Use of an enhanced surveillance system for encephalitis and aseptic meningitis for the detection of neurologic manifestations of dengue in Puerto Rico, 2003.

Dengue infection has been implicated as a cause of neurologic manifestations since the beginning of the 20th century. An enhanced surveillance system for encephalitis and aseptic meningitis developed by the Puerto Rico Department of Health in collaboration with the Dengue Branch, Centers for Disease Control and Prevention, identified eleven laboratory positive dengue patients presenting with neurologic manifestations in 2003. Anti-dengue IgM antibody was detected in serum of eight patients and in cerebrospinal fluid of one patient. DENV-2 and DENV-3 were isolated from the serum of one patient each. All patients were negative for serologic markers of West Nile Virus and St. Louis encephalitis. Nine (82%) of the 11 patients had symptoms compatible with encephalitis. Their median age was 46 years (range: 9 months - 82 years) and five were males. Symptoms included severe headache, seizures, altered mental status, confusion, and coma. A motor disorder (upper extremities weakness and Guillain Barré Syndrome, respectively) occurred in two additional patients. Most patients recovered but there were two fatalities. Neurologic manifestations of dengue were rarely reported in Puerto Rico until the institution of enhanced surveillance, which resulted in the recognition of severe and fatal cases.

Medical examiner samples: a source for dengue surveillance.

Post-mortem medical examiner samples may be useful for sentinel surveillance of disorders usually detected by antibody determinations on specimens from ill patients or from surveys. We found anti-dengue IgM positivity in 3% (23/780) and anti-dengue IgG positivity in 77% (597/777) of sera obtained at the Puerto Rico medical examiner (Institute of Forensic Sciences) in December 2000, April 2001, and October 2001. This approach may be a useful alternative for estimating the population prevalence of serologic markers for dengue and other infectious diseases.

Molecular evolution of dengue 2 virus in Puerto Rico: positive selection in the viral envelope accompanies clade reintroduction.

Dengue virus is a circumtropical, mosquito-borne flavivirus that infects 50-100 million people each year and is expanding in both range and prevalence. Of the four co-circulating viral serotypes (DENV-1 to DENV-4) that cause mild to severe febrile disease, DENV-2 has been implicated in the onset of dengue haemorrhagic fever (DHF) in the Americas in the early 1980s. To identify patterns of genetic change since DENV-2's reintroduction into the region, molecular evolution in DENV-2 from Puerto Rico (PR) and surrounding countries was examined over a 20 year period of fluctuating disease incidence. Structural genes (over 20 % of the viral genome), which affect viral packaging, host-cell entry and immune response, were sequenced for 91 DENV-2 isolates derived from both low- and high-prevalence years. Phylogenetic analyses indicated that DENV-2 outbreaks in PR have been caused by viruses assigned to subtype IIIb, originally from Asia. Variation amongst DENV-2 viruses in PR has since largely arisen in situ, except for a lineage-replacement event in 1994 that appears to have non-PR New World origins. Although most structural genes have remained relatively conserved since the 1980s, strong evidence was found for positive selection acting on a number of amino acid sites in the envelope gene, which have also been important in defining phylogenetic structure. Some of these changes are exhibited by the multiple lineages present in 1994, during the largest Puerto Rican outbreak of dengue, suggesting that they may have altered disease dynamics, although their functional significance will require further investigation.

Risk factors for infection during a dengue-1 outbreak in Maui, Hawaii, 2001.

Autochthonous dengue virus transmission, last identified in the state of Hawaii in 1945, was detected again in 2001. A seroepidemiological survey in a high-incidence community (Nahiku) and a nearby low-incidence community (Hana Subdivision) was implemented. The two communities studied differed in median household size (two vs. four persons), median lot size (2.8 vs. 0.8acres), proportion of households with mosquito larvae (81 vs. 28%) and incidence of recent infection (39% [28/72] vs. 1% [1/131]). The average number of reported anti-mosquito actions by residents of both locations remained low, and approximately 50% (42/80) of the inspected houses had larvae, evidencing the need for more effective community mosquito control. Logistic regression analysis of risk factors for infection in Nahiku identified residing in properties with birds in the house or yard as significantly associated with infection (odds ratio 7.0, 95% CI 1.7-28.5), probably as an indicator of unspecified environmental characteristics that were attractive to the vector. We documented that nearly 40% of Nahiku residents had acquired dengue locally in 2001 and that undetected dengue outbreaks had occurred in Hawaii. Our data suggest that ecological characteristics may help Hawaii health officials identify communities at increased risk of dengue infection.

Dengue fever, Hawaii, 2001-2002.

Autochthonous dengue infections were last reported in Hawaii in 1944. In September 2001, the Hawaii Department of Health was notified of an unusual febrile illness in a resident with no travel history; dengue fever was confirmed. During the investigation, 1,644 persons with locally acquired denguelike illness were evaluated, and 122 (7%) laboratory-positive dengue infections were identified; dengue virus serotype 1 was isolated from 15 patients. No cases of dengue hemorrhagic fever or shock syndrome were reported. In 3 instances autochthonous infections were linked to a person who reported denguelike illness after travel to French Polynesia. Phylogenetic analyses showed the Hawaiian isolates were closely associated with contemporaneous isolates from Tahiti. Aedes albopictus was present in all communities surveyed on Oahu, Maui, Molokai, and Kauai; no Ae. aegypti were found. This outbreak underscores the importance of maintaining surveillance and control of potential disease vectors even in the absence of an imminent disease threat.

Outbreak of dengue fever in Palau, Western Pacific: risk factors for infection.

Between January and June 1995, an outbreak of dengue fever occurred in Palau, an island nation of 32,000 inhabitants in the Western Pacific. To determine the magnitude of this outbreak and to determine modifiable risk factors to guide control strategies, we established active surveillance at the national hospital and private clinics, reviewed available clinical records, and conducted serologic and entomologic surveys. Between January 1 and July 1, 1995, 817 case-patients with acute febrile illness with body or joint aches and one of the following: headache, rash, nausea, vomiting, or hemorrhagic manifestations presented to health facilities in Palau. The epidemic peaked in the second week of April 1995. Of 338 case-patients tested, 254 (75%) had positive serologic results by an IgM capture enzyme-linked immunosorbent assay. Dengue 4 virus was isolated from 78 (51%) of 154 serum samples tested. Blood samples collected during a cross-sectional survey were tested for IgM antibody and yielded an attack ratio of 27% (95% confidence interval = 23-31%). Potential vectors included the introduced species Aedes aegypti and Ae. albopictus, and the native species Ae. hensilli. Significant risk factors (P < or = 0.05) for infection included age < 20 years, the presence of food or water pans for animals on the property, taro farming, the presence of Ae. aegypti on the property, and presence of Ae. scutellaris group mosquitoes (Ae. Hensilli, Ae. albopictus, and a native species). This was the first outbreak of dengue 4 virus in the Western Pacific, and the first documented epidemic of dengue in Palau since 1988.

Selection-driven evolution of emergent dengue virus.

In the last four decades the incidence of dengue fever has increased 30-fold worldwide, and over half the world's population is now threatened with infection from one or more of four co-circulating viral serotypes (DEN-1 through DEN-4). To determine the role of viral molecular evolution in emergent disease dynamics, we sequenced 40% of the genome of 82 DEN-4 isolates collected from Puerto Rico over the 20 years since the onset of endemic dengue on the island. Isolates were derived from years with varying levels of DEN-4 prevalence. Over our sampling period there were marked evolutionary shifts in DEN-4 viral populations circulating in Puerto Rico; viral lineages were temporally clustered and the most common genotype at a particular sampling time often arose from a previously rare lineage. Expressed changes in structural genes did not appear to drive this lineage turnover, even though these regions include primary determinants of viral antigenic properties. Instead, recent dengue evolution can be attributed in part to positive selection on the nonstructural gene 2A (NS2A), whose functions may include replication efficiency and antigenicity. During the latest and most severe DEN-4 epidemic in Puerto Rico, in 1998, viruses were distinguished by three amino acid changes in NS2A that were fixed far faster than expected by drift alone. Our study therefore demonstrates viral genetic turnover within a focal population and the potential importance of adaptive evolution in viral epidemic expansion.

Molecular evolution and phylogeny of dengue type 4 virus in the Caribbean.

We sequenced the E gene and adjacent prM/M and NS1 junctions (1940 bp) of 48 Dengue-4 (DEN-4) isolates collected between 1981 and 1999 from 8 Caribbean islands and from 7 South and Central American countries. Phylogenetic analysis confirms a single introduction in the early 1980s and a high degree of gene flow resulting in a pattern of evolution defined more by time period than geographic origin, especially within the Caribbean basin. A modern Caribbean clade consisting of four distinct lineages has arisen, comprised of isolates from Caribbean islands and nearby regions of South America. This clade is defined by three amino acid substitutions in the E (aa 163 and 351) and NS1 (aa 52) proteins. These findings highlight the importance of migration and gene flow in dengue viral change and suggest that efforts to understand disease dynamics in the Caribbean basin need to focus at regional, rather than local scales.

Molecular evolution and phylogeny of dengue type 4 virus in the Caribbean

We sequenced the E gene and adjacent prM/M and NS1 junctions (1940 bp) of 48 Dengue-4 (DEN-4) isolates collected between 1981 and 1999 from 8 Caribbean islands and from 7 South and Central American countries. Phylogenetic analysis confirms a single introduction in the early 1980s and a high degree of gene flow resulting in a pattern of evolution defined more by time period than geographic origin, especially within the Caribbean basin. A modern Caribbean clade consisting of four distinct lineages has arisen, comprised of isolates from Caribbean islands and nearby regions of South America. This clade is defined by three amino acid substitutions in the E (aa 163 and 351) and NS1 (aa 52) proteins. These findings highlight the importance of migration and gene flow in dengue viral change and suggest that efforts to understand disease dynamics in the Caribbean basin need to focus at regional, rather than local scales.

Texas lifestyle limits transmission of dengue virus.

Urban dengue is common in most countries of the Americas, but has been rare in the United States for more than half a century. In 1999 we investigated an outbreak of the disease that affected Nuevo Laredo, Tamaulipas, Mexico, and Laredo, Texas, United States, contiguous cities that straddle the international border. The incidence of recent cases, indicated by immunoglobulin M antibody serosurvey, was higher in Nuevo Laredo, although the vector, Aedes aegypti, was more abundant in Laredo. Environmental factors that affect contact with mosquitoes, such as air-conditioning and human behavior, appear to account for this paradox. We conclude that the low prevalence of dengue in the United States is primarily due to economic, rather than climatic, factors.

The reappearance of dengue-3 and a subsequent dengue-4 and dengue-1 epidemic in Puerto Rico in 1998.

In January 1998, dengue-3 (DEN)-3 (group III genotype) was detected in Puerto Rico after an absence of 20 years. Public health officials intensified education efforts to promote community participation in dengue control. Virologic surveillance revealed an unexpected paradox: DEN-4 and DEN-1 produced a large epidemic overlaying the DEN-3 epidemic. In 1998 there were 17,000 reported cases of dengue (4.8/1,000 persons), and among all virus isolations (n = 960), DEN-4 (419, 43.6%), DEN-1 (337, 35.1%), and DEN-2 (143, 14.9%) were detected much more frequently than DEN-3 (61, 6%). Age group-specific attack rates were highest for persons 10-19 years old, followed by infants less than a year of age. Nineteen fatal cases (median = 37 years old, range = 8 months to 90 years) had a positive laboratory diagnosis of dengue. Among DEN-3 cases no fatalities were documented, 50 were hospitalized, and 10 of 48 (21%) fulfilled the criteria for dengue hemorrhagic fever (four had primary infections and six had secondary infections). During 1999, DEN-3 became the predominant serotype isolated (182 of 310 isolations, 59%). The reappearance of DEN-3 and its subsequent circulation from 1999 to 2001 produced no changes in dengue incidence that could have been detected in the absence of virologic surveillance.

Enzyme-linked immunosorbent assay-format microneutralization test for dengue viruses.

A microneutralization test that measures anti-dengue antibodies was developed. Serum dilutions, neutralization reactions, and virus growth were performed in 96-well plates. After incubation, an enzyme-linked immunosorbent assay that used mouse anti-dengue antibodies and an enzyme-conjugated anti-mouse antibody was used to measure cell-associated viral antigens. The resulting optical density readings were processed and graphed automatically by a spreadsheet program. This procedure provided results that are essentially the same as those from the plaque-reduction neutralization test for serum samples from primary dengue virus infections, but results correlated poorly with results from samples from people with secondary infections. The test offers the advantages of ease of performance, ease in the calculation of results, lower cost, and increased speed.