Emergence of new genotypes and lineages of dengue viruses during the 2012–15 epidemics in southern India

Objectives: To genotypically characterize dengue virus (DENV) isolates among dengue-infected children from 2012–13/2014–15 outbreaks in southern India. Methods: Children hospitalized with suspected dengue were tested for dengue RT-PCR targeting Capsid-preMembrane (C-prM) and Envelope (Env) regions. Following virologic confirmation (n = 612), a representative selection of DENV isolates (n = 99) were sequenced for C-prM, aligned using ClustalW and subjected to phylogenetic analysis by maximum-likelihood method in MEGA6. Results: In 2012–13 (n = 113), DENV-3 (44, 38.9%) and DENV-2 (43, 38.1%) predominated; DENV-1 (22, 19.5%) and DENV-4 (1, 0.9%) were less common. The pattern changed in 2014–15 (n = 499), when DENV-1 (329, 65.7%) predominated, followed by DENV-2 (97, 21.2%), DENV-3 (36, 6.7%) and DENV-4 (10, 2.0%). Multiple-serotype co-infections occurred in 2.7% and 5.4% in 2012–13 and 2014–15, respectively. Genotype III (GIII) of DENV-1 predominated (85.7%) in 2012–13, ceding to GI predominance (80.8%) in 2014–15. Among DENV-2, 71.9% (23/32) showed distinct clustering suggesting a new lineage, 'GIVc'. All tested DENV-4 were GIC, whose clustering pattern showed the emergence of two distinct clades. Conclusions: New genotypic/lineage variations in DENV-1 and DENV-2 may have influenced the magnitude and severity of dengue epidemics in southern India during this period. These findings emphasize the role of active surveillance of DENV serotypes/genotypes in aiding outbreak control and vaccine studies. Keywords: Dengue virus, Serotyping, Sequencing, Phylogenetics, Genotypes, Lineage

Persistent circulation of a fluoroquinolone-resistant Salmonella enterica Typhi clone in the Indian subcontinent.

BACKGROUND: The molecular structure of circulating enteric fever pathogens was studied using hospital-based genomic surveillance in a tertiary care referral centre in South India as a first genomic surveillance study, to our knowledge, of blood culture-confirmed enteric fever in the region. METHODS: Blood culture surveillance was conducted at St John's Medical College Hospital, Bengaluru, between July 2016 and June 2017. The bacterial isolates collected were linked to demographic variables of patients and subjected to WGS. The resulting pathogen genomic data were also globally contextualized to gauge possible phylogeographical patterns. RESULTS: Hospital-based genomic surveillance for enteric fever in Bengaluru, India, identified 101 Salmonella enterica Typhi and 14 S. Paratyphi A in a 1 year period. Ninety-six percent of isolates displayed non-susceptibility to fluoroquinolones. WGS showed the dominant pathogen was S. Typhi genotype 4.3.1.2 (H58 lineage II). A fluoroquinolone-resistant triple-mutant clone of S. Typhi 4.3.1.2 previously associated with gatifloxacin treatment failure in Nepal was implicated in 18% of enteric fever cases, indicating ongoing inter-regional circulation. CONCLUSIONS: Enteric fever in South India continues to be a major public health issue and is strongly associated with antimicrobial resistance. Robust microbiological surveillance is necessary to direct appropriate treatment and preventive strategies. Of particular concern is the emergence and expansion of the highly fluoroquinolone-resistant triple-mutant S. Typhi clone and its ongoing inter- and intra-country transmission in South Asia, which highlights the need for regional coordination of intervention strategies, including vaccination and longer-term strategies such as improvements to support hygiene and sanitation

Laboratory and molecular surveillance of paediatric typhoidal Salmonella in Nepal: Antimicrobial resistance and implications for vaccine policy.

BACKGROUND: Children are substantially affected by enteric fever in most settings with a high burden of the disease, including Nepal. However pathogen population structure and transmission dynamics are poorly delineated in young children, the proposed target group for immunization programs. Here we present whole genome sequencing and antimicrobial susceptibility data on 198 S. Typhi and 66 S. Paratyphi A isolated from children aged 2 months to 15 years of age during blood culture surveillance at Patan Hospital, Nepal, 2008-2016. PRINCIPAL FINDINGS: S. Typhi was the dominant agent and comprised several distinct genotypes, dominated by 4.3.1 (H58). The heterogeneity of genotypes in children under five was reduced compared to data from 2005-2006, attributable to ongoing clonal expansion of H58. Most isolates (86%) were non-susceptible to fluoroquinolones, associated mainly with S. Typhi H58 lineage II and S. Paratyphi A harbouring mutations in the quinolone resistance-determining region (QRDR); non-susceptible strains from these groups accounted for 50% and 25% of all isolates. Multi-drug resistance (MDR) was rare (3.5% of S. Typhi, 0 S. Paratyphi A) and restricted to chromosomal insertions of resistance genes in H58 lineage I strains. Temporal analyses revealed a shift in dominance from H58 Lineage I to H58 Lineage II, with the latter being significantly more common after 2010. Comparison to global data sets showed the local S. Typhi and S. Paratyphi A strains had close genetic relatives in other South Asian countries, indicating regional strain circulation. Multiple imports from India of ciprofloxacin-resistant H58 lineage II strains were identified, but these were rare and showed no evidence of clonal replacement of local S. Typhi. SIGNIFICANCE: These data indicate that enteric fever in Nepal continues to be a major public health issue with ongoing inter- and intra-country transmission, and highlights the need for regional coordination of intervention strategies. The absence of a S. Paratyphi A vaccine is cause for concern, given its prevalence as a fluoroquinolone resistant enteric fever agent in this setting

Pathogen genomic surveillance of typhoidal Salmonella infection in adults and children reveals no association between clinical outcomes and infecting genotypes

Funder: Rhodes Scholarships; doi: http://dx.doi.org/10.13039/501100000697Funder: Bill and Melinda Gates Foundation; doi: http://dx.doi.org/10.13039/100000865Abstract: Background: India is endemic for enteric fever, and it is not known whether the variations in clinical manifestations between patients are due to host, environmental or pathogen factors. Blood culture surveillance was conducted at St. John’s Medical College Hospital, Bangalore, between July 2016 and June 2017. Clinical, laboratory and demographic data were collected from each case, and bacterial isolates were subjected to whole genome sequencing. Comparative analysis between adults and paediatric patients was carried out to ascertain differences between adult and paediatric disease. Results: Among the 113 cases of blood culture-confirmed enteric fever, young adults (16–30 years) and children < 15 years accounted for 47% and 37% of cases, respectively. Anaemia on presentation was seen in 46% of cases, and 19% had an abnormal leucocyte count on presentation. The majority received treatment as inpatients (70%), and among these, adults had a significantly longer duration of admission when compared with children (p = 0.002). There were atypical presentations including arthritis, acute haemolysis and a case of repeated typhoid infection with two separate S. Typhi genotypes. There was no association between infecting genotype/serovar and treatment status (outpatient vs inpatient), month of isolation, duration of admission, patient age (adult or child), antimicrobial susceptibility, Widal positivity or haematologic parameters. Conclusions: Amidst the many public health concerns of South India, enteric fever continues to contribute substantially to hospital burden with non-specific as well as uncommon clinical features in both paediatric and adult populations likely driven by host and environmental factors. Robust clinical surveillance as well monitoring of pathogen population structure is required to inform treatment and preventive strategies

The molecular epidemiology of paediatric enteric fever in Nepal between 2008 and 2016, and South India between 2016 and 2017

Enteric fever continues to affect people living in endemic settings substantially causing at least 20 million cases of febrile illnesses every year with 1% mortality. Over the last decade there has been considerable debate surrounding the burden and disease profile of enteric fever in the paediatric population. This is partially due to the similarity of the clinical features of paediatric enteric fever to most other febrile illness seen in endemic settings. The treatment of enteric fever is proving to be a challenge with the emergence of antimicrobial resistant strains, particularly the 4.3.1 genotype (H58 haplotype), which is spreading rapidly. Multi-drug resistant (MDR) enteric fever, defined as infection with typhoidal Salmonellae that exhibit a combined resistance to ampicillin, cotrimoxazole and chloramphenicol emerged in the 1990s and was mediated primarily via the 4.3.1 genotype population through the horizontal acquisition of antimicrobial resistance determinants. Subsequently, fluoroquinolones became the drug of choice and the treatment of enteric fever following which fluoroquinolone resistance emerged, again through the 4.3.1 genotype. However, these antimicrobial trends may not be uniform across endemic regions and an understanding of these differing patterns as well the temporal changes in these trends are important in planning treatment strategies. In the short and medium term work needs to be focused on achieving the greatest benefits from the prudent use of the recently WHO pre-qualified Vi-TT conjugate vaccine candidate. Whilst the long term vision towards eradicating enteric fever needs to focus on better understanding the underlying the biology of this disease through the use of contemporary technologies while simultaneously improving infrastructure for the provision of clean water, adequate sanitation and hygiene. This thesis aims to age-characterise the disease burden of typhoid fever in endemic regions of South and South-East Asia as well as the African continent. Following this, the molecular epidemiology of enteric fever in two endemic settings in the Indian subcontinent is delineated with a keen focus on the 4.3.1 genotype (H58) population as well the phenotypic patterns and molecular determinants of antimicrobial resistance. This thesis finally systematically reviews the global trends of antimicrobial resistance of S. Typhi isolates over time both from a phenotypic and molecular perspective. The key results from this thesis include; the age stratification of disease occurrence in endemic regions which showed a substantial proportion occurs in the youngest age group in both Africa and Asia, the uniform dominance of 4.3.1 genotypes conferring a high degree of fluoroquinolone resistance contrary to earlier suggestions of younger children being more susceptible to a broader range of infecting genotypes, the dissimilarities between the antimicrobial resistance carrying capabilities of lineage I and lineage II strains of the 4.3.1 genotype as well as novel AMR gene arrangements and finally the temporal trends of AMR in S. Typhi which were different between Asia an Africa. The high prevalence of lineage I strains in Africa and South-East Asia in contrast to the high prevalence of lineage II strains in the Indian subcontinent reflect the antimicrobial selection pressures as well the evolutionary characteristics of circulating pathogen populations in these regions. The implications of the data reported in this thesis have implications for treatment and prevention strategies. For the first time in history an opportunity has risen to effectively vaccinate the youngest age group (0-4 years) from typhoid through the Vi-TT conjugate vaccine. As highlighted in this thesis the youngest age group (0-4 years) have a high disease occurrence in endemic areas as seen in a meta-analysis as well as through data from two endemic sites collated and reported in this thesis. The older age groups also suffer greatly from this disease calling for a broad based vaccine strategy. The implications for treatment of enteric fever are however more relevant in the immediate term which suggest that in endemic regions in Asia, fluoroquinolones have little role to play in treatment protocols while fluoroquinolones are still relevant in the African setting. In Asia, reverting back to former first-line antimicrobials might be an option but the possibility of re-emergence of widespread resistance to these currently sensitive antimicrobials is very high exemplifying the ability of S. Typhi to adapt to changing antimicrobial pressures.</p

The molecular epidemiology of paediatric enteric fever in Nepal between 2008 and 2016, and South India between 2016 and 2017

Enteric fever continues to affect people living in endemic settings substantially causing at least 20 million cases of febrile illnesses every year with 1% mortality. Over the last decade there has been considerable debate surrounding the burden and disease profile of enteric fever in the paediatric population. This is partially due to the similarity of the clinical features of paediatric enteric fever to most other febrile illness seen in endemic settings. The treatment of enteric fever is proving to be a challenge with the emergence of antimicrobial resistant strains, particularly the 4.3.1 genotype (H58 haplotype), which is spreading rapidly. Multi-drug resistant (MDR) enteric fever, defined as infection with typhoidal Salmonellae that exhibit a combined resistance to ampicillin, cotrimoxazole and chloramphenicol emerged in the 1990s and was mediated primarily via the 4.3.1 genotype population through the horizontal acquisition of antimicrobial resistance determinants. Subsequently, fluoroquinolones became the drug of choice and the treatment of enteric fever following which fluoroquinolone resistance emerged, again through the 4.3.1 genotype. However, these antimicrobial trends may not be uniform across endemic regions and an understanding of these differing patterns as well the temporal changes in these trends are important in planning treatment strategies. In the short and medium term work needs to be focused on achieving the greatest benefits from the prudent use of the recently WHO pre-qualified Vi-TT conjugate vaccine candidate. Whilst the long term vision towards eradicating enteric fever needs to focus on better understanding the underlying the biology of this disease through the use of contemporary technologies while simultaneously improving infrastructure for the provision of clean water, adequate sanitation and hygiene. This thesis aims to age-characterise the disease burden of typhoid fever in endemic regions of South and South-East Asia as well as the African continent. Following this, the molecular epidemiology of enteric fever in two endemic settings in the Indian subcontinent is delineated with a keen focus on the 4.3.1 genotype (H58) population as well the phenotypic patterns and molecular determinants of antimicrobial resistance. This thesis finally systematically reviews the global trends of antimicrobial resistance of S. Typhi isolates over time both from a phenotypic and molecular perspective. The key results from this thesis include; the age stratification of disease occurrence in endemic regions which showed a substantial proportion occurs in the youngest age group in both Africa and Asia, the uniform dominance of 4.3.1 genotypes conferring a high degree of fluoroquinolone resistance contrary to earlier suggestions of younger children being more susceptible to a broader range of infecting genotypes, the dissimilarities between the antimicrobial resistance carrying capabilities of lineage I and lineage II strains of the 4.3.1 genotype as well as novel AMR gene arrangements and finally the temporal trends of AMR in S. Typhi which were different between Asia an Africa. The high prevalence of lineage I strains in Africa and South-East Asia in contrast to the high prevalence of lineage II strains in the Indian subcontinent reflect the antimicrobial selection pressures as well the evolutionary characteristics of circulating pathogen populations in these regions. The implications of the data reported in this thesis have implications for treatment and prevention strategies. For the first time in history an opportunity has risen to effectively vaccinate the youngest age group (0-4 years) from typhoid through the Vi-TT conjugate vaccine. As highlighted in this thesis the youngest age group (0-4 years) have a high disease occurrence in endemic areas as seen in a meta-analysis as well as through data from two endemic sites collated and reported in this thesis. The older age groups also suffer greatly from this disease calling for a broad based vaccine strategy. The implications for treatment of enteric fever are however more relevant in the immediate term which suggest that in endemic regions in Asia, fluoroquinolones have little role to play in treatment protocols while fluoroquinolones are still relevant in the African setting. In Asia, reverting back to former first-line antimicrobials might be an option but the possibility of re-emergence of widespread resistance to these currently sensitive antimicrobials is very high exemplifying the ability of S. Typhi to adapt to changing antimicrobial pressures.</p

A systematic review of antimicrobial resistance in Salmonella enterica serovar Typhi, the etiological agent of typhoid.

BACKGROUND: The temporal and spatial change in trends of antimicrobial resistance (AMR) in typhoid have not been systematically studied, and such information will be critical for defining intervention, as well as planning sustainable prevention strategies. METHODOLOGY AND FINDINGS: To identify the phenotypic trends in AMR, 13,833 individual S. Typhi isolates, reported from 1973 to 2018 in 62 publications, were analysed to determine the AMR preponderance over time. Separate analyses of molecular resistance determinants present in over 4,000 isolates reported in 61 publications were also conducted. Multi-drug resistant (MDR) typhoid is in decline in Asia in a setting of high fluoroquinolone resistance while it is on the increase in Africa. Mutations in QRDRs in gyrA (S83F, D87N) and parC (S80I) are the most common mechanisms responsible for fluoroquinolone resistance. Cephalosporin resistant S. Typhi, dubbed extensively drug-resistant (XDR) is a real threat and underscores the urgency in deploying the Vi-conjugate vaccines. CONCLUSION: From these observations, it appears that AMR in S. Typhi will continue to emerge leading to treatment failure, changes in antimicrobial policy and further resistance developing in S. Typhi isolates and other Gram-negative bacteria in endemic regions. The deployment of typhoid conjugate vaccines to control the disease in endemic regions may be the best defence

Neonatal and infant mortality associated with spina bifida: A systematic review and meta-analysis.

ObjectivesA systematic review was conducted in high-income country settings to analyse: (i) spina bifida neonatal and IMRs over time, and (ii) clinical and socio-demographic factors associated with mortality in the first year after birth in infants affected by spina bifida.Data sourcesPubMed, Embase, Ovid, Web of Science, CINAHL, Scopus and the Cochrane Library were searched from 1st January, 1990 to 31st August, 2020 to review evidence.Study selectionPopulation-based studies that provided data for spina bifida infant mortality and case fatality according to clinical and socio-demographical characteristics were included. Studies were excluded if they were conducted solely in tertiary centres. Spina bifida occulta or syndromal spina bifida were excluded where possible.Data extraction and synthesisIndependent reviewers extracted data and assessed their quality using MOOSE guideline. Pooled mortality estimates were calculated using random-effects (+/- fixed effects) models meta-analyses. Heterogeneity between studies was assessed using the Cochrane Q test and I2 statistics. Meta-regression was performed to examine the impact of year of birth cohort on spina bifida infant mortality.ResultsTwenty studies met the full inclusion criteria with a total study population of over 30 million liveborn infants and approximately 12,000 spina bifida-affected infants. Significant declines in spina bifida associated infant and neonatal mortality rates (e.g. 4.76% decrease in IMR per 100, 000 live births per year) and case fatality (e.g. 2.70% decrease in infant case fatality per year) were consistently observed over time. Preterm birth (RR 4.45; 2.30-8.60) and low birthweight (RR 4.77; 2.67-8.55) are the strongest risk factors associated with increased spina bifida infant case fatality.SignificanceSignificant declines in spina bifida associated infant/neonatal mortality and case fatality were consistently observed, advances in treatment and mandatory folic acid food fortification both likely play an important role. Particular attention is warranted from clinicians caring for preterm and low birthweight babies affected by spina bifida

Comparing COVID-19 vaccine allocation strategies in India: A mathematical modelling study

BACKGROUND: The development and widespread use of an effective SARS-CoV-2 vaccine could prevent substantial morbidity and mortality associated with COVID-19 and mitigate the secondary effects associated with non-pharmaceutical interventions. METHODS: We used an age-structured, expanded SEIR model with social contact matrices to assess age-specific vaccine allocation strategies in India. We used state-specific age structures and disease transmission coefficients estimated from confirmed incident cases of COVID-19 between 1 July and 31 August 2020. Simulations were used to investigate the relative reduction in mortality and morbidity of vaccine allocation strategies based on prioritizing different age groups, and the interactions of these strategies with concurrent non-pharmaceutical interventions. Given the uncertainty associated with COVID-19 vaccine development, we varied vaccine characteristics in the modelling simulations. RESULTS: Prioritizing COVID-19 vaccine allocation for older populations (i.e., &gt;60 years) led to the greatest relative reduction in deaths, regardless of vaccine efficacy, control measures, rollout speed, or immunity dynamics. Preferential vaccination of this group often produced relatively higher total symptomatic infections and more pronounced estimates of peak incidence than other assessed strategies. Vaccine efficacy, immunity type, target coverage, and rollout speed significantly influenced overall strategy effectiveness, with the time taken to reach target coverage significantly affecting the relative mortality benefit comparative to no vaccination. CONCLUSIONS: Our findings support global recommendations to prioritize COVID-19 vaccine allocation for older age groups. Relative differences between allocation strategies were reduced as the speed of vaccine rollout was increased. Optimal vaccine allocation strategies will depend on vaccine characteristics, strength of concurrent non-pharmaceutical interventions, and region-specific goals