A Dry-format Field-deployable Quantitative Reverse Transcriptase-polymerase Chain Reaction Assay for Diagnosis of Dengue Infections
Shuenn-Jue WuCurtis G. HayesSajeewane EkanayakeJohnny D. CallahanSilvia LaraKanakatte RaviprakashTadeusz J. KochelKevin R. PorterCurtis G. HayesWilliam M. NelsonKevin R. Porter
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Abstract:
We have systematically evaluated a dry-format, quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) assay developed by Tetracore Inc. for the Cepheid SmartCycler platform to facilitate rapid diagnosis of dengue virus infections. A panel of related flaviviruses was used to evaluate the clinical specificity of the assay, and it was found to be specific to dengue. Eighty-one clinical samples previously confirmed dengue positive by virus isolation, along with 25 dengue negative control specimens were used to validate this new diagnostic assay. Using these clinical samples, the assay exhibited 98.77% sensitivity and 100% specificity. Over 85% of the clinical specimen exhibited viral loads ranging from 10(3) to 10(7) plaque-forming units per milliliter (PFU/mL). In addition, this dry-format assay is stable at ambient temperatures and requires minimal technical expertise to perform in a small thermocycler platform. These characteristics make it a promising candidate for diagnosis of dengue in mobile laboratories in the field.Keywords:
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Dengue fever is a serious mosquito-borne viral disease caused by the dengue virus (DENV) and multiple infections by the mosquitoes could be severe and life-threatening. Currently, there is no remedy against the virus. Accurate and early detection of dengue is vital to reduce the mortality rate due to the severe type of dengue fever. This thesis identifies markers that have the potential to predict the disease. The identification of these biomarkers may aid clinicians in the prediction of patients who may progress to severe dengue so that early treatment could be undertaken to reduce the tendency of fatal dengue.
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Antibody-dependent enhancement
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Background: Dengue fever is a mosquito-borne tropical disease caused by the dengue virus. Symptoms typically begin three to fourteen days after infection. The virus has four serotypes; infection with one type usually gives lifelong immunity to that type, but only short-term immunity to the others. Subsequent infection with different dengue virus increases the risk of severe complications. A number of tests are available to confirm the diagnosis including detection via Dengue NS1Ag Microlisa and Dengue IgM/IgG Microlisa antibody by ELISA Method. Methods: There are three methods followed in particular to detect the dengue virus: (A) Dengue NS1 AG Microlisa is designed for in-vitro qualitative detection of Dengue NS1 antigen in human serum or plasma and is used as a screening test for testing of collected blood samples suspected for DENGUE. This method detects all four subtypes; DEN1, DEN2, DEN3 & DEN4 of Dengue Virus. (B) Dengue IgM Microlisa designed for in-vitro qualitative detection of Dengue IgM Antibody in human serum or plasma and is used as a screening test for testing of collected’ blood samples suspected for DENGUE. This method also detects all four subtypes; DEN1, DEN2, DEN3 & DEN4 of Dengue Virus. (C) Dengue IgG Microlisa designed for in vitro qualitative defection on Dengue IgG antibody in human serum / plasma. Duration – 6 months – July, August, September, October, November, December 2019 at KPC Medical College and Hospital, Kolkata West Bengal. Results: Among 1860 dengue samp1es, 420 sâmp1es were found to be NS1Ag positive 160 samples were found to be IgM positive and 24 samples were found to be IgG positive. Rest Samples negative for all three parameters. Conclusion: Dengue is a mosquito borne viral infection causing a severe flu like illness and sometimes causing a potentially lethal complication called severe dengue. The incidence of dengue has increased 30 fold over the last 50 years. Up to 50-100 million infections are now estimated to occur annually in over 100 endemic countries, putting almost half of the world’s population at risk. If sever dengue fever can damage the lungs, liver or heart or multiorgan failure. Blood pressure can drop to dangerous levels, causing shock and in some cases death.
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Dengue fever (DF) is associated with significant morbidity and mortality in the tropical and sub-tropical regions of the world. Since there are no effective antiviral drugs for treatment, clinicians often rely on the accurate diagnosis of dengue fever to begin supportive therapy at early stages of the illness. The objective of this study was to develop an in-house dengue virus serotype 2 (DENV-2) non-structural protein- 5 (NS5) based indirect ELISA.DENV-2 was raised in Vero cells and the viral proteins were separated and subsequently the NS5 protein was eluted. Serum samples from primary and secondary dengue fever patients; and acute and convalescent samples from Japanese encephalitis (JE) and West Nile virus (WNV) cases were used to validate the ELISA.The assay was found to be 100 per cent specific in detecting DENV-2 specific antibodies from patient's serum. However, in terms of sensitivity, the assay could detect IgM antibodies only from 90 per cent of the primary dengue samples. The IgM/IgG ratio of the primary and secondary samples was 7.24 and 0.64, respectively.The results indicate that the DENV-2 NS5 ELISA is dengue group specific and can be used to differentiate dengue infection from other circulating Flavivirus infections. This NS5 ELISA can also be used to distinguish between primary and secondary dengue fever on the basis of IgM/IgG ratios. Further studies with larger sample sizes and different DENV serotypes are required to validate the ELISA.
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Immunoglobulin M
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Introduction Dengue virus (DENV) infection is currently a major cause of morbidity and mortality in the world; it has become more common and virulent over the past half-century and has gained much attention. Thus, this review compared the percentage of severe cases of both primary and secondary infections with different serotypes of dengue virus. Methods Data related to the number of cases involving dengue fever (DF), dengue hemorrhagic fever (DHF), dengue shock syndrome (DSS) or severe dengue infections caused by different serotypes of dengue virus were obtained by using the SCOPUS, the PUBMED and the OVID search engines with the keywords "(dengue* OR dengue virus*) AND (severe dengue* OR severity of illness index* OR severity* OR DF* OR DHF* OR DSS*) AND (serotypes* OR serogroup*)", according to the MESH terms suggested by PUBMED and OVID. Results Approximately 31 studies encompassing 15,741 cases reporting on the dengue serotypes together with their severity were obtained, and meta-analysis was carried out to analyze the data. This study found that DENV-3 from the Southeast Asia (SEA) region displayed the greatest percentage of severe cases in primary infection (95% confidence interval (CI), 31.22–53.67, 9 studies, n = 598, I2 = 71.53%), whereas DENV-2, DENV-3, and DENV-4 from the SEA region, as well as DENV-2 and DENV-3 from non-SEA regions, exhibited the greatest percentage of severe cases in secondary infection (95% CI, 11.64–80.89, 4–14 studies, n = 668–3,149, I2 = 14.77–96.20%). Moreover, DENV-2 and DENV-4 from the SEA region had been found to be more highly associated with dengue shock syndrome (DSS) (95% CI, 10.47–40.24, 5–8 studies, n = 642–2,530, I2 = 76.93–97.70%), while DENV-3 and DENV-4 from the SEA region were found to be more highly associated with dengue hemorrhagic fever (DHF) (95% CI, 31.86–54.58, 9 studies, n = 674–2,278, I2 = 55.74–88.47%), according to the 1997 WHO dengue classification. Finally, DENV-2 and DENV-4 from the SEA region were discovered to be more highly associated with secondary infection compared to other serotypes (95% CI, 72.01–96.32, 9–12 studies, n = 671–2,863, I2 = 25.01–96.75%). Conclusion This study provides evidence that the presence of certain serotypes, including primary infection with DENV-3 from the SEA region and secondary infection with DENV-2, DENV-3, and DENV-4 also from the SEA region, as well as DENV-2 and DENV-3 from non SEA regions, increased the risk of severe dengue infections. Thus, these serotypes are worthy of special consideration when making clinical predictions upon the severity of the infection. Systematic Review Registration PROSPERO CRD42015026093 (http://www.crd.york.ac.uk/PROSPERO)
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Dengue virus, a mosquito-transmitted flavivirus, is the most common arbovirus infection of humans. Any of four dengue virus serotypes (1, 2, 3, and 4) may cause illness. Infection confers long-lasting protection against reinfection with the same dengue virus serotype but not against other serotypes. Hence, repeated dengue virus infections are possible if an individual is exposed sequentially to several serotypes. Infection with dengue virus may result in dengue fever or the more severe forms of disease known as dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS).
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Antibody-dependent enhancement
Dengue hemorrhagic fever
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The kinetics of total and dengue virus-specific immunoglobulin E (IgE) were studied in serial serum samples obtained from 168 patients, 41 of whom suffered from primary dengue virus infection and 127 suffered from secondary dengue virus infection. Seventy-one patients were classified as dengue fever, 30 as dengue hemorrhagic fever, and 67 as dengue shock syndrome. A control group included single serum samples from patients with a herpes virus infection (n = 14), non-dengue febrile patients (n = 10), and healthy blood donors (n = 10). Patients with dengue virus infection had higher levels of total and dengue virus-specific IgE than non-dengue patients (P < 0.05). Patients with secondary dengue virus infections had not significantly increased levels of both total and dengue virus-specific IgE in the acute phase of disease compared to patients with primary dengue virus infections. Dengue virus-specific IgE was significantly higher in dengue hemorrhagic fever and/or dengue shock syndrome patients compared to dengue fever and non-dengue patients (P < 0.05). In conclusion, this study showed elevated total and dengue virus-specific IgE serum antibody levels in the acute stage of disease. Therefore, measurement of both total and dengue virus-specific IgE serum antibodies can be used as an additional prognostic marker in the development of severe complications in dengue virus infections. In addition, the presence and increase of dengue virus-specific IgE serum antibodies in patients with dengue virus infections is suggestive of the pathogenetic role that IgE may play in the hemostatic disorders observed in dengue hemorrhagic fever and dengue shock syndrome.
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