Neutralization of African enterovirus A71 genogroups by antibodies to canonical genogroups.

Enterovirus 71 (EV-A71) is a major public health problem, causing a range of illnesses from hand-foot-and-mouth disease to severe neurological manifestations. EV-A71 strains have been phylogenetically classified into eight genogroups (A to H), based on their capsid-coding genomic region. Genogroups B and C have caused large outbreaks worldwide and represent the two canonical circulating EV-A71 subtypes. Little is known about the antigenic diversity of new genogroups as compared to the canonical ones. Here, we compared the antigenic features of EV-A71 strains that belong to the canonical B and C genogroups and to genogroups E and F, which circulate in Africa. Analysis of the peptide sequences of EV-A71 strains belonging to different genogroups revealed a high level of conservation of the capsid residues involved in known linear and conformational neutralization antigenic sites. Using a published crystal structure of the EV-A71 capsid as a model, we found that most of the residues that are seemingly specific to some genogroups were mapped outside known antigenic sites or external loops. These observations suggest a cross-neutralization activity of anti-genogroup B or C antibodies against strains of genogroups E and F. Neutralization assays were performed with diverse rabbit and mouse anti-EV-A71 sera, anti-EV-A71 human standards and a monoclonal neutralizing antibody. All the batches of antibodies that were tested successfully neutralized all available isolates, indicating an overall broad cross-neutralization between the canonical genogroups B and C and genogroups E and F. A panel constituted of more than 80 individual human serum samples from Cambodia with neutralizing antibodies against EV-A71 subgenogroup C4 showed quite similar cross-neutralization activities between isolates of genogroups C4, E and F. Our results thus indicate that the genetic drift underlying the separation of EV-A71 strains into genogroups A, B, C, E and F does not correlate with the emergence of antigenically distinct variants.

Strengthened surveillance revealed a rapid disappearance of the poliovirus serotype 2 vaccine strain in Madagascar after its removal from the oral polio vaccine.

To assess circulation of the Sabin 2 poliovirus vaccine strain in Madagascar after its withdrawal from the oral polio vaccine in April 2016, a reinforced poliovirus surveillance was implemented in three regions of Madagascar from January 2016 to December 2017. Environmental samples and stool specimens from healthy children were screened using the Global Polio Laboratory Network algorithm to detect the presence of polioviruses. Detected polioviruses were molecularly typed and their genomes fully sequenced. Polioviruses were detected during all but 4 months of the study period. All isolates were related to the vaccine strains and no wild poliovirus was detected. The majority of isolates belong to the serotype 3. The last detection of Sabin 2 occurred in July 2016, 3 months after its withdrawal. No vaccine-derived poliovirus of any serotype was observed during the study. Only few poliovirus isolates contained sequences from non-polio origin. The genetic characterization of all the poliovirus isolates did not identify isolates that were highly divergent compared to the vaccine strains. This observation is in favor of a good vaccine coverage that efficiently prevented long-lasting transmission chains between unvaccinated persons. This study underlines that high commitment in the fight against polioviruses can succeed in stopping their circulation even in countries where poor sanitation remains a hurdle.

Enterovirus detection in different regions of Madagascar reveals a higher abundance of enteroviruses of species C in areas where several outbreaks of vaccine-derived polioviruses occurred.

BACKGROUND: Poliomyelitis outbreaks due to pathogenic vaccine-derived polioviruses (VDPVs) are threatening and complicating the global polio eradication initiative. Most of these VDPVs are genetic recombinants with non-polio enteroviruses (NPEVs) of species C. Little is known about factors favoring this genetic macroevolution process. Since 2001, Madagascar has experienced several outbreaks of poliomyelitis due to VDPVs, and most of VDPVs were isolated in the south of the island. The current study explored some of the viral factors that can promote and explain the emergence of recombinant VDPVs in Madagascar. METHODS: Between May to August 2011, we collected stools from healthy children living in two southern and two northern regions of Madagascar. Virus isolation was done in RD, HEp-2c, and L20B cell lines, and enteroviruses were detected using a wide-spectrum 5'-untranslated region RT-PCR assay. NPEVs were then sequenced for the VP1 gene used for viral genotyping. RESULTS: Overall, we collected 1309 stools, of which 351 NPEVs (26.8%) were identified. Sequencing revealed 33 types of viruses belonging to three different species: Enterovirus A (8.5%), Enterovirus B (EV-B, 40.2%), and Enterovirus C (EV-C, 51.3%). EV-C species included coxsackievirus A13, A17, and A20 previously described as putative recombination partners for poliovirus vaccine strains. Interestingly, the isolation rate was higher among stools originating from the South (30.3% vs. 23.6%, p-value = 0.009). EV-C were predominant in southern sites (65.7%) while EV-B predominated in northern sites (54.9%). The factors that explain the relative abundance of EV-C in the South are still unknown. CONCLUSIONS: Whatever its causes, the relative abundance of EV-C in the South of Madagascar may have promoted the infections of children by EV-C, including the PV vaccine strains, and have favored the recombination events between PVs and NPEVs in co-infected children, thus leading to the recurrent emergence of recombinant VDPVs in this region of Madagascar.

Leveraging serology to titrate immunisation programme functionality for diphtheria in Madagascar.

Diphtheria is a potentially devastating disease whose epidemiology remains poorly described in many settings, including Madagascar. Diphtheria vaccination is delivered in combination with pertussis and tetanus antigens and coverage of this vaccine is often used as a core measure of health system functioning. However, coverage is challenging to estimate due to the difficulty in translating numbers of doses delivered into numbers of children effectively immunised. Serology provides an alternative lens onto immunisation, but is complicated by challenges in discriminating between natural and vaccine-derived seropositivity. Here, we leverage known features of the serological profile of diphtheria to bound expectations for vaccine coverage for diphtheria, and further refine these using serology for pertussis. We measured diphtheria antibody titres in 185 children aged 6-11 months and 362 children aged 8-15 years and analysed them with pertussis antibody titres previously measured for each individual. Levels of diphtheria seronegativity varied among age groups (18.9% of children aged 6-11 months old and 11.3% of children aged 8-15 years old were seronegative) and also among the districts. We also find surprisingly elevated levels of individuals seropositive to diphtheria but not pertussis in the 6-11 month old age group suggesting that vaccination coverage or efficacy of the pertussis component of the DTP vaccine remains low or that natural infection of diphtheria may be playing a significant role in seropositivity in Madagascar.

High Permissiveness for Genetic Exchanges between Enteroviruses of Species A, including Enterovirus 71, Favors Evolution through Intertypic Recombination in Madagascar.

Human enteroviruses of species A (EV-A) are the leading cause of hand-foot-and-mouth disease (HFMD). EV-A71 is frequently implicated in HFMD outbreaks and can also cause severe neurological manifestations. We investigated the molecular epidemiological processes at work and the contribution of genetic recombination to the evolutionary history of EV-A in Madagascar, focusing on the recently described EV-A71 genogroup F in particular. Twenty-three EV-A isolates, collected mostly in 2011 from healthy children living in various districts of Madagascar, were characterized by whole-genome sequencing. Eight different types were identified, highlighting the local circulation and diversity of EV-A. Comparative genome analysis revealed evidence of frequent recent intra- and intertypic genetic exchanges between the noncapsid sequences of Madagascan EV-A isolates. The three EV-A71 isolates had different evolutionary histories in terms of recombination, with one isolate displaying a mosaic genome resulting from recent genetic exchanges with Madagascan coxsackieviruses A7 and possibly A5 and A10 or common ancestors. The engineering and characterization of recombinants generated from progenitors belonging to different EV-A types or EV-A71 genogroups with distantly related nonstructural sequences indicated a high level of permissiveness for intertypic genetic exchange in EV-A. This permissiveness suggests that the primary viral functions associated with the nonstructural sequences have been highly conserved through the diversification and evolution of the EV-A species. No outbreak of disease due to EV-A has yet been reported in Madagascar, but the diversity, circulation, and evolution of these viruses justify surveillance of EV-A circulation and HFMD cases to prevent possible outbreaks due to emerging strains.IMPORTANCE Human enteroviruses of species A (EV-A), including EV-A71, are the leading cause of hand-foot-and-mouth disease (HFMD) and may also cause severe neurological manifestations. We investigated the circulation and molecular evolution of EV-A in Madagascar, focusing particularly on the recently described EV-A71 genogroup F. Eight different types, collected mostly in 2011, were identified, highlighting the local circulation and diversity of EV-A. Comparative genome analysis revealed evidence of frequent genetic exchanges between the different types of isolates. The three EV-A71 isolates had different evolutionary histories in terms of recombination. The engineering and characterization of recombinants involving progenitors belonging to different EV-A types indicated a high degree of permissiveness for genetic exchange in EV-A. No outbreak of disease due to EV-A has yet been reported in Madagascar, but the diversity, circulation, and evolution of these viruses justify the surveillance of EV-A circulation to prevent possible HFMD outbreaks due to emerging strains.

Seroprevalence of pertussis in Madagascar and implications for vaccination.

Pertussis is a highly contagious infectious disease and remains an important cause of mortality and morbidity worldwide. Over the last decade, vaccination has greatly reduced the burden of pertussis. Yet, uncertainty in individual vaccination coverage and ineffective case surveillance systems make it difficult to estimate burden and the related quantity of population-level susceptibility, which determines population risk. These issues are more pronounced in low-income settings where coverage is often overestimated, and case numbers are under-reported. Serological data provide a direct characterisation of the landscape of susceptibility to infection; and can be combined with vaccination coverage and basic theory to estimate rates of exposure to natural infection. Here, we analysed cross-sectional data on seropositivity against pertussis to identify spatial and age patterns of susceptibility in children in Madagascar. A large proportion of individuals surveyed were seronegative; however, there were patterns suggestive of natural infection in all the regions analysed. Improvements in vaccination coverage are needed to help prevent additional burden of pertussis in the country.

Revealing Measles Outbreak Risk With a Nested Immunoglobulin G Serosurvey in Madagascar.

Madagascar reports few measles cases annually and high vaccination campaign coverage. However, the underlying age profile of immunity and risk of a measles outbreak is unknown. We conducted a nested serological survey, testing 1,005 serum samples (collected between November 2013 and December 2015 via Madagascar's febrile rash surveillance system) for measles immunoglobulin G antibody titers. We directly estimated the age profile of immunity and compared these estimates with indirect estimates based on a birth cohort model of vaccination coverage and natural infection. Combining these estimates of the age profile of immunity in the population with an age-structured model of transmission, we further predicted the risk of a measles outbreak and the impact of mitigation strategies designed around supplementary immunization activities. The direct and indirect estimates of age-specific seroprevalence show that current measles susceptibility is over 10%, and modeling suggests that Madagascar may be at risk of a major measles epidemic.

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.

[Eradication of poliomyelitis and emergence of pathogenic vaccine-derived polioviruses: from Madagascar to Cameroon]. / Éradication de la poliomyélite et émergence de poliovirus pathogènes dérivés du vaccin - De Madagascar au Cameroun.

The oral poliovaccine, a live vaccine made of attenuated poliovirus strains, is the main tool of the vaccination campaigns organised for eradicating poliomyelitis. these campaigns had led to the decline and, thereafter, to the disappearance of wild poliovirus strains of the three serotypes (1-3) in most parts of the world. However, when the poliovaccine coverage becomes too low, vaccine polioviruses can circulate in insufficiently immunized populations and become then pathogenic by mutations and genetic recombination with other enteroviruses of the same species, in particular some coxsackievirus A. These mutated and recombinant vaccine strains have been implicated in several epidemics of paralytic poliomyelitis. Two polio outbreaks associated with these pathogenic circulating vaccine-derived poliovirus (cVDPV) occurred in 2001-2002 and 2005 in the South of Madagascar where vaccine coverage was low. These cVDPV, of serotype 2 or 3, were isolated from paralyzed children and some of their healthy contacts. Other cVDPV were isolated in the same region from healthy children in 2011, indicating that these viruses were circulating again. Vaccination campaigns could stop the outbreaks in 2002 and 2005, and most probably prevent another one in 2011. Therefore, the genetic plasticity of poliovaccine strains that threatens the benefit of vaccination campaigns is the target of an accurate surveillance and an important theme of studies in the virology laboratories of the Institut Pasteur international network.

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.

Cross-sectional cycle threshold values reflect epidemic dynamics of COVID-19 in Madagascar.

As the national reference laboratory for febrile illness in Madagascar, we processed samples from the first epidemic wave of COVID-19, between March and September 2020. We fit generalized additive models to cycle threshold (Ct) value data from our RT-qPCR platform, demonstrating a peak in high viral load, low-Ct value infections temporally coincident with peak epidemic growth rates estimated in real time from publicly-reported incidence data and retrospectively from our own laboratory testing data across three administrative regions. We additionally demonstrate a statistically significant effect of duration of time since infection onset on Ct value, suggesting that Ct value can be used as a biomarker of the stage at which an individual is sampled in the course of an infection trajectory. As an extension, the population-level Ct distribution at a given timepoint can be used to estimate population-level epidemiological dynamics. We illustrate this concept by adopting a recently-developed, nested modeling approach, embedding a within-host viral kinetics model within a population-level Susceptible-Exposed-Infectious-Recovered (SEIR) framework, to mechanistically estimate epidemic growth rates from cross-sectional Ct distributions across three regions in Madagascar. We find that Ct-derived epidemic growth estimates slightly precede those derived from incidence data across the first epidemic wave, suggesting delays in surveillance and case reporting. Our findings indicate that public reporting of Ct values could offer an important resource for epidemiological inference in low surveillance settings, enabling forecasts of impending incidence peaks in regions with limited case reporting.

Cross-sectional cycle threshold values reflect epidemic dynamics of COVID-19 in Madagascar.

As the national reference laboratory for febrile illness in Madagascar, we processed samples from the first epidemic wave of COVID-19, between March and September 2020. We fit generalized additive models to cycle threshold (C t ) value data from our RT-qPCR platform, demonstrating a peak in high viral load, low-C t value infections temporally coincident with peak epidemic growth rates estimated in real time from publicly-reported incidence data and retrospectively from our own laboratory testing data across three administrative regions. We additionally demonstrate a statistically significant effect of duration of time since infection onset on C t value, suggesting that C t value can be used as a biomarker of the stage at which an individual is sampled in the course of an infection trajectory. As an extension, the population-level C t distribution at a given timepoint can be used to estimate population-level epidemiological dynamics. We illustrate this concept by adopting a recently-developed, nested modeling approach, embedding a within-host viral kinetics model within a population-level Susceptible-Exposed-Infectious-Recovered (SEIR) framework, to mechanistically estimate epidemic growth rates from cross-sectional C t distributions across three regions in Madagascar. We find that C t -derived epidemic growth estimates slightly precede those derived from incidence data across the first epidemic wave, suggesting delays in surveillance and case reporting. Our findings indicate that public reporting of C t values could offer an important resource for epidemiological inference in low surveillance settings, enabling forecasts of impending incidence peaks in regions with limited case reporting.

Validation of a redesigned pan-poliovirus assay and real-time PCR platforms for the global poliovirus laboratory network.

Surveillance and detection of polioviruses (PV) remain crucial to monitoring eradication progress. Intratypic differentiation (ITD) using the real-time RT-PCR kit is key to the surveillance workflow, where viruses are screened after cell culture isolation before a subset are verified by sequencing. The ITD kit is a series of real-time RT-PCR assays that screens cytopathic effect (CPE)-positive cell cultures using the standard WHO method for virus isolation. Because ITD screening is a critical procedure in the poliovirus identification workflow, validation of performance of real-time PCR platforms is a core requirement for the detection of poliovirus using the ITD kit. In addition, the continual update and improvement of the ITD assays to simplify interpretation in all platforms is necessary to ensure that all real-time machines are capable of detecting positive real-time signals. Four platforms (ABI7500 real-time systems, Bio-Rad CFX96, Stratagene MX3000P, and the Qiagen Rotor-Gene Q) were validated with the ITD kit and a redesigned poliovirus probe. The poliovirus probe in the real-time RT-PCR pan-poliovirus (PanPV) assay was re-designed with a double-quencher (Zen™) to reduce background fluorescence and potential false negatives. The updated PanPV probe was evaluated with a panel consisting of 184 polioviruses and non-polio enteroviruses. To further validate the updated PanPV probe, the new assay was pilot tested in five Global Polio Laboratory Network (GPLN) laboratories (Madagascar, India, Philippines, Pakistan, and Democratic Republic of Congo). The updated PanPV probe performance was shown to reduce background fluorescence and decrease the number of false positives compared to the standard PanPV probe.

Efforts Towards Polio Eradication in Madagascar: 1997 to 2017.

BACKGROUND: In 1988, the World Health Assembly launched the Global Polio Eradication Initiative. WHO AFRO is close to achieve this goal with the last wild poliovirus detected in 2014 in Borno States in Nigeria. The certification of the WHO African Region requires that all the 47 member states meet the critical indicators for a polio free status. Madagascar started implementing polio eradication activities in 1996 and was declared polio free in June 2018 in Abuja. This study describes the progress achieved towards polio eradication activities in Madagascar from 1977-2017 and highlights the remaining challenges to be addressed. METHODS: Data were collected from the national routine immunization services, Country Acute Flaccid surveillance databases and national reports of SIAS and Mop Up campaign. Country complete polio and immunization related documentation provided detailed historical information's. RESULTS: From 1997 to 2017, Madagascar reported one wild poliovirus (WPV) outbreak and four circulating Vaccine Derived Polio Virus (cVDPV) oubreaks with a total of 21 polioviruses (1 WPV and 21 cVDPV). The last WPV and cVDPV were notified in 1997 in Antananarivo and 2015 in Sakaraha health districts respectively. Madagascar met the main polio surveillance indicators over the last ten years and made significant progress following the last cVDPV2 outbreak in 2014 -2015. In addition, the country successfully implemented the switch from trivalent Oral Polio Vaccine (tOPV) to bivalent Oral Polio vaccine (bOPV) and containment activities. Environmental Surveillance established since 2015 did not reveal any poliovirus. The administrative coverage of the 3rd dose of oral polio vaccine (OPV3) varied across the years from 55% in 1991 to a maximum of 95% in 2007 before a progressive decrease to 86% in 2017. The percentage of AFP cases with more than 3 doses of oral polio vaccines increased from 56% in 2014 to 88% in 2017. A total of 19 supplementary immunization activities (SIA) were conducted in Madagascar from 1997 to 2017, among which 3 were subnational immunization days (sNID) and 16 were national immunization days (NIDs). Poor routine coverage contributed to the occurrence of cVDPC outbreaks in the country; addressing this should remain a key priority for the country to maintain the polio free status.From 2015 to June 2017, Madagascar achieved the required criteria leading to the acceptance of the country's polio-free documentation in June 2018 by ARCC. However, continuous efforts will be needed to maintain a highly sensitive polio surveillance system with emphasis on security compromised areas. Finally strengthening the health system and governance at all levels will be necessary if these achievements are to be sustained. CONCLUSIONS: High national political commitment and support of the Global Polio Eradication Partnership were critical for Madagascar to achieve polio free status. Socio-political instability, weakness of the health system, sub-optimal routine immunization performance, insufficient SIA quality and existing security compromised areas remain critical program challenges to address in order to maintaining the polio free status. Continuous high-level advocacy should be kept in order to ensure that new government authorities maintain polio eradication among the top priorities of the country.

The COVID-19 epidemic in Madagascar: clinical description and laboratory results of the first wave, march-september 2020.

BACKGROUND: Following the first detection of SARS-CoV-2 in passengers arriving from Europe on 19 March 2020, Madagascar took several mitigation measures to limit the spread of the virus in the country. METHODS: Nasopharyngeal and/or oropharyngeal swabs were collected from travellers to Madagascar, suspected SARS-CoV-2 cases and contact of confirmed cases. Swabs were tested at the national reference laboratory using real-time RT-PCR. Data collected from patients were entered in an electronic database for subsequent statistical analysis. All distribution of laboratory-confirmed cases were mapped, and six genomes of viruses were fully sequenced. RESULTS: Overall, 26,415 individuals were tested for SARS-CoV-2 between 18 March and 18 September 2020, of whom 21.0% (5,553/26,145) returned positive. Among laboratory-confirmed SARS-CoV-2-positive patients, the median age was 39 years (IQR: 28-52), and 56.6% (3,311/5,553) were asymptomatic at the time of sampling. The probability of testing positive increased with age with the highest adjusted odds ratio of 2.2 [95% CI: 1.9-2.5] for individuals aged 49 years and more. Viral strains sequenced belong to clades 19A, 20A and 20B indicative of several independent introduction of viruses. CONCLUSIONS: Our study describes the first wave of the COVID-19 in Madagascar. Despite early strategies in place Madagascar could not avoid the introduction and spread of the virus. More studies are needed to estimate the true burden of disease and make public health recommendations for a better preparation to another wave.

Measles outbreak in 2018-2019, Madagascar: epidemiology and public health implications.

INTRODUCTION: In October 4th, 2018, a measles outbreak was declared in Madagascar. This study describes the epidemiology of the outbreak and determines public health implications for measles elimination in Madagascar. METHODS: Data have been collected using line list developed for the outbreak. Serum samples were collected within 30 days of rash onset for laboratory testing; confirmation was made by detection of measles immunoglobulin M (IgM) antibody. RESULTS: A total of 2,930 samples were analysed in the laboratory among which 1,086 (37%) were laboratory confirmed. Measles cases age ranged from a minimum of 1 month to a maximum of 88 years. The median and the mean were 7 years and 9 years respectively. Children between 1 to 9 years accounted for 50.6% of measles cases. Attack rate (39,014 per 1,000,000 inhabitants) and case fatality rate (1.2%) were highest among children aged 9-11 months. A total of 67.2% cases were unvaccinated. As of March 14th, 2019, all the 22 regions and 105 (92%) health districts out of 114 were affected by the measles outbreak in Madagascar. CONCLUSION: Measles outbreak in Madagascar showed that the country is not on the track to achieve the goal of measles elimination by 2020.

Development of a New Internally Controlled One-Step Real-Time RT-PCR for the Molecular Detection of Enterovirus A71 in Africa and Madagascar.

Enterovirus A71 (EV-A71) is a leading cause of hand-foot-and-mouth disease (HFMD) and can be associated with severe neurological complications. EV-A71 strains can be classified into seven genogroups, A-H, on the basis of the VP1 capsid protein gene sequence. Genogroup A includes the prototype strain; genogroups B and C are responsible of major outbreaks worldwide, but little is known about the others, particularly genogroups E and F, which have been recently identified in Africa and Madagascar, respectively. The circulation of EV-A71 in the African region is poorly known and probably underestimated. A rapid and specific assay for detecting all genogroups of EV-A71 is required. In this study, we developed a real-time RT-PCR assay with a competitive internal control (IC). The primers and TaqMan probe specifically target the genomic region encoding the VP1 capsid protein. Diverse EV-A71 RNAs were successfully amplified from the genogroups A, B, C, D, E, and F, with similar sensitivity and robust reproducibility. Neither cross reaction with other EVs nor major interference with the competitive IC was detected. Experimentally spiked stool and plasma specimens provided consistent and reproducible results, and validated the usefulness of the IC for demonstrating the presence of PCR inhibitors in samples. The analysis in an African laboratories network of 1889 untyped enterovirus isolates detected 15 EV-A71 of different genogroups. This specific real-time RT-PCR assay provides a robust and sensitive method for the detection of EV-A71 in biological specimens and for the epidemiological monitoring of EV-A71 including its recently discovered genogroups.

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

BACKGROUND: The 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 available METHODS: A 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. RESULTS: The 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. CONCLUSION: HCV 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 Enteroviruses Species A to D by High-Throughput Sequencing: Application for Viral Mixtures.

Human enteroviruses (EV) consist of more than 100 serotypes classified within four species for enteroviruses (EV-A to -D) and three species for rhinoviruses, which have been implicated in a variety of human illnesses. Being able to simultaneously amplify the whole genome and identify enteroviruses in samples is important for studying the viral diversity in different geographical regions and populations. It also provides knowledge about the evolution of these viruses. Therefore, we developed a rapid, sensitive method to detect and genetically classify all human enteroviruses in mixtures. Strains of EV-A (15), EV-B (40), EV-C (20), and EV-D (2) viruses were used in addition to 20 supernatants from RD cells infected with stool extracts or sewage concentrates. Two overlapping fragments were produced using a newly designed degenerated primer targeting the conserved CRE region for enteroviruses A-D and one degenerated primer set designed to specifically target the conserved region for each enterovirus species (EV-A to -D). This method was capable of sequencing the full genome for all viruses except two, for which nearly 90% of the genome was sequenced. This method also demonstrated the ability to discriminate, in both spiked and unspiked mixtures, the different enterovirus types present.