PHARE: a bioinformatics pipeline for compositional profiling of multiclonal Plasmodium falciparum infections from long-read Nanopore sequencing data.

BACKGROUND: The emergence of drug-resistant clones of Plasmodium falciparum is a major public health concern, and the ability to detect and track the spread of these clones is crucial for effective malaria control and treatment. However, in endemic settings, malaria infected people often carry multiple P. falciparum clones simultaneously making it likely to miss drug-resistant clones using traditional molecular typing methods. OBJECTIVES: Our goal was to develop a bioinformatics pipeline for compositional profiling in multiclonal P. falciparum samples, sequenced using the Oxford Nanopore Technologies MinION platform. METHODS: We developed the 'Finding P. falciparum haplotypes with resistance mutations in polyclonal infections' (PHARE) pipeline using existing bioinformatics tools and custom scripts written in python. PHARE was validated on three control datasets containing P. falciparum DNA of four laboratory strains at varying mixing ratios. Additionally, the pipeline was tested on clinical samples from children admitted to a paediatric hospital in the Central African Republic. RESULTS: The PHARE pipeline achieved high recall and accuracy rates in all control datasets. The pipeline can be used on any gene and was tested with amplicons of the P. falciparum drug resistance marker genes pfdhps, pfdhfr and pfK13. CONCLUSIONS: The PHARE pipeline helps to provide a more complete picture of drug resistance in the circulating P. falciparum population and can help to guide treatment recommendations. PHARE is freely available under the GNU Lesser General Public License v.3.0 on GitHub: https://github.com/Fippu/PHARE.

Development and evaluation of PlasmoPod: A cartridge-based nucleic acid amplification test for rapid malaria diagnosis and surveillance.

Malaria surveillance is hampered by the widespread use of diagnostic tests with low sensitivity. Adequate molecular malaria diagnostics are often only available in centralized laboratories. PlasmoPod is a novel cartridge-based nucleic acid amplification test for rapid, sensitive, and quantitative detection of malaria parasites. PlasmoPod is based on reverse-transcription quantitative polymerase chain reaction (RT-qPCR) of the highly abundant Plasmodium spp. 18S ribosomal RNA/DNA biomarker and is run on a portable qPCR instrument which allows diagnosis in less than 30 minutes. Our analytical performance evaluation indicates that a limit-of-detection as low as 0.02 parasites/µL can be achieved and no cross-reactivity with other pathogens common in malaria endemic regions was observed. In a cohort of 102 asymptomatic individuals from Bioko Island with low malaria parasite densities, PlasmoPod accurately detected 83 cases, resulting in an overall detection rate of 81.4%. Notably, there was a strong correlation between the Cq values obtained from the reference RT-qPCR assay and those obtained from PlasmoPod. In an independent cohort, using dried blood spots from malaria symptomatic children living in the Central African Republic, we demonstrated that PlasmoPod outperforms malaria rapid diagnostic tests based on the PfHRP2 and panLDH antigens as well as thick blood smear microscopy. Our data suggest that this 30-minute sample-to-result RT-qPCR procedure is likely to achieve a diagnostic performance comparable to a standard laboratory-based RT-qPCR setup. We believe that the PlasmoPod rapid NAAT could enable widespread accessibility of high-quality and cost-effective molecular malaria surveillance data through decentralization of testing and surveillance activities, especially in elimination settings.

Stunted children display ectopic small intestinal colonization by oral bacteria, which cause lipid malabsorption in experimental models.

Environmental enteric dysfunction (EED) is an inflammatory syndrome postulated to contribute to stunted child growth and to be associated with intestinal dysbiosis and nutrient malabsorption. However, the small intestinal contributions to EED remain poorly understood. This study aimed to assess changes in the proximal and distal intestinal microbiota in the context of stunting and EED and to test for a causal role of these bacterial isolates in the underlying pathophysiology. We performed a cross-sectional study in two African countries recruiting roughly 1,000 children aged 2 to 5 years and assessed the microbiota in the stomach, duodenum, and feces. Upper gastrointestinal samples were obtained from stunted children and stratified according to stunting severity. Fecal samples were collected. We then investigated the role of clinical isolates in EED pathophysiology using tissue culture and animal models. We find that small intestinal bacterial overgrowth (SIBO) is extremely common (>80%) in stunted children. SIBO is frequently characterized by an overgrowth of oral bacteria, leading to increased permeability and inflammation and to replacement of classical small intestinal strains. These duodenal bacterial isolates decrease lipid absorption in both cultured enterocytes and mice, providing a mechanism by which they may exacerbate EED and stunting. Further, we find a specific fecal signature associated with the EED markers fecal calprotectin and alpha-antitrypsin. Our study shows a causal implication of ectopic colonization of oral bacterial isolated from the small intestine in nutrient malabsorption and gut leakiness in vitro. These findings have important therapeutic implications for modulating the microbiota through microbiota-targeted interventions.

Putative Biomarkers of Environmental Enteric Disease Fail to Correlate in a Cross-Sectional Study in Two Study Sites in Sub-Saharan Africa.

Environmental enteric dysfunction (EED) is an elusive, inflammatory syndrome of the small intestine thought to be associated with enterocyte loss and gut leakiness and lead to stunted child growth. To date, the gold standard for diagnosis is small intestine biopsy followed by histology. Several putative biomarkers for EED have been proposed and are widely used in the field. Here, we assessed in a cross-sectional study of children aged 2-5 years for a large set of biomarkers including markers of protein exudation (duodenal and fecal alpha-1-antitrypsin (AAT)), inflammation (duodenal and fecal calprotectin, duodenal, fecal and blood immunoglobulins, blood cytokines, C-reactive protein (CRP)), gut permeability (endocab, lactulose-mannitol ratio), enterocyte mass (citrulline) and general nutritional status (branched-chain amino acids (BCAA), insulin-like growth factor) in a group of 804 children in two Sub-Saharan countries. We correlated these markers with each other and with anemia in stunted and non-stunted children. AAT and calprotectin, CRP and citrulline and citrulline and BCAA correlated with each other. Furthermore, BCAA, citrulline, ferritin, fecal calprotectin and CRP levels were correlated with hemoglobin levels. Our results show that while several of the biomarkers are associated with anemia, there is little correlation between the different biomarkers. Better biomarkers and a better definition of EED are thus urgently needed.

Factors Associated with Stunted Growth in Children Under Five Years in Antananarivo, Madagascar and Bangui, Central African Republic.

OBJECTIVES: With a fourth of all under-five children affected, stunting remains one of the biggest health challenges worldwide. Even though the main underlying factors are known, the exact pathways to stunting varying in affected regions, and interventions thus need to be tailored to the local contexts. This study aimed assessing and comparing factors associated with stunting in two understudied sub-Saharan urban contexts with some of the highest stunting prevalence globally: Bangui, Central African Republic (~ 36%) and Antananarivo, Madagascar (42%). METHODS: We performed a case-control study on 175 + 194 stunted and 237 + 230 non-stunted control children aged 2-5 years and matched for age, gender and district of residency. Factors associated with stunting were identified using a standardized, paper questionnaire delivered by trained interviewers. Statistical analysis was done using logistic regression modelling. RESULTS: In both sites, formal maternal education lowered the risk of being stunted and restricted access to soap, suffering of anaemia and low birth weight were associated with higher risk of stunting. Short maternal stature, household head different from parents, diarrhoea and coughing were associated with an increased risk and continuing breastfeeding was associated with a lower risk of stunting in Antananarivo. Previous severe undernutrition and dermatitis/ fungal skin infections were associated with higher and changes in diet during pregnancy with lower risk of stunting in Bangui. CONCLUSIONS: Our results suggest maternal education, antenatal care, iron supplementation and simple WASH interventions such as using soap and infection control as general and breastfeeding (Antananarivo) or better nutrition (Bangui) as area-specified interventions.

[Latent tuberculosis infection in children in Bangui: about 524 cases exposed at home to index cases of positive microscopy pulmonary tuberculosis]. / Infection tuberculeuse latente chez l'enfant à Bangui: à propos de 524 cas exposés à domicile aux cas index de tuberculose pulmonaire à microscopie positive.

INTRODUCTION: in endemic areas, despite BCG vaccination, the risk of developing tuberculosis (TB) in young children is high after exposure to adults with tuberculosis. The purpose of this study is to reduce the risk of active tuberculosis in children experiencing household exposure to adult index cases. METHODS: we conducted a cross-sectional multi-site study (April 2016- January 2019) of children aged 0 to 59 months experiencing household exposure to index cases. They were screened and followed up at the pediatric center in Bangui. RESULTS: five hundred twenty four children were included in the study. The average age of patients was 2 years and 1 month and sex ratio (male/female) was 1.02; more than eighty-eight percent (88.5%) of contacts had received a BCG vaccination versus 11.5% who were unvaccinated. In more than half of the cases (52%), contacts and index cases had shared the same room and daily contact time had been greater than 12h in 56% of households; more than nine percent (9.35%) of contacts had positive tuberculin skin (IDR) test. All children received chemoprophylaxis with rifampicin + isoniazid, according to the national guidelines and, despite this, 14 or 2.67% of patients developed active tuberculosis, including 13 patients with pulmonary tuberculosis and one with ganglionic tuberculosis. CONCLUSION: chemoprophylaxis of tuberculosis significantly reduced the risk of TB in children experiencing household exposure to index cases.

Escalating and sustained immunovirological dissociation among antiretroviral drug-experienced perinatally human immunodeficiency virus-1-infected children and adolescents living in the Central African Republic: A STROBE-compliant study.

Sub-Saharan Africa has the vast majority (∼90%) of new pediatric acquired immunodeficiency syndrome cases worldwide. Biologically monitoring HIV-infected pediatric populations remains challenging. The differential interest of human immunodeficiency virus (HIV)-1 RNA loads and CD4 T-cell counts is debated for the treatment of pediatric acquired immunodeficiency syndrome patients.Long-term antiretroviral treatment (ART) outcomes regarding immunological and virological surrogate markers were longitudinally evaluated between 2009 and 2014 (over 57 months) in 245 perinatally HIV-1-infected children and adolescents born from HIV-infected mothers, treated at inclusion for at least 6 months by the World Health Organization-recommended ART in Bangui, Central African Republic.Patients were monitored over time biologically for CD4 T-cell counts, HIV-1 RNA loads, and drug resistance mutation genotyping.Children lost to follow-up totaled 6%. Four categories of immunovirological responses to ART were observed. At baseline, therapeutic success with sustained immunological and virological responses was observed in 80 (32.6%) children; immunological and virologic nonresponses occurred in 32 (13.0%) children; finally, the majority (133; 54.2%) of the remaining children showed discordant immunovirological responses. Among them, 33 (13.4%) children showed rapid virological responses to ART with an undetectable viral load, whereas immunological responses remained absent after 6 months of treatment and increased progressively over time in most of the cases, suggesting slow immunorestoration. Notably, nearly half of the children (40.8% at baseline and 48.2% at follow-up) harbored discordant immunovirological responses with a paradoxically high CD4 T-cell count and HIV-1 RNA load, which are always associated with high levels of drug resistance mutations. The latter category showed a significant increase over time, with a growth rate of 1.23% per year of follow-up.Our STROBE-compliant study demonstrates the high heterogeneity of biological responses under ART in children with frequent passage from 1 category to another over time. Close biological evaluation with access to routine plasma HIV-1 RNA load monitoring is crucial for adapting the complex outcomes of ART in HIV-infected children born from infected mothers.

Purifying Selection in Human Immunodeficiency Virus-1 pol Gene in Perinatally Human Immunodeficiency Virus-1-Infected Children Harboring Discordant Immunological Response and Virological Nonresponse to Long-Term Antiretroviral Therapy.

BACKGROUND: Biological monitoring of antiretroviral treatment (ART) in human immunodeficiency virus (HIV)-infected pediatric population remains challenging. The aim of the present study was to assess the long-term HIV-1 genetic diversity in pol gene in HIV-1-infected children in virological failure under antiretroviral regimen adapted according to the successive World Health Organization (WHO) guidelines for resource-constrained settings. METHODS: HIV-1 diversity in pol gene was assessed in HIV-1-infected children and adolescents born from HIV-infected mothers (median age at follow-up: 13.8 years) in virological failure (VF+) despite long-term regimen recommended by the WHO. The numbers of nonsynonymous substitutions per potential nonsynonymous site (dN) and of synonymous substitutions at potential synonymous sites (dS) in HIV-1 pol gene and the dN/dS ratios were used to estimate the selective pressure on circulating HIV-1. RESULTS: The immunological responses to ART basically corresponded to: 1) Full therapeutic failure with immunological (I-) and virological nonresponses in one-quarter (24.6%) of study children ((I-, VF+) subgroup); 2) Discordant immunovirological responses with paradoxical high CD4 T cell counts (I+) and high HIV-1 RNA load in the remaining cohort patients (75.4%) ((I+, VF+) subgroup). The mean dS was 1.8-fold higher in (I+, VF+) than (I-, VF+) subgroup (25.9 ± 18.4 vs. 14.3 ± 10.8). In the (I+, VF+) subgroup, the mean dS was 1.6-fold higher than the mean dN. Finally, the mean dN/dS ratio was 2.1-fold lower in (I+, VF+) than (I-, VF+) subgroup (0.6 ± 0.3 vs. 1.3 ± 0.7), indicating purifying selection in the immunovirological discordant (I+, VF+) subgroup and positive selection in the immunovirological failure (I-, VF+) subgroup. CONCLUSIONS: Children and adolescents in immunovirological therapeutic failure harbor positive selection of HIV-1 strains favoring diversifying in pol-encoded amino acids. In contrast, children with persistent discordant immunovirological responses show accumulation of mutations and purifying selection in pol gene sequences, indicating limited genetic evolution and likely suggesting genetic adaptation of viruses to host functional constraints.

Molecular assessment of kelch13 non-synonymous mutations in Plasmodium falciparum isolates from Central African Republic (2017-2019).

BACKGROUND: Over the last decade, artemisinin-based combination therapy (ACT) has contributed substantially to the decrease in malaria-related morbidity and mortality. The emergence of Plasmodium falciparum parasites resistant to artemisinin derivatives in Southeast Asia and the risk of their spread or of local emergence in sub-Saharan Africa are a major threat to public health. This study thus set out to estimate the proportion of P. falciparum isolates, with Pfkelch13 gene mutations associated with artemisinin resistance previously detected in Southeast Asia. METHODS: Blood samples were collected in two sites of Bangui, the capital of the Central African Republic (CAR) from 2017 to 2019. DNA was extracted and nested PCR were carried out to detect Plasmodium species and mutations in the propeller domain of the Pfkelch13 gene for P. falciparum samples. RESULTS: A total of 255 P. falciparum samples were analysed. Plasmodium ovale DNA was found in four samples (1.57%, 4/255). Among the 187 samples with interpretable Pfkelch13 sequences, four samples presented a mutation (2.1%, 4/187), including one non-synonymous mutation (Y653N) (0.5%, 1/187). This mutation has never been described as associated with artemisinin resistance in Southeast Asia and its in vitro phenotype is unknown. CONCLUSION: This preliminary study indicates the absence of Pfkelch13 mutant associated with artemisinin resistance in Bangui. However, this limited study needs to be extended by collecting samples across the whole country along with the evaluation of in vitro and in vivo phenotype profiles of Pfkelch13 mutant parasites to estimate the risk of artemisinin resistance in the CAR.

Incidence et facteurs de risque du tétanos néonatal en milieu rural centrafricain

Objectif : Décrire le profil épidémiologique et clinique du tétanos néonatal et identifier les facteurs de risque.Méthodologie : Il s'agissait d'une étude cas-témoins portant sur les nouveau-nés atteints du tétanos ou non (témoins), réalisée entre 2012 et 2017 à l'hôpital de district de Carnot. Un questionnaire a permis de collecter les données sociodémographiques et cliniques qui ont été saisies et analysées sur Epi info. Le test de chi-carré a servi à comparer les proportions au seuil de significativité 5%.Résultats : Au total,332 nouveau-nés ont été inclus dont 166 cas de TN et 166 témoins. L'âge moyen était de 7,8 ±3,6jours. L'incidence du TN était de 2,7 à 5,2 cas pour 1000 naissances vivantes. La fréquence hospitalière était de 7,5% et celle du tétanos de classe 3 selon le score de Dakar a été de 62,4%, le taux de létalité de 54%. Les facteurs de risque de TN étaient: le sexe masculin (p=0,00), le bas niveau d'instruction des mères (p=0,001),l'éloignement de l'hôpital (p=0,000), la primiparité (p=0,000), un faible nombre de consultations prénatales (p=0,000)et de doses de vaccin antitétanique (p=0,000), l'accouchement à domicile (p=0,000), l'accouchement par un personnel non qualifié (p=0,000),la section septique du cordon ombilical (p=0,000), l'absence de pansement du cordon (p=0,000) et un pansement septique (p=0,024).Conclusion : En Centrafrique, l'incidence du tétanos néonatal reste élevée et les facteurs de risque multiples. Des mesures efficaces de promotion de la santé peuvent contribuer à l'élimination de cette maladie

High predictive efficacy of integrase strand transfer inhibitors in perinatally HIV-1-infected African children in therapeutic failure of first- and second-line antiretroviral drug regimens recommended by the WHO.

OBJECTIVES: The predictive efficacy of integrase (IN) strand transfer inhibitors (INSTIs) was investigated in HIV-infected children born to HIV-infected mothers in Africa. METHODS: Plasma was collected at the Complexe Pédiatrique of Bangui, Central African Republic, from INSTI-naive children (n = 8) and adolescents (n = 10) in virological failure (viral load >1000 copies/mL) after 5 years of first- and/or second-line combination ART (cART). IN, reverse transcriptase (RT) and protease (P) genes were genotyped and drug resistance mutations (DRMs) to INSTIs, NRTIs, NNRTIs and PIs were interpreted using the Stanford algorithm. RESULTS: Successful IN, RT and P genotypes were obtained for 18, 13 and 15 children (median age 11 years, range 5-18; 8 were female), respectively. Two (2/18; 11.1%) viruses from children treated with a first-line regimen had INSTI DRMs at codon 138 (E138K and E138T), which is known to harbour major resistance mutations, and also had the accessory mutations L74I, G140K, G140R and G163R. The majority (16/18; 88.9%) of HIV-1 IN sequences demonstrated full susceptibility to all major INSTIs with a high frequency of natural polymorphic mutations. Most (12/15; 80%) genotyped viruses harboured at least one major DRM conferring resistance to at least one of the WHO-recommended antiretroviral drugs (NNRTIs, NRTIs and PIs) prescribed in first- and second-line regimens. CONCLUSIONS: INSTIs could be proposed in first-line regimens in the majority of African children or adolescents and may constitute relevant therapeutic alternatives as second- and third-line cART regimens in HIV-infected children and adolescents living in sub-Saharan Africa.

Stunted childhood growth is associated with decompartmentalization of the gastrointestinal tract and overgrowth of oropharyngeal taxa.

Linear growth delay (stunting) affects roughly 155 million children under the age of 5 years worldwide. Treatment has been limited by a lack of understanding of the underlying pathophysiological mechanisms. Stunting is most likely associated with changes in the microbial community of the small intestine, a compartment vital for digestion and nutrient absorption. Efforts to better understand the pathophysiology have been hampered by difficulty of access to small intestinal fluids. Here, we describe the microbial community found in the upper gastrointestinal tract of stunted children aged 2-5 y living in sub-Saharan Africa. We studied 46 duodenal and 57 gastric samples from stunted children, as well as 404 fecal samples from stunted and nonstunted children living in Bangui, Central African Republic, and in Antananarivo, Madagascar, using 16S Illumina Amplicon sequencing and semiquantitative culture methods. The vast majority of the stunted children showed small intestinal bacterial overgrowth dominated by bacteria that normally reside in the oropharyngeal cavity. There was an overrepresentation of oral bacteria in fecal samples of stunted children, opening the way for developing noninvasive diagnostic markers. In addition, Escherichia coli/Shigella sp. and Campylobacter sp. were found to be more prevalent in stunted children, while Clostridia, well-known butyrate producers, were reduced. Our data suggest that stunting is associated with a microbiome "decompartmentalization" of the gastrointestinal tract characterized by an increased presence of oropharyngeal bacteria from the stomach to the colon, hence challenging the current view of stunting arising solely as a consequence of small intestine overstimulation through recurrent infections by enteric pathogens.

Identifying the etiology and pathophysiology underlying stunting and environmental enteropathy: study protocol of the AFRIBIOTA project.

BACKGROUND: Globally one out of four children under 5 years is affected by linear growth delay (stunting). This syndrome has severe long-term sequelae including increased risk of illness and mortality and delayed psychomotor development. Stunting is a syndrome that is linked to poor nutrition and repeated infections. To date, the treatment of stunted children is challenging as the underlying etiology and pathophysiological mechanisms remain elusive. We hypothesize that pediatric environmental enteropathy (PEE), a chronic inflammation of the small intestine, plays a major role in the pathophysiology of stunting, failure of nutritional interventions and diminished response to oral vaccines, potentially via changes in the composition of the pro- and eukaryotic intestinal communities. The main objective of AFRIBIOTA is to describe the intestinal dysbiosis observed in the context of stunting and to link it to PEE. Secondary objectives include the identification of the broader socio-economic environment and biological and environmental risk factors for stunting and PEE as well as the testing of a set of easy-to-use candidate biomarkers for PEE. We also assess host outcomes including mucosal and systemic immunity and psychomotor development. This article describes the rationale and study protocol of the AFRIBIOTA project. METHODS: AFRIBIOTA is a case-control study for stunting recruiting children in Bangui, Central African Republic and in Antananarivo, Madagascar. In each country, 460 children aged 2-5 years with no overt signs of gastrointestinal disease are recruited (260 with no growth delay, 100 moderately stunted and 100 severely stunted). We compare the intestinal microbiota composition (gastric and small intestinal aspirates; feces), the mucosal and systemic immune status and the psychomotor development of children with stunting and/or PEE compared to non-stunted controls. We also perform anthropological and epidemiological investigations of the children's broader living conditions and assess risk factors using a standardized questionnaire. DISCUSSION: To date, the pathophysiology and risk factors of stunting and PEE have been insufficiently investigated. AFRIBIOTA will add new insights into the pathophysiology underlying stunting and PEE and in doing so will enable implementation of new biomarkers and design of evidence-based treatment strategies for these two syndromes.

Hepatitis B and hepatitis D virus infections in the Central African Republic, twenty-five years after a fulminant hepatitis outbreak, indicate continuing spread in asymptomatic young adults.

Hepatitis delta virus (HDV) increases morbidity in Hepatitis B virus (HBV)-infected patients. In the mid-eighties, an outbreak of HDV fulminant hepatitis (FH) in the Central African Republic (CAR) killed 88% of patients hospitalized in Bangui. We evaluated infections with HBV and HDV among students and pregnant women, 25 years after the fulminant hepatitis (FH) outbreak to determine (i) the prevalence of HBV and HDV infection in this population, (ii) the clinical risk factors for HBV and/or HDV infections, and (iii) to characterize and compare the strains from the FH outbreak in the 1980s to the 2010 HBV-HDV strains. We performed a cross sectional study with historical comparison on FH-stored samples (n = 179) from 159 patients and dried blood-spots from volunteer students and pregnant women groups (n = 2172). We analyzed risk factors potentially associated with HBV and HDV. Previous HBV infection (presence of anti-HBc) occurred in 345/1290 students (26.7%) and 186/870 pregnant women (21.4%)(p = 0.005), including 110 students (8.8%) and 71 pregnant women (8.2%), who were also HBsAg-positive (p = 0.824). HDV infection occurred more frequently in pregnant women (n = 13; 18.8%) than students (n = 6; 5.4%) (p = 0.010). Infection in childhood was probably the main HBV risk factor. The risk factors for HDV infection were age (p = 0.040), transfusion (p = 0.039), and a tendency for tattooing (p = 0.055) and absence of condom use (p = 0.049). HBV-E and HDV-1 were highly prevalent during both the FH outbreak and the 2010 screening project. For historical samples, due to storage conditions and despite several attempts, we could only obtain partial HDV amplification representing 25% of the full-length genome. The HDV-1 mid-eighties FH-strains did not form a specific clade and were affiliated to two different HDV-1 African subgenotypes, one of which also includes the 2010 HDV-1 strains. In the Central African Republic, these findings indicate a high prevalence of previous and current HBV-E and HDV-1 infections both in the mid-eighties fulminant hepatitis outbreak and among asymptomatic young adults in 2010, and reinforce the need for universal HBV vaccination and the prevention of HDV transmission among HBsAg-positive patients through blood or sexual routes.

Sentinel surveillance of influenza-like illness in the Central African Republic, 2010-2015.

BACKGROUND: Influenza-like illness (ILI) is an important public health problem worldwide. In the Central African Republic, acute infectious diseases are the commonest reason for consultation. The Institut Pasteur of Bangui set up a surveillance network in 2008 to monitor the circulation of influenza viruses. We report the results of use of this surveillance system during the period 2010-2015. METHODS: The first surveillance centre covered Bangui, the capital of the country, and neighbouring areas and epidemiological data on syndromes similar to ILI. Throat and nasopharyngeal swab samples are transmitted weekly to the Institut Pasteur of Bangui, where real-time and multiplex reverse transcription polymerase chain reaction are used to detect and subtype influenza A (H1N1 and H3N2) and B viruses. The demographic characteristics of all patients and of positive cases according to age and the seasonal patterns of influenza virus circulation were analysed. RESULTS: Between January 2010 and December 2015, 5385 throat swabs were collected; 454 (8.4%) of the samples were positive. Of these, 450 yielded at least one influenza virus and four showed co-infections. Children under the age of 5 years were the most frequently infected (257/450, 57.1%), with irregular peaks of ILI. Influenza B predominated (56.2%; n = 201), with 39.0% H3N2 and 16.7%H1N1pdm09. Influenza viruses were detected mainly in the rainy season (July-December). CONCLUSION: The sentinel surveillance site is yielding important information about the seasonality and age pattern of circulating influenza virus. Nationwide distribution of sentinel sites is warranted.

Factors associated with stunting in healthy children aged 5 years and less living in Bangui (RCA).

Stunting remains a major public health concern worldwide. Although its global prevalence is slowly decreasing, the actual number of affected children is still rising in Sub-Saharan Africa. In the Central African Republic (CAR), about one third of all children below the age of five are stunted. Stunting is correlated with many long-term consequences, including poor cognitive development and a higher rate of morbidity and mortality, making stunting a major contributor to poverty. In CAR, little is known about the factors that contribute to stunting. This study aimed at analysing, in a cross-sectional study, the main factors associated with stunting in a group of 414 children recruited between December 2011 and November 2013, aged five years or less and living in Bangui. For all children, demographic, socio-economic and anthropometric data were recorded and asymptomatic enteropathogen carriage was assessed in stool samples using classical microbiological assays. The study group had a mean age of 14.2±10 months. Fifty-eight percent (292/414) were boys, and 36 percent (148/414) exhibited stunted growth. Of the stunted children, 51% (75/148) showed a moderate delay in linear growth for their age group [height-for-age z-score (HAZ) between -2 and -3 SD] while 49% (73/148) presented a severe delay (HAZ < -3). Factors significantly associated with stunting included gender (aOR: 1.67; 95% CI: 1.07; 2.62 for boys compared to girls) and age (aOR of 3.98 (95% CI: 2.45; 6.46) for toddlers and aOR 4.42 (95% CI: 2.36; 8.28) for children compared to infants). Most importantly, we identified being overweight [weight-for-height z-score (WHZ) > 2 SD; aOR: 3.21; 95% CI: 1.50; 6.90 of overweight compared to normal weight] as also being significantly associated with stunting. This is the first study showing that even in the poorest countries of the world there is an association of stunting with being overweight.

High levels of virological failure with major genotypic resistance mutations in HIV-1-infected children after 5 years of care according to WHO-recommended 1st-line and 2nd-line antiretroviral regimens in the Central African Republic: A cross-sectional study.

A large cohort of 220 HIV-1-infected children (median [range] age: 12 [4-17] years) was cared and followed up in the Central African Republic, including 198 in 1st-line and 22 in 2nd-line antiretroviral regimens. Patients were monitored clinically and biologically for HIV-1 RNA load and drug resistance mutations (DRMs) genotyping. A total of 87 (40%) study children were virological responders and 133 (60%) nonresponders. In children with detectable viral load, the majority (129; 97%) represented a virological failure. In children receiving 1st-line regimens in virological failure for whom genotypic resistance test was available, 45% displayed viruses harboring at least 1 DRM to NNRTI or NRTI, and 26% showed at least 1 major DRM to NNRTI or NRTI; more than half of children in 1st-line regimens were resistant to 1st-generation NNRTI and 24% of the children in 1st-line regimens had a major DRMs to PI. Virological failure and selection of DRMs were both associated with poor adherence. These observations demonstrate high rate of virological failure after 3 to 5 years of 1st-line or 2nd-line antiretroviral treatment, which is generally associated with DRMs and therapeutic failure. Overall, more than half (55%) of children receiving 1st-line antiretroviral treatment for a median of 3.4 years showed virological failure and antiretroviral-resistance and thus eligible to 2nd-line treatment. Furthermore, two-third (64%) of children under 2nd-line therapy were eligible to 3rd-line regimen. Taken together, these observations point the necessity to monitor antiretroviral-treated children by plasma HIV-1 RNA load to diagnose as early as possible the therapeutic failure and operate switch to a new therapeutic line.

Etiology and Epidemiology of Diarrhea in Hospitalized Children from Low Income Country: A Matched Case-Control Study in Central African Republic.

BACKGROUND: In Sub-Saharan Africa, infectious diarrhea is a major cause of morbidity and mortality. A case-control study was conducted to identify the etiology of diarrhea and to describe its main epidemiologic risk factors among hospitalized children under five years old in Bangui, Central African Republic. METHODS: All consecutive children under five years old hospitalized for diarrhea in the Pediatric Complex of Bangui for whom a parent's written consent was provided were included. Controls matched by age, sex and neighborhood of residence of each case were included. For both cases and controls, demographic, socio-economic and anthropometric data were recorded. Stool samples were collected to identify enteropathogens at enrollment. Clinical examination data and blood samples were collected only for cases. RESULTS: A total of 333 cases and 333 controls was recruited between December 2011 and November 2013. The mean age of cases was 12.9 months, and 56% were male. The mean delay between the onset of first symptoms and hospital admission was 3.7 days. Blood was detected in 5% of stool samples from cases. Cases were significantly more severely or moderately malnourished than controls. One of the sought-for pathogens was identified in 78% and 40% of cases and controls, respectively. Most attributable cases of hospitalized diarrhea were due to rotavirus, with an attributable fraction of 39%. Four other pathogens were associated with hospitalized diarrhea: Shigella/EIEC, Cryptosporidium parvum/hominis, astrovirus and norovirus with attributable fraction of 9%, 10%, 7% and 7% respectively. Giardia intestinalis was found in more controls than cases, with a protective fraction of 6%. CONCLUSIONS: Rotavirus, norovirus, astrovirus, Shigella/EIEC, Cryptosporidium parvum/hominis were found to be positively associated with severe diarrhea: while Giardia intestinalis was found negatively associated. Most attributable episodes of severe diarrhea were associated with rotavirus, highlighting the urgent need to introduce the rotavirus vaccine within the CAR's Expanded Program on Immunization. The development of new medicines, vaccines and rapid diagnostic tests that can be conducted at the bedside should be high priority for low-resource countries.

Strengthening laboratory capacity through the surveillance of rotavirus gastroenteritis in Central Africa: the Surveillance Épidémiologique en Afrique Centrale (SURVAC) Project.

OBJECTIVES: The goal of the SURVAC pilot project was to strengthen disease surveillance and response in three countries; Cameroon (CAE), Democratic Republic of the Congo (DRC) and Central African Republic (CAR). METHODS: Seven laboratories involved in rotavirus surveillance were provided with equipment, reagents and supplies. CDC and WHO staff provided on-site classroom and bench training in biosafety, quality assurance, quality control (QC), rotavirus diagnosis using Enzyme Immunoassay (EIA) and genotyping of rotavirus strains using the Reverse Transcription Polymerase-chain reaction (RT-PCR). All laboratory data were reported through WHO/AFRO. RESULTS: Twenty-three staff members were trained on RT-PCR for rotavirus genotyping which was introduced for the first time in all three countries. In CAE, the number of samples analysed by EIA and RT-PCR increased tenfold between 2007 and 2013. In DRC, this number increased fivefold, from 2009 to 2013 whereas in CAR, it increased fourfold between 2011 and 2013. All laboratories passed WHO proficiency testing in 2014. CONCLUSION: Laboratory capacity was strengthened through equipping laboratories and strengthening a subregional laboratory workforce for surveillance of rotavirus gastroenteritis. Each of the three countries generated rotavirus surveillance and genotyping data enabling the mapping of circulating genotypes. These results will help monitor the impact of rotavirus vaccination in these countries.

Molecular surveillance of rotavirus infection in Bangui, Central African Republic, October 2011-September 2013.

BACKGROUND: The World Health Organization (WHO) recommends the introduction of rotavirus vaccine in the immunization program of all countries. In the Central African Republic (CAR), sentinel surveillance for rotavirus gastroenteritis was established in 2011 by the Ministry of Health, with the support of the Surveillance en Afrique Centrale Project (SURVAC). The purpose of this study was to assess the burden of rotavirus gastroenteritis and to identify rotavirus strains circulating in CAR before the introduction of rotavirus vaccine planned for this year, 2014. METHODS: One sentinel site and one laboratory at the national level were designated by the CAR Ministry of Health to participate in this surveillance system. Stool samples were collected from children who met the WHO rotavirus gastroenteritis case definition (WHO, 2006). The samples were first screened for group A rotavirus antigen by enzyme immunoassay (EIA), and genotyping assays performed using a multiplex reverse transcriptase PCR (RT-PCR) technique. RESULTS: Between October 2011 and September 2013, 438 stool samples were collected and analyzed for detection of rotavirus antigen; 206 (47%) were positive. Among the 160 (78%) that could be genotyped, G2P[6] was the predominant strain (47%) followed by G1P[8] (25%) and G2P[4] (13%). CONCLUSIONS: Almost half of stool samples obtained from children hospitalized with gastroenteritis were positive for rotavirus. These baseline rotavirus surveillance data will be useful to health authorities considering rotavirus vaccine introduction and for evaluating the efficacy of rotavirus vaccine once it is introduced into the routine immunization system.