Sanjaya K. Shrestha

Comparison of novel MLB-clade, VA-clade and classic human astroviruses highlights constrained evolution of the classic human astrovirus nonstructural genes

Eight serotypes of human astroviruses (the classic human astroviruses) are causative agents of diarrhea. Recently, five additional astroviruses belonging to two distinct clades have been described in human stool, including astroviruses MLB1, MLB2, VA1, VA2 and VA3. We report the discovery in human stool of two novel astroviruses, astroviruses MLB3 and VA4. The complete genomes of these two viruses and the previously described astroviruses VA2 and VA3 were sequenced, affording seven complete genomes from the MLB and VA clades for comparative analysis to the classic human astroviruses. Comparison of the genetic distance, number of synonymous mutations per synonymous site (dS), number of non-synonymous mutations per non-synonymous site (dN) and the dN/dS ratio in the protease, polymerase and capsid of the classic human, MLB and VA clades suggests that the protease and polymerase of the classic human astroviruses are under distinct selective pressure.

A growing global network’s role in outbreak response: AFHSC-GEIS 2008-2009

A cornerstone of effective disease surveillance programs comprises the early identification of infectious threats and the subsequent rapid response to prevent further spread. Effectively identifying, tracking and responding to these threats is often difficult and requires international cooperation due to the rapidity with which diseases cross national borders and spread throughout the global community as a result of travel and migration by humans and animals. From Oct.1, 2008 to Sept. 30, 2009, the United States Department of Defense’s (DoD) Armed Forces Health Surveillance Center Global Emerging Infections Surveillance and Response System (AFHSC-GEIS) identified 76 outbreaks in 53 countries. Emerging infectious disease outbreaks were identified by the global network and included a wide spectrum of support activities in collaboration with host country partners, several of which were in direct support of the World Health Organization’s (WHO) International Health Regulations (IHR) (2005). The network also supported military forces around the world affected by the novel influenza A/H1N1 pandemic of 2009. With IHR (2005) as the guiding framework for action, the AFHSC-GEIS network of international partners and overseas research laboratories continues to develop into a far-reaching system for identifying, analyzing and responding to emerging disease threats.

Simultaneous and complete genome sequencing of influenza A and B with high coverage by Illumina MiSeq Platform

Active global surveillance and characterization of influenza viruses are essential for better preparation against possible pandemic events. Obtaining comprehensive information about the influenza genome can improve our understanding of the evolution of influenza viruses and emergence of new strains, and improve the accuracy when designing preventive vaccines. This study investigated the use of deep sequencing by the next-generation sequencing (NGS) Illumina MiSeq Platform to obtain complete genome sequence information from influenza virus isolates. The influenza virus isolates were cultured from 6 respiratory acute clinical specimens collected in Thailand and Nepal. DNA libraries obtained from each viral isolate were mixed and all were sequenced simultaneously. Total information of 2.6 Gbases was obtained from a 455±14 K/mm2 density with 95.76% (8,571,655/8,950,724 clusters) of the clusters passing quality control (QC) filters. Approximately 93.7% of all sequences from Read1 and 83.5% from Read2 contained high quality sequences that were ≥Q30, a base calling QC score standard. Alignments analysis identified three seasonal influenza A H3N2 strains, one 2009 pandemic influenza A H1N1 strain and two influenza B strains. The nearly entire genomes of all six virus isolates yielded equal or greater than 600-fold sequence coverage depth. MiSeq Platform identified seasonal influenza A H3N2, 2009 pandemic influenza A H1N1and influenza B in the DNA library mixtures efficiently.

Influenza A (H3N2) Outbreak, Nepal

Worldwide emergence of variant viruses has prompted a change in the 2005–2006 H3N2 influenza A vaccine strain.

Bhaktapur, Nepal: The MAL-ED Birth Cohort Study in Nepal

The Etiology, Risk Factors and Interactions of Enteric Infections and Malnutrition and the Consequences for Child Health and Development (MAL-ED) cohort study site in Nepal is located in the Bhaktapur municipality, 15 km east of Kathmandu, the capital city of Nepal. Bhaktapur, an ancient city famous for its traditional temples and buildings, is included on UNESCOs World Heritage List and is a major tourist attraction in Nepal. Nepal is a land-locked country located in South Asia between China and India with an area of 147 181 km(2), ranging from sea-level plains to Mount Everest, the worlds highest peak. The total population as of the 2011 census was 26.6 million, with an average annual population growth rate of 1.4. Nepal is one of the worlds least developed countries and is ranked 157 of 186 in the 2013 Human Development Report; one-third of the Nepali population lives below the poverty line. The current under-5 mortality rate is 54 per 1000 live births, the infant mortality rate is 46 per 1000 live births, and the neonatal mortality rate is 33 per 1000 live births. Vaccine coverage for all Expanded Program on Immunization vaccines is >80%. Among children, the most common diseases contributing to significant morbidity and mortality are acute respiratory infection and dehydration from severe diarrhea. In this article, we report on the geographic, demographic, and socioeconomic features of the Bhaktapur MAL-ED site and describe the data that informed our cohort recruitment strategy.

The MAL-ED Cohort Study: Methods and Lessons Learned When Assessing Early Child Development and Caregiving Mediators in Infants and Young Children in 8 Low- and Middle-Income Countries

More epidemiological data are needed on risk and protective factors for child development. In The Etiology, Risk Factors and Interactions of Enteric Infections and Malnutrition and the Consequences for Child Health and Development (MAL-ED) cohort study, we assessed child development in a harmonious manner across 8 sites in Bangladesh, Brazil, India, Nepal, Pakistan, Peru, South Africa, and Tanzania. From birth to 24 months, development and language acquisition were assessed via the Bayley Scales of Infant and Toddler Development and a modified MacArthur Communicative Development Inventory. Other measures were infant temperament, the childs environment, maternal psychological adjustment, and maternal reasoning abilities. We developed standard operating procedures and used multiple techniques to ensure appropriate adaptation and quality assurance across the sites. Test adaptation required significant time and human resources but is essential for data quality; funders should support this step in future studies. At the end of this study, we will have a portfolio of culturally adapted instruments for child development studies with examination of psychometric properties of each tool used.

Dynamics and Trends in Fecal Biomarkers of Gut Function in Children from 1–24 Months in the MAL-ED Study

Growth and development shortfalls that are disproportionately prevalent in children living in poor environmental conditions are postulated to result, at least in part, from abnormal gut function. Using data from The Etiology, Risk Factors, and Interactions of Enteric Infections and Malnutrition and the Consequences for Child Health and Development (MAL-ED) longitudinal cohort study, we examine biomarkers of gut inflammation and permeability in relation to environmental exposures and feeding practices. Trends in the concentrations of three biomarkers, myeloperoxidase (MPO), neopterin (NEO), and α-1-antitrypsin (AAT), are described from fecal samples collected during the first 2 years of each childs life. A total of 22,846 stool samples were processed during the longitudinal sampling of 2,076 children 0–24 months of age. Linear mixed models were constructed to examine the relationship between biomarker concentrations and recent food intake, symptoms of illness, concurrent enteropathogen infection, and socioeconomic status. Average concentrations of MPO, NEO, and AAT were considerably higher than published references for healthy adults. The concentration of each biomarker tended to decrease over the first 2 years of life and was highly variable between samples from each individual child. Both MPO and AAT were significantly elevated by recent breast milk intake. All three biomarkers were associated with pathogen presence, although the strength and direction varied by pathogen. The interpretation of biomarker concentrations is subject to the context of their collection. Herein, we identify that common factors (age, breast milk, and enteric infection) influence the concentration of these biomarkers. Within the context of low- and middle-income communities, we observe concentrations that indicate gut abnormalities, but more appropriate reference standards are needed.

Serological response to Bartonella species in febrile patients from Nepal

The Bartonella-associated illnesses are spread world-wide and involve a broad spectrum of signs and symptoms in humans. Several Bartonella species have been shown to be responsible for cases of febrile illnesses. Little information exists on distribution of Bartonella species and their role in human diseases in Nepal. Our preliminary study, a retrospective serological survey of archived specimens, suggests that Bartonella antibodies are prevalent among febrile patients in the Kathmandu Valley of Nepal.

Field evaluation of commercial immunoglobulin M antibody capture ELISA diagnostic tests for the detection of Japanese encephalitis virus infection among encephalitis patients in Nepal.

OBJECTIVESnJapanese encephalitis (JE) is a devastating disease with high rates of death and disability that occurs particularly in resource-limited, rural regions of Asia. Simple, accurate and inexpensive diagnostics tests are vital for quantifying the burden of illness. This field study evaluated two commercial JE immunoglobulin M antibody capture (MAC) ELISA kits using samples from routine JE surveillance.nnnMETHODSnPositive (n=132) and negative (n=218) sera were randomly selected from patient samples collected as part of JE surveillance in Nepal in 2005. Samples were tested in a national public health laboratory with commercial kits produced by XCyton and Inverness (Panbio). Results were compared with those of the research lab-based reference standard, the Armed Forces Research Institute of Medical Sciences JE MAC ELISA.nnnRESULTSnPositive and negative predictive values and 95% confidence intervals were 90% (82-95%) and 85% (79-89%) for Panbio1, 94% (88-98%) and 89% (87-93%) for Panbio2, and 84% (77-90%) and 96% (92-98%) for XCyton kits, respectively. Sensitivities of Panbio1, Panbio2, and XCyton kits were 71% (63-79%), 80% (72-87%), and 93% (88-97%); specificities were 95% (91-98%), 97% (94-99%), and 89% (85-93%), respectively. Overall percent agreement was 86% for Panbio1 and 91% for both Panbio2 and XCyton.nnnCONCLUSIONSnBoth commercial kits had good predictive values when single serum samples from encephalitis cases were tested in a national laboratory. Either kit can be used in similar JE-endemic settings where co-transmission of dengue virus, a flavivirus which has strong cross-reactivity with JE, is limited. These results can inform decisions by countries and the World Health Organization laboratory networks on national-level use of these kits for JE surveillance.

Evidence of West Nile virus infection in Nepal

BackgroundAcute febrile illness is common among those seeking medical care and is frequently treated empirically with the underlying illness remaining undiagnosed in resource-poor countries. A febrile illness study was conducted 2009-2010 to identify known and unknown pathogens circulating in Nepal.MethodStudy methods included diagnostic testing and preliminary ELISA screening of acute and convalescent samples for diseases both known and unknown to be circulating in Nepal, including West Nile virus (WNV). The molecular assays including Polymerase Chain Reaction (PCR), Sanger sequencing and ultra deep sequencing on MiSeq Illumina Platform were conducted to further confirm the presence of WNV.ResultsThe study enrolled 2,046 patients presenting undifferentiated febrile illness with unknown etiology. Sera from 14 out of 2,046 patients were tested positive for west nile virus (WNV) by nested Reverse Transcription-Polymerase Chain Reaction (RT-PCR). Only two out of 14 cases were confirmed for the presence of WNV by sequencing and identified as WNV lineage 1 phylogentically. The two patients were adult males with fever and no neurological symptoms from Kathmandu and Bharatpur, Nepal.ConclusionTwo out of 2,046 serum samples contained fragments of WNV genome resembling WNV lineage 1, which is evidence of the continued spread of WNV which should be considered a possible illness cause in Nepal.