Jacques Morvan

Evaluation of an Enzyme Immunoassay for Detection of Dengue Virus NS1 Antigen in Human Serum

ABSTRACT We evaluated a one-step sandwich-format microplate enzyme immunoassay for detecting dengue virus NS1 antigen (Ag) in human serum by use of Platelia Dengue NS1 Ag kits (Bio-Rad Laboratories, Marnes La Coquette, France). We collected 299 serum samples from patients with dengue disease and 50 serum samples from patients not infected with dengue virus. For the 239 serum samples from patients with acute infections testing positive by reverse transcription-PCR and/or virus isolation for one of the four dengue virus serotypes, the sensitivity of the Platelia Dengue NS1 Ag kit was 88.7% (95% confidence interval, 84.0% to 92.4%). None of the serum samples from patients not infected with dengue virus tested positive with the Platelia Dengue NS1 Ag kit. A diagnostic strategy combining the Platelia Dengue NS1 Ag test for acute-phase sera and immunoglobulin M capture enzyme-linked immunosorbent assay for early-convalescent-phase sera increased sensitivity only from 88.7% to 91.9%. Thus, NS1 antigen detection with the Platelia Dengue NS1 Ag kit could be used for first-line testing for acute dengue virus infection in clinical diagnostic laboratories.

Dengue spatial and temporal patterns, French Guiana, 2001.

To study a 2001 dengue fever outbreak in Iracoubo, French Guiana, we recorded the location of all patients’ homes and the date when symptoms were first observed. A geographic information system was used to integrate the patient-related information. The Knox test, a classic space-time analysis technique, was used to detect spatiotemporal clustering. Analysis of the relative-risk (RR) variations when space and time distances vary, highlighted the maximum space and time extent of a dengue transmission focus. The results show that heterogeneity in the RR variations in space and time corresponds to known entomologic and epidemiologic factors, such as the mosquito feeding cycle and host-seeking behavior. This finding demonstrates the relevance and potential of the use of GIS and spatial statistics for elaborating a dengue fever surveillance strategy.

Identification of Ebola virus sequences present as RNA or DNA in organs of terrestrial small mammals of the Central African Republic.

The life cycle of the Ebola (EBO) virus remains enigmatic. We tested for EBO virus in the organs of 242 small mammals captured during ecological studies in the Central African Republic. EBO virus glycoprotein or polymerase gene sequences were detected by reverse transcription PCR in RNA extracts of the organs of seven animals and by PCR in DNA extract of one animal. Neither live virus nor virus antigen was detected in any organ sample. Direct sequencing of amplicons identified the virus as being of the Zaire/Gabon subtype. Virus-like nucleocapsids were observed by electron microscopy in the cytoplasm of the spleen cells of one animal. The animals belonged to two genera of rodents (Muridae; Mus setulosus, Praomys sp1 and P. sp2) and one species of shrew (Soricidae; Sylvisorex ollula). These preliminary results provide evidence that common terrestrial small mammals living in peripheral forest areas have been in contact with the EBO virus and demonstrate the persistence of EBO virus RNA and DNA in the organs of the animals. Our findings should lead to better targeting of research into the life cycle of the EBO virus.

Rift Valley fever epizootic in the central highlands of Madagascar

Between February and April 1991, unusual numbers of bovine abortion around Antananarivo (central highlands, Madagascar) were reported by official veterinary services. Rift Valley fever (RVF) virus isolations were made from sixteen aborted foetuses and one dead calf in different foci. Using monoclonal antibodies, the isolated viruses were found to be different from the 1979 RVF strains isolated in Madagascar from mosquitoes and human laboratory infection, and closer to African RVF strains. In a bovine population--previously characterized by a negative or very low RVF antibody prevalence--a high prevalence of IgM antibodies (264/994: 26.5% positive) was revealed; the IgM prevalence in recently aborting females varied from 40 to 91%. Among 994 human sera tested by IgG-IFA (immunofluorescent antibody assay) and IgM ELISA, 8.2% and 4.5%, respectively, proved positive. A total of 11,371 mosquitoes (61% Culex antennatus) were collected in the epizootic areas and tested without any virus isolation. Extensive studies were conducted to determine the geographical extension and the impact of this epidemic on the highly susceptible livestock and human populations.

Rift Valley fever on the east coast of Madagascar.

In March 1990, a Rift Valley fever virus (RVFV) outbreak was suspected in the district of Fenerive on the east coast of Madagascar after an abnormally high incidence of abortions and disease in livestock. Sera from humans and cattle were tested for RVFV antibodies by immunofluorescence assay (IFA) and ELISA-IgM capture. Sera and mosquitoes collected in the same area were tested for virus isolation by tissue culture and suckling mouse intracerebral inoculation, and for antigen detection by an ELISA antigen capture assay. Among cattle from the area, RVFV antibody prevalence was 58.6% by IFA and 29.6% by ELISA-IgM. In contrast, human populations in the same area had a lower RVFV antibody prevalence, with 8.01% IFA and 5.4% IgM-positive sera. No RVFV antigen was detected and virus isolation was unsuccessful from the sera and mosquito pools tested. Different hypotheses concerning the emergence and diffusion of RVFV in this area and the occurrence of the outbreak are discussed.

Dengue Infection in Neotropical Forest Mammals

In South America, dengue is the arbovirus-transmitted disease with the highest incidence. Unlike other arboviruses, wild mammals have no confirmed role in the cycle of dengue in the neotropics, although serological studies have suggested a possible secondary amplification cycle involving mammals other than nonhuman primates. In French Guiana, where all four serotypes (DENV-1, DENV-2, DENV-3, DENV-4) are present, the disease is endemic with outbreak events. To determine whether wild mammals can be infected by DENV, rodents, marsupials, and bats were captured over several periods, from 2001 to 2007, at two sites. The first location is a secondary forest surrounded by an urban area where dengue is endemic. The second location is a forest edge site where the disease has not yet emerged. A total of 10,000 trap-nights were performed and 616 mammals were captured. RNAs representing the four DENV serotypes were detected at both sites by reverse-transcriptase polymerase chain reaction in the livers and/or sera of 92 mammals belonging to 14 out of 32 species distributed among all the orders investigated: Rodentia (33 positive/146 tested), Marsupialia (40/318), and Chiroptera (19/152). Sequence analyses of a portion of the capsid and premembrane junction revealed that mammal strains of DENV-1, DENV-2, DENV-3, and DENV-4 had only 92.6%, 89%, 95%, and 95.8% identity, respectively, with strains circulating in the human population during the same periods. Regarding DENV-2, strains related (99% identity) to those responsible for an epidemic event in humans in French Guiana concurrent to the capture sessions were also evidenced, suggesting that wild mammals in edge habitats can be infected by circulating human strains. Our results demonstrate, for the first time, that neotropical wild mammals can be infected with dengue virus. The question of whether mammals maintain DENV in enzootic cycles and can play a role in its reemergence in human populations remains to be answered.

Discrimination between Primary and Secondary Dengue Virus Infection by an Immunoglobulin G Avidity Test Using a Single Acute-Phase Serum Sample

ABSTRACT For clinical and epidemiological purposes, it is necessary to be able to classify serological responses during dengue virus infection. Thus, it is important to develop a test that can distinguish between primary and secondary serological responses. The hemagglutination inhibition (HI) test, which is currently recommended by the World Health Organization, is complicated to perform. We developed an enzyme-linked immunosorbent assay based on changes in the avidity of immunoglobulin G during the infectious episode. This test can discriminate between primary and secondary infections by using a single serum sample collected during the acute phase of infection. We took 1,140 avidity measurements with 118 pairs of serum samples or sequential samples taken from patients classified as having primary or secondary infection according to World Health Organization laboratory criteria. The mean percent avidity was significantly lower during primary infection (25.9%) than during secondary infection (66.3%) (Student t test, P < 0.001). The test had a sensitivity of 82.7% (95% confidence interval [CI] = 79.0 to 86.6) and a specificity of 77.5% (95% CI = 73.3 to 81.7). Based on analysis of only blood samples collected between the third and seventh days of the illness, during which most clinical complications occur, the sensitivity and specificity of the test were 95.1% (95% CI = 92.6 to 97.7) and 80.0% (95% CI = 75.3 to 84.7), respectively. This rapid and simple test appears to be an excellent alternative to the HI test for discriminating between primary and secondary dengue virus infections during the acute phase of dengue.

Ebola and Marburg virus antibody prevalence in selected populations of the Central African Republic

With the natural history of the filovirus family seemingly unknown, filovirus ecology in its natural environment remains a rudimentary field of research. In order to investigate the maintenance cycle of filovirus in Central Africa, a study was conducted within the rain forest of the Central African Republic. The epidemiological study determines the frequency and distribution of filovirus seroprevalence in a selected human population. Using an ELISA, serum samples from Pygmy and non-Pygmy populations were tested for Ebola-Zaire virus and Marburg (MBG) virus antibody. Filovirus antibody reacting sera were found in all zones investigated, and in all populations studied (Ebola virus IgG 5.3%; Marburg virus IgG 2.4%). Pygmies appeared to have a significantly higher seroprevalence (P < 0.03) against Ebola-Zaire virus (7.02%) than non-Pygmies (4.2%). MBG virus or related unknown filovirus strains also seem to be present in the western part of Central Africa. MBG virus antibodies were present in different Pygmy groups (ranging from 0.7 to 5.6%, mean 2.05%) and in several non-Pygmy populations (ranging from 0.0 to 3.9%, mean 3.4%) without an overall significant difference between the two groups (P = 0.14). The potentialities of nonpathogenic filovirus strains circulating in the Central African Republic are discussed.

Serological and Molecular Evidence That Human Herpesvirus 8 Is Endemic among Amerindians in French Guiana

We evaluated the presence of human herpesvirus 8 (HHV-8) infection among groups of Amerindians in French Guiana. The overall prevalence of antibodies against lytic HHV-8 antigens was 23.0% (180/781), increasing from 18.4% in children <6 years old to approximately 30% in older persons (>45 years). Seroprevalence was higher in Amerindians living in remote localities than it was in those living in the coastal region. Analysis of a 725-base pair fragment of the K1 gene amplified from DNA from a Wayampi Amerindian showed that the virus belonged to molecular subtype E, which has hitherto been found in only a few Amerindians in Brazil and Ecuador.

Mayaro Virus in Wild Mammals, French Guiana

A serologic survey for Mayaro virus (Alphavirus, Togaviridae) in 28 wild nonflying forest mammal species in French Guiana showed a prevalence ranging from 0% to 52% and increasing with age. Species active during the day and those who spent time in trees were significantly more infected, results consistent with transmission implicating diurnal mosquitoes and continuous infectious pressure.