Nazma Habib Khan

Leishmania tropica is the causative agent of cutaneous leishmaniasis in Pakistan. Here, intraspecific diversity of L. tropica from northern Pakistan was investigated using multilocus microsatellite typing. Fourteen polymorphic microsatellite markers were typed in 34 recently collected L. tropica isolates from Pakistan along with 158 archival strains of diverse Afro-Eurasian origins. Previously published profiles for 145 strains of L. tropica originating from different regions of Africa, Central Asia, Iran, and Middle East were included for comparison. Six consistently well-supported genetic groups were resolved: 1) Asia, 2) Morroco A, 3) Namibia and Kenya A, 4) Kenya B/Tunisia and Galilee, 5) Morocco B, and 6) Middle East. Strains from northern Pakistan were assigned to Asian cluster except for three that were placed in a geographically distant genetic group; Morocco A. Lesion variability among these Pakistani strains was not associated with specific L. tropica genetic profile. Pakistani strains showed little genetic differentiation from strains of Iraq, Afghanistan, and Syria (F_ST = 0.00–0.06); displayed evidence of modest genetic flow with India (F_ST = 0.14). Furthermore, genetic structuring within these isolates was not geographically defined. Pak–Afghan cluster was in significant linkage disequilibrium (I_A = 1.43), had low genetic diversity, and displayed comparatively higher heterozygosity (F_IS = −0.62). Patterns of genetic diversity observed suggest dominance of a minimally diverse clonal lineage within northern Pakistan. This is surprising as a wide clinical spectrum was observed in patients, suggesting the importance of host and other factors. Further genotyping studies of L. tropica isolates displaying different clinical phenotypes are required to validate this potentially important observation.

Dracunculiasis or guinea worm disease causes severe morbidity and disability among effected populations. Pakistan was declared dracunculaisis-free in 1996. The present study is a follow-on to the declared eradication of dracunculiasis in Dera Ismail Khan district of Khyber Pakhtunkhwa (KP) Province, Pakistan through investigating disease recurrences and Cyclops species in drinking water sources. A cross-sectional household survey was carried out using in-person questionnaire evaluation along with microscopic assessment of water samples for Cyclops species. Based on previous clinical records of dracunculiasis, five villages of Dera Ismail Khan including Kath Garh, Wanda Dost Ali, Chah Mundar Wala, Ketch and Gara Baloch were surveyed for a total of 206 randomly selected households, where the household heads (106 males and 100 females) were interviewed for any previous dracunculiasis lesion scars or active infections among household members. No active cases of dracunculiasis were observed in the present study. However, 11 individuals, had history of affliction by the disease during 1972-1992. All the water samples from drinking water sources tested negative for the presence of Cyclops species. No recurrence of dracunculiasis to an extent corroborates the proclaimed eradication by Guinea Worm Eradication Program (GWEP) from Dera Ismail Khan District in Khyber Pakhtunkhwa (KP) province.

Despite their medical importance, natural breeding sites of sand flies have been poorly explored. Detecting such sites is imperative in vector control strategies. This study aimed to identify potential breeding and resting microhabitats of sand flies and household risk factors associated with cutaneous leishmaniasis transmission in Dir districts, Khyber Pakhtunkhwa, Pakistan. Survey of indoor and outdoor habitats in four tehsils (subdistricts) of upper and lower Dir districts was carried out using sticky traps, flit method, and aspirator. To assess risk factors associated with cutaneous leishmaniasis (CL), questionnaires were administered to household heads. Mud houses and plants belonging to Cucurbitaceae family sheltered highest number of sand flies. Excessive number of flies were collected from indoor sites (common rooms used for both people and animals) compared to outdoor habitats (cattle dungs). Phlebotomus salangensis Artemiev, 1978 and Phlebotomus sergenti were the most widely distributed species, while Sergentomyia bailyi peaked in caves with high organic content in soil. Roof material and ownership of fans were associated with significant risk of CL. Findings of this study present a list of potential breeding sites for sand flies and CL risk factors that can be targeted and implemented in control programs for CL. Further studies are required to elucidate the breeding behavior and preferences of these medically important vectors.

This study primarily aimed to identify the causative species of cutaneous leishmaniasis (CL) in the Khyber Pakhtunkhwa Province of Pakistan and to distinguish any species-specific variation in clinical manifestation of CL. Diagnostic performance of different techniques for identifying CL was assessed. Isolates of Leishmania spp. were detected by in vitro culture, polymerase chain reaction (PCR) on DNA extracted from dried filter papers and microscopic examination of direct lesion smears from patients visiting three major primary care hospitals in Peshawar. A total of 125 CL patients were evaluated. Many acquired the disease from Peshawar and the neighboring tribal area of Khyber Agency. Military personnel acquired CL while deployed in north and south Waziristan. Leishmania tropica was identified as the predominant infecting organism in this study (89.2%) followed by Leishmania major (6.8%) and, unexpectedly, Leishmania infantum (4.1%). These were the first reported cases of CL caused by L. infantum in Pakistan. PCR diagnosis targeting kinetoplast DNA was the most sensitive diagnostic method, identifying 86.5% of all samples found positive by any other method. Other methods were as follows: ribosomal DNA PCR (78.4%), internal transcribed spacer 2 region PCR (70.3%), culture (67.1%), and microscopy (60.5%). Clinical examination reported 14 atypical forms of CL. Atypical lesions were not significantly associated with the infecting Leishmania species, nor with “dry” or “wet” appearance of lesions. Findings from this study provide a platform for species typing of CL patients in Pakistan, utilizing a combination of in vitro culture and molecular diagnostics. Moreover, the clinical diversity described herein can benefit clinicians in devising differential diagnosis of the disease.