Population genetics of Leishmania (Leishmania) major DNA isolated from cutaneous leishmaniasis patients in Pakistan based on multilocus microsatellite typing.

BACKGROUND: Cutaneous leishmaniasis (CL) is a major and fast increasing public health problem, both among the local Pakistani populations and the Afghan refugees in camps. Leishmania (Leishmania) major is one of the etiological agents responsible for CL in Pakistan. Genetic variability and population structure have been investigated for 66 DNA samples of L. (L.) major isolated from skin biopsy of CL patients. METHODS: Multilocus microsatellite typing (MLMT), employing 10 independent genetic markers specific to L. (L.) major, was used to investigate the genetic polymorphisms and population structures of Pakistani L. (L.) major DNA isolated from CL human cases. Their microsatellite profiles were compared to those of 130 previously typed strains of L. (L.) major from various geographical localities. RESULTS: All the markers were polymorphic and fifty-one MLMT profiles were recognized among the 66 L. (L.) major DNA samples. The data displayed significant microsatellite polymorphisms with rare allelic heterozygosities. A Bayesian model-based approach and phylogenetic analysis inferred two L. (L.) major populations in Pakistan. Thirty-four samples belonged to one population and the remaining 32 L. (L.) major samples grouped together into another population. The two Pakistani L. (L.) major populations formed separate clusters, which differ genetically from the populations of L. (L.) major from Central Asia, Iran, Middle East and Africa. CONCLUSIONS: The considerable genetic variability of L. (L.) major might be related to the existence of different species of sand fly and/or rodent reservoir host in Sindh province, Pakistan. A comprehensive study of the epidemiology of CL including the situation or spreading of reservoirs and sand fly vectors in these foci is, therefore, warranted.

Zoophilic feeding behaviour of phlebotomine sand flies in the endemic areas of cutaneous leishmaniasis of Sindh Province, Pakistan.

Leishmania (Leishmania) major has been identified as the major causative agent of cutaneous leishmaniasis in Sindh Province of southern Pakistan. To make a rational approach for understanding the pathogen transmission cycles, the sand fly species and their natural blood meals in the endemic areas were examined. Total DNA was individually extracted from sand flies collected in four villages in Sindh Province. PCR-RFLP (restriction fragment length polymorphism) and sequence analysis of the 18S ribosomal RNA gene revealed that female sand flies identified were Sergentomyia clydei/Sergentomyia ghesquierei/Sergentomyia magna (68.6%), Sergentomyia dubia (17.1%), Phlebotomus papatasi (7.4%), Phlebotomus alexandri-like sand flies (3.4%) and Sergentomyia dentata (3.4%). PCR amplification of leishmanial kinetoplast DNA did not result in positive signals, suggesting that all 175 tested female sand flies were not infected with leishmanial parasites or contained undetectable levels of leishmanial DNA. Amplification and sequencing of the vertebrate cytochrome b gene in 28 blood-fed sand flies revealed that P. papatasi fed on cattle and wild rat whereas P. alexandri-like specimens fed on human, cattle, goat and dog. Although Sergentomyia sand flies are generally known to feed on cold-blooded animals, S. clydei, S. dubia and S. ghesquierei preferred humans, cattle, goat, sheep, buffalo, dog, donkey, wild rat and Indian gerbil. The epidemiological significance of the zoophilic feeding on various host species by Phlebotomus and Sergentomyia sand flies in Pakistan is further required to study for better understanding the zoonotic transmission of sand-fly-borne pathogens and for appropriate management of the vectors.

Cutaneous leishmaniasis caused by Leishmania (L.) major infection in Sindh province, Pakistan.

Leishmaniasis is endemic in Pakistan and is wide-spread throughout the country. Polymerase chain reaction (PCR) was performed to identify the Leishmania species present in cutaneous leishmaniasis (CL) patients from new endemic areas of the central part of Sindh province, Pakistan. The PCR primers used were designed for the identification and differentiation of Leishmania (Leishmania) major and Leishmania (Leishmania) tropica species, and PCR bands at 620 and 830 bp of the parasite-specific kinetoplast DNA sequences was identified for L. (L.) major and L. (L.) tropica, respectively. Among a total of 144 DNA samples purified from the skin biopsies of clinically suspected CL patients, 108 (75%) were positive for PCR amplification. Out of the 108 cases, 105 (97.2%) were determined to be positive for L. (L.) major infection, and 3 (2.8%) were positive for L. (L.) tropica infection. It was concluded that CL caused by L. (L.) major is the main source of infection in the central part of Sindh province in Pakistan. This rapid screening technique could be used for the diagnosis of a large number of samples from skin lesions, which commonly contain other bacterial and fungal infections.