Samantha N. Hammond

The WHO dengue classification and case definitions: time for a reassessment

Dengue is the most prevalent mosquito-borne viral disease in people. It is caused by four dengue virus serotypes (DEN-1 DEN-2 DEN-3 and DEN-4) of the genus Flavivirus and transmitted by Aedes aegypti mosquitoes. Infection provides life-long immunity against the infecting viral serotype but not against the other serotypes. Although most of the estimated 100 million dengue virus infections each year do not come to the attention of medical staff of those that do the most common clinical manifestation is non-specific febrile illness or classic dengue fever. About 250 000--500 000 patients developing more severe disease. The risk of severe disease is several times higher in sequential than in primary dengue virus infections. Despite the large numbers of people infected with the virus each year the existing WHO dengue classification scheme and case definitions have some drawbacks. In addition the widely used guidelines are not always reproducible in different countries--a quality that is crucial to effective surveillance and reporting as well as global disease comparisons. And as dengue disease spreads to different parts of the globe several investigators have reported difficulties in using the system and some have had to create new categories or new case definitions to represent the observed patterns of disease more accurately. (excerpt)

Phenotyping of peripheral blood mononuclear cells during acute dengue illness demonstrates infection and increased activation of monocytes in severe cases compared to classic dengue fever.

In vitro studies have attempted to identify dengue virus (DEN) target cells in peripheral blood; however, extensive phenotyping of peripheral blood mononuclear cells (PBMCs) from dengue patients has not been reported. PBMCs collected from hospitalized children suspected of acute dengue were analyzed for DEN prM, CD32, CD86, CD14, CD11c, CD16, CD209, CCR7, CD4, and CD8 by flow cytometry to detect DEN antigen in PBMCs and to phenotype DEN-positive cells. DEN prM was detected primarily in activated monocytes (CD14(+), CD32(+), CD86(+), CD11c(+)). A subset of samples analyzed for DEN nonstructural protein 3 (NS3) confirmed that approximately half of DEN antigen-positive cells contained replicating virus. A higher percentage of PBMCs from DHF patients expressed prM, CD86, CD32, and CD11c than did those from DF patients. Increased activation of monocytes and greater numbers of DEN-infected cells were associated with more severe dengue, implicating a role for monocyte activation in dengue immunopathogenesis.

High seroprevalence of antibodies against dengue virus in a prospective study of schoolchildren in Managua, Nicaragua

To investigate the incidence of dengue virus (DENV) infection in Nicaragua, a 2‐year prospective study was conducted in schoolchildren 4–16 years old in the capital city of Managua. Blood samples were collected before the rainy season in 2001, 2002 and 2003, and were assayed for DENV‐specific antibodies. Participants were monitored for dengue‐like illness, and acute and convalescent blood samples were collected from suspected dengue cases. In 2001 and 2002, 602 and 397 students were recruited, respectively, and paired annual serum samples were available from 467 and 719 participants in 2001–2002 and 2002–2003, respectively. The overall seroprevalence of anti‐DENV antibodies was 91%, increasing from 75% at age 4 to 100% at age 16. The incidence of DENV infection was 12% in Year 1 and 6% in Year 2 (P < 0.001). During Year 1, four laboratory‐confirmed dengue cases were detected, with one DENV2 isolate; during Year 2, there were six confirmed dengue cases, with one DENV1 isolate. These and additional circulating serotypes were confirmed by plaque reduction neutralisation test. This study demonstrates surprisingly high transmission of DENV in urban Nicaragua.

Trends in Patterns of Dengue Transmission over 4 Years in a Pediatric Cohort Study in Nicaragua

BACKGROUND Dengue is the most prevalent mosquito-borne viral disease in humans and a major urban public health problem worldwide. METHODS A prospective cohort study of approximately 3800 children initially aged 2-9 years was established in Managua, Nicaragua, in 2004 to study the natural history of dengue transmission in an urban pediatric population. Blood samples from healthy subjects were collected annually prior to the dengue season, and identification of dengue cases occurred via enhanced passive surveillance at the study health center. RESULTS Over the first four years of the study, seroprevalence of anti-dengue virus (DENV) antibodies increased from 22%-40% in the 2-year-old cohort and 90%-95% in the 9-year-old cohort. The incidence of symptomatic dengue cases and the ratio of inapparent to symptomatic DENV infection varied substantially from year to year. The switch in dominant transmission from DENV-1 to DENV-2 was accompanied by an increase in disease severity but, paradoxically, a decrease in transmission. Phylogeographic analysis of full-length DENV-2 sequences revealed strong geographic clustering of dengue cases. CONCLUSIONS This large-scale cohort study of dengue in the Americas demonstrates year-to-year variation of dengue within a pediatric population, revealing expected patterns in transmission while highlighting the impact of interventions, climate, and viral evolution.

Diagnosis of Dengue Virus Infection by Detection of Specific Immunoglobulin M (IgM) and IgA Antibodies in Serum and Saliva

ABSTRACT To evaluate alternative approaches to the serological diagnosis of dengue virus (DEN) infection, the detection of DEN-specific immunoglobulin M (IgM) and IgA antibodies in serum and saliva specimens was assessed in 147 patients with symptoms of DEN infection seen at the Ministry of Health in Nicaragua. Seventy-two serum samples were determined to be positive for anti-DEN antibodies by IgM capture enzyme-linked immunosorbent assay, the routine diagnostic procedure. Serum and saliva specimens were obtained from 50 healthy adults as additional controls. IgM was detected in the saliva of 65 of the 72 serum IgM-positive cases, 6 of the 75 serum IgM-negative cases, and none of the control group, resulting in a sensitivity of 90.3% and a specificity of 92.0% and demonstrating that salivary IgM is a useful diagnostic marker for DEN infection. Detection of IgA in serum may be another feasible alternative for the diagnosis of DEN infection, with serum IgA found in 68 (94.4%) of the IgM-positive cases. In contrast, detection of IgA in saliva was not found to be a useful tool for DEN diagnosis in the present study. Further studies of the kinetics of antibody detection in another set of 151 paired acute- and convalescent-phase serum samples showed that DEN-specific IgA antibodies were detected in more acute-phase samples than were IgM antibodies. Thus, we conclude that DEN-specific IgA in serum is a potential diagnostic target. Furthermore, given that saliva is a readily obtainable, noninvasive specimen, detection of DEN-specific salivary IgM should be considered a useful, cheaper diagnostic modality with similar sensitivity and specificity to IgM detection in serum.

The Nicaraguan Pediatric Dengue Cohort Study: Study Design, Methods, Use of Information Technology, and Extension to Other Infectious Diseases

Dengue is a mosquito-borne viral disease that is a major public health problem worldwide. In 2004, the Pediatric Dengue Cohort Study was established in Managua, Nicaragua, to study the natural history and transmission of dengue in children. Here, the authors describe the study design, methods, and results from 2004 to 2008. Initially, 3,721 children 2–9 years of age were recruited through door-to-door visits. Each year, new children aged 2 years are enrolled in the study to maintain the age structure. Children are provided with medical care through the study, and data from each medical visit are recorded on systematic study forms. All participants presenting with suspected dengue or undifferentiated fever are tested for dengue by virologic, serologic, and molecular biologic assays. Yearly blood samples are collected to detect inapparent dengue virus infections. Numerous information and communications technologies are used to manage study data, track samples, and maintain quality control, including personal data assistants, barcodes, global information systems, and fingerprint scans. Close collaboration with the Nicaraguan Ministry of Health and use of almost entirely local staff are essential components for success. This study is providing critical data on the epidemiology and transmission of dengue in the Americas needed for future vaccine trials.

Evaluation of immunological markers in serum, filter-paper blood spots, and saliva for dengue diagnosis and epidemiological studies

BACKGROUND Numerous immunological approaches exist to diagnose dengue or detect dengue virus (DENV) infections. OBJECTIVES To determine the best immunological markers and specimen types for dengue diagnosis and for measuring incidence of DENV infection in community-based studies. STUDY DESIGN In one study, acute- and convalescent-phase samples were collected from hospitalized suspected pediatric dengue cases in Managua, Nicaragua, from September 2003 to February 2004. A second study examined specimens collected in a community setting in Managua before and after the 2003-2004 dengue season to measure incidence of DENV infection. In both studies, detection of anti-DENV IgM, IgA, and IgG in serum, filter-paper blood spots, and saliva was compared to a gold standard performed on serum samples. RESULTS For dengue diagnosis, the highest sensitivity and specificity was obtained by measuring IgM or IgA in serum or filter-paper blood spots; intermediate and poor results were obtained in saliva for IgM and IgA, respectively. Detection of IgG alone in serum, filter-paper blood spots, or saliva functioned best for measuring DENV infection. CONCLUSIONS Detection of IgM and IgA in serum and filter-paper blood spots yielded optimal results for diagnosis of dengue cases, whereas IgG was the best marker for measuring incidence of DENV infection.

Characterization of Aedes aegypti (Diptera: Culcidae) production sites in urban Nicaragua.

Abstract To characterize the production patterns of the dengue virus vector Aedes aegypti (L.) (Diptera: Culcidae), pupal surveys were conducted in selected neighborhoods of two major cities in Nicaragua. In León, 833 houses were visited in July and September 2003, corresponding to the beginning and middle of the dengue season; in Managua, 1,365 homes were visited in July 2003. In total, 7,607 containers were characterized, of which 11% were positive for Ae. aegypti larvae and 4% for pupae. In addition to barrels, potted plants and superficial water on tarps and in puddles were identified as highly productive sites. Univariate and multivariate analysis revealed frequency of container use, use of a lid, and rainwater filling as key variables affecting pupal positivity. Importantly, this survey demonstrated the risk associated with the presence of lids, the limited temporal efficacy of temephos, and the lack of association of water availability with risky water storage practices. Finally, we introduce the concept of an efficiency value and an accompanying graphical display system that can facilitate development of targeted pupal control strategies. These data underscore the importance of entomological surveillance of pupal productivity to gather information from which to derive streamlined, efficient, and effective vector control measures to reduce the density of Aedes mosquito larvae and pupae and thus the risk for dengue.

Gene expression patterns of dengue virus-infected children from nicaragua reveal a distinct signature of increased metabolism.

Background Infection with dengue viruses (DENV) leads to a spectrum of disease outcomes. The pathophysiology of severe versus non-severe manifestations of DENV infection may be driven by host responses, which could be reflected in the transcriptional profiles of peripheral blood immune cells. Methodology/Principal Findings We conducted genome-wide microarray analysis of whole blood RNA from 34 DENV-infected children in Nicaragua collected on days 3–6 of illness, with different disease manifestations. Gene expression analysis identified genes that are differentially regulated between clinical subgroups. The most striking transcriptional differences were observed between dengue patients with and without shock, especially in the expression of mitochondrial ribosomal proteins associated with protein biosynthesis. In the dengue hemorrhagic fever patients, one subset of differentially expressed genes encode neutrophil-derived anti-microbial peptides associated with innate immunity. By performing a meta-analysis of our dataset in conjunction with previously published datasets, we confirmed that DENV infection in vivo is associated with large changes to protein and nucleic acid metabolism. Additionally, whereas in vitro infection leads to an increased interferon signature, this was not consistently observed from in vivo patient samples, suggesting that the interferon response in vivo is relatively transient and was no longer observed by days 3–6 of illness. Conclusions/Significance These data highlight important differences between different manifestations of severity during DENV infection as well as identify some commonalities. Compilation of larger datasets in the future across multiple studies, as we have initiated in this report, may well lead to better prediction of disease manifestation via a systems biology approach.

IDENTIFICATION OF AEDES ALBOPICTUS IN URBAN NICARAGUA

ABSTRACT Larvae of Aedes albopictus, a mosquito known for transmitting dengue virus, were identified in the city of León, Nicaragua, in 2003. Mosquito larvae were collected from a total of 2,225 residences in the 2 largest cities in Nicaragua during the period from June to September of 2003, and larval Ae. albopictus were identified in 4 homes in León. This represents the 1st detection of Ae. albopictus in a major Nicaraguan urban center, and increased control efforts appear to have eliminated the mosquito subsequently from Le León. The presence of Ae. albopictus in urban Nicaragua highlights the need for surveillance of areas thought to be free of the mosquito so that early detection and control activities can prevent its spread.