Diagnosis and genetic characteristics of potential pathogens in children under five years of age with diarrhea

Diarrheal diseases collectively constitute a serious public health challenge globally. The causative agents of diarrheal disease include adenovirus (serotypes 40 and 41), Aeromonas spp, Entamoeba histolytica, Cryptosporidium spp., Escherichia coli strains, norovirus (NoV), non-typhoidal Salmonella spp., rotavirus A (RVA), Shigella spp., Vibrio cholerae and Clostridium difficile. Among these, rotavirus, Cryptosporidium spp., and Shigella spp. are the three aetiological agents responsible for most deaths in children under 5 years old. The distributions of these pathogens are overlapping and they coexist in many endemic areas, particularly in low-income countries. Accurate diagnosis and molecular characterization of diarrheal pathogens are necessary for surveillance, prevention, and control of diarrhea. To obtain accurate epidemiological data,to support diarrheal disease control and elimination intervention strategies in Gabon, four studies were conducted as part of this thesis. First, the prevalence of pathogens found in stool samples in outpatient Gabonese children < 5 years with diarrhea was for the first time systematically investigated to describe the local spectrum of infectious agents. The most frequently identified were enteroinvasive Escherichia coli (EIEC) /Shigella and Enterotoxigenic Escherichia coli (ETEC), followed by Giardia lamblia, Cryptosporidium spp. and rotavirus. The emergent ETEC, EIEC /Shigella, Cryptosporidium spp. and rotavirus were frequently detected in combination. The most frequently observed combinations of pathogens were EIEC/Shigella and ETEC, ETEC and rotavirus as well as Cryptosporidium and EIEC/Shigella. This information serves as baseline for recommendation for interventions and diagnostic algorithms to public health stakeholders. The second study was nested in a broader community-based project and aimed to evaluate cryptosporidiosis diagnosis by a rapid diagnostic test (CerTest Crypto RDT) against a composite reference of quantitative polymerase chain reaction (qPCR) and restriction fragment length polymorphism (RFLP)-PCR in African children from four countries (Gabon, Ghana, Madagascar, and Tanzania) admitted to a hospital with diarrhea. The performance of this RDT varied across the four study sites. Overall, the test showed a low sensitivity for the detection of Cryptosporidium parvum and C. hominis. This work included the comprehensive investigation of rotavirus genotypes and antigenic epitope variability in the VP7 and VP4 proteins of circulating rotavirus A strains compared to vaccine strains, as the third part of this dissertation. Rotavirus A was detected in 55 % (98/177) of hospitalized children with gastroenteritis and 21 % (14/67) of the control children. The most common genotypes were G1, G3, G8, G9, G12, with G8 and G9 being reported for the first time in Gabon. All of these G genotypes were associated either with P[6] or P[8] genotypes. Several amino acid mutations associated with immune evasion were detected on antigenic epitopes of VP7 (sites 94, 147) and VP8* (sites 89, 116, 146, 150) of Gabonese strains, which may lead to reduced efficacy of available RotaTeq and Rotarix vaccines. The fourth study was designed to determine the prevalence and genetic diversity of four main enteric viruses (Norovirus, Sapovirus, Astrovirus and Aichvirus A) in hospitalized children <5 years with gastroenteritis and community controls without gastroenteritis. Norovirus (14.7 %; 26/177) and astrovirus (7.3 %; 13/177) were the most prevalent in children with diarrhea, while in the healthy group norovirus (9 %; 6/67) followed by aichivirus A (6 %; 4/67) were predominant. The predominant norovirus genogroup was GII, consisting mostly of genotype GII.P31-GII.4 Sydney. This study provides the first report on the detection of Aichvirus A in Gabon and Central Africa. This thesis provides the epidemiological and genetic baseline data that will be essential for advocating a much-needed management of diarrhea as recommended by the WHO. Moreover, these results show that the implementation of a national vaccination program against rotavirus is necessary and urgent in Gabon. The findings related to the performance of CerTest Crypto RDT in Africa will support implementation of diarrhea control and elimination efforts in endemic areas. Future work should focus on the development of laboratory methods to improve the sensitivity of cryptosporidiosis detection and on expanding the use of routine diagnosis

Genetic Diversity of Enteric Viruses in Children under Five Years Old in Gabon

Enteric viruses are the leading cause of diarrhea in children globally. Identifying viral agents and understanding their genetic diversity could help to develop effective preventive measures. This study aimed to determine the detection rate and genetic diversity of four enteric viruses in Gabonese children aged below five years. Stool samples from children <5 years with (n = 177) and without (n = 67) diarrhea were collected from April 2018 to November 2019. Norovirus, astrovirus, sapovirus, and aichivirus A were identified using PCR techniques followed by sequencing and phylogenetic analyses. At least one viral agent was identified in 23.2% and 14.9% of the symptomatic and asymptomatic participants, respectively. Norovirus (14.7%) and astrovirus (7.3%) were the most prevalent in children with diarrhea, whereas in the healthy group norovirus (9%) followed by the first reported aichivirus A in Gabon (6%) were predominant. The predominant norovirus genogroup was GII, consisting mostly of genotype GII.P31-GII.4 Sydney. Phylogenetic analysis of the 3CD region of the aichivirus A genome revealed the presence of two genotypes (A and C) in the study cohort. Astrovirus and sapovirus showed a high diversity, with five different astrovirus genotypes and four sapovirus genotypes, respectively. Our findings give new insights into the circulation and genetic diversity of enteric viruses in Gabonese children.Peer Reviewe

The evolving SARS-CoV-2 epidemic in Africa: Insights from rapidly expanding genomic surveillance

INTRODUCTION Investment in Africa over the past year with regard to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) sequencing has led to a massive increase in the number of sequences, which, to date, exceeds 100,000 sequences generated to track the pandemic on the continent. These sequences have profoundly affected how public health officials in Africa have navigated the COVID-19 pandemic. RATIONALE We demonstrate how the first 100,000 SARS-CoV-2 sequences from Africa have helped monitor the epidemic on the continent, how genomic surveillance expanded over the course of the pandemic, and how we adapted our sequencing methods to deal with an evolving virus. Finally, we also examine how viral lineages have spread across the continent in a phylogeographic framework to gain insights into the underlying temporal and spatial transmission dynamics for several variants of concern (VOCs). RESULTS Our results indicate that the number of countries in Africa that can sequence the virus within their own borders is growing and that this is coupled with a shorter turnaround time from the time of sampling to sequence submission. Ongoing evolution necessitated the continual updating of primer sets, and, as a result, eight primer sets were designed in tandem with viral evolution and used to ensure effective sequencing of the virus. The pandemic unfolded through multiple waves of infection that were each driven by distinct genetic lineages, with B.1-like ancestral strains associated with the first pandemic wave of infections in 2020. Successive waves on the continent were fueled by different VOCs, with Alpha and Beta cocirculating in distinct spatial patterns during the second wave and Delta and Omicron affecting the whole continent during the third and fourth waves, respectively. Phylogeographic reconstruction points toward distinct differences in viral importation and exportation patterns associated with the Alpha, Beta, Delta, and Omicron variants and subvariants, when considering both Africa versus the rest of the world and viral dissemination within the continent. Our epidemiological and phylogenetic inferences therefore underscore the heterogeneous nature of the pandemic on the continent and highlight key insights and challenges, for instance, recognizing the limitations of low testing proportions. We also highlight the early warning capacity that genomic surveillance in Africa has had for the rest of the world with the detection of new lineages and variants, the most recent being the characterization of various Omicron subvariants. CONCLUSION Sustained investment for diagnostics and genomic surveillance in Africa is needed as the virus continues to evolve. This is important not only to help combat SARS-CoV-2 on the continent but also because it can be used as a platform to help address the many emerging and reemerging infectious disease threats in Africa. In particular, capacity building for local sequencing within countries or within the continent should be prioritized because this is generally associated with shorter turnaround times, providing the most benefit to local public health authorities tasked with pandemic response and mitigation and allowing for the fastest reaction to localized outbreaks. These investments are crucial for pandemic preparedness and response and will serve the health of the continent well into the 21st century