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PubMed2006

Dengue virus circulation and evolution in Mexico: a phylogenetic perspective.

BACKGROUND: Dengue is the most important arthropod-borne viral infection in the Americas. In the last decades a progressive increment in dengue severity has been observed in Mexico and other countries of the region. METHODS: Molecular epidemiological studies were conducted to investigate the viral determinants of the emergence of epidemic dengue, dengue hemorrhagic fever and dengue shock syndrome as major public health problems in Mexico. Bayesian phylogenetic analyses were conducted to determine the origin, persistence and geographical dispersion of the four serotypes of dengue virus (DENV) isolated in Mexico between 1980 and 2002. Tests for natural selection were also conducted. RESULTS: The origin of some, but not all, strains circulating in Mexico could be inferred. Frequent lineage replacements were observed and were likely due to stochastic events. In situ evolution was detected but not associated with natural selection. Recent changes in the incidence and severity of dengue were temporally associated with the introduction and circulation of different serotypes and genotypes of DENV. CONCLUSIONS: Introduction of new DENV genotypes and serotypes is a major risk factor for epidemic dengue and severe disease. Increased surveillance for such introductions is critical to allow public health authorities to intervene in impending epidemics.

Aedes↗
PubMed2005

Serological evidence for the co-circulation of multiple dengue virus serotypes in Singapore.

We did a seroepidemiological study to determine the circulating dengue virus serotypes and the extent to which the Singapore population has been exposed to multiple dengue virus serotypes, using the plaque reduction neutralization assay (PRNT). Of 164 enrolled subjects aged between 18-30 years, 49 subjects (29.8%) were PRNT positive for at least one dengue serotype. The seroprevalence was 39 (23.8%) for dengue virus serotype 1, 37 (22.6%) for type 2, 43 (26.2%) for type 3, and 30 (18.3%) for type 4. Of the 49 subjects with PRNT-positive dengue virus results, 28 (57.1%) were positive to all four virus serotypes, seven (14.3%) to three serotypes, two (4%) to two serotypes, and 12 (24.5%) to a single serotype. All four dengue virus serotypes circulate in Singapore, and a substantial proportion of the adult population in Singapore had exposure to more than one dengue virus serotype. In spite of multiple circulating types, the rate of dengue haemorrhagic fever is low in Singapore.

Adolescent↗
PubMed1991

Development of cross-reactive antibodies to plasminogen during the immune response to dengue virus infection.

The four serotypes of dengue virus (a mosquito-borne flavivirus) cause an acute febrile illness (dengue fever) or a more prolonged illness with plasma leakage resulting in hypovolemia (dengue hemorrhagic fever). Hemorrhage may accompany either. Epidemiologic data suggest a role for dengue antibodies in pathogenesis. Computer analysis revealed a 20-residue region of similarity in amino acid sequence between the dengue type 4 envelope glycoprotein (E) and a family of clotting factors, including plasminogen, the prime mediator of fibrinolysis. By use of synthetic peptides in ELISA, E antibodies that potentially bind plasminogen were detected in 75% of 40 Thai patients acutely infected with dengue virus type 1, 2, 3, or 4. Plasminogen cross-reactivity of dengue antibodies was shown to be specific for the related sites in E and plasminogen. The dengue E sequence with similarity to plasminogen is largely conserved within the currently known flavivirus E sequences. However, 15 Thai patients hospitalized for illness caused by Japanese encephalitis virus (a flavivirus not associated with hemorrhage) did not develop plasminogen-cross-reactive antibodies, and this finding correlated with failure of Japanese encephalitis virus antibodies to bind to the plasminogen-cross-reactive site in E.

Adolescent↗
PubMed2006

Chimeric Japanese encephalitis virus/dengue 2 virus infectious clone: biological properties, immunogenicity and protection against dengue encephalitis in mice.

A molecular clone of Japanese encephalitis virus (JE virus) was derived from the JE virus Nakayama strain and used to produce infectious JE virus in cell culture. The engineered JE virus resembled the parental JE virus in cell-culture properties and was related closely to other JE virus strains based on nucleotide sequence analysis. The JE virus clone was used as a genetic background for construction of a chimeric virus containing the structural proteins prM and E of Dengue virus, serotype 2. The chimeric JE/dengue 2 virus generated authentic dengue 2 structural proteins as assessed by immunoassays for the dengue E protein. It exhibited a small plaque size and less efficient growth in various cell lines than the parental JE virus. JE/dengue 2 virus was non-neuroinvasive for young adult mice, but displayed partial neurovirulence at doses up to 4 log p.f.u. given intracerebrally. Immunization of 3-week-old mice with JE/dengue 2 virus yielded neutralizing-antibody titres against dengue 2 virus and conferred protection against dengue encephalitis caused by neuroadapted dengue 2 virus. A rise in post-challenge neutralizing-antibody titres against dengue 2 virus in surviving mice suggests that immunization is associated with establishment of a memory antibody response in this model. This study demonstrates the capacity of JE virus to serve as a vector for expression of heterologous flavivirus structural proteins. Similar to previous studies with other chimeric flaviviruses, this approach may be useful as a genetic system for engineering experimental vaccines against Dengue virus and other medically important flaviviruses.

Animals↗
PubMed1993

Applications of polymerase chain reaction for identification of dengue viruses isolated from patient sera.

A simple and sensitive procedure of reverse transcriptase polymerase chain reaction (RT-PCR) was developed previously such that all 4 serotypes of dengue viruses could be detected and their serotypes identified simultaneously in a single-step procedure. In this study we compared the RT-PCR with a conventional immunoperoxidase (PAP) staining method for the identification of dengue viruses currently isolated from patient sera. Sixty-six sera taken from dengue hemorrhagic fever (DHF) patients were subjected to virus isolation by inoculating onto C6/36 cell cultures. Screening for the presence of dengue viruses in culture fluids was done after 7 days of incubation by PAP staining using hyperimmune rabbit anti-dengue virus antibody as the primary reagent. Dengue viruses in positive cultures were further identified for their serotypes by PAP using type-specific monoclonal antibodies (MAb) and by RT-PCR. Thirty-two out of the 66 serum specimens tested (48.5%) were positive for dengue viruses. Of these, 5 were type 1 (DEN-1), 25 were type 2 (DEN-2) and 2 contained both DEN-1 and DEN-2. All cultures that were positive by PAP method were also positive by RT-PCR and vice versa. Thus, the results obtained by RT-PCR were in good agreement with those by PAP. It is important to point out that while all 5 DEN-1 isolates reacted readily with the MAb 1F1, only 2 of them could be identified by the MAb 15F3. Our data suggest that antigenic variation among DEN-1 isolates occur frequently and this should be taken into consideration in the selection of appropriate type-specific MAb for serotyping of dengue viruses.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗
PubMed1990

The dengue viruses.

Dengue, a major public health problem throughout subtropical and tropical regions, is an acute infectious disease characterized by biphasic fever, headache, pain in various parts of the body, prostration, rash, lymphadenopathy, and leukopenia. In more severe or complicated dengue, patients present with a severe febrile illness characterized by abnormalities of hemostasis and increased vascular permeability, which in some instances results in a hypovolemic shock. Four distinct serotypes of the dengue virus (dengue-1, dengue-2, dengue-3, and dengue-4) exist, with numerous virus strains found worldwide. Molecular cloning methods have led to a greater understanding of the structure of the RNA genome and definition of virus-specific structural and nonstructural proteins. Progress towards producing safe, effective dengue virus vaccines, a goal for over 45 years, has been made.

Dengue↗
PubMed2000

An antigen capture enzyme-linked immunosorbent assay reveals high levels of the dengue virus protein NS1 in the sera of infected patients.

We describe the development of a capture enzyme-linked immunosorbent assay for the detection of the dengue virus nonstructural protein NS1. The assay employs rabbit polyclonal and monoclonal antibodies as the capture and detection antibodies, respectively. Immunoaffinity-purified NS1 derived from dengue 2 virus-infected cells was used as a standard to establish a detection sensitivity of approximately 4 ng/ml for an assay employing monoclonal antibodies recognizing a dengue 2 serotype-specific epitope. A number of serotype cross-reactive monoclonal antibodies were also shown to be suitable probes for the detection of NS1 expressed by the remaining three dengue virus serotypes. Examination of clinical samples demonstrated that the assay was able to detect NS1 with minimal interference from serum components at the test dilutions routinely used, suggesting that it could form the basis of a useful additional diagnostic test for dengue virus infection. Furthermore, quantitation of NS1 levels in patient sera may prove to be a valuable surrogate marker for viremia. Surprisingly high levels of NS1, as much as 15 microg/ml, were found in acute-phase sera taken from some of the patients experiencing serologically confirmed dengue 2 virus secondary infections but was not detected in the convalescent sera of these patients. In contrast, NS1 could not be detected in either acute-phase or convalescent serum samples taken from patients with serologically confirmed primary infection. The presence of high levels of secreted NS1 in the sera of patients experiencing secondary dengue virus infections, and in the context of an anamnestic antibody response, suggests that NS1 may contribute significantly to the formation of the circulating immune complexes that are suspected to play an important role in the pathogenesis of severe dengue disease.

Acute Disease↗
PubMed2001

Development and evaluation of serotype- and group-specific fluorogenic reverse transcriptase PCR (TaqMan) assays for dengue virus.

Five fluorogenic probe hydrolysis (TaqMan) reverse transcriptase PCR (RT-PCR) assays were developed for serotypes 1 to 4 and group-specific detection of dengue virus. Serotype- and group-specific oligonucleotide primers and fluorogenic probes were designed against conserved regions of the dengue virus genome. The RT-PCR assay is a rapid single-tube method consisting of a 30-min RT step linked to a 45-cycle PCR at 95 and 60 degrees C that generates a fluorogenic signal in positive samples. Assays were initially evaluated against cell culture-derived dengue stock viruses and then with 67 dengue viremic human sera received from Peru, Indonesia, and Taiwan. The TaqMan assays were compared to virus isolation using C6/36 cells followed by an immunofluorescence assay using serotype-specific monoclonal antibodies. Viral titers in sera were determined by plaque assay in Vero cells. The serotype-specific TaqMan RT-PCR assay detected 62 of 67 confirmed dengue virus-positive samples, for a sensitivity of 92.5%, while the group-specific assay detected 66 of 67 confirmed dengue virus-positive samples, for a sensitivity of 98.5%. The TaqMan RT-PCR assays have a specificity of 100% based on the serotype concordance of all assays compared to cell culture isolation and negative results obtained when 21 normal human sera and plasma samples were tested. Our results demonstrate that the dengue virus TaqMan RT-PCR assays may be utilized as rapid, sensitive, and specific screening and serotyping tools for epidemiological studies of dengue virus infections.

Animals↗
PubMed2001

Detection of dengue viral RNA using a nucleic acid sequence-based amplification assay.

Faster techniques are needed for the early diagnosis of dengue fever and dengue hemorrhagic fever during the acute viremic phase of infection. An isothermal nucleic acid sequence-based amplification (NASBA) assay was optimized to amplify viral RNA of all four dengue virus serotypes by a set of universal primers and to type the amplified products by serotype-specific capture probes. The NASBA assay involved the use of silica to extract viral nucleic acid, which was amplified without thermocycling. The amplified product was detected by a probe-hybridization method that utilized electrochemiluminescence. Using normal human plasma spiked with dengue viruses, the NASBA assay had a detection threshold of 1 to 10 PFU/ml. The sensitivity and specificity of the assay were determined by testing 67 dengue virus-positive and 21 dengue virus-negative human serum or plasma samples. The "gold standard" used for comparison and evaluation was the mosquito C6/36 cell culture assay followed by an immunofluorescent assay. Viral infectivity titers in test samples were also determined by a direct plaque assay in Vero cells. The NASBA assay was able to detect dengue viral RNA in the clinical samples at plaque titers below 25 PFU/ml (the detection limit of the plaque assay). Of the 67 samples found positive by the C6/36 assay, 66 were found positive by the NASBA assay, for a sensitivity of 98.5%. The NASBA assay had a specificity of 100% based on the negative test results for the 21 normal human serum or plasma samples. These results indicate that the NASBA assay is a promising assay for the early diagnosis of dengue infections.

Animals↗
PubMed2003

Development of group- and serotype-specific one-step SYBR green I-based real-time reverse transcription-PCR assay for dengue virus.

A quantitative one-step SYBR Green I-based reverse transcription (RT)-PCR system was developed for the detection and differentiation of four different dengue virus serotypes in acute-phase serum samples. A set of group- and serotype-specific primer pairs was designed against conserved sequences in the core region and evaluated for clinical diagnosis. A linear relationship was obtained between the amount of input RNA and cycle threshold (Ct) value over a range of 10 to 10(7) PFU per ml of cell culture-derived dengue viruses. The detection limit of the group-specific primer pair was between 4.1 and 43.5 PFU/ml for four dengue serotypes. The detection limit of each of the serotype-specific primer pairs was calculated to be 10 PFU/ml for dengue virus serotype 1 (DEN-1), 4.6 PFU/ml for DEN-2, 4.1 PFU/ml for DEN-3, and 5 PFU/ml for DEN-4. Comparisons between the one-step SYBR Green-based RT-PCR assay and the conventional cell culture method in the clinical diagnosis of dengue virus infection from acute-phase serum samples of confirmed dengue patients were performed. The results showed that 83 and 67% of 193 acute-phase serum samples tested were positive by the one-step SYBR Green-based RT-PCR method and cell culture method, respectively. Further analysis showed that the one-step SYBR Green-based RT-PCR method could detect twice as many acute-phase serum samples with positive dengue-specific immunoglobulin M (IgM) and/or IgG antibodies than cell culture method. Our results demonstrate the potential clinical application of the one-step SYBR Green I-based RT-PCR assay for the detection and differentiation of dengue virus RNA.

Acute Disease↗
PubMed2005

Dengue virus nonstructural protein NS5 induces interleukin-8 transcription and secretion.

Elevated circulating levels of chemokines have been reported in patients with dengue fever and are proposed to contribute to the pathogenesis of dengue disease. To establish in vitro models for chemokine induction by dengue 2 virus (DEN2V), we studied a variety of human cell lines and primary cells. DEN2V infection of HepG2 and primary dendritic cells induced the production of interleukin-8 (IL-8), RANTES, MIP-1alpha, and MIP-1beta, whereas only IL-8 and RANTES were induced following dengue virus infection of HEK293 cells. Chemokine secretion was accompanied by an increase in steady-state mRNA levels. No chemokine induction was observed in HEK293 cells treated with poly(I:C) or alpha interferon, suggesting a direct effect of virus infection. To determine the mechanism(s) involved in the induction of chemokine production by DEN2V, individual dengue virus genes were cloned into plasmids and expressed in HEK293 cells. Transfection of a plasmid expressing NS5 or a dengue virus replicon induced IL-8 gene expression and secretion. RANTES expression was not induced under these conditions, however. Reporter assays showed that IL-8 induction by NS5 was principally through CAAT/enhancer binding protein, whereas DEN2V infection also induced NF-kappaB. These results indicate a role for the dengue virus NS5 protein in the induction of IL-8 by DEN2V infection. Recruitment and activation of potential target cells to sites of DEN2V replication by virus-induced chemokine production may contribute to viral replication as well as to the inflammatory components of dengue virus disease.

CCAAT-Enhancer-Binding Proteins↗
PubMed2003

E/NS1 modifications of dengue 2 virus after serial passages in mammalian and/or mosquito cells.

OBJECTIVE: Dengue viruses are routinely maintained in nature by transmission cycles involving the passage of virus between humans and AEDES mosquitoes. The number of dengue virus lineages has been increasing over time. The aim of this study was to identify the genetic diversity of dengue 2 virus serially transferred in mammalian and/or mosquito cells. METHODS: The E/NS1 gene of dengue 2 virus variants derived from serial passages in Vero or C6/36 cells, or alternately in both cell systems, was amplified and sequenced in order to observe gene modification after serial passages. RESULTS: Three nucleotides (two in E and one in NS1) or two amino acids (one each in E and NS1) changed in the virus that was continuously cultured in Vero cells for 20 passages, whereas four nucleotides (two each in E and NS1) or three amino acids (one in E and two in NS1) changed in the virus cultured for 30 passages. The genome of dengue 2 virus remained stable even when the virus was serially transferred in C6/36 cells for 30 generations. However, there was one amino acid substitution (E46 I-->V) resulting from a single nucleotide change in the E region of dengue 2 virus alternately transferred in C6/36 and Vero cells for either 20 or 30 passages. In addition, dengue 2 virus obtained from serially cultured Vero cells usually replicated better when it reinfected Vero cells, reflecting its high adaptation fitness to the host cell. CONCLUSIONS: It is concluded that genetic changes of dengue 2 virus are constrained in Vero (mammalian) cells, resulting in a variety of genome-related quasispecies populations. Some populations of the virus are subsequently selected by and genetically (at least in the E/NS1 portion of the viral genome) maintained in C6/36 (mosquito) cells during replicative competition.

Aedes↗
PubMed2005

Suckling mice were used to detect infectious dengue-2 viruses by intracerebral injection of the full-length RNA transcript.

OBJECTIVE: In this study we established a mouse brain injection system to detect infectious dengue-2 virus produced from the full-length RNA transcripts. METHODS: In vitro transcription was used to synthesize full-length dengue-2 virus RNA from the plasmid pRS424FLDEN2NGC, which was intracerebrally injected into the 6-day-old suckling mice (ICR strains). Engineered dengue-2 viruses were detected in the brain sections using immunohistochemistry staining. RT-PCR followed by restriction endonuclease BstEII digestion was used to confirm the mosquito C6/36 cells cocultured with the mouse brain extract. RESULTS: The mice inoculated with the full-length dengue-2 viral RNA transcript showed paralysis symptoms and died between day 10 and 13 postinjection. The dengue-2 virus-specific antigens (E, Core and NS1) were detected in all the brain and part of the liver sections of the paralyzed mice by immunohistochemistry staining, indicating the existence of dengue-2 virus in these tissues of the suckling mice. The viruses detected in the brains of suckling mice were indeed infectious, which was further confirmed by coculturing mosquito C6/36 cells with the brain extract of the injected mice. CONCLUSIONS: We developed an in vivo approach to detect and produce engineered dengue viruses with infectivity from the full-length plasmid cDNA. This suckling mice system will also aid in screening the infectious viruses that are created by site-directed mutagenesis and is useful for the studies of dengue virus gene function and pathogenesis in the host.

Animals↗
PubMedDate not supplied

Detection and identification of dengue virus isolates from Brazil by a simplified reverse transcription-polymerase chain reaction (RT-PCR) method.

We show here a simplified RT-PCR for identification of dengue virus types 1 and 2. Five dengue virus strains, isolated from Brazilian patients, and yellow fever vaccine 17DD as a negative control, were used in this study. C6/36 cells were infected and supernatants were collected after 7 days. The RT-PCR, done in a single reaction vessel, was carried out following a 1/10 dilution of virus in distilled water or in a detergent mixture containing Nonidet P40. The 50 microliters assay reaction mixture included 50 pmol of specific primers amplifying a 482 base pair sequence for dengue type 1 and 210 base pair sequence for dengue type 2. In other assays, we used dengue virus consensus primers having maximum sequence similarity to the four serotypes, amplifying a 511 base pair sequence. The reaction mixture also contained 0.1 mM of the four deoxynucleoside triphosphates, 7.5 U of reverse transcriptase, 1U of thermostable Taq DNA polymerase. The mixture was incubated for 5 minutes at 37 degrees C for reverse transcription followed by 30 cycles of two-step PCR amplification (92 degrees C for 60 seconds, 53 degrees C for 60 seconds) with slow temperature increment. The PCR products were subjected to 1.7% agarose gel electrophoresis and visualized by UV light after staining with ethidium bromide solution. Low virus titer around 10(3, 6) TCID50/ml was detected by RT-PCR for dengue type 1. Specific DNA amplification was observed with all the Brazilian dengue strains by using dengue virus consensus primers. As compared to other RT-PCRs, this assay is less laborious, done in a shorter time, and has reduced risk of contamination.

Brazil↗
PubMed2006

Activated peripheral lymphocytes with increased expression of cell adhesion molecules and cytotoxic markers are associated with dengue fever disease.

The immune mechanisms involved in dengue fever and dengue hemorrhagic/dengue shock syndrome are not well understood. The ex vivo activation status of immune cells during the dengue disease in patients was examined. CD4 and CD8 T cells were reduced during the acute phase. Interestingly, CD8 T cells co-expressing activation marker HLA-DR, Q, P, and cytolytic granule protein-Tia-1 were significantly higher in dengue patients than in controls. Detection of adhesion molecules indicated that in dengue patients the majority of T cells (CD4 and CD8) express the activation/memory phenotype, characterized as CD44HIGH and lack the expression of the naïve cell marker, CD62L LOW. Also, the levels of T cells co-expressing ICAM-1 (CD54), VLA-4, and LFA-1 (CD11a) were significantly increased. CD8 T lymphocytes expressed predominantly low levels of anti-apoptotic molecule Bcl-2 in the acute phase, possibly leading to the exhibition of a phenotype of activated/effector cells. Circulating levels of IL-18, TGF-b1 and sICAM-1 were significantly elevated in dengue patients. Early activation events occur during acute dengue infection which might contribute to viral clearance. Differences in expression of adhesion molecules among CD4 and CD8 T cells might underlie the selective extravasation of these subsets from blood circulation into lymphoid organs and/or tissues. In addition, activated CD8 T cells would be more susceptible to apoptosis as shown by the alteration in Bcl-2 expression. Cytokines such as IL-18, TGF-b1, and sICAM-1 may be contributing by either stimulating or suppressing the adaptative immune response, during dengue infection, thereby perhaps establishing a relationship with disease severity.

Acute Disease↗
PubMed2003

Dengue severity in the elderly in Puerto Rico.

OBJECTIVE: Severe dengue affects all age groups in the Americas, but little detailed information is available about this disease in the elderly. The objective of this article is to describe the disease in this age group. METHODS: We reviewed suspected dengue-case investigation forms submitted with diagnostic samples as well as clinical reports from infection control nurses in Puerto Rico, for the period of 1994 through 1999. RESULTS: We assigned the laboratory-positive case-patients to four age groups: infants (1 year: 554), youth (2 to 18 years: 6 857), adults (19 to 64 years: 9 433), and elderly (>/= 65 years: 822). Regardless of infecting serotype, the elderly were more likely to have been hospitalized (48% vs. 33%) (P < 0.01) and were less likely to show hemorrhage (26% vs. 33%) (P < 0.01). On multivariate analysis, controlling for gender and the presence of hemorrhage, the elderly had a higher risk for hospitalization and death than did the youths and the adults. CONCLUSIONS: The elderly appear to be more likely than youth and younger adults to develop severe illness when infected with the dengue virus, in a pattern similar to that of infants. The clinical evaluation of elderly patients with dengue must include a careful assessment of increased capillary permeability and occult hemorrhage in order to avoid complications from delayed identification and treatment of severe dengue infection. These findings are of increasing importance for dengue epidemiology and medical care in view of the expanding nature of dengue and dengue hemorrhagic fever in a world that also has a growing number and proportion of elderly persons.

Adolescent↗
PubMedDate not supplied

A dengue outbreak among camp participants in a Caribbean island, 1995.

BACKGROUND: Dengue, a mosquito-transmitted viral disease, is a risk for visitors in tropical and subtropical areas. Several participants in a community-assistance program in Tortola, British Virgin Islands, in August, 1995, reported dengue-like symptoms either before or soon after leaving the island. METHODS: We conducted a retrospective cohort study to determine the extent of the outbreak, risk factors for illness, and the proportion of inapparent infections. Program participants were interviewed by telephone or mail, and asked to submit a serum sample for dengue diagnosis. A clinically-diagnosed case of dengue was defined as a person with fever and two or more of the following: headache, retro-orbital pain, myalgia, arthralgia, rash, or hemorrhagic manifestations. Serum specimens were tested for virus isolation, polymerase chain reaction (PCR), plaque-reduction neutralization (PRNT) or anti-dengue IgM and IgG antibody. RESULTS: Thirty-two (97%) of the 33 program participants responded; 21 of the 32 (66%) provided at least one serum sample for study. The median age was 17 years; 20 (62%) were women. Of 32 respondents, 22 (69%) met the clinical case definition for dengue: 15 of them (68%) had a positive IgM antibody response and 7 did not submit a serum sample. Dengue 1 virus (DEN-1) was identified by PCR in one case and all 11 positive PRNT results. No asymptomatic infections were identified. No respondent used effective mosquito repellent, and only 2 (6%) used bednets. CONCLUSIONS: A DEN-1 outbreak with a high attack rate (69%) occurred in a group of young short-term community aid workers. There were no asymptomatic infections documented. Participants' rare use of bednets or effective mosquito repellent highlights the importance of providing travelers to tropical areas with information about dengue fever and the recommended precautions to protect against infection.

Adolescent↗
PubMed2006

A prospective clinical study on the use of reverse transcription-polymerase chain reaction for the early diagnosis of Dengue fever.

Laboratory testing for dengue virus is used to confirm the diagnosis of dengue virus infection and to differentiate dengue from other febrile tropical illnesses. There are few data on the clinical use of reverse transcription-polymerase chain reaction (RT-PCR) for diagnosis of dengue virus infection. We prospectively evaluated 121 consecutive patients with possible dengue who had samples submitted for RT-PCR, IgM serology, and virus culture. Results were compared with the final discharge diagnosis. Semi-nested RT-PCR was performed using genus- and serotype-specific NS3 consensus primers. Results of 112 patients were available for the final analysis. The RT-PCR was positive in 40 of 62 patients with dengue. Patients who were RT-PCR-positive alone showed a mean of 4.4 days to RT-PCR positivity compared with 5.9 days in patients who were RT-PCR-negative and IgM serology-positive (P = 0.03, Mann-Whitney U-test). The sensitivity, specificity, negative predictive value, and positive predictive value were 70, 100, 84, and 100%, respectively, for samples analyzed within 5 days of illness onset. The RT-PCR also provided epidemiological data regarding the prevailing dengue virus serotypes: 25 with Den-2, eight with Den-3, and seven with Den-1 infection. We propose an algorithm of dengue testing that uses RT-PCR within 5 days of illness onset, whereas IgM capture enzyme-linked immunosorbent assay is preferred for those presenting later.

Dengue↗