Abstract

Leishmaniasis, a neglected tropical disease prevalent in 98 countries (Alvar et al. 2012Alvar J, Velez ID, Bern C, Herrero M, Desjeux P, Cano J, Jannin J, den Boer M, WHO Leishmaniasis Control Team 2012. Leishmaniasis worldwide and global estimates of its incidence. PLoS One 1: e114.), has new emerging foci in Sri Lanka (Karunaweera et al. 2003Karunaweera ND, Pratlong F, Siriwardana HV, Ihalamulla RL, Dedet JP 2003. Sri Lankan cutaneous leishmaniasis is caused by Leishmania donovani zymodeme MON-37. Trans Roy Soc Trop Med Hyg 97: 380-381.). It has been two decades since the first endemic case of cutaneous leishmaniasis (CL) was reported from Southern Sri Lanka (Athukorale et al. 1992Athukorale DN, Seneviratne JK, Ihalamulla RL, Premaratne UN 1992. Locally acquired cutaneous leishmaniasis in Sri Lanka. J Trop Med Hyg 95: 432-433.). CL remained sporadic until the year 2000, when more CL cases were reported in soldiers engaged in the jungles in the northern part of the country (Siriwardana et al. 2003Siriwardana HV, Udagedara CU, Karunaweera ND 2003. Clinical features, risk factors and efficacy of cryotherapy in cutaneous leishmaniasis in Sri Lanka. Ceylon Med J 48: 10-12., 2010Siriwardana HV, Thalagala N, Karunaweera ND 2010. Clinical and epidemiological studies on the cutaneous leishmaniasis caused byLeishmania (Leishmania) donovani in Sri Lanka. Ann Trop Med Parasitol 104: 213-223., Karunaweera 2009Karunaweera ND 2009. Leishmania donovani causing cutaneous leishmaniasis in Sri Lanka: a wolf in sheep’s clothing? Trends Parasitol 25: 458-463.). In 2008, CL was declared a notifiable disease in the country (MS Sri Lanka 2009MS - Ministry of Health Sri Lanka 2009. Improving quality of disease surveillance. Available from: epid.gov.lk/web/pdf/wer_2009/vol_36_no_05_english.pdf.
epid.gov.lk/web/pdf/wer_2009/vol_36_no_0... ). The annual case incidence of CL rose from 674 in December 2009 to 1,365 in December 2014 (MS Sri Lanka 2010MS - Ministry of Health Sri Lanka 2010. Flashback 2009. Available from: epid.gov.lk/web/images/pdf/wer/2010/vol_37_no_01_english.pdf.
epid.gov.lk/web/images/pdf/wer/2010/vol_... ,2015aMS - Ministry of Health Sri Lanka 2015a. Flashback 2014. Available from: epid.gov.lk/web/images/pdf/wer/2015/vol_42_no_01-english.pdf.
epid.gov.lk/web/images/pdf/wer/2015/vol_... ), probably due to more efficient case detection and reporting measures, and increased numbers of infected patients (Alvar et al. 2012Alvar J, Velez ID, Bern C, Herrero M, Desjeux P, Cano J, Jannin J, den Boer M, WHO Leishmaniasis Control Team 2012. Leishmaniasis worldwide and global estimates of its incidence. PLoS One 1: e114.). The cumulative CL case incidence from January 2015-March 2015 was 154 (MS Sri Lanka 2015bMS - Ministry of Health Sri Lanka 2015b. Pneumococcal disease. Available from: epid.gov.lk/web/images/pdf/wer/2015/vol_42_no_10-english.pdf.
epid.gov.lk/web/images/pdf/wer/2015/vol_... ). Visceral leishmaniasis (VL) could be under reported or under diagnosed in the country since only three confirmed cases of endogenous VL had been reported from Sri Lanka to date (Abeygunasekara et al. 2007Abeygunasekara PH, Costa YJ, Seneviratne N, Ratnatunga N, Wijesundera M de S 2007. Locally acquired visceral leishmaniasis in Sri Lanka.Ceylon Med J 52: 30-31., Ranasinghe et al. 2012Ranasinghe S, Zhang WW, Wickremasinghe R, Abeygunasekera P, Chandrasekharan V, Athauda S, Mendis S, Hulangamuwa S, Matlashewski G, Pratlong F 2012. Leishmania donovani zymodeme MON-37 isolated from an autochthonous visceral leishmaniasis patient in Sri Lanka. Pathog Glob Health 106: 421-424.). Genetically different Leishmania donovani zymodeme MON-37 strains have been suggested to cause autochthonous CL and VL in Sri Lanka (Karunaweera et al. 2003Karunaweera ND, Pratlong F, Siriwardana HV, Ihalamulla RL, Dedet JP 2003. Sri Lankan cutaneous leishmaniasis is caused by Leishmania donovani zymodeme MON-37. Trans Roy Soc Trop Med Hyg 97: 380-381., Ranasinghe et al. 2012Ranasinghe S, Zhang WW, Wickremasinghe R, Abeygunasekera P, Chandrasekharan V, Athauda S, Mendis S, Hulangamuwa S, Matlashewski G, Pratlong F 2012. Leishmania donovani zymodeme MON-37 isolated from an autochthonous visceral leishmaniasis patient in Sri Lanka. Pathog Glob Health 106: 421-424., Zhang et al. 2014Zhang WW, Ramasamy G, McCall LI, Haydock A, Ranasinghe S, Abeygunasekara P, Sirimanna G, Wickremasinghe R, Myler P, Matlashewski G 2014. Genetic analysis of Leishmania donovani tropism using a naturally attenuated cutaneous strain. PLoS Pathog 10: e1004244.).

In spite of the Public Health importance of CL in Sri Lanka, diagnosis continues to be mostly based on clinical features and traditional diagnostic techniques, i.e., detection of amastigotes in Giemsa stained slit skin smears (SSSs), and punch biopsy and histology (Ranawaka et al. 2012Ranawaka RR, Abeygunasekara PH, Weerakoon HS 2012. Correlation of clinical, parasitological and histopathological diagnosis of cutaneous leishmaniasis in an endemic region in Sri Lanka. Ceylon Med J 57: 149-152.). However, the sensitivity of microscopic SSS and histological diagnosis from Sri Lankan patients was in the 33-45.6% range (Ranawaka et al. 2012Ranawaka RR, Abeygunasekara PH, Weerakoon HS 2012. Correlation of clinical, parasitological and histopathological diagnosis of cutaneous leishmaniasis in an endemic region in Sri Lanka. Ceylon Med J 57: 149-152.), greatly lower than the 60-70% sensitivity range reported for patients from CL foci in other countries (Al-Hucheimi et al. 2009Al-Hucheimi SN, Sultan BA, Al-Dhalimi MA 2009. A comparative study of the diagnosis of Old World cutaneous leishmaniasis in Iraq by polymerase chain reaction and microbiologic and histopathologic methods. Int J Dermatol 48: 404-408., Chouihi et al. 2009Chouihi E, Amri F, Bouslimi N, Siala E, Selmi K, Zallagua N, Ben Abdallah R, Bouratbine A, Aoun K 2009. Cultures on NNN medium for the diagnosis of leishmaniasis. Pathol Biol (Paris) 57: 219-224.). Furthermore, the reported sensitivity of in vitro cultures of Sri Lankan strains of L. donovaniwas only 40% (Ihalamulla et al. 2008Ihalamulla RL, Siriwardana HV, Karunaweera ND 2008. Efficacy of RPMI-1640 and M 199 media in the isolation of Leishmania from cutaneous lesions. Ann Trop Med Parasitol 102: 173-175.).

Different DNA-polymerase chain reaction (PCR) assays have been described to detectLeishmania in clinical samples and to determine the disease causative species (El Tai et al. 2000El Tai NO, Osman OF, el Fari M, Presber W, Schönian G 2000. Genetic heterogeneity of ribosomal internal transcribed spacer in clinical samples ofLeishmania donovani spotted on filter paper as revealed by single-strand conformation polymorphisms and sequencing. Trans R Soc Trop Med Hyg 94: 575-579., Kuhls et al. 2005Kuhls K, Mauricio IL, Pratlong F, Presber W, Schönian G 2005. Analysis of ribosomal DNA internal transcribed spacer sequences of theLeishmania donovani complex. Microbes Infect 7: 1224-1234., Mosleh et al. 2015Mosleh IM, Schönian G, Geith E, Al-Jawabreh A, Natsheh L 2015. The Jordanian Mid Jordan Valley is a classic focus of Leishmania major as revealed by RFLP of 56 isolates and 173 ITS-1-PCR-positive clinical samples. Exp Parasitol 148: 81-85., Rodríguez-Brito et al. 2015Rodríguez-Brito S, Camacho E, Mendoza M, Niño-Vega GA 2015. Differential identification of Sporothrix spp andLeishmania spp by conventional PCR and qPCR in multiplex format. Med Mycol 53: 22-27.). KDNA PCR has shown to have > 90% sensitivity and 100% specificity in detecting Leishmania DNA in clinical samples (Salotra et al. 2001Salotra P, Sreenivas G, Pogue GP, Lee N, Nakhasi HL, Ramesh V, Negi NS 2001. Development of a species-specific PCR assay for detection ofLeishmania donovani in clinical samples from patients with kala-azar and post-kala-azar dermal leishmaniasis. J Clin Microbiol 39: 849-854., Nasreen et al. 2012Nasreen SA, Hossain MA, Paul SK, Mahmud MC, Ahmed S, Ghosh S, Kobayashi N 2012. PCR-based detection of Leishmania DNA in skin samples of post kala-azar dermal leishmaniasis patients from an endemic area of Bangladesh. Jpn J Infect Dis 65: 315-317.). Internal transcribed spacer (ITS)1 PCR followed by restriction fragment length polymorphism (RFLP) analysis has also been widely used to identifyLeishmania parasite to species and subspecies level (Akhoundi et al. 2013Akhoundi M, Mohebali M, Asadi M, Mahmodi MR, Amraei K, Mirzaei A 2013. Molecular characterization of Leishmania spp in reservoir hosts in endemic foci of zoonotic cutaneous leishmaniasis in Iran. Folia Parasitol (Praha) 60: 218-224., Hajjaran et al. 2013Hajjaran H, Mohebali M, Mamishi S, Vasigheh F, Oshaghi MA, Naddaf SR, Teimouri A, Edrissian GH, Zarei Z 2013. Molecular identification and polymorphism determination of cutaneous and visceral leishmaniasis agents isolated from human and animal hosts in Iran. Biomed Res Int ID 789326: 7 pp., Mosleh et al. 2015Mosleh IM, Schönian G, Geith E, Al-Jawabreh A, Natsheh L 2015. The Jordanian Mid Jordan Valley is a classic focus of Leishmania major as revealed by RFLP of 56 isolates and 173 ITS-1-PCR-positive clinical samples. Exp Parasitol 148: 81-85.).

A spectrum of skin lesions have been reported in Sri Lankan CL patients ranging from papules, nodules, dry, and wet ulcers, to plaques (Ranawaka et al. 2012Ranawaka RR, Abeygunasekara PH, Weerakoon HS 2012. Correlation of clinical, parasitological and histopathological diagnosis of cutaneous leishmaniasis in an endemic region in Sri Lanka. Ceylon Med J 57: 149-152.). This observed clinical polymorphism raises the need to re-examine parasite contribution to clinical outcome and the existence of moreLeishmania species in addition to L. donovani in Sri Lanka. Furthermore, differential diagnosis of infective causes of chronic granulomatous skin lesions in Sri Lanka implies distinguishing between CL, lupus vulgaris, and scrofuloderma [cutaneous tuberculosis (TB)] and leprosy. However, no systematic evaluation of DNA based diagnostic tools to detectLeishmania DNA in cutaneous lesions in Sri Lanka has been reported to date. In this study, three different Leishmania DNA PCR assays were compared regarding sensitivity and specificity for identifyingLeishmania to the species level in 38 skin biopsy samples from patients from an endemic CL focus in the north-central province of Sri Lanka where cutaneous TB and leprosy are also endemic (Ranawaka et al. 2010Ranawaka RR, Abeygunasekara PH, Perera E, Weerakoon HS 2010. Clinico-histopathological correlation and the treatment response of 20 patients with cutaneous tuberculosis. Dermatol Online J 16: 13., Rathnayake et al. 2010Rathnayake D, Ranawake RR, Sirimanna G, Siriwardhane Y, Karunaweera N, De Silva R 2010. Co-infection of mucosal leishmaniasis and extra pulmonary tuberculosis in a patient with inherent immune deficiency. Int J Dermatol 49: 549-551.,Wijesinghe & Wijesinghe 2013Wijesinghe TS, Wijesinghe PR 2013. Integration of leprosy services into the General Health Service in Sri Lanka: overcoming challenges to implementation in a remote district. WHO South-East Asia J Public Health 2: 63-68., MS Sri Lanka 2014MS - Ministry of Health Sri Lanka 2014. Leprosy situation in Sri Lanka. Available from: epid.gov.lk/web/images/pdf/wer/2014/vol_41_no_40-english.pdf.
epid.gov.lk/web/images/pdf/wer/2014/vol_... ).

SUBJECTS, MATERIALS AND METHODS

Patients and ethics - Clinically suspected CL cases living or working in the north-central province of Sri Lanka who were passively reported to the dermatology clinics of the Teaching Hospital Anuradhapura and the National Hospital of Sri Lanka between January-June 2010 were included in the study. Clinical diagnosis was made by the consultant dermatologists as described (Siriwardana et al. 2003Siriwardana HV, Udagedara CU, Karunaweera ND 2003. Clinical features, risk factors and efficacy of cryotherapy in cutaneous leishmaniasis in Sri Lanka. Ceylon Med J 48: 10-12., Bari 2012Bari AU 2012. Clinical spectrum of cutaneous leishmaniasis: an overview from Pakistan. Dermatol Online J 18: 4., Ranawaka et al. 2012Ranawaka RR, Abeygunasekara PH, Weerakoon HS 2012. Correlation of clinical, parasitological and histopathological diagnosis of cutaneous leishmaniasis in an endemic region in Sri Lanka. Ceylon Med J 57: 149-152.). Ethical approval was obtained from the Ethical Review Committee (ERC), Faculty of Medical Sciences, University of Sri Jayewardenepura, Sri Lanka. Written informed consent was obtained from every literate adult participant and from either of the parents or the guardian before including a child into the study. Since informed consent was oral for illiterate participants their ERC approved thumbprint consent was used in this study. All information collected was kept under confidential cover. Any patient having foreign exposure to a leishmaniasis endemic country during any time of their life and/or who did not give written informed consent was excluded from the study.

Sample size calculation - Three pre-describedLeishmania DNA PCR assays were selected and tested for their robustness in diagnosing CL of Sri Lankan origin (Rodgers et al. 1990Rodgers MR, Popper SJ, Wirth DF 1990. Amplification of kinetoplast DNA as a tool in the detection and diagnosis of Leishmania.Exp Parasitol 71: 267-275., El Tai et al. 2000El Tai NO, Osman OF, el Fari M, Presber W, Schönian G 2000. Genetic heterogeneity of ribosomal internal transcribed spacer in clinical samples ofLeishmania donovani spotted on filter paper as revealed by single-strand conformation polymorphisms and sequencing. Trans R Soc Trop Med Hyg 94: 575-579., Salotra et al. 2001Salotra P, Sreenivas G, Pogue GP, Lee N, Nakhasi HL, Ramesh V, Negi NS 2001. Development of a species-specific PCR assay for detection ofLeishmania donovani in clinical samples from patients with kala-azar and post-kala-azar dermal leishmaniasis. J Clin Microbiol 39: 849-854.). The sample size was calculated as follows: the standard normal deviation for two-sided α with a 95% confidence level was 1.96, the estimated proportion (sensitivity) was 0.9 based on the published data (Salotra et al. 2001Salotra P, Sreenivas G, Pogue GP, Lee N, Nakhasi HL, Ramesh V, Negi NS 2001. Development of a species-specific PCR assay for detection ofLeishmania donovani in clinical samples from patients with kala-azar and post-kala-azar dermal leishmaniasis. J Clin Microbiol 39: 849-854.), and the width of confidence interval was 0.2. The calculated sample size was n = 35 (Hulley et al. 2001Hulley SB, Cummings SR, Browner WS, Grady DG, Newman TB 2001. Designing clinical research. In: Estimating sample size and power, 3rd ed., Lippincott Williams & Wilkins, Philadelphia, p. 65-71.). In this study, n = 38 samples from 38 patients were used.

SSS and punch biopsy - SSSs from 38 clinically suspected previously undiagnosed and untreated CL lesions were obtained by a clinician. The slides were stained with Giemsa and examined under a light microscope under oil immersion in the Department of Parasitology, Faculty of Medical Sciences, University of Sri Jayewardenepura. Amastigotes were identified as having round to ovoid shape and characterised by a distinctive nucleus and adjacent kinetoplast. A diameter of 2-4 mm punch biopsy samples from the active edge of the same suspected lesion, taken by a clinician under local anaesthesia and sterile conditions, were stored in Net buffer [150 mM NaCl, 15 mM Tris-HCL (pH 8.3), 1 mM ethylenediamine tetraacetic acid (EDTA)] prior to DNA extraction (Salotra et al. 2001Salotra P, Sreenivas G, Pogue GP, Lee N, Nakhasi HL, Ramesh V, Negi NS 2001. Development of a species-specific PCR assay for detection ofLeishmania donovani in clinical samples from patients with kala-azar and post-kala-azar dermal leishmaniasis. J Clin Microbiol 39: 849-854.). These punch biopsy samples were initially stored at -40°C at the Teaching Hospital Anuradhapura, transported on ice to the Department of Parasitology, Faculty of Medical Sciences, University of Sri Jayewardenepura, and stored at -80°C until DNA was extracted. Samples collected from the National Hospital of Sri Lanka were brought in Net buffer on ice and frozen at -80°C as described above.

DNA extraction from positive control culture and skin biopsy samples - An endogenous in vitro cultured CL causing strain from one of the patients was identified as L. donovani MON-37 by Sanger partial DNA sequencing of the 6-phosphogluconate dehydrogenase gene at the McGill University Genome Quebec Innovation Centre, Canada (McCall et al. 2013McCall LI, Zhang WW, Ranasinghe S, Matlashewski G 2013. Leishmanization revisited: immunization with a naturally attenuated cutaneousLeishmania donovani isolate from Sri Lanka protects against visceral leishmaniasis. Vaccine 31: 1420-1425.). This same strain was later confirmed by whole genome sequencing in a different study (Zhang et al. 2014Zhang WW, Ramasamy G, McCall LI, Haydock A, Ranasinghe S, Abeygunasekara P, Sirimanna G, Wickremasinghe R, Myler P, Matlashewski G 2014. Genetic analysis of Leishmania donovani tropism using a naturally attenuated cutaneous strain. PLoS Pathog 10: e1004244.). DNA from this pure culture (at a parasite count of 1 x 10⁶/mL) was extracted using QIAGEN DNeasy blood and tissue kits according to manufacturers’ guidelines, quantified by spectrophotometry (Thermo Fisher Scientific, USA), and used as a positive control. Two millimetre diameter samples from the punch biopsy specimens obtained from SSS positive CL patients were cut on a sterile glass slide using a sterile scalpel blade and DNA was extracted using the same DNA extraction kit.

PCR - Previously described JW11/12 primer set amplifies a 120 bp fragment of KDNA of genus Leishmania (Rodgers et al. 1990Rodgers MR, Popper SJ, Wirth DF 1990. Amplification of kinetoplast DNA as a tool in the detection and diagnosis of Leishmania.Exp Parasitol 71: 267-275.), the LdF/LdR primer set amplifies a 600 bp fragment in L. donovani species-specific KDNA (Salotra et al. 2001Salotra P, Sreenivas G, Pogue GP, Lee N, Nakhasi HL, Ramesh V, Negi NS 2001. Development of a species-specific PCR assay for detection ofLeishmania donovani in clinical samples from patients with kala-azar and post-kala-azar dermal leishmaniasis. J Clin Microbiol 39: 849-854.), and the LITSR/L5.8S set amplifies a 320 bp fragment of ITS1 region of Leishmaniagenus-specific DNA (El Tai et al. 2000El Tai NO, Osman OF, el Fari M, Presber W, Schönian G 2000. Genetic heterogeneity of ribosomal internal transcribed spacer in clinical samples ofLeishmania donovani spotted on filter paper as revealed by single-strand conformation polymorphisms and sequencing. Trans R Soc Trop Med Hyg 94: 575-579.). SSS positivity was taken as the gold standard. For each PCR assay, any SSS positive sample that became negative by first PCR was repeated three times on three consecutive days. Similarly, any sample that became positive by one set of primers and negative with a different set of primers was subjected to three repeat PCRs to conclude a negative PCR result given by the specific primer pair. Sensitivity and specificity assays were performed with all three sets of primers.

PCR conditions - A volume of 2 µL of extracted DNA from punch biopsy samples or from pure L. donovani culture was amplified with 100 pmol of each forward and reverse primers in the presence of 1.5 mM MgCl₂, 25 mM Tris-HCL (pH 9.0), 25 mM NaCl, 200 µM each deoxynucleotide triphosphate, 50 units/mL Taq DNA polymerase (Thermus acquaticus) (PCR master mix; Promega, USA) in a final volume of 10 µL. Primer sequences and relevant PCR conditions are described in the Table. A volume of 5 µL from the PCR products were run on 1-1.75% (w/v) wide range agarose gel stained with 0.2 µg/mL ethidium bromide (EtBr) (Sigma Aldrich) in 1X tris-acetate-EDTA buffer at 100 V for 45 min, visualised under ultraviolet light and images captured by a computerised gel documentation unit (Quantum ST5; Vilber Lourmat, Germany).

Sensitivity assays - Sensitivity assays were performed by carrying out PCR with the three selected sets of primers in the presence of 10-fold serial dilutions, ranging from 100 ng to 1 fg of CL in vitro culture L. donovani DNA prepared in molecular biology grade water.

Negative controls and specificity assays - Each endpoint PCR reaction contained a PCR reaction with no DNA. The selectedLeishmania primers were tested for cross amplification with five samples of each Mycobacterium tuberculosis andMycobacterium leprae, and human DNA. The M. leprae samples were extracted from smear positive leprosy skin biopsy samples and were tested with previously described M. leprae-specific primers for the presence of M. leprae DNA (Banerjee et al. 2008Banerjee S, Ray D, Bandyopadhyay D, Gupta S, Gupta S, Ghosal C, Biswas N, Bhattacharya S, Dutta RN, Bhattacharya B 2008. Development and application of a new efficient and sensitive multiplex polymerase chain reaction (PCR) in diagnosis of leprosy. J Indian Med Assoc 106: 436-440.). Pre-tested DNA samples extracted from five different M. tuberculosis in vitro cultures were kindly supplied by Prof Jennifer Perera, Department of Microbiology, Faculty of Medicine, University of Colombo, Sri Lanka. All PCR positive samples, i.e., the samples that were positive with Leishmania genus-specific primers (JW11/12 & LITSR/L5.8S) (n = 35) and negative with L. donovani-specific primers (LdF/LdR) (n = 8), were subjected to ITS1 PCR (LITSR/L5.8S), HaeIII digestion, and RFLP analysis. The restriction sites for HaeIII were obtained from RestrictionMapper v.3 (restrictionmapper.org/). A 10 µL volume of the selected ITS1 PCR products was digested with 10 U of HaeIII at 37°C for 2 h. A volume of 10 µL of the digested products was then run for 45 min at 100 V on a wide range 1.75% (w/v) agarose gel (Invitrogen) in 0.5 x Tris-boric acid-EDTA followed by staining with 0.2 µg/mL EtBr (Sigma Aldrich). The gel images were captured as described above.

RESULTS

Spectrum of clinical presentations - The spectrum of clinical presentations in the collected CL samples varied from papules, nodules, noduloulcerative (volcano-type) lesions, dry ulcers, wet ulcers to scaly plaques. These lesions were located in exposed areas of the body. A number of lesions had a depigmented halo (Fig. 1).

Sensitivity assays - Genus Leishmania-specific PCR with KDNA JW11/12 primers (Fig. 2A) andL. donovani species-specific LdF/LdR primers (Fig. 2B) detected as little as 100 fg (1 parasite) Leishmania DNA in the positive control L. donovani DNA serial dilutions. ITS1 PCR (LITSR/L5.8S) primers detected as little as 10 fg (0.1 parasite) in the serial dilutions (Fig. 2C).

Thirty-five from the 38 study samples became positive with both JW11/12 KDNA primers and ITS1 (LITSR/L5.8S) primers yielding similar sensitivity of 92.1% for pairs of both primers (Fig. 3A, B). Occasional samples that showed negative PCR in the first experiment became positive with a repeat PCR (sample 12, Lane 7 in Fig. 3B). However, three PCR negative samples showed amastigote-like forms with a nucleus and a kinetoplast like morphology and absent cytoplasm in the SSS. The possible parasitological grading was 2+ in all three PCR negative samples (Fig. 3C). These three negative samples were tested for the presence of DNA by spotting 2 µL of each sample with ~250 ng (2 µL) of quantified CL DNA as a positive control on a 1% thin agarose gel. The results showed the presence of DNA in the three PCR negative samples (results not shown). Furthermore, these PCR-negative samples (n = 3) did not show any PCR inhibition when tested by spiking with 1 pg of L. donovani DNA from pure cultures and amplified with primers described above (results not shown).

Out of the 35 samples that were tested positive with JW11/12 and LITSR/L5.8S primers, eight samples became negative with LdF/LdR primers yielding a sensitivity of only 71.1% (27/38) (Fig. 3D).

Specificity assays - LdF/LdR primer assays [reportedly L. donovani-specific, Salotra et al. (2001)Salotra P, Sreenivas G, Pogue GP, Lee N, Nakhasi HL, Ramesh V, Negi NS 2001. Development of a species-specific PCR assay for detection ofLeishmania donovani in clinical samples from patients with kala-azar and post-kala-azar dermal leishmaniasis. J Clin Microbiol 39: 849-854.] amplified 500 bp and 700 bp bands in four and two out of five tested M. tuberculosis DNA samples, respectively (Fig. 4A). However, these primers did not show any cross amplification in the presence of M. leprae or human DNA (results not shown). Specificity assays in the presence of and M. tuberculosis, M. leprae and human DNA showed that there was no cross-amplification in Leishmania KDNA JW11/12 (Fig. 4B) and LITSR/L5.8S ITS1 primer assays (Fig. 4C).

ITS1 PCR HaeIII RFLP analysis was used to increase species-specificity of the highly sensitive ITS1 PCR assay (Figs 2C, ⁵ 5 Keele University Keele University Centre for Applied Entomology and Parasitology Staffordshire UK Keele University, Centre for Applied Entomology and Parasitology, Staffordshire, UK ). The calculated sizes of the expected fragments after digestion withHaeIII were 54, 75, and 187 bp according to the L. donovani ITS1 sequence (GenBank accession AM901448.1). All 35 samples including the eight LdF/LdR PCR negative samples gave a similar L. donovani banding pattern upon ITS1 PCR HaeIII RFLP (Fig. 5).

DISCUSSION

This study for the first time systematically evaluated DNA based methods to detectLeishmania DNA in skin biopsy samples taken from Sri Lankan CL lesions. Out of the three sets of primers tested both the JW11/12 KDNA (Rodgers et al. 1990Rodgers MR, Popper SJ, Wirth DF 1990. Amplification of kinetoplast DNA as a tool in the detection and diagnosis of Leishmania.Exp Parasitol 71: 267-275.) and the LITSR/L5.8S ITS1 primer pairs (El Tai et al. 2000El Tai NO, Osman OF, el Fari M, Presber W, Schönian G 2000. Genetic heterogeneity of ribosomal internal transcribed spacer in clinical samples ofLeishmania donovani spotted on filter paper as revealed by single-strand conformation polymorphisms and sequencing. Trans R Soc Trop Med Hyg 94: 575-579.) showed high sensitivity (92%) with 100% specificity when compared to 71% sensitivity shown by the third LdF/LdR primer set (Salotra et al. 2001Salotra P, Sreenivas G, Pogue GP, Lee N, Nakhasi HL, Ramesh V, Negi NS 2001. Development of a species-specific PCR assay for detection ofLeishmania donovani in clinical samples from patients with kala-azar and post-kala-azar dermal leishmaniasis. J Clin Microbiol 39: 849-854.) (Fig. 4). Although a clinical polymorphism was detected in the CL presentations in Sri Lanka, the L. donovani specific LdF/LdR PCR and the ITS1 PCR- RFLP revealed that the only causative organism in all 35 PCR positive tested samples was L. donovani. Although parasite virulence factors might account for strain or clone-specific clinical presentations (WHO 2010WHO - World Health Organization 2010. Control of the leishmaniases. Available from: whqlibdoc.who.int/trs/WHO_TRS_949_eng.pdf.
whqlibdoc.who.int/trs/WHO_TRS_949_eng.pd... , McCall & McKerrow 2014McCall LI, McKerrow JH 2014. Determinants of disease phenotype in trypanosomatid parasites. Trends Parasitol 30: 342-349.), it has been also shown that different host cytokines, chemokines may contribute to different clinical outcomes in leishmaniasis (Carvalho et al. 2012Carvalho LP, Passos S, Schriefer A, Carvalho EM 2012. Protective and pathologic immune responses in human tegumentary leishmaniasis. Front Immunol 3: 301., Hartley et al. 2012Hartley MA, Ronet C, Zangger H, Beverley SM, Fasel N 2012.Leishmania RNA virus: when the host pays the toll.Front Cell Infect Microbiol 2: 99.).

The specificity assays carried out in this study revealed unexpected 500 bp and 700 bp bands in four and two out of five tested M. tuberculosis DNA samples, isolated from five different cultures when amplified with L. donovani-specific LdF/LdR primers. Thus, the use of LdF/LdR primers would produce complex diagnostic banding patterns in geographic regions including Sri Lanka where cutaneous TB is one of the differential diagnoses of infective causes of chronic skin lesions with leishmaniasis. Salotra et al. (2001)Salotra P, Sreenivas G, Pogue GP, Lee N, Nakhasi HL, Ramesh V, Negi NS 2001. Development of a species-specific PCR assay for detection ofLeishmania donovani in clinical samples from patients with kala-azar and post-kala-azar dermal leishmaniasis. J Clin Microbiol 39: 849-854. reported no LdF/LdR primers cross-amplification with M. tuberculosis DNA using blood samples from patients with pulmonary TB. The reported sensitivity of PCR in detecting M. tuberculosis DNA in blood of pulmonary and extra-pulmonary TB patients was low (< 50%) (Crump et al. 2012Crump JA, Tuohy MJ, Morrissey AB, Ramadan HO, Njau BN, Maro VP, Reller LB, Procop GW 2012. Performance of nucleic acid amplification following extraction of 5 milliliters of whole blood for diagnosis ofMycobacterium tuberculosis bacteremia. J Clin Microbiol 50: 138-141., Kashyap et al. 2013Kashyap RS, Nayak AR, Gaherwar HM, Bhullar SS, Husain AA, Shekhawat SD, Jain RK, Gaikwad SS, Satav AR, Purohit HJ, Taori GM, Daginawala HF 2013. Laboratory investigations on the diagnosis of tuberculosis in the malnourished tribal population of Melghat, India. PLoS One 12: e74652.). Therefore, the negative results reported could have been most likely due to false negatives or absence ofM. tuberculosis DNA in the tested blood samples (true negatives). It was also observed that LdF/LdR primers give bands in 400 bp and 300 bp region in some CL positive samples in addition to the 600 bp region (Lane 5 inFig. 3D, Lanes 3, 5-7 in Fig. 4A). This could be due to intraspecies polymorphism in the KDNA as described by Srivastava et al. (2011)Srivastava P, Singh T, Sundar S 2011. Genetic heterogeneity in clinical isolates of Leishmania donovani from India. J Clin Microbiol 49: 3687-3690., and the presence of mini-circle subclasses, and sequence heterogeneity (Ceccarelli et al. 2014Ceccarelli M, Galluzzi L, Migliazzo A, Magnani M 2014. Detection and characterization of Leishmania (Leishmania) and Leishmania (Viannia) by SYBR green-based real-time PCR and high resolution melt analysis targeting kinetoplast minicircle DNA. PLoS One9: e88845.).

However, it should be emphasised that in the present study LdF/LdR primers did not yield a L. donovani specific 600 bp amplicon in any of the negative controls including M. tuberculosis, M. leprae or human DNA. Thus, L. donovani-specific LdF/LdR primers (Salotra et al. 2001Salotra P, Sreenivas G, Pogue GP, Lee N, Nakhasi HL, Ramesh V, Negi NS 2001. Development of a species-specific PCR assay for detection ofLeishmania donovani in clinical samples from patients with kala-azar and post-kala-azar dermal leishmaniasis. J Clin Microbiol 39: 849-854.) could be used successfully to detect L. donovani DNA in skin biopsy samples in low income setups.

Although ITS1 PCR-HaeIII RFLP analysis appeared to be the best molecular diagnostic tool evaluated in this study to detect L. donovani DNA in skin biopsy samples, it will be costlier than end point LdF/LdR PCR. When the diagnostic purpose is to detect Leishmaniagenus-specific DNA in skin biopsy samples, either ITS1 PCR with LITSR/L5.8S primers or Leishmania KDNA primers JW11/12 would be successful due to a 92% sensitivity and 100% specificity shown in the study. Our study emphasises the importance of laboratory-based in addition to clinical diagnosis of CL as suggested by Siriwardana et al. (2015Siriwardana HV, Senarath U, Chandrawansa PH, Karunaweera ND 2015. Use of a clinical tool for screening and diagnosis of cutaneous leishmaniasis in Sri Lanka. Pathog Glob Health 109: 174-183.). An interesting conclusion, to be confirmed by larger sample size studies, is that L. donovani is the only species causing polymorphic clinical presentations in Sri Lanka. However, the relative contributions of strain polymorphism and host-related immune factors to the observed clinical spectrum deserve further investigation.

It is important to note that leishmaniasis is a spreading disease in the country warranting the implementation of active case detection, early treatment and integrated vector control at national level. In this context, 1% SSS positive newly diagnosed CL case incidence was reported in a cross sectional study in a high CL endemic area in the north-central province of Sri Lanka (Ranasinghe et al. 2013Ranasinghe S, Wickremasinghe R, Munasinghe A, Hulangamuwa S, Sivanantharajah S, Seneviratne K, Bandara S, Athauda I, Navaratne C, Silva O, Wackwella H, Matlashewski G, Wickremasinghe R 2013. Cross-sectional study to assess risk factors for leishmaniasis in an endemic region in Sri Lanka.Am J Trop Med Hyg 89: 742-749.), indicating the need for active case detection measures. A recent study described the successful application of a PCR method along with SSS and culture for active case detection in another CL endemic area in southern Sri Lanka (Kariyawasam et al. 2015Kariyawasam KK, Edirisuriya CS, Senerath U, Hensmen D, Siriwardana HV, Karunaweera ND 2015. Characterisation of cutaneous leishmaniasis in Matara district, southern Sri Lanka: evidence for case clustering. Pathog Glob Health 7: 2047773215Y0000000032.). Furthermore, limited data on epidemiology show evidence of geographical overlap of cutaneous TB, leprosy, and CL in Sri Lanka (Ranawaka et al. 2010Ranawaka RR, Abeygunasekara PH, Perera E, Weerakoon HS 2010. Clinico-histopathological correlation and the treatment response of 20 patients with cutaneous tuberculosis. Dermatol Online J 16: 13., Yasaratne & Madegedara 2010Yasaratne BMGD, Madegedara DM 2010. Tuberculosis of the skin.J Cey Coll Phy 41: 83-88., MS Sri Lanka 2014MS - Ministry of Health Sri Lanka 2014. Leprosy situation in Sri Lanka. Available from: epid.gov.lk/web/images/pdf/wer/2014/vol_41_no_40-english.pdf.
epid.gov.lk/web/images/pdf/wer/2014/vol_... , 2015aMS - Ministry of Health Sri Lanka 2015a. Flashback 2014. Available from: epid.gov.lk/web/images/pdf/wer/2015/vol_42_no_01-english.pdf.
epid.gov.lk/web/images/pdf/wer/2015/vol_... ). Coinfections of different clinical forms of TB and leprosy with leishmaniasis have also been reported in endemic geographic regions including Sri Lanka (Delobel et al. 2003Delobel P, Launois P, Djossou F, Sainte-Marie D, Pradinaud R 2003. American cutaneous leishmaniasis, lepromatous leprosy, and pulmonary tuberculosis coinfection with down regulation of the T-helper 1 cell response.Clin Infect Dis 37: 628-633., Rathnayake et al. 2010Rathnayake D, Ranawake RR, Sirimanna G, Siriwardhane Y, Karunaweera N, De Silva R 2010. Co-infection of mucosal leishmaniasis and extra pulmonary tuberculosis in a patient with inherent immune deficiency. Int J Dermatol 49: 549-551.). Therefore, these highly sensitive and specific ITS1 (LITSR/L5.8S) and JW11/12 PCR methods evaluated with local samples in this study would be valuable and cost-effective tools for active case detection of clinically suspected CL patients with negative SSS and culture and for epidemiological studies even in areas where skin TB and leprosy coexist with leishmaniasis.

ACKNOWLEDGEMENTS

To Mr Nuwan Dediwalage (IT Unit, Faculty of Medical Sciences, University of Sri Jayewardenepura), for preparation of Figures.

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