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PubMed1998

[Dengue in Martinique in 1995-1996].

The incidence of dengue increased sharply in Martinique from the end of 1995 into 1996. Virological tests performed jointly on 36 serum samples by the Pasteur Institute in French Guyana and the Center for Disease Control in Puerto Rico led to identification of serogroups 1, 2, and 4 for six dengue virus. Between January 1995 and December 1996, the Departmental Hygiene Laboratory of Martinique carried out screening tests to detect specific IgM by the immunocapture method (MAC ELISA) in patients with suspected dengue. Results were positive in 701 of the 2,143 patients tested (32.7%). Symptoms were studied in 421 of these positive cases. The most frequent presentation was a flu-like syndrome with hyperalgia. Nausea, vomiting, joint pain, and retroocular pain were frequent. At least one clinical sign of coagulation disturbance was noted in 83 patients (19.7%). Dengue hemorrhagic syndrome was diagnosed according to the criteria of the World Health Organization in six patients including one who developed circulatory collapse and died. This fatality was the first to be reported in Martinique. The incidence of typical dengue as well as of the hemorrhagic form is probably underestimated in Martinique because specific serological tests are not routinely requested and application of WHO criteria for diagnosis of hemorrhagic forms is often impractical.

Adolescent↗
PubMed2006

[Complications associated to severe thrombocytopenia in patients with dengue].

BACKGROUND: Thrombocytopenia is a distinctive characteristic of dengue; however, few studies have related thrombocytopenia with its clinical severity. AIM: To identify the complications of dengue, associated to severe thrombocytopenia, defined as a platelets count below 50,000/mm3). PATIENTS AND METHODS: Patients with clinical and serologic (positive IgM) diagnosis of dengue, consulting at hospitals located in Santander, Colombia, during the period 1993-1998, were studied. Clinical findings and laboratory tests (including hematocrit and platelets counts) were registered. The association between severe thrombocytopenia and the presence of complications, such as hemorrhagic manifestations (positive tourniquet test, petechiae, ecchymoses, bleeding from gums, epistaxis, hematemesis, hematuria and metrorrhagia) and signs of plasma leakage (pleural effusions and haemoconcentration), were evaluated using a univariate and multivariate analysis. RESULTS: Of the 790 patients enrolled, 502 had a platelet count below 50,000/mm3. Severe thrombocytopenia was associated with hemorrhagic manifestations (OR= 3.16; 95% CI: 2.09-4.76; p <0.0001) and signs of plasma leakage (OR= 2.67; 95% CI: 1.86-3.84; p <0.0001). The associations between severe thrombocytopenia and the complications of dengue (with exception of bleeding from gums, hematuria and metrorrhagia), were confirmed with the multivariated analysis (p <0.05). CONCLUSION: There was a strong association between deep thrombocytopenia and the severity of dengue.

Adult↗
PubMed1998

Elevated levels of IL-8 in dengue hemorrhagic fever.

Dengue virus causes dengue fever, a mild febrile illness, and at times dengue hemorrhagic fever (DHF), a severe illness the pathogenesis of which is not fully understood. Given the crucial roles played by interleukin-8 (IL-8) as a chemoattractant cytokine and in inflammatory processes, levels of circulating IL-8 in the sera and IL-8 mRNA in the peripheral blood mononuclear cells (PBMC) were measured in 99 patients of a recent dengue epidemic that occurred in India in 1996 and in 21 normal healthy controls. Twenty-six of the patients had dengue fever (DF) and the remaining 73 were diagnosed as having different grades of DHF. All the control normal sera were negative for IL-8, so were their PBMC for IL-8 mRNA. Increased levels of IL-8 in the sera and IL-8 mRNA in their PBMC were observed in patients with severe illness of DHF grades III and IV. Only two out of 26 patients of DF and one out of 10 DHF grade I patient were positive for IL-8 and all three deteriorated to DHF grade IV within 24 hr. All six patients of DHF grade IV who died had higher serum level of IL-8 above 200 pg/ml, the highest being 5,568 pg/ml in one patient; the presence of mRNA for IL-8 was very high in all patients. A striking correlation was observed between increased levels of IL-8 and severe DHF, with greater levels in patients with increased grade of the disease and death. These results suggest that IL-8 may have an important role and may be an indicator of increasing severity of the disease and death.

Adolescent↗
PubMed1998

Molecular and in vitro analysis of eight dengue type 2 viruses isolated from patients exhibiting different disease severities.

Potential genetic determinants of dengue virulence were studied by sequencing the entire genomes of eight dengue 2 virus strains isolated from patients exhibiting different disease severities during an epidemic season in northeastern Thailand in 1993. The isolates came from one dengue shock syndrome (ThNH-7/93), three dengue hemorrhagic fever, and four dengue fever patients. Phylogenetic analysis showed that the isolates belonged to the Southeast Asian genotype. The 3' noncoding regions showed distinctive secondary structures, with one specific structure for the isolate ThNH-7/93. Analysis of the predicted polyprotein showed several amino acid (aa) changes scattered mostly in the nonstructural region. Of 30 positions with aa changes, 7 were unique to the isolate ThNH-7/93 and 3 of those led to radical alterations in aa character. Several aa changes coincided with previous studies relating genome sequence and virulence. Minimal changes in computer-predicted protein secondary structures were observed. Infective particles in the inoculum for all isolates were approximately equal as measured by focus formation on BHK-21 cells, but this did not correlate with the number of plaques formed on LLC-MK2 cells. Isolates from patients that experienced secondary infection were shown to have significantly larger plaques than the isolates from primary infection patients.

Adolescent↗
PubMed1993

Genetic and biological differentiation of dengue 3 isolates obtained from clinical cases in Java, Indonesia, 1976-1978.

Previous epidemiological, virological and clinical studies have documented a series of outbreaks of dengue fever and dengue haemorrhagic fever/dengue shock syndrome which occurred in Java, Indonesia in 1976-1978. In the current study we compare growth characteristics in cell culture, and nucleotide sequence data for the viral prM and E genes, of five low passage DEN-3 isolates obtained during these epidemics from clinically defined cases. All isolates had the same passage history: human sera were passed twice in mosquitoes and three times in a mosquito cell line (Aedes albopictus, C 6/36 cells). Growth differences were observed between individual isolates in Vero cells; growth differences were not observed in C 6/36 cells. Nucleotide sequencing of the prM and E gene region indicated that no two isolates were identical (sequence divergence ranged from 0.4 to 1.6% in pairwise comparisons) but that they were closely enough related to present a single genetic type. There were one or two differences in deduced amino acid sequence in E between isolates. Differences were at residues 65, 187, 298 or 443. One isolate differed from all others at residue 16 in the M protein. No relationship was apparent between the amino acid sequence of M or E and the nature of the disease profile, the year of isolation or the geographic region of isolation. The isolates showed 3.5 to 4.4% nucleotide sequence divergence from the highly-adapted H 87 prototype, isolated in the Philippines in 1956. The isolates showed a total of twelve common amino acid differences in prM and E proteins from H 87. Ten of these twelve residues were at positions which differed between the four dengue serotypes. Two differences (at residues 37 in M and 293 in E) were at positions which are conserved in sequence between the four dengue serotypes. The data are discussed in relation to the dengue outbreaks in Java in the period 1976-1978.

Aedes↗
PubMed2005

Capsid-targeted viral inactivation can destroy dengue 2 virus from within in vitro.

Capsid-targeted viral inactivation (CTVI) has emerged as a conceptually powerful antiviral strategy that exploits viral structural proteins to target a destructive enzyme specifically into progeny virions. We have recently demonstrated the principle of CTVI against dengue virus infection and observed a modest therapeutic effect in vitro (Arch Virol 2005, 150: 659-669). Here we tested a prophylactic model of CTVI, in which mammalian cells stably expressing the dengue 2 virus capsid protein fused to a nuclease were infected with dengue virus and determined the effects on progeny virion infectivity. CTVI efficiently destroyed dengue 2 virus from within and decreased the infectious titers by 10(3)- to 10(4)-fold, suggesting that CTVI has potential in the prophylactic application for dengue virus infection.

Animals↗
PubMed2002

Use of a rapid immunochromatographic test for early diagnosis of dengue virus infection.

Acute serum samples collected from 92 patients were analysed for the presence of immunoglobulin M and G antibodies against dengue viruses using a rapid immunochromatographic (IC) test. Of 52 patients with dengue virus infection diagnosed using a standard hemagglutination inhibition test, 41 patients demonstrated dengue virus infection using the IC test. Half of the serum samples collected on day 3 after the onset of fever were positive for dengue antibodies, and all were positive on day 6 after symptom onset. In comparison with the hemagglutination inhibition test, the rapid IC test showed 79% sensitivity and 95% specificity. The efficiency of the test was 86%. The IC test showed good agreement with the hemagglutination inhibition test (k=0.72). The IC test is quick to perform and provides early diagnosis of dengue virus infection.

Adolescent↗
PubMed2003

Virus isolation for diagnosing dengue virus infections in returning travelers.

Dengue fever is recognized as one of the most frequent imported acute febrile illnesses affecting European tourists returning from the tropics. In order to assess the value of virus isolation for the diagnosis of dengue fever, 70 cases of dengue fever confirmed in German travelers during the period 1993-2001 were analyzed retrospectively. In 26 patients who had developed acute febrile illness within 2 weeks following their return from a trip to a dengue-endemic area, 9 of 13 attempts to isolate the virus were successful in sera drawn 1-5 days and 2 of 13 sera drawn 6-10 days after the onset of illness. DEN-1 was the most frequent serotype isolated. If performed early, virus isolation is a reliable tool for detecting dengue virus in returning travelers.

Adult↗
PubMed1987

Serological survey on dengue virus infection in Singapore.

A serological study on dengue infection conducted in Singapore during the period 1982 to 1984 showed that 54.4% of the healthy population between 6 months and over 50 years of age surveyed possessed no haemagglutination-inhibition antibody to dengue type 2 virus. Children below 10 years of age showed the lowest antibody prevalence and were at the greatest risk, with 96.6% susceptible to infection, whereas virtually all adults over 40 showed evidence of prior dengue infection. The geometric mean titre showed a rising trend indicating continuing acquisition of infection in the older age groups. The seropositivity rate of dengue infection of males was twice that of females. Among the 3 major ethnic groups, no significant difference in seropositivity was noted between the Malays and Indians, but the differences between Malays and Chinese and between Indians and Chinese were statistically significant. The study confirmed that the successful implementation of the nation-wide Aedes control programme is reducing endemic dengue virus transmission in the country.

Adolescent↗
PubMedDate not supplied

Dengue 1 epidemic in French Polynesia, 1988-1989: surveillance and clinical, epidemiological, virological and serological findings in 1752 documented clinical cases.

An epidemic of dengue 1 occurred in French Polynesia in December 1988 and June 1989. This paper records (i) the trend of the outbreak and its surveillance and (ii) the clinical, epidemiological and virological data obtained from 1752 documented cases. The epidemic reached its peak in February in Tahiti Island, 7 weeks after its recognition. Among 6034 suspect cases reported by sentinel physicians, 60.3% were < 20 years old. The illness was classical dengue. No fatality or case of dengue haemorrhagic fever/dengue with shock syndrome was reported. Of 4792 patients subjected to laboratory testing, 41% were confirmed as positive. The serological attack rate was c. 40%. The estimated number of dengue infections in the Windward Islands was about 20,000. Transmission was associated with Aedes aegypti. Study of documented cases showed a higher confirmation rate in both the civilian population < 15 years old (46.5%) and the susceptible French military population (47.6%) than in older civilians (31.1%, P < 0.05). Furthermore, primary dengue infections were predominant in both of the first 2 groups. The diagnosis was mostly confirmed (i) by virus isolation on day < 5 of illness and (ii) by detection of immunoglobulin (Ig) M on day > or = 5 of illness. The study showed that adequate surveillance of an epidemic requires both clinically and laboratory-based systems.

Dengue↗
PubMed2005

Elevated levels of plasma VEGF in patients with dengue hemorrhagic fever.

Vascular endothelial growth factor (VEGF) can be produced by monocytes and endothelial cells. It plays important role in angiogenesis and vascular permeability. The phenomenon of extensive plasma leakage into various serous cavities of the body is a cardinal symptom of dengue hemorrhagic fever (DHF). This study was performed to investigate the role of VEGF in patients with DHF. Plasma samples collected from the 53 dengue fever (DF) patients (including 14 patients with DHF), and 5 additional subjects with non-dengue febrile illness as controls were tested for VEGF levels using commercial enzyme-linked immunosorbent assay (ELISA) kits. The results showed that median plasma levels of VEGF in the patients with DHF (54.6 pg ml(-1)) were significantly higher than those of DF (14.6 pg ml(-1)) and control group (27.1 pg ml(-1)) (P<0.05). In addition, VEGF levels in DF patients were not significantly different from those of control patients with non-dengue febrile illness (P=0.17). Multiple regression analysis was used to analyze the clinical variables independently associated with VEGF levels. The data showed that D-dimer levels were significantly associated with VEGF levels. In this study, plasma VEGF levels in patients with DHF were significantly higher than values from DF patients. The association between increased plasma VEGF levels and increased plasma D-dimer levels in the patients with dengue illness suggests that activation of the fibrinolytic system may play a role in VEGF production in the patients with DF.

Adult↗
PubMed2006

Kinetics of antibodies in sera, saliva, and urine samples from adult patients with primary or secondary dengue 3 virus infections.

OBJECTIVES: The kinetics of three serological markers (IgM, IgA, and IgG) in serum, saliva, and urine samples from adult patients with primary or secondary dengue infection were studied. DESIGN: Serum, saliva, and urine samples were collected from 22 patients with clinical and confirmed dengue 3 virus infection during the outbreak in Havana City in 2001. They were tested by capture IgM (MAC-ELISA), IgA (AAC-ELISA), and IgE (EAC-ELISA) and IgG ELISA inhibition method (EIM) to detect specific dengue antibodies. RESULTS: Similar kinetics were observed in IgM, IgA, and IgG antibodies in saliva and IgA and IgG in urine samples from secondary cases compared with kinetics in serum samples, although the values were lower. No IgG antibody was detected in saliva and urine samples in primary cases and IgM antibody was not detected in urine samples from either primary or secondary infection. All secondary cases were positive for IgG in saliva and urine samples at day 7. The kinetics of specific IgE antibodies in primary and secondary cases were different. CONCLUSIONS: The kinetics of three serological markers (IgM, IgA, and IgG) in serum, saliva, and urine samples from adult patients with primary or secondary dengue 3 virus infection were studied for the first time, showing its behavior and usefulness in dengue virus diagnosis. The specific IgE could play a role as a serological marker in secondary infections.

Adult↗
PubMed2006

Modelling the relationship between antibody-dependent enhancement and immunological distance with application to dengue.

When antibodies raised in response to a particular pathogen bind with immunologically similar pathogens it may facilitate infection through a phenomenon known as antibody-dependent enhancement (ADE). This process occurs between the four serotypes of dengue virus and, furthermore, secondary infection is a major risk factor in dengue hemorrhagic fever (DHF). Theory has suggested that ADE may be responsible for the large immunological distance between dengue serotypes. We investigate this hypothesis using an epidemic model for dengue in which immunological distance and the strength of immune cross-reaction are expressed separately. Cross-enhancement is considered in three alternative forms acting on susceptibility, transmission and mortality. Previous models have shown that transmission and mortality enhancement can lead to periodicity or chaos. We confirm this result for reasonable levels of susceptibility and transmission enhancement but not for mortality enhancement. We also show that when the two strains have identical basic reproductive numbers no form of enhancement leads to competitive exclusion. When the two strains have different basic reproductive numbers susceptibility or transmission enhancement allow strains with greater immunological similarity to stably coexist but mortality enhancement forces strains to be more distinct. All three forms of enhancement can be associated with DHF and we conclude that mortality enhancement must be dominant if ADE really is responsible for the immunological distance between dengue serotypes.

Antibody-Dependent Enhancement↗
PubMed2005

PCR detection of dengue virus using dried whole blood spotted on filter paper.

Whole blood dried onto filter paper constitutes a potentially useful material for molecular testing of viruses, including dengue. In order to assess the stability of viral RNA, we carried out dengue-RNA detection in whole blood infected with dengue virus that had been previously spotted onto filter paper. Filter papers were stored at room temperature, 4 and -70 degrees C and processed for PCR assay at intervals of 2, 4, 6 and 9 weeks. Our results demonstrated that dengue-RNA was stable in filter paper for 9 weeks at all tested temperatures. Furthermore, we evaluated these conditions using frozen sera and dried blood samples onto filter paper from 52 patients with confirmed clinical diagnosis of dengue infection. PCR results showed a 100% specificity and 93% sensitivity for dried blood samples. This storage method facilitates the transportation and analysis by nucleic acid amplification techniques even when freezing conditions are not available.

Blood↗
PubMed2005

Microarray and real-time RT-PCR analyses of a novel set of differentially expressed human genes in ECV304 endothelial-like cells infected with dengue virus type 2.

The cellular and molecular pathways of dengue infection have not been as intensively studied compared to the host immunological responses. Changes in mRNA expression levels of ECV304 human endothelial-like cells following infection with the virulent New Guinea C strain of dengue virus type 2 were analyzed by a microarray system comprising 7600 oligonucleotide cDNAs. After normalization against the uninfected control using two independent software programs, 111 genes exhibited at least a 1.5-fold difference in expression level. Out of these, 21 mRNAs were upregulated while 90mRNAs were downregulated. Quantitative real-time RT-PCR was then performed to determine the expression patterns of 15 selected genes of interest involved in the cell cycle (MAD3), apoptosis (RIPK3, PDCD8), cellular receptors (H963, CCR7, KLRC3), transcriptional regulation (RUNX3, HNF4G, MIZ1), signal transduction (HSP27, TRIP, MAP4K4), enzymes (angiotensinogen), protein transport (AP4M1), and cytoskeleton (ACTA2). Dengue virus infection resulted in the downregulation of the C-terminal alternatively spliced p53 variant, the pro-apoptotic IG20 and IG20-SV2 isoforms, and the Fas apoptosis inhibitory molecule (FAIM). Most of the real-time RT-PCR data showed concordance with the normalized microarray data. Hence, real-time RT-PCR validation of high-throughput gene microarray screening is important and necessary before further conclusions on gene expression can be drawn. This study elucidated novel information on the complex responses at the transcriptional level in susceptible human endothelial-like cells induced by a virulent dengue virus strain implicated in the pathogenesis of dengue and/or its complications.

Apoptosis↗
PubMed2006

Rapid detection, serotyping and quantitation of dengue viruses by TaqMan real-time one-step RT-PCR.

The use of the polymerase chain reaction (PCR) in molecular diagnosis is now accepted worldwide and has become an essential tool in the research laboratory. In the laboratory, a rapid detection, serotyping and quantitation, one-step real-time RT-PCR assay was developed for dengue virus using TaqMan probes. In this assay, a set of forward and reverse primers were designed targeting the serotype conserved region at the NS5 gene, at the same time flanking a variable region for all four serotypes which were used to design the serotype-specific TaqMan probes. This multiplex one-step RT-PCR assay was evaluated using 376 samples collected during the year 2003. These groups included RNA from prototype dengue virus (1-4), RNA from acute serum from which dengue virus was isolated, RNA from tissue culture supernatants of dengue virus isolated, RNA from seronegative acute samples (which were culture and IgM negative) and RNA from samples of dengue IgM positive sera. The specificity of this assay was also evaluated using a panel of sera which were positive for other common tropical disease agents including herpes simplex virus, cytomegalovirus, measles virus, varicella-zoster virus, rubella virus, mumps virus, WWF, West Nile virus, Japanese encephalitis virus, S. typhi, Legionella, Leptospira, Chlamydia, and Mycoplasma. The sensitivity, specificity and real-time PCR efficiency of this assay were 89.54%, 100% and 91.5%, respectively.

Base Sequence↗
PubMed2004

Ocular manifestations of dengue fever.

PURPOSE: To evaluate ocular manifestations associated with dengue fever. DESIGN: Retrospective case series and literature review. METHODS: Clinical records of patients with dengue fever who subsequently had ocular symptoms and signs develop were reviewed. The clinical presentation and ocular complications were evaluated. RESULTS: Six patients, 5 females and 1 male, were seen with a sudden decrease in vision 6 to 7 days after the initial manifestations of dengue fever were identified. The diagnosis was confirmed by detection of dengue-specific IgM antibodies (IgM enzyme-linked immunoassay). The presenting best-corrected visual acuity ranged from 20/30 to counting fingers, and ocular involvement was bilateral but asymmetric in 5 cases and unilateral in 1 case. Fundus findings included small, intraretinal, whitish lesions, with localized retinal and retinal pigment epithelium (RPE) disturbance, small dot hemorrhages, and vascular sheathing around the macula and the papillomacular bundle. Fluorescein angiography showed arteriolar focal knobby hyperfluorescence at the macula with mild staining of the vascular walls and leakage at the level of the RPE. All 5 cases that had indocyanine green angiography done showed early diffuse choroidal hyperfluorescence with late silhouetting of the larger choroidal vessels. Five patients received steroid therapy: 1 topical, 2 periocular, and 2 oral. Over 2 to 4 months, RPE discoloration was observed over the affected areas. After the acute episode, 3 patients showed partial recovery of vision, and in the remaining patients, the visual acuity remained stable. CONCLUSIONS: Ocular complications associated with dengue fever are rare but may result in permanent visual impairment.

Adult↗
PubMed2005

The performance of RT-PCR compared with a rapid serological assay for acute dengue fever in a diagnostic laboratory.

The laboratory diagnosis of dengue has largely relied on serological assays, although many different RT-PCR protocols have been reported. Owing to its limited use, the value of RT-PCR in the clinical laboratory has not been fully evaluated. During the outbreak of severe acute respiratory syndrome (SARS) in Singapore in 2003, RT-PCR to detect dengue viral RNA was used as a rapid diagnostic tool to differentiate dengue from SARS among patients who presented to a hospital designated to manage and quarantine SARS cases. A total of 343 results for RT-PCR and 439 results for serology were analysed and compared with the final discharge diagnosis. Our experience indicates that RT-PCR for dengue can be set up rapidly in a clinical laboratory, with very sensitive and specific results for the diagnosis of dengue, particularly in the first 5 days from onset of symptoms.

Dengue↗