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Data collection was implemented through an initiative by the Child Health and Mortality Prevention Surveillance (CHAMPS) Network to assess whether lockdowns and other social distancing policies during COVID-19 had implications for household economic status, maternal and child health, and healthcare accessibility for pregnant women and children. The data were collected from April 2021 until February 2022 from a population living in a rural community of Mozambique. This rural community is located within a Health and Demographic Surveillance System (HDSS) that operates in the Manhiça district of Maputo province. The survey instrument used for data collection was specifically designed to examine household awareness, knowledge, and prevalence of COVID-19; it was also designed to document hardships experienced by households during the pandemic period such as food insecurity, job losses and/or business closures of household members, and access to healthcare. The data are generalizable to a contiguous community in Manhiça, Mozambique of approximately 200,000 inhabitants.

Introduction Determining aetiology of severe illness can be difficult, especially in settings with limited diagnostic resources, yet critical for providing life-saving care. Our objective was to describe the accuracy of antemortem clinical diagnoses in young children in high-mortality settings, compared with results of specific postmortem diagnoses obtained from Child Health and Mortality Prevention Surveillance (CHAMPS).Methods We analysed data collected during 2016–2022 from seven sites in Africa and South Asia. We compared antemortem clinical diagnoses from clinical records to a reference standard of postmortem diagnoses determined by expert panels at each site who reviewed the results of histopathological and microbiological testing of tissue, blood, and cerebrospinal fluid. We calculated test characteristics and 95% CIs of antemortem clinical diagnostic accuracy for the 10 most common causes of death. We classified diagnostic discrepancies as major and minor, per Goldman criteria later modified by Battle.Results CHAMPS enrolled 1454 deceased young children aged 1–59 months during the study period; 881 had available clinical records and were analysed. The median age at death was 11 months (IQR 4–21 months) and 47.3% (n=417) were female. We identified a clinicopathological discrepancy in 39.5% (n=348) of deaths; 82.3% of diagnostic errors were major. The sensitivity of clinician antemortem diagnosis ranged from 26% (95% CI 14.6% to 40.3%) for non-infectious respiratory diseases (eg, aspiration pneumonia, interstitial lung disease, etc) to 82.2% (95% CI 72.7% to 89.5%) for diarrhoeal diseases. Antemortem clinical diagnostic specificity ranged from 75.2% (95% CI 72.1% to 78.2%) for diarrhoeal diseases to 99.0% (95% CI 98.1% to 99.6%) for HIV.Conclusions Antemortem clinical diagnostic errors were common for young children who died in areas with high childhood mortality rates. To further reduce childhood mortality in resource-limited settings, there is an urgent need to improve antemortem diagnostic capability through advances in the availability of diagnostic testing and clinical skills.

Introduction The burden of severe maternal morbidity is highest in sub-Saharan Africa, and its relative contribution to maternal (ill) health may increase as maternal mortality continues to fall. Women’s perspective of their long-term recovery following severe morbidity beyond the standard 42-day postpartum period remains largely unexplored.Methods This woman-centred, grounded theory study was nested within the Pregnancy Care Integrating Translational Science Everywhere (PRECISE) study in Kilifi, Kenya. Purposive and theoretical sampling was used to recruit 20 women who experienced either a maternal near-miss event (n=11), potentially life-threatening condition (n=6) or no severe morbidity (n=3). Women were purposively selected between 6 and 36 months post partum at the time of interview to compare recovery trajectories. Using a constant comparative approach of line-by-line open codes, focused codes, super-categories and themes, we developed testable hypotheses of women’s postpartum recovery trajectories after severe maternal morbidity.Results Grounded in women’s accounts of their lived experience, we identify three phases of recovery following severe maternal morbidity: ‘loss’, ‘transition’ and ‘adaptation to a new normal’. These themes are supported by multiple, overlapping super-categories: loss of understanding of own health, functioning and autonomy; transition in women’s identity and relationships; and adaptation to a new physical, psychosocial and economic state. This recovery process is multidimensional, potentially cyclical and extends far beyond the standard 42-day postpartum period.Conclusion Women’s complex needs following severe maternal morbidity require a reconceptualisation of postpartum recovery as extending far beyond the standard 42-day postpartum period. Women’s accounts expose major deficiencies in the provision of postpartum and mental healthcare. Improved postpartum care provision at the primary healthcare level, with reach extended through community health workers, is essential to identify and treat chronic mental or physical health problems following severe maternal morbidity.

In resource-limited settings where vital registration and medical death certificates are unavailable or incomplete, verbal autopsy (VA) is often used to attribute causes of death (CoD) and prioritize resource allocation and interventions. We aimed to determine the CoD concordance between InterVA and CHAMPS's method. The causes of death (CoDs) of children

The COVID-19 pandemic has prompted countries to swiftly implement rigorous preventive measures on a population-wide scale worldwide. However, in low-income countries like Mozambique this was difficult, coupled with a generalised lack of knowledge on how the population understood and complied with these measures. This study assessed community perceptions and implementation of anti-COVID-19 measures recommended by Mozambican authorities in Manhiça and Quelimane districts, including confinement, social distancing, frequent handwashing, mask wearing, and quarantine as the key practices to evaluate. We conducted a cross-sectional quantitative survey in October 2020 and February 2021, interviewing heads of households, face-to-face. The data collected included self-evaluation of compliance and existence of handwashing facilities and face-masks in the households, aided by observations. We present descriptive statistics on perceptions and compliance at individual and household levels. Out of the 770 participants, nearly all (98.7%) were aware of Coronavirus disease, including the term COVID-19 (89.2%). Knowledge varied between districts, with Manhiça participants showing higher levels of sufficient ability to define the disease. The symptoms most mentioned were dry cough (17.8%), fever (15.7%), flu-like symptoms (14.2%), breathing difficulties (13.6%), and headache (13.1%). Participants recognized various transmission modes, including touching infected objects and inhaling infected air. Preventive measures like handwashing with soap or sanitizing hands with alcohol, wearing masks, and social distancing were acknowledged, but the understanding varied. Compliance with these measures was generally low, with fewer than half of respondents reporting adherence to them. Only 30.4% of households had handwashing facilities (of which only 41.0% had water), and masks were often limited to one per person aged 6 years or more. Community members in Manhica and Quelimane were aware of COVID-19 but had limited understanding of what the preventive measures meant, and had lower levels of compliance. Understanding and addressing the factors affecting the proper implementation of these measures is crucial for improving community adherence in preventing infectious diseases with epidemic potential.

BackgroundSeroprevalence studies are an alternative approach to estimating the extent of transmission of SARS-CoV-2 and the evolution of the pandemic in different geographical settings. We aimed to determine the SARS-CoV-2 seroprevalence from March 2020 to March 2022 in a rural and urban setting in Kilifi County, Kenya.MethodsWe obtained representative random samples of stored serum from a pregnancy cohort study for the period March 2020 to March 2022 and tested for antibodies against the spike protein using a qualitative SARS-CoV-2 ELISA kit (Wantai, total antibodies). All positive samples were retested for anti-SARS-CoV-2 anti-nucleocapsid antibodies (Euroimmun, ELISA kits, NCP, qualitative, IgG) and anti-spike protein antibodies (Euroimmun, ELISA kits, QuantiVac; quantitative, IgG).ResultsA total of 2,495 (of 4,703 available) samples were tested. There was an overall trend of increasing seropositivity from a low of 0% [95% CI 0–0.06] in March 2020 to a high of 89.4% [95% CI 83.36–93.82] in Feb 2022. Of the Wantai test-positive samples, 59.7% [95% CI 57.06–62.34] tested positive by the Euroimmun anti-SARS-CoV-2 NCP test and 37.4% [95% CI 34.83–40.04] tested positive by the Euroimmun anti-SARS-CoV-2 QuantiVac test. No differences were observed between the urban and rural hospital but villages adjacent to the major highway traversing the study area had a higher seroprevalence.ConclusionAnti-SARS-CoV-2 seroprevalence rose rapidly, with most of the population exposed to SARS-CoV-2 within 23 months of the first cases. The high cumulative seroprevalence suggests greater population exposure to SARS-CoV-2 than that reported from surveillance data.

BackgroundStaphylococcus aureus is one of the main causes of bacteraemia, associated with high mortality, mainly due to the occurrence of multidrug resistant (MDR) strains. Data on antibiotic susceptibility and genetic lineages of bacteraemic S. aureus are still scarce in Mozambique. The study aims to describe the antibiotic susceptibility and clonality of S. aureus isolated from blood cultures of children admitted to the Manhiça District Hospital over two decades (2001–2019).MethodsA total of 336 S. aureus isolates detected in blood cultures of children aged

Understanding community members' knowledge of SARS-CoV-2 transmission and prevention is essential for directing public health interventions to reduce disease spread and improve vaccination coverage. Here, we describe knowledge of COVID-19 transmission, prevention, and symptoms among community residents in Mozambique. We conducted a cross-sectional survey among 33,087 households in a Health and Demographic Surveillance System in Manhiça, Mozambique. Participants were recruited in April 2021 before the Delta variant wave to the peak of Omicron cases in February 2022. Principal components analysis was used to create scores representing knowledge of COVID-19 symptoms, transmission, and prevention. Multiple imputation and quasi-Poisson regression were used to examine associations between demographic characteristics and sources of COVID-19 information, and knowledge of COVID-19 symptoms, transmission, and prevention. We examined whether sources of COVID-19 information mediated the relationship between educational attainment and knowledge of symptoms, transmission, and prevention. Across this rural community, 98.2%, 97.0%, and 85.1% of respondents reported knowing how COVID-19 could be prevented, that SARS-CoV-2 can cause disease, and how SARS-CoV-2 is transmitted, respectively. The most recognized COVID-19 symptoms were cough (51.2%), headaches (44.9%), and fever (44.5%); transmission mechanisms were saliva droplets (50.5%) or aerosol (46.9%) from an infected person; and prevention measures were handwashing (91.9%) and mask-wearing (91.8%). Characteristics associated with greater knowledge of symptoms, transmission, and prevention included having at least primary education, older age, employment, higher wealth, and Christian religion. Respondents who had experienced COVID-19 symptoms were also more likely to possess knowledge of symptoms, transmission, and prevention. Receiving information from television, WhatsApp, radio, and hospital, mediated the relationship between educational attainment and knowledge scores. These findings support the need for outreach and for community-engaged messaging to promote prevention measures, particularly among people with low education.

Each year, 2.4 million children die within their first month of life. Child Health and Mortality Prevention Surveillance (CHAMPS) established in 7 countries aims to generate accurate data on why such deaths occur and inform prevention strategies. Neonatal deaths that occurred between December 2016 and December 2021 were investigated with MITS within 24-72 hours of death. Testing included blood, cerebrospinal fluid and lung cultures, multi-pathogen PCR on blood, CSF, nasopharyngeal swabs and lung tissue, and histopathology examination of lung, liver and brain. Data collection included clinical record review and family interview using standardized verbal autopsy. The full set of data was reviewed by local experts using a standardized process (Determination of Cause of Death) to identify all relevant conditions leading to death (causal chain), per WHO recommendations. For analysis we stratified neonatal death into 24-hours of birth, early (1-

(1) Background: Laboratories supporting the invasive bacteria preventable disease (IB-VPD) network are expected to demonstrate the capacity to identify the main etiological agents of pediatric bacterial meningitis (PBM) (Neisseria meningitidis, Streptococcus pneumoniae and Haemophilus influenzae) on Gram stains and in phenotypic identification. Individual reports of sentinel site (SSL), national (NL) and regional reference (RRL) laboratories participating in the World Health Organization (WHO)-coordinated external quality assessment, distributed by the United Kingdom National External Quality Assessment (EQA) Services (UK NEQAS) for Microbiology between 2014 and 2019 were analyzed. (2) Methods: The panels consisted of (1) unstained bacterial smears for Gram staining, (2) viable isolates for identification and serotyping/serogrouping (ST/SG) and (3) simulated cerebral spinal fluid (CSF) samples for species detection and ST/SG using polymerase chain reaction (PCR). SSLs and NLs tested for Gram staining and species identification (partial panel). RRLs, plus any SSLs and NLs (optionally) also analyzed the simulated CSF samples (full panel). The passing score was ≥75% for NLs and SSLs, and ≥90% for RRLs and NLs/SSLs testing the full panel. (3) Results: Overall, 63% (5/8) of the SSLs and NLs were able to correctly identify the targeted pathogens, in 2019; but there were challenges to identify Haemophilus influenzae either on Gram stains (35% of the labs failed 2014), or in culture. Individual performance showed inconsistent capacity, with only 39% (13/33) of the SSLs/NLs passing the EQA exercise throughout all surveys in which they participated. RRLs performed well over the study period, but one of the two failed to reach the minimal passing score in 2016 and 2018; while the SSLs/NLs that optionally tested the full panel scored between 75% and 90% (intermediate pass category). (4) Conclusions: We identified a need for implementing a robust quality management system for timely identification of the gaps and then implementing corrective and preventive actions, in addition to continuous refresher training in the SSLs and NLs supporting the IB-VPD surveillance in the World Health Organization, Regional Office for Africa (WHO AFRO).

Background: Following the World Health Organization (WHO) recommendation, 38/47 countries have introduced rotavirus vaccines into the program of immunization in the WHO Regional Office for Africa (WHO/AFRO). Initially, two vaccines (Rotarix and Rotateq) were recommended and recently two additional vaccines (Rotavac and Rotasiil) have become available. However, the global supply challenges have increasingly forced some countries in Africa to switch vaccine products. Therefore, the recent WHO pre-qualified vaccines (Rotavac, Rotasiil) manufactured in India, offer alternatives and reduce global supply challenges related to rotavirus vaccines; Methods: Using a questionnaire, we administered to the Program Managers, Expanded Program for Immunization, we collected data on vaccine introduction and vaccine switch and the key drivers of the decisions for switching vaccines products, in the WHO/AFRO. Data was also collected fromliterature review and the global new vaccine introduction status data base maintained by WHO and other agencies. Results: Of the 38 countries that introduced the vaccine, 35 (92%) initially adopted Rotateq or Rotarix; and 23% (8/35) switched between products after rotavirus vaccine introduction to either Rotavac (n = 3), Rotasiil (n = 2) or Rotarix (n = 3). Three countries (Benin, Democratic Republic of Congo and Nigeria) introduced the rotavirus vaccines manufactured in India. The decision to either introduce or switch to the Indian vaccines was predominately driven by global supply challenges or supply shortage. The withdrawal of Rotateq from the African market, or cost-saving for countries that graduated or in transition from Gavi support was another reason to switch the vaccine; Conclusions: The recently WHO pre-qualified vaccines have offered the countries, opportunities to adopt these cost-effective products, particularly for countries that have graduated or transitioning from full Gavi support, to sustain the demand of vaccines products.

Background: Soil-transmitted helminths (STH) are targeted for control through mass drug-administration campaigns to prevent morbidity affecting at-risk groups in endemic regions. Although broadly successful, the use of albendazole and mebendazole achieved variable progress, with deficiencies against Trichuris trichiura and a predictable low efficacy against Strongyloides stercoralis. Novel drug combinations offer a potential solution, providing they can be delivered safely and maintain efficacy against all STH species. Here we present the protocol of a clinical trial to evaluate a fixed-dose combination (FDC) tablet containing albendazole and ivermectin that will be compared against albendazole against STH. Methods: An adaptive phase II/III randomized controlled trial will be undertaken in STH endemic sites in Ethiopia, Kenya and Mozambique to evaluate an oral FDC of 400 mg albendazole and either 9- or 18 mg ivermectin. FDC will be administered as a single dose or single doses over three-consecutive days and assessed against a single dose of 400 mg albendazole. In the phase II trial, 126 T. trichiura-infected children weighting 15 to 45 kg will be treated in a dose-escalation manner to determine safety objectives. In the phase III trial, 1097 participants aged 5 to 18 years old infected with T. trichiura, hookworm and S. stercoralis will be recruited to determine safety and efficacy. The trial will be open-label with blinded outcome assessors. Cure rate measured 21-days after-treatment in duplicate Kato-Katz is the primary efficacy outcome. Secondary objectives include efficacy evaluation by quantitative polymerase chain reaction (PCR) as an outcome measurement, description of pharmacokinetic parameters, palatability and acceptability evaluations, and monitoring of anthelmintic resistance. Conclusions: This trial with registrational goals seeks to evaluate an innovative fixed-dose combination of albendazole and ivermectin co-formulated tablets, with the goal of providing an anthelmintic regimen with improved efficacy and spectrum of coverage against STH. ClinicalTrials.gov registration: NCT05124691 (18/11/2021).

Background: Globally, mortality in children younger than 5 years has been decreasing over the past few decades, but high under-5 mortality persists across regions of sub-Saharan Africa and southern Asia. Interventions—such as improved quality of clinical and antenatal care, better access to emergency obstetrical procedures, better triage and risk stratification, better immunisation coverage, or infection control measures—could substantially reduce deaths, but it is unclear which strategies could save the most lives. We aimed to use data from the Child Health and Mortality Prevention Surveillance (CHAMPS) network to examine which health-care and public health improvements could have prevented the most deaths. Methods: We used standardised, population-based, mortality surveillance data collected by CHAMPS from seven sites (Bangladesh, Ethiopia, Kenya, Mali, Mozambique, Sierra Leone, and South Africa) to understand preventable causes of death in children younger than 5 years. Deaths were investigated with minimally invasive tissue sampling, a post-mortem approach using biopsy needles for sampling key organs and body fluids. For each death, an expert panel reviewed case data to determine whether the death was preventable and (if preventable) provided recommendations as to how the death could have been avoided. We evaluated which health system improvements could have prevented the most deaths among those who underwent minimally invasive tissue sampling for each age group: stillbirths, neonatal deaths (aged

IntroductionThe high burden of stillbirths and neonatal deaths is driving global initiatives to improve birth outcomes. Discerning stillbirths from neonatal deaths can be difficult in some settings, yet this distinction is critical for understanding causes of perinatal deaths and improving resuscitation practices for live born babies.MethodsWe evaluated data from the Child Health and Mortality Prevention Surveillance (CHAMPS) network to compare the accuracy of determining stillbirths versus neonatal deaths from different data sources and to evaluate evidence of resuscitation at delivery in accordance with World Health Organization (WHO) guidelines. CHAMPS works to identify causes of stillbirth and death in children ResultsOf 1967 deaths ultimately classified as stillbirth, only 28 (1.4%) were initially reported as livebirths. Of 845 cases classified as very early neonatal death, 33 (4%) were initially reported as stillbirth. Of 367 cases with post-mortem examination showing delivery weight >1000g and no maceration, the maternal clinical record documented that resuscitation was not performed in 161 cases (44%), performed in 14 (3%), and unknown or data missing for 192 (52%).ConclusionThis analysis found that CHAMPS cases assigned as stillbirth or neonatal death after DeCoDe expert panel review were generally consistent with the initial report of the case as a stillbirth or neonatal death. Our findings suggest that more frequent use of resuscitation at delivery and improvements in documentation around events at birth could help improve perinatal outcomes.

BackgroundThe current burden of >5 million deaths yearly is the focus of the Sustainable Development Goal (SDG) to end preventable deaths of newborns and children under 5 years old by 2030. To accelerate progression toward this goal, data are needed that accurately quantify the leading causes of death, so that interventions can target the common causes. By adding postmortem pathology and microbiology studies to other available data, the Child Health and Mortality Prevention Surveillance (CHAMPS) network provides comprehensive evaluations of conditions leading to death, in contrast to standard methods that rely on data from medical records and verbal autopsy and report only a single underlying condition. We analyzed CHAMPS data to characterize the value of considering multiple causes of death.Methods and findingsWe examined deaths identified from December 2016 through November 2020 from 7 CHAMPS sites (in Bangladesh, Ethiopia, Kenya, Mali, Mozambique, Sierra Leone, and South Africa), including 741 neonatal, 278 infant, and 241 child ConclusionsIncluding conditions that appear anywhere in the causal chain, rather than considering underlying condition alone, markedly changed the proportion of deaths attributed to various diagnoses, especially LRI, sepsis, and meningitis. While CHAMPS methods cannot determine when 2 conditions cause death independently or may be synergistic, our findings suggest that considering the chain of events leading to death can better guide research and prevention priorities aimed at reducing child deaths.

Background Global malaria mortality estimates are hindered by the low reliability of the verbal autopsy (VA) and the clinical records, the most common sources of information used to estimate malaria-specific mortality. We aimed to determine the accuracy of these tools, as well as of the minimally invasive autopsy (MIA), a needle-based postmortem sampling method, to identify malaria-specific mortality in a large series of deceased patients from Mozambique, using complete autopsy as the gold standard.Methods Observational study that included 264 deaths, occurring at a tertiary level hospital in Mozambique, from 1 November 2013 to 31 March 2015 (17 months-long period). Clinical data were abstracted, a computer coded VA was completed using the clinical data as source of information, and an MIA followed by a complete autopsy were performed. Screening for malaria infection was conducted postmortem to all participants using molecular and histological techniques (PCR and immunohistochemistry).Findings Malaria infection was considered the cause of death in 6/264 (2.3%) cases: 2/54 children (3.7%, both less than 5 years old) and 4/57 (7.0%) maternal deaths. The sensitivity and specificity of the VA, the clinical data and the MIA to identify malaria-specific deaths were 33.3% and 96.1%, 66.7% and 96.1%, and 100% and 100%, respectively. In addition, malaria was identified as a possible contributor in 14 additional patients who died of other diseases. These cases were also accurately identified by the MIA (sensitivity 82.4%, specificity 100%).Interpretation The high sensitivity and specificity of the MIA in identifying malaria may help to improve current estimates of malaria-specific mortality in endemic areas.

Background: Accurate information on causes of death (CoD) is essential to estimate burden of disease, track global progress, prioritize cost-effective interventions, and inform policies to reduce mortality. In low-income settings, where a significant proportion of deaths take place at home or in poorly-resourced peripheral health facilities, data on CoD often relies on verbal autopsies (VAs). Validations of VAs have been performed against clinical diagnosis, but never before against an acceptable gold standard: the complete diagnostic autopsy (CDA). Methods: We have validated a computer-coded verbal autopsy method –the InterVA- using individual and population metrics to determine CoD against the CDA, in 316 deceased patients of different age groups who died in a tertiary-level hospital in Maputo, Mozambique between 2013 and 2015. Results: We found a low agreement of the model across all age groups at the individual (kappa statistic ranging from -0.030 to 0.232, lowest in stillbirths and highest in adults) and population levels (chance-corrected cause-specific mortality fraction accuracy ranging from -1.00 to 0.62, lowest in stillbirths, highest in children). The sensitivity in identifying infectious diseases was low (0% for tuberculosis, diarrhea, and disseminated infections, 32% for HIV-related infections, 33% for malaria and 36% for pneumonia). Of maternal deaths, 26 were assigned to eclampsia but only four patients actually died of eclampsia. Conclusions: These findings do not lead to building confidence in current estimates of CoD. They also call to the need to implement autopsy methods where they may be feasible, and to improve the quality and performance of current VA techniques.

Abstract Background In rhesus macaques, simian immunodeficiency virus infection is followed by expansion of enteric viruses but has a limited impact on the gut bacteriome. To understand the longitudinal effects of HIV-1 infection on the human gut microbiota, we prospectively followed 49 Mozambican subjects diagnosed with recent HIV-1 infection (RHI) and 54 HIV-1-negative controls for 9–18 months and compared them with 98 chronically HIV-1-infected subjects treated with antiretrovirals (n = 27) or not (n = 71). Results We show that RHI is followed by increased fecal adenovirus shedding, which persists during chronic HIV-1 infection and does not resolve with ART. Recent HIV-1 infection is also followed by transient non-HIV-specific changes in the gut bacterial richness and composition. Despite early resilience to change, an HIV-1-specific signature in the gut bacteriome—featuring depletion of Akkermansia, Anaerovibrio, Bifidobacterium, and Clostridium—previously associated with chronic inflammation, CD8+ T cell anergy, and metabolic disorders, can be eventually identified in chronically HIV-1-infected subjects. Conclusions Recent HIV-1 infection is associated with increased fecal shedding of eukaryotic viruses, transient loss of bacterial taxonomic richness, and long-term reductions in microbial gene richness. An HIV-1-associated microbiome signature only becomes evident in chronically HIV-1-infected subjects.

Background: Accurate information on causes of death (CoD) is essential to estimate burden of disease, track global progress, prioritize cost-effective interventions, and inform policies to reduce mortality. In low-income settings, where a significant proportion of deaths take place at home or in poorly-resourced peripheral health facilities, data on CoD often relies on verbal autopsies (VAs). Validations of VAs have been performed against clinical diagnosis, but never before against an acceptable gold standard: the complete diagnostic autopsy (CDA). Methods: We have validated a computer-coded verbal autopsy method –the InterVA- using individual and population metrics to determine CoD against the CDA, in 316 deceased patients of different age groups who died in a tertiary-level hospital in Maputo, Mozambique between 2013 and 2015. Results: We found a low agreement of the model across all age groups at the individual (kappa statistic ranging from -0.030 to 0.232, lowest in stillbirths and highest in adults) and population levels (chance-corrected cause-specific mortality fraction accuracy ranging from -1.00 to 0.62, lowest in stillbirths, highest in children). The sensitivity in identifying infectious diseases was low (0% for tuberculosis, diarrhea, and disseminated infections, 32% for HIV-related infections, 33% for malaria and 36% for pneumonia). Of maternal deaths, 26 were assigned to eclampsia but only four patients actually died of eclampsia. Conclusions: These findings do not lead to building confidence in current estimates of CoD. They also call to the need to implement autopsy methods where they may be feasible, and to improve the quality and performance of current VA techniques.

Primary HIV infection (PHI) and subsequent chronic infection alter B-cell compartment. However, longitudinal analysis defining the dynamics of B-cell alterations are still limited. We longitudinally studied B-cell subsets in individuals followed for 1 year after PHI (n = 40). Treated and untreated chronic HIV infected (n = 56) and HIV-uninfected individuals (n = 58) were recruited as reference groups at the Manhiça District in Mozambique. B cells were analyzed by multicolor flow-cytometry. Anti-HIV humoral response and plasma cytokines were assessed by ELISA or Luminex-based technology. A generalized activation of B cells induced by HIV occurs early after infection and is characterized by increases in Activated and Tissue-like memory cells, decreases in IgM-IgD- (switched) and IgM-only B cells. These alterations remain mostly stable until chronic infection and are reverted in part by ART. In contrast, other parameters followed particular dynamics: PD-1 expression in memory cells decreases progressively during the first year of infection, Transitional B cells expand at month 3–4 after infection, and Marginal zone-like B cells show a late depletion. Plasmablasts expand 2 months after infection linked to plasma viral load and anti-p24 IgG3 responses. Most of well-defined changes induced by HIV in B-cell activation and memory subsets are readily observed after PHI, lasting until ART initiation. However, subsequent changes occur after sustained viral infection. These data indicate that HIV infection impacts B cells in several waves over time, and highlight that early treatment would result in beneficial effects on the B-cell compartment.

Background Technological advances and high throughput biological assays can facilitate discovery science in biobanks from population cohorts, including pregnant women. Biological pathways associated with health outcomes differ depending on geography, and high-income country data may not generalise to low-resource settings. We conducted a systematic review to identify prospective pregnancy cohorts in sub-Saharan Africa (SSA) that include biobanked samples with potential to enhance discovery science opportunity.Methods Inclusion criteria were prospective data collection during pregnancy, with associated biobanking in SSA. Data sources included: scientific databases (with comprehensive search terms), grey literature, hand searching applicable reference lists and expert input. Results were screened in a three-stage process based on title, abstract and full text by two independent reviewers. The review is registered on PROSPERO (CRD42019147483).Results Fourteen SSA studies met the inclusion criteria from database searches (n=8), reference list searches (n=2) and expert input (n=4). Three studies have ongoing data collection. The most represented countries were South Africa and Mozambique (Southern Africa) (n=3), Benin (Western Africa) (n=4) and Tanzania (Eastern Africa) (n=4); including an estimated 31 763 women. Samples commonly collected were blood, cord blood and placenta. Seven studies collected neonatal samples. Common clinical outcomes included maternal and perinatal mortality, malaria and preterm birth.Conclusions Increasingly numerous pregnancy cohorts in SSA that include biobanking are generating a uniquely valuable resource for collaborative discovery science, and improved understanding of the high regional risks of maternal, fetal and neonatal morbidity and mortality. Future studies should align protocols and consider their added value and distinct contributions.

Although enteroaggregative E. coli (EAEC) has been implicated as a common cause of diarrhea in multiple settings, neither its essential genomic nature nor its role as an enteric pathogen are fully understood. The current definition of this pathotype requires demonstration of cellular adherence; a working molecular definition encompasses E. coli which do not harbor the heat-stable or heat-labile toxins of enterotoxigenic E. coli (ETEC) and harbor the genes aaiC, aggR, and/or aatA. In an effort to improve the definition of this pathotype, we report the most definitive characterization of the pan-genome of EAEC to date, applying comparative genomics and functional characterization on a collection of 97 EAEC strains isolated in the course of a multicenter case-control diarrhea study (Global Enteric Multi-Center Study, GEMS). Genomic analysis revealed that the EAEC strains mapped to all phylogenomic groups of E. coli. Circa 70% of strains harbored one of the five described AAF variants; there were no additional AAF variants identified, and strains that lacked an identifiable AAF generally did not have an otherwise complete AggR regulon. An exception was strains that harbored an ETEC colonization factor (CF) CS22, like AAF a member of the chaperone-usher family of adhesins, but not phylogenetically related to the AAF family. Of all genes scored, sepA yielded the strongest association with diarrhea (P = 0.002) followed by the increased serum survival gene, iss (p = 0.026), and the outer membrane protease gene ompT (p = 0.046). Notably, the EAEC genomes harbored several genes characteristically associated with other E. coli pathotypes. Our data suggest that a molecular definition of EAEC could comprise E. coli strains harboring AggR and a complete AAF(I-V) or CS22 gene cluster. Further, it is possible that strains meeting this definition could be both enteric bacteria and urinary/systemic pathogens.

Background: Accurate information on causes of death (CoD) is essential to estimate burden of disease, track global progress, prioritize cost-effective interventions, and inform policies to reduce mortality. In low-income settings, where a significant proportion of deaths take place at home or in poorly-resourced peripheral health facilities, data on CoD often relies on verbal autopsies (VAs). Validations of VAs have been performed against clinical diagnosis, but never before against an acceptable gold standard: the complete diagnostic autopsy (CDA). Methods: We have validated a computer-coded verbal autopsy method –the InterVA- using individual and population metrics to determine CoD against the CDA, in 316 deceased patients of different age groups who died in a tertiary-level hospital in Maputo, Mozambique between 2013 and 2015. Results: We found a low agreement of the model across all age groups at the individual (kappa statistic ranging from -0.030 to 0.232, lowest in stillbirths and highest in adults) and population levels (chance-corrected cause-specific mortality fraction accuracy ranging from -1.00 to 0.62, lowest in stillbirths, highest in children). The sensitivity in identifying infectious diseases was low (0% for tuberculosis, diarrhea, and disseminated infections, 32% for HIV-related infections, 33% for malaria and 36% for pneumonia). Of maternal deaths, 26 were assigned to eclampsia but only four patients actually died of eclampsia. Conclusions: These findings do not lead to building confidence in current estimates of CoD. They also call to the need to implement autopsy methods where they may be feasible, and to improve the quality and performance of current VA techniques.

Summary: Background: The Global Enteric Multicenter Study (GEMS) was a 3-year case-control study that measured the burden, aetiology, and consequences of moderate-to-severe diarrhoea (MSD) in children aged 0–59 months. GEMS-1A, a 12-month follow-on study, comprised two parallel case-control studies, one assessing MSD and the other less-severe diarrhoea (LSD). In this report, we analyse the risk of death with each diarrhoea type and the specific pathogens associated with fatal outcomes. Methods: GEMS was a prospective, age-stratified, matched case-control study done at seven sites in Africa and Asia. Children aged 0–59 months with MSD seeking care at sentinel health centres were recruited along with one to three randomly selected matched community control children without diarrhoea. In the 12-month GEMS-1A follow-on study, children with LSD and matched controls, in addition to children with MSD and matched controls, were recruited at six of the seven sites; only cases of MSD and controls were enrolled at the seventh site. We compared risk of death during the period between enrolment and one follow-up household visit done about 60 days later (range 50–90 days) in children with MSD and LSD and in their respective controls. Approximately 50 pathogens were detected using, as appropriate, classic bacteriology, immunoassays, gel-based PCR and reverse transcriptase PCR, and quantitative real-time PCR (qPCR). Specimens from a subset of GEMS cases and controls were also tested by a TaqMan Array Card that compartmentalised probe-based qPCR for 32 enteropathogens. Findings: 223 (2·0%) of 11 108 children with MSD and 43 (0·3%) of 16 369 matched controls died between study enrolment and the follow-up visit at about 60 days (hazard ratio [HR] 8·16, 95% CI 5·69–11·68, p

We report a case of Sneathia amnii as the causative agent of maternal chorioamnionitis and congenital pneumonia resulting in a late fetal death in Mozambique, with strong supportive postmortem molecular and histopathologic confirmation. This rare, fastidious gram-negative coccobacillus has been reported to infrequently cause abortions, stillbirths, and neonatal infections.

Abstract Acute HIV infection (AHI) is the period prior to seroconversion characterized by high viral replication, hyper-transmission potential and commonly, non-specific febrile illness. AHI detection requires HIV-RNA viral load (VL) determination, which has very limited access in low-income countries due to restrictive costs and implementation constraints. We sought to identify a biomarker that could enable AHI diagnosis in scarce-resource settings, and to evaluate the feasibility of its implementation. HIV-seronegative adults presenting at the Manhiça District Hospital, Mozambique, with reported-fever were tested for VL. Plasma levels of 49 inflammatory biomarkers from AHI (n = 61) and non-HIV infected outpatients (n = 65) were determined by Luminex and ELISA. IP-10 demonstrated the best predictive power for AHI detection (AUC = 0.88 [95%CI 0.80–0.96]). A cut-off value of IP-10 ≥ 161.6 pg/mL provided a sensitivity of 95.5% (95%CI 85.5–99.5) and a specificity of 76.5% (95%CI 62.5–87.2). The implementation of an IP-10 screening test could avert from 21 to 84 new infections and save from US$176,609 to US$533,467 to the health system per 1,000 tested patients. We conclude that IP-10 is an accurate biomarker to screen febrile HIV-seronegative individuals for subsequent AHI diagnosis with VL. Such an algorithm is a cost-effective strategy to prevent disease progression and a substantial number of further HIV infections.

Abstract Clonal clusters and gene repertoires of Staphylococcus aureus are essential to understand disease and are well characterized in industrialized countries but poorly analysed in developing regions. The objective of this study was to compare the molecular-epidemiologic profiles of S. aureus isolates from Sub-Saharan Africa and Germany. S. aureus isolates from 600 staphylococcal carriers and 600 patients with community-associated staphylococcal disease were characterized by DNA hybridization, clonal complex (CC) attribution, and principal component (PCA)-based gene repertoire analysis. 73% of all CCs identified representing 77% of the isolates contained in these CCs were predominant in either African or German region. Significant differences between African versus German isolates were found for alleles encoding the accessory gene regulator type, enterotoxins, the Panton-Valentine leukocidin, immune evasion gene cluster, and adhesins. PCA in conjunction with silhouette analysis distinguished nine separable PCA clusters, with five clusters primarily comprising of African and two clusters of German isolates. Significant differences between S. aureus lineages in Africa and Germany may be a clue to explain the apparent difference in disease between tropical/(so-called) developing and temperate/industrialized regions. In low-resource countries further clinical-epidemiologic research is warranted not only for neglected tropical diseases but also for major bacterial infections.

Summary: Background: Diarrheal diseases remain a leading cause of illness and death among children younger than 5 years in low-income and middle-income countries. The Global Enteric Multicenter Study (GEMS) has described the incidence, aetiology, and sequelae of medically attended moderate-to-severe diarrhoea (MSD) among children aged 0–59 months residing in censused populations in sub-Saharan Africa and south Asia, where most child deaths occur. To further characterise this disease burden and guide interventions, we extended this study to include children with episodes of less-severe diarrhoea (LSD) seeking care at health centres serving six GEMS sites. Methods: We report a 1-year, multisite, age-stratified, matched case-control study following on to the GEMS study. Six sites (Bamako, Mali; Manhiça, Mozambique; Basse, The Gambia; Mirzapur, Bangladesh; Kolkata, India; and Bin Qasim Town, Karachi, Pakistan) participated in this study. Children aged 0–59 months at each site who sought care at a sentinel hospital or health centre during a 12-month period were screened for diarrhoea. New (onset after ≥7 diarrhoea-free days) and acute (onset within the previous 7 days) episodes of diarrhoea in children who had sunken eyes, whose skin lost turgor, who received intravenous hydration, who had dysentery, or who were hospitalised were eligible for inclusion as MSD. The remaining new and acute diarrhoea episodes among children who sought care at the same health centres were considered LSD. We aimed to enrol the first eight or nine eligible children with MSD and LSD at each site during each fortnight in three age strata: infants (aged 0–11 months), toddlers (aged 12–23 months), and young children (aged 24–59 months). For each included case of MSD or LSD, we enrolled one to three community control children without diarrhoea during the previous 7 days. From patients and controls we collected clinical and epidemiological data, anthropometric measurements, and faecal samples to identify enteropathogens at enrolment, and we performed a follow-up home visit about 60 days later to ascertain vital status, clinical outcome, and interval growth. Primary outcomes were to characterise, for MSD and LSD, the pathogen-specific attributable risk and population-based incidence values, and to assess the frequency of adverse clinical consequences associated with these two diarrhoeal syndromes. Findings: From Oct 31, 2011, to Nov 14, 2012, we recruited 2368 children with MSD, 3174 with LSD, and one to three randomly selected community control children without diarrhoea matched to cases with MSD (n=3597) or LSD (n=4236). Weighted adjusted population attributable fractions showed that most attributable cases of MSD and LSD were due to rotavirus, Cryptosporidium spp, enterotoxigenic Escherichia coli encoding heat-stable toxin (with or without genes encoding heat-labile enterotoxin), and Shigella spp. The attributable incidence per 100 child-years for LSD versus MSD, by age stratum, for rotavirus was 22·3 versus 5·5 (0–11 months), 9·8 versus 2·9 (12–23 months), and 0·5 versus 0·2 (24–59 months); for Cryptosporidium spp was 3·6 versus 2·3 (0–11 months), 4·3 versus 0·6 (12–23 months), and 0·3 versus 0·1 (24–59 months); for enterotoxigenic E coli encoding heat-stable toxin was 4·2 versus 0·1 (0–11 months), 5·2 versus 0·0 (12–23 months), and 1·1 versus 0·2 (24–59 months); and for Shigella spp was 1·0 versus 1·3 (0–11 months), 3·1 versus 2·4 (12–23 months), and 0·8 versus 0·7 (24–59 months). Participants with both MSD and LSD had significantly more linear growth faltering than controls at follow-up. Interpretation: Inclusion of participants with LSD markedly expands the population of children who experience adverse clinical and nutritional outcomes from acute diarrhoeal diseases. Since MSD and LSD have similar aetiologies, interventions targeting rotavirus, Shigella spp, enterotoxigenic E coli producing heat-stable toxin, and Cryptosporidium spp might substantially reduce the diarrhoeal disease burden and its associated nutritional faltering. Funding: Bill & Melinda Gates Foundation.

BACKGROUND:Clinico-pathological discrepancies are more frequent in settings in which limited diagnostic techniques are available, but there is little information on their actual impact. AIM:We assessed the accuracy of the clinical diagnoses in a tertiary referral hospital in sub-Saharan Africa by comparison with post-mortem findings. We also identified potential risk factors for misdiagnoses. METHODS:One hundred and twelve complete autopsy procedures were performed at the Maputo Central Hospital (Mozambique), from November 2013 to March 2015. We reviewed the clinical records. Major clinico-pathological discrepancies were assessed using a modified version of the Goldman and Battle classification. RESULTS:Major diagnostic discrepancies were detected in 65/112 cases (58%) and were particularly frequent in infection-related deaths (56/80 [70%] major discrepancies). The sensitivity of the clinical diagnosis for toxoplasmosis was 0% (95% CI: 0-37), 18% (95% CI: 2-52) for invasive fungal infections, 25% (95% CI: 5-57) for bacterial sepsis, 34% (95% CI: 16-57), for tuberculosis, and 46% (95% CI: 19-75) for bacterial pneumonia. Major discrepancies were more frequent in HIV-positive than in HIV-negative patients (48/73 [66%] vs. 17/39 [44%]; p = 0.0236). CONCLUSIONS:Major clinico-pathological discrepancies are still frequent in resource constrained settings. Increasing the level of suspicion for infectious diseases and expanding the availability of diagnostic tests could significantly improve the recognition of common life-threatening infections, and thereby reduce the mortality associated with these diseases. The high frequency of clinico-pathological discrepancies questions the validity of mortality reports based on clinical data or verbal autopsy.

IntroductionPrimary HIV infection (PHI) is the initial phase after HIV acquisition characterized by high viral replication, massive inflammatory response and irreversible immune-damage, particularly at the gastrointestinal level. In this study we aimed to characterize the dynamics of gastrointestinal damage biomarkers during the different phases of HIV infection and assess their association with HIV-disease markers and their accuracy to differentiate PHI from chronic HIV infection (CHI).MethodsPHI-individuals (n = 57) were identified as HIV-seronegative/HIV-RNA positive and were followed up for one year at the Manhiça District Hospital in Mozambique. Ten plasma and 12 stool biomarkers were quantified by Luminex or ELISA and levels were compared to CHI-naive (n = 26), CHI on antiretroviral-treatment (ART; n = 30) and HIV-uninfected individuals (n = 58). Regression models adjusted by time point were used to estimate the association of the biomarkers with HIV-disease markers. Receiver operating curves were compared for the best accuracy to distinguish PHI from CHI.ResultsSoluble (s)CD14 was significantly associated with the CD4/CD8 ratio (P < 0.05) and viremia levels (P < 0.0001) during PHI. Plasma zonulin and stool lactoferrin were significantly higher in PHI as compared to CHI-individuals (P < 0.05). Plasma zonulin demonstrated the best accuracy to identify PHI among HIV-infected individuals (AUC = 0.85 [95% CI 0.75-0.94]). Using a cutoff value of plasma zonulin ≥ 8.75 ng/mL the model identified PHI with 87.7% sensitivity (95% CI 76.3-94.9) and 69.2% specificity (95% CI 48.2-85.7). An adjusted multivariate model including age, plasma zonulin and sCD14 further increased the classification performance (AUC = 0.92 [95% CI 0.86-0.99]).ConclusionsWhile the stool biomarkers did not provide any predictive ability to distinguish PHI from CHI-individuals, plasma sCD14 and zonulin were significantly associated with HIV-disease markers and PHI identification, respectively. These inflammatory biomarkers may be useful to monitor changes in gastrointestinal integrity during HIV infection.

BackgroundEnterotoxigenic Escherichia coli (ETEC) encoding heat-stable enterotoxin (ST) alone or with heat-labile enterotoxin (LT) cause moderate-to-severe diarrhea (MSD) in developing country children. The Global Enteric Multicenter Study (GEMS) identified ETEC encoding ST among the top four enteropathogens. Since the GEMS objective was to provide evidence to guide development and implementation of enteric vaccines and other interventions to diminish diarrheal disease morbidity and mortality, we examined colonization factor (CF) prevalence among ETEC isolates from children age Methodology/principal findingsMSD cases enrolled at healthcare facilities over three years and matched controls were tested in a standardized manner for many enteropathogens. To identify ETEC, three E. coli colonies per child were tested by polymerase chain reaction (PCR) to detect genes encoding LT, ST; confirmed ETEC were examined by PCR for major CFs (Colonization Factor Antigen I [CFA/I] or Coli Surface [CS] antigens CS1-CS6) and minor CFs (CS7, CS12, CS13, CS14, CS17, CS18, CS19, CS20, CS21, CS30). ETEC from 806 cases had a single toxin/CF profile in three tested strains per child. Major CFs, components of multiple ETEC vaccine candidates, were detected in 66.0% of LT/ST and ST-only cases and were associated with MSD versus matched controls by conditional logistic regression (p≤0.006); major CFs detected in only 25.0% of LT-only cases weren't associated with MSD. ETEC encoding exclusively CS14, identified among 19.9% of 291 ST-only and 1.5% of 259 LT/ST strains, were associated with MSD (p = 0.0011). No other minor CF exhibited prevalence ≥5% and significant association with MSD.Conclusions/significanceMajor CF-based efficacious ETEC vaccines could potentially prevent up to 66% of pediatric MSD cases due to ST-encoding ETEC in developing countries; adding CS14 extends coverage to ~77%.

During primary HIV infection (PHI), there is a striking cascade response of inflammatory cytokines and many cells of the immune system show altered frequencies and signs of extensive activation. These changes have been shown to have a relevant role in predicting disease progression; however, the challenges of identifying PHI have resulted in a lack of critical information about the dynamics of early pathogenic events. We studied soluble inflammatory biomarkers and changes in T-cell subsets in individuals at PHI (n = 40), chronic HIV infection (CHI, n = 56), and HIV-uninfected (n = 58) recruited at the Manhiça District Hospital in Mozambique. Plasma levels of 49 biomarkers were determined by Luminex and ELISA. T-cell immunophenotyping was performed by multicolor flow cytometry. Plasma HIV viremia, CD4, and CD8 T cell counts underwent rapid stabilization after PHI. However, several immunological parameters, including Th1-Th17 CD4 T cells and activation or exhaustion of CD8 T cells continued decreasing until more than 9 months postinfection. Importantly, no sign of immunosenescence was observed over the first year of HIV infection. Levels of IP-10, MCP-1, BAFF, sCD14, tumor necrosis factor receptor-2, and TRAIL were significantly overexpressed at the first month of infection and underwent a prompt decrease in the subsequent months while, MIG and CD27 levels began to increase 1 month after infection and remained overexpressed for almost 1 year postinfection. Early levels of soluble biomarkers were significantly associated with subsequently exhausted CD4 T-cells or with CD8 T-cell activation. Despite rapid immune control of virus replication, the stabilization of the T-cell subsets occurs months after viremia and CD4 count plateau, suggesting persistent immune dysfunction and highlighting the potential benefit of early treatment initiation that could limit immunological damage.

BackgroundDespite global health efforts to reduce maternal mortality, rates continue to be unacceptably high in large parts of the world. Feasible, acceptable, and accurate postmortem sampling methods could provide the necessary evidence to improve the understanding of the real causes of maternal mortality, guiding the design of interventions to reduce this burden.Methods and findingsThe validity of a minimally invasive autopsy (MIA) method in determining the cause of death was assessed in an observational study in 57 maternal deaths by comparing the results of the MIA with those of the gold standard (complete diagnostic autopsy [CDA], which includes any available clinical information). Concordance between the MIA and the gold standard diagnostic categories was assessed by the kappa statistic, and the sensitivity, specificity, positive and negative predictive values and their 95% confidence intervals (95% CI) to identify the categories of diagnoses were estimated. The main limitation of the study is that both the MIA and the CDA include some degree of subjective interpretation in the attribution of cause of death. A cause of death was identified in the CDA in 98% (56/57) of cases, with indirect obstetric conditions accounting for 32 (56%) deaths and direct obstetric complications for 24 (42%) deaths. Nonobstetric infectious diseases (22/32, 69%) and obstetric hemorrhage (13/24, 54%) were the most common causes of death among indirect and direct obstetric conditions, respectively. Thirty-six (63%) women were HIV positive, and HIV-related conditions accounted for 16 (28%) of all deaths. Cerebral malaria caused 4 (7%) deaths. The MIA identified a cause of death in 86% of women. The overall concordance of the MIA with the CDA was moderate (kappa = 0.48, 95% CI: 0.31-0.66). Both methods agreed in 68% of the diagnostic categories and the agreement was higher for indirect (91%) than for direct obstetric causes (38%). All HIV infections and cerebral malaria cases were identified in the MIA. The main limitation of the technique is its relatively low performance for identifying obstetric causes of death in the absence of clinical information.ConclusionsThe MIA procedure could be a valuable tool to determine the causes of maternal death, especially for indirect obstetric conditions, most of which are infectious diseases. The information provided by the MIA could help to prioritize interventions to reduce maternal mortality and to monitor progress towards achieving global health targets.

BackgroundIn recent decades, the world has witnessed unprecedented progress in child survival. However, our knowledge of what is killing nearly 6 million children annually in low- and middle-income countries remains poor, partly because of the inadequacy and reduced precision of the methods currently utilized in these settings to investigate causes of death (CoDs). The study objective was to validate the use of a minimally invasive autopsy (MIA) approach as an adequate and more acceptable substitute for the complete diagnostic autopsy (CDA) for pediatric CoD investigation in a poor setting.Methods and findingsIn this observational study, the validity of the MIA approach in determining the CoD was assessed in 54 post-neonatal pediatric deaths (age range: ≥1 mo to 15 y) in a referral hospital of Mozambique by comparing the results of the MIA with those of the CDA. Concordance in the category of disease obtained by the two methods was evaluated by the Kappa statistic, and the sensitivity, specificity, and positive and negative predictive values of the MIA diagnoses were calculated. A CoD was identified in all cases in the CDA and in 52/54 (96%) of the cases in the MIA, with infections and malignant tumors accounting for the majority of diagnoses. The MIA categorization of disease showed a substantial concordance with the CDA categorization (Kappa = 0.70, 95% CI 0.49-0.92), and sensitivity, specificity, and overall accuracy were high. The ICD-10 diagnoses were coincident in up to 75% (36/48) of the cases. The MIA allowed the identification of the specific pathogen deemed responsible for the death in two-thirds (21/32; 66%) of all deaths of infectious origin. Discrepancies between the MIA and the CDA in individual diagnoses could be minimized with the addition of some basic clinical information such as those ascertainable through a verbal autopsy or clinical record. The main limitation of the analysis is that both the MIA and the CDA include some degree of expert subjective interpretation.ConclusionsThe MIA showed substantial concordance with CDA for CoD identification in this series of pediatric deaths in Mozambique. This minimally invasive approach, simpler and more readily acceptable than the more invasive CDA, could provide robust data for CoD surveillance, especially in resource-limited settings, which could be helpful for guiding child survival strategies in the future.

BackgroundOver 5 million stillbirths and neonatal deaths occur annually. Limited and imprecise information on the cause of these deaths hampers progress in achieving global health targets. Complete diagnostic autopsies (CDAs)-the gold standard for cause of death determination-are difficult to perform in most high-burden settings. Therefore, validation of simpler and more feasible methods is needed.Methods and findingsIn this observational study, the validity of a minimally invasive autopsy (MIA) method in determining the cause of death was assessed in 18 stillbirths and 41 neonatal deaths by comparing the results of the MIA with those of the CDA. Concordance between the categories of diseases obtained by the 2 methods was assessed by the Kappa statistic, and the sensitivity, specificity, positive, and negative predictive values of the MIA diagnoses were calculated. A cause of death was identified in 16/18 (89%) and 15/18 (83%) stillborn babies in the CDA and the MIA, respectively. Fetal growth restriction accounted for 39%, infectious diseases for 22%, intrapartum hypoxia for 17%, and intrauterine hypoxia for 11% of stillborn babies. Overall, the MIA showed in this group a substantial concordance with the CDA (Kappa = 0.78, 95% CI [0.56-0.99]). A cause of death was identified in all (100%) and 35/41 (85%) neonatal deaths in the CDA and the MIA, respectively. In this group, the majority of deaths were due to infectious diseases (66%). The overall concordance of the MIA with the CDA in neonates was moderate (Kappa = 0.40, 95% CI [0.18-0.63]). A high percentage of accuracy was observed for the MIA in all the diagnostic categories in both stillbirths and neonates (>75%). The main limitation of this study is that some degree of subjective interpretation is inherent to cause-of-death attribution in both the MIA and the CDA; this is especially so in stillbirths and in relation to fetal growth restriction.ConclusionsThe MIA could be a useful tool for cause-of-death determination in stillbirths and neonatal deaths. These findings may help to accelerate progress towards meeting global health targets by obtaining more accurate information on the causes of death in these age groups, which is essential in guiding the design of new interventions and increasing the effectiveness of those already implemented.

BackgroundThere is an urgent need to identify tools able to provide reliable information on the cause of death in low-income regions, since current methods (verbal autopsy, clinical records, and complete autopsies) are either inaccurate, not feasible, or poorly accepted. We aimed to compare the performance of a standardized minimally invasive autopsy (MIA) approach with that of the gold standard, the complete diagnostic autopsy (CDA), in a series of adults who died at Maputo Central Hospital in Mozambique.Methods and findingsIn this observational study, coupled MIAs and CDAs were performed in 112 deceased patients. The MIA analyses were done blindly, without knowledge of the clinical data or the results of the CDA. We compared the MIA diagnosis with the CDA diagnosis of cause of death. CDA diagnoses comprised infectious diseases (80; 71.4%), malignant tumors (16; 14.3%), and other diseases, including non-infectious cardiovascular, gastrointestinal, kidney, and lung diseases (16; 14.3%). A MIA diagnosis was obtained in 100/112 (89.2%) cases. The overall concordance between the MIA diagnosis and CDA diagnosis was 75.9% (85/112). The concordance was higher for infectious diseases and malignant tumors (63/80 [78.8%] and 13/16 [81.3%], respectively) than for other diseases (9/16; 56.2%). The specific microorganisms causing death were identified in the MIA in 62/74 (83.8%) of the infectious disease deaths with a recognized cause. The main limitation of the analysis is that both the MIA and the CDA include some degree of expert subjective interpretation.ConclusionsA simple MIA procedure can identify the cause of death in many adult deaths in Mozambique. This tool could have a major role in improving the understanding and surveillance of causes of death in areas where infectious diseases are a common cause of mortality.

Cytoadhesion of Plasmodium falciparum infected erythrocytes to gC1qR has been associated with severe malaria, but the parasite ligand involved is currently unknown. To assess if binding to gC1qR is mediated through the P. falciparum erythrocyte membrane protein 1 (PfEMP1) family, we analyzed by static binding assays and qPCR the cytoadhesion and var gene transcriptional profile of 86 P. falciparum isolates from Mozambican children with severe and uncomplicated malaria, as well as of a P. falciparum 3D7 line selected for binding to gC1qR (Pf3D7gC1qR). Transcript levels of DC8 correlated positively with cytoadhesion to gC1qR (rho = 0.287, P = 0.007), were higher in isolates from children with severe anemia than with uncomplicated malaria, as well as in isolates from Europeans presenting a first episode of malaria (n = 21) than Mozambican adults (n = 25), and were associated with an increased IgG recognition of infected erythrocytes by flow cytometry. Pf3D7gC1qR overexpressed the DC8 type PFD0020c (5.3-fold transcript levels relative to Seryl-tRNA-synthetase gene) compared to the unselected line (0.001-fold). DBLβ12 from PFD0020c bound to gC1qR in ELISA-based binding assays and polyclonal antibodies against this domain were able to inhibit binding to gC1qR of Pf3D7gC1qR and four Mozambican P. falciparum isolates by 50%. Our results show that DC8-type PfEMP1s mediate binding to gC1qR through conserved surface epitopes in DBLβ12 domain which can be inhibited by strain-transcending functional antibodies. This study supports a key role for gC1qR in malaria-associated endovascular pathogenesis and suggests the feasibility of designing interventions against severe malaria targeting this specific interaction.

BACKGROUND:The importance of Cryptosporidium as a pediatric enteropathogen in developing countries is recognized. METHODS:Data from the Global Enteric Multicenter Study (GEMS), a 3-year, 7-site, case-control study of moderate-to-severe diarrhea (MSD) and GEMS-1A (1-year study of MSD and less-severe diarrhea [LSD]) were analyzed. Stools from 12,110 MSD and 3,174 LSD cases among children aged

The relevance of vaginal colonization of pregnant women by Escherichia coli is poorly understood, despite these strains sharing a similar virulence profile with other extraintestinal pathogenic E. coli producing severe obstetric and neonatal infections. We characterized the epidemiology, antimicrobial susceptibility and virulence profiles of 84 vaginal E. coli isolates from pregnant women from Rabat (Morocco) and Manhiça (Mozambique), two very distinct epidemiological settings. Low levels of antimicrobial resistance were observed to all drugs tested, except for trimethoprim-sulfamethoxazole in Manhiça, where this drug is extensively used as prophylaxis for opportunistic HIV infections. The most prevalent virulence factors were related to iron acquisition systems. Phylogroup A was the most common in Rabat, while phylogroups E and non-typeable were the most frequent in Manhiça. Regardless of the apparently "low virulence" of these isolates, the frequency of infections is higher and the outcomes more devastating in constrained-resources conditions, especially among pregnant women and newborns.

BACKGROUND:During pregnancy, syncytiotrophoblast vesicles contribute to maternal tolerance towards the fetus, but also to pathologies such as pre-eclampsia. The aim of the study was to address whether Plasmodium falciparum and HIV infections in pregnancy affect the secretion, microRNA content and function of trophoblast microparticles. METHODS:Microparticles were isolated and characterized from 122 peripheral plasmas of Mozambican pregnant women, malaria- and/or HIV-infected and non-infected. Expression of placenta-related microRNAs in microparticles was analysed by qPCR and the effect of circulating microparticles on dendritic cells assessed by phenotype analysis and cytokine/chemokine measurement. RESULTS:Concentrations of total and trophoblast microparticles detected by flow cytometry were higher in HIV-positive (P = 0.005 and P = 0.030, respectively) compared to non-infected mothers, as well as in women delivering low birthweight newborns (P = 0.032 and P = 0.021, respectively). miR-517c was overexpressed in mothers with placental malaria (P = 0.034), compared to non-infected. Microparticles from HIV-positive induced a higher expression of MHCII (P = 0.021) and lower production of MCP1 (P = 0.008) than microparticles from non-infected women. CONCLUSIONS:In summary, alterations in total and trophoblast microparticles associated with malaria and HIV in pregnant women may have an immunopathogenic role. The potential for placental-derived vesicles and microRNAs as biomarkers of adverse outcomes during pregnancy and malaria infection should be confirmed in future studies.

BACKGROUND:Vibrio cholerae is endemic in South Asia and Africa where outbreaks of cholera occur widely and are particularly associated with poverty and poor sanitation. Knowledge of the genetic diversity of toxigenic V. cholerae isolates, particularly in Africa, remains scarce. The constraints in improving this understanding is not only the lack of regular cholera disease surveillance, but also the lack of laboratory capabilities in endemic countries to preserve, store and ship isolates in a timely manner. We evaluated the use of simplified sample preservation methods for molecular characterization using multi-locus variable-number tandem-repeat analysis (MLVA) for differentiation of Vibrio cholerae genotypes. METHODS AND FINDINGS:Forty-seven V. cholerae isolates and 18 enriched clinical specimens (e.g. stool specimens after enrichment in broth) from cholera outbreaks in Cameroon were preserved on Whatman filter paper for DNA extraction. The samples were collected from two geographically distinct outbreaks in the Far North of Cameroon (FNC) in June 2014 and October 2014. In addition, a convenience sample of 14 isolates from the Philippines and 8 from Mozambique were analyzed. All 87 DNAs were successfully analyzed including 16 paired samples, one a cultured isolate and the other the enriched specimen from which the isolate was collected. Genotypic results were identical between 15 enriched specimens and their culture isolates and the other pair differed at single locus. Two closely related, but distinct clonal complexes were identified among the Cameroonian specimens from 2014. CONCLUSIONS:Collecting V. cholerae using simplified laboratory methods in remote and low-resource settings allows for subsequent advanced molecular characterization of V. cholerae O1. These simplified DNA preservation methods identify V. cholerae and make possible timely information regarding the genetic diversity of V. cholerae; our results set the stage for continued molecular epidemiological research to better understand the transmission and dissemination of V. cholerae in Africa and elsewhere worldwide.

INTRODUCTION:Early diagnosis and initiation to appropriate treatment is vital for tuberculosis (TB) control. The XpertMTB/RIF (Xpert) assay offers rapid TB diagnosis and quantitative estimation of bacterial burden through Cycle threshold (Ct) values. We assessed whether the Xpert Ct value is associated with delayed TB diagnosis as a potential monitoring tool for TB control programme performance. MATERIALS AND METHODS:This analysis was nested in a prospective study under the routine TB surveillance procedures of the National TB Control Program in Manhiça district, Maputo province, Mozambique. Presumptive TB patients were tested using smear microscopy and Xpert. We explored the association between Xpert Ct values and self-reported delay of Xpert-positive TB patients as recorded at the time of diagnosis enrolment. Patients with >60 days of TB symptoms were considered to have long delays. RESULTS:Of 1,483 presumptive TB cases, 580 were diagnosed as TB of whom 505 (87.0%) were due to pulmonary TB and 302 (94.1%) were Xpert positive. Ct values (range, 9.7-46.4) showed a multimodal distribution. The median (IQR) delay was 30 (30-45) days. Ct values showed no correlation with delay (R2 = 0.001, p = 0.621), nor any association with long delays: adjusted odds ratios (AOR) (95% confidence interval [CI]) comparing to >28 cycles 0.99 (0.50-1.96; p = 0.987) for 23-28 cycles, 0.93 (0.50-1.74; p = 0.828) for 16-22 cycles; and 1.05 (0.47-2.36; p = 0.897) for

BACKGROUND:Typical enteropathogenic Escherichia coli (tEPEC) strains were associated with mortality in the Global Enteric Multicenter Study (GEMS). Genetic differences in tEPEC strains could underlie some of the variability in clinical outcome. METHODS:We produced draft genome sequences of all available tEPEC strains from GEMS lethal infections (LIs) and of closely matched EPEC strains from GEMS subjects with non-lethal symptomatic infections (NSIs) and asymptomatic infections (AIs) to identify gene clusters (potential protein encoding sequences sharing ≥90% nucleotide sequence identity) associated with lethality. RESULTS:Among 14,412 gene clusters identified, the presence or absence of 392 was associated with clinical outcome. As expected, more gene clusters were associated with LI versus AI than LI versus NSI. The gene clusters more prevalent in strains from LI than those from NSI and AI included those encoding proteins involved in O-antigen biogenesis, while clusters encoding type 3 secretion effectors EspJ and OspB were among those more prevalent in strains from non-lethal infections. One gene cluster encoding a variant of an NleG ubiquitin ligase was associated with LI versus AI, while two other nleG clusters had the opposite association. Similar associations were found for two nleG gene clusters in an additional, larger sample of NSI and AI GEMS strains. CONCLUSIONS:Particular genes are associated with lethal tEPEC infections. Further study of these factors holds potential to unravel the mechanisms underlying severe disease and to prevent adverse outcomes.

Diarrheal disease remains a leading cause of illness and death, particularly in low-income countries. Its burden, microbiological causes and risk factors were examined in children aged 0-59 months living in Manhiça, rural southern Mozambique.Trends of diarrhea-related burden of disease were estimated during the period 2001-2012. A prospective, age-stratified and matched (by age, gender and geographical origin), case-control study was conducted during 2007-2011. Clinical, epidemiology, anthropometric measurement and fecal samples obtained from recruited children were used to estimate moderate-to-severe diarrhea (MSD) weighted attributable fractions.Over the last decade the incidence of acute diarrhea has dropped by about 80%. Incidence of MSD per 100 child years at risk for the period 2007-2011 was 9.85, 7.73 and 2.10 for children aged 0-11, 12-23 and 24-59 months respectively. By adjusted population attributable fractions, most cases of MSD were due to rotavirus, Cryptosporidium, ETEC ST (ST only or ST/LT), Shigella and Adenovirus 40/41. Washing hands and having facilities to dispose child's stools were associated with a reduced risk of MSD, while giving stored water to the child was associated with an increased risk of MSD.Despite the predominantly decreasing trends observed throughout the last decade, diarrheal diseases remain today a major cause of morbidity among children aged 0-59 months living in this rural Mozambican area. Rotavirus, cryptosporidium, Shigella, ETEC ST and Adenovirus 40/41 were the most important aetiologies of MSD. Thus, well-known preventive strategies such as washing hands, improving the treatment of stored water, having facilities to dispose children stools, and accelerating the introduction of the rotavirus vaccine should be promoted on a wider scale to reduce the current burden of diarrheal diseases.

Enteroaggregative Escherichia coli (EAEC) is a leading cause of acute and persistent diarrhea worldwide. A recently emerged Shiga-toxin-producing strain of EAEC resulted in significant mortality and morbidity due to progressive development of hemolytic-uremic syndrome. The attachment of EAEC to the human intestinal mucosa is mediated by aggregative adherence fimbria (AAF). Using X-ray crystallography and NMR structures, we present new atomic resolution insight into the structure of AAF variant I from the strain that caused the deadly outbreak in Germany in 2011, and AAF variant II from archetype strain 042, and propose a mechanism for AAF-mediated adhesion and biofilm formation. Our work shows that major subunits of AAF assemble into linear polymers by donor strand complementation where a single minor subunit is inserted at the tip of the polymer by accepting the donor strand from the terminal major subunit. Whereas the minor subunits of AAF have a distinct conserved structure, AAF major subunits display large structural differences, affecting the overall pilus architecture. These structures suggest a mechanism for AAF-mediated adhesion and biofilm formation. Binding experiments using wild type and mutant subunits (NMR and SPR) and bacteria (ELISA) revealed that despite the structural differences AAF recognize a common receptor, fibronectin, by employing clusters of basic residues at the junction between subunits in the pilus. We show that AAF-fibronectin attachment is based primarily on electrostatic interactions, a mechanism not reported previously for bacterial adhesion to biotic surfaces.

BACKGROUND: The epidemiology of meningococcal disease in Mozambique and other African countries located outside the "meningitis belt" remains widely unknown. With the event of upcoming vaccines microbiological and epidemiological information is urgently needed. METHODS: Prospective surveillance for invasive bacterial infections was conducted at the Manhiça District hospital (rural Mozambique) among hospitalized children below 15 years of age. Available Neisseria meningitidis isolates were serogrouped and characterized by Multilocus Sequence Typing (MLST). Antibiotic resistance was also determined. RESULTS: Between 1998 and 2008, sixty-three cases of confirmed meningococcal disease (36 meningitis, 26 sepsis and 1 conjunctivitis) were identified among hospitalized children. The average incidence rate of meningococcal disease was 11.6/100,000 (8/100,000 for meningitis and 3.7/100,000 for meningococcemia, respectively). There was a significant rise on the number of meningococcal disease cases in 2005-2006 that was sustained till the end of the surveillance period. Serogroup was determined for 43 of the 63 meningococcal disease cases: 38 serogroup W-135, 3 serogroup A and 2 serogroup Y. ST-11 was the most predominant sequence type and strongly associated with serogroup W-135. Two of the three serogroup A isolates were ST-1, and both serogroup Y isolates were ST-175. N. meningitidis remained highly susceptible to all antibiotics used for treatment in the country, although the presence of isolates presenting intermediate resistance to penicillin advocates for continued surveillance. CONCLUSIONS: Our data show a high rate of meningococcal disease in Manhiça, Mozambique, mainly caused by serogroup W-135 ST-11 strains, and advocates for the implementation of a vaccination strategy covering serogroup W-135 meningococci in the country.

The RTS,S/AS02(D) vaccine has been shown to have a promising safety profile, to be immunogenic and to confer protection against malaria in children and infants.We did a randomized, controlled, phase I/IIb trial of RTS,S/AS02(D) given at 10, 14 and 18 weeks of age staggered with routine immunization vaccines in 214 Mozambican infants. The study was double-blind until the young child completed 6 months of follow-up over which period vaccine efficacy against new Plasmodium falciparum infections was estimated at 65.9% (95% CI 42.6-79.8, p

Current recommendations to prevent malaria in African pregnant women rely on insecticide treated nets (ITNs) and intermittent preventive treatment (IPTp). However, there is no information on the safety and efficacy of their combined use.1030 pregnant Mozambican women of all gravidities received a long-lasting ITN during antenatal clinic (ANC) visits and, irrespective of HIV status, were enrolled in a randomised, double blind, placebo-controlled trial, to assess the safety and efficacy of 2-dose sulphadoxine-pyrimethamine (SP). The main outcome was the reduction in low birth weight.Two-dose SP was safe and well tolerated, but was not associated with reductions in anaemia prevalence at delivery (RR, 0.92 [95% CI, 0.79-1.08]), low birth weight (RR, 0.99 [95% CI, 0.70-1.39]), or overall placental infection (p = 0.964). However, the SP group showed a 40% reduction (95% CI, 7.40-61.20]; p = 0.020) in the incidence of clinical malaria during pregnancy, and reductions in the prevalence of peripheral parasitaemia (7.10% vs 15.15%) (p