Séverine Matheus

Evaluation of Two New Commercial Tests for the Diagnosis of Acute Dengue Virus Infection Using NS1 Antigen Detection in Human Serum

Background We compared the performance of two new commercial tests for the detection of dengue NS1 protein during the clinical phase of dengue virus (DENV) infection—an immunochromatographic test allowing rapid detection of the NS1 antigen, Dengue NS1 Ag STRIP (Bio-Rad Laboratories - Marnes La Coquette, France), and a two-step sandwich-format microplate enzyme-linked immunosorbent assay (ELISA), pan-E Dengue Early ELISA (Panbio - Brisbane, Australia)—with a one-step sandwich-format microplate ELISA, the Platelia Dengue NS1 Ag test (Bio-Rad). Methods We tested 272 serum samples from patients with dengue disease. Of these, 222 were from patients with acute infection of one of the four dengue serotypes, detected by RT-PCR and/or virus isolation. Forty-eight acute-phase serum samples from patients not infected with dengue virus were also included. Results The sensitivity of the Platelia Dengue NS1 Ag test on acute serum samples (n = 222) was 87.4% (95% confidence interval: 82.3% to 91.5%); that of Dengue NS1 Ag STRIP was 81.5% (95% CI: 75.8% to 86.4%) after 15 minutes and 82.4% (95% CI: 76.8% to 87.2%) after 30 minutes. Both tests had a specificity of 100% (97.5% CI, one-sided test: 92.6% to 100.0%). The pan-E Dengue Early ELISA had a sensitivity of 60.4% (95% CI: 53.4% to 66.8%) and a specificity of 97.9% (95% CI: 88.9% to 99.9%). Conclusion Our findings support the use of diagnostic tools based on the NS1 antigen detection for the diagnosis of acute DENV infection. The immunochromatographic test, Dengue NS1 Ag STRIP—the first rapid diagnostic test for DENV infection—was highly sensitive and specific, and would therefore be a suitable first-line test in the field. The pan-E Dengue Early ELISA was less sensitive than the Platelia test; this two-step ELISA should be combined with DENV IgM antibody detection for the diagnosis of DENV infection.

Chikungunya outbreak in the Caribbean region, December 2013 to March 2014, and the significance for Europe

On 6 December 2013, two laboratory-confirmed cases of chikungunya without a travel history were reported on the French part of the Caribbean island of Saint Martin, indicating the start of the first documented outbreak of chikungunya in the Americas. Since this report, the virus spread to several Caribbean islands and French Guiana, and between 6 December 2013 and 27 March 2014 more than 17,000 suspected and confirmed cases have been reported. Further spread and establishment of the disease in the Americas is likely, given the high number of people travelling between the affected and non-affected areas and the widespread occurrence of efficient vectors. Also, the likelihood of the introduction of the virus into Europe from the Americas and subsequent transmission should be considered especially in the context of the next mosquito season in Europe. Clinicians should be aware that, besides dengue, chikungunya should be carefully considered among travellers currently returning from the Caribbean region.

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.

Early clinical and biological features of severe clinical manifestations of dengue in Vietnamese adults

BACKGROUND Dengue is a major public health problem in both children and adults in Vietnam, but dengue severity in adults has been poorly investigated. OBJECTIVES To describe severe manifestations of dengue in Vietnamese adults and to identify early indicators of these manifestations. STUDY DESIGN A prospective longitudinal study was carried out from July to October, 2007, in Peoples Hospital 115, Ho Chi Minh City, Vietnam. RESULTS One hundred ninety-five clinically suspected dengue patients were enrolled. 151 of these were laboratory-confirmed using serum samples on day 3 after onset of fever for RT-PCR and/or virus isolation. Dengue was associated with plasma leakage in 51 patients (33.8%), gallbladder thickening in 30 patients (20%), spontaneous bleeding in 127 patients (84.1%), and internal bleeding in 37 patients (24.5%). Several early indicators were associated with severe manifestations of dengue. Frequent vomiting (> or =3 times a day), marked lymphopenia, thrombocytopenia, and elevated liver enzymes on day 3 after onset of fever were significantly associated with plasma leakage and gallbladder thickening. Increased alanine aminotransferase level in plasma on day 3 was significantly associated with internal bleeding. Gallbladder thickening and internal bleeding were more common with specific serotypes. CONCLUSION Several severe manifestations of dengue were observed in Vietnamese adults. These manifestations were associated with early clinical and biological indicators. This information may be useful for clinicians to better monitor adult dengue patients, particularly in tropical areas where health resources may be limited.

Concurrent Dengue and Malaria in Cayenne Hospital, French Guiana

Dengue-malaria co-infection reports are scarce. Of 1,723 consecutive febrile patients in Cayenne Hospital, 238 had dengue (174 early dengue fever cases) and 393 had malaria (371 acute malaria); 17 had both. Diagnosis of 1 of these 2 infections should not rule out testing for the other infection.

Use of Capillary Blood Samples as a New Approach for Diagnosis of Dengue Virus Infection

ABSTRACT We evaluated the use of capillary blood samples stored on filter papers for diagnosis of dengue virus infection. Venous and capillary blood samples were collected from 130 patients suspected of having dengue fever. We compared the performances of standard reference methods using capillary blood samples absorbed onto filter papers versus venous blood samples. The resulting sensitivity, specificity, and positive predictive value of tests performed on filter paper compared to those performed on venous blood samples were 81.6% (62/76; 95% confidence interval [CI], 74.9% to 88.3%), 90.7% (49/54; 95% CI, 85.7% to 95.7%), and 92.5% (62/67; 95% CI, 86.2% to 98.8%), respectively. During the acute phase of dengue virus infection (day 1 to day 4), the tests performed on capillary blood samples had a sensitivity of 88.5% (95% CI, 82.0% to 95.0%) and a specificity of 93.8% (95% CI, 88.9% to 98.7%). During the convalescent phase of infection, this method allowed the viral serotype to be determined for 4 of 15 (27%) dengue virus-infected patients for whom virological diagnosis using venous samples was negative. Capillary blood samples could therefore be a good alternative for the diagnosis of dengue virus infection in tropical areas. Indeed, these samples are convenient for storage and transport without the need for a cold chain and simplify the collection of samples from children. Moreover, our results suggest that viral particles persist longer in capillary blood than in peripheral blood. Analysis of the viability of viral particles under these conditions may give new insights into the physiopathology of dengue virus infection and the transmission of dengue virus during outbreaks.

From serological surveillance to the identification of native human cases of hantavirus pulmonary syndrome in French Guiana

Hantaviruses are rodent-borne negative-sense RNA viruses belonging to the Bunyaviridae family, genus Hantavirus. These emerging viruses cause cardiopulmonary syndrome in North and South America, which is a respiratory illness following the inhalation of dust contaminated by infectious rodent feces or urine. Until recently, no information was available related to the presence of hantavirus in French Guyana, a French department in South America. Nevertheless, the description of atypical pneumonia cases unrelated to any known etiological agent and the identification of hantavirus reservoirs in neighboring countries led us to conduct a serological study in a collection of sera from patients who had presented compatible symptoms: the prevalence of IgG antibodies to hantavirus in this population was 1.42%. After those retroactive results, systematic hantavirus serology screening was implemented in every newcoming patient with suggestive etiology. This led us to identify a native case in French Guyana. After this first case, a second case was registered 1 year later in December 2009 (in a periurban area). Molecular analyses were conducted to characterize genetically these two strains of hantavirus. Complete sequences of the S segment were obtained and phylogenetic analyses confirmed that strains isolated in French Guyana and tentatively named Maripa virus belong to the Rio Mamore species. Human hantavirus epidemics are associated with fluctuations of rodent populations, caused by climatic, ecological and environmental changes, or growing human activities associated with nature or agriculture. In Guyana, 90% of the land is still tropical rain forest, but economic development results in growing pressures on natural habitats. Continuous surveillance of the virus in the human population would be beneficial. Furthermore, surveys of potential reservoirs may help to understand hantavirus dispersion and to reduce the risk of viral emergence.

Whole-Genome Sequencing Analysis from the Chikungunya Virus Caribbean Outbreak Reveals Novel Evolutionary Genomic Elements.

Background Chikungunya virus (CHIKV), an alphavirus and member of the Togaviridae family, is capable of causing severe febrile disease in humans. In December of 2013 the Asian Lineage of CHIKV spread from the Old World to the Americas, spreading rapidly throughout the New World. Given this new emergence in naïve populations we studied the viral genetic diversity present in infected individuals to understand how CHIKV may have evolved during this continuing outbreak. Methodology/Principle Findings We used deep-sequencing technologies coupled with well-established bioinformatics pipelines to characterize the minority variants and diversity present in CHIKV infected individuals from Guadeloupe and Martinique, two islands in the center of the epidemic. We observed changes in the consensus sequence as well as a diverse range of minority variants present at various levels in the population. Furthermore, we found that overall diversity was dramatically reduced after single passages in cell lines. Finally, we constructed an infectious clone from this outbreak and identified a novel 3’ untranslated region (UTR) structure, not previously found in nature, that led to increased replication in insect cells. Conclusions/Significance Here we preformed an intrahost quasispecies analysis of the new CHIKV outbreak in the Caribbean. We identified novel variants present in infected individuals, as well as a new 3’UTR structure, suggesting that CHIKV has rapidly evolved in a short period of time once it entered this naïve population. These studies highlight the need to continue viral diversity surveillance over time as this epidemic evolves in order to understand the evolutionary potential of CHIKV.

Use of Four Dengue Virus Antigens for Determination of Dengue Immune Status by Enzyme-Linked Immunosorbent Assay of Immunoglobulin G Avidity

ABSTRACT We used an enzyme-linked immunosorbent assay (ELISA) of immunoglobulin G avidity to determine the dengue immune status of 105 pairs of serum samples from patients infected with dengue virus. This study shows that a simple avidity test, for which only one acute-phase serum sample is required, is potentially more useful than the hemagglutination inhibition test for the discrimination of primary from secondary dengue virus infection, whatever the type of dengue antigen used.