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PubMed2003

Molecular epidemiology of dengue virus type 3 in Venezuela.

During the past 40 years, dengue haemorrhagic fever and dengue shock syndrome (DHF/DSS) have emerged in humans, with approximately 3 million cases reported and over 58 000 deaths. Dengue virus serotypes 1, 2 and 4 (DENV-1, -2 and -4) have been co-circulating in Venezuela for at least the past 10 years, causing minor or major outbreaks of dengue fever (DF) and DHF/DSS. The first recorded outbreak due to DENV-3 in Venezuela dates to 1964 and the virus then seems to have disappeared. However, DENV-3 re-appeared recently (in July, 2000) in Venezuela after 32 years of absence and produced a prolonged major outbreak, which, by the end of 2001, involved 83 180 cases of dengue, mostly DF (92 %). Previous phylogenetic studies revealed that the DENV-3 circulating during the 1960s Latin American outbreak was a genotype V virus. To gain a better understanding of the nature of the current epidemic, the complete sequence was determined of the envelope (E) gene of 15 Venezuelan DENV-3 viruses isolated during 2000 and 2001 from patients presenting with different disease severity. Sequence data were used in phylogenetic comparisons with global samples of DENV-3. Analysis revealed that the strain circulating in Venezuela is closely related to isolates that were previously present in Panama and Nicaragua in 1994 and since then have spread through Central American countries and Mexico. This study also confirms previous reports showing that the DENV-3 strain currently circulating in the Americas is related to the strain that caused DHF epidemics in Sri Lanka and India in 1989-1991 (genotype III). Finally, no evidence of the re-emergence of the strain that circulated in Venezuela in the late 1960s and 1970s (genotype V) was found.

Americas↗
PubMed2004

Association of increased platelet-associated immunoglobulins with thrombocytopenia and the severity of disease in secondary dengue virus infections.

Severe thrombocytopenia and increased vascular permeability are two major characteristics of dengue haemorrhagic fever (DHF). To develop a better understanding of the roles of platelet-associated IgG (PAIgG) and IgM (PAIgM) in inducing thrombocytopenia and its severity of disease in patients with secondary dengue virus infection, the relationship between the PAIgG or PAIgM levels and disease severity as well as thrombocytopenia was examined in 78 patients with acute phase secondary infection in a prospective hospital-based study. The decrease in platelet count during the acute phase recovered significantly during the convalescent phase. In contrast, the increased levels of PAIgG or PAIgM that occurred during the acute phase of these patients decreased significantly during the convalescent phase. An inverse correlation between platelet count and PAIgG or PAIgM levels was found in these patients. Anti-dengue virus IgG and IgM activity was found in platelet eluates from 10 patients in an acute phase of secondary infection. Increased levels of PAIgG or PAIgM were significantly higher in DHF than those in dengue fever (DF). An increased level of PAIgM was associated independently with the development of DHF, representing a possible predictor of DHF with a high specificity. Our present data suggest that platelet-associated immunoglobulins involving antidengue virus activity play a pivotal role in the induction of thrombocytopenia and the severity of the disease in secondary dengue virus infections.

Adolescent↗
PubMed1997

First record in America of Aedes albopictus naturally infected with dengue virus during the 1995 outbreak at Reynosa, Mexico.

Mosquito collections were conducted during a dengue outbreak in Reynosa, Tamaulipas, Mexico, July-December 1995. A total of 6694 adult mosquitoes (four genera and nine species) were captured, of which 2986 (78.3% females and 21.7% males) were Aedes albopictus and 2339 (39.7% females and 60.3% males) were Ae.aegypti. These two species comprised 84.2% of the total collection. Specimens were grouped into pools, nearly 50% of them processed for detection of virus by cythopathic effect in C6-36 and VERO cell cultures and by haemagglutination test. Five pools gave positive haemagglutination reactions and were examined by immunofluorescence using monoclonal antibodies to flavivirus and to dengue virus. One pool of ten Ae.albopictus males was positive for dengue virus: serotypes 2 and 3 were identified by serotype-specific monoclonal antibodies and confirmed by RT-PCR. This is the first report of Ae.albopictus naturally infected with dengue virus in America. Also, it is the very first time Ae.albopictus males have been found infected with dengue virus in the wild.

Aedes↗
PubMed2004

Dengue vector prevalence and virus infection in a rural area in south India.

We conducted a 2-year (1997-1999) longitudinal, entomological and virological study in three dengue endemic villages in Vellore district, Tamil Nadu, to understand the dynamics of dengue transmission. Aedes aegypti (Linn.), Ae. albopictus (Skuse) and Ae. vittatus (Bigot) were the prevalent vector species. Aedes aegypti was breeding throughout the year with a Breteau index ranging from 9.05 to 45.49. Aedes albopictus and Ae. vittatus were prevalent mainly in the rainy season. Small water holding containers (cemented tanks/cisterns) were the perennial breeding source of Ae. aegypti, and its abundance was significantly higher in semi-urbanized central areas than the peripheral areas of the villages. From 271 pools (4016 specimens) of adult females, eight dengue virus (DENV) isolates were obtained of which seven were from Ae. aegypti and one from Ae. albopictus. This is the first report of DENV isolation from Ae. albopictus in rural India. Infection rates in the two species were comparable. However, due to higher and perennial prevalence, Ae. aegypti is considered as primary vector with Ae. albopictus playing a secondary role. Despite circulation of all four serotypes (DENV 1-4) detected mainly during the transmission season, the high anthropophilic index of the vectors and their abundance, no human dengue case was reported, suggesting silent dengue transmission.

Aedes↗
PubMed2006

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.

Adolescent↗
PubMed1980

Inhibition of dengue virus replication by amantadine hydrochloride.

The effect of amantadine hydrochloride (1-adamantanamine hydrochloride) on dengue virus replication was examined in vitro. Amantadine decreased the titers of all four types of dengue viruses grown in LLC-MK2 cells by greater than 90% at concentrations of 50 micrograms/ml. There was no evidence for any cytopathic effect of the drug at concentrations less than 100 micrograms/ml. Studies of the time of addition showed that the antiviral effect was maximal when drug was added to virus cultures immediately after the viral adsorption period. In addition, amantadine caused a marked reduction in the growth of dengue virus type 2 in both human and rhesus peripheral blood leukocytes without affecting cell viabili ty. These findings demonstrate that amantadine significantly inhibits the replication of dengue viruses in vitro and indicate a need to determine the efficacy of this drug against dengue virus infections in vivo.

Amantadine↗
PubMed2005

Randomized, placebo-controlled trial of nonpegylated and pegylated forms of recombinant human alpha interferon 2a for suppression of dengue virus viremia in rhesus monkeys.

Dengue fever and dengue hemorrhagic fever are caused by infection with any one of the four dengue viruses (DVs) and are significant public health burdens throughout the tropics. Higher viremia levels are associated with greater dengue disease severity. A therapeutic intervention to suppress viremia early in DV infection could potentially ameliorate severe disease. Recombinant alpha interferon 2a (rIFN-alpha-2a, Roferon-A) suppressed DV replication in human peripheral blood mononuclear cells in vitro. We therefore examined the effects of rIFN-alpha-2a and pegylated recombinant IFN-alpha-2a (PEG-rIFN-alpha-2a, PEGASYS) on DV serotype 2 (DV-2) viremia in rhesus monkeys. Flavivirus-naïve monkeys were inoculated with DV-2 and randomized to receive a single dose of rIFN-alpha-2a (10 million international units/m2) versus placebo or PEG-rIFN-alpha-2a (6 microg/kg) versus placebo 1 day after the onset of viremia. Serial daily viremia levels were measured, and convalescent-phase DV-2 neutralizing antibody titers were determined. Compared to placebo, a single injection of rIFN-alpha-2a temporarily suppressed DV-2 replication and delayed the time to peak viremia by a median of 3 days. However, measures of total viral burden were not different between the two groups. A single injection of PEG-rIFN-alpha-2a significantly lowered daily viremia levels and improved virus clearance, starting 48 h after administration. There were no significant differences in DV-2 neutralizing antibody titers between the treatment and placebo groups at 30 and 90 days postinfection. Based on their individual effects, future studies should investigate a combination of rIFN-alpha-2a and PEG-rIFN-alpha-2a for suppression of dengue virus viremia and as a potential therapeutic intervention.

Animals↗
PubMed2000

Evaluation of six immunoassays for detection of dengue virus-specific immunoglobulin M and G antibodies.

The performance of six commercially available immunoassay systems for the detection of dengue virus-specific immunoglobulin M (IgM) and IgG antibodies in serum was evaluated. These included two IgM and IgG enzyme immunoassays (EIA) from MRL Laboratories and PanBio, a rapid immunochromatographic test (RIT) from PanBio, immunofluorescence assays (IFA) from Progen, a dot blot assay from Genelabs, and a dipstick EIA from Integrated Diagnostics (INDX). For this study a panel of 132 serum samples, including 90 serum samples from patients with suspected dengue virus infection and 42 serum samples from patients with other viral infections, was used. In addition, serial serum samples from two monkeys experimentally immunized and challenged with dengue virus type 2 were used. Results were considered conclusive when concordant results were obtained with four of the six antibody-specific assays. Based on this definition, the calculated overall agreement for the human serum samples for the respective IgM immunoassays was 97% (128 of 132), with 34% (45 of 132) positive serum samples, 63% (83 of 132) negative samples, and 3% of samples (4 of 132) showing discordant results. The calculated overall agreement for the IgG assays was 94% (124 of 132), with 49% (65 of 132) positive, 45% (59 of 132) negative, and 6% (8 of 132) discordant results, respectively. The sensitivities of the dengue virus-specific assays evaluated varied between 71 and 100% for IgM and between 52 and 100% for IgG, with specificities of 86 to 96% and 81 to 100%, respectively. The relative sensitivities of the respective IgM assays measured with the monkey serum samples were comparable with those obtained with 12 serial serum samples from humans. Overall performance, based on the sum of the agreement, sensitivity, specificity, and Kappa statistics of the IgM and IgG immunoassays, showed that the antibody detection systems from INDX and Genelabs and the MRL and PanBio EIA are useful and reliable assays for dengue virus serodiagnosis.

Animals↗
PubMed2001

Use of recombinant envelope proteins for serological diagnosis of Dengue virus infection in an immunochromatographic assay.

An immunochromatographic test that incorporates recombinant antigens (Dengue Duo Rapid Strip Test; PanBio, Brisbane, Australia) has recently become commercially available. This assay is performed in 15 min and detects both immunoglobulin M (IgM) and IgG in a capture format. The four recombinant proteins used represent the N-terminal 80% of the viral envelope glycoproteins of dengue viruses 1, 2, 3, and 4, respectively. The sensitivity and specificity of the recombinant-antigen-based assay were 90 and 86%, respectively. The similar diagnostic performance of these antigens to that of enzyme-linked immunosorbent assays using whole dengue virus suggests that they mimic whole dengue viruses in primary structure and epitope conformation. These results suggest that recombinant proteins can be used in diagnostic assays for dengue to overcome safety issues associated with the use of whole virus.

Antigens, Viral↗
PubMed1992

Rapid detection and typing of dengue viruses from clinical samples by using reverse transcriptase-polymerase chain reaction.

We report on the development and application of a rapid assay for detecting and typing dengue viruses. Oligonucleotide consensus primers were designed to anneal to any of the four dengue virus types and amplify a 511-bp product in a reverse transcriptase-polymerase chain reaction (PCR). First, we produced a cDNA copy of a portion of the viral genome in a reverse transcriptase reaction in the presence of primer D2 and then carried out a standard PCR (35 cycles of heat denaturation, annealing, and primer extension) with the addition of primer D1. The resulting double-stranded DNA product of the RT-PCR was typed by two methods: dot blot hybridization of the 511-bp amplified product to dengue virus type-specific probes or a second round of PCR amplification (nested PCR) with type-specific primers, yielding DNA products the unique sizes of which were diagnostic for each dengue virus serotype. The accumulated data demonstrated that dengue viruses can be accurately detected and typed from viremic human serum samples.

Animals↗
PubMed1998

Detection of specific antibodies in saliva during dengue infection.

Saliva was collected prospectively from patients presenting with suspected dengue infection 4 to 8 days after the onset of symptoms and assayed by a commercial dengue immunoglobulin M (IgM) and IgG capture enzyme-linked immunosorbent assay (ELISA) (PanBio Dengue Duo ELISA). Laboratory diagnosis was based on virus isolation and on hemagglutination inhibition (HAI) assay and an in-house IgM and IgG capture ELISA. With a positive result defined as either salivary IgM or IgG levels above the cutoff value, an overall sensitivity of 92% was obtained for both primary- and secondary-dengue patients (22 of 24), while no patients with non-flavivirus infections (n = 11) and no healthy laboratory donors (n = 17) showed elevation of salivary antidengue antibody (100% specificity). Salivary IgG levels correlated well with serum HAI titer (r = 0.78), and salivary IgG levels could be used to distinguish between primary- and secondary-dengue virus infections.

Antibodies, Viral↗
PubMed2005

Rapid detection and differentiation of dengue virus serotypes by a real-time reverse transcription-loop-mediated isothermal amplification assay.

The development and validation of a one-step, real-time, and quantitative dengue virus serotype-specific reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay targeting the 3' noncoding region for the rapid detection and differentiation of dengue virus serotypes are reported. The RT-LAMP assay is very simple and rapid, wherein the amplification can be obtained in 30 min under isothermal conditions at 63 degrees C by employing a set of four serotype-specific primer mixtures through real-time monitoring in an inexpensive turbidimeter. The evaluation of the RT-LAMP assay for use for clinical diagnosis with a limited number of patient serum samples, confirmed to be infected with each serotype, revealed a higher sensitivity by picking up 100% samples as positive, whereas 87% and 81% of the samples were positive by reverse transcription-PCR and virus isolation, respectively. The sensitivity and specificity of the RT-LAMP assay for the detection of viral RNA in patient serum samples with reference to virus isolation were 100% and 93%, respectively. The optimal assay conditions with zero background and no cross-reaction with other closely related members of the Flavivirus family (Japanese encephalitis, West Nile, and St. Louis encephalitis viruses) as well as within the four serotypes of dengue virus were established. None of the serum samples from healthy individuals screened in this study showed any cross-reaction with the four dengue virus serotype-specific RT-LAMP assay primers. These findings demonstrate that RT-LAMP assay has the potential clinical application for detection and differentiation of dengue virus serotypes, especially in developing countries.

Dengue↗
PubMed2006

Development of real-time reverse transcriptase PCR assays to detect and serotype dengue viruses.

Serotyping dengue virus (DENV) from suspect human specimens is crucial for developing sound epidemiological control measurements early in the transmission season and for effective patient management. We modified DENV consensus D1 (mD1) and serotype-specific TS2 (mTS2) and redesigned serotype-specific TS1 (rTS1) and TS4 (rTS4) as described previously in the conventional capsid and premembrane gene (C-prM) protocol (R. S. Lanciotti, C. H. Calisher, D. J. Gubler, G.-J. Chang, A. V. Vorndam, J. Clin. Microbiol. 30:545-551, 1992). In addition, we designed two new sets of amplimers and probes, located at nonstructural protein 5 (NS5) and the 3' noncoding region (3'NC) of DENV. The NS5 protocol utilizes two flaviviral consensus outer amplimers (mFU1 and CFD2) and four dengue virus serotype-specific TaqMan fluorogenic probes. The 3'NC protocol uses two DENV consensus amplimers, DC10418 and CDC10564. The conventional gel-based, heminested detection method was adapted for the C-prM protocol for detecting and serotyping dengue viruses. In addition, we developed the real-time SYBR green I and postamplification melting temperature curve analysis for the mD1/TS and 3'NC protocols using identical amplification conditions. The NS5 amplimer/probe set was formulated as a one-tube, multiplex, real-time reverse transcriptase PCR for serotype identification. Three sets of amplimers and probes were verified for their specificity in tests with yellow fever, Japanese encephalitis, St. Louis encephalitis, and West Nile viruses; optimized against 109 DENV strains; and validated for detection of the virus in sera from two different panels of acute-phase human dengue serum specimens and one panel of virus isolates from dengue patients' serum specimens. Clinical evaluation by two separate laboratories indicated that the C-prM was more sensitive (100%) than the NS5 (91%) or the 3'NC (91%) protocol.

3' Untranslated Regions↗
PubMed1994

Recognition of envelope protein by dengue virus serotype-specific human CD4+ CD8- cytotoxic T-cell clones.

We analyzed dengue virus-specific CD4+ CD8- cytotoxic T lymphocytes (CTL) at the clonal level to further understand their role in dengue virus infections. Stimulation of peripheral blood mononuclear cells from two dengue virus type 4 (D4V)-immune donors with live D4V or noninfectious D4V antigen generated 17 HLA class II-restricted CD4+ CTL capable of specific lysis of dengue virus antigen-treated autologous lymphoblastoid cell lines. Thirteen clones were D4V specific, three clones were cross-reactive for D2V and D4V, and one clone was cross-reactive for D1V, D3V, and D4V. Antigen recognition by six D4V-specific clones and three D2V- and D4V-cross-reactive clones was restricted by HLA-DR7. Five D4V-specific CD4+ CTL clones lysed autologous lymphoblastoid cell lines infected with a dengue virus-vaccinia virus recombinant containing the E gene of D4V, whereas three serotype-cross-reactive CTL clones did not. These results indicate that E-specific clones are serotype specific and HLA-DR7 restricted in these two donors and suggest that a common epitope on E protein may be recognized. E protein-specific CD4+ CTL may be important mediators of virus clearance especially during reinfection with the same serotype as that in primary infection by providing help for virus-specific antibody production and lysis of virus-infected cells.

Antigens, Viral↗
PubMed2005

[Effectiveness of early dipyrone administration on severity of dengue virus infection in a prospective cohort].

OBJECTIVE: To evaluate the impact of dipyrone administration in the first days of the disease on the severity of dengue infection. MATERIALS AND METHODS: Prospective cohort study in adults (> 12 years) with dengue virus infection, confirmed by specific IgM titers in paired samples or isolation of the virus. The patients were enrolled in the first 96 hours of the disease. Basic demographic details, symptoms, drugs received and examination findings were recorded at admittance. A detailed clinical examination and hematocrit determination were done every day by a member of the study team until day 7 of disease. Platelet count was performed at baseline and with the development of spontaneous bleeding or evidence of plasma leakage. The association between dipyrone use in the first 96 hours of disease and the risk of dengue hemorrhagic fever (DHF) was evaluated. RESULTS: Seven of the 110 patients enrolled developed DHF. At admission, 17 patients had received dipyrone and administration of this drug was not related to the clinical manifestations of dengue, or the use of other medication. Dipyrone was associated with an increased risk of presenting DHF (RR = 7.29; 95% CI: 1.79-29.34; P = 0.0016). Minimum platelet count in the dipyrone group (median: 105,588.2 plat/microl), was significantly smaller than that of the control group (median: 145,698.9 plat/microl): difference = 40,110.69 plat/microl; 95% CI: 1597.36-78,624.02; P = 0.0414. CONCLUSIONS: Our data suggest that early administration of dipyrone in patients with disease caused by dengue virus is associated with lower platelet counts and an increased risk of developing DHF.

Adolescent↗
PubMed2000

Efficient diagnosis of dengue infections using patients' peripheral blood leukocytes and serum/plasma.

OBJECTIVE: Dengue fever has been one of the most important health problems in Taiwan since a large outbreak during 1987 and 1988. It is critically necessary to have a diagnostic approach that can detect early infections in an outbreak or even find infections existing in silent transmission of the disease. METHODS: To develop an efficient diagnostic protocol, 105 plasma/serum and 35 peripheral blood leukocyte (PBL) specimens from the 1994 outbreak in southern Taiwan were collected for assessment by various diagnostic techniques in this study. RESULTS: In acute blood samples, dengue viruses were isolated from 19.4% (14/72) and 33.3% (14/42) of reported and confirmed cases, respectively. Viral RNA in serum/plasma was detected from 20.0% (12/60) of acute samples, which was significantly higher than that from convalescent samples (3/44; 6.8%). However, viral RNA in PBLs, detected by reverse transcription polymerase chain reaction (PBL-RT-PCR), could be observed in 73.2% (19/26) and 66.7% (6/9) of acute and convalescent samples, respectively. The persistence of dengue viruses in PBLs was also evidenced by the presence of viral antigens in 42.9% (4/7) of confirmed convalescent samples by the immunofluorescence antibody test. In addition, IgM antibodies were detected in 43.8% (46/105) of reported cases and 85.2% (46/54) of confirmed cases by the IgM antibody capture enzyme-linked immunosorbent assay (MAC-ELISA). CONCLUSIONS: Although IgM antibody detection achieved the highest detection rate among techniques assessed in this study, no individual test can actually reach full efficiency for early diagnosis of dengue infections. Here, we propose a protocol which applies MAC-ELISA and PBL-RT-PCR in sequence, by which 22 confirmed cases were definitely proved as dengue positive. High levels of both sensitivity and specificity were shown in this protocol.

Antibodies, Viral↗
PubMed2003

Improved detection of dengue-1 virus from IgM-positive serum samples using C6/36 cell cultures in association with RT-PCR.

Dengue is the most important arboviral disease in the world, and its diagnosis is primarily made by serology. Virus isolation has been successful mainly in clinical samples obtained during the acute phase of illness, and is carried out through inoculation of clinical samples into C6/36 cell monolayers followed by the detection of infection by indirect immunofluorescence assay (IFA). We compared the efficiency of RT-PCR and IFA in the detection of dengue-1 virus after inoculation of C6/36 cells with samples obtained in the convalescent period of dengue infection. Out of 75 IgM-positive samples inoculated into C6/36 cells, 2 were positive by IFA while 17 were positive by RT-PCR. The 2 IFA-positive samples were collected during the acute phase of the illness; 17 positive samples were found by RT-PCR, including the 2 detected by IFA. For both methods, we also investigated the time necessary for viral detection using a fixed dose of 1 x 10(4) viruses/ml. RT-PCR and IFA detected the dengue virus 1 and 4 days after virus inoculation, respectively. The results obtained here indicate that RT-PCR is the most sensitive method in the detection of dengue viruses using C6/36 cells for viral isolation.

Animals↗
PubMedDate not supplied

Clinical profile of dengue hemorrhagic fever cases in Mexico.

OBJECTIVE: Dengue hemorrhagic fever is a public health problem in Mexico since 1994. With four serotypes circulating the risk of epidemic dengue hemorrhagic fever is increasing. MATERIAL AND METHODS: We describe the clinical features of confirmed cases in the social security health system (IMSS) from 1995 to 2003. Clinical picture and epidemiological features were compared and a multivariate model was fitted to evaluate associations. RESULTS: Cases were divided into two groups: 438 patients with dengue fever, including 109 cases with hemorrhagic manifestations without thrombocytopenia, and 977 cases with dengue hemorrhagic fever, including 79 deaths. The main risk factors associated with mortality were hematemesis (RR 2.6; CI 95% 1.4-4.6) and melena (RR 2.2; Cl 95% 1.2-3.7). CONCLUSIONS: Our results characterize the clinical profile of dengue hemorrhagic fever cases in Mexico and identify prognostic factors to alert clinician for the prevention of a fatal evolution.

Adult↗