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.

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.

Geographical distribution and relative risk of Anjozorobe virus (Thailand orthohantavirus) infection in black rats (Rattus rattus) in Madagascar.

BACKGROUND: Hantavirus infection is a zoonotic disease that is associated with hemorrhagic fever with renal syndrome and cardiopulmonary syndrome in human. Anjozorobe virus, a representative virus of Thailand orthohantavirus (THAIV), was recently discovered from rodents in Anjozorobe-Angavo forest in Madagascar. To assess the circulation of hantavirus at the national level, we carried out a survey of small terrestrial mammals from representative regions of the island and identified environmental factors associated with hantavirus infection. As we were ultimately interested in the potential for human exposure, we focused our research in the peridomestic area. METHODS: Sampling was achieved in twenty districts of Madagascar, with a rural and urban zone in each district. Animals were trapped from a range of habitats and examined for hantavirus RNA by nested RT-PCR. We also investigated the relationship between hantavirus infection probability in rats and possible risk factors by using Generalized Linear Mixed Models. RESULTS: Overall, 1242 specimens from seven species were collected (Rattus rattus, Rattus norvegicus, Mus musculus, Suncus murinus, Setifer setosus, Tenrec ecaudatus, Hemicentetes semispinosus). Overall, 12.4% (111/897) of Rattus rattus and 1.6% (2/125) of Mus musculus were tested positive for THAIV. Rats captured within houses were less likely to be infected than rats captured in other habitats, whilst rats from sites characterized by high precipitation and relatively low seasonality were more likely to be infected than those from other areas. Older animals were more likely to be infected, with infection probability showing a strong increase with weight. CONCLUSIONS: We report widespread distribution of THAIV in the peridomestic rats of Madagascar, with highest prevalence for those living in humid areas. Although the potential risk of infection to human may also be widespread, our results provide a first indication of specific zone with high transmission. Gathered data will be helpful to implement policies for control and prevention of human risk infection.

Prevalence of chronic hepatitis B virus infection and infrastructure for its diagnosis in Madagascar: implication for the WHO's elimination strategy.

BACKGROUND: WHO developed a global strategy to eliminate hepatitis B by 2030 and set target to treat 80% of people with chronic hepatitis B virus (HBV) infection eligible for antiviral treatment. As a first step to achieve this goal, it is essential to conduct a situation analysis that is fundamental to designing national hepatitis plans. We therefore estimated the prevalence of chronic HBV infection, and described the existing infrastructure for HBV diagnosis in Madagascar. METHODS: We conducted a stratified multi-stage serosurvey of hepatitis B surface antigen (HBsAg) in adults aged ≥18 years using 28 sentinel surveillance sites located throughout the country. We obtained the list of facilities performing HBV testing from the Ministry of Health, and contacted the person responsible at each facility. RESULTS: A total of 1778 adults were recruited from the 28 study areas. The overall weighted seroprevalence of HBsAg was 6.9% (95% CI: 5.6-8.6). Populations with a low socio-economic status and those living in rural areas had a significantly higher seroprevalence of HBsAg. The ratio of facilities equipped to perform HBsAg tests per 100,000 inhabitants was 1.02 in the capital city of Antananarivo and 0.21 outside the capital. There were no facilities with the capacity to perform HBV DNA testing or transient elastography to measure liver fibrosis. There are only five hepatologists in Madagascar. CONCLUSION: Madagascar has a high-intermediate level of endemicity for HBV infection with a severely limited capacity for its diagnosis and treatment. Higher HBsAg prevalence in rural or underprivileged populations underlines the importance of a public health approach to decentralize the management of chronic HBV carriers in Madagascar by using simple and low-cost diagnostic tools.

Genetic diversity of hepatitis B virus (HBV) in Madagascar.

Hepatitis B virus (HBV) is a DNA virus belonging to Hepadnaviridae family. Chronic infection with HBV is one major risk factor of hepatic disease. In Madagascar, former studies classified the country as part of high endemic area, as HBV prevalence can reach 23% in general population. However, this prevalence differs largely between urban and rural areas and is estimated to be, respectively, 5% and 26%. The aims of the present study were to describe the genetic diversity of HBV strains from different regions of Madagascar, and to describe the viral gene flow throughout the country by using phylogenetic analysis. This is the first large-scale molecular and phylogenetic study analyzing HBV sequences from 28 different Malagasy areas, never sampled in the past. In this study, the most prevalent genotype/sub-genotypes was E. Migration analysis showed a gene flow from zone 3 (rural) to zone 2 (suburban), and a greater gene flow from the middle part of Madagascar to the north than to the south. It is important to study the HBV infections in Madagascar and to monitor the potential spread of this viral strain inside this country. J. Med. Virol. 88:2138-2144, 2016. © 2016 Wiley Periodicals, Inc.

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.

Integrated Analysis of Environment, Cattle and Human Serological Data: Risks and Mechanisms of Transmission of Rift Valley Fever in Madagascar.

BACKGROUND: Rift Valley fever (RVF) is a vector-borne disease affecting ruminants and humans. Madagascar was heavily affected by RVF in 2008-2009, with evidence of a large and heterogeneous spread of the disease. The identification of at-risk environments is essential to optimize the available resources by targeting RVF surveillance in Madagascar. Herein, the objectives of our study were: (i) to identify the environmental factors and areas favorable to RVF transmission to both cattle and human and (ii) to identify human behaviors favoring human infections in Malagasy contexts. METHODOLOGY/PRINCIPAL FINDINGS: First, we characterized the environments of Malagasy communes using a Multiple Factor Analysis (MFA). Then, we analyzed cattle and human serological data collected at national level using Generalized Linear Mixed Models, with the individual serological status (cattle or human) as the response, and MFA factors, as well as other potential risk factors (cattle density, human behavior) as explanatory variables. Cattle and human seroprevalence rates were positively associated to humid environments (p<0.001). Areas with high cattle density were at risk (p<0.01; OR = 2.6). Furthermore, our analysis showed that frequent contact with raw milk contributed to explain human infection (OR = 1.6). Finally, our study highlighted the eastern-coast, western and north-western parts as high-risk areas for RVF transmission in cattle. CONCLUSIONS/SIGNIFICANCE: Our integrated approach analyzing environmental, cattle and human datasets allow us to bring new insight on RVF transmission patterns in Madagascar. The association between cattle seroprevalence, humid environments and high cattle density suggests that concomitant vectorial and direct transmissions are critical to maintain RVF enzootic transmission. Additionally, in the at-risk humid environment of the western, north-western and the eastern-coast areas, suitable to Culex and Anopheles mosquitoes, vectorial transmission probably occurs in both cattle and human. The relative contribution of vectorial or direct transmissions could be further assessed by mathematic modelling.

Correction: Integrated Analysis of Environment, Cattle and Human Serological Data: Risks and Mechanisms of Transmission of Rift Valley Fever in Madagascar.

[This corrects the article DOI: 10.1371/journal.pntd.0004827.].

Dried-blood spots: a cost-effective field method for the detection of Chikungunya virus circulation in remote areas.

BACKGROUND: In 2005, there were outbreaks of febrile polyarthritis due to Chikungunya virus (CHIKV) in the Comoros Islands. CHIKV then spread to other islands in the Indian Ocean: La Réunion, Mauritius, Seychelles and Madagascar. These outbreaks revealed the lack of surveillance and preparedness of Madagascar and other countries. Thus, it was decided in 2007 to establish a syndrome-based surveillance network to monitor dengue-like illness. OBJECTIVE: This study aims to evaluate the use of capillary blood samples blotted on filter papers for molecular diagnosis of CHIKV infection. Venous blood samples can be difficult to obtain and the shipment of serum in appropriate temperature conditions is too costly for most developing countries. METHODOLOGY AND PRINCIPAL FINDINGS: Venous blood and dried-blood blotted on filter paper (DBFP) were collected during the last CHIKV outbreak in Madagascar (2010) and as part of our routine surveillance of dengue-like illness. All samples were tested by real-time RT-PCR and results with serum and DBFP samples were compared for each patient. The sensitivity and specificity of tests performed with DBFP, relative to those with venous samples (defined as 100%) were 93.1% (95% CI:[84.7-97.7]) and 94.4% (95% CI:[88.3-97.7]), respectively. The Kappa coefficient 0.87 (95% CI:[0.80-0.94]) was excellent. CONCLUSION: This study shows that DBFP specimens can be used as a cost-effective alternative sampling method for the surveillance and monitoring of CHIKV circulation and emergence in developing countries, and probably also for other arboviruses. The loss of sensitivity is insignificant and involved a very small number of patients, all with low viral loads. Whether viruses can be isolated from dried blood spots remains to be determined.

Reemergence of recombinant vaccine-derived poliovirus outbreak in Madagascar.

BACKGROUND: After the 2001-2002 poliomyelitis outbreak due to recombinant vaccine-derived polioviruses (VDPVs) in the Toliara province of Madagascar, another outbreak reoccurred in the same province in 2005. METHODS: We conducted epidemiological and virological investigations for each polio case patient and for their contacts. RESULTS: From May to August 2005, a total of 5 cases of acute flaccid paralysis were reported among unvaccinated or partially vaccinated children 2-3 years old. Type-3 or type-2 VDPV was isolated from case patients and from healthy contacts. These strains were classified into 4 recombinant lineages that showed complex mosaic genomic structures originating from different vaccine strain serotypes and probably from human enterovirus C (HEV-C) species. Genetic relatedness could be observed among these 4 lineages. Vaccination coverage of the population was very low (<50%). CONCLUSIONS: The broad distribution of VDPVs in the province and their close genetic relationship indicate intense and rapid cocirculation and coevolution of the vaccine strains and of their related HEV-C strains. The occurrence of an outbreak due to VDPV 3 years after a previous outbreak indicates that a short period with low vaccination coverage is enough to create favorable conditions for the emergence of VDPV in this setting.