Deciphering Bordetella pertussis epidemiology through culture-independent multiplex amplicon and metagenomic sequencing.

Whooping cough (pertussis) has re-emerged despite high vaccine coverage in Australia and many other countries worldwide, partly attributable to genetic adaptation of the causative organism, Bordetella pertussis, to vaccines. Therefore, genomic surveillance has become essential to monitor circulating strains for these genetic changes. However, increasing uptake of PCR for the diagnosis of pertussis has affected the availability of cultured isolates for typing. In this study, we evaluated the use of targeted multiplex PCR (mPCR) amplicon sequencing and shotgun metagenomic sequencing for culture-independent typing of B. pertussis directly from respiratory swabs. We developed a nine-target mPCR amplicon assay that could accurately type major lineages [ptxP3/non-ptxpP3, fim3A/B, fhaB3/non-fhaB3, and epidemic lineages (ELs) 1-5] circulating in Australia. Validation using DNA from isolates and 178 residual specimens collected in 2010-2012 (n = 87) and 2019 (n = 91) showed that mPCR amplicon sequencing was highly sensitive with a limit of detection of 4.6 copies [IS481 cycle threshold (Ct) 27.3]. Shotgun metagenomic sequencing was successful in genotyping B. pertussis in 84% of clinical specimens with PCR Ct < 24 and was concordant with mPCR typing results. The results revealed an expansion of EL4 strains from 2010 to 2012 to 2019 in Australia and identified unrecognized co-circulating cases of Bordetella holmesii. This study provides valuable insight into the circulating lineages in Australia prior to the COVID-19 pandemic during which border closure and other interventions reduced pertussis cases to an all-time low, and paves the way for the genomic surveillance of B. pertussis in the era of culture-independent PCR-based diagnosis. IMPORTANCE: In this paper, we evaluated the use of targeted multiplex PCR (mPCR) amplicon sequencing and shotgun metagenomic sequencing for culture-independent typing of Bordetella pertussis directly in respiratory swabs. We first developed a novel targeted mPCR amplicon sequencing assay that can type major circulating lineages and validated its accuracy and sensitivity on 178 DNA extracts from clinical swabs. We also demonstrate the feasibility of using deep metagenomic sequencing for determining strain lineage and markers of virulence, vaccine adaptation, macrolide resistance, and co-infections. Our culture-independent typing methods applied to clinical specimens revealed the expansion of a major global epidemic lineage in Australia (termed EL4) just prior to the COVID-19 pandemic. It also detected cases of previously hidden co-infections from another Bordetella species called Bordetella holmesii. These findings offer valuable insight into the circulating pertussis lineages in Australia prior to the COVID-19 pandemic during which border closure and other interventions reduced pertussis cases to an all-time low. It also provides comparative data for future surveillance as pertussis resurgence after the COVID-19 pandemic has been reported this year in Australia and many other countries. Overall, our paper demonstrates the utility, sensitivity, and specificity of mPCR amplicon and metagenomic sequencing-based culture-independent typing of B. pertussis, which not only paves the way for culture-independent genomic surveillance of B. pertussis but also for other pathogens in the era of PCR-based diagnosis.

Effects of a Water, Sanitation, and Hygiene Mobile Health Program on Diarrhea and Child Growth in Bangladesh: A Cluster-randomized Controlled Trial of the Cholera Hospital-based Intervention for 7 Days (CHoBI7) Mobile Health Program.

BACKGROUND: The Cholera Hospital-Based Intervention for 7 Days (CHoBI7) mobile health (mHealth) program was a cluster-randomized controlled trial of diarrhea patient households conducted in Dhaka, Bangladesh. METHODS: Patients were block-randomized to 3 arms: standard message on oral rehydration solution use; health facility delivery of CHoBI7 plus mHealth (no home visits); and health facility delivery of CHoBI7 plus 2 home visits and mHealth. The primary outcome was reported diarrhea in the past 2 weeks collected monthly for 12 months. The secondary outcomes were stunting, underweight, and wasting at a 12-month follow-up. Analysis was intention-to-treat. RESULTS: Between 4 December 2016 and 26 April 2018, 2626 participants in 769 households were randomly allocated to 3 arms: 849 participants to the standard message arm, 886 to mHealth with no home visits arm, and 891 to the mHealth with 2 home visits. Children <5 years had significantly lower 12-month diarrhea prevalence in both the mHealth with 2 home visits arm (prevalence ratio [PR]: 0.73 [95% confidence interval {CI}, .61-.87]) and the mHealth with no home visits arm (PR: 0.82 [95% CI, .69-.97]). Children <2 years were significantly less likely to be stunted in both the mHealth with 2 home visits arm (33% vs 45%; odds ratio [OR]: 0.55 [95% CI, .31-.97]) and the mHealth with no home visits arm (32% vs 45%; OR: 0.54 [95% CI, .31-.96]) compared with children in the standard message arm. CONCLUSIONS: The CHoBI7 mHealth program lowered pediatric diarrhea and stunting among diarrhea patient households. CLINICAL TRIALS REGISTRATION: NCT04008134.

Child mouthing of soil and presence of animals in child sleeping spaces are associated with growth faltering among young children in Dhaka, Bangladesh (CHoBI7 Program).

OBJECTIVE: The objective of the study was to investigate potential risk factors for growth faltering among children under 5 years of age. METHOD: We conducted a prospective cohort study of 553 children under 5 years from diarrhoea patient households in urban Dhaka, Bangladesh. Height and weight measurements were obtained at baseline and at a 12-month follow-up. Caregivers of young children were administered a monthly questionnaire on household sociodemographic characteristics and hygiene practices. RESULTS: Children with caregiver reports of mouthing soil at the majority of household visits had a significant reduction in their height-for-age z-scores (HAZ) from baseline to the 12-month follow-up (ΔHAZ: -0.28 (95% confidence interval (CI): -0.51, -0.05)). A significant reduction in HAZ was also observed for children in households with animals in their sleeping space (ΔHAZ: -0.37 (95% CI: -0.71, -0.04)). CONCLUSION: These findings provide further evidence to support the hypothesis that child mouthing of soil and the presence of animals in the child's sleeping space are potential risk factors for growth faltering among young children. Interventions are urgently needed to provide clean play and sleeping spaces for young children to reduce exposure to faecal pathogens through child mouthing.

OBJECTIF: L'objectif de l'étude était d'examiner les facteurs de risque potentiels de retard de croissance chez les enfants de moins de 5 ans. MÉTHODE: Nous avons mené une étude de cohorte prospective sur 553 enfants de moins de 5 ans provenant de ménages avec des patients diarrhéiques dans la ville de Dhaka, au Bangladesh. Les mesures de taille et de poids ont été obtenues au départ et à 12 mois de suivi. Les personnes s'occupant de jeunes enfants (les gardiens) ont reçu un questionnaire mensuel sur les caractéristiques sociodémographiques des ménages et les pratiques d'hygiène. RÉSULTATS: Les enfants pour lesquels les gardiens ont déclaré qu'ils mâchouillaient de la terre lors de la majorité des visites à domicile présentaient une diminution du score Z de taille pour l'âge (TAZ) de manière significative de l'âge de référence au 12 mois de suivi (ΔTAZ −0,28 (intervalle de confiance (IC) à 95%: −0,51, −0,05)). Une réduction significative des TAZ a également été observée pour les enfants des ménages ayant des animaux dans leur espace de couchage (ΔTAZ −0,37 (IC95%: −0,71, −0,04)). CONCLUSION: Ces résultats apportent des données supplémentaires pour appuyer l'hypothèse selon laquelle le fait que l'enfant mâchouille de la terre et la présence d'animaux dans son espace de couchage sont des facteurs de risque potentiels de retard de croissance chez les jeunes enfants. Des interventions sont urgemment nécessaires pour fournir des espaces de jeu et de sommeil sains aux jeunes enfants afin de réduire l'exposition aux agents pathogènes fécaux par le mâchouillement des enfants.

Transmission of Infectious Vibrio cholerae through Drinking Water among the Household Contacts of Cholera Patients (CHoBI7 Trial).

Recurrent cholera causes significant morbidity and mortality among the growing population of Dhaka, the capital city of Bangladesh. Previous studies have demonstrated that household contacts of cholera patients are at >100 times higher risk of cholera during the week after the presentation of the index patient. Our prospective study investigated the mode of transmission of Vibrio cholerae, the cause of cholera, in the households of cholera patients in Dhaka city. Out of the total 420 rectal swab samples analyzed from 84 household contacts and 330 water samples collected from 33 households, V. cholerae was isolated from 20%(17/84) of household contacts, 18%(6/33) of stored drinking water, and 27%(9/33) of source water samples. Phenotypic and molecular analyses results confirmed the V. cholerae isolates to be toxigenic and belonging to serogroup O1 biotype El Tor (ET) possessing cholera toxin of classical biotype (altered ET). Phylogenetic analysis by pulsed-field gel electrophoresis (PFGE) showed the V. cholerae isolates to be clonally linked, as >95% similarity was confirmed by sub-clustering patterns in the PFGE (NotI)-based dendrogram. Mapping results showed cholera patients to be widely distributed across 25 police stations. The data suggesting the transmission of infectious V. cholerae within the household contacts of cholera patients through drinking water underscores the need for safe water to prevent spread of cholera and related deaths in Dhaka city.