Genomic epidemiology of SARS-CoV-2 within households in coastal Kenya: a case ascertained cohort study

BackgroundAnalysis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genomic sequence data from household infections should aid its detailed epidemiological understanding. Using viral genomic sequence data, we investigated household SARS-CoV-2 transmission and evolution in coastal Kenya households. MethodsWe conducted a case-ascertained cohort study between December 2020 and February 2022 whereby 573 members of 158 households were prospectively monitored for SARS-CoV-2 infection. Households were invited to participate if a member tested SARS-CoV-2 positive or was a contact of a confirmed case. Follow-up visits collected a nasopharyngeal/oropharyngeal (NP/OP) swab on days 1, 4 and 7 for RT-PCR diagnosis. If any of these were positive, further swabs were collected on days 10, 14, 21 and 28. Positive samples with an RT-PCR cycle threshold of <33.0 were subjected to whole genome sequencing followed by phylogenetic analysis. Ancestral state reconstruction was used to determine if multiple viruses had entered households. ResultsOf 2,091 NP/OP swabs that were collected, 375 (17.9%) tested SARS-CoV-2 positive. Viral genome sequences (>80% coverage) were obtained from 208 (55%) positive samples obtained from 61 study households. These genomes fell within 11 Pango lineages and four variants of concern (Alpha, Beta, Delta and Omicron). We estimated 163 putative transmission events involving members of the sequenced households, 40 (25%) of which were intra-household transmission events while 123 (75%) were infections that likely occurred outside the households. Multiple virus introductions (up-to-5) were observed in 28 (47%) households with the 1-month follow-up period. ConclusionsWe show that a considerable proportion of SARS-CoV-2 infections in coastal Kenya occurred outside the household setting. Multiple virus introductions frequently occurred into households within the same infection wave in contrast to observations from high income settings, where single introduction appears to be the norm. Our findings suggests that control of SARS-CoV-2 transmission by household member isolation may be impractical in this setting.

Spike protein cleavage-activation mediated by the SARS-CoV-2 P681R mutation: a case-study from its first appearance in variant of interest (VOI) A.23.1 identified in Uganda

Based on its predicted ability to affect transmissibility and pathogenesis, surveillance studies have highlighted the role of a specific mutation (P681R) in the S1/S2 furin cleavage site of the SARS-CoV-2 spike protein. Here we analyzed A.23.1, first identified in Uganda, as a P681R-containing virus several months prior to the emergence of B.1.617.2 (Delta variant). We performed assays using peptides mimicking the S1/S2 from A.23.1 and B.1.617 and observed significantly increased cleavability with furin compared to both an original B lineage (Wuhan-Hu1) and B.1.1.7 (Alpha variant). We also performed cell-cell fusion and functional infectivity assays using pseudotyped particles and observed an increase in activity for A.23.1 compared to an original B lineage spike. However, these changes in activity were not reproduced in the B lineage spike bearing only the P681R substitution. Our findings suggest that while A.23.1 has increased furin-mediated cleavage linked to the P681R substitution, this substitution needs to occur on the background of other spike protein changes to enable its functional consequences.

Genomic surveillance reveals the spread patterns of SARS-CoV-2 in coastal Kenya during the first two waves

BackgroundThe transmission networks of SARS-CoV-2 in sub-Saharan Africa remain poorly understood. MethodsWe undertook phylogenetic analysis of 747 SARS-CoV-2 positive samples collected across six counties in coastal Kenya during the first two waves (March 2020 - February 2021). Viral imports and exports from the region were inferred using ancestral state reconstruction (ASR) approach. ResultsThe genomes were classified into 35 Pango lineages, six of which accounted for 79% of the sequenced infections: B.1 (49%), B.1.535 (11%), B.1.530 (6%), B.1.549 (4%), B.1.333 (4%) and B.1.1 (4%). Four identified lineages were Kenya specific. In a contemporaneous global subsample, 990 lineages were documented, 261 for Africa and 97 for Eastern Africa. ASR analysis identified >300 virus location transition events during the period, these comprising: 69 viral imports into Coastal Kenya; 93 viral exports from coastal Kenya; and 191 inter-county import/export events. Most international viral imports (58%) and exports (92%) occurred through Mombasa City, a key touristic and commercial Coastal Kenya center; and many occurred prior to June 2020, when stringent local COVID-19 restriction measures were enforced. After this period, local virus transmission dominated, and distinct local phylogenies were seen. ConclusionsOur analysis supports moving control strategies from a focus on international travel to local transmission. FundingThis work was funded by Wellcome (grant#: 220985) and the National Institute for Health Research (NIHR), project references: 17/63/and 16/136/33 using UK aid from the UK Government to support global health research, The UK Foreign, Commonwealth and Development Office.

The Vietnam Initiative on Zoonotic Infections (VIZIONS): A Strategic Approach to Studying Emerging Zoonotic Infectious Diseases.

The effect of newly emerging or re-emerging infectious diseases of zoonotic origin in human populations can be potentially catastrophic, and large-scale investigations of such diseases are highly challenging. The monitoring of emergence events is subject to ascertainment bias, whether at the level of species discovery, emerging disease events, or disease outbreaks in human populations. Disease surveillance is generally performed post hoc, driven by a response to recent events and by the availability of detection and identification technologies. Additionally, the inventory of pathogens that exist in mammalian and other reservoirs is incomplete, and identifying those with the potential to cause disease in humans is rarely possible in advance. A major step in understanding the burden and diversity of zoonotic infections, the local behavioral and demographic risks of infection, and the risk of emergence of these pathogens in human populations is to establish surveillance networks in populations that maintain regular contact with diverse animal populations, and to simultaneously characterize pathogen diversity in human and animal populations. Vietnam has been an epicenter of disease emergence over the last decade, and practices at the human/animal interface may facilitate the likelihood of spillover of zoonotic pathogens into humans. To tackle the scientific issues surrounding the origins and emergence of zoonotic infections in Vietnam, we have established The Vietnam Initiative on Zoonotic Infections (VIZIONS). This countrywide project, in which several international institutions collaborate with Vietnamese organizations, is combining clinical data, epidemiology, high-throughput sequencing, and social sciences to address relevant one-health questions. Here, we describe the primary aims of the project, the infrastructure established to address our scientific questions, and the current status of the project. Our principal objective is to develop an integrated approach to the surveillance of pathogens circulating in both human and animal populations and assess how frequently they are exchanged. This infrastructure will facilitate systematic investigations of pathogen ecology and evolution, enhance understanding of viral cross-species transmission events, and identify relevant risk factors and drivers of zoonotic disease emergence.

Novel porcine-like human G26P[19] rotavirus identified in hospitalized paediatric diarrhoea patients in Ho Chi Minh City, Vietnam.

During a hospital-based diarrhoeal disease study conducted in Ho Chi Minh City, Vietnam from 2009 to 2010, we identified four symptomatic children infected with G26P[19] rotavirus (RV)--an atypical variant that has not previously been reported in human gastroenteritis. To determine the genetic structure and investigate the origin of this G26P[19] strain, the whole genome of a representative example was characterized, revealing a novel genome constellation: G26-P[19]-I5-R1-C1-M1-A8-N1-T1-E1-H1. The genome segments were most closely related to porcine (VP7, VP4, VP6 and NSP1) and Wa-like porcine RVs (VP1-3 and NSP2-5). We proposed that this G26P[19] strain was the product of zoonotic transmission coupled with one or more reassortment events occurring in human and/or animal reservoirs. The identification of such strains has potential implications for vaccine efficacy in south-east Asia, and outlines the utility of whole-genome sequencing for studying RV diversity and zoonotic potential during disease surveillance.

Identification of possible virulence marker from Campylobacter jejuni isolates.

A novel protein translocation system, the type-6 secretion system (T6SS), may play a role in virulence of Campylobacter jejuni. We investigated 181 C. jejuni isolates from humans, chickens, and environmental sources in Vietnam, Thailand, Pakistan, and the United Kingdom for T6SS. The marker was most prevalent in human and chicken isolates from Vietnam.

Tracking the establishment of local endemic populations of an emergent enteric pathogen.

Shigella sonnei is a human-adapted pathogen that is emerging globally as the dominant agent of bacterial dysentery. To investigate local establishment, we sequenced the genomes of 263 Vietnamese S. sonnei isolated over 15 y. Our data show that S. sonnei was introduced into Vietnam in the 1980s and has undergone localized clonal expansion, punctuated by genomic fixation events through periodic selective sweeps. We uncover geographical spread, spatially restricted frontier populations, and convergent evolution through local gene pool sampling. This work provides a unique, high-resolution insight into the microevolution of a pioneering human pathogen during its establishment in a new host population.

Endemic norovirus infections in children, Ho Chi Minh City, Vietnam, 2009-2010.

We performed a case-control investigation to identify risk factors for norovirus infections among children in Vietnam. Of samples from 1,419 children who had diarrhea and 609 who were asymptomatic, 20.6% and 2.8%, respectively, were norovirus positive. Risk factors included residential crowding and symptomatic contacts, indicating person-to-person transmission of norovirus.

The dynamics of GII.4 Norovirus in Ho Chi Minh City, Vietnam.

Norovirus (NoV) is a major cause of epidemic gastroenteritis in industrialized countries, yet the epidemiological significance of NoV in industrializing countries remains poorly understood. The spatiotemporal distribution of NoV genotypes identified in 2054 enrolled children was investigated between May 2009 and December 2010, in Ho Chi Minh City (HCMC), Vietnam. A total of 315 NoV extracted from stool samples were genotyped and GPS mapped to their source. Genogroup II NoV, particularly GII.4, were predominant, and the GII.4 strains could be subgrouped into GII.4-2006b (Minerva) and GII.4-2010 (New Orleans) variants. There was no spatiotemporal structure among the endemic GII strains; yet a significant spatiotemporal signal corresponding with the novel introduction of GII.4-2010 variant was detected. These data show that NoV GII.4 variants are highly endemic in HCMC and describe a scenario of rapid NoV strain replacement occurring in HCMC in early 2010.

The validation and utility of a quantitative one-step multiplex RT real-time PCR targeting rotavirus A and norovirus.

Rotavirus (RoV) and Norovirus (NoV) are the main causes of viral gastroenteritis. Currently, there is no validated multiplex real-time PCR that can detect and quantify RoV and NoV simultaneously. The aim of the study was to develop, validate, and internally control a multiplex one-step RT real-time PCR to detect and quantify RoV and NoV in stool samples. PCR sensitivity was assessed by comparing amplification against the current gold standard, enzyme immunoassay (EIA), on stool samples from 94 individuals with diarrhea and 94 individuals without diarrhea. PCR detected 10% more RoV positive samples than EIA in stools samples from patients with diarrhea. PCR detected 23% more NoV genogroup II positive samples from individuals with diarrhea and 9% more from individuals without diarrhea than EIA, respectively. Genotyping of the PCR positive/EIA negative samples suggested the higher rate of PCR positivity, in comparison to EIA, was due to increased sensitivity, rather than nonspecific hybridization. Quantitation demonstrated that the viral loads of RoV and NoV in the stools of diarrheal patients were an order of magnitude greater than in individuals without diarrhea. This internally controlled real-time PCR method is robust, exhibits a high degree of reproducibility, and may have a greater utility and sensitivity than commercial EIA kits.

The emergence of rotavirus G12 and the prevalence of enteric viruses in hospitalized pediatric diarrheal patients in southern Vietnam.

Diarrhea is a major cause of childhood morbidity and mortality in developing countries, and the majority of infections are of viral etiology. We aimed to compare the etiological prevalence of the major enteric viruses in an urban and a rural setting in southern Vietnam. We simultaneously screened fecal specimens from 362 children in Ho Chi Minh City and Dong Thap province that were hospitalized with acute diarrhea over a 1-month-long period for four viral gastrointestinal pathogens. Rotavirus was the most common pathogen identified, but there was a differential prevalence of rotavirus and norovirus between the urban and rural locations. Furthermore, rotavirus genotyping and phylogenetic analysis again differentiated the genotypes by the sampling location. Our data show a disproportional distribution of enteric viral pathogens in urban and rural locations, and we provide evidence of continual importation of new rotavirus strains into southern Vietnam and report the emergence of rotavirus genotype G12.

The burden and characteristics of enteric fever at a healthcare facility in a densely populated area of Kathmandu.

Enteric fever, caused by Salmonella enterica serovars Typhi and Paratyphi A (S. Typhi and S. Paratyphi A) remains a major public health problem in many settings. The disease is limited to locations with poor sanitation which facilitates the transmission of the infecting organisms. Efficacious and inexpensive vaccines are available for S. Typhi, yet are not commonly deployed to control the disease. Lack of vaccination is due partly to uncertainty of the disease burden arising from a paucity of epidemiological information in key locations. We have collected and analyzed data from 3,898 cases of blood culture-confirmed enteric fever from Patan Hospital in Lalitpur Sub-Metropolitan City (LSMC), between June 2005 and May 2009. Demographic data was available for a subset of these patients (n = 527) that were resident in LSMC and who were enrolled in trials. We show a considerable burden of enteric fever caused by S. Typhi (2,672; 68.5%) and S. Paratyphi A (1,226; 31.5%) at this Hospital over a four year period, which correlate with seasonal fluctuations in rainfall. We found that local population density was not related to incidence and we identified a focus of infections in the east of LSMC. With data from patients resident in LSMC we found that the median age of those with S. Typhi (16 years) was significantly less than S. Paratyphi A (20 years) and that males aged 15 to 25 were disproportionately infected. Our findings provide a snapshot into the epidemiological patterns of enteric fever in Kathmandu. The uneven distribution of enteric fever patients within the population suggests local variation in risk factors, such as contaminated drinking water. These findings are important for initiating a vaccination scheme and improvements in sanitation. We suggest any such intervention should be implemented throughout the LSMC area.

A changing picture of shigellosis in southern Vietnam: shifting species dominance, antimicrobial susceptibility and clinical presentation.

BACKGROUND: Shigellosis remains considerable public health problem in some developing countries. The nature of Shigellae suggests that they are highly adaptable when placed under selective pressure in a human population. This is demonstrated by variation and fluctuations in serotypes and antimicrobial resistance profile of organisms circulating in differing setting in endemic locations. Antimicrobial resistance in the genus Shigella is a constant threat, with reports of organisms in Asia being resistant to multiple antimicrobials and new generation therapies. METHODS: Here we compare microbiological, clinical and epidemiological data from patients with shigellosis over three different periods in southern Vietnam spanning 14 years. RESULTS: Our data demonstrates a shift in dominant infecting species (S. flexneri to S. sonnei) and resistance profile of the organisms circulating in southern Vietnam. We find that there was no significant variation in the syndromes associated with either S. sonnei or S. flexneri, yet the clinical features of the disease are more severe in later observations. CONCLUSIONS: Our findings show a change in clinical presentation of shigellosis in this setting, as the disease may be now more pronounced, this is concurrent with a change in antimicrobial resistance profile. These data highlight the socio-economic development of southern Vietnam and should guide future vaccine development and deployment strategies. TRIAL REGISTRATION: Current Controlled Trials ISRCTN55945881.