Gemykibivirus detection in acute encephalitis patients from Nepal.

Acute encephalitis syndrome (AES) causes significant morbidity and mortality worldwide. In Nepal, Japanese encephalitis virus (JEV) accounts for ~5-20% of AES cases, but ~75% of AES cases are of unknown etiology. We identified a gemykibivirus in CSF collected in 2020 from an 8-year-old male patient with AES using metagenomic next-generation sequencing. Gemykibiviruses are single stranded, circular DNA viruses in the family Genomoviridae. The complete genome of 2,211 nucleotides was sequenced, which shared 98.69% nucleotide identity to its closest relative, Human associated gemykibivirus 2 isolate SAfia-449D. Two real-time PCR assays were designed, and screening of 337 cerebrospinal fluid (CSF) and 164 serum samples from AES patients in Nepal collected in 2020 and 2022 yielded 11 CSF and 1 serum sample that were positive in both PCR assays. Complete genomes of seven of the positives were sequenced. These results identify a potential candidate etiologic agent of encephalitis in Nepal. IMPORTANCE: Viral encephalitis is a devastating disease, but unfortunately, worldwide, the causative virus in many cases is unknown. Therefore, it is important to identify viruses that could be responsible for cases of human encephalitis. Here, using metagenomic sequencing of CSF, we identified a gemykibivirus in a male child from Nepal with acute encephalitis syndrome (AES). We subsequently detected gemykibivirus DNA in CSF or serum of 12 more encephalitis patients by real-time PCR. The virus genomes we identified are highly similar to gemykibiviruses previously detected in CSF of three encephalitis patients from Sri Lanka. These results raise the possibility that gemykibivirus could be an underrecognized human pathogen.

Genomic sequencing and neutralizing serological profiles during acute dengue infection: A 2017 cohort study in Nepal.

Dengue virus (DENV) is a mosquito-borne flavivirus that poses a threat to nearly 50% of the global population. DENV has been endemic in Nepal since 2006; however, little is known about how DENV is evolving or the prevalence of anti-DENV immunity within the Nepalese population. To begin to address these gaps, we performed a serologic and genetic study of 49 patients from across Nepal who presented at central hospitals during the 2017 dengue season with suspected DENV infection. Of the 49 subjects assessed, 21 (43%) were positive for DENV NS1 antigen; of these; 5 were also anti-DENV IgM + IgG + ; 7 were DENV IgM + IgG - , 2 were IgM - IgG + , and 7 were IgM - IgG - by specific ELISAs. Seven of the 21 NS1+ sera were RNA+ by RT-PCR (six DENV2, one DENV3), suggesting that DENV2 was the dominant serotype in our cohort. Whole-genome sequencing of two DENV2 isolates showed similarity with strains circulating in Singapore in 2016, and the envelope genes were also similar to strains circulating in India in 2017. DENV-neutralizing antibodies (nAbs) were present in 31 of 47 sera tested (66%); among these, 20, 24, 26, and 12 sera contained nAbs against DENV1, 2, 3, and 4 serotypes, respectively. Serology analysis suggested that 12 (26%) and 19 (40%) of the 49 subjects were experiencing primary and secondary DENV infections, respectively. Collectively, our results provide evidence for current and/or past exposure to multiple DENV serotypes in our cohort, and the RNA analyses further indicate that DENV2 was the likely dominant serotype circulating in Nepal in 2017. These data suggest that expanded local surveillance of circulating DENV genotypes and population immunity will be important to effectively manage and mitigate future dengue outbreaks in Nepal.

Dengue Virus Surveillance in Nepal Yields the First On-Site Whole Genome Sequences of Isolates from the 2022 Outbreak.

Background: The 4 serotypes of dengue virus (DENV1-4) can each cause potentially deadly dengue disease, and are spreading globally from tropical and subtropical areas to more temperate ones. Nepal provides a microcosm of this global phenomenon, having met each of these grim benchmarks. To better understand DENV transmission dynamics and spread into new areas, we chose to study dengue in Nepal and, in so doing, to build the onsite infrastructure needed to manage future, larger studies. Methods and Results: During the 2022 dengue season, we enrolled 384 patients presenting at a hospital in Kathmandu with dengue-like symptoms; 79% of the study participants had active or recent DENV infection (NS1 antigen and IgM). To identify circulating serotypes, we screened serum from 50 of the NS1 + participants by RT-PCR and identified DENV1, 2, and 3 - with DENV1 and 3 codominant. We also performed whole-genome sequencing of DENV, for the first time in Nepal, using our new on-site capacity. Sequencing analysis demonstrated the DENV1 and 3 genomes clustered with sequences reported from India in 2019, and the DENV2 genome clustered with a sequence reported from China in 2018. Conclusion: These findings highlight DENV's geographic expansion from neighboring countries, identify China and India as the likely origin of the 2022 DENV cases in Nepal, and demonstrate the feasibility of building onsite capacity for more rapid genomic surveillance of circulating DENV. These ongoing efforts promise to protect populations in Nepal and beyond by informing the development and deployment of DENV drugs and vaccines in real time.

Gemykibivirus detection in acute encephalitis patients from Nepal.

Acute Encephalitis Syndrome (AES) causes significant morbidity and mortality worldwide. In Nepal, Japanese encephalitis virus (JEV) accounts for ~ 5-20% of AES cases, but ~75% of AES cases are of unknown etiology. We identified a gemykibivirus in CSF collected in 2020 from a male child with AES using metagenomic next-generation sequencing. Gemykibiviruses are single stranded, circular DNA viruses in the family Genomoviridae. The complete genome of 2211 nucleotides was sequenced which shared 98.69% nucleotide identity to its closest relative, Human associated gemykibivirus 2 isolate SAfia-449D. Two real-time PCR assays were designed, and screening of 337 CSF and 164 serum samples from AES patients in Nepal collected in 2020 and 2022 yielded 11 CSF and 1 serum sample that were positive in both PCR assays. Complete genomes of 7 of the positives were sequenced. These results identify a candidate etiologic agent of encephalitis in Nepal.

Effect of ABCA1-R219K Polymorphism in Serum Lipid Parameters in Patients under Statin Therapy Visiting Tertiary Cardiac Center of Nepal.

Introduction ATP-binding cassette transporter A1 (ABCA1) encoded by ABCA1 gene is one of the important protein involved in lipid metabolism. The effect of statin therapy on dyslipidemia varies among individuals and it may be due to different genetic polymorphism. The R219K polymorphism of ABCA1 gene is found to have a significant role in the response of statin. Objective This study was designed to evaluate the effect of R219K polymorphism in lipid-lowering action of statin in patients with dyslipidemia. Material and Methods This study was conducted in 88 patients. Blood samples were taken from patients before and at the end of 3 months of statin use and were analyzed for lipid profile. Whole blood was analyzed for R219K Polymorphism using polymerase chain reaction-restriction fragment length polymorphism. Results R219K polymorphism was associated with significant percentage reduction of serum triglyceride/high-density lipoprotein (TG/HDL) ratio and total cholesterol/high-density lipoprotein (TC/HDL) ratio in atorvastatin users. However, there was no significant association of polymorphism with change in serum TC, HDL-C, LDL-C, TG, and very low-density lipoprotein (VLDL). Among KK genotype individuals, value of TG, VLDL, TG/HDL, and TC/HDL were significantly lower than in RR genotypes. Also, TG/HDL and TC/HDL were significantly lower in RK genotype than in RR. Treatment of dyslipidemia with statin was found to be comparatively better in patients having the genotypes KK and RK. Conclusion Our study demonstrated association of R219K polymorphism with the significant reduction of TG/HDL and TC/HDL and particularly the KK genotype was associated with significant improvement of lipid parameters following atorvastatin treatment.

Leishmania donovani persistence and circulation causing cutaneous leishmaniasis in unusual-foci of Nepal.

Cutaneous leishmaniasis cases have increased dramatically in recent years in Nepal. The study offers molecular identification of the Leishmania species using 40 patient's aspiration biopsy samples, targeting markers kinetoplast minicircle DNA (kDNA) and internal transcribed spacer-1 (ITS1). Among molecularly diagnosed 22 cutaneous leishmaniasis cases, L. donovani complex was identified in 13 instances and L. major in 9 cases. The ITS1 PCR was positive in 12 of the positive nested- kDNA PCR cases (12/22), confirming L. donovani complex in seven of the cases and L. major in five of the cases. In addition, the study conclude that concurrent occurrence of atypical cutaneous infections caused by L. donovani parasite in 59.1% of cases and typical cutaneous infections caused by L. major parasite in 40.9% of cases. A Phylogentic analaysis showed that the detected L. donovani species present null genetic distances from seven references of L. donovani, but slight differences between ITS1 sequences and not grouped into a significant monophyletic cluster.

Multiple Orientia clusters and Th1-skewed chemokine profile: a cross-sectional study in patients with scrub typhus from Nepal.

OBJECTIVES: Scrub typhus is an emerging infectious disease in Asia caused by Orientia tsutsugamushi (Ot). From Nepal, only scant data on the genetic epidemiology of this agent is available, and determinants of immunoregulation are poorly understood. METHODS: Patients (n = 238) referred to the National Public Health Laboratory (Kathmandu, Nepal) from all over Nepal for suspected scrub typhus were enrolled upon positive immunoglobulin (Ig)M testing between July and October 2015. From Ot 16S and 47 kD polymerase chain reaction (PCR)-positive samples, the variable domain I of the 56 kD gene was sequenced and phylogenetically analyzed. T helper (Th) cell-associated cytokines (n = 13) and chemokines (n = 12) were quantified by multiplex bead arrays. RESULTS: In 93/238 (39.1%) IgM-positive samples, Ot DNA was detected by quantitative PCR. Phylogenetic analysis of 56 kD sequences revealed seven distinct clusters, six of them with high homologies to strains detected in other countries. The Th1-related cytokines interferon-γ and C-X-C motif chemokine ligand 10 were strongly upregulated and correlated with bacteremia, while levels of Th2-associated chemokines were reduced. Bacteremia also correlated with concentrations of interleukin (IL)-6 and IL-10 but not tumor necrosis factor-α. CONCLUSION: We identified a considerable genetic heterogeneity of human-pathogenic Ot strains circulating in Nepal. Acute Nepalese scrub typhus patients showed strong Th1 but impaired Th2 responses, especially on the chemokine level.

Pyridine-Based NNS Tridentate Chitosan Thiosemicarbazones and Their Copper(II) Complexes: Synthesis, Characterization, and Anticancer Activity.

Chitosan-functionalized pyridine-based thiosemicarbazones and their copper(II) complexes have been found to own a substantial antiproliferative activity against the tumorigenic Madin Darby canine kidney (MDCK) and MCF-7 cancer cell lines. In the current study, chitosan oligosaccharide (CS) (87% DDA, Mw < 3000 Da) and crab shell chitosan (CCS) (67% DDA, M w 350 kDa) were functionalized as chitosan pyridine-2-thiosemicarbazones and chitosan 2-acetyl pyridine-2-thiosemicarbazones, and their copper(II) complexes were synthesized. The formation of chitosan thiosemicarbazones and their NNS tridentate behavior to give the square planar copper(II) chitosan thiosemicarbazone complexes were established by spectroscopic studies, powder X-ray diffraction, elemental analysis, and magnetic moment measurements. The thermal study showed a marked stability of these derivatives before the outset of chitosan backbone degradation at 200 °C. The colorimetric MTT assay revealed a higher activity of CS thiosemicarbazones, viz., CSTSC series (IC50 375-381 µg mL-1 in the MDCK cell line and 281-355 µg mL-1 in the MCF-7 cell line) than that of high-molecular-weight CCS thiosemicarbazones, viz., CCSTSC series (IC50 335-400 µg mL-1 in the MDCK cell line and 365-400 µg mL-1 in the MCF-7 cell line), showing an enhanced activity with a decrease in Mw and an increase in DDA of constituent chitosan, a higher activity of both of these series of thiosemicarbazones than that of their native chitosan, viz., CS (IC50 370 µg mL-1 in the MCF-7 cell line and >400 µg mL-1 in the MDCK cell line) and CCS (IC50 > 400 µg mL-1 in both cell lines), and a higher activity of the Cu-CSTSC complexes (IC50 322-342 µg mL-1 in the MDCK cell line and 278-352 µg mL-1 in the MCF-7 cell line) and Cu-CCSTSC complexes (IC50 274-400 µg mL-1 in the MDCK cell line and 231-352 µg mL-1 in the MCF-7 cell line) than that of their respective ligands.

Isolation and Characterization of Lytic Bacteriophage Against Multi-drug Resistant Pseudomonas aeruginosa.

BACKGROUND: Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen frequently causing healthcare-associated infections. The apocalyptic rise of antimicrobial resistance has rekindled interest in age-old phage therapy that uses phages (viruses that infect bacteria) to kill the targeted pathogenic bacteria. Because of its specificity, phages are often considered as potential personalized therapeutic candidate for treating bacterial infections. METHODS: In this study, we isolated and purified lytic phages against multi-drug resistant P. aeruginosa using soft agar overlay technique. Phage characteristics like thermal and pH stability, latent period and burst size were determined using one-step growth assay while multiple host range spectrum was determined by spot assay. The phages were further characterized using protein profiling. RESULTS: Three Pseudomonas phages (øCDBT-PA31, øCDBT-PA56 and øCDBT-PA58) were isolated from the holy rivers of Kathmandu valley. Among 3 phages, øCDBT-PA31 demonstrated multiple host range and could lyse multi-drug resistant strain of P. aeruginosa. Further, øCDBT-PA31 showed latent period of 30 minutes with corresponding burst sizes of 423-525 PFU/cell. Interestingly, øCDBT-PA31 also tolerated a wide range of adverse conditions, such as high temperature (50°C) and pH 3-11. Further, protein profiling revealed that øCDBT-PA31 has 4 and øCDBT-PA11 had 3 distinct bands in the gradient gel ranging from approximately 3.5-29 kilodaltons (kDa) suggesting them to be morphologically distinct from each other. CONCLUSIONS: As multi-drug resistant bacteria are emerging as a global problem, lytic phages can be an alternative treatment strategy when all available antibiotics fail.

Accuracy of lymphocyte counts from UniCel DxH 800 in ß-thalassemia/HbE patients having various numbers of nucleated red blood cells.

BACKGROUND: The UniCel® DxH-800 is an automated cell counter widely used in laboratories. However, the effects of increased nucleated red blood cells (NRBCs) on the lymphocyte counts obtained using the UniCel DxH-800 have not been fully elucidated. OBJECTIVE: The study's objective was to compare lymphocyte counts obtained using the DxH-800 and those obtained using flow cytometry in various ranges of NRBCs. METHODS: This cross-sectional study analyzed 25 healthy volunteers and 69 ß-thalassemia/HbE patients. The numbers of lymphocytes were determined using a UniCel DxH-800 and a standard flow cytometer using counting beads. RESULTS: In healthy volunteers, regression analysis of the lymphocyte counts using the two approaches showed an r2 0.85 and a p < 0.0001, and a Bland-Altman plot showed mean bias of +264 cells/µL. In ß-thalassemia/HbE patients, regression analysis of the lymphocyte counts obtained using an automated cell counter and a flow cytometer showed an r2 of 0.06, a p = 0.028, and a Bland-Altman plot showed the mean bias of +1,509 cells/µL. In addition, a high degree of discrepancy in the lymphocyte counts was observed in ß-thalassemia/HbE patients who had NRBCs > 100,001 cells/µL. CONCLUSIONS: The present study demonstrated that the UniCel DxH-800 performed well in enumerating lymphocytes in specimens that contained various numbers of NRBCs. However, a high number of NRBCs may interfere with lymphocyte counts obtained using the counter.

First report on the molecular detection of Entamoeba bovis from the endangered wild water buffalo (Bubalus arnee) in Nepal.

BACKGROUND: The Asiatic wild water buffalo (Bubalus arnee) is an endangered species that is conserved in the Koshi Tappu Wildlife Reserve (KTWR), Nepal, and was recently translocated to the Chitwan National Park (CNP). Gastrointestinal (GI) parasites are the cause of significant negative health and production impacts on animals worldwide. METHODS: A coprological survey of GI parasites of wild water buffalo was carried out in the CNP in 2020. Fresh dung samples (n = 25) were collected from wild water buffaloes and analysed using sedimentation and flotation techniques for morphological identification of parasite cysts, oocysts and eggs. RESULTS: Nine different GI parasites were recorded of which Entamoeba spp. (20 samples, 80%) were the most common. The presence of Entamoeba spp. was further validated using polymerase chain reaction (PCR) analysis and DNA sequencing. The PCR results were positive for all of the microscopically positive samples, and the species was identified as Entamoeba bovis. Three samples were sequenced and formed a cluster of E. bovis, which was separated from other Entamoeba spp. in phylogenetic analysis. CONCLUSION: This is the first report for molecular detection of E. bovis from wild water buffaloes in Nepal. Future work should focus on the prevalence of such infections in water buffaloes in forest environments.

Whole Genome Sequencing of Dengue Virus Serotype 2 from Two Clinical Isolates and Serological Profile of Dengue in the 2015-2016 Nepal Outbreak.

Dengue virus (DENV) is the cause of one of the most prevalent neglected tropical diseases, and up to half of the world's population is at risk for infection. Recent results from clinical trials have shown that DENV vaccination can induce the development of severe dengue disease and/or prolong hospitalization after natural infection in certain naive populations. Thus, it is crucial that vaccine development takes into account the history of DENV exposure in the targeted population. In Nepal, DENV infection was first documented in 2004, and despite the increasing prevalence of DENV infection, the population remains relatively naive. However, it is not known which of the four DENV serotypes circulate in Nepal or whether there is evidence of repeated exposure to DENV in the Nepali population. To address this, we studied 112 patients who presented with symptomology suspicious for DENV infection at clinics throughout Nepal during late 2015 and early 2016. Of the 112 patients examined, 39 showed serological and/or genetic evidence of primary or secondary DENV infection: 30 were positive for DENV exposure by IgM/IgG ELISA, two by real-time reverse-transcription PCR (RT-PCR), and seven by both methods. Dengue virus 1-3, but not DENV4, serotypes were detected by RT-PCR. Whole genome sequencing of two DENV2 strains isolated from patients with primary and secondary infections suggests that DENV was introduced into Nepal through India, with which it shares a porous border. Further study is needed to better define the DENV epidemic in Nepal, a country with limited scientific resources and infrastructure.

Serological markers and molecular analysis of hepatitis B infection in a tertiary care hospital at Kathmandu, Nepal.

AIM: To analyze the serology and molecular markers of the hepatitis B-infected patients from the tertiary care hospital at Kathmandu in Nepal. METHODS: A total of 399 blood samples of patients from Sukraraj Tropical and Infectious Disease Hospital, Teku, Kathmandu, were collected. Samples were tested for HBsAg, HBeAg, and IgM anti-HBc using ELISA method. The samples were further categorized as acute and chronic. The genotyping was performed by real-time polymerase chain reaction (real-time PCR) and further validated by sequencing. RESULTS: Out of 399 samples that were collected, 271 and 128 samples were acute and chronic cases respectively. Fifty-six samples were genotyped by qPCR, out of which 40 samples belonged to genotype D, 4 to C/D recombinant, 5 to genotype C, 3 to genotype B, and 4 were genotype A respectively. From these, 15 samples were used for sequencing of P (polymerase) gene and S (surface) genes. Thus, obtained sequences were used to construct neighbor-joining tree using Tamura-Nei model evolution and further validated by Bayesian analysis. A total of four sub-genotypes namely A1, C1, D1, and D5 were detected. CONCLUSION: Hepatitis B virus infection is a global health problem affecting about 257 million people worldwide. In Nepal, there are few reports on the molecular and phylogenetic analysis of this virus. In this study, we report the circulation of seropositive occult hepatitis as well as CD-recombinant genotype in Nepalese population.

Cutaneous Leishmaniasis of Lip and Role of Polymerase Chain Reaction: A Case Report.

The diagnosis of cutaneous leishmaniasis is mostly confirmed by the identification of parasite in a skin smear or biopsy. However, this method may not always be sensitive enough to detect the disease when parasitic load is low. Molecular test such as polymerase chain reactions can be useful in such circumstances. Here, we report a case of cutaneous leishmaniasis diagnosed by a polymerase chain reaction test when both smear and biopsy failed to confirm the diagnosis. A 17-years-old female from mountainous district of Nepal, presented with a crusted plaque over the upper lip for a duration of 6 months. Both skin smear and biopsy from the lesion failed to demonstrate Leishmania parasite but a polymerase chain reaction test was positive for Leishmania donovani. This case emphasizes on the importance of molecular testing such as polymerase chain reaction when commonly performed diagnostics test fails to support confirmation of clinical diagnosis.

Molecular phylogeny and distribution of dengue virus serotypes circulating in Nepal in 2017.

Dengue virus (DENV) infection is endemic in Nepal. Although infection rates are reported annually, little information is available about the circulating viral serotypes and genotypes. Here, we report the results of a multicentre cross-sectional study of DENV serotypes and genotypes sampled from individuals with suspected DENV infection in Nepal in 2017. Of the 50 patients sampled, 40 were serologically positive for DENV NS1, 29 for anti-DENV IgM, 21 for anti-DENV IgG and 14 were positive by qRT-PCR. The three serotypes DENV-1, 2 and 3 were detected and there was no DENV-4. Positive samples from serotyping were subjected to PCR amplification by envelope (E) gene specific primer and subsequent bidirectional sequencing of 5 samples. A time to most recent common ancestor phylogenetic tree was constructed from the new sequences obtained here together with historical DENV-1 and DENV-2 E gene sequences. The DENV-1 isolates (n = 2) from Nepalese individuals were closely related to Indian genotype V, whereas DENV-2 isolates (n = 3) belonged to Cosmopolitan genotype IVa, which is closely related to Indonesian isolates. Historical DENV isolates obtained between 2004 and 2013 clustered with Cosmopolitan IVb, Cosmopolitan IVa, and Asian II genotypes. All Nepalese isolates had different lineages with distinct ancestries. With the exception of isolates obtained in 2004, all other previously published isolates had ancestry to geographically distant part of the world. Molecular analysis revealed dengue epidemics to be comprised of different genotypes of serotype 1 and 2 raising concerns on potential role of different genotypes causing Dengue hemorrhagic fever. Also, our result indicated spread of DENV-2 in non-endemic area such as hilly region of Nepal which was considered to be free of dengue due to high altitude and cold weather.

Overview of seasonal influenza and recommended vaccine during the 2016/2017 season in Nepal.

BACKGROUND: Influenza is a highly contagious viral respiratory infection caused by influenza viruses whose epidemic and pandemic have resulted in significant morbidity and mortality. The annual epidemic of influenza results in an estimated 3-5 million cases of severe illness and about 290000-650000 deaths globally. The vaccination program has been successful to control the epidemic however, it further needs improvement. This study was aimed to investigate the types of influenza viruses prevailing in Nepal during 2016 and, to match the recommended vaccine for use during the same season. METHODS: A descriptive cross sectional study was carried out at National Public Health Laboratory, Kathmandu, Nepal for the period of one year (Jan-Dec 2016). A total of 1683 throat swab specimen was collected from patients of different age group referred to NPHL for influenza testing. The specimen was primarily stored at 4 °C and processed using ABI 7500 RT PCR system for the identification of influenza viruses. RESULTS: Of the total 1683 patients suspected of having influenza infection, influenza viruses were isolated from 614 (36.5%) patients with male predominance. The highest number of infection was caused by influenza A/H3 strain (51.0%) followed by influenza B (40.4%) and influenza A (H1N1) pdm09 (8.6%). Two peaks of infection were observed during the year 2016. The widely available trivalent vaccine during the season did not match the prevailing strain because of the dominance of B/Yamagata lineage over B/Victoria lineage. CONCLUSION: We concluded that Nepal experiences semiannual cycle of influenza infection, firstly during the month of January-February and secondly during the month of July-August. The vaccine to be introduced in Nepal need to be decided by national authority based on prevailing influenza types to confer effective immunization.

A case of high altitude cutaneous leishmaniasis in a non-endemic region in Nepal.

A case of cutaneous leishmaniasis was discovered in a 32-year old man with a persistent erythematous plaque. The patient resides in a high altitude (~2000 m above sea level) area that is not endemic for cutaneous leishmaniasis in the Dunai village of Dolpa, Nepal. The patient's lesion was initially misdiagnosed as lupus vulgaris. After response failure to initial treatment, additional testing by histological microscopy revealed the presence of Leishmania amastigotes in tissue from the lesion, and the diagnosis of cutaneous leishmaniasis was confirmed by nested PCR DNA assay of tissue from the lesion, and by a positive rK39 test in blood. Sequencing of the kinetoplast region confirmed the presence of Leishmania donovani complex. The patient responded well to treatments for cutaneous leishmaniasis and the skin lesions regressed after 6 months. This is the first known case of cutaneous leishmaniasis in a patient in Nepal who resides at high altitude in a non-endemic region. Increasing temperatures in this region of Nepal may be expanding the range of vectors that transmit cutaneous leishmaniasis.

Molecular evidence supports the expansion of visceral leishmaniasis towards non-program districts of Nepal.

BACKGROUND: Visceral Leishmaniasis (VL) is caused by a protozoan parasite Leishmania donovani that is transmitted to humans by an infected female sandfly, Phlebotomus argentipes. VL is common in the Indian sub-continent including Nepal and efforts for its elimination are ongoing. However, expansion of disease towards the higher altitude areas, previously considered as VL free in Nepal, may impact the ability to achieve the elimination target by 2020. METHODS: This was an exploratory study, where VL suspected patients living exclusively in the non-program districts of Nepal and presenting with fever > 2 weeks and splenomegaly was included. The patients' blood samples were collected, and DNA was extracted. DNA was subjected to PCR amplification and subsequent sequencing. Additionally, past 10 years data of VL cases from the national databases were analysed to see the trends of the disease in program and non program districts. RESULTS: Analysis of the past 10 years data revealed that trend of VL cases significantly decreased in the program districts (p = 0.001) while it increased in the non-program districts (p = 0.002). The national trend for overall incidence of VL also significantly decreased over this time period. Limited number of patients' samples (n = 14) were subjected to molecular investigation, and four patients were found to be positive for Leishmania species by PCR. Interestingly, these cases in non-program districts were indeed also L. donovoni complex. All four patients were male with age ranges from 10 to 68 years. GenBank BLAST of the obtained DNA sequences confirmed identified specimens as L. donovani complex. We identified additional VL cases from non-program districts (including the high lands) of Nepal, indicating that the infection could be an emerging threat for the non-program areas of Nepal. CONCLUSION: The demonstration of VL cases in areas initially considered non-endemic has raised concern about on-going transmission in those regions and may trigger subsequent government plan and action to include those areas in the elimination program. Thus, the government should consider revising the disease control programs to accommodate non-program districts for achieving the VL elimination goal set for 2020.

Visceral leishmaniasis from a non-endemic Himalayan region of Nepal.

Visceral leishmaniasis (VL) is endemic to the southern plains of Nepal. Here, we report the first case of VL from a non-endemic Himalayan region of Nepal. The patient presented with a history of high-grade fever, splenomegaly, and anemia but had not traveled to a VL-endemic region. Visceral leishmaniasis was diagnosed following microscopic detection of the Leishmania species amastigote in a bone marrow aspirate, positive result for the rK39 test, and further validation by nested polymerase chain reaction (PCR). The patient was treated with 5 mg/kg liposomal amphotericin B and was clinically improved upon discharge. Our result suggests that VL is expanding towards non-endemic regions of Nepal, and it should therefore be considered that VL surveillance systems be strengthened, particularly for non-program districts and VL be included as a differential diagnosis in febrile illnesses.

Dengue periodic outbreaks and epidemiological trends in Nepal.

Dengue is a global health problem and expansion of its endemics towards new territories in the hilly regions in Nepal is a serious concern. It appeared as a new disease in Nepal in 2004 from Japanese traveler with sporadic cases every year and massive outbreaks in 2010, 2013 and 2016. The serotype was responsible for outbreak in particular year was dengue virus serotype-1 (DENV-1) in 2010, 2016; and DENV-2 in 2013. Nepal lacks basic health related infrastructure in rural areas and does not have a stringent health care policy. With severances of epidemic like dengue, a new surveillance or an upgrading of existing one are direly needed to better challenge the possible outbreaks. This review paper aims to explain the dengue trend in last one decade in Nepal and warrants concerted and timely public health interventions to minimize the deleterious effects of the disease.