to effectivedichlorodiphenyltrichloroethane (DDT) sprays.However, in 1974 the disease unfortunatelyreappeared in Bihar state. Since then, the diseasehas become rampant and endemic with a shift intrend from East to West. It is now localized toEastern parts of India and no new cases of VL arereported from Southern and Western India [6].

Of all cases of VL in the world, 90% casesoccur in India, Bangladesh, Sudan and Nepal[1,2]. Another major source of Old World VLis the Mediterranean region (mainly Spain,

CONTENTS

Leishmaniasis in AIDS patients

Diagnosis

PCR

Nucleic acid sequence-based amplification

Proteomics & proteogenomics

Expert opinion

Five-year view

Information resources

Key issues

References

Affiliations

http://www.future-drugs.com

Singh, Dey & Sivakumar

252 Expert Rev. Mol. Diagn. 5(2), (2005)

Portugal, France and Italy). Both sexes are vulnerable; how-ever, males are predominantly susceptible to disease due togreater exposure to sandfly biting. The disease is a poor man’sdisease and more common in socially backward communitiesdue to poor nutrition and increased exposure to infection.Young children are more vulnerable and suffer severe diseasemanifestations due to malnutrition and weak immune defensesystems [7]. The disease is characterized by irregular bouts offever, substantial weight loss, swelling of the spleen and liver,and anemia. It is primarily caused by three species, all groupedunder the Leishmania donovani complex. This complex con-sists of L. donovani, which causes visceral disease in the Indiansubcontinent, oriental China, Sudan and Kenya. Leishmaniainfantum is the causative agent of a similar disease in Centraland Southern Asia, Northeastern China, North Africa andMediterranean Europe, while Leishmania chagasi causes Amer-ican visceral leishmaniasis (AVL), or New World VL. Thereare also reports of VL caused by Leishmania amazonensis in aparticular region of the Bahia state of Brazil [4]. Regarding thetaxonomic position of three visceralizing species, opinion isdivided on the separate identity of L. infantum. One schoolstrongly feels that L. chagasi is in fact L. infantum [8], whichwas introduced to the New World in the post-Columbian eravia domestic dogs. However, the other group does not agreewith this hypothesis and believes that the two species differgenetically and phenotypically [4].

Cutaneous leishmaniasis (CL), which is also known as orientalsore, delhi boil or baghdad boil in the Old World and chicleroulcer or chiclero’s ear if it involves the ear (common in NewWorld). Approximately 90% of all cases of VL occur in Afghan-istan, Brazil, Peru, Iran, Iraq, Saudi Arabia, Syria, Pakistan andparts of India, with 1–1.5 million new cases reported annuallyworldwide [1,2,4,9]. Recently, an outbreak of CL occurred inAfghanistan affecting more than 200,000 persons and an esti-mated 67,500 cases were in Kabul only, the largest centre of CLin the world [101]. Recently, a very unusually cold VL climaticregion of sub-Himalayan India had a minor outbreak of CL [10].It is primarily caused by Leishmania tropica (West and CentralAsia), Leishmania major (Palaearctic and Aethiopian regions ofAfrica, Iran, Iraq, some parts of Russia and Afghanistan, Paki-stan and Northwest India) and Leishmania aethiopica (EastAfrica, Ethiopia and Kenya). Leishmania klilicki has also occa-sionally been reported to cause Old World VL. New World CLis prevalent from Southern USA to Northern Argentina and atleast 14 species belong to two subgenera Viannia and Leishma-nia (L. mexicana and L. amazonensis) in New World. [11]. Thedisease is usually characterized by large numbers of skin ulcers,as many as 200 in some cases, on the exposed parts of the bodysuch as the face, arms and legs. This causes serious disability andmay leave the patient permanently scarred. Natural resolutionleads to partial resistance to reinfection [3,4,11].

MVL, or American leishmaniasis or espundia (also referred toulcera de Baura, ferida seca bouba, buba), produces lesions thatcan lead to extensive and disfiguring destruction of mucousmembranes of the nose, mouth and throat cavities. The disease

is found in Mexico and South and Central Americas. Muco-cutaneous disease is a chronic inflammatory process involvingthe nasal, pharyngeal and laryngeal mucosa, which can lead toextensive tissue destruction. MVL develops as a complication ofCL as parasites disseminate from the primary cutaneous lesionvia lymphatic vessels and blood to reach the upper respiratorytract mucosa. Such metastatic spread more commonly occurswith L. (viannia) braziliensis and L. (viannia) guyanensis species.These species are present in tropical forested areas of Centraland South America. On occasions, the disease is reported intourists from nonendemic areas who have traveled to endemicareas [3,4,12].

Diffuse CL (DVL) is an anergic variant of localized CL. Thelesions of this form are disseminated, resemble lepromatousleprosy and are commonly caused by L. (mexicana) amazonensisand L. aethiopica [4]. Another form of manifestations known aspost-KA dermal leishmaniasis (PKDL) is a sequel of KA and isprimarily limited to India and Sudan. However, the VL clinicalpattern of PKDL in these two geographic regions varies signifi-cantly. It occurs in 50–60% of cases following VL in Sudan,while only 5–10% Indian VL cases will develop PKDL. Also,the interval between VL and PKDL development is0–6 months in Sudan and 2–3 years (up to 32 years) in India.In Sudan, the children are more affected while in India theadults are. This difference is not solely limited to genetic mani-festations as the L. donovani of Sudan and Indian origin havebeen found to be different [13]. The lesions of PKDL are charac-teristic but variable, ranging from hypopigmented macules toerythematous papules and from nodules to plaques [13,14].

Leishmania is a parasite that has adapted to varied andheterogenous environments, such as [14]:

• Temperature: from 37°C in mammalian hosts to ambienttemperatures in sandfly and in vitro

• pH: from neutral to highly acidic in the sandfly stomach andthe macrophage phagolysosome

• Nutrients and oxygen contents

• Attack by the immune system – complement, antibodies andT-lymphocytes

This rapid adaptation to the environment must have beendue to the ability of Leishmania to modulate gene expression.Gene (over-) expression in Leishmania most likely occurs byspecific gene amplification or through having several tandemrepeats. The best examples of tandem repeats in this parasiteinclude glycoprotein (gp)63, promastigote surface antigen(PSA)2, β-tubulin and cysteine proteinase, which are involvedin host–parasite interactions. The copy number of these locihas been reported from 20 to 80 on chromosome 10 [14]. Otherloci exist but their copy number is less, such as 39-amino acidtandem repeats in the kinesin gene (K39). The copies of theserepeats have been reported to vary in different species of Leish-mania, seven in L. chagasi and four to six in Indian L. donovani[15]. The exact functions of the expressed proteins are not yetexactly known but the antigen has been found to be highlyantigenic and is being used in diagnostics [16].

Molecular methods for Leishmania control

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Leishmaniasis in AIDS patientsLeishmania–HIV coinfection has emerged as a major complica-tion of leishmaniasis. Most of the Leishmania endemic countriesare also facing HIV epidemics, which results in a high rate ofcoinfection. Of the first 1700 cases of Leishmania–HIV coinfec-tion reported to the WHO from 33 countries up to 1998, 1440cases were from the Mediterranean region: Spain (835), Italy(229), France (259) and Portugal (117). It is very important toknow that HIV modifies the clinical presentation of leishmaniasisin coinfected patients [17]. Several atypical etiologic agents havebeen described in leishmanial syndromes affecting HIV-infectedsubjects. In HIV-associated leishmaniasis caused by nonvisceraliz-ing species, the parasite may disseminate to the reticuloendothelialsystem and various other organs, and conversely the visceralizingspecies can manifest in an atypical manner [5,17]. Fulminant pres-entation of VL is possible in patients with AIDS. Relapses are alsousual. VL is now the fourth most common opportunistic parasiticdisease in HIV-positive individuals in Spain and 20–40% caseshad absence of splenomegaly. In Africa, particularly Ethiopia andSudan, and Southern Europe, Leishmania-HIV coinfection isregarded as an emerging disease. As many as 70% of adults withVL also have HIV infection [2]. To date, 34 countries have alreadyreported Leishmania-HIV coinfection. Dissemination is commonand gastrointestinal involvement is commonest. The Leishmaniaamastigotes can be seen in gastrointestinal mucosal biopsy speci-mens. Leishmanial amastigotes are commonly found in Kaposi’ssarcoma cutaneous lesions concomitant with VL. Leishmania par-asites were recently found in herpes zoster lesions in a HIV-posi-tive patient. Leishmaniasis has also been reported presenting as adermatomyositis-like eruption in threepatients with AIDS [17].

The oldest drug developed to treatpatients was urea stibamine. This drug wasdeveloped in India in 1922; however, it hadsevere toxic effects. Its pentavalent com-pounds were later prepared, whichremained the sole treatment modality for allforms of leishmaniasis for several decadesand saved millions of lives. However,reports of unresponsiveness to the pentava-lent sodium antimony gluconate (SAG)began emerging in 1970s mainly from someparts of India. It is also feared that resistanceagainst new drugs including miltefosine willnot take long if these drugs are continued tobe given as monotherapy [5]. The main issuein Leishmania drug research has beenunderstanding the mechanism of drugresistance. To date, no gene or plasmidresponsible for drug resistance has beenfound in Leishmania. However, researchersare optimistic that the Leishmania genomeproject and functional genomics will shedlight on the mechanisms of action ofantileishmanial drugs and drug targeting.

DiagnosisThe clinical signs and symptoms of VL or CL are not patho-gnomonic. KA may be confused with other similar conditionssuch as malaria, tropical splenomegaly, schistosomiasis or cir-rhosis with portal hypertension, African trypanosomiasis, mil-liary tuberculosis, brucellosis, typhoid fever, bacterial endocar-ditis, histoplasmosis, malnutrition, lymphoma and leukemia.Similarly, numerous primary and secondary skin conditions arefrequently overdiagnosed as early lesions of CL in endemiccountries and in nonendemic countries VL is misdiagnosed asother diseases. Some of the common conditions that should bedifferentiated from CL include tropical ulcers, impetigo,infected insect bites, leprosy, lupus vulgaris, tertiary syphilis,yaws, blastomycosis and skin cancer [1,3]. Whenever there issuspicion of leishmaniasis, only the laboratory diagnosis canprovide a definite answer (FIGURE 1).

For VL or KA, several surrogate nonspecific markers are usedfor diagnosis in countries where sophisticated and specific testsare not available or where situations do not permit one to under-take invasive methods. In active visceral diseases, a significantdecrease in hemoglobin and total protein are common findings.Low total white blood cell count and high erythrocyte sedimen-tation rate with elevated liver enzymes are often used to suspectthe disease in resource-limited settings. The albumin fraction ofthe serum protein is often used as a prognostic marker. As thedisease gets cured, serum globulin decreases and albumin fractionand total serum protein increases. This also increases the albu-min/globulin ratio. The aldehyde, sia or serum water tests, whichwere used for a long time in India for KA diagnosis, are based on

Directevidence

Indirectevidence

Parasitedemonstration

Nucleicaciddetection

Antibodydetection

Microscopy

Antigendetection

In vitroculture

Animalinnoculation

Othersurrogatemarkers

Hemogram

Interleukins

TNF-α

Symptoms, signs and geographicbackground

Figure 1. Summary of the methods commonly used for diagnosing leishmaniasis. Molecular methods are described in detail in the text.TNF: Tumor necrosis factor.



the principle of high globulinemia [6]. Several interleukins andtumor necrosis factor (TNF)-α are also used as prognostic mark-ers. TNF-α is significantly raised in acute persistent cases of KA;however, levels decrease sharply after parasitologic cure [18].

Parasitologic diagnosisThe confirmatory diagnosis of leishmaniasis relies on either themicroscopic demonstration of Leishmania amastigotes in theaspirates from the tissues of relevance such as bone marrow,spleen, lymph nodes or liver, skin slit smears or biopsies [16], orin the peripheral blood buffy coat [19]. The smears can bestained with Romnowsky’s and hemotoxyline and eosine orimmunoperoxidase stains. The latter stain provided improvedsensitivity in cases of cutaneous and MVL [1,3,4]. For immuno-competent patients with VL, the best samples are thoseobtained from spleen aspirations (sensitivity >94%), liver biop-sies (sensitivity 76–90%) and bone marrow biopsies (sensitivity76–90%) [1,9,16,20]. In tissue smears the amastigotes of Leishma-nia appeared ovoid in shape and measured approximately3 × 5 µm in size. However, their size varies. The authors haveobserved that amastigotes which are short and stout, measuringas large as 4.5 × 5 µm, are resistant to SAG, while patients whoshow elongated amastigotes in their specimen are sensitive tostandard doses of SAG [UNPUBLISHED OBSERVATIONS]. This mor-phologic observation requires further study. The parasitesappear blue-purple and have a rod-shaped structure at rightangle to the nucleus, both pink in color. This rod-shaped struc-ture is an extrachromosomal DNA mass known as the kineto-plast. The parasite load on the patient can be estimated bycounting the amastigotes in the spleen or bone marrow smearsin relation to the white blood cell counts [16,20] using a logarith-mic scale from 0 (no parasites in 1000 microscopic fields) to 6+(greater than 100 parasites per microscopic field) [21]. The pro-mastigote form can also be culture isolated from these speci-mens on solid Novy-MacNeal-Nicolle medium having20–30% rabbit blood or liquid Schneider’s insect medium.

However, these procedures are invasive and difficult to performas well as being less sensitive [16]. The splenic aspirate is a moresensitive method than other tissue aspirations and the iatro-genic splenic bleeding can be minimized in experienced hands.However, despite these safety precautions, the risk of fatalbleeding is two in 10,000 patients, in spite of prior precautionsin the form of more than 40,000/µl platelet count and goodprothrombin time control measures are adopted [20]. Alter-native methods to isolate the parasite can be used. Inoculationof the clinical material obtained into either a susceptibleBALB/c mouse or a hamster footpad or nose may improve theyield. Histopathologic evaluation of biopsy samples of lesionsmay be characteristic. However, it is rarely specific enough tomake a diagnosis without identification of the amastigote.

Immunologic methods of diagnosisThe hallmark of VL is hyperimmunoglobulinemia, while incases of cutaneous and MVL, the humoral immune response isextremely poor. To exploit this host–parasite interaction for thediagnosis of VL, a number of antibody detection methods havebeen developed. Some of these tests incude indirect hemaggluti-nation, counter current immunoelectrophoresis, immunodiffu-sion and several others [6,9,16,20,22–24]. These tests are cumbersomeand lack sensitivity and specificity and hence are not commonlyused [9,16,20]. A summary of the comparative sensitivities of thesetests is shown in TABLE 1.

Fluorescent antibody test

The indirect fluorescent antibody test is one of the most com-monly used tests for antileishmanial antibody detection usingfixed promastigotes (FIGURE 2). The test is based on detecting anti-bodies, which are produced during the very early stages of infec-tion and are undetectable 6–9 months after cure. Titers above1:20 are significant and those above 1:128 are diagnostic. How-ever, the possibility of a crossreaction with trypanosomal seraexists [16]. The sensitivity of these tests is extremely variable, from

Table 1. Sensitivity and specificity of ELISA using various recombinant Leishmania proteins: rKE16, rK39, rH2A, rH2B, rGBP, rLACK, rgp63, rP20, rPSA-2-GST, rPSA-2-TRI-GST and rPSA-2-MBP, and purified Leishmania LPG. Confidence interval 95%.

Parameters Various recombinant antigens evaluated by ELISA

rKE16(%)

rK39(%)

rH2A(%)

rH2B(%)

rGBP(%)

rLACK(%)

rgp63(%)

rP20(%)

rPSA-2(%)

rPSA-2(%)

rPSA-2(%)

Purified(%)

GST TRI-GST MBP LPG

Sensitivity 100 100 100 100 97 97 86 68 47 36 57 92

Specificity 100 97 91 92 92 84 90 95 96 85 97 92

PPV 100 92 80 82 82 68 76 83 81 46 88 81

NPV 100 100 100 100 99 88 96 89 83 79 86 97

For details of the authors’ KE16 antigen refer to [201] and for other antigens see [28]. ELISA: Enzyme-linked immunosorbent assay; GST: Glutathione S-transferase; LPG: Lipophosphoglycan; NPV: Negative predictive value; PPV: Positive predictive value; PSA: Promastigote surface antigen.



as low as 28.4% [22] to 86.6% [24]. This can be overcome by usingLeishmania amastigotes as the antigen instead of the promastig-otes. To detect the antigen (amastigotes) in the tissue sections orsmears, fluorescent dye-conjugated antibodies can be used astracers. This is known as the direct fluorescent test. The directfluorescence test is more useful in the diagnosis of VL, MVL andPKDL. In lieu of fluorescence, horseradish peroxidase can beused to tag the antibody. This will not require a fluorescencemicroscope and the stained slides can be stored for a long time.

Direct agglutination test

The direct agglutination test (DAT) is a highly specific andsensitive test. It is cheap and simple to perform, making itideal for both field and laboratory use. In various studies DAThas been reported as 91–100% sensitive and 72–100% specific[9,20]. The test can be carried out on plasma and serum. For along time DAT remained the first-line diagnostic tool inresource-poor countries. The method uses whole, stained pro-mastigotes either as a suspension or in a freeze-dried form. Thefreeze-dried form is heat stable and facilitates the use of DAT inthe field [25]. However, the major disadvantage of DAT is therelatively long incubation time of 18 h and the need for serialdilutions of blood or serum. Also, the DAT has no prognosticvalue for evaluating the parasitologic cure of the disease, as thetest may remain positive for several years after cure. Recently,Schoone and coworkers have developed a fast agglutinationscreening test (FAST) for the rapid detection (<3 h) of anti-Leishmania antibodies in serum samples and on blood collectedon filter paper [26]. FAST utilizes only one serum dilution lead-ing to qualitative results. FAST offers advantages over DAT asit uses freeze-dried antigen, which gives more antigen stability,reproducibility, specificity and sensitivity.

Enzyme-linked immunosorbent assay

The enzyme-linked immunosorbant assay (ELISA) is a valuabletool and one of the most sensitive tests for the serodiagnosis ofVL. The test is useful for laboratory analysis or field applicationsand in screening a large number of samples at a rapid rate. Withadvances in automation, ELISA can be easily performed and isadaptable for use with various antigens such as whole cytoplas-mic (soluble antigen [SA]), purified antigens such as fucose-manose [27], defined, synthetic peptides and recombinant anti-gens [28]. The sensitivity and specificity of ELISA is greatly influ-enced by the antigen used. Besides the most commonly used sol-uble promastigote antigen, several antigenic molecules haverecently been reported and their negative and positive predictivevalues (NPV/PPV) were compared [28]. However, in this com-parison the reviewer had no control over the gold standards usedby the authors of the original findings. Excretory, secretory andmetabolic antigens released by L. donovani promastigotes into aprotein-free medium were used for the serodiagnosis of VL byELISA [29]. This antigen has been reported to be 100% specificand sensitive, the PPV was 99.99% and NPV was 95.45%.However, further retrospective and prospective multisite evalua-tion is required to validate these findings. Lately, a variety ofrecombinant antigens have been developed. A L. major gene Bencoding a hydrophilic protein (gene B protein, rGBP)expressed on the surface of both promastigotes and amastigotesand characterized by an amino acid repeating motif of 5.5 copiesof a 14-amino acid sequence has been identified and also shownto be expressed in L. donovani. The GBP homolog encoded byL. donovani contains up to 22 copies of a repetitive element inwhich nine out of 14 residues are completely conserved betweenthe two species. An ELISA using this antigen is reported to bespecific for L. donovani infections only [30]. Another recom-binant protein, recombinant ORFF (rORFF) of L. infantumorigin, has been developed by Raj and coworkers the for thediagnosis of VL in India [31]. The ORFF protein is encoded inthe LD1 locus of chromosome 35 of L. infantum. An ELISA-based test using this antigen (5 ng/well) was found to be highlysensitive and specific. Its sensitivity and specificity was com-pared with DAT and SA ELISA. The sensitivity of rORFFELISA was found to be significantly higher (TABLE 1). However, italso showed mild crossreactivity in 40% cases of confirmed VLcases from Turkey caused by L. major or L. tropica. Furthermore,the antigen is in an infant stage; it needs to be evaluated widelyand its utility for field diagnosis is yet to be studied. Recom-binant gp63, a major surface antigen of Leishmania, was clonedin 1988 but subsequently failed in early evaluation [16].

The recombinant antigen rK39 has been shown to be specificfor antibodies arising during VL caused by members of the L.donovani complex. It is highly sensitive and predictive for theonset of acute disease and high antibody titres have been dem-onstrated in VL patients [32]. In addition, rK39 ELISA has ahigh predictive value for detecting VL in immunocompromisedpersons (e.g., AIDS patients) [16,20]. This antigen is now com-mercially available in the form of antigen-impregnated nitro-cellulose paper strips adapted for use under field conditions.

Figure 2. Indirect fluorescence test. Note bright apple green promastigotes as seen under ultraviolet light microscopy. The promastigotes are fixed on a glass slide incubated with patients serum. The unbound antibody is then washed off. They are then probed with fluorescein isothiocyanate-conjugated antihuman antibody. If the serum contains antileishmanial antibody, the promastigotes will emit green fluorescent light while antibody-negative sera will remain colorless (nonfluorescent).



Though the rK39 strip test has been found a highly sensitiveand reliable test in India [16,20], recent reports from Sudan andother countries reveal that this antigen shows decreased sensitiv-ity and specificity. In Sudan, the rK39 ELISA test is reported tomiss 7% of parasitologically proven cases [33]. In its strip test for-mat, the sensitivity is further compromised to only 67% inSudan, 71.4% in Southern Europe and 60–90% in Brazil [33–36].Therefore, a need was felt to clone the kinesin antigen from anyOld World species of Leishmania. Furthermore, the cost of thetest per sample is significantly high due to import. Later, besidesrK39, two more recombinant proteins (rK26 and rK9) havebeen cloned from the L. chagasi kinesin gene [37]. A significantdifference between K9 and K26 is the presence of 11 copies of a14-amino acid repeat in the open reading frame of k26. Theregion flanking the repeats of k26 shares 69% identity with theopen reading frame of k9. However, none of these antigens werefound useful on Indian sera [UNPUBLISHED OBSERVATIONS]. Veryrecently, the authors have cloned and characterized a recom-binant antigen from an Indian isolate of L. donovani strainKE16. The antigen (rKE16) is found to be 100% sensitive andspecific compared with the 98% sensitivity of the rK39 antigenfor the diagnosis of Indian KA and PKDL. It also showed 100%concordance with rK39 in leishmaniasis sera from China, Paki-stan and Turkey (TABLE 1). Hence, this antigen holds tremendouspotential for the serologic diagnosis of VL worldwide [201].

Immunoblotting

Serodiagnosis using immunoblotting of SAs has beenattempted and reported to be highly sensitive and specific.The band pattern can correlate with disease stages [38]. Usingcytoplasmic fractions, SAs from five Indian strains of L. dono-vani and three Pakistani L. major strains were separated bysodium dodecyl sulfate (SDS) polyacrylamide gel electro-phoresis (PAGE) and electrotransferred on nylon membrane.This was followed by western blotting with extracts fromIndian PKDL patients, which revealed an approximately72–74-kDA antigen band to be the most predominant [6,16].The commercially available electrochemiluminiscent kit (EVL,Amersham Biosciences) enhances its sensitivity several fold. Italso has an added advantage of permanent documentation.

Rapid antibody detection methods

In most Leishmania-endemic areas, resources are limited interms of poor or nonavailability of electricity, poor laboratoryset up and lack of equipment. Therefore, a need for rapid, sim-ple and easy-to-perform tests has always existed. With thatobjective, two rapid tests have been developed.

Dipstick test

A rapid, easy-to-perform and simple dipstick test has beendeveloped by Corixa Corp. and InBios International, Inc. usingrK39 [16,20]. The test is based on the immunochromatographicprinciple and uses purified rK39 antigen. The antigen is immo-bilized on the nitrocellulose membrane in a band form along-side an antihuman antibody (total) band, which serves as test

control. Serum (5–10 µl) from the patient is taken onto themiddle of the absorbent pad below the indicator. Subsequently,2–3 drops of wash buffer is applied below the blood spot toensure that the antibodies (if present in the blood) are carriedup by the siphoning action of membrane along with the buffer.The results are read after 5 min. The appearance of two pinklines (one control and one test) indicates a positive result whilethe appearance of only one line (control) indicates a negativeresult. The rK39 dipstick test (Corixa Corp.) has been shownto be highly specific (98%) and sensitive (98–100%) for thediagnosis of Indian VL and PKDL [16,20]. However, in othercountries it has shown limited sensitivity. Its sensitivity isreported to be only 67% in Sudan, 71.4% in Southern Europeand 60–90% in Brazil [33–36]. These studies indicate that rK39is not universally acceptable as the test of choice.

Latex agglutination test

Recently, successful development of the latex agglutination testhas been reported using recombinant antigen for the diagnosis ofVL [32]. The test does not require any equipment or electricityand can be read with the naked eye. The sensitivity of the latexagglutination test was found to be 80% compared with ELISAand did not miss any clinically active KA cases. A total of 20% ofcases were missed due to low antibody levels. The latter test hashigh antibody-detection power compared with the latex aggluti-nation test, which could detect the antibodies only if their titerswere 10–4, or more in ELISA. The study reported no false-posi-tive results and the test was stable at 4oC for 9 months and atroom temperature in the field for 3 months [32].

Antigen detection

An antigen detection test would, in principle, provide a superiormeans of diagnosis of active leishmaniasis. Since antigen levels aretheoretically expected to correlate with parasite load, antigendetection may be an ideal test in immunocompromised patientswhere antibody response is very poor. The detection of antigen inthe patient’s serum is complicated due to the presence of high lev-els of antibodies, circulating immune complexes, serum amyloid,rheumatoid factor and auto-antibodies, all of which may maskimmunologically important antigenic determinants or competi-tively inhibit the binding of free antigen. Recently, a latex aggluti-nation test (KAtex, Kalon Biological Ltd) for the detection ofleishmanial antigens in the VL patient’s urine has been developed[39]. The results obtained with KAtex using samples collectedfrom different foci of VL indicate that the test performs wellregardless of the geographic origin of samples. The test had 100%specificity and sensitivity between 68 and 100%. Whether thetest has applications for the detection of asymptomatic cases ofVL and monitoring therapy is yet to be confirmed.

Diagnosis using amastigote-specific antigen

The in vivo parasitic stage of Leishmania in humans is the amas-tigote form. Antigens specific or prepared from this stage ofLeishmania donovani, which are stage specific, would be moreideal. However, due to difficulty in maintaining and culturing



the amastigote stages in bulk quantity, few studies are availablein the literature. Otherwise, the sensitivity and specificity ofcrude antigens prepared from amastigotes has not been foundto be superior to the recombinant antigen rK39 [40].

Leishmanin skin test

Delayed hypersensitivity is an important feature of cutaneousforms of human leishmaniasis and can be measured by theleishmanin test, also known as the Montenegro reaction. Nocrossreaction occurs with Chagas’ disease, although some cross-reactivity is found with cases of glandular tuberculosis and lep-romatous leprosy. The leishmanin skin test (LST) is usuallyused as an indicator of the prevalence of cutaneous and MVLin human and animal populations and for the successful cureof VL. During active KA, there will be no or negligible cell-mediated immune response [9–12]. However, the leishmaninantigen is not commercially available and no field study hasbeen carried out in the Indian subcontinent.

Despite the large number of serologic tests that are available,no serologic method can be considered as gold standard in termsof specificity. Furthermore, the serologic methods are helpfulonly in VL due to immense humoral response shown by the hostagainst visceralizing species. In contrast, serology is unhelpful forcutaneous and MVL since antibodies tend to be undetectable orpresent in low titer due to poor humoral response [11,12].

Molecular methodsMolecular biology is becoming increasingly relevant to the diag-nosis and control of infectious diseases. Information on DNAsequences has been extensively exploited for the development ofPCR-based assays for various applications in the understandingof the parasite and the disease leishmaniasis. With the advent ofnucleic acid engineering and recombinant technology, a numberof strategies have been developed to produce recombinant pro-teins for diagnostic purposes [9,16,20]. A variety of nucleic aciddetection methods targeting DNA and RNA genes have beendeveloped. However, amongst all the molecular advances, geneamplification techniques have been most rewarding as far asdiagnosis and disease management is concerned.

PCRPCR in disease diagnosisAmongst the molecular methods used for clinical diagnosis,PCR has proved to be the most sensitive and specific technique,albeit limited to tertiary care hospitals and research laborato-ries. The specificity of PCR can be adapted to specific needs bytargeting conserved regions of the gene. Gene amplificationthrough PCR has several advantages compared with traditionaltechniques. This is due to its extremely high sensitivity, rapidityand its ability to be performed with a broad range of clinicalspecimens [9]. Detection or identification of the causative agentis also possible directly from the clinical specimens. Unlike theELISA-based antibody detection methods, no host species-spe-cific reagents are required and the same reagents can be used forspecimens from humans, dogs or any other animal host [41].

Various gene targets and nucleic acids can be used in PCR. Theimportant gene targets are 18S-ribosomal RNA (rRNA), smallsubunit (SSU) rRNA, a repetitive genomic sequence of DNA,the mini-exon (spliced ladder) gene repeat, the β-tubulin generegion, gp63 gene locus, internal transcribed spacer (ITS)regions and microsatellite DNAs (e.g., maxi- and minicircles ofkDNA) [42–50].

Several studies have reported that PCR could detect para-sitemia a few weeks prior to the appearance of any clinical signsor symptoms [51–59]. Recently, Martin-Sanchez and coworkersfound 24% of asymptomatic individuals carrying LeishmaniakDNA in their blood, using PCR-ELISA [52]. It was pointedout that these individuals could be potential sources of trans-mitting the infections in the community. The authors alsofound a good correlation between the antibody titers, skin testand PCR positivity. PCR methods could also be useful wheretransfusion transmitted KA is a potential threat in multiplytransfused patients in whom serology has a limited role [53].

For the diagnosis of VL, bone marrow, lymph node aspirates,skin biopsy, skin scrape/exudates and blood samples have beenused in PCR in several studies [41–59]. The specificity of PCR onbone marrow aspirates has been reported up to 100% [52] andsensitivity 80–93.3% compared with 50–60% sensitivity ofsmear and culture examination [9]. Additionally, a modified formof PCR, such as nested PCR, proves its predictive values in diag-nosis of PKDL. In a recent study, nested PCR was positive in 27of 29 (93%) samples while only 20 of 29 (69%) samples werepositive in the primary PCR assay [54]. Using PCR methodology,it is no longer essential to undergo invasive methods such asbone marrow biopsy, splenic punctures, lymph node biopsy, liverbiopsy or collecting large volumes of blood samples. Even a fewdrops of blood on filter paper may be sufficient [42].

The chronic VL patients prove the greatest diagnostic chal-lenge and are easily misdiagnosed by clinical criteria since theyare often atypical. These patients often have low or no Leishma-nia antibodies, and thus serologic tests are not rewarding. There-fore, PCR has proved to be the most important diagnostic toolfor its diagnosis. The sensitivity of PCR in VL has been reportedas 100%. In this study, de Oliveira and coworkers could confirmdiagnosis in all 50 dogs using PCR [56]. Various types of speci-mens may be used such as skin biopsies and dermal scrapingsfrom the bottom of the ulcer, as well as exudates and syringe-sucked fluid taken from ulcerative lesions [57]. Recently, the sam-pling method has been further improved using cotton swab fordiagnosis of VL [58]. Collection of the exudates material withswabs is easy, painless and convenient for both the patients aswell as the collectors. The cotton swabs had no inhibitory effecton PCR. The authors recommend that exudates collected bycotton swab may be a good alternative to biopsy samples, partic-ularly in field conditions [58]. In MVL, PCR was capable ofdetecting parasites in 17 of 24 (71%) patients, whereas only four(17%) patients could be diagnosed by conventional techniques[59]. ELISA-based detection of PCR products further increasedsensitivity and specificity. Using this technique the percentage ofdetection was found to be 83.3% on blood samples [60].



A recent development in PCR technology, such as fluoro-genic probes and automation, takes care of nonspecific amplifi-cation, speed and interoperator variability. In this technique,two fluorogenic dyes (i.e., a reporter and quencher dye) areattached to the 5´ and 3´ ends of the probes. Real-time PCR isused qualitatively and quantitatively, as the fluorescence isdirectly proportional to the number of amplicons, or in otherwords the parasite load, in the given specimen [45,46]. The auto-mization of real-time PCR with comprehensive portable unitshas made these tools field friendly. Multiplex PCR can be usedwhen double or mixed infections are suspected, such as inAIDS patients [45].

PCR in strain identification & molecular epidemiologyPCR can also be used for species, strain and genotype identi-fication. Genetic heterogeneity within the same species oramongst various Leishmania strains has been documentedextensively utilizing various molecular methods. The conven-tional restriction fragment length polymorphism (RFLP) fol-lowed by Southern hybridization applied on nuclear orkDNA and isoenzyme analysis methods are slow, expensiveand more laborious [9,20]. Furthermore, these methodsrequire culturing of the parasite [51]. With the advances inmolecular techniques, detection or identification of the caus-ative agent is directly possible from clinical and even archivalspecimens [61]. Various modifications and manipulation ofthe PCR-amplified products can reveal valuable geneticinformation. Methods may include the enzymatic digestionof PCR products and resolution through various gradientssuch as agarose (PCR-RFLP) [62], SDS-PAGE to check thesingle strand conformational polymorphism (SSCP) [63],

Southern hybridization [64], multiplex PCR [46] or PCRsequencing [64]. The limitation of conventional PCR is that itcannot identify multiple causative species simultaneously.Recently, development of a simple, sensitive and specific one-step multiplex PCR has been reported for differentiating thethree complexes of New World Leishmania. PCR uses themulticopy spliced ladder RNA (mini-exon gene) as a target.This assay generates complex-specific products of differentsizes for L. braziliensis, L. mexicana and L. donovani. Inanother study, Leishmania strains were characterized using asingle 5´ primer and two 3´ primers combined in a singlemultiplex reaction [65,66].

In a study comparing three molecular techniques, PCR fin-gerprinting, mini-exon PCR-RFLP and PCR-SSCP, the PCR-SSCP technique was superior to the other two for the detec-tion of minor sequence variations in rRNA genes usingsequence-confirmed amplified regions analysis [67]. Schulz andcoworkers applied manipulations in fluorescence energy trans-fer to plot a melting curve analysis of three Leishmania species[64]. Using these curves, this group could differentiate thesespecies by real-time PCR. Species-specific primers fromL. donovani telomeric sequences increase specificity comparedwith kDNA-based PCR and this PCR was found species spe-cific with minimal or no crossreactivity with L. major and

L. tropica strains [47]. Marfurt and coworkers found that PCR-RFLP targeting the mini-exon sequences was a very usefultechnique for the differentiation of New and Old World Leish-mania species [49]. The assay can identify the Leishmaniadirectly from clinical samples. Mauricio and coworkers haddemonstrated the diagnostic potential of intergenic region typ-ing by restriction analysis of the ribosomal ITS and mini-exonfor visceralizing leishmaniasis [68]. In addition, the authorsfound restriction analysis of these targets a useful tool for thegenotyping and phylogenetic analysis within the L. donovanicomplex. Recently, primers developed by the authors in whichan Alu-like PCR was developed could distinguish VL fromPKDL strains of L. donovani [UNPUBLISHED OBSERVATIONS].PCR-based random amplification of polymorphic DNA(RAPD) allows the amplification of fragments of the genomewithout previous knowledge of their sequences. Since Leishma-nia present a great genetic variability and gene sequences ofmost Leishmania spp. are not known, RAPD-PCR may be avery useful tool. A single 10-mer long primer was used toamplify the genes from L. braziliensis, L. mexicana, L. infan-tum, L. tropica, L. chagasi, L. amazonensis and L. major. Themethod could differentiate all these species and also helpeddesign some primers for L. braziliensis DNA amplificationexclusively [69]. However, the RAPD method has some draw-backs: it can only be used on cultured parasites free of hostDNA, which will give false-positive results as the sequences arenot Leishmania specific. The problem of reproducibility amonglaboratories is another issue of relevance to RAPD-PCR [70,71].

PCR for detecting Leishmania–HIV coinfectionAs previously mentioned, HIV modifies the clinical presenta-tion of leishmaniasis in the coinfected patient. In SouthernEurope, people whose CD4 cell counts are less than200 cells/cmm account for more cases of HIV and leishmania-sis coinfection. Lack of anti-Leishmania antibodies is a charac-teristic feature seen in such patients [16]. The various techniquesavailable for immunologic, parasitologic and molecular diagno-sis was evaluated by Deniau and coworkers and the authorsreport that PCR is the only promising tool in Leishmania–HIVcoinfections [72]. Bossolasco and coworkers evaluated the use ofreal-time PCR for Leishmania DNA in the diagnosis and fol-low-up of patients with HIV Type 1 and Leishmania coinfec-tion [73]. These authors found that Leishmania DNA levels cor-related with the clinical course and their measurement before,during and after treatment were useful in clinical management,particularly in the highly active antiretroviral therapy era. Inanother study, Cruz and coworkers reported that Leishmania-specific nested PCR performed on blood samples was the tech-nique of choice for diagnosis, monitoring the success of treat-ment and predicting relapses in patients with Leishmania–HIVcoinfection [74]. PCR assay using the SSU rRNA gene targetwas assessed for post-therapeutic follow-up and the detection ofrelapses could be predicted in 97% of cases with peripheralblood and in 100% of cases with bone marrow samples fromHIV-infected patients [44].



PCR for determining the drug responseProphylactic vaccination is considered the best option todevelop an effective strategy in controlling leishmaniasis; how-ever, no suitable vaccine has been found thus far. Hence,chemotherapy is the only effective method to treat the disease.Pentavalent antimonials have been used as the first-line andmost cost-effective treatment of choice for more than 50 years.However, resistance or unresponsiveness to antimonials isincreasing, which is most evident in India [5]. The mechanismsunderlying drug resistance in leishmaniasis are just beginningto be understood [75]. Potential ability of real-time PCR toaccurately measure the circulating parasite load provided a valu-able tool for predicting disease outcome. Recently, Bossolascoand coworkers examined 25 Leishmania–HIV-coinfectedpatients by real-time PCR using SSU rRNA gene target [73]. Allpatients received liposomal amphotericin B and responded clin-ically. However, Leishmania DNA was cleared from peripheralblood in only one case after completion of therapy. In theremaining patients, PCR still detected low levels of circulatingparasite DNA and seven of these eight (87.5%) patients hadclinical relapse. No patient with negative PCR relapsed. Thisstudy also shows that molecular diagnosis is very promising formonitoring therapy. In another recent study, PCR was appliedto the peripheral blood samples to monitor drug effectiveness.kDNA was undetectable after 37 days of SAG treatment in94.1% patients [76].

PCR has also been used for assessing the success of treatmentin nonvisceral forms of leishmaniasis. In Brazil, scars of 32patients who were treated and clinically cured of AVL were ana-lyzed by PCR and other diagnostic methods. DNA specific forLeishmania (Viannia) was detected in scars of 30 (93.7%)patients. This study provides an insight that clinical cure ofAVL is rarely associated with sterile cure [77]. Molecular meth-ods have also helped to understand the mechanism of resist-ance. Recently, clinically SAG-resistant field isolates of L. dono-vani were studied and the results showed that mechanisms ofresistance, as seen in laboratory mutants, are not operating infield isolates. Instead, a novel gene at chromosome 9 was ampli-fied in drug-resistant parasites [78]. More information isexpected on this aspect in the next few years.

Besides all the above applications, real-time PCR has beenapplied to estimate the positivity thresholds of various in vitrodiagnostic methods. In a recent study, real-time PCR could beoptimized to a sensitivity of 0.0125 parasites/ml of blood and thecomparative sensitivity of other tests was estimated to be in therange of 18–42 parasites/ml for microscopy, 0.7–42 for cultureand 0.12–22.5 for conventional PCR [79].

Nucleic acid sequence-based amplificationAlthough PCR has certainly proved its merit in detecting Leish-mania parasites and the diagnosis of leishmaniasis, it has certainlimitations. For example, amplification of DNA from non-viable parasites may be wrongly interpreted as ongoing infec-tion. Nucleic acid sequence-based amplification (NASBA)technology is a new method in which specific mRNA

sequences are amplified even in the background of DNA.Therefore, it only provides an indication of viable parasites.The reaction is isothermal and quick (90 min). Its superior sen-sitivity has been extensively reported in other infectious diseasesincluding HIV and Plasmodium falciparum and as little as tentarget molecules in a sample can be amplified [80]. Furthermore,NASBA can be used for the accurate quantification of mRNAlevels. Quantitative analysis of mRNA levels during and afterdrug treatment could be a useful method to assess the efficacyof anti-Leishmania treatment to predict the prognosis of thedisease [80]. However, application of NASBA is yet to bereported on a wide scale for leishmaniasis.

RiboprintingRiboprinting is used for taxonomic classification of eukaryotes,which are characterized by the presence of unique and specificsmall subunits of rRNA sequences that are not found in bacteria.The technique was first applied for Entamoeba histolytica sincethese amoebae were also suspected to have several symbiotic bac-teria inside that could have resulted in false PCR results.Recently, this technique has also been applied for more preciseLeishmania spp. differentiation. RFLP profiles derived fromdigestion of PCR products of the 18S rRNA from Leishmaniaspp. yield a typical riboprint profile on gel electrophoresis thatcan vary intraspecifically. Nimri and Schallig analyzed 76 stocksof L. major and L. tropica isolated from patients with CL to assessdivergence within and between these two species [81]. This groupfound that L. major and L. tropica could be easily differentiatedfrom each other. The riboprinting results were in agreement withthe zymodeme analysis. The restriction patterns produced weresimple, reproducible and easy to interpret.

Peptide nucleic acid fluorescence in situ hybridizationFluorescence in situ hybridization (FISH) using peptide nucleicacid (PNA) probes (PNA FISH) is a novel diagnostic techniquecombining the simplicity of traditional staining procedureswith the unique performance of molecular methods to providerapid and accurate diagnosis of infectious diseases, a featurethat makes PNA FISH well suited for routine application. Thistechnique has recently been applied for diagnosing Trypano-somiasis. The assay is based on the use of PNA probes withFISH. The probes are pseudopeptides that hybridize to com-plementary nucleic acid targets (the counterpart of the sugarphosphate backbone of DNA and RNA), which is a polyamideformed by repetitive units of N-2-aminoethyl glycine units towhich nucleases are covalently attached. The samples in ques-tion are fixed at 4°C for 1 h with 1 ml of 1.5% formaldehyde-phosphate buffered saline. After centrifugation, pellets areresuspended and treated with 50% ethanol–water solution and10 µl of sample suspension is applied onto a microscope slideand dried. Genus or species-specific 18S ribosomal DNAsequences are searched and 13- to 15-nucleotide-long PNAprobes are designed and fluorescein labeled. The smears onglass slides are hybridized with PNA probes and examinedusing a fluorescence microscope. The positive samples will give



a clear and strong fluorescence signal. The authors could detecteven a single parasite in a 2-µl blood smear. Therefore, thedetection limit was reported to be 100-times more than conven-tional microscopy and matched the PCR for low cost. Advan-tages of the PNA probes include specificity, no effect of anti-genic variation and crossreactivity (as seen in direct fluorescentantibody method), and preservation of morphology [82].

The Leishmania genome projectFor designing PCR primers, probes for DNA microarrays andcloning specific recombinant antigens, prior knowledge of thetargeted gene and its functions is crucial. Therefore, it hasbecome imperative to explore the Leishmania genome and func-tions of each gene (functional genomics). To understand thesedetails, the Leishmania Genome Network (LGN) has been estab-lished [102]. The sequence data of the reference strain L. majorFriedlin [83,84] and L. infantum is now available in the GeneDBdatabase [103] and the L. major Friedlin genome project [104]. Onthe basis of information obtained from the genome project, theLeishmania genome is found to be relatively small, with an esti-mated size of 34.7 Mbp and a karyotype of 36 chromosomes. Itis estimated to encode approximately 10,000 genes [85].

The postgenomic era is expected to address the global analysisof gene expression using a battery of techniques to resolve, iden-tify, quantify and characterize proteins. The genome network willstore and interlink protein and DNA sequences and mappinginformation from the genome projects. The genome informationshall be utilized to identify the species- and stage-specific mole-cular determinants of virulence in Leishmania and new strategiesthat can be exploited to improve the existing diagnostic methods.It will also facilitate the search for new molecular diagnosticmethods, therapeutic molecules and vaccine candidates [41].

MicroarraysTraditional molecular methods work on a one gene-to-oneexperiment basis and due to the limited throughput of suchmethods, the whole picture of gene function is difficult toobtain. Rapid advances in genomic technology open the possi-bility of discovering new genes that can contribute to vaccineinitiatives or serve as targets for development of new drugs.Also, even if gene sequences are available, interpretation ofsequence information requires knowledge of RNA transcrip-tion, protein expression and metabolic pathways. DNA andprotein microarrays are genomic technologies. In DNA micro-arrays, the nucleic acid molecules representing many genes areplaced in thousands of discrete spots on a microscope slide.This allows scientists to look at many genes at once and deter-mine which ones are expressed in a particular cell type. Thus,the expression of thousands of Leishmania genes is being ana-lyzed at the same time by DNA microarrays and numerous pro-tein-coding genes have been identified that show significantlevels of differential expression between various life cycle stages.It is expected that the application of microarray technology incombination with the Leishmania genome project will allow theidentification of many new drug and vaccine targets. Recently,

some enzymatic targets such as DNA topoisomerase II havebeen identified as eminent targets for anti-Leishmania treat-ment. These enzymes are essential for cellular replication of theparasite but differences between Leishmania topoisomerase IIand human DNA topoisomerase II genes are being exploitedfor this aim [86]. The DNA microarrays may also reveal antigen-coding genes suitable for diagnostic purposes [41]. Both comple-mentary DNA (cDNA) and genomic microarrays for L. majorhave identified a number of new genes that are expressed in astage-specific fashion and preliminary results from a L. dono-vani genomic microarray also demonstrated new gene discov-ery. Very recently, using massive cDNA sequencing, proteomicsand customized computational biology approaches, researchershave isolated and identified the most abundant secreted pro-teins from the salivary glands of the sandfly Lutzomyia longi-palpis, thereby indicating the stage-specific diagnostic potentialof these techniques [87]. A microarray of Trypanosoma bruceigenomic fragments identified new genes whose expression dif-fers between the insect borne stage and the human infectiousstage of the parasite. CustomArrays™ (CombiMatrix) plat-forms have now been made commercially available that willallow the users to maintain critical research momentum andflexibility to change array contents rapidly [105]. In the nextfew years, building on this foundational work should witnessthe most exciting stage as microarrays are applied to Leishmaniaspecies to solve questions such as:

• What is the mechanism of drug resistance for various drugs?

• Why do only 2–5% of VL cases develop PKDL?

• Is it due to in vivo hybrid formation or are strains different?

• If it is due to reactivation of the visceral form, why docured patients that migrated to nonendemic countries notget reactivation?

• What are the gene regulatory mechanisms that control differ-entiation to amastigote and promastigote stages?

• Why do some species cause visceral disease and others causethe cutaneous and mucocutaneous form of disease?

• Can we expect development of genetically defined parasitesthat may have potential as live attenuated vaccines [88]

Gene expression profilingGene expression analysis or expression profiling is a new mole-cular method of determining the levels or quantity at which thetargeted gene is expressed under the given conditions. Geneexpression analysis is determined by microarrays or expressionchips in which thousands of copies of cDNA from the mRNAof the specific genes is immobilized on solid phase and the con-trol and sample DNAs are hybridized with fluorogenic probes.Following specific hybridization, specific fluorescent signals areproduced and the intensity of these signals which is directly pro-portional to the gene expression is measured [89]. These expres-sions may be stage or function specific indicating the specificrole of that gene in particular host–parasite or drug–parasiteinteractions. Quantitative imaging and data processing then



allows direct visualization of the mRNA levels. Work on devel-opment of microarray expression profiling for the Leishmaniagenome had already begun. In one study, 11,000 cDNAs wereused to generate a 11k microarray analysis of L. major. Statisticalmethods and analysis were used to apply results emerging fromthese studies to the task of identifying genes whose expressionchanges at the level of mRNA abundance [90].

Proteomics & proteogenomicsIt is widely recognized that in order to understand organisms,we must understand not only the organization of genes andmRNA expression patterns, but also the abundance, modifica-tions and interactions of the encoded proteins. This field ofresearch is known as proteomics. However, it offers some sig-nificant challenges as, unlike nucleic acids, proteins are rela-tively fickle and individualistic in their experimental needs.One approach in proteomics could be the application of gene-fusion techniques to study the regulation at the protein level.In this approach, libraries of genes fused to convenient reporterproteins (e.g., β-galactosidase or green fluorescent protein) aregenerated and then scored for expression. While most gene-fusion libraries emphasize regulation at the mRNA or tran-scriptional level, one can design fusions that only work at theprotein or translational level. Such fusions are ideal for system-atically studying expression broadly across the entire proteome.The generation of libraries of gene fusions in Leishmania posessome challenges. When DNA constructs are introduced intoLeishmania, they appear to take one of two paths: they may cir-cularize (if not already circular) and replicate autonomously asan episome, or they may integrate into homologous chromo-somal loci. Thus, one cannot generate gene-fusion librariesin vivo by simply transfecting parasites with reporter DNA cas-settes, and identifying fusions arising from numerous non-HIregion events. Transposon methodologies, recently describedby Beverly and coworkers, provide a simple method of generat-ing gene fusions independent of host recombinational pathways[90]. They have shown that it is possible to import the transposonmariner from Drosophila into Leishmania. The authors havetaken a shuttle mutagenesis approach to generating gene-fusionlibraries. In this approach, transpositions are obtained in largesegments of Leishmania genomic DNA, carried in the shuttlevector VLHYG, and then introduced into Leishmania by trans-fection. To test the feasibility of the shuttle mutagenesis system, aLeishmania cosmid bearing a 40-kb fragment of genomic DNAarising from the H region was chosen. This region of DNA isfrequently amplified in drug-resistant parasites and encodes atleast two genes, one of them implicated in drug resistance toarsenite and animonials [90]. This approach therefore helps oneto identify the drug resistant strains and thus find effective thera-peutic molecules [91]. Recently, researchers have attemptedmRNA interference approaches to study the gene functions inL. major and L. donovani but surprisingly no effect of α-tubulinsmall interfering RNA was observed in these eukaryotes. Thissuggested that Leishmania are probably naturally deficient forRNA interference activity [92].

Expert opinionA significant advancement has been made in the understand-ing of Leishmania genomics. These advances must now beapplied for rapid, sensitive and specific diagnosis of the dis-eases and for anti-Leishmanial drug design. Using molecularmethods such as PCR and cloning and expression of species-specific recombinant proteins, it is possible to diagnose thedisease specifically and even predict the disease in asympto-matic, latently infected patients several weeks before clinicalmanifestations. These tools have not remained limited tospecialized laboratories; however, simple methods such aslatex agglutination and dipstick methods developed usingrecombinant antigens have successfully been used underfield conditions. The recombinant antigens are now pre-pared from both New and Old World Leishmania and PCRprimers specific for VL and PKDL strains of L. donovanihave also been developed. Molecular methods have also ena-bled us to understand a great deal of genetic heterogeneity inall species of Leishmania. The sensitivity of PCR is unques-tionably extremely high, and it can be applied on variousclinical samples and be used under variable conditions fordiagnosis, species identification and gene sequencing. Port-able and comprehensive real-time PCR systems have becomereality and these developments will definitely have an impacton rapid and accurate disease diagnosis and management.The Leishmania genome project has helped us to understandthe genetic variation and pathophysiology of this parasite aswell as various drug targets. However, all these developmentsneed to be made cost effective and user friendly for widerapplication.

Five-year viewWith the help of the Leishmania genome project and func-tional proteomics it may be expected that within the next5–8 years, specific genes for drug resistance will be identi-fied and gene knockout experiments will help in our under-standing and development of new drugs. The next few yearsmay also be very exciting as DNA and protein microarraysare applied to Leishmania species to solve many questionssuch as:

• Basis of drug resistance

• Whether VL and PKDL strains are the same or different. Ifsimilar, under what conditions do they manifest differently?

• Regulation and differentiation to amastigote and promastig-ote stages, and development of genetically defined parasitesas live attenuated vaccine candidates

Also, we can expect a recombinant DNA vaccine to tacklethis disease soon. The experimental work for this has alreadystarted in the authors’ and other laboratories. However, withthe exception of rapid serologic methods, most of the molecularmethods are beyond the reach of scientists in areas where thisdisease is endemic due to unaffordable cost and lack of techni-cal skills required. Molecular methods such as PCR need to bemade field friendly.



Information resources

• PubMed Central www.ncbi.nih.gov/entrez/query.fcgi (Viewed February 2005)

• Google www.google.co.in/search (Viewed February 2005)

• Leishmania Genome Network www.ebi.ac.uk/parasites/leish.html (Viewed February 2005)

• Leishmania major genome project www.sanger.ac.uk/Projects/L_major (Viewed February 2005)

• World Health Organization www.who.int/topics/Leishmaniasis/en (Viewed February 2005)

• University of East London, UKhttp://homepages.uel.ac.uk/D.P.Humber/akhter/cont.htm(Viewed February 2005)

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

1 Herwaldt BL. Leishmaniasis. Lancet 354(9185), 1191–1199 (1999).

•• Comprehensive review covering parasitology, epidemiology, clinical manifestations with variations, diagnosis and treatment.

2 Desjeux P. The increase in risk factors for leishmaniasis worldwide. Trans. R. Soc. Trop. Med. Hyg. 95(3), 239–243 (2001).

3 Ashford RW, Bates PA. Leishmaniasis in the Old World. In: Topey & Wilson’s Microbiology and Microbial Infections (volume 5), ninth edition. Parasitology. Cox FEG, Kreier JP, Wakelin D (Eds), Oxford University Press, NY, USA, 215–240 (1998).

4 Lainson R, Shaw JJ. New World leishmaniasis –The Neotropical Leishmania species. In: Topey & Wilson’s Microbiology and Microbial Infections (volume 5), ninth edition. Parasitology. Cox FEG, Kreier JP, Wakelin D (Eds). Oxford University Press, NY, USA, 241–282(1998).

5 Singh S, Sivakumar R. Challenges and new discoveries in the treatment of leishmaniasis: review. J. Infect. Chemother. 10(6), 307–315 (2004).

• Provides up-to-date information on antileishmanial treatment.

6 Singh S. Diagnostic and prognostic markers of antikala-azar therapy and vaccination. In: Proceeding of V Round Table Conference Series. No. 5. Gupta S, Sood OP (Eds), Ranbaxy Science Foundation, New Delhi, India, 95–114 (1999).

7 Kafetzis DA. An overview of pediatric leishmaniasis. J. Postgrad. Med. 49, 31–38 (2003).

•• Comprehensive review on pediatric leishmaniasis.

8 Mauricio IL, Stothard JR, Miles MA. The strange case of Leishmania chagasi. Parasitol. Today 16, 188–189 (2000).

9 Tavares CAP, Fernandes AP, Melo MN. Molecular diagnosis of leishmaniasis. Expert Rev. Mol. Diagn. 3(5), 657–667 (2003).

•• Comprehensive review providing updates up to 2003.

10 Sharma RC, Mahajan VK, Sharma NC, Sharma A. A new focus of cutaneous leishmaniasis in Himachal Pradesh (India). Indian J. Dermatol. Venerol. Leprol. 69, 170–172 (2003).

11 Amaral V, Pirmez C, Goncalves A, Ferreira V, Grimaldi G Jr. Cell populations in lesions of cutaneous leishmaniasis of Leishmania (L.) amazonensis-infected rhesus macaques, Macaca mulatta. Mem. Inst. Oswaldo Cruz. 95(2), 209–216 (2000).

12 Ahluwalia S, Lawn SD, Kanagalingam J, Grant H, Lockwood DN. Mucocutaneous leishmaniasis: an imported infection among travellers to central and South America. Br. Med. J. 329(7470), 842–844 (2004).

13 Zijlstra EE, Musa AM, Khalil EA, el-Hassan IM, el-Hassan AM. Post-kala-azar-dermal-leiishmaniasis. Lancet Infect. Dis. 3(2), 87–98 (2003).

•• Concise review on post-kala-azar-dermal leishmaniasis describing various clinical forms of post-kala-azar-dermal leishmaniasis and its management.

14 Victoir K, Dujardin J-C. How to succeed in parasitic life without sex? Asking Leishmania. Trends Parasitol. 18(2), 81–85 (2002).

Key issues

• Leishmaniasis is major health problem of the Indian subcontinent, Mediterranean region and South America. Visceral leishmaniasis or kala-azar is highly fatal if untreated and 90% of visceral leishmaniasis cases occur in India, Bangladesh, Nepal and Sudan. A total of 90% of cases of nonvisceral leishmaniasis occur in South America. The recent emergence of drug-resistant strains in India has posed a challenge for researchers to find effective and less toxic medicines and to understand the mechanism of drug resistance, which is still unknown.

• Diagnosis is conventionally achieved by parasitologic demonstration of amastigotes in the tissues of relevance. However, these methods are invasive and hazardous.

• In recent years, the most promising recombinant antigens from the kinesin-related gene of Leishmania chagasi (New World) and Leishmania donovani (Old World) for antibody detection have been prepared and field evaluated with highest accuracy.

• Antigen and Leishmania nucleic acid detection methods give direct evidence of active infections and have been used in several studies. PCR-based methods for DNA and RNA detection have shown the most promising results in terms of sensitivity, rapidity, species identification and molecular epidemiology.

• However, the most important advances have been in the Leishmania genome project, microarrays and functional genomics, which are expected to solve several unanswered questions in the near future.

Dr.Sarman Singh

Note

Topley

Dr.Sarman Singh

Note

Topley



15 Sivakumar R. Studies on the kinesin related antigen gene of Leishmania donovani and its diagnostic application. All India Institute of Medical Sciences, New Delhi, India, PhD Thesis (2004).

16 Singh S, Sivakumar R. Recent advances in the diagnosis of leishmaniasis. J. Postgrad. Med. 49(1), 55–60 (2003).

17 Paredes R, Munoz J, Diaz I, Domingo P, Gurgui M, Clotet B. Leishmania in HIV Infection. J. Postgrad. Med. 49(1), 39–49 (2003).

•• Comprehensive review on Leishmania–HIV coinfection.

18 Singh S, Singh N, Handa R. High serum levels of TNF in malaria and kala-azar. J. Assoc. Physic. India 45, 526–529 (1997).

19 Liarte DB, Mendonca IL, Luz FCO et al. QBC® for the diagnosis of human and canine American visceral leishmaniasis: preliminary data. Rev. Soc. Bras. Med. Trop. 34, 577–581 (2001).

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

21 Chulay JD, Bryceson AD. Quantitation of amastigotes of Leishmania donovani in smears of splenic aspirates from patients with visceral leishmaniasis. Am. J. Trop. Med. Hyg. 32(3), 475–479 (1983).

22 Boelaert M, Rijal S, Regmi S et al. Comparative study of the effectiveness of diagnostic tests for visceral leishmaniasis. Am. J. Trop. Med. Hyg. 70(1), 72–77 (2004).

23 Schallig HD, Schoone GJ, Kroon CC, Hailu A, Chappuis F, Veeken H. Development and application of ‘simple’ diagnostic tools for visceral leishmaniasis. Med. Microbiol. Immunol. (Berl.) 190(1–2), 69–71 (2001).

24 Iqbal J, Hira PR, Saroj G et al. Imported visceral leishmaniasis: diagnostic dilemmas and comparative analysis of three assays. J. Clin. Microbiol. 40(2), 475–479 (2002).

25 Abdallah KA, Nour BY, Schallig HD et al. Evaluation of the direct agglutination test based on freeze-dried Leishmania donovani promastigotes for the serodiagnosis of visceral leishmaniasis in Sudanese patients. Trop. Med. Int. Health 9(10), 1127–1131 (2004).

26 Schoone GJ, Hailu A, Kroon CC, Nieuwenhuys JL, Schallig HD, Oskam L. A fast agglutination-screening test (FAST) for the detection of antiLeishmania antibodies. Trans. R. Soc. Trop. Med. Hyg. 95(4), 400–401 (2001).

27 Palatnik-de-Sousa CB, Gomes EM, Paraguai-de-Souza E, Palatnik M, Luz K, Borojevic R. Leishmania donovani: titration of antibodies to the fucose-mannose ligand as an aid in diagnosis and prognosis of visceral leishmaniasis. Trans. R. Soc. Trop. Med. Hyg. 89(4), 390–393 (1995).

28 Maalej IA, Chenik M, Louzir H et al. Comparative evaluation of ELISAs based on ten recombinant or purified Leishmania antigens for the serodiagnosis of Mediterranean visceral leishmaniasis. Am. J. Trop. Med. Hyg. 68(3), 312–320 (2003).

• Reports sensitivity, specificity, positive and negative predictive values of various antigens reported until 2002.

29 Martin SK, Thuita-Harun L, Adoyo-Adoyo M, Wasunna KM. A diagnostic ELISA for visceral leishmaniasis, based on antigen from media conditioned by Leishmania donovani promastigotes. Ann. Trop. Med. Parasitol. 92(5), 571–577 (1998).

30 Jensen AT, Gasim S, Moller T et al. Serodiagnosis of Leishmania donovani infections: assessment of enzyme-linked immunosorbent assays using recombinant L. donovani gene B protein (GBP) and a peptide sequence of L. donovani GBP. Trans. R. Soc. Trop. Med. Hyg. 93(2), 157–160 (1999).

31 Raj VS, Ghosh A, Dole VS, Madhubala R, Myler PJ, Stuart KD. Serodiagnosis of leishmaniasis with recombinant ORFF antigen. Am. J. Trop. Med. Hyg. 61(3), 482–487 (1999).

32 Singh S, Kumari V, Singh N. Predicting Kala-Azar disease manifestations in asymptomatic patients with latent Leishmania donovani infection by detection of antibody against recombinant K39 antigen. Clin. Diagn. Lab. Immunol. 9, 568–572 (2002).

33 Zijlstra EE, Nur Y, Desjeux P, Khalil EA, El-Hassan AM, Groen J. Diagnosing visceral leishmaniasis with the recombinant K39 strip test: experience from the Sudan. Trop. Med. Int. Health 6(2), 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(9), 669–670 (1999).

35 Schallig HD, Canto-Cavalheiro M, da Silva ES. Evaluation of the direct agglutination test and the rK39 dipstick test for the sero-diagnosis of visceral leishmaniasis. Mem. Inst. Oswaldo. Cruz. 7, 1015–1018 (2002).

36 Carvalho SF, Lemos EM, Corey R, Dietze R. Performance of recombinant K39 antigen in the diagnosis of Brazilian visceral leishmaniasis. Am. J. Trop. Med. Hyg. 68(3), 321–324 (2003).

37 Bhatia A, Daifalla NS, Jen S, Badaro R, Reed SG, Skeiky YA. Cloning, characterization and serological evaluation of K9 and K26: two related hydrophilic antigens of Leishmania chagasi. Mol. Biochem. Parasitol. 102(2), 249–261 (1999).

38 Ravindran R, Anam K, Bairagi BC et al. Characterization of immunoglobulin G and its subclass response to Indian kala-azar infection before and after chemotherapy. Infect. Immun. 72(2), 863–670 (2004).

39 Attar ZJ, Chance ML, el-Safi S et al. Latex agglutination test for the detection of urinary antigens in visceral leishmaniasis. Acta Trop. 78(1), 11–16 (2001).

40 Sreenivas G, Ansari NA, Singh R et al. Diagnosis of visceral leishmaniasis: comparative potential of amastigote antigen, recombinant antigen and PCR. Br. J. Biomed. Sci. 59(4), 218–222 (2002).

41 Schallig HD, Oskam L. Molecular biological applications in the diagnosis and control of leishmaniasis and parasite identification. Trop. Med. Int. Health 7(8), 641–651 (2002).

42 da Silva ES, Gontijo CM, Pacheco Rda S, Brazil RP. Diagnosis of human visceral leishmaniasis by PCR using blood samples spotted on filter paper. Genet. Mol. Res. 3(2), 251–257 (2004).

43 El Tai NO, El Fari M, Mauricio I et al. Leishmania donovani: intraspecific polymorphisms of Sudanese isolates revealed by PCR-based analyses and DNA sequencing. Exp. Parasitol. 97(1), 35–44 (2001).

44 Pizzuto M, Piazza M, Senese D et al. Role of PCR in diagnosis and prognosis of visceral leishmaniasis in patients co-infected with human immunodeficiency virus Type 1. J. Clin. Microbiol. 39(1), 357–361 (2001).

45 Wortman G, Sweeney C, Houng H-S et al. Rapid diagnosis of leishmaniasis by fluorogenic polymerase chain reaction. Am. J. Trop. Med. Hyg. 65, 583–587 (2001).

46 Monroy Ostria, Sanchez-Tezeda G. Molecular probes and the polymerase chain reaction for detection and typing of Leishmania species in Mexico. Trans. R. Soc. Trop. Med. Hyg. 96(Suppl. 1), S101–S104 (2002).



47 Chiurillo MA, Sachdeva M, Dole VS et al. Detection of Leishmania causing visceral leishmaniasis in the Old and New Worlds by a polymerase chain reaction assay based on telomeric sequences. Am. J. Trop. Med. Hyg. 65(5), 573–582 (2001).

48 Davila AM, Momen H. Internal-transcribed-spacer (ITS) sequences used to explore phylogenetic relationships within Leishmania Ann. Trop. Med. Parasitol. 94(6), 651–654 (2000).

49 Marfurt J, Niederwieser I, Makia ND, Beck HP, Felger I. Diagnostic genotyping of Old and New World Leishmania species by PCR-RFLP. Diagn. Microbiol. Infect. Dis. 46(2), 115–124 (2003).

50 Gangneux JP, Menotti J, Lorenzo F et al. Prospective value of PCR amplification and sequencing for diagnosis and typing of old world Leishmania infections in an area of nonendemicity. J. Clin. Microbiol. 41(4), 1419–1422 (2003).

• Reports that isoenzyme characterization and PCR-sequencing have 100% concordance.

51 Schonian G, El Fari M, Lewin S, Schweynoch C, Presber W. Molecular epidemiology and population genetics in Leishmania Med. Microbiol. Immunol. (Berl.) 190(1–2), 61–63 (2001).

52 Martin-Sanchez J, Pineda JA, Morillas-Marquez F, Garcia-Garcia JA, Acedo C, Macias J. Detection of Leishmania infantum kinetoplast DNA in peripheral blood from asymptomatic individuals at risk for parenterally transmitted infections: relationship between polymerase chain reaction results and other Leishmania infection markers. Am. J. Trop. Med. Hyg. 70(5), 545–548 (2004).

53 Otero AC, da Silva VO, Luz KG et al. Short report: occurrence of Leishmania donovani DNA in donated blood from seroreactive Brazilian blood donors. Am. J. Trop. Med. Hyg. 62(1), 128–131 (2000).

54 Gannavaram S, Ansari NA, Kataria J, Salotra P. Nested PCR assay for detection of Leishmania donovani in slit aspirates from post kala-azar dermal leishmaniasis lesions. J. Clin. Microbiol. 42, 1777–1778 (2004).

55 Strauss-Ayali D, Jaffe VL, Burshtain O, Gonen L, Baneth G. Polymerase chain reaction using noninvasively obtained samples, for the detection of Leishmania infantum DNA in dogs. J. Infect. Dis. 189(9), 1729–1733 (2004).

• Reports the high sensitivity of PCR and its field application.

56 de Oliveira CI, Bafica A, Oliveira F et al. Clinical utility of polymerase chain reaction-based detection of Leishmania in

the diagnosis of American cutaneous leishmaniasis. Clin. Infect. Dis. 37(11), E149–E153 (2003).

57 Matsumoto T, Hashiguchi Y, Gomez EA et al. Comparison of PCR results using scrape/exudate, syringe-sucked fluid and biopsy samples for diagnosis of cutaneous leishmaniasis in Ecuador. Trans. R. Soc. Trop. Med. Hyg, 93(6), 606–607 (1999).

58 Mimori T, Matsumoto T, Calvopina MH et al. Usefulness of sampling with cotton swab for PCR-diagnosis of cutaneous leishmaniasis in the New World. Acta Trop. 81(3), 197–202 (2002).

59 Pirmez C, da Silva Trajano V, Paes-Oliveira Neto M et al. Use of PCR in diagnosis of human American tegumentary leishmaniasis in Rio de Janeiro, Brazil. J. Clin. Microbiol. 37(6), 1819–1823 (1999).

60 Pinero J, Martinez E, Pacheco R et al. PCR-ELISA for diagnosis of mucocutaneous leishmaniasis. ActaTrop. 73(1), 21–29 (1999).

61 Victoir K, de Doncker S, Cabrera L et al. Direct identification of Leishmania species in biopsies from patients with American tegumentary leishmaniasis. Trans. R. Soc. Trop. Med. Hyg. 97(1), 80–87 (2003).

62 Volpini AC, Passos VM, Oliveira GC, Romanha AJ. PCR-RFLP to identify Leishmania (Viannia) braziliensis and L. (Leishmania) amazonensis causing American cutaneous leishmaniasis. Acta Trop. 90(1), 31–37 (2004).

63 el Tai NO, Osman OF, el Fari M, Presber W, Schonian G. Genetic heterogeneity of ribosomal internal transcribed spacer in clinical samples of Leishmania donovani spotted on filter paper as revealed by single strand conformation polymorphisms and sequencing. Trans. R. Soc. Trop. Med. Hyg. 94, 575–579 (2000).

64 Schulz A, Mellenthin K, Schonian G, Fleischer B, Drosten C. Detection, differentiation, and quantitation of pathogenic Leishmania organisms by a fluorescence resonance energy transfer-based real-time PCR assay. J. Clin. Microbiol. 41, 1529–1535 (2003).

•• Reports the modifications in the PCR protocol to detect, differentiate and quantitate the Leishmania.

65 Harris E, Kropp G, Belli A, Rodriguez B, Agabian N. Single-step multiplex PCR assay for characterization of New World Leishmania complexes. J. Clin. Microbiol. 36(7), 1989–1995 (1998).

66 Belli A, Rodriguez B, Aviles H, Harris E. Simplified polymerase chain reaction detection of new world Leishmania in clinical specimen of cutaneous leishmaniasis. Am. J. Trop. Med. Hyg. 58(1), 102–109 (1998).

67 Lewin S, Schonian G, El Tai N, Oskam L, Bastien P, Presber W. Strain typing in Leishmania donovani by using sequence-confirmed amplified region analysis. Int. J. Parasitol. 32(10), 1267–1276 (2002).

68 Mauricio IL, Stothard JR, Miles MA. Leishmania donovani complex: genotyping with the ribosomal internal transcribed spacer and the mini-exon. Parasitology 128(Pt 3), 263–267 (2004).

69 Martinez E, Alonso V, Quispe A et al. RAPD method useful for distinguishing Leishmania species: design of specific primers for L. braziliensis. Parasitology 127(Pt 6), 513–517 (2003).

70 Williams JG, Hanafey MK, Rafalski JA, Tingey SV. Genetic analysis using random amplified polymorphic DNA markers. Methods Enzymol. 218, 704–740 (1993).

71 Hanafi R, Barhoumi M, Ali SB. Guizani I. Molecular analyses of Old World Leishmania RAPD markers and development of a PCR assay selective for parasites of the L. donovani species complex. Exp. Parasitol. 98(2), 90–99 (2001).

72 Deniau M, Canavate C, Faraut-Gambarelli F, Marty P. The biological diagnosis of leishmaniasis in HIV-infected patients. Ann. Trop. Med. Parasitol. 297(Suppl. 1), 115–133 (2003).

73 Bossolasco S, Gaiera G, Olchini D et al. Real-time PCR assay for clinical management of human immunodeficiency virus-infected patients with visceral leishmaniasis. J. Clin. Microbiol. 41(11), 5080–5084 (2003).

74 Cruz I, Morales MA, Noguer I, Rodriguez A, Alvar J. Leishmania in discarded syringes from intravenous drug users. Lancet 359(9312), 1124–1125 (2002).

75 Croft SL. Monitoring drug resistance in leishmaniasis. Trop. Med. Intern. Health 6, 899–905 (2001).

76 Disch J, Oliveira MC, Orsini M, Rabello A. Rapid clearance of circulating Leishmania kinetoplast DNA after treatment of visceral leishmaniasis. Acta Trop. 92(3), 279–283 (2004).

77 Mendonca MG, de Brito ME, Rodrigues EH, Bandeira V, Jardim ML, Abath FG. Persistence of Leishmania parasites in scars after Clinical cure of American cutaneous leishmaniasis: is there a sterile cure? J. Infect. Dis. 189(6), 1018–1023 (2004).



78 Singh N, Singh RT, Sundar S. Novel mechanism of drug resistance in kala-azar field isolates. J. Infect. Dis. 188(4), 600–607 (2003).

79 Mary C, Faraut F, Lascombe L, Dumon H. Quantification of Leishmania infantum DNA by a real-time PCR assay with high sensitivity. J. Clin. Microbiol. 42(11), 5249–5255 (2004).

80 Schoone GJ, Oskam L, Kroon NCM, Schallig HDFH, Omar SA. Detection and quantification of Plasmodium falciparum blood samples using quantitative nucleic acid sequence-based amplification. J. Clin. Microbiol. 38, 4072–4075 (2000).

81 Nimri LF, Schallig HD. Application of riboprinting for the identification of isolates of cutaneous Leishmania spp. Parasitology 127(Pt 3), 201–215 (2003).

82 Radwanska M, Magez S, Perry-O’Keefe H et al. Direct detection and identification of African trypanosomes by fluorescence in situ hybridization with peptide nucleic acid probes. J. Clin. Microbiol. 40(11), 4295–4297 (2002).

• Reports application of peptide nucleic acid fluorescence in situ hybridization.

83 Myler PJ, Stuart KD. Recent developments from the Leishmania genome project. Curr. Opin. Microbiol. 3(4), 412–416 (2000).

84 Sunkin SM, Kiser P, Myler PJ et al. The size difference between Leishmania major Friedlin chromosome one homologues is localized to sub-telomeric repeats at one chromosomal end. Mol. Biochem. Parasit. 109, 1–15 (2000).

85 Zhou S, Kile A, Kvikstad E et al. Shotgun optical mapping of the entire Leishmania major Friedlin genome. Mol. Biochem. Parasitol. 138(1), 97–106 (2004).

86 Valenzuela JG, Garfield M, Rowton ED, Pham VM. Identification of the most abundant secreted proteins from the salivary glands of the sand fly Lutzomyia

longipalpis, vector of Leishmania chagasi. J. Exp. Biol. 207(Pt 21), 3717–3729 (2004).

87 de Sousa JM, Lareau SM, Pearson RD, Carvalho EM, Mann BJ, Jeronimo SM. Characterization of Leishmania chagasi DNA topoisomerase II: a potential chemotherapeutic target. Scand. J. Infect. Dis. 35(11–12), 826–829 (2003).

88 Duncan RC, Salotra P, Goyal N, Akopyants NS, Beverley SM, Nakhasi HL. The application of gene expression microarray technology to kinetoplastid research. Curr. Mol. Med. 4(6), 611–621 (2004).

•• Recent review on the application of functional genomics of Leishmania.

89 Brown PO, Botstein D. Exploring the new world of the genome with DNA microarrays. Nature Genet. 21(1 Suppl.), 33–37 (1999).

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

91 Radtke OA, Kiderlen AF, Kayser O, Kolodziej H. Gene expression profiles of inducible nitric oxide synthase and cytokines in Leishmania major-infected macrophage-like RAW 264.7 cells treated with gallic acid. Planta. Med. 70(10), 924–928 (2004).

92 Robinson KA, Beverley SM. Improvements in transfection efficiency and tests of RNA interference (RNAi) approaches in the protozoan parasite Leishmania. Mol. Biochem. Parasitol. 128(2), 217–228 (2003).

Websites

101 WHO www.who.int/mediacentre/releases/2004/pR55/en(Viewed February 2005)

102 Leishmania Genome Network www.ebi.ac.uk/parasites/leish.html (Viewed February 2005)

103 Leishmania major GeneDBwww.genedb.org/genedb/leish/index.jsp (Viewed February 2005)

104 Leishmania major Friedlin Genome Project www.sanger.ac.uk/Projects/L_major/(Viewed February 2005)

105 CombiMatrix www.CombiMatrix.com (Viewed February 2005)

Patent

201 Singh S, Sivakumar R. Patent titled Polypeptides for the diagnosis and therapy of leishmaniasis. PCT/IN03/00400 (2003).

•• Claiming cloning, expressing and evaluating a recombinant antigen from Leishmania donovani.

Affiliations

• Sarman Singh, MD

Head, Division of Clinical Microbiology, All India Institute of Medical Sciences, New Delhi-110029, IndiaTel.: +91 112 658 8484Fax: +91 112 658 [email protected]

• Ayan Dey, MSc

Research fellow, Division of Clinical Microbiology, All India Institute of Medical Sciences, New Delhi-110029, IndiaTel.: +91 112 658 8700 ext. 4397Fax: +91 112 658 [email protected]

• Ramu Sivakumar, PhD

The University of Kansas Medical Center, Department of Microbiology, Molecular Genetics & Immunology, 3901 Rainbow Blvd, 4004 Hixon, MSN 3029, Kansas City, KS 66160, USATel.: +1 913 588 7295Fax: +1 913 588 [email protected]

Applications of molecular methods for Leishmania control

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