Dataset Information

Polymerase chain reaction detection of Leishmania DNA in skin biopsy samples in Sri Lanka where the causative agent of cutaneous leishmaniasis is Leishmania donovani.

ABSTRACT: Leishmania donovani is the known causative agent of both cutaneous (CL) and visceral leishmaniasis in Sri Lanka. CL is considered to be under-reported partly due to relatively poor sensitivity and specificity of microscopic diagnosis. We compared robustness of three previously described polymerase chain reaction (PCR) based methods to detect Leishmania DNA in 38 punch biopsy samples from patients presented with suspected lesions in 2010. Both, Leishmania genus-specific JW11/JW12 KDNA and LITSR/L5.8S internal transcribed spacer (ITS)1 PCR assays detected 92% (35/38) of the samples whereas a KDNA assay specific forL. donovani (LdF/LdR) detected only 71% (27/38) of samples. All positive samples showed a L. donovani banding pattern upon HaeIII ITS1 PCR-restriction fragment length polymorphism analysis. PCR assay specificity was evaluated in samples containing Mycobacterium tuberculosis, Mycobacterium leprae, and human DNA, and there was no cross-amplification in JW11/JW12 and LITSR/L5.8S PCR assays. The LdF/LdR PCR assay did not amplify M. leprae or human DNA although 500 bp and 700 bp bands were observed in M. tuberculosis samples. In conclusion, it was successfully shown in this study that it is possible to diagnose Sri Lankan CL with high accuracy, to genus and species identification, using Leishmania DNA PCR assays.

SUBMITTER: Ranasinghe S

PROVIDER: S-EPMC4708022 | biostudies-literature | 2015 Dec

REPOSITORIES: biostudies-literature

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Polymerase chain reaction detection of Leishmania DNA in skin biopsy samples in Sri Lanka where the causative agent of cutaneous leishmaniasis is Leishmania donovani.

Ranasinghe Shalindra S Wickremasinghe Renu R Hulangamuwa Sanjeeva S Sirimanna Ganga G Opathella Nandimithra N Maingon Rhaiza D C RD Chandrasekharan Vishvanath V

Memorias do Instituto Oswaldo Cruz 20151211 8

Leishmania donovani is the known causative agent of both cutaneous (CL) and visceral leishmaniasis in Sri Lanka. CL is considered to be under-reported partly due to relatively poor sensitivity and specificity of microscopic diagnosis. We compared robustness of three previously described polymerase chain reaction (PCR) based methods to detect Leishmania DNA in 38 punch biopsy samples from patients presented with suspected lesions in 2010. Both, Leishmania genus-specific JW11/JW12 KDNA and LITSR/L ...[more]

PMID: 26676321

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Project description:Sri Lanka is a recent focus having Leishmania donovani induced cutaneous leishmaniasis (CL) as the main clinical entity. A separate clinical entity within profile of CL was described in this study. Laboratory confirmed cases of CL (n= 950, 2002-2014) were analysed. Most lesions showed known classical developmental stages of CL (CCL) observed in other CL endemic settings while few cases (13%, 122/950) showed atypical skin manifestations (ACL). Clinical, geographical, and treatment response patterns of ACL were different from those of CCL. ACL was mainly found among males (68.0%), in 21-40 year age group (51.6%), and reported delayed treatment seeking (23.5% vs 16.3% in CCL), more nonclassical onset (lesions other than acne form <1cm sized papules), (12.1 vs 2.7%, P<0.05.), more head and neck lesions (41.5%. vs 27.2%), more large lesions (>4cm), (18.6 vs 9.9%), and poor laboratory positivity rates (65.6% vs 88.2%) when compared to CCL. When compared to lesions reporting a typical onset, lesions reporting nonclassical onset were more likely to develop ACL later on (50.1% vs 10.7%). As compared to lesions on limbs, those on head and neck and trunk were more likely to be ACL (7.0%, 16.3%, and 22.8%, respectively, P<0.05). ACL features were not age or gender dependent. Highest proportion within ACL category (32.8%) and small proportion of CCL (10.1%) originated from less leishmaniasis prevalent areas (other regions) (P<0.05). North reported more ACL than South (15.9% vs 7.4%). A total of 95 CL cases with a significant travel history were further analyzed. Residents of other regions when acquired infection from North or South developed more ACL than residents in North or South (60.9% vs 15.9% and 42.9% vs 7.4% respectively). Patients in other regions when travelled to North developed more ACL than when they travelled to South (60.9%, 42.9%). ACL and CCL required an average of 18 doses over 16.7 months and 10 doses over 12 weeks, respectively, to achieve a complete clinical cure. Underlying host immunological factors, parasite strain variations and regional variations of both could be underlying etiologies. Established independent transmission within less leishmaniasis prevalent regions combined with an unusual clinical picture leading to poor clinical suspicion and low laboratory confirmation rate will pose potential difficulties in early case detection in these highly populated and commercialized areas. This in turn will further facilitate silent and high disease transmission.

Project description:Among the most central questions in Leishmania research is why some species remain in the skin dermis at the site of infection by the sand fly vector whereas other species migrate to visceral organs where they cause fatal visceral leishmaniasis. Although L. donovani is the species typically responsible for visceral leishmaniasis, an atypical L. donovani strain is the etiologic agent for cutaneous leishmaniasis in Sri Lanka. To identify molecular determinants for visceral disease, we have analysed the phenotype and genotype of two L. donovani clinical isolates from Sri Lanka where one isolate was derived from a cutaneous leishmaniasis patient (CL) and the other from a visceral leishmaniasis patient (VL). These isolates cause dramatically different pathology when introduced into mice; notably the CL isolate has lost the ability to survive in visceral organs while the VL isolate was highly virulent in visceral organs of BALB/c mice. Whole genome sequencing of the CL and VL isolates revealed that these genomes were very similar as there were no gene deletions and few individual gene amplifications. Indels resulting in frame shifts and loss/gain of stop codons resulted in 13 distinct pseudogenes present in each of the CL and VL isolates. There were 154 non-synonymous SNPs specific to the CL isolate and 193 non-synonymous SNPs specific to the VL isolate. Genome wide gene expression analysis revealed several transcript level differences, including the A2 virulence gene resulting in higher expression of A2 proteins in the VL isolate than in the CL isolate. Genotypic variations relevant to pathology and tropism in Leishmania can be interrogated by reverse genetics. Experimentally increasing A2 expression in the CL isolate through gene transfer significantly increased itM-bM-^@M-^Ys ability to survive in the spleen of BALB/c mice and conversely, down-regulating A2 expression in the VL isolate abrogated attenuated its survival in BALB/c mice. These observations reveal that there are relatively few genetic differences between the CL and VL isolates apart from the A2 genes, but collectively these have profound effects on human disease and experimentally infected mice. 6 Samples in total, 3 each from VL and CL causing isolates were analyzed by Splice Leader RNASeq. These three samples from each of the isolates were grown to form one of the following three lifestages, Promastigotes, Macrophage derived Amastigotes, Axenic Amastigotes.

Project description:Among the most central questions in Leishmania research is why some species remain in the skin dermis at the site of infection by the sand fly vector whereas other species migrate to visceral organs where they cause fatal visceral leishmaniasis. Although L. donovani is the species typically responsible for visceral leishmaniasis, an atypical L. donovani strain is the etiologic agent for cutaneous leishmaniasis in Sri Lanka. To identify molecular determinants for visceral disease, we have analysed the phenotype and genotype of two L. donovani clinical isolates from Sri Lanka where one isolate was derived from a cutaneous leishmaniasis patient (CL) and the other from a visceral leishmaniasis patient (VL). These isolates cause dramatically different pathology when introduced into mice; notably the CL isolate has lost the ability to survive in visceral organs while the VL isolate was highly virulent in visceral organs of BALB/c mice. Whole genome sequencing of the CL and VL isolates revealed that these genomes were very similar as there were no gene deletions and few individual gene amplifications. Indels resulting in frame shifts and loss/gain of stop codons resulted in 13 distinct pseudogenes present in each of the CL and VL isolates. There were 154 non-synonymous SNPs specific to the CL isolate and 193 non-synonymous SNPs specific to the VL isolate. Genome wide gene expression analysis revealed several transcript level differences, including the A2 virulence gene resulting in higher expression of A2 proteins in the VL isolate than in the CL isolate. Genotypic variations relevant to pathology and tropism in Leishmania can be interrogated by reverse genetics. Experimentally increasing A2 expression in the CL isolate through gene transfer significantly increased it’s ability to survive in the spleen of BALB/c mice and conversely, down-regulating A2 expression in the VL isolate abrogated attenuated its survival in BALB/c mice. These observations reveal that there are relatively few genetic differences between the CL and VL isolates apart from the A2 genes, but collectively these have profound effects on human disease and experimentally infected mice.

Project description:Sri Lanka reports a large focus of Leishmania donovani-induced cutaneous leishmaniasis (CL) with CL as the main clinical entity. Two independent, long existed, and clinicoepidemiologically different transmission foci in the northern region (NR) and southern region (SR) were recently reported. Current project is an extension to this previous study. Clinical diversity within a profile of classical cutaneous leishmaniasis (CCL) in a focus of L. donovani-induced CL is described for the first time. Patients with laboratory confirmed CCL (n = 550) from NF and SF were evaluated. Lesions in both foci were found to have all classical developmental stages (small and large nodules, ulcerating nodules, and ulcers) and other identified changes (multiplication, ulceration, and enlargement). Main difference was in the proportions of lesions progressing in to each different stages, proportions of lesion undergoing the main changes, and in timing of these changes during the course of a lesion. Northern focus reported a smaller proportion of lesions showing enlargement and ulceration, and a longer period of time was also required for these changes when compared to same in southern focus. In northern focus, most lesions remained small and nonulcerating and showed a higher tendency to multiply while most lesions reported in southern focus enlarged and ulcerated rapidly and remained single. Current study also evidenced a wider spectrum in the rate and pattern of progression of a skin lesion and high individual variation which could mask these region-based differences. Parasitic, vector-related, or a host etiology is suggested. Slow progressing nonulcerating infections in North may be the result of a well-adopted parasite strain that coevolved with its host for a long period while inducing only a minimal host response. This could be one among many reasons for previously observed silent expansion in northern focus while southern focus remained more confined and stable over time. Small nonprogressive, nondisturbing lesions can play a major role as silent parasite reservoirs in a community. In addition, the laboratory detection rate declined significantly when lesions multiplied and enlarged indicating the need for early laboratory confirmation. Usefulness of identified features in clinical screening and management needs to be considered.

Dataset Information

Polymerase chain reaction detection of Leishmania DNA in skin biopsy samples in Sri Lanka where the causative agent of cutaneous leishmaniasis is Leishmania donovani.

Publications

Polymerase chain reaction detection of Leishmania DNA in skin biopsy samples in Sri Lanka where the causative agent of cutaneous leishmaniasis is Leishmania donovani.

Similar Datasets

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