IgM Antibody Capture ELISA Research Articles

Laboratory Evaluation and Field Testing of Dengue NS1 and IgM/IgG Rapid Diagnostic Tests in an Epidemic Context in Senegal.

In Senegal, the burden of dengue is increasing and expanding. As case management and traditional diagnostic techniques can be difficult to implement, rapid diagnostic tests (RDTs) deployed at point of care are ideal for investigating active outbreaks. The aim of this study was to evaluate the diagnostic performance of the Dengue NS1 and Dengue IgM/IgG RDTs on the serum/plasma samples in a laboratory setting and in the field. During laboratory evaluation, performance of the NS1 RDT was assessed using NS1 ELISA as the gold standard. Sensitivity and specificity were 88% [75-95%] and 100% [97-100%], respectively. Performance of the IgM/IG RDT was assessed using the IgM Antibody Capture (MAC) ELISA, indirect IgG, and PRNT as gold standards. The IgM and IgG test lines respectively displayed sensitivities of 94% [83-99%] and 70% [59-79%] and specificities of 91% [84-95%] and 91% [79-98%]. In the field, the Dengue NS1 RDT sensitivity and specificity was 82% [60-95%] and 75% [53-90%], respectively. The IgM and IgG test lines displayed sensitivities of 86% [42-100%] and 78% [64-88%], specificities of 85% [76-92%] and 55% [36-73%], respectively. These results demonstrate that RDTs are ideal for use in a context of high prevalence or outbreak setting and can be implemented in the absence of a confirmatory test for acute and convalescent patients.

Trend analysis in seroprevalence of dengue, chikungunya and malaria: A seven-year serological study from a tertiary care hospital of Maharashtra, India.

Dengue, chikungunya and malaria are mosquito-borne infections, which have shared endemicity and similar clinical presentation. Simultaneous co-infection with more than one infectious agent complicates the diagnosis and further course of treatment. This study aims to determine the seroprevalence and trend of malaria, dengue and chikungunya from 2014-2020 in a tertiary care hospital of western India. The present study was retrospective descriptive record-based. Serum samples from clinically suspected dengue and chikungunya were subjected to both IgM antibody capture ELISA kits produced by National Institute of Virology (NIV), Pune, India. They were also subjected to ELISA based NS1Ag testing. In Suspected malaria cases, blood collected in EDTA tubes was subjected for Rapid Malaria antigen testing. Statistical analysis was performed using MS Excel and JMP Software. Seropositivity of malaria was comparatively higher in 2014 (5.53%) and a decreasing trend was observed in subsequent years. Majority of malarial infections were caused by Plasmodium vivax (81.67%). There is drastic increase in seropositivity of chikungunya from 2016 (23.67%) and thereafter as compared to 2014 (6.57%) and 2015 (7.29%) indicating its re-emergence. The dengue seropositivity in 2019 (40.19%) was highest in last seven years. Males were predominantly affected, and most affected age group was 21-30 years. Peak transmission was observed in post-monsoon seasons. Dengue and chikungunya co-infection was observed to be 5.79%. This study emphasizes the importance of surveillance studies to understand the trend of vector-borne diseases for prompt diagnosis, management of patients in hospital setup and for early detection and curtailment of outbreaks and epidemics by public health sectors through appropriate vector control programs.

Capacity of a Multiplex IgM Antibody Capture ELISA to Differentiate Zika and Dengue Virus Infections in Areas of Concurrent Endemic Transmission.

ABSTRACT.Serological cross-reactivity has proved to be a challenge to diagnose Zika virus (ZIKV) infections in dengue virus (DENV) endemic countries. Confirmatory testing of ZIKV IgM positive results by plaque reduction neutralization tests (PRNTs) provides clarification in only a minority of cases because most individuals infected with ZIKV were previously exposed to DENV. The goal of this study was to evaluate the performance of a ZIKV/DENV DUO IgM antibody capture ELISA (MAC-ELISA) for discriminating between DENV and ZIKV infections in endemic regions. Our performance evaluation included acute and convalescent specimens from patients with real-time reverse transcription polymerase chain reaction (RT-PCR)-confirmed DENV or ZIKV from the Sentinel Enhanced Dengue Surveillance System in Ponce, Puerto Rico. The ZIKV/DENV DUO MAC-ELISA specificity was 100% for DENV (N = 127) and 98.4% for ZIKV (N = 275) when specimens were tested during the optimal testing window (days post-onset of illness [DPO] 6–120). The ZIKV/DENV DUO MAC-ELISA sensitivity of RT-PCR confirmed specimens reached 100% for DENV by DPO 6 and for ZIKV by DPO 9. Our new ZIKV/DENV DUO MAC-ELISA was also able to distinguish ZIKV and DENV regardless of previous DENV exposure. We conclude this novel serologic diagnostic assay can accurately discriminate ZIKV and DENV infections. This can potentially be useful considering that the more labor-intensive and expensive PRNT assay may not be an option for confirmatory diagnosis in areas that lack PRNT capacity, but experience circulation of both DENV and ZIKV.

Incidence of Japanese encephalitis virus among patients presenting with acute encephalitis syndrome in a tertiary care hospital, West Bengal, India

Background: Japanese encephalitis virus (JEV) is a flavivirus related to dengue, yellow fever and West Nile viruses, and is spread by mosquitoes. JEV is the main cause of viral encephalitis in many countries of Asia with an estimated 68000 clinical cases every year. Although symptomatic Japanese encephalitis (JE) is rare, the case-fatality rate among those with encephalitis can be as high as 30%. Permanent neurologic or psychiatric sequelae can occur in 30-50% of those with encephalitis. This study was conducted to find the incidence of JEV IgM in patients presenting with acute encephalitis syndrome (AES) in Raiganj govt. medical college and hospital, Uttar Dinajpur, West Bengal, India.Methods: Blood and CSF samples were collected from patients presenting with AES. IgM antibody capture ELISA was performed on the CSF and serum samples by JE virus MAC ELISA kit.Results: The overall prevalence of JEV IgM was 13.8%. Among the positive cases male comprised of 71.4% and female 28.5%. The most common age group affected was above 16 years of age. JEV prevalence was present throughout the year with high number of cases between the monsoon and post monsoon seasons.Conclusions: This study demonstrates the endemicity of JEV in Uttar Dinajpur district of West Bengal, India. As most of the JE cases are asymptomatic strengthening the existing surveillance system is required to find out the actual scenario of JEV in West Bengal. Control of vectors, early diagnosis and treatment, vaccinations are the key to decrease the morbidity and mortality caused by JEV.

Development and validation of an IgM antibody capture ELISA for early detection of Hendra virus

Zoonotic transmission of Hendra virus (HeV) from primary hosts (pteropid bats) to horses, and, occasionally, onward adventitious spread to humans, is associated with high mortality rates in both affected secondary species. The introduction of an effective recombinant G protein vaccine for use in horses has been a major advance for the suppression of disease risk. However, equine HeV vaccination induces neutralising antibody that is indistinguishable from a post infection immune response when using most first line serology assays (eg. VNT and some ELISAs). We have constructed and evaluated an IgM antibody capture (MAC) ELISA which employs yeast expressed HeV nucleoprotein (N). All other serology tests use the G protein which does not detect early infection and is present in the current Hendra virus vaccine and may cause ambiguity in interpretation of results. Thus, this is the first test developed using a N protein which can successfully detect a recent (primarily within the last four weeks) infection of horses with HeV and is not affected by vaccination induced antibody. Testing a limited panel (21 samples) of post infection sera, a normal serum panel (288 samples) and a post vaccination panel (163 samples), we have estimated DSe to be 100 % (95 % CI, 83.9–100.0 %) and DSp to be 98.4 % (95 % CI, 96.8–99.4 %) relative to assigned serology results (VNT, ELISA and Luminex) for the test panels. The HeV IgM MAC ELISA is intended to supplement other molecular and serology test results, with selective use, and is the only serology test which can provide an indication for recent infection which is otherwise not available.

Standardization and Evaluation of an Anti-ZIKV IgM ELISA Assay for the Serological Diagnosis of Zika Virus Infection.

ABSTRACT.Here, we describe the development of the in-house anti-Zika virus (ZIKV) IgM antibody capture ELISA (in-house ZIKV IgM ELISA) for the detection and diagnosis of acute ZIKV infections. We compared the in-house ZIKV IgM ELISA assay performance against two commercial kits, Euroimmun ZIKV IgM and InBios 2.0 ZIKV IgM ELISA. We tested the assays’ ability to detect anti-ZIKV IgM using a well-defined serum sample panel. This panel included 80 ZIKV negative samples (20 negative, 20 found to be primary dengue virus [DENV][ infections, 20 secondary DENV infections, and 20 Japanese encephalitis virus [JEV] infections) and 67 ZIKV reverse transcriptase–polymerase chain reaction–positive acute serum samples. The OD values were calculated to enzyme immunoassay (EIA) unts by comparing them to weak positive controls. The results demonstrated the high sensitivity (88.06%) and specificity (90.00%) of our in-house ZIKV IgM ELISA and its 89.12% overall percentage agreement. The kappa values were deemed to be within excellent range and comparable to the InBios ZIKV IgM ELISA. Some cross-reactivity was observed among secondary DENV and JEV samples, and to a much lower extent, among primary DENV samples. These data indicate that our in-house ZIKV IgM ELISA is a reliable assay for the detection of anti-ZIKV IgM antibodies in serum.

Persistence of IgM Antibodies after Vaccination with Live Attenuated Japanese Encephalitis Vaccine.

.Japanese encephalitis (JE) is a vaccine-preventable, mosquito-borne disease. Substantial progress with JE control in Asia has been made during the past decade, with most endemic countries now having JE vaccination programs, commonly using live attenuated SA14-14-2 JE vaccine (trade name CD-JEV). If a child develops encephalitis during the weeks to months following CD-JEV vaccination and anti-JE virus IgM (JE IgM) antibody is detected in serum, the question arises if this is JE virus infection indicating vaccine failure, or persistent JE IgM antibody postvaccination. To better understand JE IgM seropositivity following vaccination, sera from 268 children from a previous CD-JEV study were tested by two different JE IgM assays to determine JE IgM antibody frequency on days 28, 180, and 365 postvaccination. With the CDC JE IgM antibody capture ELISA (MAC-ELISA), 110 children (41%) had JE IgM positive or equivocal results on their day 28 sample, and eight (3%) and two (1%) had positive or equivocal results on day 180 and day 365 samples, respectively. With the InBios JE Detect™ MAC-ELISA (Seattle, WA), 118 (44%) children had positive or equivocal results on day 28 sample, and three (1%) and one (0.4%) had positive or equivocal results on day 180 and day 365 samples, respectively. Our results indicate that more than 40% children vaccinated with CD-JEV can have JE IgM antibodies in their serum at 1 month postvaccination but JE IgM antibody is rare by 6 months. These data will help healthcare workers assess the likelihood that JE IgM antibodies in the serum of a child with encephalitis after vaccination are vaccine related.

Seroprevalence of Dengue Viral Infection in Patients Attending To a Tertiary Care Hospital in Kota, South–East Rajasthan, India

Background: Dengue virus (DENV) is the most important cause of mosquito-borne viral disease in humans world-wide. Dengue fever is caused by four serotypes of dengue virus (DENV)-DENV1, DENV2, DENV3 and DENV4 .Infections is transmitted mainly by Aedes aegypti. In India, a dengue virus infection has been frequently encountered in epidemic proportions in several states. Objectives: This study was conducted to report the seroprevalence of Dengue virus infection in South-East Rajasthan. The ultimate goal of this study was to identify Dengue infection in early phase of disease and prevent future complications. Methodology: The present study was conducted in the central laboratory of Govt. Medical College, Kota from period of 1st January 2018 to 31th December 2018 to evaluate the seroprevalence of Dengue virus infection in South-East Rajasthan. Sera were tested by SD dengue IgM antibody capture ELISA for IgM antibody detection as per kit insert. Results: Out of total 16945 serum samples tested, 1064(6.28%) were found positive for dengue virus infection. Of all the patients tested, 12031(71%) were males and 4914(29%) were females. 659(61.94%) positive samples were of male patients and 405(38.06%) positive samples were of female patients. Dengue affected males and females in a ratio of about 1.6:1. Out of the total positive for dengue, Age group mostly affected were 21-30 years, 31-40 years and 11-20 year 38.53%, 27.54% and 18.70% respectively. Seasonal trend showed a gradual increase in dengue positives started from August with a peak in October (30.73%) and November (21.43 %).Conclusion: Dengue is an important emerging disease of the tropical and subtropical regions today. Epidemics of dengue are increasing in frequency. Dengue virus activity is high during monsoon and post monsoon period which coincides with increased vector breeding. This study calls attention to the Dengue infection detected in early phase of disease and prevents morbidity, mortality and future complications. Keywords: Dengue virus, Dengue fever, Mosquito-borne viral disease, Dengue IgM antibody, ELISA. Cite this Article Bhupendra Kumar Mandawat, Preetam Singh Mandawat. Seroprevalence of Dengue Viral Infection in Patients Attending To a Tertiary Care Hospital in Kota, South–East Rajasthan, India. Research & Reviews: A Journal of Microbiology & Virology. 2020; 10(1): 1–9p.

A study on seroprevalence of dengue viral infection using IgM antibody capture ELISA for the Early diagnosis in Kalaburagi district, North-Eastern part of Karnataka, India

Introduction: Dengue is an acute viral illness caused by RNA virus of the family Flaviviridae and spread by Aedes mosquitoes which occur mainly in four closely related serotypes (DENV1- 4). It has developed into emergent threat in the India and Karnataka state now. The most challenging difficulty associated with patient administration in dengue infection is early diagnosis. Objective: To determine the Prevalence of Dengue infection in the Kalaburagi district by serological test IgM ELISA. The detection of Immunoglobulin IgM is the core method for the laboratory diagnosis of dengue. The present study considered the relevance of a screening test for dengue pertaining to new cases and assessment of the epidemic attack rate. Materials and Methods: This evocative study was conducted at our Gulbarga Institute of Medical Sciences hospital, Kalaburagi for the period between January to December 2018. Serum samples were collected from suspected dengue patients attended in and out patient department, Sera were analysed by ELISA test for the presence of dengue IgM antibodies. Results: Over the study period, out of 1,807 samples, 123 were showed positive for dengue virus infection. Both male 61(49.59%) and female 62 (50.40%) patients predominantly affected. The positive peak values were noticed between April to December 2018. The most positivity was observed between the age group of 1-20 years. Conclusion: the present results revealed that the study region is epidemic for dengue viral infection and there is an urgent need for the constant monitoring to control further spreading of the infection in the community, hence serological test have important role in the early diagnosis. Therefore IgM ELISA is recommended in all the suspected dengue patients so as to instigate essential treatment and assessment of morbidity and mortality rate during an outbreak. Keywords: IgM ELISA, Dengue viral infection, Aedes aegypti.

Coinfection of chikungunya and dengue viruses: A serological study from North Western region of Punjab, India.

INTRODUCTION:Dengue and chikungunya (CHIK) infections appear to be increasing in all parts of India. Aedes aegypti mosquitoes are common vectors for dengue virus (DENV) and CHIK virus (CHIKV). In areas where both viruses cocirculate, they can be transmitted together. There are very few studies discussing the dengue-chik coinfection from Punjab region of India. The present study was undertaken to study the clinical features of dengue–CHIK coinfection and compare with monoinfection.MATERIALS AND METHODS:IgM antibody capture (MAC) ELISA for dengue IgM and CHIK IgM and ELISA for nonstructural protein 1 antigen was performed on serum samples obtained from suspected patients.RESULTS:Out of total 3160 samples from suspected patients for dengue infection, 2178 (68.92%) samples were positive for DENV while CHIK IgM antibodies were positive in 127 patients out of the total suspected 373 cases (34.04%). In addition to this, 283 samples were tested for both viruses, out of which 27 sera were positive (9.54%) for coinfection of dengue and CHIK. The comparison of signs and symptoms showed that the coinfected patients had fever in all cases while rash was seen in only 30% cases. Arthralgia (79%) and thrombocytopenia (77%) was seen in significant number of coinfected cases thus revealing overlapping nature of dengue–CHIK coinfection.CONCLUSION:Increase in the number of Dengue and Chikungunya infections and their cocirculation is an important public health concern which warrants the implementation of strict control measures.

Dengue IgM antibody sero-status assessment: a current experience in a teaching institution

Background: In 2015, Delhi city experienced the worst outbreak of dengue infection since 1996 and our laboratory received a very high number of cases for IgM ELISA testing following clinicians’ referrals. The present study intends to correlate IgM antibody response with duration of fever, platelets counts and ‘with age and gender distribution’ of such cases attending OPDs and subsequently admitted to the medicine and pediatric wards in our tertiary care hospital, New Delhi, albeit, with some useful conclusions and suggestions.Methods: A hospital based observational study was conducted enrolling a total of 500 cases, clinically suspected of DF/DHF/DSS that either reported directly or were referred to Safdarjung Hospital from region in and around Delhi and NCR and subsequently admitted to Emergency Medicine and Paediatrics wards. A total of 500 cases and 50 controls were tested for IgM antibody by ‘IgM antibody capture ELISA (MAC ELISA).Results: Of total 500 serum samples tested, 169 i.e. 33.8% were found to be sero positive. Seropositivity was more among the males 99/169 (58.57%) than the females 70/169 (41.43%). The highest numbers of cases were seen in the age group 41-60 years (46.15%). Amongst seropositive cases, 146 (86.39%) cases had fever of >5 days duration while remaining 23 (13.60%) presented with fever of <5 days. Low platelet count between 20,000-1L cells/mm3 was a feature in all the seropositive cases and statistically it was found significant (p-value 0.001).Conclusions: Detection of IgM specific antibodies by MAC ELISA in cases with high index of clinical suspicion continues to be an effective tool providing a supportive criterion to clinical diagnosis, especially during outbreak periods.

English

Introduction: Dengue is a most significant febrile illness caused by a single stranded enveloped RNA virus belongs to the family Flaviviridae, genus Flavivrus. Dengue infection is caused by any one of four distinctive antigenically related dengue virus serotypes: DENV-1, DENV-2, DENV-3 and DENV-4. In India, a dengue virus infection has been frequently encountered in epidemic proportions in several states. There is no specific treatment for dengue/ severe dengue, but early detection and access to proper medical care lowers fatality rates below 1%. MATERIALS AND METHODS: This study was undertaken at a tertiary care teaching hospital at Kota from January 2013 to December 2013. Blood samples were received from patients of all age group suspected of dengue, DHF and Dengue Shock Syndrome. Sera were separated and preserved at 4°C till the time of testing. Sera were tested by dengue IgM antibody capture ELISA (MAC ELISA) (received from NIV, Pune) for IgM antibody detection as per kit insert. RESULT: During the study period, a total of 13077 blood samples were tested for dengue. Of all the patients tested, 9066 were males and 4011 were females. Dengue affected males and females in a ratio of 3.7:1. During this study, out of the total positive for dengue, positivity was highest (n=566, 31.65%) in the adult age group of >30 yrs followed by the age group of 15 to 30 yrs (n=408, 22.8%). DISCUSSION: Dengue is an important emerging disease of the tropical and subtropical regions today. Epidemics of dengue are increasing in frequency. The prevalence of dengue seropositivity among clinically suspected cases during the study period was 13.67%. Our study results call attention to the need for continuous surveillance and individual and community action for dengue control.

A pilot study of varicella zoster infection among patients with fever and rash in a tertiary care centre

Results All enrolled patients had fever with rash without evidence of vesicles. Majority, (n=29) were adults and 18 were male. Of the 14 (38.9%) that were positive by IgM ELISA for VZV, 5 (35.7%) belonged to the age group of 19-30 years, 4 (28.5%) were 1.5 to < 2 lakh /cumm). All sera that were tested were negative for Dengue NS1 antigen, IgM and IgG antibody capture ELISA (Panbio, Australia).

Dengue and Typhoid Co-infection- Study from a Government Hospital in North Delhi.

Concurrent infection with two agents can result in an illness having overlapping symptoms creating a diagnostic dilemma for the treating physician. The symptoms of dengue may mimic other diseases such as leptospirosis, influenza A, Salmonella Typhi, Japanese encephalitis, chikungunya and malaria. There is paucity of data regarding dengue and typhoid co- infection both in the developed and developing countries. This study attempts to find the current co- infection rates in North Delhi. This retrospective study was done between August and November 2013. Medical records of 659 patients exhibiting febrile illness who visited Kasturba Hospital were studied. Dengue specific IgM antibodies were detected by Dengue IgM antibody capture ELISA test. Serodiagnosis of Salmonella infection was conducted by Widal test in the hospital. Of the 659 febrile sera samples tested here, 141 (21.39%) tested positive for dengue. Of these 91 were females and 50 males. Of the dengue cases, eleven were co-infected with enteric fever (11/141= 7.8%). Maximum number of dengue positive cases seen in age group 0-10 y. Case Fatality Rate (CFR) was zero. Age groups of patients co-infected with dengue and typhoid were as follows: 0-10 y: 5, 11-20 y: 3, 21-30 y: 2, >60 y: 1. Co-infection should always be kept in mind while dealing with cases of dengue or enteric fever with or without atypical features. In order to reduce the burden of disease, along with improvement of sanitation and personal hygiene, emphasis should be given on vaccination against typhoid.

Emergence of Dengue Virus Infection in Nepal

This article reviews Dengue, a common viral disease in humans and is an emerging public health problem in Tarai Region of Nepal. The most affected are among the poorest populations living in remote, rural areas and urban slums who have even no access for medical treatment, acquired by bite of infected mosquito. Aedes Aegypti infected with dengue virus is the major source of infections for humans and cannot be transmitted from person-to-person because human are the dead end host. DENV-1 was first isolated by Ren Kimura and Susumu Hotta in Japan in 1943. An epidemic of DF involving at least 200,000 cases had occurred between 1942 and 1944 during World War II in Japanese port cities such as Nagasaki, Kobe, and Osaka. First case of dengue was reported in 2004 in Nepal. The seroprevalence study were done in different part of Nepal by IgM antibody capture ELISA and positive rate was highest (50.0%) in Biratnagar, and lowest (19.6%) in Chitwan male to female ratio was 2:1. IgM-positive rate was 29.0% at ages 21-30, 25.4% at ages 11-20 and 23.6% at ages 0-10, but 10.9% at ages 31-40, and ages over 40. There was not significant association between occupation of the patients and positive rate among farmer, labour, service, business and student. The epidemiological studies of Dengue virus infection and the knowledge of the pattern of the disease outbreak can guide therapy and effective preventive measures against this disease.DOI: http://dx.doi.org/10.3126/ijasbt.v1i3.8384 Int J Appl Sci Biotechnol, Vol. 1(3) 2013 : 79-84

Cross-reactivities between human IgMs and the four serotypes of dengue virus as probed with artificial homodimers of domain-III from the envelope proteins

BackgroundDengue fever is the most important vector-borne viral disease. Four serotypes of dengue virus, DENV1 to DENV4, coexist. Infection by one serotype elicits long-lasting immunity to that serotype but not the other three. Subsequent infection by a different serotype is a risk factor for severe dengue. Domain III (ED3) of the viral envelope protein interacts with cell receptors and contains epitopes recognized by neutralizing antibodies. We determined the serotype specificity and cross-reactivity of human IgMs directed against ED3 by using a well-characterized collection of 90 DENV-infected and 89 DENV-uninfected human serums.MethodsThe recognitions between the four serotypes of ED3 and the serums were assayed with an IgM antibody-capture ELISA (MAC-ELISA) and artificial homodimeric antigens. The results were analyzed with Receiving Operator Characteristic (ROC) curves.ResultsThe DENV-infected serums contained IgMs that reacted with one or several ED3 serotypes. The discrimination by ED3 between serums infected by the homotypic DENV and uninfected serums varied with the serotype in the decreasing order DENV1 > DENV2 > DENV3 > DENV4. The ED3 domain of DENV1 gave the highest discrimination between DENV-infected and DENV-uninfected serums, whatever the infecting serotype, and thus behaved like a universal ED3 domain for the detection of IgMs against DENV. Some ED3 serotypes discriminated between IgMs directed against the homotypic and heterotypic DENVs. The patterns of cross-reactivities and discriminations varied with the serotype.ConclusionsThe results should help better understand the IgM immune response and protection against DENV since ED3 is widely used as an antigen in diagnostic assays and an immunogen in vaccine candidates.

Estimating the True Accuracy of Diagnostic Tests for Dengue Infection Using Bayesian Latent Class Models

BackgroundAccuracy of rapid diagnostic tests for dengue infection has been repeatedly estimated by comparing those tests with reference assays. We hypothesized that those estimates might be inaccurate if the accuracy of the reference assays is not perfect. Here, we investigated this using statistical modeling.Methods/Principal FindingsData from a cohort study of 549 patients suspected of dengue infection presenting at Colombo North Teaching Hospital, Ragama, Sri Lanka, that described the application of our reference assay (a combination of Dengue IgM antibody capture ELISA and IgG antibody capture ELISA) and of three rapid diagnostic tests (Panbio NS1 antigen, IgM antibody and IgG antibody rapid immunochromatographic cassette tests) were re-evaluated using Bayesian latent class models (LCMs). The estimated sensitivity and specificity of the reference assay were 62.0% and 99.6%, respectively. Prevalence of dengue infection (24.3%), and sensitivities and specificities of the Panbio NS1 (45.9% and 97.9%), IgM (54.5% and 95.5%) and IgG (62.1% and 84.5%) estimated by Bayesian LCMs were significantly different from those estimated by assuming that the reference assay was perfect. Sensitivity, specificity, PPV and NPV for a combination of NS1, IgM and IgG cassette tests on admission samples were 87.0%, 82.8%, 62.0% and 95.2%, respectively.ConclusionsOur reference assay is an imperfect gold standard. In our setting, the combination of NS1, IgM and IgG rapid diagnostic tests could be used on admission to rule out dengue infection with a high level of accuracy (NPV 95.2%). Further evaluation of rapid diagnostic tests for dengue infection should include the use of appropriate statistical models.

Virological surveillance for early warning of dengue epidemics in the State of Rio de Janeiro, Brazil

The state of Rio de Janeiro has been important since 1986 as a portal for the introduction of dengue virus (DENV) into Brazil and dissemination of the virus throughout the country. This study describes an active surveillance of DENV in the state of Rio de Janeiro from 2004 to 2008. A total of 14408 samples from patients suspected to be infected with DENV were tested by virus isolation, and nested reverse transcriptase (RT)-PCR assay or anti-DENV dengue IgM antibody capture ELISA (MAC-ELISA), or both. By the use of these different methods, a total of 2324 (16.1%) cases were confirmed as dengue infection. The study covers an inter-epidemic period (2004-2005), the DENV-3 circulation in 2006, the re-emergence of DENV-2 in 2007 and the severe epidemic caused by DENV-2 in the summer of 2008. During the period, 69 dengue fatal cases were reported, 14 (20.2%) deaths being attributable to DENV-3 and 55 (79.7%) to DENV-2. Our results emphasize the role of the laboratory in the early detection of dengue virus transmission and provide information on the dynamics of DENV introduction and spread, important for the assessment of intervention strategies.

Incidence of Japanese Encephalitis among Acute Encephalitis Syndrome Cases in West Bengal, India

Background and Objectives. Japanese encephalitis (JE) is the most important cause of acute and epidemic viral encephalitis. Every year sporadic JE cases are reported from the various districts of West Bengal, indicating its endemicity in this state. JE vaccination programme has been undertaken by the State Health Department of West Bengal. This study was aimed at seeing the present scenario of JE among acute encephalitis syndrome (AES) cases in West Bengal. Materials and Methods. Blood and/or CSF samples were referred from suspected AES cases to the referral virology laboratory of the Calcutta School of Tropical Medicine from different hospitals of Kolkata. IgM antibody capture ELISA was performed on the CSF and serum samples by JE virus MAC ELISA kit supplied by the National Institute of Virology, Pune. Results. The present study reveals that 22.76% and 5% of the AES cases were positive for JE IgM in 2011 and 2012, respectively. JE is mainly prevalent in children and adolescents below 20 years of age with no gender predilection. Although the percentages of JE positive cases were high in 2011, it sharply decreased thereafter possibly due to better awareness programs, due to mass vaccination, or simply due to natural epidemiological niche periodicity due to herd immunity.

Optimization of Dengue-3 recombinant NS1 protein expression in E. coli and in vitro refolding for diagnostic applications

Dengue non-structural protein (NS1) is known to be protective antigen and also has immense application for serodiagnosis. Several serodiagnostic assays available for dengue viral infection are dependent on tissue culture-grown viral proteins. This task is unsafe, laborious, more expensive that makes it unsuitable for routine large-scale production. Although bacterial expression is relatively simple and easy for recombinant protein expression, it is more challenging to make NS1 protein with native structural and immunological features using bacterial expression system. We have successfully developed a method leading to the purification and refolding of recombinant dengue virus type 3 (DENV3) NS1. The gene encoding NS1 was amplified and cloned in pET28a (+) vector. In order to increase the purity of the recombinant NS1, the transgene was engineered to carry 6× Histidine tags at both N and C-terminal ends. The recombinant construct (pETNS1) was transformed into E. coli Rosetta-gami cells and the expression conditions viz IPTG concentration, media type, temperature, and harvest time were optimized. The size of the expressed protein was found to be ~45 kDa and the authenticity of the expressed protein was confirmed using anti-His and anti-NS1 monoclonal antibodies. The NS1 protein was purified under denaturing conditions, to attain the native conformation, NS1 protein was in vitro refolded and dialyzed. The refolded NS1 protein was detected by commercial Immuno chromatographic strip and NS1 specific monoclonal antibodies. IgM antibody capture ELISA was performed using refolded recombinant NS1 protein which recognized the IgM antibodies in dengue-positive samples of acute phase of infection. Our result suggests that rNS1 protein has immense diagnostic potential and can be used in developing point of care diagnostic assays.