Dengue hemorrhagic fever in Cuba, 1981: a retrospective seroepidemiologic study

In Cuba, 2 epidemics of dengue virus occurred: 1 caused by DEN-1 in 1977 and 1 caused by DEN-2 in 1981. The latter was associated with cases of dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS). To study viral risk factors for DHF/DSS, a retrospective seroepidemiological survey was conducted in Cerro, a densely populated district in Havana City. The prevalence of plaque reduction neutralizing antibodies to DEN-1 and DEN-2 viruses was measured in 1,295 individuals (children and adults). Of these, 43.7% were immune to DEN-1 virus and 23.6% to DEN-2 virus. Of those individuals who were immune, 26.1% were immune to DEN-1 virus only, 6% to DEN-2 virus only, and 17.6% to both viruses. The DEN-2 virus infection rate in DEN-1 immune individuals was 3.8 times higher than in non-immune individuals. The 5 DHF/DSS cases in the sample had evidence of DEN-1 virus plus DEN-2 virus infections. Three were children and 2 were young adults. No cases were found in individuals infected with DEN-1 virus or DEN-2 virus only. Children infected by DEN-1 virus followed by DEN-2 virus had a high risk of acquiring DHF/DSS. Blacks and whites were equally infected with DEN-1 and DEN-2 viruses.     

Double infection of heteroserotypes of dengue viruses in field populations of Aedes aegypti and Aedes albopictus (Diptera: Culicidae) and serological features of dengue viruses found in patients in southern Thailand

In order to understand more about the epidemiology of DHF, a study of the type of dengue viruses and vectors under natural conditions was carried out. Mosquito vectors in the field and the serum of DHF patients in southern Thailand were examined. The two mosquito species are abundant and DHF incidence remains high in this region. Dengue viruses were examined in field-caught mosquitoes by RT-PCR technique. The mosquitoes were caught in 4 provinces: Krabi, Phuket, Phang-Nga and Surat Thani during the late dry season until the early rainy season in 2005. Three dengue serotypes (DEN-2, DEN-3, DEN-4) were detected in Ae. aegypti males and females, and 2 (DEN-2, DEN-3) were detected in Ae. albopictus females. Double infection with 2 serotypes of dengue viruses (DEN-2 and DEN-3) were detected in Ae. aegypti males and females and Ae. albopictus females. DEN-2 and DEN-1 were the most prevalent serotypes found in the serum of the patients in this area, followed by DEN-4 and DEN-3. The prevalence of the predominant dengue serotype varied from province to province. Detection of viruses in adult male mosquitoes reveals the role of transovarial transmission of dengue viruses in field populations of DHF vectors and elucidates circulation of dengue viruses in vectors in the natural environment of endemic areas. The incidence of multiple serotypes of dengue virus in Ae. aegypti and Ae. albopictus in the same area points toward a high risk for an epidemic of DHF. These findings provide greater understanding of the relationship among mosquito vectors, virus transmission and DHF epidemiology in endemic areas.     

Dengue viremia titer, antibody response pattern, and virus serotype correlate with disease severity

Viremia titers in serial plasma samples from 168 children with acute dengue virus infection who were enrolled in a prospective study at 2 hospitals in Thailand were examined to determine the role of virus load in the pathogenesis of dengue hemorrhagic fever (DHF). The infecting virus serotype was identified for 165 patients (DEN-1, 46 patients; DEN-2, 47 patients; DEN-3, 47 patients, DEN-4, 25 patients). Patients with DEN-2 infections experienced more severe disease than those infected with other serotypes. Eighty-one percent of patients experienced a secondary dengue virus infection that was associated with more severe disease. Viremia titers were determined for 41 DEN-1 and 46 DEN-2 patients. Higher peak titers were associated with increased disease severity for the 31 patients with a peak titer identified (mean titer of 107.6 for those with dengue fever vs. 108.5 for patients with DHF, P=.01). Increased dengue disease severity correlated with high viremia titer, secondary dengue virus infection, and DEN-2 virus type.     

Use of nucleotide sequencing of the genomic cDNA fragments of the capsid/premembrane junction region for molecular epidemiology of dengue type 2 viruses

The recent emergence of dengue hemorrhagic fever/dengue shock syndrome (DHF/ DSS) in India has been a source of concern. In the present study a quantitative comparison of 406 nucleotide long sequence from the capsid-premembrane junction region (C-PrM) of 9 dengue virus type 2 (DEN-2) isolates from Delhi with 10 DEN-2 isolates from diverse geographic areas provided sufficient information for estimating genetic relationships. The data indicated that the 1996 epidemic of DHF in Delhi was caused by genotype IV strains of DEN-2. This genotype, perhaps, displaced genotype V strains of DEN-2, which was circulating genotype in 1967. The period during which this displacement had occurred is not clear from the present study. Nonetheless, similar experience in four countries in Latin America and in Sri Lanka suggest that the introduction of new genotypes of DEN-2 displacing the circulating genotype may be associated with the appearance of DHF/DSS. More work is required to elucidate this hypothesis. Transitions at nucleotide positions 406 and 431 resulted in amino acid substitutions near (aa position 104, methionine --> valine) and at the hinge region (aa position 112, valine --> alanine) of C-PrM, respectively in all/most genotypes of group III and IV DEN-2 viruses analysed. Most of these virus strains have been isolated from DHF/DSS outbreaks. Significance of this observation is discussed. The data presented in this study suggest the utility of C-PrM sequence analysis for molecular epidemiology of dengue viruses.     

Possible dengue sequential infection: dengue spread in a neighbourhood during the 1996/97 dengue-2 epidemic in French Polynesia

A DEN-2 epidemic occurred in French Polynesia from August 1996 to April 1997 after 7 years of DEN-3 circulation. The susceptible population constituted all expatriates and Polynesians under 21. In August 1996, two successive DEN-2 cases occurred in Teroma, a Tahitian neighbourhood close to the international airport of Tahiti. A serological prospective study of persons < 21 years living in Teroma was conducted. The study population was bled in September 1996, October 1996 and June 1997. Analysis of dengue spread in Teroma confirmed that dengue transmission occurs primarily in the house, thus vector control campaigns should incorporate focal insecticide spraying and systematic daily use of insecticide in houses. The evolution in time of the disease demonstrated that among a susceptible population, prevalence and incidence rates are related to the time of exposure, and consequently to age. Comparison of dengue incidence or dengue prevalence between populations therefore requires adjusted age rates. Most studies did not adjust for age, leading to the conclusion that DHF is more frequent during secondary than during primary dengue infection. Prospective studies taking into account the time of dengue exposure are necessary to confirm the sequential infection hypothesis.     

福建省1株登革Ⅱ型病毒的鉴定

目的对2005年福建省分离的1株登革病毒(DV)进行鉴定,并从分子水平追踪其可能的传染源。方法采用酶联免疫吸附试验(ELISA)检测疑似登革热患者血清中DV(IgM、IgG抗体;同时应用C6/36细胞、单克隆抗体间接免疫荧光(McAb-IFA)、逆转录(套式PCR法分别进行病毒的分离和鉴定,并对分离株的部分基因进行核苷酸序列分析。结果患者血清登革病毒特异性IgM抗体阳性、IgG抗体可疑,表明该患者在近期感染过登革病毒。患者血清接种C6/36细胞,观察到登革病毒特有的CPE。受感染的C6/36细胞能与登革病毒Ⅱ型单克隆抗体反应,表明分离的病毒株为登革Ⅱ型病毒。分离株的核酸提取物经RT(PCR扩增,登革病毒通用引物可扩增出511bp的特异性条带,型特异性引物扩增出119bp的特异性条带,进一步证实分离的病毒株为登革Ⅱ型病毒。分离株RT(PCR扩增产物的核苷酸序列与30株不同地域来源的登革Ⅱ型病毒相应序列构建的系统发生树表明,此毒株与东南亚地区的毒株比较接近。此次分离株的序列与1999年登革Ⅱ型病毒福建株的对应序列在亲缘关系上有一定程度距离。结合流行病学调查资料,进一步确定此病例为输入性感染病例。结论福建省首次从输入性登革热患者血清中分离出登革Ⅱ型病毒,该病毒来源于东南亚地区。     

Epidemiology of dengue and dengue hemorrhagic fever in a cohort of adults living in Bandung, West Java, Indonesia

A prospective study of dengue fever (DF) and dengue hemorrhagic fever (DHF) was conducted in a cohort of adult volunteers from two textile factories located in West Java, Indonesia. Volunteers in the cohort were bled every three months and were actively followed for the occurrence of dengue (DEN) disease. The first two years of the study showed an incidence of symptomatic DEN disease of 18 cases per 1,000 person-years and an estimated asymptomatic/ mild infection rate of 56 cases per 1,000 person-years in areas of high disease transmission. In areas where no symptomatic cases were detected, the incidence of asymptomatic or mild infection was 8 cases per 1,000 person-years. Dengue-2 virus was the predominant serotype identified, but all four serotypes were detected among the cohort. Four cases of DHF and one case of dengue shock syndrome (DSS) were identified. Three of the four DHF cases were due to DEN-3 virus. The one DSS case occurred in the setting of a prior DEN-2 virus infection, followed by a secondary infection with DEN-1 virus. To our knowledge, this is the first report of a longitudinal cohort study of naturally acquired DF and DHF in adults.     

Emergence of dengue virus type-3 in northern India

During the last few decades dengue has reemerged in several parts of Southeast Asia, including India. A major outbreak of dengue infection occurred in northern India during October to December 2003. To determine the etiology, we carried out serological, virological and molecular investigations of this outbreak. A total of 76 dengue suspected patient blood samples were collected from Gwalior, Madhya Pradesh and Delhi, India. Serological investigations carried out using an in-house Dipstick ELISA protocol revealed the presence of anti-dengue antibodies in 53 patients. Twelve of them (22%) had a positive IgM response, indicative of primary infection, and 22 of them (42%) revealed only IgG antibodies, indicative of secondary infection. RT-PCR analysis employing dengue group specific amplimer revealed the presence of dengue specific RNA in four acute phase samples. These four RT-PCR positive samples were further processed for virus isolation in C6/36 cells and suckling mice, yielding four dengue virus isolates. The Nested PCR analysis employing serotype specific amplimer revealed the presence of dengue-3 specific 389 bp amplicon. This study confirmed the reemergence of dengue virus type-3 in a dominant form in India after a gap of nine years. Earlier, dengue virus type-2 was implicated as the etiology of a major dengue epidemic in Delhi in 1996 and Gwalior in 2001. The implication of dengue type-3 as etiology of a DHF epidemic in neighboring Sri Lanka and Bangladesh recently confirms the reemergence of dengue type-3 as the dominant form on the Indian subcontinent.     

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.     

Molecular characterization and clinical evaluation of dengue outbreak in 2002 in Bangladesh

During the febrile illness epidemic in Bangladesh in 2002, 58 people died out of the 6,132 affected. Two hundred hospitalized patients were analyzed clinically, serologically and virologically to determine the features of this dengue infection. Among the 10- to 70-year-old age group of the 200 clinically suspected dengue patients, 100 (50%) were confirmed as dengue cases by virus isolation and dengue IgM-capture ELISA. Of the 100 dengue-confirmed cases, the mean age was 29.0 (+/-12.4). The possible dengue secondary infection rate determined by Flavivirus IgG-indirect ELISA was 78% in 2002. Eight dengue virus strains were isolated, representing the first dengue virus isolation in the country, and all of the strains were dengue virus type-3 (DEN-3). Sequence data for the envelope gene of the DEN-3 Bangladeshi isolates were used in a phylogenetic comparison with DEN-3 from other countries. A phylogenetic analysis revealed that all 8 strains of DEN-3 were clustered within a well-supported independent sub-cluster of genotype II and were closely related to the Thai isolates from the 1990s. Therefore, it is likely that the currently circulating DEN-3 viruses entered Bangladesh from neighboring countries.     

Relationship of preexisting dengue virus (DV) neutralizing antibody levels to viremia and severity of disease in a prospective cohort study of DV infection in Thailand

BACKGROUND: Infection with any 1 of the 4 dengue viruses (DVs) can produce several illnesses, ranging from a mild febrile illness to classic dengue fever (DF) to dengue hemorrhagic fever (DHF), a potentially life-threatening disease. Most DHF cases occur after sequential heterotypic DV infections. The role of preexisting humoral immunity in modifying severity of dengue disease is not well understood. METHODS: We conducted a prospective cohort study of children in a region where dengue disease is hyperendemic and examined the role of preexisting neutralizing anti-DV antibodies (Abs) in modifying secondary dengue-3 virus (D3V), dengue-2 virus (D2V), and dengue-1 virus (D1V) infections. RESULTS: In secondary D3V infection, higher levels of preexisting neutralizing Ab directed against D3V (reference virus strain and patient's virus isolate) were associated with lower viremia levels and milder disease. Preexisting neutralizing Ab levels against D2V were not associated with severity of secondary D2V infection. The levels of preexisting neutralizing Ab against the infecting virus isolates were not associated with viremia levels in secondary D2V or D1V infections. CONCLUSIONS: Cross-reactive memory humoral immune responses appear to be beneficial in symptomatic secondary D3V infection, but not in secondary D2V or D1V infection. These results may have important implications for the development of live attenuated tetravalent dengue vaccines.     

High circulating levels of the dengue virus nonstructural protein NS1 early in dengue illness correlate with the development of dengue hemorrhagic fever

Infection with any 1 of 4 dengue viruses produces a spectrum of clinical illness ranging from a mild undifferentiated febrile illness to dengue fever (DF) to dengue hemorrhagic fever (DHF), a potentially life-threatening disease. The morbidity and mortality of DHF can be reduced by early hospitalization and careful supportive care. To determine its usefulness as a predictor of DHF, plasma levels of the secreted dengue virus nonstructural protein NS1 (sNS1) were measured daily in 32 children with dengue-2 virus infections participating in a prospective, hospital-based study. Free sNS1 levels in plasma correlated with viremia levels and were higher in patients with DHF than in those with DF. An elevated free sNS1 level (> or =600 ng/mL) within 72 h of illness onset identified patients at risk for developing DHF.     

Surveillance épidémiologique specifique de la dengue: Méthode et intérêt lors de l'épidémie de dengue 2 en Polynésie française en 1996

La dengue est une arbovirose présente dans toute la zone intertropicale. Son extension géographique et l'augmentation simultanée de sa létalité sont préoccupantes. En zone d'endémie ou d'épidémie, une surveillance épidémiologique spécifique de la dengue permet d'identifier et de confirmer au plus vite la circulation d'un nouveau sérotype viral de la dengue: avec un délai d'alerte épidémique raccourci, l'efficacité des stratégies de lutte est renforcée. En Polynésie française, alors que seul le sérotype 3 circulait depuis 1989, la menace d'une épidémie de dengue 4 a déclenché la mise en place d'une surveillance épidémiologique spécifique en mai 1996. Devant chaque cas suspect de dengue, 18 médecins répartis sur le territoire polynésien effectuaient un prélèvement sanguin et rédigeaient une fiche de renseignements cliniques. Entre mai et novembre 1996, le laboratoire de virologie de l'Institut Malardé a mis en évidence 21 virus (2 de dengue 3 et 19 de dengue 2) soit par mise en culture, soit par RT-PCR, sur les 302 prélèvements traités. La surveillance spécifique a démontré la circulation d'un virus dengue 2 et elle a permis de raccourcir de 12 à 16 semaines le délai d'alerte épidémiologique par rapport aux précédentes épidémies. Tenant compte du délai de prélèvement, une régression logistique effectuée sur les variables cliniques a montré que seule l'absence de toux était prédictive de la dengue. Construits autour d'objectifs pragmatiques, la performance des systèmes épidémiologiques de surveillance spécifique de la dengue doit encourager leur mise en ?uvre dans tous les pays concernés, en passant, si nécessaire, par une collaboration avec des laboratoires de référence. Dengue fever is present in tropical and subtropical regions and its geographical extension and the simultaneous increase of its mortality are worrisome. In endemic or epidemic countries, the aim of dengue-specific epidemiological surveillance is to confirm as soon as possible the circulation of a new viral dengue serotype, i.e. the beginning of an epidemic. The efficiency of the control strategy is improved by an earlier epidemic alert. In French Polynesia, dengue-3 virus circulated since 1989 at low level and, in May 1996, a specific epidemiological surveillance was undertaken because of the threat of a dengue-4 epidemic. From each suspected dengue case reported by 18 Polynesian physicians located in the Société Islands, a blood sample was taken for virological assay and clinical data were reported. Between May and November 1996, the virology unit of the Institut Malardé isolated 21 viruses (2 dengue-3 and 19 dengue-2) from 302 suspected cases. The dengue-specific epidemiological surveillance confirmed that dengue-2 virus was circulating and reduced the time of the epidemiological alert by 2 or 3 months compared to previous epidemics. Taking into account the day of illness, a logistic regression undertaken on the clinical data showed that the absence of cough was the only predictive sign of dengue diagnosis. The performance of this dengue-specific epidemiological surveillance system led us to consider its implementation in all concerned countries. A collaboration with international reference laboratories could be a solution for the developing countries.     

Do escape mutants explain rapid increases in dengue case-fatality rates within epidemics?

During the Cuban dengue epidemics of 1981 and 1997, significant monthly increases were observed in the proportion of total cases that presented as dengue haemorrhagic fever or dengue shock syndrome (DHF/DSS), and in case-fatality rates for both dengue fever and DHF/DSS. We believe that theses increases can be explained by the hypothesis that some of the population of antibodies against dengue 1 virus raised after natural primary infections react with "neutralisation" determinants found on dengue 2 viruses. These heterotypic antibodies do not prevent secondary dengue 2 infections, but serve to down-regulate the disease to mild illness or symptomless infections. A population of dengue 2 viruses that replicates in dengue-1-immune hosts escape heterotypic neutralisation. When inoculated into a new dengue-1-immune host, these viruses are free to interact with the more abundant infection-enhancing antibodies to produce severe disease.     

Differing influences of virus burden and immune activation on disease severity in secondary dengue-3 virus infections

Dengue hemorrhagic fever (DHF), the most severe form of illness following infection with a dengue virus, is characterized by plasma leakage, thrombocytopenia, and hepatic inflammation. The interrelationships among virus burden, immune activation, and development of DHF were examined in 54 children with secondary dengue-3 virus infections participating in a prospective, hospital-based study. DHF was associated with higher mean plasma viremia early in illness and earlier peak plasma interferon-gamma levels. Maximum plasma viremia levels correlated with the degree of plasma leakage and thrombocytopenia. Maximum plasma levels of interleukin (IL)-10 and soluble tumor necrosis factor receptor-II correlated with the degree of thrombocytopenia, independently of viremia levels. Hepatic transaminase elevation correlated with plasma soluble IL-2 receptor levels and not with viremia levels. Quantitative differences in virus burden and host immune responses, and the timing of type 1 cytokine responses, have differing influences on the severity of disease manifestations during secondary dengue-3 virus infections.     

Effect of age on outcome of secondary dengue 2 infections.

OBJECTIVE: Dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS) is a growing global health problem. It is not known how age affects the outcome of secondary dengue infections. In an island setting, a large DHF/DSS outbreak in Cuba occurred in 1981. Involved were individuals, 3-40 year old, whose only lifetime dengue exposure was to DEN-1 in 1977 and DEN-2 in 1981. In this report we calculate age-specific DHF/DSS hospitalization and death rates based on secondary DEN 2 infections. METHODS: Published and unpublished hospital and seroepidemiologic data from the 1981 DHF/DSS outbreak were used for the analysis. RESULTS: Children, aged 3 and 4 years, with secondary DEN-2 infections were found to have a high death rate (25.4/10 000 secondary DEN-2 infections). The death rate fell with increasing age, being 15.9-fold lower in the 10-14-year age group. The death rate for children aged 3-14 years was 14.5-fold higher than in young adults aged 15-39 years. The death rate rose somewhat in adults aged 50 years and older. DHF/DSS hospitalization rates showed the same trend as death rates. CONCLUSIONS: Age is an important variable in the outcome of secondary DEN-2 infections. DHF/DSS case fatality and hospitalization rates are highest in young infants and the elderly. The risk that a child will die during a secondary DEN-2 infection is nearly 15-fold higher than the risk in adults.     

Clinical profile and outcome of hospitalized patients during first outbreak of dengue in Makkah, Saudi Arabia

OBJECTIVE: To describe clinical profile of patients with dengue virus infection hospitalized at a single center during the first outbreak of dengue in Makkah, Saudi Arabia from April to July 2004. METHODS: Clinical information and laboratory abnormalities of patients with suspected dengue infection were collected by a standardized data collection sheet and review of medical records. Dengue virus infection was confirmed by a positive IgM capture ELISA or RT-PCR. RESULTS: Of the 160 clinically suspected patients, 91 were confirmed (64 by IgM ELISA, 14 by RT-PCR and 13 by both) to have dengue virus infection. Dengue serotypes 2 and 3 were identified in 19 and 4 patients respectively. Most patients were young adults with median age of 26 (range=6-94) years and male:female ratio of 1.5:1. The common symptoms were fever (100%), malaise (83%), musculoskeletal pain (81%), headache (75%), nausea (69%), vomiting (65%) and abdominal pain (48%). According to World Health Organization (WHO) classification (10 patients were excluded due to lack of serial hematocrits), 75 (93%) had dengue fever (DF) and 6 (7%) had dengue hemorrhagic fever (DHF). Only one patient with DHF was in pediatric age group. Twenty-one patients (5 with DHF and 16 with DF) developed one or more clinical complications that included bleeding (14), shock (4), seizures (3), acute renal failure (2), meningo-encephalitis (1), and secondary bacterial infection (1). Only one patient with shock had dengue shock syndrome (DSS) by WHO classification. Development of clinical complications was significantly associated with absence of musculoskeletal pain (p-value=0.03), lower platelet counts (p-value=0.03) and higher serum aspartate aminotransferase levels (p-value=0.04). The median duration of symptoms and hospitalization was 8 days (range=3-18) and 4 days (range=1-10) respectively. No mortality was noted. CONCLUSION: Occurrence of dengue virus infection in Makkah, Saudi Arabia is documented. Continued surveillance and effective vector control programs are warranted due to unique population dynamics of Makkah that receives millions of pilgrims annually from all over the world.     

Validation of probability equation and decision tree in predicting subsequent dengue hemorrhagic fever in adult dengue inpatients in Singapore

We developed a probability equation and a decision tree from 1,973 predominantly dengue serotype 1 hospitalized adult dengue patients in 2004 to predict progression to dengue hemorrhagic fever (DHF), applied in our clinic since March 2007. The parameters predicting DHF were clinical bleeding, high serum urea, low serum protein, and low lymphocyte proportion. This study validated these in a predominantly dengue serotype 2 cohort in 2007. The 1,017 adult dengue patients admitted to Tan Tock Seng Hospital, Singapore had a median age of 35 years. Of 933 patients without DHF on admission, 131 progressed to DHF. The probability equation predicted DHF with a sensitivity (Sn) of 94%, specificity (Sp) 17%, positive predictive value (PPV) 16%, and negative predictive value (NPV) 94%. The decision tree predicted DHF with a Sn of 99%, Sp 12%, PPV 16%, and NPV 99%. Both tools performed well despite a switch in predominant dengue serotypes.