Punita Gauchan

Front-Loading Sputum Microscopy Services: An Opportunity to Optimise Smear-Based Case Detection of Tuberculosis in High Prevalence Countries

Setting. Ethiopia, Nepal, Nigeria, and Yemen. Objective. To reduce the time to complete sputum microscopy. Design. Cross-sectional surveys enrolling 923 patients with chronic cough in the 4 countries and using similar protocols. Spot-morning-spot sputum specimens were collected. An additional sputum specimen (Xspot) was collected one hour after the first, and the yields of the first two or the three specimens collected as spot-morning-spot or spot-Xspot-morning were compared. Results. 216 patients had ≥ one positive smear. 210 (97%) were identified by the spot-morning-spot, and 210 (97%) were identified by the spot-Xspot-morning specimens, with 203 and 200 identified by the first 2 specimens of each approach, respectively. Neither difference was significant. Conclusions. The time to complete smear microscopy could be reduced.

Molecular epidemiology of Rotavirus A, causing acute gastroenteritis hospitalisations among children in Nha Trang, Vietnam, 2007–2008: Identification of rare G9P[19] and G10P[14] strains

Rotavirus A (RVA) causes acute diarrhea in children as well as animals. As part of a cross‐sectional study of children less than 5 years of age hospitalized for acute diarrhea in Vietnam during a 15‐month period (2007–2008), 322 (43.5%) of 741 fecal specimens contained RVA with 92% either G1P[8] or G3P[8]. This study was undertaken to further characterize strains that remained untypeable to complete the G and P genotypes of the 322 rotavirus‐positive specimens. While 307 (95.3%) strains possessed the common human RVA genotypes: G1P[8] (45.0%), G2P[4] (2.8%), G3P[8] (46.9%), and G9P[8] (0.6%), sequencing of initially untypeable specimens revealed the presence of two unusual strains designated NT0073 and NT0082 possessing G9P[19] and G10P[14], respectively. The genotype constellation of NT0073 (G9‐P[19]‐I5‐R1‐C1‐M1‐A8‐N1‐T7‐E1‐H1) and the phylogenetic trees suggested its origin as a porcine RVA strain causing diarrhea in a 24‐month‐old girl whereas the genotype constellation of NT0082 (G10‐P[14]‐I2‐R2‐C2‐M2‐A3‐N2‐T6‐E2‐H3) and the phylogenetic trees suggested its origin as an RVA strain of artiodactyl origin (such as cattle, sheep and goats) causing diarrhea in a 13‐month‐old boy. This study showed that RVA strains of animal host origin were not necessarily attenuated in humans. A hypothesis may be postulated that P[19] and P[14] VP4 spike proteins helped the virus to replicate in the human intestine but that efficient onward human‐to‐human spread after crossing the host species barrier may require the virus to obtain some additional features as there was no evidence of widespread transmission with the limited sampling performed over the study period. J. Med. Virol. 89:621–631, 2017.

Evolution of DS-1-like G1P[8] double-gene reassortant rotavirus A strains causing gastroenteritis in children in Vietnam in 2012/2013

Rotavirus A (RVA) strains, a leading cause of severe gastroenteritis in children worldwide, commonly possess the Wa or DS-1 genotype constellations. During a hospital-based study conducted in Hanoi, Vietnam, in the 2012-2013 rotavirus season, G1P[8] strains with a virtually identical short RNA migration pattern were detected in 20 (14%) of 141 rotavirus-positive samples. Two representatives of these strains were shown by whole-genome sequencing to be double-gene reassortants possessing the genotype constellation of G1-P[8]-I2-R2-C2-M2-A2-N2-T2-E2-H2. Sequencing and a database search revealed that these Vietnamese G1P[8] double-gene reassortant strains shared an immediate ancestor with a locally circulating G2P[4] strain in all of the inner-capsid and non-structural protein genes, whereas they were more closely related in the VP7 and VP4 genes to a Chinese G1P[8] strain and a Chinese G3P[8] strain, respectively, than to locally circulating G1P[8] strains. Despite the marked similarity between Japanese and Thai G1P[8] double-gene reassortant strains, phylogenetic analysis suggested that the Vietnamese and Japanese/Thai G1P[8] double-gene reassortant strains originated from independent reassortment events. Clinically, children infected with Vietnamese G1P[8] double-gene reassortant strains experienced severe diarrhoea, but it was not more severe than that in children infected with ordinary G1P[8] strains. In conclusion, Vietnamese G1P[8] double-gene reassortant strains originated from a locally circulating G2P[4] strain and caused severe diarrhoea, but there was no evidence of increased virulence.

Continued circulation of G12P[6] rotaviruses over 28 months in Nepal: successive replacement of predominant strains.

Rotavirus A causes severe diarrhoea in infants and young children worldwide. The migration pattern (electropherotype) of the double-stranded RNA genome upon polyacrylamide gel electrophoresis has been used to define “strains” in molecular epidemiology. In temperate countries, distinct electropherotypes (strains) appear after the annual, off-seasonal interruption of rotavirus circulation. In Nepal, rotavirus circulated year-round and an uncommon genotype G12P[6] predominated and persisted, providing a unique opportunity to examine whether the same electropherotype (the same strain) persisted or new electropherotypes (new strains) emerged successively under the same G12P[6] predominance. A total of 147 G12P[6] rotaviruses, collected from diarrhoeal children in Nepal between 2007 and 2010, were classified into 15 distinct electropherotypes (strains). Of these, three electropherotypes (strains), LP1, LP24, and LP27, accounted for 10%, 32% and 38% of the G12P[6] rotaviruses, respectively. Each of the three major strains successively appeared, dominated, and disappeared. This study provided new evidence for the hypothesis that rotavirus constantly changes its strains to predominate in the local population even under conditions where a single genotype predominates and persists. Such dynamic strain replacement, the constant takeover of one predominant strain by another, fitter strain, is probably gives a competitive edge to the survival of rotavirus in nature.

Whole genotype constellation of prototype feline rotavirus strains FRV-1 and FRV64 and their phylogenetic relationships with feline-like human rotavirus strains

Feline rotaviruses, members of the species Rotavirus A, are an infrequent source of zoonotic infections, and were previously shown by RNA-RNA hybridization assays to possess two distinct genomic RNA constellations, represented by strains FRV-1 and FRV64. Due to the lack of whole genome sequence information for FRV-1, human rotavirus strain AU-1 has been used as a surrogate for the genotype constellation of feline rotaviruses. The aim of this study was to determine the whole genome sequence of FRV-1 and FRV64 to help understand the genetic relationships among existing feline rotaviruses from the evolutionary perspective. The genotype constellations of FRV-1 and FRV64 were G3-P[9]-I3-R3-C3-M3-A3-N3-T3-E3-H3 and G3-P[3]-I3-R3-C2-M3-A9-N2-T3-E3-H6, respectively. FRV-1 has a genotype constellation identical to that of the AU-1 strain. Although for individual genes they shared lineages, with the exception of genes encoding VP2, VP6 and VP7, the sequence identity between FRV-1 and AU-1 was considered to be sufficiently high for the AU-1 to be regarded as an example of the direct transmission of a feline rotavirus to a child. On the other hand, the FRV64 strain was not only similar in all the 11 genome segments to another feline rotavirus strain, Cat97, but also to canine rotavirus strains (K9 and CU-1) and feline/canine-like human rotavirus strains (Ro1845 and HCR3A). In conclusion, this study revealed intermingled sharing of genotypes and lineages among feline rotaviruses, suggesting the occurrence of frequent reassortment events over the course of evolution to emerge in four genotype constellations represented by FRV-1, FRV64/Cat97, Cat2 and BA222 strains.

A High Incidence of Intussusception Revealed by a Retrospective Hospital-Based Study in Nha Trang, Vietnam between 2009 and 2011

Rotavirus is a leading cause of severe diarrhea among children worldwide. Thus, the World Health Organization recommended including rotavirus vaccines in national immunization programs. One concern about rotavirus vaccine, however, is a possible association with intussusception. Thus, it is crucial to know the baseline incidence of intussusception in the first year of life. A study conducted in Hanoi, Vietnam showed that the incidence of intussusception was the highest in the world. This retrospective cross-sectional study was undertaken to determine the incidence of intussusception among children <5 years of age in Nha Trang, Vietnam. Hospital charts between 2009 and 2011 were reviewed in Khanh Hoa Provincial General Hospital where virtually all cases of intussusception occurring in the city were assumed to have been encountered. The incidence of intussusception among children <1 year of age was 296 per 100,000 person-years (95% confidence interval [CI]: 225–382), and that among children <5 years of age was 196 per 100,000 person-years (95% CI: 169–226), confirming the high incidence of intussusception in Vietnam. Nevertheless, there was no intussusception in the first three months of life. We therefore recommend that the first dose of any rotavirus vaccine be administered to infants between 6 and 12 weeks of age.

The First Identification of Rotavirus B from Children and Adults with Acute Diarrhoea in Kathmandu, Nepal

Rotavirus B (RVB) in the genus Rotavirus of the family Reoviridae is known to be a cause of acute gastroenteritis among children and adults in parts of Asia including China, India, Bangladesh and Myanmar. In a 15-month surveillance programme between March 2007 and May 2008, 3,080 stool specimens were collected from children and adults with acute gastroenteritis in an infectious disease hospital in Kathmandu, Nepal. In 25 (0.8%) specimens RVB was detected, for the first time in Nepal, by the use of polyacrylamide gel electrophoresis followed by confirmation with reverse-transcription PCR and sequencing. The strains detected in this study had very similar electropherotypes, and their VP7 sequences were almost identical and phylogenetically belonged to the Indo-Bangladeshi lineage which was distinct from the Chinese lineage. Thus, this study showed the circulation of RVB strains belonging to the Indo-Bangladeshi lineage in a broader region than previously documented, suggesting that this phylogenetic divide corresponded to the geographic divide created by the Himalayan Mountains. Further studies may be warranted to identify and characterize the RVB strains in northern Vietnam which is adjacent to southern China with a long and less mountainous border.

Whole genome characterisation of a porcine-like human reassortant G26P[19] Rotavirus A strain detected in a child hospitalised for diarrhoea in Nepal, 2007

A rare G26 Rotavirus A strain RVA/Human-wt/NPL/07N1760/2007/G26P[19] was detected in a child hospitalised for acute diarrhoea in Kathmandu, Nepal. The complete genome of 07N1760 was determined in order to explore its evolutionary history as well as examine its relationship to a Vietnamese strain RVA/Human-wt/VNM/30378/2009/G26P[19], the only G26 strain whose complete genotype constellation is known. The genotype constellation of 07N1760 was G26-P[19]-I12-R1-C1-M1-A8-N1-T1-E1-H1, a unique constellation identical to that of the Vietnamese 30378 except the VP6 gene. Phylogenetic analysis revealed that both strains were unrelated at the lineage level despite their similar genotype constellation. The I12 VP6 gene of 07N1760 was highly divergent from the six currently deposited I12 sequences in the GenBank. Except for its NSP2 gene, the remaining genes of 07N1760 shared lineages with porcine and porcine-like human RVA genes. The NSP2 gene belonged to a human RVA N1 lineage which was distinct from typical porcine and porcine-like human lineages. In conclusion, the Nepali G26P[19] strain 07N1760 was a porcine RVA strain which derived an NSP2 gene from a human Wa-like RVA strain by intra-genotype reassortment probably after transmission to the human host.

Molecular identification of a G2 rotavirus that provided a G1P[4] mono‐reassortant with a DS‐1‐like genotype constellation

The segmented nature of the rotavirus genome provides an opportunity for the virus to reassort upon co‐infection of more than one rotavirus strain. Previously, two G1P[4] strains isolated from children with diarrhoea, AU64, and AU67, were shown by RNA–RNA hybridization to be a VP7 mono‐reassortant possessing a DS‐1 genogroup background. However, the origin of the parental G2 strain was not sought for at that time. The aim of this study, therefore, was to identify the G2 strain that provided AU64/AU67 with the DS‐1‐like genotype constellation. By carefully comparing the genomic RNA migration patterns on polyacrylamide gels of strains circulating at the same and nearby geographic locations around the time of the mono‐reassortant detection (1989), a G2 strain designated 88H449 was shown to possess 10 genome segments co‐migrating with those of AU64. Sequencing of all genome segments of AU64 and 88H449 showed that those two strains were 99.6–100% identical at the nucleotide level in all except the VP7 gene, indicating that the parental G2 strain that provided AU64 with the 10 genome segments was a sibling of 88H449. Sequencing of the VP7 genes of 36 G1P[8] strains circulating locally between 1981 and 1990 revealed the presence of strains bearing diverse VP7 sequences among which the closest nucleotide identity to AU64 was 98.6% and the closest amino acid identity in the major antigenic regions was 100%. This unusual mono‐reassortant was concluded to be the result of a contemporary reassosrtment event occurring between locally co‐circulating strains. J. Med. Virol. 87:694–701, 2015.

Whole genome analysis of Vietnamese G2P[4] rotavirus strains possessing the NSP2 gene sharing an ancestral sequence with Chinese sheep and goat rotavirus strains

Because imminent introduction into Vietnam of a vaccine against Rotavirus A is anticipated, baseline information on the whole genome of representative strains is needed to understand changes in circulating strains that may occur after vaccine introduction. In this study, the whole genomes of two G2P[4] strains detected in Nha Trang, Vietnam in 2008 were sequenced, this being the last period during which virtually no rotavirus vaccine was used in this country. The two strains were found to be >99.9% identical in sequence and had a typical DS‐1 like G2‐P[4]‐I2‐R2‐C2‐M2‐A2‐N2‐T2‐E2‐H2 genotype constellation. Analysis of the Vietnamese strains with >184 G2P[4] strains retrieved from GenBank/EMBL/DDBJ DNA databases placed the Vietnamese strains in one of the lineages commonly found among contemporary strains, with the exception of the NSP2 and NSP4 genes. The NSP2 genes were found to belong to a previously undescribed lineage that diverged from Chinese sheep and goat rotavirus strains, including a Chinese rotavirus vaccine strain LLR with 95% nucleotide identity; the time of their most recent common ancestor was 1975. The NSP4 genes were found to belong, together with Thai and USA strains, to an emergent lineage (VIII), adding further diversity to ever diversifying NSP4 lineages. Thus, there is a need to enhance surveillance of locally‐circulating strains from both children and animals at the whole genome level to address the effect of rotavirus vaccines on changing strain distribution.