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

Abstract

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.