Ionela Gouandjika-Vasilache

Molecular Characterization of Human Enteroviruses in the Central African Republic: Uncovering Wide Diversity and Identification of a New Human Enterovirus A71 Genogroup

ABSTRACT Human enteroviruses (HEV) are among the most common viruses infecting humans. Their circulation has been widely studied in most parts of the world but not in sub-Saharan Africa, where poliomyelitis remains prevalent. We report here the molecular characterization of 98 nonpoliovirus (non-PV) HEV strains isolated from 93 randomly selected cell culture-positive supernatants from stool samples collected from 1997 through 2006 from children with acute flaccid paralysis living in the Central African Republic (CAR). The isolates were typed by sequencing the VP1 coding region and sequenced further in the VP2 coding region, and phylogenetic studies were carried out. Among the 98 VP1 sequences, 3, 74, 18, and 3 were found to belong to the HEV-A, -B, -C, and -D species, respectively. Overall, 42 types were detected. In most cases, the VP2 type was correlated with that of the VP1 region. Some of the isolates belonged to lineages that also contain viruses isolated in distant countries, while others belonged to lineages containing viruses isolated only in Africa. In particular, one isolate (type EV-A71) did not fall into any of the genogroups already described, indicating the existence of a previously unknown genogroup for this type. These results illustrate the considerable diversity of HEV isolates from the stools of paralyzed children in the CAR. The presence of diverse HEV-C types makes recombination between poliovirus and other HEV-C species possible and could promote the emergence of recombinant vaccine-derived polioviruses similar to those that have been implicated in repeated poliomyelitis outbreaks in several developing countries.

Characterization of Hepatitis Delta Virus in Sub-Saharan Africa

ABSTRACT Hepatitis D virus (HDV) is a satellite of hepatitis B virus (HBV), and infection with this virus aggravates acute and chronic liver disease. While HBV seroprevalence is very high across sub-Saharan Africa, much less is known about HDV in the region. In this study, almost 2,300 blood serum samples from Burkina Faso (n = 1,131), Nigeria (n = 974), Chad (n = 50), and the Central African Republic (n = 118) were screened for HBV and HDV. Among 743 HBsAg-positive serum samples, 74 were positive for HDV antibodies and/or HDV RNA, with considerable differences in prevalence, ranging from <2% (pregnant women from Burkina Faso) to 50% (liver patients from Central African Republic). HDV seems to be much more common in chronic liver disease patients in the Central African Republic (CAR) than in similar cohorts in Nigeria. In a large nested mother-child cohort in Burkina Faso, the prevalence of HDV antibodies was 10 times higher in the children than in their mothers, despite similar HBsAg prevalences, excluding vertical transmission as an important route of infection. The genotyping of 16 full-length and 8 partial HDV strains revealed clade 1 (17/24) in three of the four countries, while clades 5 (5/24) and 6 (2/24) were, at least in this study, confined to Central Nigeria. On the amino acid level, almost all our clade 1 strains exhibited a serine at position 202 in the hepatitis D antigen, supporting the hypothesis of an ancient African HDV-1 subgroup. Further studies are required to understand the public health significance of the highly varied HDV prevalences in different cohorts and countries in sub-Saharan Africa.

Characterization of the genome of human enteroviruses: Design of generic primers for amplification and sequencing of different regions of the viral genome

Human 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. Biodiversity and evolution of human enterovirus genomes are shaped by frequent recombination events. Therefore, identification and characterization of circulating strains of enteroviruses require partial determination of different genomic regions. The development is described of a simple method allowing amplification and partial sequencing of the P1, P2 and P3 genomic regions of field human enterovirus strains isolated in cell cultures, by performing PCR on cDNAs generated through a single RT reaction. A set of generic primers were designed and tested on a panel of 90 field and prototype viruses belonging to the five species of human enteroviruses. This assay was shown to amplify efficiently the targeted regions of all the 90 genomes tested. The generated amplicons were sequenced successfully without the need for gel purification. This assay could be a valuable tool for laboratories interested in molecular epidemiology and evolution studies implicating a great number of human enterovirus strains isolated from human or environmental samples.

No evidence of prolonged enterovirus excretion in HIV-seropositive patients

Mutations frequently occur in oral poliovirus vaccine (OPV) strains upon replication in the human intestine. These strains occasionally revert to being neurovirulent. The more prolonged the excretion of OPV, the higher the risk of reversion. OPV strains can be secreted for several months in humans presenting humoral immune system deficiencies. The duration of excretion of OPV strains or other enteroviruses in individuals infected with the human immunodeficiency virus (HIV) is unknown. We investigated whether HIV infection, which is very prevalent in the Central African Republic, causes prolonged excretion of enteroviruses and, in particular, of OPV strains in adults. We studied 28 HIV‐infected adults living with children who were immunized with OPV during national immunization days (NIDs). Blood samples were collected to confirm HIV status and to evaluate immunodeficiency before the NIDs. Stool samples for enterovirus isolation were also collected before the NIDs, between the two rounds of immunization and 2, 4 and 6 months after the second round of immunization. No poliovirus was isolated from any stool sample. Eight enteroviruses were isolated from eight adults (maximum one strain per patient). Enteroviruses were not more frequently isolated from severely immunodeficient patients. Thus, HIV‐infected adults do not appear to be at high risk of infection with OPV strains and the excretion of enteroviruses (and thus of polioviruses) does not seem to be prolonged in HIV‐infected adults.

Seroprevalence of measles and natural rubella antibodies among children in Bangui, Central African Republic

BackgroundPassively acquired maternal antibodies are necessary to protect infants against circulating measles virus until they reach the eligible age of vaccination. Likewise, high levels of population immunity must be achieved and maintained to reduce measles virus transmission. This study was undertaken to (1) assess the presence of maternally acquired measles-specific IgG antibodies among infants less than 9 months of age in Bangui, Central African Republic and (2) determine the immune status of vaccination-age children and the concordance with reported vaccination status. A secondary objective was to describe the presence of rubella-specific IgG antibody in the study population.MethodsVaccination history and blood samples were collected from 395 children using blotting paper. Samples were analyzed for the presence of measles-specific IgG antibodies using commercial ELISA kits.ResultsMeasles-specific IgG antibodies were detected in 51.3% of vaccinated children and 27.6% of non-vaccinated children. Maternally derived measles IgG antibodies were present in only 14.8% of infants aged 0-3 months and were absent in all infants aged 4-8 months. The presence of IgG-specific measles antibodies varied among children of vaccination age, from 57.3% for children aged 9 months to 5 years, to 50.6% for children aged 6-9 years and 45.6% for chidren aged 10 years and above. The overall prevalence of rubella-specific IgG was 55.4%, with a high prevalence (87.4%) among children over 10 years of age.ConclusionThe findings suggest that despite efforts to accelerate measles control by giving a second dose of measles vaccine, a large number of children remain susceptible to measles virus. Further research is required to determine the geographic extent of immunity gaps and the factors that influence immunity to measles virus in the Central African Republic.

Molecular surveillance of rotavirus infection in Bangui, Central African Republic, October 2011–September 2013

BACKGROUND The World Health Organization (WHO) recommends the introduction of rotavirus vaccine in the immunization program of all countries. In the Central African Republic (CAR), sentinel surveillance for rotavirus gastroenteritis was established in 2011 by the Ministry of Health, with the support of the Surveillance en Afrique Centrale Project (SURVAC). The purpose of this study was to assess the burden of rotavirus gastroenteritis and to identify rotavirus strains circulating in CAR before the introduction of rotavirus vaccine planned for this year, 2014. METHODS One sentinel site and one laboratory at the national level were designated by the CAR Ministry of Health to participate in this surveillance system. Stool samples were collected from children who met the WHO rotavirus gastroenteritis case definition (WHO, 2006). The samples were first screened for group A rotavirus antigen by enzyme immunoassay (EIA), and genotyping assays performed using a multiplex reverse transcriptase PCR (RT-PCR) technique. RESULTS Between October 2011 and September 2013, 438 stool samples were collected and analyzed for detection of rotavirus antigen; 206 (47%) were positive. Among the 160 (78%) that could be genotyped, G2P[6] was the predominant strain (47%) followed by G1P[8] (25%) and G2P[4] (13%). CONCLUSIONS Almost half of stool samples obtained from children hospitalized with gastroenteritis were positive for rotavirus. These baseline rotavirus surveillance data will be useful to health authorities considering rotavirus vaccine introduction and for evaluating the efficacy of rotavirus vaccine once it is introduced into the routine immunization system.

Measles outbreak in Northern Central African Republic 3 years after the last national immunization campaign.

BackgroundDespite huge efforts to promote widespread vaccination, measles remains an important cause of morbidity and mortality worldwide, especially in African children. In March 2011, an abnormally high number of cases were reported from the Ouham Prefecture, Central African Republic to the national measles case-based surveillance system. In response, reactive vaccination activities were implemented. The aims of this study were to investigate this outbreak and describe the response.MethodsMeasles cases were defined according to WHO recommendations. In the first weeks of the outbreak, blood samples were collected and sent to the Institut Pasteur in Bangui for laboratory confirmation by detection of IgM antibodies against measles virus. In addition, a portion of viral RNA was amplified from 5 IgM positive patient samples and the amplicons were sequenced for phylogenetic analysis.ResultsBetween March and September 2011, 723 clinical cases originated from the Ouham Prefecture, including 2 deaths, were reported. Amongst 59 blood samples collected, 49 were positive for the detection of IgM. A high number of self-declared vaccinated subjects (31%) were found amongst the cases. Most of the cases were under 5 years. The causative virus was found to belong to genotype B3.1. In response, 2 sub-national supplementary immunization activities were quickly conducted and limited this outbreak to mainly 2 sub-prefectures.ConclusionsThis outbreak was the largest epidemic of measles in CAR since 2002. Its occurrence, 3 years after the last national immunization campaign, highlights the necessity to pursue efforts and improve and extend immunization programs in order to reach measles elimination goal in Africa.

OPV strains circulation in HIV infected infants after National Immunisation Days in Bangui, Central African Republic

BackgroundHumans are the only host of polioviruses, thus the prospects of global polio eradication look reasonable. However, individuals with immunodeficiencies were shown to excrete vaccine derived poliovirus for long periods of time which led to reluctance to prolong the vaccination campaign for fear of this end result. Therefore, we aimed to assess the duration of excretion of poliovirus after the 2001 National Immunization Days according to Human immunodeficiency virus status.FindingsFifty three children were enrolled. Sequential stool samples were collected in between National Immunisation Days rounds and then every month during one year. Children were classified into 2 groups: no immunodepression (n = 38), immunodepression (n = 15) according to CD4+ lymphocytes cells count. Thirteen poliovirus strains were isolated from 11 children: 5 Human immunodeficiency virus positive and 6 Human immunodeficiency virus negative. None of the children excreted poliovirus for more than 4 weeks. The restriction fragment length polymorphism analysis showed that all strains were of Sabin origin including a unique Polio Sabine Vaccine types 2 and 3 (S2/S3) recombinant.ConclusionsFrom these findings we assume that Human immunodeficiency virus positive children are not a high risk population for long term poliovirus excretion. More powerful studies are needed to confirm our findings.

Factors associated with stunting in healthy children aged 5 years and less living in Bangui (RCA)

Stunting remains a major public health concern worldwide. Although its global prevalence is slowly decreasing, the actual number of affected children is still rising in Sub-Saharan Africa. In the Central African Republic (CAR), about one third of all children below the age of five are stunted. Stunting is correlated with many long-term consequences, including poor cognitive development and a higher rate of morbidity and mortality, making stunting a major contributor to poverty. In CAR, little is known about the factors that contribute to stunting. This study aimed at analysing, in a cross-sectional study, the main factors associated with stunting in a group of 414 children recruited between December 2011 and November 2013, aged five years or less and living in Bangui. For all children, demographic, socio-economic and anthropometric data were recorded and asymptomatic enteropathogen carriage was assessed in stool samples using classical microbiological assays. The study group had a mean age of 14.2±10 months. Fifty-eight percent (292/414) were boys, and 36 percent (148/414) exhibited stunted growth. Of the stunted children, 51% (75/148) showed a moderate delay in linear growth for their age group [height-for-age z-score (HAZ) between -2 and -3 SD] while 49% (73/148) presented a severe delay (HAZ < -3). Factors significantly associated with stunting included gender (aOR: 1.67; 95% CI: 1.07; 2.62 for boys compared to girls) and age (aOR of 3.98 (95% CI: 2.45; 6.46) for toddlers and aOR 4.42 (95% CI: 2.36; 8.28) for children compared to infants). Most importantly, we identified being overweight [weight-for-height z-score (WHZ) > 2 SD; aOR: 3.21; 95% CI: 1.50; 6.90 of overweight compared to normal weight] as also being significantly associated with stunting. This is the first study showing that even in the poorest countries of the world there is an association of stunting with being overweight.

Wild Poliovirus Importation, Central African Republic

To the Editor: Since the Global Polio Eradication Initiative was launched in 1988, indigenous transmission of wild poliovirus (WPV) has been interrupted in all countries except Afghanistan, Pakistan, and Nigeria (1). However, during 2003–2011, outbreaks resulting from importation of WPV occurred in 29 previously polio-free countries in Africa, including Central African Republic (CAR) (1–3). In 2011, 350 WPV cases were reported from 12 countries in Africa, a 47% decrease from the 657 cases reported by 12 countries in Africa in 2010 (1). In CAR, the last case of poliomyelitis caused by indigenous transmission of wild poliovirus was reported in 2000, but importation of WPV type 1 has been reported (4). We describe the importation of WPV1 and WPV3 into CAR during successive events in 2008, 2009, and 2011. To investigate importation of WPV into CAR, we conducted a study using fecal samples collected from patients in CAR who had acute flaccid paralysis (AFP) during 2008–2011. The samples were analyzed for virus isolation, typing, and intratypic differentiation at the Regional Reference Laboratory for Polio, Institut Pasteur de Bangui, using World Health Organization (WHO) standard procedures (5). Isolated WPV strains were sent to the Centers for Disease Control and Prevention (Atlanta, Georgia, USA) or the National Institute for Communicable Diseases (Johannesburg, South Africa) for sequencing according to WHO guidelines (6–8). Cases were classified as laboratory confirmed or polio-compatible according to WHO recommendations; a polio-compatible case was defined as AFP for which stool samples were not adequate or a situation in which the patient was lost to follow up or had residual paralysis 60 days after testing. Of 141 AFP cases from 2008, three, from Bangui, Ouham, and Ouaka districts, were laboratory confirmed as WPV1; this cluster was designated B2D1B (Figure). Sequencing results showed that the virus in this cluster belonged to the South Asia A (Indian) genotype, which was circulating in Angola and Democratic Republic of Congo at that time (Figure). Figure Clusters of polio cases caused by wild poliovirus importations, Central African Republic, 2008–2011. Each circle represents 1 case of acute flaccid paralysis confirmed as polio. Black circles, cluster B2D1B, 2008 poliovirus (PV) type 1 SOAS importation ... Of 163 AFP cases from 2009, 14 in Ouham-Pende district were laboratory confirmed as WPV3; this cluster was designated D2B2B1. Sequencing results showed that the virus in this cluster belonged to West Africa B genotype, which was circulating in Nigeria and southern Chad at that time (Figure). Of 142 AFP cases from 2011, four in Ouham district were laboratory confirmed as WPV1; this cluster was designated I6C2B4C1A2. Sequencing results showed that the virus in this cluster belonged to West Africa B genotype, which was circulating in south Chad and Nigeria at the time (Figure). The importation of wild poliovirus strains into CAR appeared to follow 3 different routes. In 2008, WPV1 originated from Democratic Republic of Congo and was first detected in the capital, Bangui, which is located in the southern part of the country. Two more cases were detected in 2 other districts, in the north (Ouham) and in the middle (Ouaka) of the country. During that year, 2 AFP cases were classified as polio-compatible; these cases originated from Haute Kotto and Ouham districts. In 2008, routine coverage of oral polio vaccine (OPV) was 45%, 33%, and 57% for Bangui and Sanitary Regions 3 and 4, respectively. (Sanitary regions are equivalent to provinces and have several districts under their jurisdiction; Bangui is considered a Sanitary Region containing 8 districts.) To interrupt wild poliovirus circulation, health authorities implemented 4 rounds of national immunization days, 2 using monovalent OPV (mOPV) type 1 and 2 using trivalent OPV; 1 local immunization day using mOPV1 was also instituted. In 2009, WPV3 was imported from southern Chad to the Ouham-Pende district in CAR. This insecure district is difficult to access, but routine OPV coverage was reported as 61% for 2009, compared with the country’s official OPV coverage of 55%. The apparently higher coverage in areas of insecurity is likely the result of inaccurate target population estimates. Five additional AFP cases were classified as polio-compatible; these occurred in Ouham (1), Ouham Pende (2), Mambere Kadei (1), and Mbomou (1) districts. To interrupt wild poliovirus circulation, 8 supplementary immunization activities were organized. The 2011 polio outbreak occurred in the district of Ouham and was caused by WPV1 poliovirus imported from southern Chad (Figure). Fourteen additional cases of AFP were classified as polio-compatible; these occurred in Ouham-Pende (6), Ouham (1), Ombela M’Poko (1), Kemo (1), Ouaka (1), Haute Kotto (2), and Mbomou (2) districts. Routine OPV coverage for this insecure and difficult-to-access region was 55% for 2010 and 58% for 2011. CAR is one of the countries with the highest predicted risk for WPV circulation after importation (9). The 3 WPV importation events we report demonstrate that current immunization levels in this country are insufficient to guard against polio. High, sustained levels of routine OPV coverage in every district, supplemented by high-quality supplementary immunization activities, will help prevent future outbreaks. Surveillance standards must also be maintained to ensure the rapid detection of WPV importation, thus enabling timely response and containment.