[Overview of the Ebola vaccines in pre-clinical and clinical development]. / Aperçu des vaccins Ebola en phase clinique et préclinique de développement.

The Ebola epidemic that occurred in West Africa between 2013-2016 significantly accelerated the research and development of Ebola vaccines. Few dozens of clinical trials have been recently conducted leading to opportunities to test several new vaccine candidates. Other vaccines are still in early development phases (table 1). This paper provides an overview of the new developments in that area.

Chikungunya.

Chikungunya is an acute viral disease characterised by fever and painful arthralgia. The arthritic symptoms associated with chikungunya can be debilitating and may persist for months or even years in some patients. Severe neurological complications such as encephalitis have also been reported during recent large outbreaks. The disease is caused by chikungunya virus (CHIKV), a mosquito-borne alphavirus from the Togaviridae family, which has recently emerged to become one of the most important exotic viral threats worldwide. Chikungunya is endemic throughout Africa, and over the past decade, it has also spread throughout the Indian Ocean, Asia, the South Pacific, southern Europe, the Caribbean and Central America. The rapid emergence of CHIKV has been linked to expansion of the mosquito vector species, Aedes aegypti and Ae. albopictus, throughout most tropical and subtropical regions of the world. Furthermore, mutations in some strains of CHIKV have been associated with increased transmissibility of the virus. The lack of a commercial vaccine and the failure of vector control strategies to limit the expansion of chikungunya have prompted the need for further options to prevent the spread of this disease.

Epidemiological Analysis of Influenza A Infection in Cambodian Pigs and Recommendations for Surveillance Strategies.

This study analysed the available data of seroprevalence to human influenza viruses in pigs in Cambodia using generalized linear mixed models in order to improve understanding of factors underlying the spread of human influenza viruses in Cambodian pigs. The associations between seroprevalence against seasonal H1N1 influenza virus in pigs and the population density of humans and pigs were not significant. However, a positive association between anti-H3 antibodies in pigs and the human population density was identified. In contrast, there was a negative association between seroprevalence of H3N2 in pigs and the pig population density. Our study has highlighted the difficulty in identifying epidemiological risk factors when a limited data set is used for analyses. We therefore provide recommendations on data collection for future epidemiological analyses that could be improved by collecting metadata related to the animals sampled. In addition, serosurveillance for influenza A viruses in pigs in high-risk areas or at slaughterhouses is recommended in resource-limited countries.

Two clustered cases of confirmed influenza A(H5N1) virus infection, Cambodia, 2011.

In February 2011, a mother and her child from Banteay Meanchey Province, Cambodia, were diagnosed, postmortem, with avian influenza A(H5N1) virus infection. A field investigation was conducted by teams from the Cambodian Ministry of Health, the World Health Organization and the Institut Pasteur in Cambodia. Nasopharyngeal, throat and serum specimens collected from 11 household or three neighbour contacts including two suspect cases tested negative by reverse transcriptase-polymerase chain reaction (RT-PCR) for A(H5N1). Follow-up sera from the 11 household contacts also tested negative for A(H5N1) antibodies. Twenty-six HCW who were exposed to the cases without taking adequate personal protective measures self-monitored and none developed symptoms within the two following weeks. An unknown number of passengers travelling with the cases on a minibus while they were symptomatic could not be traced but no clusters of severe respiratory illnesses were detected through the Cambodian surveillance systems in the two weeks after that. The likely cause of the fatal infection in the mother and the child was common-source exposure in Preah Sdach District, Prey Veng Province. Human-to-human transmission of A(H5N1) virus was unlikely but genetic susceptibility is suspected. Clusters of A(H5N1) virus infection should be systematically investigated to rule out any human-to-human transmission.

A specific and sensitive antigen capture assay for NS1 protein quantitation in Japanese encephalitis virus infection.

Japanese encephalitis virus (JEV) is a human pathogenic, mosquito-borne flavivirus that is endemic/epidemic in Asia. JEV is rarely detected or isolated from blood or cerebrospinal fluid (CSF), and detection of IgM is generally diagnostic of the infection. The flavivirus nonstructural glycoprotein NS1 is released transiently during flavivirus replication. The aim of this study was to set up a quantitative JEV NS1 antigen capture assay. A soluble hexameric form of JEV NS1 protein was produced in a stable Drosophila S2 cell clone and purified from supernatant fluids. Two IgG1 monoclonal antibodies (MAbs) with high affinity against two different epitopes of JEV NS1 antigen were used to develop an antigen-capture assay with a limit of detection of 0.2ngml(-1) NS1. Up to 1µgml(-1) JEV NS1 protein was released in supernatants of mammalian cells infected with JEV but <10ngml(-1) was released in sera of virus-infected mice before the onset of encephalitis and death. Moreover, NS1 protein was detected at low levels (<10ngml(-1)) in 23.8% of sera and in 10.5% of CSF of patients diagnosed as IgM-positive for JEV. This quantitative test of NS1 protein is proposed for highly specific diagnosis of acute infection with JEV genotypes I to IV.

Under-recognition and reporting of dengue in Cambodia: a capture-recapture analysis of the National Dengue Surveillance System.

Robust disease burden estimates are important for decision-making concerning introduction of new vaccines. Dengue is a major public health problem in the tropics but robust disease burden estimates are lacking. We conducted a two-sample, capture-recapture study in the largest province in Cambodia to determine disease under-recognition to the National Dengue Surveillance System (NDSS). During 2006-2008, community-based active surveillance for acute febrile illness was conducted in 0- to 19-year-olds in rural and urban areas combined with testing for dengue virus infection. Of 14 354 individuals under active surveillance (22 498 person-seasons), the annual incidence ranged from 13·4 to 57·8/1000 person-seasons. During the same period, NDSS incidence rates ranged from 1·1/1000 to 5·7/1000, which was 3·9- to 29·0-fold lower than found in the capture-recapture study. In hospitalized cases, the rate of under-recognition was 1·1- to 2·4-fold. This study shows the substantial degree of under-recognition/reporting of dengue and that reported hospitalized cases are not a good surrogate for estimating dengue disease burden.

Neuraminidase inhibitor sensitivity and receptor-binding specificity of Cambodian clade 1 highly pathogenic H5N1 influenza virus.

The evolution of the highly pathogenic H5N1 influenza virus produces genetic variations that can lead to changes in antiviral susceptibility and in receptor-binding specificity. In countries where the highly pathogenic H5N1 virus is endemic or causes regular epidemics, the surveillance of these changes is important for assessing the pandemic risk. In Cambodia between 2004 and 2010, there have been 26 outbreaks of highly pathogenic H5N1 influenza virus in poultry and 10 reported human cases, 8 of which were fatal. We have observed naturally occurring mutations in hemagglutinin (HA) and neuraminidase (NA) of Cambodian H5N1 viruses that were predicted to alter sensitivity to neuraminidase inhibitors (NAIs) and/or receptor-binding specificity. We tested H5N1 viruses isolated from poultry and humans between 2004 and 2010 for sensitivity to the NAIs oseltamivir (Tamiflu) and zanamivir (Relenza). All viruses were sensitive to both inhibitors; however, we identified a virus with a mildly decreased sensitivity to zanamivir and have predicted that a V149A mutation is responsible. We also identified a virus with a hemagglutinin A134V mutation, present in a subpopulation amplified directly from a human sample. Using reverse genetics, we verified that this mutation is adaptative for human α2,6-linked sialidase receptors. The importance of an ongoing surveillance of H5N1 antigenic variance and genetic drift that may alter receptor binding and sensitivities of H5N1 viruses to NAIs cannot be underestimated while avian influenza remains a pandemic threat.

[Dengue and other arboviral diseases in South-East Asia]. / Dengue et autres arboviroses en Asie du Sud-Est.

The most medically significant arboviruses causing human illness in south-east Asia belong to the genera Flavivirus (dengue, Japanese encephalitis, Kunjin, Zika, etc.) and Alphavirus (Chikungunya, Sindbis, Getah, etc.). All of these arboviral diseases are transmitted by mosquitoes. Dengue virus is the most prevalent arbovirus in south-east Asia and constitutes a major public health problem. Japanese encephalitis virus is also widespread in the region and symptomatic infection is associated with high mortality and severe neurological morbidity. Chikungunya virus causes mild but extremely painful illness. The number of Chikungunya cases has been increasing since early 2009. Epidemiological data show a steady, sometimes exponential, increase in the number of arbovirus infections in Asia. The spread of these viral infections can be linked to a number of complex factors.

[Asia: avian influenza H5N1]. / La grippe H5N1.

The emergence of the first human cases of avian influenza in Hong Kong in 1997 has raised fears of a new pandemic originating from the Asian continent. Despite unprecedented international mobilization, first to stop then to limit its diffusion, the highly pathogenic avian virus A/H5N1 has successfully spread in Asia, Europe and Africa by successive epizootic outbreaks affecting migratory birds and poultry, Transmission from animals to humans is uncommon but severe with a fatality rate exceeding 60% in confirmed cases. Nearly half of the countries struck by the disease is in Asia. With 87% of confirmed cases and 91% of deaths, Asia is also one of the continent most heavily affected. Like the neighbouring countries in South-East Asia, Cambodia has been repeatedly hit by avian flu in the recent years. The measures implemented to prevent the spread of A/H5N1 virus in farms, to improve the behaviour of farmers and to clean up the poultry markets emphasize the multiple difficulties to control this zoonosis. Meanwhile, influenza surveillance is being carried out in humans, based on a reporting system and a network of sentinel hospitals connected to the Institut Pasteur du Cambodge. Thus, Cambodia takes an active part in the wide international network which should quickly detect any mutational event among avian flu viruses that might lead to the emergence of a pandemic.

Natural variation can significantly alter the sensitivity of influenza A (H5N1) viruses to oseltamivir.

Geographic spread of highly pathogenic avian H5N1 influenza viruses may give rise to an influenza pandemic. During the first months of a pandemic, control measures would rely mainly on antiviral drugs, such as the neuraminidase (NA) inhibitors oseltamivir and zanamivir. In this study, we compare the sensitivities to oseltamivir of the NAs of several highly pathogenic H5N1 viruses isolated in Asia from 1997 to 2005. The corresponding 50% inhibitory concentrations were determined using a standard in vitro NA inhibition assay. The K(m) for the substrate and the affinity for the inhibitor (K(i)) of NA were determined for a 1997 and a 2005 virus, using an NA inhibition assay on cells transiently expressing the viral enzyme. Our data show that the sensitivities of the NAs of H5N1 viruses isolated in 2004 and 2005 to oseltamivir are about 10-fold higher than those of earlier H5N1 viruses or currently circulating H1N1 viruses. Three-dimensional modeling of the N1 protein predicted that Glu248Gly and Tyr252His changes could account for increased sensitivity. Our data indicate that genetic variation in the absence of any drug-selective pressure may result in significant variations in sensitivity to anti-NA drugs. Although the clinical relevance of a 10-fold increase in the sensitivity of NA to oseltamivir needs to be investigated further, the possibility that sensitivity to anti-NA drugs could increase (or possibly decrease) significantly, even in the absence of treatment, underscores the need for continuous evaluation of the impact of genetic drift on this parameter, especially for influenza viruses with pandemic potential.

[Prevalence of hepatitis A, B, C and E virus markers among patients with elevated levels of Alanine aminotransferase and Aspartate aminotransferase in Phnom Penh (Cambodia) and Nha Trang (Central Vietnam)]. / Prévalence de marqueurs d'infection des hépatites virales A, B, C et E chez des patients ayant une hypertransaminasémie a Phnom Penh (Cambodge) et Nha Trang (Centre Vietnam).

In order to describe the respective part of viral hepatitis in liver diseases observed in Cambodia and Vietnam, ninety consecutive patients with Aspartate aminotransferase (AST) and Alanine aminotransferase (ALT) > or = 100 Ul/l were tested for hepatitis A, B, C and E markers in Phnom Penh and Nha Trang. The markers were IgM antibodies to hepatitis A virus (anti-HAV IgM), hepatitis B surface antigen (HBsAg), antibodies to hepatitis C virus (anti-HCVAb) and IgG antibodies to hepatitis E virus (anti-HEV IgG). Recruited patients were predominantly adults and male (sex ratio 76%). Among these patients, 81% were tested positive to at least one marker in Nha Trang and 79% in Phnom Penh. In Nha Trang, HBsAg was more frequent (73%) than anti-HCV Ab (9%) while in Phnom Penh both markers were closely similar (HBsAg: 41%, anti-HCV Ab: 39%). In both population samples, HBsAg was more prevalent among young people whereas anti-HCV Ab were only detected in adults. No case of acute HAV infection was diagnosed in Nha Trang while anti-HAV IgM were detected in 20% of Cambodian patients. Anti-HEV IgG were infrequent (2% in Nha Trang, 5.5% in Phnom Penh). Only one case was notified, a male Vietnamese patient probably suffering from acute hepatitis E. More studies would be useful to improve the control measures against viral hepatitis in the public health programs.

[Intestinal parasitoses in the Mahajanga region, west coast of Madagascar]. / Les parasitoses digestives dans la région de Mahajanga, côte Ouest de Madagascar.

A study on human intestinal parasites was carried out from November 1996 until January 1997, in Mahajanga's hospital, on the western coast of Madagascar. We collected the faeces from 401 patients and the sera from 112 of them. Faecal examination using direct examination and MIF method revealed that 67.6% of the stools contained at least one parasite. The frequency of the protozoa was high (47.7%). The prevalence of the nematodosis reached 23.4%. Hymenolepis nana, Taenia saginata or solium and Schistosoma mansoni were less frequent (respectively 2.5%, 0.75% and 3.7%). More than 50% of the sera contained antibodies anti-Ascaris lumbricoides and anti-Strongyloides stercoralis. Serology by IFI using Schistosoma antigen was positive in 15.2% of the cases. The serological and microscopical exams showed that Entamoeba histolytica was present in this region and that amoebiasis should be considered as one of the etiologies of diarrhoea. The study pointed out also the frequency of the transmitted fecal infections. Preventive measures as water distribution, sanitary installations, hygiene education should be implemented.

[Serological study of toxoplasmosis in Vietnam in a population of drug users (Ho Chi Minh city) and pregnant women (Nha Trang)]. / Etude sérologique de la toxoplasmose au Vietnam dans une population de toxicomanes (Ho Chi Minh ville) et de femmes enceintes (Nha Trang).

Toxoplasmosis is a neglected disease in Vietnam particularly in populations with a high risk of developing complications. The seroprevalence of Toxoplasma gondii was calculated by testing blood samples for Toxoplasma specifically immunoglobulin G and immunoglobulin M on 300 intravascular drug users and on 300 pregnant women. Among intravascular drug users, the seroprevalence of IgG and IgM was 7.7% and 0.08%, respectively. In pregnant women the prevalence of anti-toxoplasmosis IgG and IgM was respectively 11.2% and 0%. 0.28% of all estimated pregnancies in Vietnam are affected with toxoplasmosis, i.e. around 4800 pregnancies per year. In conclusion, a screening of Toxoplasma infections should be recommended in HIV/AIDS patients.

Intérêts et difficultés de la microscopie pour le diagnostic de présomption de la peste

Le diagnostic bactériologique de la peste est basé sur la microscopie et l'isolement de Yersinia pestis à partir de prélèvements suspects. Lorsque la culture et l'inoculation à la souris sont négatives, l'examen direct du frottis permet de poser un diagnostic de présomption de peste. Examen simple à réaliser a priori, la microscopie pose cependant de réelles difficultés de lecture et de reproductibilité entre deux laboratoires. Cette étude compare les résultats obtenus au Centre Hospitalier Universitaire (CHU) de Mahajanga et au Laboratoire Central de la Peste (LCP), lors des deux épidémies successives de peste en 1995 et 1996.En prenant la culture comme référence, la microscopie réalisée à Mahajanga est plus sensible mais moins spécifique que celle réalisée au LCP. Entre l'année 1995 (350 patients) et 1996 (245 patients), la concordance entre les 2 laboratoires s'est améliorée, passant de 55% à 75%. Malgré une sensibilité et une spécificité assez faibles par rapport à la culture (elle-même peu sensible aussi en raison des conditions de transport des prélèvements), la microscopie conserve toute sa valeur pour le diagnostic de présomption de la peste lorsqu'elle est faite par un personnel entraîné. La solution idéale à terme sera un test immunodiagnostique simple et rapide, réalisable par des agents de santé non biologistes

Field evaluation of an immunoglobulin G anti-F1 enzyme-linked immunosorbent assay for serodiagnosis of human plague in Madagascar.

Bacteriological isolation of Yersinia pestis is the reference test for confirming plague infection, but recovery of the pathogen from human samples is usually very poor. When the etiology of the disease cannot be bacteriologically confirmed, it may be useful to possess alternative tests such as detection of specific circulating antibodies to help guide the diagnosis. In the present study, the immunoglobulin G (IgG) anti-F1 enzyme-linked immunosorbent assay (ELISA) has been applied to various human sera to evaluate its large-scale applicability in the high-endemicity plague foci of Madagascar. The sensitivity of the test was found to be 91.4%, and its specificity was 98.5%. The positive and negative predictive values were 96 and 96.6%, respectively. Seroconversion was observed on day 7 after onset of the disease. Patients with a positive ELISA result could be separated into high (82%) and low (18%) IgG anti-F1 responders. Cross-reactions with eight other infectious diseases prevalent in Madagascar were scarce and were found in 1 of 27 Mycobacterium tuberculosis-, 3 of 34 Schistosoma haematobium-, and 1 of 41 Salmonella-infected patients. Finally, the efficiency of the IgG anti-F1 ELISA was evaluated during the Mahajanga, Madagascar, plague outbreak of 1995. When the number of ELISA-positive patients was added to the number of bacteriologically confirmed and probable cases, the number of positive patients was increased by 35%. In conclusion, although it does not replace bacteriology, IgG anti-F1 ELISA is a useful and powerful tool for retrospective diagnosis and epidemiological surveillance of plague outbreaks.