Entomological assessment in 'hotspots' of four lymphatic filariasis endemic districts, Central Nepal during post-MDA surveillance.

BACKGROUND OBJECTIVES: Annual mass drug administration (MDA) is the main strategy to interrupt the transmission of lymphatic filariasis (LF) in the community. The main aim of monitoring the MDA program, for its effectiveness and interruption of LF is the post-MDA surveillance using antigen survey in children born after MDA. The latest technique of new research suggests that xenomonitoring is an effective tool for monitoring LF intervention. The objective of this study was to assess the W. bancrofti infection/or infectivity in vector mosquitoes by xenomonitoring during post-MDA surveillance. METHODS: A descriptive cross-sectional study was conducted in the hotspots of selected four districts of Central Nepal. A gravid trap technique was used for sampling mosquitoes. Infection/or infectivity was determined via the dissection of vector mosquitoes. Anopheles, Aedes, Armigerus and Culex species were collected from hotspots of four endemic districts, two from the hilly region (Lalitpur and Dhading) and two from Terai region (Bara and Mahottari) of Central Nepal. RESULTS: A total of 4450 mosquitoes belonging to four genera, Anopheles, Culex, Armigeres, and Aedes were collected from four hotspots. The distribution of Culex quinquefasciatus was found to be the highest, 88.9% (n=3955/4450) followed by Cx. vishnui (4.5%), Armigeres sp (5.8%), An. culicifascies (0.2%), Aedes spp (0.8%). The proportion of female mosquitoes trapped is significantly higher. A total of 3344 parous Cx. quinquefasciatus mosquitoes were dissected for any larval stage of W. bancrofti. We could not find any filarial infection in dissected mosquito samples. INTERPRETATION CONCLUSION: We conclude that the gravid trap is an efficient tool for the collection of gravid Cx. quinquefasciatus mosquitoes for xenomonitoring studies of filariasis endemic regions. Vector composition indicated a maximum number of vector mosquitoes of lymphatic filariasis were trapped compared with the other three species. Distribution and density of Cx. quinquefasciatus was found highest in four hotspots of endemic districts. None of the Cx. quinquefasciatus dissected were found to be infected by larval forms of filaria. Since the low levels of infection persistence in the human population in these hot spots, vector infection and infectivity can't be ignored. Microscopic xenomonitoring at a low level of infection persistent is less likely to be efficient so molecular xenomonitoring along with a large sample should be required in each of the hot spots of the districts. Additionally, area is receptive so further vector control intervention should be required to reduce the risk of resurgence of infection.

Assessment of microfilaremia in 'hotspots' of four lymphatic filariasis endemic districts of Nepal during post-MDA surveillance.

BACKGROUND: The lymphatic filariasis (LF) elimination program in all sixty-three endemic districts of Nepal was based on annual mass drug administration (MDA) using a combination of diethylcarbamazine (DEC) and albendazole for at least 5 years. The MDA program was started in the Parsa district of the Terai region and at least six rounds of MDA were completed between 2003 and 2017 in all filariasis endemic districts of Central Nepal. Transmission Assessment Survey (TAS) report indicated that circulating filarial antigen (CFA) prevalence was below the critical value i.e., ≤ 2% in selected LF endemic districts of Central Nepal. Based on the TAS report, antigen-positive cases were found clustered in the foci of those districts which were considered as "hotspots". Hence the present study was designed to assess microfilaremia in hotspots of four endemic districts of Central Nepal after the MDA program. METHODOLOGY AND PRINCIPAL FINDINGS: The present study assessed microfilaremia in hotspots of four endemic districts i.e. Lalitpur and Dhading from the hilly region and Bara and Mahottari from the Terai region of Central Nepal. Night blood samples (n = 1722) were collected by finger prick method from the eligible sample population irrespective of age and sex. Community people's participation in the MDA program was ensured using a structured questionnaire and chronic clinical manifestation of LF was assessed using standard case definition. Two districts one each from the hilly region (Lalitpur district) and Terai region (Bara district) showed improved microfilaria (MF) prevalence i.e. below the critical level (<1%) while the other two districts are still over the critical level. There was a significantly high prevalence of MF in male (p = <0.05) and ≥41 years of age group (p = <0.05) community people in the hotspots of four endemic districts. People who participated in the previous rounds of the MDA program have significantly low MF prevalence. The upper confidence limit of MF prevalence in all hotspots of four districts was above the critical level (>1%). Chronic clinical manifestation of LF showed significant association with the older age group (≥41 years) but not with sex. CONCLUSIONS: The study revealed LF transmission improved in hotspots of two districts while continued in others but the risk of LF resurgence cannot be ignored since the upper confidence level of MF prevalence is over 1% in all the hotspots studied districts. High MF prevalence is well correlated with the number of MDA rounds but not with the MDA coverage. Community people involved in MDA drug uptake in any previous and last rounds have significantly less MF infection. Hence it is recommended that before deciding to stop the MDA rounds it is essential to conduct the MF survey at the hotspots of the sentinel sites.

Assessment of antigenemia among children in four hotspots of filarial endemic districts of Nepal during post-MDA surveillance.

BACKGROUND: Sixty-three out of 77 districts reported lymphatic filariasis (LF) endemic in Nepal. Mass drug administration (MDA) with diethylcarbamazine (DEC) and albendazole (ALB) treatment program was continued for 6 to 11 rounds in these districts. Nepal government has stopped the MDA program based on the transmission assessment survey (TAS) report of 2014 and 2018 indicating Wuchereria bancrofti antigenemia prevalence < 2%. But the persistence of low levels of the circulating filarial antigen (CFA) in some foci of four endemic districts of Central Nepal, i.e., 0.4% in Dhading, 0.7% in Mahottari, 0.21% in Lalitpur and 1.2% in Bara district could responsible for enhancing the risk of infection resurgence. Hence the present study was designed to assess antigenic prevalence using Filariasis Test Strip (Alere, Scarborough ME) in children born after MDA in hotspot areas of four endemic districts of Central Nepal. RESULTS: The present study covers 70% children of the eligible population. The result revealed significantly high CFA prevalence in hotspots of Mahottari district belonging to the Terai region and Dhading district belonging to the hilly region, i.e., 13% and 10%, respectively, compared to baseline prevalence and TAS report. While in Lalitpur district and Bara district CFA prevalence was still found to be less than 2%. A higher number of MDA rounds covered in hotspots were found significantly associated with the low antigenic prevalence of W. bancrofti. Whereas median treatment coverage and inter-quartile range (IQR) in study districts were not found significantly associated with CFA prevalence. Although the clinical manifestation of hydrocele (1%) was found in all four study districts, it was not due to the W. bancrofti infection. CONCLUSIONS: Two hotspot regions, one each from the Terai (Mahottari) and hilly (Dhading) districts were found highly prevalent with CFA and significantly associated with the number of MDA rounds but were not associated with treatment coverage and IQR. Higher CFA prevalence was observed in hotspots where baseline prevalence was high together indicating that rounds of MDA program need to be extended further in these hotspot regions of endemic districts.

Minimum risk of intestinal parasitic infection to the visitors of Ghandruk, Kaski, Nepal: a pilot survey.

Ghandruk is one of the famous destinations of both international and domestic tourists situated in Kaski, Nepal. Travel-related diseases are an important aspect that one should consider before making a travel plan. Among diseases, zoonotically important ones make serious worries among visitors. In order to assess the existence of the zoonotically important parasitic disease in Ghandruk, a pilot survey was carried out by examining representative stool samples (n=51) of local residence, domestic animals, pet animals in Ghandruk. Samples were examined using direct smear as well as concentration methods. A questionnaire survey was conducted to see the associated risk factors among residents and their livestock of Ghandruk. None of the faecal samples from residents (n=14) found positive for any kind of intestinal parasites (IPs), while samples from most of the livestock: chicken (86%, 6/7), pigeons (75%, 3/4), cow (66%, 2/3), mule (60%, 3/5), and dog (60%, 3/5) showed heavy infection, except goat and buffalo indicated no infection. Eimeria spp., Ascardia spp. and cestodes spp. were the most prevalent IPs in livestock. Periodic deworming, walking outdoor with sandals/shoes, frequent use of soap and water for handwashing as reported by most of the residents (>80%) could be the main reason behind zero prevalence of IPs in them. The heavy infection among livestock may be incriminated to the contaminated vicinity and free-range livestock and poultry which were noticed in contact with river, sludge, and toilets during our field observation. Conclusively, it indicates that the residents of Ghandruk are conscious about their health, but have not paid satisfactory attention to the hygiene of their domestic animals including livestock, poultry and even pet. Though the observed parasites in livestock are of minimal zoonotic importance regarding safety of visitors, it is deemed necessary for at least to apply some preventive measures to mitigate the burden of parasites in their animals.

Prevalence and risk factors of gastrointestinal parasites in the Chepangs in Nepal.

Gastrointestinal (GI) infection is predominant globally, especially in people with low socio-economic status with existing illiteracy, ignorance, poor housing and lifestyle, and the surrounding environment. It has been implicated as a significant public health concern in the rural parts of many developing countries like Nepal. This study aimed to determine the diversity and prevalence of GI parasites in the highly marginalized indigenous Chepang communities in central Nepal. One hundred fresh stool samples of Chepangs were collected and preserved in 2.5% potassium dichromate solution. The samples were analyzed by direct wet mount, sedimentation, flotation, and acid-fast techniques and examined under the microscope at the total magnifications of 100Å~, 400Å~, and 1000Å~. We detected 97% prevalence rates with eight protozoan and six helminth parasites. Considering the infection's concurrency, mixed pattern of infection was found to be higher than single pattern. Mixed infections from two to seven parasitic species were differently recorded in different samples. We also found that people's eating habits were associated with GI parasitism (p=0.0034). "One Health" Approach/Principle accompanied by the detailed molecular and epidemiologic studies of parasitic transmission is required to identify the causal evidence critical in controlling and preventing parasitic infections.

Sex-Biased Prevalence of Intestinal Parasitic Infections and Gender Inequality in Rural Nepal.

OBJECTIVES: Intestinal parasitic infections (IPIs) are related to poverty and socioeconomic disparities. In rural Nepal, IPIs are highly endemic and gender inequality is still deeply rooted. This study provides a novel epidemiological assessment of IPIs in Nepal, juxtaposing spatial, age and sex stratification of prevalence. METHODS: A PRISMA and PICO-based systematic review of peer-reviewed and grey literature provided IPI prevalence data for the Nepalese population. Demographic and environmental variables were considered to investigate differences related to dwelling area and sex. Variations among prevalence rates were calculated with 95% CI and significance level for odds ratio evaluated with chi-square test. RESULTS: The IPI prevalence rate for the Nepalese general population was 37.6%. Moreover, IPI prevalence (52.3%) was significantly higher in rural areas than in urban areas (32.4%), and school-age girls (55.2%) were more infected than boys (48.6%). CONCLUSIONS: The IPI infection rate appeared to be enhanced among young women living in rural Nepal, where they experienced low school attendance and heavy enrolment in agricultural work, as result of gender discrimination. Plausibly, these dynamics affect both girls' environmental exposure and fruition of periodic school-based preventive chemotherapy, thus increasing the chances of IPI infection.

Prevalence of Gastrointestinal Parasites in the Frugivorous and the Insectivorous Bats in Southcentral Nepal.

Bats are the only active flying placental mammals and are traditionally classified into mega- and microbats, which are, respectively, herbivorous and insectivorous in feeding habit. Though deforestation, habitat destruction, natural calamities, illegal hunting, and climate changes are the challenging threats for bats, the role of existing gastrointestinal (GI) parasites have not been evaluated yet in Nepal. Thus, the current study aims to determine the prevalence of GI parasites in bats from the Shaktikhor area at the Chitwan district of Southcentral Nepal. From July 2018 to February 2019, a total of 60 fecal samples of bats (30 from frugivorous bats and 30 from the insectivorous bats) were collected. These samples were preserved at 2.5% potassium dichromate solution. The fecal examination was carried out by the direct wet mount, concentrations, acid-fast staining, and sporulation techniques. Overall results showed the prevalence rate of 80% GI parasites. The parasites detected in the insectivorous bats were Ascarid spp., Capillarid sp., Cryptosporidium sp., Eimeria spp., Entamoeba sp., Giardia sp., Hymenolepis spp., Isospora sp., Oxyurid sp., Strongyle, and Strongyloides sp. In contrast, Eimeria sp., Entamoeba sp., and Hymenolepis sp. were detected in the frugivorous bats. Based on a wide diversity of parasite richness and parasitic concurrency measured by the prevalence rates, we suggest that GI parasitism might be a threatening factor in the insectivorous bats in the current study area.

Chewing lice from the white-rumped vulture in Nepal, with description of a new species of Aegypoecus.

The purpose of this study was to record the species of chewing lice parasitising Gyps bengalensis (Gmelin, 1788), the white-rumped vulture, in Pithauli, Nawalparasi District, Nepal, from February to June 2017. Three louse species were identified: Cuculiphilus (Aegypiphilus) gypsis (Eichler, 1944), Colpocephalum sp., and the new species Aegypoecus bengalensis, described herein.

Soil-transmitted helminthiases in Nepal: Transmission boundaries and implications for local communities and international travelers.

Soil transmitted helminthiasis (STHs) are endemic in Nepal but since the country territory is extremely heterogeneous, a multidisciplinary assessment is required to evaluate the risk of infection in different areas. Prevalence data were retrieved with systematic literature search and geographical coordinates were assigned to each survey site. Spatial stratification was then implemented considering land use and ecological parameters. STH transmission boundaries were tested after reference criteria and elevation was evaluated as additional predictor: the elevation threshold of 3057 m above sea level is demarking the STH transmission limit. Areas above this threshold are safe, but since lodging in at-risk sites is a compulsory stopover for international travelers, STH risk exposure should be considered for every travel to the country.

Ticks (Acari: Ixodidae) of Nepal: First record of Amblyomma varanense (Supino), with an update of species list.

Males and females specimens of Amblyomma were collected from Orthriophis hodgsoni (Squamata: Colubridae) caught during routine herpetological work in Kathmandu. Morphological characteristics led to the diagnosis of A. varanense, constituting the second Nepalese species for the genus after the collection of Amblyomma gervaisi, also from a colubrid snake, almost 100 years ago. Amblyomma varanense is the 55th species found in the country, and preceded for the collection of 21 species of Haemaphysalis, 14 Ixodes, 6 Rhipicephalus, 6 Hyalomma, 4 Dermacentor, 1 Amblyomma, 1 Anomalohimalaya and 1 species of Nosomma. Eleven and 19 species have been found on humans and domestic mammals, respectively, evidencing the medical and economic importance of Nepalese Ixodidae.

Assessing aquaglyceroporin gene status and expression profile in antimony-susceptible and -resistant clinical isolates of Leishmania donovani from India.

OBJECTIVES: Clinical resistance to pentavalent antimonials results from an interplay between uptake, efflux and sequestration in Leishmania. Aquaglyceroporins (AQPs) have been shown to facilitate uptake of trivalent metalloids. Down-regulation of AQP1 in Leishmania results in resistance to trivalent antimony, whereas overexpression of AQP1 in drug-resistant parasites can reverse the resistance. The present work investigates the role of AQP1 in monitoring antimonial resistance in Indian leishmaniasis. METHODS AND RESULTS: Susceptibility to trivalent antimony as determined in vitro with intracellular amastigotes from both visceral leishmaniasis (VL) and post-kala-azar dermal leishmaniasis (PKDL) patients correlated well with the clinical response. Higher accumulation of trivalent antimony (SbIII) was observed in all susceptible isolates compared with resistant isolates. Reduced accumulation of SbIII correlated, with a few exceptions, with down-regulation of AQP1 RNA as determined by real-time PCR. Cloning and sequencing of the AQP1 gene from both VL and PKDL isolates showed sequence variation in four of the clinical isolates. None of the isolates had an alteration of Glu152 and Arg230, which have been previously shown to affect metalloid transport. Transfection of the AQP1 gene in a sodium antimony gluconate-resistant field isolate conferred susceptibility to the resistant isolate. CONCLUSIONS: Our studies indicate genetic variation in VL and PKDL isolates. Down-regulation of AQP1 correlates well with clinical drug resistance in a majority of Indian VL and PKDL isolates. AQP1 gene expression at both the genetic and transcriptional level showed positive correlation with SbIII accumulation, with some exceptions.

High-throughput screening of amastigotes of Leishmania donovani clinical isolates against drugs using a colorimetric beta-lactamase assay.

A simple colorimetric beta-lactamase assay for quantifying Leishmania amastigotes in macrophages grown in microtiter plates has been reported. The beta-lactamase gene was integrated into the rRNA region of the genome, thereby allowing for high-level stable expression of the enzyme. Both visceral leishmaniasis (VL) and post-kala azar dermal leishmaniasis (PKDL) isolates were transfected with beta-Lactamase gene. These beta-lactamase-expressing promastigotes were used for infecting intracellular J774A.1 macrophages in vitro. Quantification was done by a colorimetric readout with CENTA beta-lactamase as substrate and with an optical density plate reader. The assay was carried out in 96-well plates. Results obtained demonstrate that this methodology could be a valuable high-throughput screening assay for checking efficacy of anti-leishmanial drugs in the clinical isolates.

Role of aquaglyceroporin (AQP1) gene and drug uptake in antimony-resistant clinical isolates of Leishmania donovani.

Antimonial-containing drugs are the first line of treatment against Leishmaniasis. Resistance to antimonials in Leishmania is proposed to be due to reduced uptake of trivalent antimony (SbIII) through the aquaglyceroporin (AQP1). We investigated the uptake of SbIII and involvement of aquaglyceroporin in developing antimony resistance phenotype in Leishmania donovani clinical isolates. SbIII accumulation, copy number of AQP1 gene, and transcript levels were compared in antimony-sensitive versus -resistant isolates. Antimony-resistant field isolates showed reduced uptake of SbIII. The copy number of AQP1 gene showed higher copy number in the antimony-resistant isolates when compared with the sensitive isolates and did not correlate to the reduced uptake of SbIII. Downregulation of AQP1 RNA levels was not consistently found in the antimony-resistant isolates. Our studies indicate that while downregulation of AQP1 may be one of the mechanisms of antimony resistance, it is however not an invariable feature.

Prevalence of Salmonella species in various raw meat samples of a local market in Kathmandu.

A cross-sectional study of raw meat samples from the local meat market of Kathmandu Metropolitan City was carried out during September 2002 to May 2003 with special emphasis on isolation and identification of Salmonella bacteria. A total of 123 raw meat samples (55 chicken, 37 buffalo, and 31 goat) were collected and analyzed relative to season. Salmonella spp was found in 11.4% (14/123) meat samples. Eight samples of chicken, that is, 14.5%, five samples of buffalo (13.5%), and one sample of goat (3.3%) were found to be positive for Salmonella. Salmonella prevalence revealed Salmonella (S.) pullorum in 3.3% samples, S. gallinarum in 0.8%, S. typhi in 1.6%, S. choleraesuis in 0.8%, and Salmonella of subgenus I or II group in 4.9% samples. More than 80% meat samples microbiologically processed indicated coliform contamination. Seasonal prevalence of Salmonella was highest in the months of April/May. Surveys revealed unsatisfactory conditions of sanitation in the local meat markets of Kathmandu.

Trypanosomiasis in domestic animals of Makwanpur district, Nepal.

Trypanosomiasis is an infectious emerging hemoprotozoan parasitic disease in domestical animals of Nepal. The prevalence of disease was found in 16 of 240 (6.67%) in domestic animals of Makawanpur district, out of which 9 of 105 were (8.57%) cattle; 5 of 75 (6.67%) buffalos, and 2 of 15 (13.3%) dogs, while none of the goats and pigs acquired infection. The disease was found maximum during rainy season 9 of 82 (10.98%) with higher prevalence among cross breeds than that of local breeds.

Improving meat inspection and control in resource-poor communities: the Nepal example.

Meat is an important source of protein and a valuable commodity in resource-poor communities. In many developing countries, lack of appropriate slaughtering facilities and unsatisfactory slaughtering techniques are causing unnecessary losses of meat as well as invaluable by-products from animal carcasses. Slaughtering places are frequently contaminated and may not be protected against dogs, rodents and insects. Meat products coming from such conditions are often deteriorated due to bacterial infection or contaminated, which may cause food poisoning or diseases in consumers. In many developing countries, regulations concerning meat inspection and/or control are inadequate or non-existent allowing consumers to be exposed to pathogens including zoonotic parasites. In Nepal, buffaloes contribute about 64% of the meat consumed, followed by goat meat (20%), pork (7%), poultry (6%) and mutton (2%). Goat and poultry meat is acceptable to all castes of people while buffalo meat is consumed mainly by the Newar ethnic group. Previously, pork was consumed only by people belonging to low castes, however, in recent years, the consumption of pork has increased in higher castes as the caste system has become more relaxed. Until recently, there were no official meat inspection regulations in the country, however, in 1999, the national government legislated an as-yet-to-be implemented Animal Slaughtering and Meat Inspection Act which mandates slaughterhouse construction and meat inspection and control. Due to the lack of implementation of the Meat Inspection Act and resultant absence of meat inspection, meat from sick or parasite-infected animals is serving as a source of infection to humans as well as other animals. In addition, meat quality is adversely affected by careless handling conditions in the slaughtering places as well as in the meat markets or shops. For improvement in animal slaughtering and meat inspection in both rural and urban areas of Nepal, several strategies are to be recommended. Sustainable capacity building should be introduced including training of veterinarians, meat inspectors and butchers as well as building of slaughter facilities. Government policies on slaughter procedures including ante-mortem examination, meat inspection and stamping of meat should be implemented. Programmes should be instituted with strong focus on prevention and control of meat-borne diseases to reduce infection risk of consumers and meat handlers and to avoid contamination of the environment. Lastly, emphasis should be put on improving the animal husbandry system in Nepal. These same actions can be undertaken in other developing countries to assist with improving meat inspection and control, thus helping with prevention and control of cysticercosis as well as other important meat-borne diseases.