Imported cutaneous leishmaniasis: molecular investigation unveils Leishmania major in Bangladesh
The main clinical forms of leishmaniasis in Bangladesh are visceral leishmaniasis and post-kala-azar dermal leishmaniasis, which are caused by Leishmania donovani. Imported cutaneous leishmaniasis (CL) is emerging globally due mainly to increased human mobility. In recent years, several imported CL cases have also been reported in Bangladesh. Sporadic atypical cases of CL can be challenging for diagnosis and clinical management, while occurrence of infection on a frequent basis can be alarming. We report of a case of a Bangladeshi temporary-migrant worker who, upon return, presented development of skin lesions that are characteristic of CL.
A serum sample was collected and tested with an rK39 immunochromatographic test. Nucleic acid from skin biopsy derived culture sample was extracted and screened with a real-time PCR assay which targets the conserved REPL repeat region of L. donovani complex. The internal transcribed spacer 2 region of the ribosomal RNA gene cluster was amplified and sequenced.
The suspect had a history of travel in both CL and VL endemic areas and had a positive rK39 test result. Based on clinical presentation, travel history and demonstration of the parasite in the skin biopsy, CL was diagnosed and the patient underwent a combination therapy with Miltefosine and liposomal amphotericin B. While typical endemic species were not detected, we identified Leishmania major, a species that, to our knowledge, has never been reported in Bangladesh.
Proper monitoring and reporting of imported cases should be given careful consideration for both clinical and epidemiological reasons. Molecular tests should be performed in diagnosis to avoid dilemma, and identification of causative species should be prioritized.
KeywordsCutaneous leishmaniasis Bangladesh Leishmania major Imported infection Sequencing
basic local alignment search tool
internal transcribed spacer 2
kala-azar elimination program
Kingdom of Saudi Arabia
post kala-azar dermal leishmaniasis
- rk39 ICT
recombinant k39 immunochromatographic test
real time polymerase chain reaction
Leishmaniasis is a group of insidious infectious diseases caused by species of the protozoan genus Leishmania transmitted through the bite of sand flies. It is classified into three clinical forms based upon the affected tissue, namely cutaneous (CL), mucocutaneous (MCL) and visceral (VL) leishmaniasis. The disease is endemic in many parts of the world. Bangladesh belongs to the endemic zones for VL as well as its skin complication known as post-kala-azar dermal leishmaniasis (PKDL), both of which are caused by Leishmania donovani. A regional initiative for VL elimination, known as the regional kala-azar Elimination Programme (KAEP) have contributed to a remarkable decline in the incidence rate of VL cases over recent years in Bangladesh and other endemic regions of the Indian subcontinent; it is now approaching the maintenance phase of elimination . Patients affected by CL or MCL, on the other hand, are not usually found in Bangladesh, which might be due to the absence of specific transmitting vectors . The diagnostic and clinical practices are well-defined in the local health care centres for VL and PKDL, which is not the case for CL or MCL. Upon occurrence, the atypical disease forms may cause diagnostic and clinical dilemmas with respect to clinical presentation, cross-reaction in serological tests, and treatment strategies [3, 4]. Systematic investigation of atypical cases and identification of the causative Leishmania species are also important for epidemiological reasons. Here, we report of a temporary-migrant worker who was diagnosed with CL after his return to Bangladesh. We identified L. major as the causative agent. To our knowledge, this is the first report of a L. major infected CL case in Bangladesh.
Parasite culture and DNA extraction
The additional skin snip (~ 3.0 mm in diameter) collected from a nodular lesion was inoculated into RPMI-1640 culture medium with 10% FBS supplemented with penicillin-streptomycin. Two volumes each of stationary phase culture promastigotes were inactivated, stored in buffer AL (Qiagen) at a 1:1 ratio and sent to the Emerging Infections and Parasitology laboratory of International Center for Diarrheal Disease Research (Dhaka, Bangladesh). DNA was extracted by using a QIAmp Blood DNA Mini Kit (Qiagen).
Real-time PCR and sequencing
A TaqMan probe based real-time (RT)-PCR assay, which targets the conserved region of Leishmania REPL repeats (L42486.1) of L. donovani complex, was carried out . A threshold cycle (Cq) of 40- in a 45-cycle assay was considered positive. For species identification by sequencing, amplicons of the internal transcribed spacer region 2 (ITS2) were generated by PCR . The amplicons were purified and prepared for Sanger sequencing by Microsynth Seqlab (Goettingen, Germany). Nucleotide BLAST search (NCBI) was used to estimate pairwise similarity of the tested sequence with reference Leishmania spp. genomes. A Tamura-Nei genetic distance model and neighbour-joining phylogenetic tree method for the derived ITS2 sequence was constructed together with sequences for Leishmania spp. with GENEIOUS v.9.1.6 (Biomatters Ltd., Auckland, New Zealand) using the incorporated tree builder at default settings.
Results of NCBI online nucleotide BLAST search of the newly generated sequence (Leish 17-832) using the NCBI Genomic Reference Sequences Database
Pairwise identity (%)
Query cover (%)
Leishmania major strain Friedlin complete genome, chromosome 27
Leishmania infantum JPCM5 WGS CACT00000000 data, contig 37, whole genome shotgun sequence
Leishmania donovani BPK282A1 complete genome, chromosome 27
Leishmania mexicana MHOM/GT/2001/U1103 WGS CADB00000000 data, contig 148, whole genome shotgun sequence
In Bangladesh, VL and PKDL are prevalent in endemic areas, whereas CL, a localized manifestation of nodular or popular lesion with ulceration, is not regarded endemic. Because the presentation of CL mimics common diseases like tuberculosis, anthrax and fungal infections , it may cause a diagnostic dilemma, especially in non-endemic regions, which can lead to inappropriate clinical management. In this report, national guidelines were followed to define the clinical CL suspect, and diagnosis was confirmed parasitologically . Since the patient was rK39 ICT positive and had previously visited a VL endemic area, RT-PCR assay positivity would have suggested a chance of mixed infection  with L. donovani. On the other hand, although rK39 ICT is a specific antibody marker test for active VL detection, its cross-reactivity with sera from CL patients is also evident to some extent. Hence, the rK39 ICT test positivity could be associated either with an already healed infection with species that causes VL, species-specific cross-reactivity against CL-causing parasites [9, 10, 11], greater duration and severity of cutaneous infection  or region-specific phylogenetic proximity among species . Finally, sequencing analysis of a species discriminatory segment of the ITS2 spacer  revealed that the obtained sequence (GenBank: MK034756) had almost 100% similarity with the reference genome sequence of L. major for absolute query cover (Table 1). The phylogenetic tree indicates that the test sequence shares common ancestry with L. major strains that originated in Iran (Fig. 2). This is consistent with case travel history, as L. major and L. tropica are the main dermotropic species in the CL endemic regions of countries of the Middle East including KSA. Phlebotomus papatasi (vector of L. major) and P. sergenti (vector of L. tropica) are the proven vectors of the parasite in this region . In Bangladesh, however, P. argentipes is the only known vector of L. donovani. Although a possible variant of L. donovani has also been prevalently causing CL in neighbouring countries, India and Sri Lanka [14, 15], no such evidence is found in Bangladesh so far, and the other case reports of CL also indicated that the disease was imported from middle-eastern regions [16, 17]. Thus, CL can still be regarded as only an imported disease in Bangladesh.
Imported leishmaniasis has been becoming a globally emerging infectious disease in returned travellers; a 24-year analysis showed that more than 80% of those cases concerned CL . Assessment of the risk of contracting CL, especially by Bangladeshi travellers, should be given careful consideration because the middle-eastern countries including KSA comprise one of the largest stocks of Bangladeshi migrants and temporary-migrant workers (> 3.0 million) . Due to the self-healing nature of CL, many of them may go underreported upon return, and be asymptomatic or subclinical. Subsequently, the chances of genetic exchange between parasites can be relevant within the context, because Leishmania is capable of cross-species and intra-clonal mating, which may increase parasite fitness . Moreover, P. argentipes is not competent to only L. donovani, but rather permissive to other pathogenic species including L. major . Natural adaptation of a new Leishmania species to the endemic vector  or co-existence of species and/or genetic variants  in endemic zones are not unusual. More importantly, considering that co-endemic zones of VL and CL are emerging in neighbouring countries [15, 23], screening and examination of imported CL suspects will be crucial to estimate the occurrence rate, and address whether such atypical cases can potentially become a new challenge for the control initiatives against leishmaniasis in Bangladesh.
In Bangladesh, imported cutaneous leishmaniasis is becoming more apparent. The imported CL case reported herein is, to our knowledge, the first evidence of L. major-derived pathology occurring in a Bangladeshi citizen. Our investigation indicates that presence of atypical cases in VL endemic areas can represent a diagnostic challenge, especially with antibody-based tests specific for active VL detection. Molecular tests should be performed in the diagnosis of such atypical cases to avoid dilemma. Furthermore, such cases should not be left out of epidemiological concern.
The publication of this study has been sponsored by Georg-August-Universität Göttingen.
MAAK overviewed laboratory methods, analysed the results and drafted the manuscript. RC performed the real-time PCR assay. RN performed microscopy and maintained parasite culture. SH performed the sequencing study. PN and SM managed the clinical case and revised the manuscript. AAEW supervised sequencing, data analysis and revised the manuscript. DM designed and coordinated the study and critically reviewed the manuscript. All authors read and approved the final manuscript.
The publication fees was covered by the Open Access Publication Funds of the University of Goettingen, Germany.
Ethics approval and consent to participate
Written informed consent was obtained from the patient to take additional skin specimen and photographs of the affected area for use in research and publication without revealing identity.
Consent for publication
Patient agreed for the publication of the study.
The authors declare that they have no competing interests.
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