Kishor Pandey

Molecular characterization of Blastocystis isolates from children and rhesus monkeys in Kathmandu, Nepal.

To investigate the possible transmission of Blastocystis organisms between local rhesus monkeys and children in Kathmandu, Nepal, we compared the subtype (ST) and sequence of Blastocystis isolates from children with gastrointestinal symptoms and local rhesus monkeys. Twenty and 10 Blastocystis isolates were established from 82 and 10 fecal samples obtained from children and monkeys, respectively. Subtype analysis with seven sequence-tagged site (STS) primers indicated that the prevalence of Blastocystis sp. ST1, ST2 and ST3 was 20%, 20% and 60% in the child isolates, respectively. In contrast to human isolates, ST3 was not found in monkey isolates and the prevalence of ST1 and ST2 was 50% and 70%, respectively, including three mixed STs1 and 2 and one isolate not amplified by any STS primers, respectively. Since Blastocystis sp. ST2 has been reported as the most dominant genotype in the survey of Blastocystis infection among the various monkey species, sequence comparison of the 150bp variable region of the small subunit rRNA (SSU rRNA) gene was conducted among ST2 isolates of humans and monkeys. Sequence alignment of 24 clones developed from ST2 isolates of 4 humans and 4 monkeys showed three distinct subgroups, defined as ST2A, ST2B and ST2C. These three subgroups were shared between the child and monkey isolates. These results suggest that the local rhesus monkeys are a possible source of Blastocystis sp. ST2 infection of humans in Kathmandu.

First Isolation of Dengue Virus from the 2010 Epidemic in Nepal

Dengue is an emerging disease in Nepal and was first observed as an outbreak in nine lowland districts in 2006. In 2010, however, a large epidemic of dengue occurred with 4,529 suspected and 917 serologically-confirmed cases and five deaths reported in government hospitals in Nepal. The collection of demographic information was performed along with an entomological survey and clinical evaluation of the patients. A total of 280 serum samples were collected from suspected dengue patients. These samples were subjected to routine laboratory investigations and IgM-capture ELISA for dengue serological identification, and 160 acute serum samples were used for virus isolation, RT-PCR, sequencing and phylogenetic analysis. The results showed that affected patients were predominately adults, and that 10% of the cases were classified as dengue haemorrhagic fever/ dengue shock syndrome. The genetic characterization of dengue viruses isolated from patients in four major outbreak areas of Nepal suggests that the DENV-1 strain was responsible for the 2010 epidemic. Entomological studies identified Aedes aegypti in all epidemic areas. All viruses belonged to a monophyletic single clade which is phylogenetically close to Indian viruses. The dengue epidemic started in the lowlands and expanded to the highland areas. To our knowledge, this is the first dengue isolation and genetic characterization reported from Nepal.

Molecular detection of Leishmania parasites from whole bodies of sandflies collected in Nepal

Visceral leishmaniasis is endemic in the southern part of the Terai region of Nepal. Natural infections of Phlebotomus species with Leishmania parasites in these endemic areas were analyzed by a polymerase chain reaction (PCR) amplification-based assay. A total of 401 Phlebotomus argentipes and 202 P. papatasi female sandflies were captured in the Dhanusa district from 2004 to 2006 and analyzed. It was found that 6.7% of P. argentipes, but no P. papatasi, were positive for Leishmania parasites, suggesting that P. argentipes is a major vector in these areas. The infectivity of P. argentipes with Leishmania was consistent with the infection rates reported from other areas of the world. This is the first report of naturally infected Leishmania parasites in sandflies collected from Nepal.

Characterization of Leishmania isolates from Nepalese patients with visceral leishmaniasis

In Nepal, visceral leishmaniasis (VL) is endemic in 13 districts of the central and eastern regions. A total of 166 bone-marrow aspirates were obtained from patients with suspected VL. Ninety-seven were identified as positive by microscopy, and 29 of those were successfully isolated and cultured. We characterized these isolates by molecular analysis and by their ability to infect mice. PCR-restriction fragment length polymorphism analysis of the mini-exon and the cysteine proteinase b gene showed that all isolates were Leishmania donovani, and the restriction pattern of the Nepalese isolates corresponded to the standard Indian strain of L. donovani but differed from that of the Kenyan strain. The single-strand conformation polymorphism analysis of ribosomal internal transcribed spacer showed no genetic heterogeneity within Nepalese isolates. Intraperitoneal inoculation with the promastigotes of all isolates resulted in amastigote proliferation in the spleen of 20 nude mice, of which ten isolates were highly infective, and ten were moderately infective, including one BALB/c mouse. Of the 20 amastigotes isolated from the spleen of nude mice, only the ten highly infective isolates infected BALB/c mice, of which, two isolates were considered to have low infectivity, three isolates were considered to be moderately infective, and five isolates were considered to be highly infective.

Prevalence of Entamoeba nuttalli infection in wild rhesus macaques in Nepal and characterization of the parasite isolates.

We have recently resurrected the name Entamoeba nuttalli Castellani, 1908 for a potentially virulent ameba isolate, P19-061405, obtained from a rhesus macaque in Kathmandu, Nepal. The ameba was morphologically indistinguishable from Entamoeba histolytica/Entamoeba dispar/Entamoeba moshkovskii, but located phylogenetically between E. histolytica and E. dispar. To evaluate the prevalence of E. nuttalli infection in wild rhesus macaques, 112 fecal samples were collected in four locations of the Kathmandu Valley. PCR analysis of DNA extracted from the feces showed positive rates of E. nuttalli, E. dispar, E. histolytica and E. moshkovskii of 51%, 12%, 0% and 0%, respectively. A total of 14 E. nuttalli isolates were obtained from four locations, of which 6 were established as axenic cultures. The sequences of the serine-rich protein gene of E. nuttalli isolates differed among four locations although no differences were found in the composition of sequence motifs. Isoenzyme pattern was analyzed in 8 isolates obtained from three locations. In hexokinase, the mobility of the slower migrating band was located between E. histolytica and E. dispar regardless of the culture conditions. These results demonstrate that E. nuttalli is highly prevalent in wild rhesus macaques in Nepal. Rhesus macaques appear to be one of the natural hosts and heterogeneity of the serine-rich protein gene might be useful for geographical typing of isolates.

Diagnosis of visceral leishmaniasis by polymerase chain reaction of DNA extracted from Giemsa's solution-stained slides.

Visceral leishmaniasis (VL) is caused by the protozoan parasite Leishmania donovani and is a potentially fatal disease in endemic areas of the world. Nepal is an endemic area in which VL causes major public health problems in the lowland areas of the southeast regions. The aim of the present study was to evaluate the sensitivity of polymerase chain reaction (PCR) amplification for the detection of Leishmania DNA from Giemsas solution-stained bone marrow slides. Bone marrow samples were aspirated from a total of 115 VL suspected patients and used to prepare smears on glass slides and for the initiation of in vitro culture. Bone marrow slides were used for microscopic observation, DNA extraction, and subsequent PCR amplification. PCR analysis showed that all the positive samples were of Leishmania parasites. The PCR assay also showed a higher sensitivity (69%) than microscopic examination (57%) and culture (21%). In addition, PCR was able to detect VL in 12% of samples which were negative by microscopy. PCR of DNA extracted from Giemsas solution-stained bone marrow slides is a suitable tool for confirming diagnosis in patients with VL and may also be useful in the diagnosis of difficult cases. Bone marrow smears are easily stored and can be easily sent to research centers where PCR is available. This makes PCR a good option for diagnosis in the field.

Case Report : Expansion of Visceral Leishmaniasis to the Western Hilly Part of Nepal

We report the first case of visceral leishmaniasis (VL) from the non-endemic western hilly region of Nepal. The patient presented with a history of high-grade fever, abdominal distension, anemia, and weight loss. The case was confirmed as VL by microscopical detection of the Leishmania species amastigote in bone marrow aspiration and by a positive result for the rK39 test. The patient was treated with 0.5-1.0 mg/kg of Amphotericin B for 14 days (total of 405 mg), and amastigotes were negative on discharge. Five months later, this patient again developed fever, abdominal distension, and anemia. Clinical and hematological examinations suggested a relapse of VL. The patient was treated with 1 mg/kg of Amphotericin B for 18 days (total of 515 mg) and was clinically improved on discharge.

Detection of Chikungunya Virus in Nepal

Chikungunya virus (CHIKV) is an emerging alphaviral disease and a public health problem in South Asia including Nepal in recent years. In this study, sera were collected from patients presenting with fever, headache, muscular pain, fatigue, and joint pain of both upper and lower extremities. A total of 169 serum samples were tested for CHIKV and dengue virus (DENV) by using Immunoglobulin M (IgM) and Immunoglobulin G (IgG) antibody using enzyme-linked immunosorbent assay (ELISA) method during August to November 2013. Results showed that 3.6% and 27.8% samples were positive for CHIKV and DENV IgM positive, respectively. Similarly, results of IgG showed 3.0% samples were positive for CHIKV IgG and 29.0% were for DENV IgG positive. Further, a 50% focal reduction neutralization test (FRNT50) was performed to confirm the presence of CHIKV, which demonstrated that 8.9% of CHIKV IgM and/or IgG ELISA positive possessed neutralizing anti-CHIK antibodies. To our knowledge, this is the first report in which the presence of CHIKV is confirmed in Nepalese patients by FRNT50. Basic scientists and clinicians need to consider CHIKV as a differential diagnosis in febrile Nepalese patients, and policy makers should consider appropriate surveillance and actions for control strategies.

A Series of Case Reports of Autochthonous Visceral Leishmaniasis, Mostly in Non-Endemic Hilly Areas of Nepal

The government of Nepal has committed to eliminate visceral leishmaniasis (VL) by 2015. The expansion of VL into new areas would constitute a major obstacle to achieving this goal. We report a series of autochthonous VL cases from areas currently considered non-endemic, mostly in hilly regions of Nepal.

Ca2+ monitoring in Plasmodium falciparum using the yellow cameleon-Nano biosensor

Calcium (Ca2+)-mediated signaling is a conserved mechanism in eukaryotes, including the human malaria parasite, Plasmodium falciparum. Due to its small size (<10 μm) measurement of intracellular Ca2+ in Plasmodium is technically challenging, and thus Ca2+ regulation in this human pathogen is not well understood. Here we analyze Ca2+ homeostasis via a new approach using transgenic P. falciparum expressing the Ca2+ sensor yellow cameleon (YC)-Nano. We found that cytosolic Ca2+ concentration is maintained at low levels only during the intraerythrocytic trophozoite stage (30 nM), and is increased in the other blood stages (>300 nM). We determined that the mammalian SERCA inhibitor thapsigargin and antimalarial dihydroartemisinin did not perturb SERCA activity. The change of the cytosolic Ca2+ level in P. falciparum was additionally detectable by flow cytometry. Thus, we propose that the developed YC-Nano-based system is useful to study Ca2+ signaling in P. falciparum and is applicable for drug screening.