Richter Razafindratsimandresy

Molecular comparison and evolutionary analyses of VP1 nucleotide sequences of new African human enterovirus 71 isolates reveal a wide genetic diversity.

Most circulating strains of Human enterovirus 71 (EV-A71) have been classified primarily into three genogroups (A to C) on the basis of genetic divergence between the 1D gene, which encodes the VP1 capsid protein. The aim of the present study was to provide further insights into the diversity of the EV-A71 genogroups following the recent description of highly divergent isolates, in particular those from African countries, including Madagascar. We classified recent EV-A71 isolates by a large comparison of 3,346 VP1 nucleotidic sequences collected from GenBank. Analysis of genetic distances and phylogenetic investigations indicated that some recently-reported isolates did not fall into the genogroups A-C and clustered into three additional genogroups, including one Indian genogroup (genogroup D) and 2 African ones (E and F). Our Bayesian phylogenetic analysis provided consistent data showing that the genogroup D isolates share a recent common ancestor with the members of genogroup E, while the isolates of genogroup F evolved from a recent common ancestor shared with the members of the genogroup B. Our results reveal the wide diversity that exists among EV-A71 isolates and suggest that the number of circulating genogroups is probably underestimated, particularly in developing countries where EV-A71 epidemiology has been poorly studied.

High Frequency of Human Enterovirus Species C Circulation in Madagascar

ABSTRACT Four poliomyelitis outbreaks caused by vaccine-derived polioviruses have been reported recently, including one in Madagascar in 2002. In all cases, the viral strains involved were recombinant between poliovirus vaccine strains and nonpoliovirus strains, probably enterovirus species C. Nevertheless, little is known about the circulation and epidemiology of enteroviruses in the regions where these outbreaks occurred. To assess the circulation of enteroviruses (particularly enterovirus species C) in Madagascar, we genetically characterized 55 enterovirus strains isolated between 1994 and 2002. The strains were identified and compared by partially sequencing the region encoding the VP1 capsid protein. Phylogenetic analysis and pairwise comparison with prototype enterovirus strains distinguished two different species: 25 isolates belonged to human enterovirus B species, and 30 isolates were identified as coxsackievirus A13, A15, A17, A18, A20, A21, and A24, belonging to the human enterovirus species C. The relatively high frequency and the wide distribution of species C coxsackie A viruses in different regions of Madagascar suggest that they had been silently and widely circulating in the country during the whole study period. The circulation of coxsackie A viruses, combined with the low routine oral polio vaccine coverage, may have played a role in the emergence of the recent outbreak in Madagascar.

Case-control study of the etiology of infant diarrheal disease in 14 districts in Madagascar.

Background Acute diarrhea is a major cause of childhood morbidity and mortality worldwide. Its microbiological causes and clinico-epidemiological aspects were examined during the rainy seasons from 2008 to 2009 in 14 districts in Madagascar. Methods Stool specimens of 2196 children with acute diarrhea and 496 healthy children were collected in a community setting. Intestinal parasites were diagnosed by microscopy and bacteria by culturing methods. Rota-, astro and adenoviruses were identified using commercially available ELISA kits and rotaviruses were confirmed using reverse transcriptase polymerase chain reaction (RT-PCR). Results Intestinal microorganisms were isolated from 54.6% of diarrheal patients and 45.9% of healthy subjects (p = <0.01). The most common pathogens in diarrheic patients were intestinal parasites (36.5%). Campylobacter spp. and Rotavirus were detected in 9.7% and 6.7% of diarrheic patients. The detection rates of Entamoeba histolytica, Trichomonas intestinalis and Giardia lamblia were much greater in diarrheal patients than in non diarrheal subjects (odds ratios of 5.1, 3.2, 1.7 respectively). The abundance of other enteropathogens among the non diarrheal group may indicate prolonged excretion or limited pathogenicity. Conclusion In developing countries, where the lack of laboratory capacities is great, cross sectional studies of enteropathogens and their spatial distribution, including diarrheal and non diarrheal subjects, are interesting tools in order to advise regional policies on treatment and diarrheic patient management.

Common and Diverse Features of Cocirculating Type 2 and 3 Recombinant Vaccine-Derived Polioviruses Isolated From Patients With Poliomyelitis and Healthy Children

BACKGROUND Five cases of poliomyelitis due to type 2 or 3 recombinant vaccine-derived polioviruses (VDPVs) were reported in the Toliara province of Madagascar in 2005. METHODS We sequenced the genome of the VDPVs isolated from the patients and from 12 healthy children and characterized phenotypic aspects, including pathogenicity, in mice transgenic for the poliovirus receptor. RESULTS We identified 6 highly complex mosaic recombinant lineages composed of sequences derived from different vaccine polioviruses and other species C human enteroviruses (HEV-Cs). Most had some recombinant genome features in common and contained nucleotide sequences closely related to certain cocirculating coxsackie A virus isolates. However, they differed in terms of their recombinant characteristics or nucleotide substitutions and phenotypic features. All VDPVs were neurovirulent in mice. CONCLUSIONS This study confirms the genetic relationship between type 2 and 3 VDPVs, indicating that both types can be involved in a single outbreak of disease. Our results highlight the various ways in which a vaccine-derived poliovirus may become pathogenic in complex viral ecosystems, through frequent recombination events and mutations. Intertypic recombination between cocirculating HEV-Cs (including polioviruses) appears to be a common mechanism of genetic plasticity underlying transverse genetic variability.

Genetic relationship between cocirculating Human enteroviruses species C.

Recombination events between human enteroviruses (HEV) are known to occur frequently and to participate in the evolution of these viruses. In a previous study, we reported the isolation of a panel of viruses belonging to the Human enterovirus species C (HEV-C) that had been cocirculating in a small geographic area of Madagascar in 2002. This panel included type 2 vaccine-derived polioviruses (PV) that had caused several cases of acute flaccid paralysis in humans. Previous partial sequencing of the genome of these HEV-C isolates revealed considerable genetic diversity, mostly due to recombination. In the work presented herein, we carried out a more detailed characterization of the genomes of viruses from this collection. First, we determined the full VP1 sequence of 41 of these isolates of different types. These sequences were compared with those of HEV-C isolates obtained from other countries or in other contexts. The sequences of the Madagascan isolates of a given type formed specific clusters clearly differentiated from those formed by other strains of the same type isolated elsewhere. Second, we sequenced the entire genome of 10 viruses representing most of the lineages present in this panel. All but one of the genomes appeared to be mosaic assemblies of different genomic fragments generated by intra- and intertypic recombination. The location of the breakpoints suggested potential preferred genomic regions for recombination. Our results also suggest that recombination between type HEV-99 and other HEV-C may be quite rare. This first exhaustive genomic analysis of a panel of non-PV HEV-C cocirculating in a small human population highlights the high frequency of inter and intra-typic genetic recombination, constituting a widespread mechanism of genetic plasticity and continually shifting the HEV-C biodiversity.

Partial molecular characterization of alphaherpesviruses isolated from tropical bats.

Herpesviruses have previously been isolated from African and South-American bats. Recently, herpesviruses detected from European insectivorous bats (family Vespertilionidae) were classified molecularly as betaherpesviruses and gammaherpesviruses. In the current study, we performed PCR analyses targeting the UL30 catalytic subunit region of the DNA polymerase gene of the African and South American herpesviruses and new Malagasy and Cambodian herpesviruses isolated from bats, especially frugivorous bats from the families Pteropodidae and Phyllostomidae. The sequences obtained from the amplified products indicated that these isolates belonged to the genus Simplexvirus of the subfamily Alphaherpesvirinae. These results extend the taxonomic range of bat herpesviruses with the description of four members in the subfamily Alphaherpesvirinae. Furthermore, these data confirm and extend the geographical distribution of herpesvirus in bats to three more continents (Africa, South America and Asia) and indicate the presence of these viruses in frugivorous bats of the families Pteropodidae and Phyllostomidae.

Seroprevalence of hepatitis C and associated risk factors in urban areas of Antananarivo, Madagascar

BackgroundThe risk factors for the transmission of HCV vary substantially between countries and geographic regions. The overall prevalence in south and east Africa region has been estimated to be 1.6% but limited information about the epidemiology of HCV infection in Madagascar is availableMethodsA cross-sectional survey for hepatitis C antibodies was conducted in 2,169 subjects of the general population of Antananarivo to determine seroprevalence of hepatitis C and associated risk factors.ResultsThe overall seroprevalence was 1.2% (25/2,169). The prevalence did not differ significantly according to gender but it increased with age (Chi2 tendency test, p < 10-5). The variable history of hospitalization, previous therapeutic injections, dental treatment, intravenous drug use, and abnormal ALT and AST were statistically significantly related with the presence of HCV antibodies. No relationship with past history of blood transfusion was observed.ConclusionHCV prevalence in Madagascar seems to be similar to that in most other east African countries. Age appears to be an important risk factor. Iatrogenic causes of HCV transmission need to be further evaluated because all HCV cases had a history of receiving therapeutic injections and data suggested a cumulative effect in relation with therapeutic injections.

Whole Genome Sequencing of Enterovirus species C Isolates by High-Throughput Sequencing: Development of Generic Primers

Enteroviruses are among the most common viruses infecting humans and can cause diverse clinical syndromes ranging from minor febrile illness to severe and potentially fatal diseases. Enterovirus species C (EV-C) consists of more than 20 types, among which the three serotypes of polioviruses, the etiological agents of poliomyelitis, are included. Biodiversity and evolution of EV-C genomes are shaped by frequent recombination events. Therefore, identification and characterization of circulating EV-C strains require the sequencing of different genomic regions. A simple method was developed to quickly sequence the entire genome of EV-C isolates. Four overlapping fragments were produced separately by RT-PCR performed with generic primers. The four amplicons were then pooled and purified prior to being sequenced by a high-throughput technique. The method was assessed on a panel of EV-Cs belonging to a wide-range of types. It can be used to determine full-length genome sequences through de novo assembly of thousands of reads. It was also able to discriminate reads from closely related viruses in mixtures. By decreasing the workload compared to classical Sanger-based techniques, this method will serve as a precious tool for sequencing large panels of EV-Cs isolated in cell cultures during environmental surveillance or from patients, including vaccine-derived polioviruses.

Rotavirus genotypes in children in the community with diarrhea in Madagascar.

In the context of the possible introduction of a preventive vaccine against rotaviruses in Madagascar, the G and P genotypes distribution of the rotaviruses circulating in the children in Madagascar was studied, and the presence of emerging genotypes and unusual strains were assessed. From February 2008 to May 2009, 1,679 stools specimens were collected from children ≤5 years old with diarrhea. ELISA was used for antigen detection, and molecular amplification of VP7 and VP4 gene fragments was used for genotyping. Rotavirus antigen was detected in 104 samples (6.2%). Partial sequences of VP7 and VP4 genes were obtained from 81 and 80 antigen‐positive stools, respectively. The most frequent G and P types combinations detected were G9P[8] (n = 51; 64.6%), followed by G1P[8] (n = 15; 18.9%), and G1P[6] (n = 8; 10.1%). A few unusual G‐P combinations, such as G4P[6] (n = 3; 3.8%), G9P[6] (n = 1; 1.3%), and G3P[9] reassortant feline human virus (n = 1; 1.3%) were identified. Both VP4 and VP7 sequences in one of the three G4P[6] isolates were closely related to those in porcine strains, and one was a reassortant human porcine virus. These findings give an overview of the strains circulating in Madagascar and should help public health authorities to define a vaccine strategy. J. Med. Virol. 85:1652–1660, 2013.

Etiologies, Risk Factors and Impact of Severe Diarrhea in the Under-Fives in Moramanga and Antananarivo, Madagascar.

Background Diarrheal disease remains a leading cause of death in children in low-income countries. We investigated the etiology, risk factors and effects on nutritional status of severe diarrhea in children from two districts in Madagascar. Methods We performed a matched case-control study in 2011 to 2014, on children under the age of five years from Moramanga and Antananarivo. The cases were children hospitalized for severe diarrhea and the controls were children without diarrhea selected at random from the community. Stool samples were collected from both groups. Anthropometric measurements were made during follow-up visits about one and two months after enrolment. Results We enrolled 199 cases and 199 controls. Rotavirus infection was the most frequently detected cause of diarrhea. It was strongly associated with severe diarrhea (OR: 58.3; 95% CI: 7.7–439.9), accounting for 42.4% (95% CI: 37.6–43.1) of severe diarrhea cases. At the household level, possession of cattle (OR = 0.3; 95% CI: 0.1–0.6) and living in a house with electricity (OR = 0.4; 95% CI: 0.2–0.8) were protective factors. The presence of garbage around the house was a risk factor for severe diarrhea (OR = 3.2; 95% CI: 1.9–5.4). We found no significant association between severe diarrhea and the nutritional status of the children at follow-up visits, but evident wasting at enrolment was associated with a higher risk of severe diarrhea (OR = 9; 95% CI: 4.5–17.9). Conclusions Severe childhood diarrhea is mostly caused by rotavirus infection. An anti-rotavirus vaccine has already been introduced in Madagascar and should be promoted more widely. However, post-licensing surveillance is required. Interventions to improve the nutritional status of children, preventive measures focused on household and personal hygiene and nutritional rehabilitation during severe diarrheal disease should be reinforced.