Heterogeneous malaria transmission in long-term Afghan refugee populations: a cross-sectional study in five refugee camps in northern Pakistan.

BACKGROUND: Afghan refugees in northern Pakistan have been resident for over 30 years and current information on malaria in this population is sparse. Understanding malaria risk and distribution in refugee camps is important for effective management both in camps and on return to Afghanistan. METHODS: Cross-sectional malariometric surveys were conducted in five Afghan refugee camps to determine infection and exposure to both Plasmodium falciparum and Plasmodium vivax. Factors associated with malaria infection and exposure were analysed using logistic regression, and spatial heterogeneity within camps was investigated with SatScan. RESULTS: In this low-transmission setting, prevalence of infection in the five camps ranged from 0-0.2 to 0.4-9 % by rapid diagnostic test and 0-1.39 and 5-15 % by polymerase chain reaction for P. falciparum and P. vivax, respectively. Prevalence of anti-malarial antibodies to P. falciparum antigens was 3-11 and 17-45 % for P. vivax antigens. Significant foci of P. vivax infection and exposure were detected in three of the five camps. Hotspots of P. falciparum were also detected in three camps, only one of which also showed evidence of P. vivax hotspots. CONCLUSIONS: There is low and spatially heterogeneous malaria transmission in the refugee camps in northern Pakistan. Understanding malaria risk in refugee camps is important so the malaria risk faced by these populations in the camps and upon their return to Afghanistan can be effectively managed.

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.

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.

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.

Bionomics of the unexplored sand flies fauna of District Mohmand, Khyber Pakhtunkhwa, Pakistan: assessing risk factors associated with cutaneous leishmaniasis.

BACKGROUND: Sand flies are of considerable public health importance in Pakistan because these insects are vectors of leishmaniasis. The current study explores the bionomics of sand flies, their spatial distribution pattern and cutaneous leishmaniasis-associated risk factors in District Mohmand, Khyber Pakhtunkhwa, Pakistan. METHODS: Sand flies were collected from indoor and outdoor habitats in 69 villages of five tehsils in Mohmand during July-October 2019. Risk factor data were recorded for 829 households in 94 villages. RESULTS: In total, 2065 sand flies were captured. Phlebotomus (Paraphlebotbmus) sergenti was the most abundant species. Relative density for P. sergenti and Phlebotomus papatasi was highest in Prang Ghar and lowest in Safi. Sand flies abundance peaked in August and September, corresponding to maximum relative humidity, temperature and rainfall. Relative density for P. sergenti and P. papatasi was highest in combined dwellings (indoor) and cattle corrals (outdoor). Phlebotomus sergenti and P. papatasi were abundant at an elevation of 283-1140 m on agricultural land and rangelands. Both species were recorded abundantly on Carbontites, Mesozoic and Indus suture Melange rock formations. Presence of domestic animals, ownership of pet dogs, presence of muddy dunes in the village, knowledge of sandflies and use of mosquitoes spray remained significant risk factors. CONCLUSIONS: The study reports sand fly bionomics in District Mohmand. Risk identified for cutaneous leishmaniasis are significant in strategising control methods for Health authorities can allocate localized control means to high-risk areas using these findings.

SYSTEMATIC REVIEW OF LEISHMANIASIS IN PAKISTAN: EVALUATING SPATIAL DISTRIBUTION AND RISK FACTORS.

Both cutaneous (zoonotic cutaneous leishmaniasis [ZCL] and anthroponotic cutaneous leishmaniasis [ACL]) and visceral leishmaniasis (VL) are prevalent in Pakistan, although the cutaneous form is comparatively more widespread. The present study aimed to spatially map different forms of leishmaniasis in Pakistan. In addition, the risk for acquiring leishmaniasis was assessed in different locations of the country. Previously published literature on leishmaniasis distribution and associated risk factors in Pakistan was obtained from PubMed/NCBI and GoogleScholar using specific keywords. Further, 50 articles that focused on geographical distribution and risk factors of the disease in Pakistan were obtained and systematically reviewed. Data regarding location (used in ArcGIS for spatial analysis), number of cases, and risk factors were identified. The present study revealed a high burden of cutaneous leishmaniasis in the northern and western foci of Pakistan. Leishmania major, chiefly a causative agent of ZCL, was observed to be prevalent in the arid and semi-arid climatic zones of the country with elevations between 45 and 2,837 m (maximum number at low elevation). Leishmania tropica cases, associated with ACL, were recorded at elevations ranging from 551 to 2,837 m (majority cases at high elevation). Visceral leishmaniasis (VL) (reportedly caused by Leishmania infantum) was sporadically distributed in the northern areas at elevations 1,432-2,873 m. For VL, particularly, mountainous communities, farming communities, and the presence of dogs were the most commonly reported risk factors. On the other hand, poor preventive measures, migration, presence of domesticated animals, presence of cattle dung, living in mud houses, and outdoor sleeping habits were the principal risk factors associated with cutaneous leishmaniasis (CL). This investigation can guide the health experts and medical entomologists toward targeted and cost-effective surveillance and control of leishmaniasis in Pakistan.

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.

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.

Assessing Incidence Patterns and Risk Factors for Cutaneous Leishmaniasis in Peshawar Region, Khyber Pakhtunkhwa, Pakistan.

Pakistan faces critical challenges pertaining to cutaneous leishmaniasis (CL), where it's distribution is more or less patchy. The goal of this study was to assess the incidence of CL as well as to identify potential risk factors in Peshawar region, Khyber Pakhtunkhwa, Pakistan. The study was conducted in the dermatology outpatient unit at Kuwait Teaching Hospital, Peshawar, Khyber Pakhtunkhwa, Pakistan. Longitudinal out-patient department visit data for 9,631 CL patients spanning a 42-mo (April 2011-October 2014) period was analyzed using autoregressive integrated moving average (ARIMA) time series models ARIMA(1,0,0)(0,1,0)12 and ARIMA(0,0,0)(0,1,0)12. The ARIMA concluded that the number of patients was increasing over time. Over the duration, frequency of male patients (58.2%) was higher. The mean age of CL patients was 16.4 (confidence interval = 16.14-16.70) yr and the majority of the patients were aged 5-20 yr (52.6%). Inflow of CL patients peaked close to February and March, followed by a decline until its lowest point in the months of August and September (P < 0.001). Two hundred individuals, including 88 cases and 112 controls, were matched by gender and age categories (<5, 5-20, >20 yr) to derive 63 matched pairs. Using univariate conditional logistic regression analyses of the matched pairs, we found that living in congested rooms (>6 persons), having family members with lesions (active/scars), keeping cattle inside dwellings at night, and having in-door vegetation were established as factors that significantly increased the risk of CL. On the other hand, living in houses constructed with bricked walls or wooden roofs (thatched/beam), ownership of treated bed nets, and having meshed windows were proven to be protective against CL. It was evident that the disease incidence has been on a gradual rise over the past few years. It was concluded that household clustering, house construction, and conventional behavioral practices (living with cattle) greatly impact the epidemiology of CL in the region. Conclusions from this study have significant implications for prospective control programs.

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.

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.

Population dynamics of mosquitoes and malaria vector incrimination in district Charsadda, Khyber Pakhtunkhwa (KP) Pakistan.

This study aimed to obtain updated information on mosquito diversity and malaria vector incrimination in Charsadda Khyber Pakhtunkhwa to help in devising effective control and preventive measures in the area. Monthly survey of indoor mosquitoes for one year was carried out in three villages, Dhaki Totaqi and Mathra. Female Anopheline were used to detect Circumsporozoites protein (CSP) using Enzyme Linked Immunosorbent Assay. Among 17 mosquito species, Culex quinquefasciatus, Anopheles splendidus, Anopheles stephensi, Anopheles fluviatilus, Anopheles culicifacies and Culex tritaeniorhynchus were predominant. Dhaki village had the highest mosquito species diversity (1.015) and similar species richness (0.7) and evenness (0.5) with village Mathra. Slide positivity rate (SPR) shows that the rate of malaria transmission increases with mosquito population. Four anopheline species i.e. A. stephensi, A. fluviatilis, A. splendidus, and A. culicifacies were CSP positive. The CSP rate was 0.8%, where two specimens of A. splendidus and one of A. fluviatilus were positive for Plasmodium falciparum, Plasmodium vivax was represented by two variants 210 and 247. Among the 20 CSP positive specimens, variant 210 was found in 12 (one belonging to A. culicifacies, two each of A. stephensi and A. fluviatilus, seven specimens of A. splendidus) and 247 in 8 specimens (two of A. stephensi, three each of A. fluviatilus and A. splendidus). The number of infected mosquitoes collected from animal sheds was higher (15) though non-significant (P>0.05) than that from bedrooms (8).