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.     

Serotype-specific dengue virus circulation and dengue disease in Bangkok,Thailand from 1973 to 1999

Dengue virus circulation and association with epidemics and severe dengue disease were studied in hospitalized children with suspected dengue at the Queen Sirikit National Institute of Child Health in Bangkok, Thailand, from 1973 to 1999. Dengue serology was performed on all patients and viral isolation attempted on laboratory-confirmed patients. Acute dengue was diagnosed in 15,569 children and virus isolated from 4,846. DEN-3 was the most frequent serotype in primary dengue (49% of all isolates), DEN-2 in secondary and in dengue hemorrhagic fever (37% and 35%, respectively). The predominant dengue serotype varied by year: DEN-1 from 1990-92, DEN-2 from 1973-86 and 1988-89; DEN-3 in 1987 and 1995-99; and DEN-4 from 1993-94. Only DEN-3 was associated with severe outbreak years. Our findings illustrate the uniqueness of each serotype in producing epidemics and severe disease and underscore the importance of long-term surveillance of dengue serotypes in understanding the epidemiology of these viruses.     

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.     

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.     

Comparison of dengue-1 virus envelope glycoprotein gene sequences from French Polynesia

Dengue (DEN) is the leading arboviral infection of humans, with 100 million cases annually in the tropical areas of the world. The recent severe DEN-1 epidemic in French Polynesia in 2001, with an incidence rate of 16% and more than 45% of the cases with dengue hemorrhagic fever/dengue shock syndrome among 1,400 hospitalized children and eight fatalities, led us to study this new circulating strain. The entire envelope (E) gene of two French Polynesian DEN-1 virus isolates from the two epidemics of 1988-1989 (FP89) and 2001 (FP01) were sequenced and compared with 29 published DEN-1 virus E gene sequences. Phylogenetic relationships showed that the FP89 strain belonged to genotype V and the FP01 strain to genotype IV based on studies on the same region of DEN-1 virus genome (1,485 nucleotides). The recent dengue epidemic in French Polynesia in 2001 was probably due to the introduction of a new DEN-1 virus from Southeast Asia, since the minimum nucleotide divergence was 3.3% with A88, the Indonesian strain isolated in 1988 in Jakarta.     

A dot enzyme immunoassay for dengue 3 virus: comparison with the haemagglutination inhibition test

A dot enzyme immunoassay (DEIA) was used to determine the levels of antibody to dengue 3 virus in the acute and convalescent sera of febrile patients with a clinical diagnosis of dengue fever or dengue haemorrhagic fever. The antibody titres were compared with titres determined by the haemagglutination inhibition (HI) test. The results of the study showed that, besides being more simple to perform, the DEIA is in order of magnitude more sensitive than the HI test. Furthermore, the data suggest that it is possible to use a single dilution as a cutoff point to predict with reasonable accuracy, if a patient has had a recent dengue infection. The DEIA test for antibodies to dengue virus is an appropriate technology highly suitable for rapid diagnosis and surveillance in developing countries.     

The first report on human cases serologically diagnosed as Japanese encephalitis in Indonesia

Although Japanese encephalitis (JE) virus was isolated from mosquitos in 1974, human JE cases have never been reported in Indonesia in spite of the prevalence of anti-JE antibodies among human and pig populations as well as abundant JE vector mosquitos. In this report, we describe serological diagnosis of JE cases in Bali. Indonesia. using IgM-capture ELISA both on serum and cerebrospinal fluid (CSF) of the patients. In the first series of our investigation (Series 1), we examined serum specimens from 12 patients with clinical diagnosis of viral encephalitis, meningitis or dengue hemorrhagic fever (DHF), and found 2 possible JE cases. In the next series (Series 2), we examined both serum and CSF from encephalitis patients and gave laboratory diagnosis of JE. One of them was suspected to have concomitant or recent infection with dengue virus, probably type 3. These results strongly indicated that JE has been prevalent in Bali, Indonesia.     

云南西双版纳州勐腊县一起登革热暴发疫情调查分析

目的分析云南省西双版纳州勐腊县2013年1起登革热暴发疫情的流行病学特征,为登革热控制提供依据。方法对所有登革热病例进行流行病学个案调查,疑似病例血清标本采用登革病毒NS1抗原法进行检测,用RT-PCR进行登革热病毒型别鉴定,采用布雷图指数法进行蚊媒密度监测。结果本次疫情流行历时35d,共发现病例44例,其中本地感染病例34例,输入性病例10例(景洪市7例、缅甸2例、老挝1例);病例主要集中在勐腊县城区,共28例,占63.64%(28/44);男女性别比为1.44:1,发病年龄最小4岁、最大75岁,以20~49岁年龄组为主,共33例,占75.00%;职业以农民、商业服务和家政及待业居多;共检出3个登革血清型(Ⅰ型、Ⅱ型和Ⅲ型),其中老挝输入病例为登革病毒Ⅱ型,缅甸输入病例为登革病毒Ⅰ型,其余为登革病毒Ⅲ型。结论该起疫情属于以登革病毒Ⅲ型为主,传播媒介白纹伊蚊和埃及伊蚊并存的暴发疫情。提示今后应进一步加强登革热输入病例的监测和蚊媒控制工作。     

Absence of dengue 2 infection enhancement in human sera containing Japanese encephalitis antibodies

Sera from 52 young adults resident in a rural area in North Thailand were studied for plaque-reducing neutralizing antibodies against dengue (DEN) viruses types 1-4 and Japanese encephalitis (JE), and for DEN-2 infection-enhancing antibodies using a newly described microtest in the human monocyte cell line, U-937. Infection-enhancing antibody titers in U-937 cells using a simplified micromethod were similar to results obtained by published methods using human peripheral blood leukocytes and a macrotest using U-937 cells. In the sample, there were 23 with antibodies to one or more DEN viruses with or without accompanying JE antibodies; 16 sera demonstrated antibodies only to JE and 13 had no detectable antibodies to any flavivirus. All but two DEN antibody-containing sera enhanced DEN-2 infections in U-937 cells, often to titers of 1:10,000 or greater. By contrast, only one of 16 JE-immune sera enhanced DEN-2 infection in monocytes, and that at a dilution of 1:100. None of the flavivirus-negative sera had DEN-2 enhancing activity. The failure of human anti-JE contrasts with the ability of rabbit anti-JE to enhance DEN-2 infections, but correlates with the absence of recorded instances of dengue shock syndrome in human beings sequentially infected with JE and then a DEN virus. This report seemingly reconciles in vitro and in vivo phenomena, and may provide an opportunity to study mechanisms involved.     

An enzyme-linked immunosorbent assay to characterize dengue infections where dengue and Japanese encephalitis co-circulate

The diagnostic sensitivity and specificity of detection of anti-dengue IgM by antibody capture enzyme-linked immunosorbent assay (ELISA) was investigated in dengue infections in a variety of clinical settings. Sera from uninfected controls were uniformly negative. Serial specimens from experimental and natural infections showed that viremia and fever terminated as anti-dengue IgM became detectable. Anti-dengue IgM appeared in most cases by the 3rd afebrile day of illness and declined to undetectable levels after 30-60 days. Assay sensitivity was 78% in admission sera (924/1,183; 95% CI = 75-81%) and 97% in paired sera (1,030/1,062; 95% CI = 96-98%) thus exceeding or matching the performance of the hemagglutination-inhibition assay. Measurement of the anti-dengue IgM to anti-Japanese encephalitis IgM ratio correctly identified all sera from 112 patients with strictly defined Japanese encephalitis and 98% (307/312; 95% CI = 96-99%) of sera from patients whose dengue infections were confirmed by virus isolation. Dengue infections could be classified as primary or secondary by determining the ratio of units of dengue IgM to IgG antibody. We propose that measurement of dengue and Japanese encephalitis IgM and IgG antibodies upon admission and discharge from hospital care should replace the hemagglutination inhibition assay as the standard dengue serologic technique in regions where these 2 viruses co-circulate.     

Detection of dengue infection by combining the use of an NS1 antigen based assay with antibody detection

We analyzed the utility of a commercial NS1 antigen based ELISA (Panbio Dengue Early ELISA) for detection of dengue infection during the early acute phase and anti-Dengue IgM capture ELISA for detecting dengue infection in patients in dengue endemic settings. A total of 145 serum samples collected from febrile suspected dengue patients were tested for the presence of anti-dengue IgM antibody using IgM antibody Capture ELISA (MAC ELISA) and the presence of dengue virus antigen using PanBio Dengue NS1 Antigen Capture ELISA. Of the 145 patient samples tested, 88 (60.7%) were positive for either NS1 antigen or IgM antibody by MAC ELISA. Dengue NS1 antigen-capture ELISA gave an overall positivity rate of 65.9% (58/88), and IgM ELISA gave an overall positivity rate of 60.2% (53/88). Only NS1 antigen can be used to test during the first two days of fever. MAC ELISAbegins to show positive by the third day of illness and gradually its positivity increases. From Day 3 to Day 7, no significant difference in detection rates was seen between the NS1 assay and MAC ELISA. The NS1 antigen assay may be a useful tool for detecting dengue infection during first few days of fever.     

Acute undifferentiated fever caused by infection with Japanese encephalitis virus

To determine the proportion of acute undifferentiated fevers without neurologic deficits related to infection with Japanese encephalitis (JE) virus, flavivirus serology (dengue and JE) was performed in a cohort of 156 adults presenting to a hospital in Chiangrai, Thailand. Recent flavivirus infection was diagnosed for any individual with an IgM result > 40 units. A ratio of dengue virus IgM to JE virus IgM < 0.91 defined a JE virus infection. Diagnostic criteria for Japanese encephalitis were met in 22 individuals (14%), and were unequivocal in 8 patients. The admission findings in these eight subjects were similar to those described for other flavivirus infections. Thrombocytopenia was the most striking laboratory abnormality (median platelet count = 119,000/mm3, range = 44,000-236,000/mm3). Headache (75%), nausea (50%), myalgia (38%), rash (38%), and diarrhea (25%) were the most frequently encountered signs and symptoms. Infection with Japanese encephalitis virus is an underappreciated cause of acute undifferentiated fever in Asia.     

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.     

Use of dengue NS1 antigen for early diagnosis of dengue virus infection

Accurate and timely diagnosis of dengue virus is important for early detection of dengue virus infection. In this study, the usefulness of the dengue NS1 antigen test was evaluated as a routine, rapid diagnostic test for dengue virus infection. A total of 208 sera from patients suspected of having dengue virus infection were collected and tested for dengue antibody, dengue genome and dengue NS1 antigen. Dengue antibody test, dengue PCR test and dengue antigen test were able to detect dengue virus infection from Days 1 to 8 in 72.8, 52.8 and 44.0% of samples, respectively. Of the 208 sera tested, 69.2% (144/208) of the acute sera were positive for dengue virus infection based on IgM antibody, IgG antibody, NS1 antigen and PCR tests. Thirty-two point two percent of the samples (67/208) were found positive for dengue NS1 antigen, 38.5% (80/208) were PCR positive, 40.9% (85/208) were IgM positive and 36.1% (75/208) were IgG positive for dengue virus. The results reveal the detection rate of dengue virus infection was similar for PCR and dengue antibody (65.9%) and for NS1 antigen and dengue antibody (62.0%) combinations. Therefore, the dengue NS1 antigen test can be used to complement the current antibody test used in peripheral laboratories. Thus, the combination of the NS1 antigen and antibody tests could increase the diagnostic efficiency for early diagnosis of dengue infection.     

Antibody-dependent enhancement of dengue virus growth in human monocytes as a risk factor for dengue hemorrhagic fever

Serum specimens collected during a prospective study of dengue infections among schoolchildren in Bangkok were tested for their ability to enhance dengue 2 (DEN-2) virus growth in human monocytes in vitro. Two groups of dengue-immune sera were compared: 32 dengue antibody positive serum specimens from children who subsequently developed asymptomatic secondary dengue infections; and 9 dengue antibody positive serum specimens from children who subsequently developed severe symptomatic secondary dengue infections, 8 of which were clinically diagnosed as dengue hemorrhagic fever. Antibody-dependent enhancement of virus growth was quantitated by measurement of virus yields in supernatant fluids of normal human monocyte cultures that were infected with DEN-2 virus in the presence of undiluted test serum. Only 4 of 32 (12%) preinfection sera from asymptomatic children, but 6 of 9 (67%) preinfection sera from symptomatic children, had significant enhancing activity (P less than 0.001). High serum DEN-2 antibody dependent enhancing activity is a significant (relative risk = 6.2) risk factor for severe illness among children in a dengue hemorrhagic fever endemic region. Dengue antibodies can be neutralizing and therefore protective, or they can be enhancing and increase the risk of dengue hemorrhagic fever.     

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.     

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。