NS1 ELISA Research Articles

CO-INFECTION OF BRUCELLA AND DENGUE VIRUS

ABSTRACTObjective: Brucellosis is an important zoonotic infection having a worldwide distribution. In South East Asian developing countries like India, brucellosisis yet well-known but often neglected disease. Although dengue fever has a global distribution, South East Asian region together with the WesternPacific region represents 75% of the current global disease burden. Frequent dengue fever outbreaks have been encountered in different parts of India.Both brucellosis and dengue fever may present with many nonspecific symptoms. This study was designed to identify the trend of their co-infection.Methods: The study was carried out from February 2015 to July 2016 in Eastern India. Blood samples collected from clinically suspected denguefever cases were confirmed serologically by dengue specific NS1, IgM, and IgG capture ELISA methods. The confirmed dengue samples were furthersubjected to Brucella specific serological and molecular investigations.Results: Out of 37 serologically confirmed dengue cases, 8 (21.6%) showed co-infection of brucellosis and dengue fever. Female predominance wasobserved among the co-infection cases. Most of these cases belonged to the age group 21-40 years and resided in and around Kolkata.Conclusion: This study portrays the simultaneous acquisition of these two pathogens in a few patients. Co-infection of these two pathogens, whilefeasible, has not been accounted previously in this part of India. Thus, this report highlights the significance of studying the possibility of occurrenceof dual infection of these two diseases and its outcome in patients with febrile illness thereby providing future scope of preventing case fatalities.Keywords: Brucellosis, Dengue, Co-infection, ELISA, Polymerase chain reaction.

Accuracy of clinical criteria and an immunochromatographic strip test for dengue diagnosis in a DENV-4 epidemic.

BackgroundEarly diagnosis of dengue infection is important for decision-making and timely implementation of therapeutic measures. Although rapid NS1 assays have been used for dengue diagnosis since 2008, their performance in DENV-4 cases has not yet been fully assessed.MethodsWe evaluated the accuracy of NS1 Bioeasy™ immunochromatographic strip test and of three clinical criteria for dengue diagnosis. Patients presenting at an emergency care center within 72 h of an acute febrile illness during the 2013 DENV-4 epidemic in Rio de Janeiro were consecutively enrolled for clinical and laboratory evaluation. We classified patients as suspected dengue or not according to three clinical criteria: WHO 2009, WHO 1997, and INI-FIOCRUZ. Dengue diagnosis was defined by RNA detection using RT-PCR and the negative cases were negative for all dengue serotypes and also Platelia™ NS1 ELISA. We obtained accuracy indices for NS1 Bioeasy™ alone and in combination with the clinical criteria.ResultsRT-PCR for DENV-4 was positive in 148 out of 325 patients. Positive likelihood ratio, sensitivity, and specificity of NS1 Bioeasy™ with WHO 2009, WHO 1997, and INI-FIOCRUZ criteria were 22.6 (95 % CI 7.2–70.6), 40.6 % (95 % CI 32.3–49.3), and 98.2 % (95 % CI 94.9–99.6); 18.3 (95 % CI 6.8–49.2), 44.2 (95 % CI 35.8–52.9), 97.6 (95 % CI 94.0–99.3); 26.2 (95 % CI 6.5–106.5), 29.7 (95 % CI 22.4–37.8), 98.9 (95 % CI 96.0–99.9), respectively. WHO 1997 clinical criteria presented high sensitivity to rule out disease, but extremely low specificity. INI-FIOCRUZ had moderate sensitivity and specificity, and could target a group to a more specific test.ConclusionsAlthough the large rates of false negative results using NS1 Bioeasy™ rapid test advise against its use for triaging (rule out) purposes in DENV-4 epidemics, it could be used as a confirmatory tool in a bedside algorithm.

Value of Routine Dengue Diagnostic Tests in Urine and Saliva Specimens.

BackgroundDengue laboratory diagnosis is essentially based on detection of the virus, its components or antibodies directed against the virus in blood samples. Blood, however, may be difficult to draw in some patients, especially in children, and sampling during outbreak investigations or epidemiological studies may face logistical challenges or limited compliance to invasive procedures from subjects. The aim of this study was to assess the possibility of using saliva and urine samples instead of blood for dengue diagnosis.Methodology/Principal FindingsSerial plasma, urine and saliva samples were collected at several time-points between the day of admission to hospital until three months after the onset of fever in children with confirmed dengue disease. Quantitative RT-PCR, NS1 antigen capture and ELISA serology for anti-DENV antibody (IgG, IgM and IgA) detection were performed in parallel on the three body fluids. RT-PCR and NS1 tests demonstrated an overall sensitivity of 85.4%/63.4%, 41.6%/14.5% and 39%/28.3%, in plasma, urine and saliva specimens, respectively. When urine and saliva samples were collected at the same time-points and tested concurrently, the diagnostic sensitivity of RNA and NS1 detection assays was 69.1% and 34.4%, respectively. IgG/IgA detection assays had an overall sensitivity of 54.4%/37.4%, 38.5%/26.8% and 52.9%/28.6% in plasma, urine and saliva specimens, respectively. IgM were detected in 38.1% and 36% of the plasma and saliva samples but never in urine.ConclusionsAlthough the performances of the different diagnostic methods were not as good in saliva and urine as in plasma specimens, the results obtained by qRT-PCR and by anti-DENV antibody ELISA could well justify the use of these two body fluids to detect dengue infection in situations when the collection of blood specimens is not possible.

Epidemiological and Molecular Characterization of Dengue Virus Circulating in Bhutan, 2013-2014.

Dengue is one of the most significant public health problems in tropical and subtropical countries, and is increasingly being detected in traditionally non-endemic areas. In Bhutan, dengue virus (DENV) has only recently been detected and limited information is available. In this study, we analyzed the epidemiological and molecular characteristics of DENV in two southern districts in Bhutan from 2013–2014. During this period, 379 patients were clinically diagnosed with suspected dengue, of whom 119 (31.4%) were positive for DENV infection by NS1 ELISA and/or nested RT-PCR. DENV serotypes 1, 2 and 3 were detected with DENV-1 being predominant. Phylogenetic analysis of DENV-1 using envelope gene demonstrated genotype V, closely related to strains from northern India.

English

The performance of a commercial rapid immunochromatographic dengue NS1, IgG/IgM assay device was evaluated against an in-place dengue IgM-capture ELISA in the KPC Medical College & Hospital, Jadavpur. Of the 932 serum samples from patients with clinical diagnosis of acute dengue illness, 542, 237 and 90 samples were tested positive respectively for NS1 ELISA, anti-dengue IgM (MAC ELISA) and anti-dengue IgG ELISA. Comparatively, 476, 205 and 79 samples were tested positive respectively by the immunochromatographic method, has less sensitivity of 13% and a relative specificity of 12% compared to the ELISA method. Though the rapid immunochromato graphic assay device has the advantages of rapid testing which simultaneously detects both IgG and IgM and can also be performed with whole blood, serum or plasma, the user has to exercise extreme caution with the interpretation of the test result.

Molecular characterization of dengue and chikungunya virus strains circulating in New Delhi, India.

Dengue and chikungunya are acute viral infections with overlapping clinical symptoms. Both diseases are transmitted by common mosquito vectors resulting in their co-circulation in a region. Molecular and serological tests specific for both dengue and chikungunya infections were performed on 87 acute phase blood samples collected from patients with suspected dengue/chikungunya infections in Delhi from September to December, 2011. RT-PCR and IgM ELISA were performed to detect dengue virus (DENV) and chikungunya virus (CHIKV). NS1 and IgG ELISA were also performed to detect DENV specific antigen and secondary DENV infection. DENV infection was detected in 49%, CHIKV infection in 29% and co-infection with DENV and CHIKV in 10% of the samples by RT-PCR. DENV serotypes 1, 2 and 3 were detected in this study. Nine DENV-1 strains, six DENV-2 strains and 20 CHIKV strains were characterized by DNA sequencing and phylogenetic analysis of their respective envelope protein genes. DENV-1 strains grouped in the American African genotype, DENV-2 strains in the Cosmopolitan genotype and CHIKV strains in the East Central South African genotype by phylogenetic analysis. This is one of the few studies reporting the phylogeny of two dengue virus serotypes (DENV-1 and DENV-2) and CHIKV. Surveillance and monitoring of DENV and CHIKV strains are important for design of strategies to control impending epidemics.

Evaluation of a dengue NS1 antigen detection assay sensitivity and specificity for the diagnosis of acute dengue virus infection.

BackgroundCurrently, no dengue NS1 detection kit has regulatory approval for the diagnosis of acute dengue fever. Here we report the sensitivity and specificity of the InBios DEN Detect NS1 ELISA using a panel of well characterized human acute fever serum specimens.Methodology/Principal FindingsThe InBios DENV Detect NS1 ELISA was tested using a panel composed of 334 serum specimens collected from acute febrile patients seeking care in a Bangkok hospital in 2010 and 2011. Of these patients, 314 were found to have acute dengue by either RT-PCR and/or anti-dengue IgM/IgG ELISA. Alongside the InBios NS1 ELISA kit, we compared the performance characteristics of the BioRad Platelia NS1 antigen kit. The InBios NS1 ELISA Ag kit had a higher overall sensitivity (86% vs 72.8%) but equal specificity (100%) compared to the BioRad Platelia kit. The serological status of the patient significantly influenced the outcome. In primary infections, the InBios NS1 kit demonstrated a higher sensitivity (98.8%) than in secondary infections (83.5%). We found significant variation in the sensitivity of the InBios NS1 ELISA kit depending on the serotype of the dengue virus and also found decreasing sensitivity the longer after the onset of illness, showing 100% sensitivity early during illness, but dropping below 50% by Day 7.Conclusion/SignificanceThe InBios NS1 ELISA kit demonstrated high accuracy when compared to the initial clinical diagnosis with greater than 85% agreement when patients were clinically diagnosed with dengue illness. Results presented here suggest the accurate detection of circulating dengue NS1 by the InBios DENV Detect NS1 ELISA can provide clinicians with a useful tool for diagnosis of early dengue infections.

The Diagnostic and Prognostic Value of Dengue Non-Structural 1 Antigen Detection in a Hyper-Endemic Region in Indonesia

As dengue fever is undifferentiated from other febrile illnesses in the tropics and the clinical course is unpredictable, early diagnosis is important. Several commercial assays to detect dengue NS1 antigen have been developed; however, their performances vary and data is lacking from hyper-endemic areas where all four serotypes of dengue are equally represented. To assess the sensitivity of the Bio-Rad platelia Dengue NS1 antigen assay according to virus serotype, immune status, gender, and parameters of severe disease, acute sera from 220 individuals with confirmed dengue and 55 individuals with a non-dengue febrile illness were tested using the Bio-Rad platelia Dengue NS1 antigen assay. The overall sensitivity of the NS1 ELISA was 46.8% and the specificity was 100%. The sensitivity in primary infections was significantly higher than in secondary infections (100% vs. 35.7%). In secondary infections, the sensitivity of NS1 detection was highest in DENV-3 (47.1%), followed by DENV-1 (40.9%), DENV-2 (30%) and DENV-4 (27%) infections. NS1 was less frequently detected in sera with high titers of HI antibodies or in acute samples from patients whose pre-illness sera showed neutralizing antibodies to more than one serotype. The detection of NS1 was higher in females, severe cases, and individuals with lower platelet counts (<100,000/mm3). While the overall sensitivity of this NS1 ELISA is poor, our data suggest that in secondary infections, detection may be predictive of a more severe illness.

A clinico-microbiological study of dengue fever cases in a tertiary care center of navi Mumbai..

Dengue is an endemic viral disease affecting tropical and subtropical regions around the world, predominantly in urban and semiurban areas. Dengue fever (DF) and its more serious forms, dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS), are becoming important public health problems and were formally included within the disease portfolio of the Indian Development. During 2010, there was an increase in dengue virus infections in Mumbai as compared to 2009. This study determined the socio demographic status of suspected dengue cases from 2010 to 2012 in a tertiary care centre of Navi Mumbai. The comparative outcome of dengue NS1, IgM and IgG ICT, dengue NS1, IgM and IgG ELISA, and RT-PCR for dengue NS1 antigen to assess which of them is better for patient management and prevention of complications like DHF and DSS. Distribution in 320 positive samples were NS1 positive 51.56%, IgM positive 22.81%, IgG positive 12.2%, NS1 + IgM positive10.31%, IgM + IgG positive 2.19% and NS1 + IgM + IgG positive 0.93%.In 100 randomly selected ICT positive cases, there was a gain of 6 (3+3) positive samples by the NS1 Microlisa test as compared to the NS1 immuno chromatography test. In 66 ICT positive samples, there was a gain of 7 (2+5) positive samples by IgM Microlisa and a gain of 1 sample by IgG Microlisa. Out of 30 samples subjected to RT-PCR, all 15 positive Microlisa samples were positive, while 4 out of 15 Microlisa negative samples were positive by the RT-PCR. Overall there was a gain of 4 positive samples by the RT-PCR. As we found RT-PCR to be the gold standard, sensitivity and specificity of NS1 ICT were found to be 91.2% and 78.4%, while that of NS1 ELISA were found to be 75% and 88%. For the early diagnosis of dengue infection, Immuno chromatography test is a good screening test with good sensitivity, while dengue ELISA is more specific and can be used as confirmatory tests in routine laboratory setup. RT-PCR for dengue antigen is the most confirmatory test with highest sensitivity and specificity but is expensive. A combination of the three tests is recommended for dengue diagnosis in laboratories.

A Comprehensive Study on the 2012 Dengue Fever Outbreak in Kolkata, India

Background. Dengue viruses (DV) belong to the family Flaviviridae, with four serotypes referred to as DV-1, DV-2, DV-3, and DV-4. A large-scale outbreak of dengue fever occurred in 2012 involving several districts of West Bengal. Objective. To present a comprehensive picture of the dengue fever outbreak in 2012 and to identify the prevailing serotypes. Materials and Methods. Serum samples were collected from suspected dengue fever cases. Samples from fever cases &lt;5 days duration were tested for dengue NS1 antigen employing Pan Bio (Australia) NS1 ELISA kit. Serum samples of ≥5 days fever were tested for dengue-specific IgM by MAC ELISA test kit prepared by the National Institute of Virology Pune, India. Serotyping of dengue samples was done by dengue-specific reverse transcriptase polymerase chain reaction (RT-PCR). Results. The number of dengue cases in 2012 clearly outnumbered the dengue cases in 2010 and 2011. The majority of the cases were in the age group 11–30 years with a male preponderance. Outbreak occurred during the months of Aug.–Nov. indicating increased vector transmission in the monsoon and postmonsoon periods. The prevailing serotypes in this outbreak were Den1, Den3, and Den4.

Evaluation of laboratory tests for dengue diagnosis in clinical specimens from consecutive patients with suspected dengue in Belo Horizonte, Brazil

BackgroundDengue is a widely spread arboviral disease in tropical and subtropical regions of the world. Dengue fever presents clinical characteristics similar to other febrile illness. Thus laboratory diagnosis is important for adequate management of the disease. ObjectivesThe present study was designed to evaluate the diagnostic performance of real-time PCR and serological methods for dengue in a real epidemic context. Study designClinical data and blood samples were collected from consecutive patients with suspected dengue who attended a primary health care unit in Belo Horizonte, Brazil. Serologic methods and real-time PCR were performed in serum samples to confirm dengue diagnosis. ResultsAmong the 181 consecutive patients enrolled in this study with suspected dengue, 146 were considered positive by serological criteria (positive NS1 ELISA and/or anti-dengue IgM ELISA) and 138 were positive by real-time PCR. Clinical criteria were not sufficient for distinguishing between dengue and non-dengue febrile illness. The PCR reaction was pre-optimized using samples from patients with known viral infection. It had similar sensitivity compared to NS1 ELISA (88% and 89%, respectively). We also evaluated three commercial lateral flow immunochromatographic tests for NS1 detection (BIOEASY, BIORAD and PANBIO). All three tests showed high sensitivity (94%, 91% and 81%, respectively) for dengue diagnosis. ConclusionAccording to our results it can be suggested that lateral flow tests for NS1 detection are the most feasible methods for early diagnosis of dengue.

Validation of diagnostic tests for detection of avian influenza in vaccinated chickens using Bayesian analysis

Vaccination is an attractive tool for the prevention of outbreaks of highly pathogenic avian influenza in domestic birds. It is known, however, that under certain circumstances vaccination may fail to prevent infection, and that the detection of infection in vaccinated birds can be problematic. Here, we investigate the characteristics of three serological tests (immunofluorescent antibody test (iIFAT), neuraminidase inhibition (NI) assay, and NS1 ELISA) that are able to differentiate infected from vaccinated animals. To this end, data of H7N7 infection experiments are analyzed using Bayesian methods of inference. These Bayesian methods enable validation of the tests in the absence of a gold standard, and allow one to take into account that infected birds do not always develop antibodies after infection. The results show that the N7 iIFAT and the NI assay have sensitivities for detecting antibodies of 0.95 (95% CI: 0.89–0.98) and 0.93 (95% CI: 0.78–0.99), but substantially lower sensitivities for detecting infection: 0.64 (95% CI: 0.52–0.75) and 0.63 (95% CI: 0.49–0.75). The NS1 ELISA has a low sensitivity for both detecting antibodies 0.55 (95% CI: 0.34–0.74) and infection 0.42 (95% CI: 0.28–0.56). The estimated specificities of the N7 iIFAT and the NI assay are 0.92 (95% CI: 0.87–0.95) and 0.91 (95% CI: 0.85–0.95), and 0.82 (95% CI: 0.74–0.87) for the NS1 ELISA. Additionally, our analyses suggest a strong association between the duration of virus excretion of infected birds and the probability to develop antibodies.

Diagnostic Accuracy of NS1 ELISA and Lateral Flow Rapid Tests for Dengue Sensitivity, Specificity and Relationship to Viraemia and Antibody Responses

BackgroundDengue is a public health problem in many countries. Rapid diagnosis of dengue can assist patient triage and management. Detection of the dengue viral protein, NS1, represents a new approach to dengue diagnosis.Methodology/Principal FindingsThe sensitivity and specificity of the Platelia NS1 ELISA assay and an NS1 lateral flow rapid test (LFRT) were compared against a gold standard reference diagnostic algorithm in 138 Vietnamese children and adults. Overall, the Platelia NS1 ELISA was modestly more sensitive (82%) than the NS1 LFRT (72%) in confirmed dengue cases. Both ELISA and LFRT assays were more sensitive for primary than secondary dengue, and for specimens collected within 3 days of illness onset relative to later time points. The presence of measurable DENV-reactive IgG and to a lesser extent IgM in the test sample was associated with a significantly lower rate of NS1 detection in both assays. NS1 positivity was associated with the underlying viraemia, as NS1-positive samples had a significantly higher viraemia than NS1-negative samples matched for duration of illness. The Platelia and NS1 LFRT were 100% specific, being negative in all febrile patients without evidence of recent dengue, as well as in patients with enteric fever, malaria, Japanese encephalitis and leptospirosis.Conclusions/SignificanceCollectively, these data suggest NS1 assays deserve inclusion in the diagnostic evaluation of dengue patients, but with due consideration for the limitations in patients who present late in their illness or have a concomitant humoral immune response.