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.

Hyperlocal Variation in Soil Iron and the Rhizosphere Bacterial Community Determines Dollar Spot Development in Amenity Turfgrass.

Dollar spot, caused by the fungal pathogen Clarireedia spp., is an economically important foliar disease of amenity turfgrass in temperate climates worldwide. This disease often occurs in a highly variable manner, even on a local scale with relatively uniform environmental conditions. The objective of this study was to investigate mechanisms behind this local variation, focusing on contributions of the soil and rhizosphere microbiome. Turfgrass, rhizosphere, and bulk soil samples were collected from within a 256-m2 area of healthy turfgrass, transported to a controlled environment chamber, and inoculated with Clarireedia jacksonii Bacterial communities were profiled by targeting the 16S rRNA gene, and 16 different soil chemical properties were assessed. Despite their initial uniform appearance, the samples differentiated into highly susceptible and moderately susceptible groups following inoculation in the controlled environment chamber. The highly susceptible samples harbored a unique rhizosphere microbiome with suggestively lower relative abundance of putative antibiotic-producing bacterial taxa and higher predicted abundance of genes associated with xenobiotic biodegradation pathways. In addition, stepwise regression revealed that bulk soil iron content was the only significant soil characteristic that positively regressed with decreased dollar spot susceptibility during the peak disease development stage. These findings suggest that localized variation in soil iron induces the plant to select for a particular rhizosphere microbiome that alters the disease outcome. More broadly, further research in this area may indicate how plot-scale variability in soil properties can drive variable plant disease development through alterations in the rhizosphere microbiome.IMPORTANCE Dollar spot is the most economically important disease of amenity turfgrass, and more fungicides are applied targeting dollar spot than any other turfgrass disease. Dollar spot symptoms are small (3 to 5 cm), circular patches that develop in a highly variable manner within plot scale even under seemingly uniform conditions. The mechanism behind this variable development is unknown. This study observed that differences in dollar spot development over a 256-m2 area were associated with differences in bulk soil iron concentration and correlated with a particular rhizosphere microbiome. These findings provide interesting avenues for future research to further characterize the mechanisms behind the highly variable development of dollar spot, which may inform innovative control strategies. Additionally, these results suggest that small changes in soil properties can alter plant activity and hence the plant-associated microbial community, which has important implications for a broad array of agricultural and horticultural plant pathosystems.

Analysis of soft rot Pectobacteriaceae population diversity in US potato growing regions between 2015 and 2022.

Introduction: Soft rot Pectobacteriaceae (SRP) bacteria are globally dispersed pathogens that cause significant economic loss in potato and other crops. Our understanding of the SRP species diversity has expanded in recent years due to advances and adoption of whole-genome sequence technologies. There are currently 34 recognized SRP species that belong to the Dickeya and Pectobacterium genera. Methods: We used whole-genome sequencing based analysis to describe the current distribution and epidemiology of SRP isolated from diseased potato samples obtained from commercial potato cropping systems in the United States. Our primary objectives in the present study were to: (1) identify the species of these SRP isolates recovered from potato samples across 14 states in the US, (2) describe the variation among SRP isolates from various US locations and track their temporal changes, and (3) evaluate the evolutionary relationships among these SRP isolates to deduce their source. We collected 118 SRP strains from diseased potato plants and tubers in 14 states between 2015 and 2022. Results: We identified three Dickeya and eight Pectobacterium species from diseased potato samples. Dickeya dianthicola, Pectobacterium parmentieri, P. carotovorum, and P. versatile appeared to be the predominant species, constituting 83% of the isolates. Furthermore, all D. dianthicola strains studied here as well as 90% of US D. dianthicola isolates sequenced to date exhibit significant clonality. Discussion: The prevalence of this specific group of D. dianthicola, temporally and geographically, aligns with the occurrence of blackleg and soft rot outbreaks in the northeastern US after 2014. The genomic diversity observed in P. parmentieri implies multiple introductions to the US from at least four distinct sources, earlier than the arrival of the predominant group of D. dianthicola. In contrast, P. carotovorum and P. versatile appear to be widespread, long-term endemic strains in the US.

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.

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.

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.

Advance Age Pregnancy in a Tertiary Care Centre: A Descriptive Cross-sectional Study.

INTRODUCTION: Women who conceive at advance age are at risk of pregnancy complications and adverse foetal outcome. This study aims to find out the prevalence of pregnancy at advance age in a teaching hospital. METHODS: A descriptive cross-sectional study was conducted between October 2019 to August 2020 at department of obstetrics and gynaecology of a tertiary care centre of Nepal, after obtaining ethical clearance from Institutional Review Committee (dated 03/09/2019 with ref no. 266) and informed consent from patient. Convenience sampling was done. All the patient who were ≥35 years and >28 weeks of gestation without any chronic illness were selected. Data and descriptive analysis were done using Statistical Package for the Social Sciences version 16. Point estimate at 95% Confidence Interval was calculated along with frequency and percentage for binary data. RESULTS: Women aged 35 years and above constituted 104 (5.73%) of the total deliveries of study period. Most were multigravida 72 (69.23%) and 23 (22.12%) had preterm delivery. Rate of caesarean section were higher in advance maternal age 69 (66.35%). Maternal complications such as Hypertensive disorder of pregnancy 9 (8.65%), and mal-presentation 15 (14.42%) were higher among them. Perinatal outcome in form of low birth weight 9 (8.65%) and perinatal death 5 (4.80%) were increased in those women. CONCLUSIONS: From this study, it can be concluded that prevalence of advanced age at pregnancy was lower than study done in developed country but it was similar to study in India and is increasing in Nepal.

Microbiological patterns of endophthalmitis in a tertiary level hospital of Kathmandu, Nepal.

INTRODUCTION: Endophthalmitis is a serious ocular infection that can result in blindness and therefore is a major ophthalmic concern. The study was conducted to find out the microbial etiology in clinically diagnosed cases of endophthalmitis and to determine the antibacterial susceptibilities of bacterial isolates in vitro. MATERIALS AND METHODS: A retrospective analysis was carried out of all patients presenting between 15th January 2012 to 15th January 2013 with clinically diagnosed Endophthalmitis at Tilganga Institute of Ophthalmology (TIO). Intraocular specimens (aqueous or vitreous fluids) were collected from 102 patients. Along with intraocular aspirates, blood and urine specimens, chest X-ray from endogenous endophthalmitis and corneal scrapes from corneal ulcer leading to endophthalmitis were also collected. The intraocular specimens were then subjected to microbiological evaluation. Antibiotic susceptibility of isolates were determined for different ocular antibiotics using the Kirby-Bauer disk-diffusion test. RESULTS: Samples from 102 patients diagnosed with endophthalmitis underwent microbiological analysis, of which culture positive 34(33.33%). Out of 34, 29 (85.29%) had bacterial growth and the remaining 5 (14.70%) had fungal growth. Most of them (40.8%) were secondary to cataract surgery(postoperative). A total of 29 bacterial culture reports, 72.41% were gram-positive and 27.58% were gram-negative. Streptococcus pneumoniae (37.93%) was the most frequently isolated organism. The antimicrobial sensitivity for isolates was as follows: Ofloxacin - 86.2%, Chloramphenicol - 82.75%,Cefazolin- 82.7%, Gentamicin - 75.8%, Ceftazidime - 58.6%, Amikacin - 55.17% and Vancomycin - 51.7%. CONCLUSION: The data highlights low culture positivity and a predominance of gram positive bacteria as the major causes of infectious endophthalmitis, usually following cataract surgery. The most common isolate was Streptococcus pneumoniae. Ofloxacin and Chloramphenicol demonstrated greatest efficacy against these bacterial isolates.

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.

Outcome of cataract surgery in eyes with uveitis.

PURPOSE: To assess the outcome of cataract surgery in patients with uveitis without the use of prophylactic high dose (> 5-10 mg/day) systemic steroid. METHOD: A hospital based prospective study enrolling 64 eyes of 60 patients with uveitis and cataract from May 2013 to April 2014 having intraocular inflammation under control for at least 3 months preoperatively and underwent phacoemulsification with in bag placement of foldable acrylic intraocular lens (IOL). RESULTS: Twenty six male and 34 female were included with mean age of 47.23 ± 16.85SD (16-85) years. In 43.75 % of eyes the uveitis was idiopathic followed by sarcoiduveitis (18.7%), Herpetic uveitis (15.6%), Tubercular uveitis (6.2%), VKH (4.6%), HLAB 27(4.6%), Behcet's, endogenous endophthalmitis, Wegener's granulomatosis and lepromatous uveitis (1.5% each). Anterior chamber cell count was grade 1+ in 33 eyes (51.56%) on 1st post-operative day and in 29 eyes (45.31%) on second follow up. Out of total 11 eyes (17.18%) that developed fibrin, 7 eyes were treated with subconjunctival injection of dexamethasone with half hourly topical steroid drops. Other 4 eyes that developed fibrin responded to half hourly topical steroid. Dose of oralprednisolone increased in 6 patients in early post operative duration. At the final follow up, 50 eyes (92.58% ) had improvement in best corrected visual acuity and cystoidmacular edema (CME) in 5% (n=3) eyes. CONCLUSION: Even without the use of preoperative high dose of oral steroid, inflammation was under control with significant improvement in visual acuity 3 months postoperatively.

Molecular Confirmation of G1138A Mutation in FGFR gene in Achondroplasia.

INTRODUCTION: Achondroplasia is the most common form of skeletal dysplasia of genetic origin in humans which is characterized by disproportionate rhizomelic dwarfism. Heterozygous mutation in the transmembrane domain of the FGFR3 gene (4p16.3) occurs as a de novo mutation in most of the cases. METHODS: DNA was isolated from seven samples, out of which, five had clinical features of Achondroplasia while one was dwarf but did not show symptoms of the disorder and one as negative control. PCR was performed for the region incorporating the hotspot region viz. 1138th nucleotide. PCR amplicon of size 164 bp was obtained from all the samples, and was sequenced. RESULTS: Sequence analysis showed the presence of mutation (G to A transition) in all of the five samples. The five samples that showed the clinical features of Achondroplasia had mutation in the region being analyzed while the single patient who had no clinical manifestations of the disorder despite being dwarf had no such mutation. Among the five patients studied, one patient had a family history of Achondroplasia as observed through pedigree analysis while the remaining four cases were sporadic in nature. CONCLUSIONS: This study further supports that the G1138A mutation is the one of the most common point mutation among Achondroplasia cases. Genetic diagnosis can be useful to identify the disease prenatally and differentiate other life threatening dwarfism for the safety of both mother and fetus.

Emergence of dengue in Nepal.

Dengue virus is a major health problem in Nepal. The endogenous dengue appeared in 2006 in the country with reported outbreaks in 2010, 2013 and 2016. Eleven years vertical data show there were sporadic cases in all the years and mostly adults between 25 and 40 years of age were infected with dengue virus. Compared with primary infections, secondary infections were observed in relatively larger numbers during the period of 2008-2016. Most of the cases had symptoms of dengue fever; while 7 and 19 cases demonstrated dengue hemorrhagic fever/dengue shock syndrome in 2010 and 2013 respectively. The proportion of dengue hemorrhagic fever amongst all cases of dengue fever was 2.5:4.7% in 2010 and 2013. We found there is shift of serotype from dengue virus serotype-1 (DENV-1) in 2010, DENV-2 in 2013 and DENV-1 in 2016. We feel there is urgent need for better community, hospital and laboratory based surveillance system capable of monitoring the circulating dengue virus (DENV) serotypes in different districts of Nepal. With improvement in surveillance system and efficient management of cases, the case fatality rate due to severe dengue can be reduced.

Knowledge, Attitude and Practice Regarding Cervical Cancer Screening Among Women Attending a Teaching Hospital, Bharatpur, Chitwan.

Objective: To find out the knowledge, attitude and practice regarding cervical cancer screening among women. Materials and methods: A descriptive cross-sectional study design was used to collect data from 96 women. Each woman was selected alternately from Gynae Out-Patient Department of Teaching Hospital. Data was collected by using semi-structured interview schedule to find out knowledge and practice and Likert scale to find out the attitude regarding cervical cancer screening. Data was analyzed by using SPSS version 20.0 and interpreted in terms of descriptive and inferential statistics. Results: Out of 96 women, mean age was 38.83 ± 6.57 and 90.6% respondents followed Hinduism. More than three fourth (85.4%) were literate and 59.4% were housewife. Only 9.4% were involved in cervical cancer prevention and screening awareness programme and 2.1% had family history of cervical cancer. As per the findings, only 34.4% and 27.8% had adequate knowledge and practice respectively whereas cent percent women had favorable attitude. Only education level of women was statistically significant with level of knowledge regarding cervical cancer screening (p = 0.041). There was strong negative correlation between knowledge score and practice score regarding cervical cancer screening among women (r = -0.194). Conclusion: Considerable proportions of women have inadequate knowledge and practice regarding cervical cancer screening. Therefore cervical cancer screening health camps and awareness program should be conducted at community level for women to increase the level of knowledge and practice regarding cervical cancer screening.

First report of hepatitis E virus viremia in healthy blood donors from Nepal.

The aim of this study was to detect the prevalence of hepatitis E virus (HEV) in healthy blood donors so as to decipher the maintenance of (HEV) reservoir if any. Five hundred and eighty-one blood samples along with clinical information were collected from central blood bank, Kathmandu between February and March 2014. Samples were tested for hepatitis B virus surface antigen, anti-hepatitis C virus antibodies, anti-hepatitis A virus IgM, HEV antigen, HEV viral load and anti-HEV antibodies (IgM and IgG) by ELISA. Only those samples positive with anti-HEV IgM were tested for HEV RNA by reverse transcriptase nested PCR. Age adjusted prevalence of IgM anti HEV and IgG anti HEV were 3.6 and 8.3 % respectively. No significant difference in Median ALT levels was noted between HEV RNA positive and negative subjects. Sequence analysis of HEV shows all genotype belongs to genotype 1a. Phylogenetic analysis shows the virus has homology of 95 % with strain from India and Nepal outbreak of April 2014. This study sheds light on how inter epidemic reservoirs can be maintained in healthy population with asymptomatic cases. This raises an important question regarding nature of HEV as well as its tendency to circulate in blind sight and also cause periodic outbreaks in endemic setting like Kathmandu.

Public water sources in rural watersheds of Nepal's Middle Mountains: issues and constraints.

Inadequacy and poor quality of water supply for domestic purposes is increasingly becoming a concern in rural catchments of the Middle Mountains of Nepal. Water quantity is an issue in pocket areas of these catchments, while water quality is subject to concern in most of the water sources. Microbiological contamination in particular poses a risk to human health. In addition, sediment pollution during the monsoon season is perceived as an issue by the local residents. Elevated phosphate and nitrate levels in many water sources indicate intensive interaction with surface water hailing from agricultural areas and human settlements. These water quantity and quality concerns in two watersheds of Nepal, the Jhikhu Khola and the Yarsha Khola watersheds, are not isolated cases. Similar problems are reported from other watersheds monitored under the People and Resource Dynamics in Mountain Watersheds of the Hindu Kush-Himalayas (PARDYP) project in China, India, and Pakistan and the literature of this region.