Environmental and socio-demographic factors associated with cutaneous leishmaniasis in district Khyber, Pakistan; alarming spread of the disease to new foci.

Cutaneous leishmaniasis (CL) is a sand fly-borne infection of significant public health concern in Pakistan (endemic for CL). This study aimed to scrutinize the environmental and socio-economic risk factors for CL in district Khyber (located on Pak-Afghan border), Pakistan. Clinically confirmed 2881 CL case records for (January-December) 2017 and 2020 were obtained from district hospital. In addition, a questionnaire for CL risk factors assessment was administered to 525 households (175 in 2017 and 350 in 2020) in 40 villages throughout the district in a household survey. Higher number of CL cases were recorded in 2020 (N = 1824 belonging to 90 villages) compared to 2017 (N = 1057 from 42 villages). Highest number of CL patient cases were recorded in tehsil Jamrud (N = 2248, 39.01 %), followed by Landi Kotal (N = 398, 6.91 %) and Bara (N = 235, 4.08 %). Records showed higher number of CL cases in males (N = 1,659, 57.58 %) compared to females (N = 1,222, 42.41 %). In 2017 and 2020 the disease burden peaked in January. GIS-based spatial analyses of hospital records revealed that CL cases were abundant at 294-1,916 m elevation, in agriculture and range lands. Univariate logistic regression model analysis of risk factors suggested that higher education, family size >15, knowledge of CL, having family member/s that suffered from CL in the past, knowledge about biting time of sand flies, use of mosquito spray, presence of Afghan refugees in the village/s and living in mud-made houses increased the risk of acquiring CL. The multivariable logistic regression model showed none of the risk factors to be statistically significant. Findings of the study are crucial towards effective and targeted control of CL in district Khyber.

Insecticide susceptibility and detection of kdr-gene mutations in Aedes aegypti of Peshawar, Pakistan.

Dengue is an endemic disease in Peshawar, Pakistan and its primary vector is Aedes aegypti mosquito. Due to absence of vaccines and proper treatment for dengue, vector control becomes a necessary tool for disease management. Reported insecticide resistance in vectors is a serious threat to the control of dengue. This study presents the susceptibility status of Ae. aegypti to eight insecticides in district Peshawar along with one of the first attempts to screen mutations in the vector's knock down resistant gene (kdr). Local Ae. aegypti was found to be highly resistant to DDT and Deltamethrin while they were susceptible to Cyfluthrin and Bendiocarb. DNA sequencing of domains II and III of kdr-gene detected four SNPs in domain IIS6 at positions S989P and V1016G while two mutations were reported at position T1520I and F1534C in domain IIIS6. Lowest allele frequency was observed for S989P and V1016G positions while it was highest for F1534C. Among mutational combinations SSVVTICC (43%) was evidently the most predominant combination, where T1520I was heterozygous and F1534C was homozygous mutant. The study concludes instecticide resistance in local dengue population of Peshawar, Pakistan. The resistance observed is to some extent also corroborated in the molecular study of kdr gene. Findings herein can be utilized in designing dengue vector control strategies in Peshawar.

Characterization of sand fly breeding sites in district Malakand, Khyber Pakhtunkhwa, Pakistan, and evaluation of risk factors for cutaneous leishmaniasis in the region.

Present study was carried to determine the sand fly species composition, breeding sites ecology, seasonal abundance, and spatial distribution in district Malakand, Khyber Pakhtunkhwa, Pakistan. In addition, risk factors associated with cutaneous leishmaniasis (CL) were also evaluated. Survey of indoor and outdoor habitats was carried out using sticky traps in 31 villages of Dargai and Batkhela tehsils of Malakand. Soil from habitats of adult and immature sand flies was analysed. Questionnaire-based household survey was also performed in these villages to assess risk factors associated with CL. Soil samples from selected CL positive households were analysed for its contents. Additionally, clinicoepidemiological data from local health centres was examined for the year 2019. Total of 3,140 sand flies belonging to 18 species were collected. Phlebotomus sergenti was the most abundant species (38.16%). Its abundance had a strong positive correlation with mean monthly relative humidity and negative correlation with average temperature. Phlebotomus sergenti and Phlebotomus papatasi were abundant at an elevation ranging from 320 to 1,120 m above sea level and in agricultural lands near human settlements. Flight height preference apparatus collected maximum sand flies at 30 cm (1ft) above the ground and all species associated negatively with height. Soil analysis from habitats of adult and immature flies showed that highest mean number of adults and immatures were recorded from silt loam which carried highest concentrations of K2 O, Mg, Ca, and Zn. Number of immature sand flies correlated moderately (r = .7, p < .05) with K2 O soil concentrations. There was significant similarity between organic matter contents in soil samples from positive breeding sites and CL households (Wilcoxon rank-sum test, p = .1976). In multivariate analysis model for CL risk factors, age (26-35 and >35 years), knowledge of leishmaniasis, living in a middle and upper class, preachers visit to villages, and assumption that Afghan refugees are more prone to CL were significant. CL patient's archived data from health centres showed that majority of patients had lesions on face and hands. Patient's influx was highest in February and March.

Sand Fly (Diptera: Psychodidae) Species Diversity, Habitat Preferences, and Ecological Aspects of Distribution in Bajaur District, Khyber Pakhtunkhwa, Pakistan.

The species composition, distribution, and bionomics of sand flies was investigated from January to December 2017 in four tehsils (20 villages) of district Bajaur (formally Bajaur agency), Khyber Pakhtunkhwa, Pakistan. In total, 4,173 adult specimens comprising of seven species of Phlebotomus (Diptera: Psychodidae) and three species of Sergentomyia (Diptera: Psychodidae) were collected by oral aspirator, insecticide spray, and sticky traps from June to October. Highest numbers of flies were collected indoors in July and August when relative humidity and temperature were highest. Phlebotomus sergenti Parrot (Diptera: Psychodidae) was the most abundant species from all the collection sites, making approximately 70% of the total capture. Combined dwellings and chicken cages harbored the highest number of adult and immature flies (unrotated external genitalia). Abundance of P. sergenti had a significant (P ≤ 0.05) positive correlation with monthly average temperature, relative humidity, and average rainfall in indoor collections. On the other hand, P. papatasi Scopoli displayed similar correlation in the indoor and nighttime collections. Both the species were apparently abundant in the elevation range ≤ 419 m above sea level and on rangelands. Phlebotomus papatasi, however, also occurred on agricultural lands. The number of cutaneous leishmaniasis (CL) cases did not significantly correlate with the abundance of sand fly vectors across the villages surveyed. The results reported herein can serve as a baseline for an all-extensive future work in Bajaur. Our investigations will benefit the public health experts and medical entomologists for improved control and surveillance vectors of leishmaniasis.

Habitat characterization of sand fly vectors of leishmaniasis in Khyber Pakhtunkhwa, Pakistan.

Leishmaniasis is currently prevalent in 98 countries including Pakistan, where its wide-spread. Phlebotomus papatasi Scopoli and Phlebotomus sergenti Parrot are suspected vectors of cutaneous leishmaniasis (CL) while P. Phlebotomus major Yakimoff and Phlebotomus hindustanicus Theodor are vectors of visceral leishmaniasis (VL) in the Khyber Pakhtunkhwa (KP) province of Pakistan. The present study identifies the complexity of environmental factors affecting the distribution of these vector sand fly species in KP. Sand fly habitats in the province were characterized from relevant published sources and analyzed using geographic information system (GIS) tools. Specific spatial analysis indicated that P. sergenti and P. papatasi were ecological generalists, found in diverse ecological settings, while P. major and P. hindustanicus were less frequent and had restricted foci in Khyber Pakhtunkhwa. We specifically observed a close association between elevation and existance of several sand fly species. P. sergenti primarily presented at high elevation while P. papatasi had distribution at low altitudinal ranges. P. major and P. hindustanicus were apparently limited to high altitudes in the northern areas of the province. This GIS-based spatial investigation of Phlebotomine vectors of leishmaniasis aims to provide a beneficial tools to medical entomologists, epidemiologists and public health workers for regional control of leishmaniasis. Outcomes from such spatial studies can be utilized in planning vector control initiatives so as to ensure proper allocation of funds for the region's integrated vector control program budget.

Malaria epidemiology and comparative reliability of diagnostic tools in Bannu; an endemic malaria focus in south of Khyber Pakhtunkhwa, Pakistan.

The present study was aimed at elucidation of malaria epidemiology and comparing performance of several diagnostic procedures in Bannu, a highly endemic district of Khyber Pakhtunkhwa, Pakistan. Dried blood spots were collected from patients suspected of malaria visiting a hospital and two private laboratories in district Bannu and processed for species-specific PCR (rRNA). Patients were also screened for malaria through microscopy and RDT. A well-structured questionnaire was used to collect patient information to assess risk factors for malaria. Of 2033 individuals recruited, 21.1% (N = 429) were positive for malaria by at least one method. Overall, positivity detected by PCR was 30.5% (95/311) followed by 17.7% by microscopy (359/2033) and 16.4% by RDT (266/1618). Plasmodium vivax (16.9%, N = 343) was detected as the dominant species followed by Plasmodium falciparum (2.3%, N = 47) and mixed infections (1.2%, N = 39). Microscopy and RDT (Cohen's kappa k = 0.968, p = <0.0001, McNemar test p = 0.069) displayed significant agreement with each other. Satisfactory health, sleeping indoors, presence of health-care facility in vicinity (at an accessible range from home), living in upper middle class and in concrete houses significantly reduced malaria risk; whereas, low literacy level, presence of domestic animals indoors and malaria diagnosis recommended by clinician increased the disease risk. Overall, findings from the study provide reasonable basis for use of RDT as a cost-effective screening tool in field and for clinicians who can proceed with timely treatment of malaria patients. Appropriate management of identified risk factors could contribute to reduction of malaria prevalence in Bannu and its peripheries.

Geospatial and Climatic Patterns Associated With the Distribution of Sand Fly (Diptera: Psychodidae) Vectors of Leishmaniasis in Pakistan.

We provide a comprehensive and updated review on the effects of elevation and climatic factors on distribution of sand fly (Diptera: Psychodidae) vectors of cutaneous leishmaniasis (CL) and visceral leishmaniasis (VL) across Pakistan. Our study was undertaken in view of the increasing risk of leishmaniasis, particularly resulting from climatic deviations and political instability in the region. Literature (published and unpublished) on suspected vectors of CL (Phlebotomus sergenti Parrot and Phlebotomus papatasi Scopoli) and VL (Phlebotomus major Yakimoff and Schokhor and Phlebotomus hindustanicus Theodor) was collected, sorted, and utilized in geospatial analysis. P. papatasi and P. sergenti co-occurred in majority of the referenced localities and were generally distributed toward the western and southern parts across a wide range of elevation, whereas P. major and P. hindustanicus were limited to high elevation in cool and humid northern areas of Pakistan. Conversely P. papatasi and P. sergenti exploited diverse ecological settings across the country, being particularly abundant in warm/hot and arid regions.

Variability of Cutaneous Leishmaniasis Lesions Is Not Associated with Genetic Diversity of Leishmania tropica in Khyber Pakhtunkhwa Province of Pakistan.

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 (FST = 0.00-0.06); displayed evidence of modest genetic flow with India (FST = 0.14). Furthermore, genetic structuring within these isolates was not geographically defined. Pak-Afghan cluster was in significant linkage disequilibrium (IA = 1.43), had low genetic diversity, and displayed comparatively higher heterozygosity (FIS = -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.

Potential Resting and Breeding Sites of Sand Flies (Diptera: Psychodidae) and Their Habitat Characteristics in Leishmaniasis Foci of Dir Districts, Khyber Pakhtunkhwa, Pakistan.

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.

Cutaneous Leishmaniasis in Khyber Pakhtunkhwa Province of Pakistan: Clinical Diversity and Species-Level Diagnosis.

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.

Phylogenetic position of Leishmania isolates from Khyber Pakhtunkhwa province of Pakistan.

Several species of the genus Leishmania are causative agents of cutaneous leishmaniasis in Pakistan. This study aimed to determine phylogenetic placement of Leishmania species causing cutaneous leishmaniasis in Khyber Pakhtunkhwa province, Pakistan (34 Leishmania tropica, 3 Leishmania infantum), in-relation to species from other geographical areas using gene sequences encoding cytochrome b (cytb) and internal transcribed spacer 2 (its2). Based on cytochrome b sequence analysis, L. tropica strains from Pakistan and other geographical regions were differentiated into two genotype groups, A and B. Within the province, five distinct L. tropica genotypes were recognized; two in group A, three in group B. Two L. infantum isolates from the province were closely associated with both Afro-Eurasian and American species of the Leishmania donovani complex, including Leishmania chagasi, L. infantum and L. donovani from Sudan and Ethiopia; while a third L. infantum isolate could not be differentiated from visceralizing Kenyan and Indian L. donovani. We observed apposite phylogenetic placement of CL-causing L. tropica and L. infantum from Khyber Pakhtunkhwa. Affinities ascribed to Leishmania spp. From the region are valuable in tracing potential importation of leishmaniasis.

Species composition and activity patterns of sand flies (Psycodidae: Phlebotomine) in four tehsils of Dir Districts, Khyber Pakhtunkhwa, Pakistan.

The present study reports sand flies species composition, fauna diversity and seasonal variations from four tehsils of Dir Districts, Khyber Pakhtunkhwa, Pakistan. Collection was made using sticky traps, flit method and aspiration where highest number of sand flies was captured through sticky traps. Digitalized sand flies distribution maps were produced using geographic information system ArcGIS. A total of 7292 specimens were captured between January to December 2014, comprised of 11 Sergentomyia and 9 Phlebotomus. Phlebotomus salengensis was the most abundant species followed by Phlebotomus sergenti. Overall, male to female ratio observed was 3:1 and species diversity varied among the studied tehsils. Highest abundance was recorded in July and August, whereas the flies disappeared in the colder months (November-April) of the year. Information about insect vector behaviour in natural setting is required to understand the status of disease caused by them. This study is a thorough account of biodiversity of sand flies in the region and provides a useful insight in to identifying potential breeding preferences of sand flies and recognition of active and potential vector species in the Dir districts. Further large scale studies are needed to determine the behaviour, infection rate, and the natural reservoir hosts of sand fly vectors in the region.

In-vitro sensitivity of Pakistani Leishmania tropica field isolate against buparvaquone in comparison to standard anti-leishmanial drugs.

In this study, in vitro anti-leishmanial activity of buparvaquone was evaluated against promastigotes and intracellular amastigotes of Pakistani Leishmania tropica isolate KWH23 in relation to the current standard chemotherapy for leishmaniasis (sodium stibogluconate, sodium stibogluconate, amphotericin B and miltefosine). For buparvaquone, mean % inhibition in intracellular amastigotes at four different concentrations (1.35 µM, 0.51 µM, 0.17 µM and 0.057 µM) was 78%, 44%, 20% and 14% respectively, whereas, against promastigotes it was 89%, 77%, 45% and 35% respectively. IC50 values calculated to estimate the anti-leishmanial activity of buparvaquone against intra-cellular amastigotes and promastigotes was 0.53 µM (95% C.I. = 0.32-0.89) and 0.15 µM (95% C.I. = 0.01-1.84) respectively. Amphotericin B was the most potent in-vitro drug tested, with an IC50 of 0.075 µM (95% C.I. = 0.006-0.907) against promastigotes, and 0.065 µM (95% C.I. = 0.048-0.089) against intra-cellular amastigotes. Amphotericin B was more cytotoxic against THP1 cells, with an IC50 of 0.15 µM (95% C.I. = 0.01-0.95) and an apparent in-vitro therapeutic index of 2.0, than was buparvaquone, with an IC50 of 12.03 µM (95% C.I. = 5.36-26.96) against THP1 cells and a therapeutic index of 80.2. The study proposes that buparvaquone may be further investigated as a candidate drug for treatment of cutaneous leishmaniasis.