IgM Capture Research Articles

Suggestive Serological Evidence of Infection with Shrew-Borne Imjin Virus (Hantaviridae) in Humans.

The pathogenicity of the shrew-borne Imjin virus (MJNV) is unknown. The objective of our study was to find serological evidence of MJNV infection in humans. Partial MJNV nucleocapsid protein (NP) was cloned and expressed as an antigen for double-antigen sandwich ELISA, IgM capture ELISA, and dot blot to detect MJNV specific antibodies in hemorrhagic fever with renal syndrome (HFRS) patients’ and healthy persons’ sera from endemic areas in China. The purified recombinant NP reacted with neither the 90 healthy individuals’ sera from non-endemic areas of MJNV nor the 100 antisera to HFRS-causing virus, indicating that the MJNV NP had no cross-reaction with normal human sera and HFRS-causing viral antibodies. As determined by screening ELISA and dot blot analysis, IgG antibodies against MJNV NP were detected in sera from two of 385 healthy individuals from MJNV-endemic areas, suggesting infection with MJNV or MJNV-like thottimvirus. Based on the suggestive evidence, healthcare workers should be alert to febrile diseases occurring among individuals with exposure to shrew-infested habitats.

Prevalence of dengue antibodies in healthy children and adults in different Colombian endemic areas

ObjectivesColombia is a dengue hyperendemic country; however, the prevalence of antibodies against dengue in the general population including the inhabitants of rural areas is unknown. This study aimed to determine the prevalence of dengue IgM and IgG antibodies in healthy children and adults in urban and rural areas of seven different endemic regions in Colombia between 2013 and 2015. Design or methodBlood samples from healthy volunteers (1,318) were processed by serology (by indirect IgG and capture IgM and IgG ELISA) and molecular tests to detect viral RNA and circulating serotypes. ResultsThe seroprevalence of IgG for dengue were 85% in children and over 90% for adults. In addition to the high IgM positive rate (14.9%) and secondary recent infection marker rate (capture IgG, 16%), 8.4% of the healthy volunteers were positive for dengue virus (DENV) RNA. ConclusionThis study confirmed the broad and permanent circulation of DENV in Colombia and the high rates of infection and reinfection suffered by its inhabitants. This information can be used by the health authorities to strengthen vector control and vaccine policies and review the algorithms of diagnosis and disease management in children and adults.

Evaluation of Crimean-Congo Hemorrhagic Fever Orthonairovirus AviTagged Nucleoprotein for Potential Application in Diagnosis

Background:Crimean-Congo hemorrhagic fever (CCHF) is an acute viral zoonotic disease, with a mortality rate of 30-50%. There is no approved vaccine or any specific antiviral treatment for CCHF; therefore, the rapid diagnosis seems to be crucial for both efficient supportive therapy and control of infection spread. In this study, the potency of recombinant nucleoprotein of virus expressed in prokaryotic system was investigated for diagnosis of the infection.Methods: The DNA sequence of complete nucleoprotein ORF was codon optimized based on E. coli codon usage and synthesized commercially. The gene was subcloned in pCA4 vector and expressed in E. coli BL21 (DE3). Refolding and simultaneous purification of nucleoprotein were performed using protein folding liquid chromatography method. The recombinant nucleoprotein was analyzed by Western blotting, ELISA, immunofluorescence assay, and circular dichroism. Forty eight human samples, in three IgM positive and three negative control groups, were evaluated using recombinant nucleoprotein in a capture ELISA setting. Serum from healthy individuals, those suspected to viral hemorrhagic fevers, and positive samples of Chikungunya and Dengue were considered as negative controls. Results:The existence and structure of recombinant nucleoprotein were verified and confirmed. Capture IgM ELISA detected all positive samples (sensitivity of 100%), but none of the 25 negative samples was detected as positive (specificity of 100%). The test also detected all the included genotypes of virus. Conclusion:Our recombinant nucleoprotein can be used in IgM capture ELISA for easy and efficient detection of CCHF in any lab in endemic regions.

Epidemiology, clinical profile and laboratory parameters of scrub typhus cases in West Bengal

Context: Scrub typhus is an acute febrile illness caused by Orientia tsutsugamushi. It is a re-emerging rickettsial disease known to occur all over India. Also no data exists as to what proportion of fever cases are due to scrub typhus in West Bengal. Aims: This study was aimed at identifying the proportion of Orientia tsutsugamughi infection among the patients presenting with acute febrile illness and their epidemiological characteristics, clinical profile and laboratory parameters. Methods and Material: Blood samples of patients suffering from acute undifferentiated febrile illness for more than five days duration were collected from different hospitals of Kolkata and IgM capture Enzyme Linked Immuno-Sorbant Assay (ELISA) was performed at referral virology laboratory of School of Tropical Medicine, Kolkata over the period, April 2017 to March 2018. Results: 89 cases were found reactive for scrub typhus IgM out of 259 suspected cases (34.36%). Scrub typhus was mainly prevalent in children with slight male predilection. Most of the patients were from rural belt with a history of poor housing conditions. The common symptoms associated with fever were headache, nausea/vomiting, cough and skin rashes. Common signs were hepatomegaly, splenomegaly, followed by eschar and lymphadenopathy. Important laboratory findings were anaemia, leucocytosis, thrombocytopenia, and elevated liver enzymes whereas serum urea/creatinine level was almost within normal limits. There was no mortality. Conclusions: In this study 34.36% of febrile patients (duration of fever ? five days) were positive for scrub typhus. So it needs to be included in the differential diagnosis of febrile illness in West Bengal. Keywords: Acute febrile illness, Scrub typhus, West Bengal, Orientiatsutsugamushi.

Comparison of Two Commercial ELISA Kits for the Detection of Anti-Dengue IgM for Routine Dengue Diagnosis in Laos.

The endemicity of Dengue virus (DENV) infection remains a major public health problem in Lao PDR. In this study, we compared two commercial anti-dengue IgM ELISA kits, Panbio® Dengue IgM Capture ELISA (Panbio Kit, Alere, Waltham, MA, USA) and DEN DetectTM MAC-ELISA (InBios kit, InBios International, Inc., Seattle, WA, USA), in the context of diagnosis of patients admitted to hospital with clinical dengue presentation. Two panels of paired blood samples were tested. Panel A was composed of 54 dengue confirmed patients (by DENV real-time RT-PCR) and 11 non-dengue dengue patients (other infections confirmed by corresponding PCR results). Panel B included 74 patients randomly selected from consecutive patients admitted to Mahosot Hospital in 2008 with suspicion of dengue fever according to WHO criteria. Results from panel A showed significantly better sensitivity for Panbio kit (64.8%; 95%CI: 50.6–77.3%) than for InBios kit (18.5%; 95%CI: 9.3–31.4%) when testing admission sera. Sensitivity was increased for both kits when combining results from admission and convalescent sera. Concordant results were obtained from panel B with fair agreement (κ = 0.29) between both kits when testing single admission samples, and moderate agreement (κ = 0.5) when combining results from admission and convalescent sera.

A study presenting the analysis of co-infection of chikungunya and dengue virus in a microbiology laboratory at tertiary care hospital in western India

Coinfection means simultaneous infection by two or more virus. Dengue and Chikungunya are viral infections caused by RNA virus (arbovirus). The transmitting vector for both viruses is Aediesaegypti. Human co-infection with DENV and CHIKV have been reported in India since 1967. DENV and CHIKV share a number of similarities in clinical presentation which includes seasonal transmission cycle. This study was carried out in Virology Department, PDU Medical College, Rajkot. A total of 3810, 2122 and 1605 hospitalized patients were serologically screened for DENV, CHIKV and both, respectively between July and October 2017 by IgM Capture ELISA. Co-infection was defined as a positive IgM assay for both infections.60 (3.73%) were IgM positive for both DENV and CHIKV. 474 (12.44%) were mono-infected with DENV and 721 (33.97%) were mono-infected with CHIKV. Simultaneous infection with Dengue and Chikungunya viruses is having more severity than single infection. Early detection and time bound Notification of the positive cases help to reduce the morbidity and mortality rate and help the health authorities to execute effective measures for prevention and control of diseases. This will also help in increasing vigilance before any epidemic. Keywords: Co infection, Dengue, Chikungunya, ELISA.

Performance of InBios ZIKV Detect™ 2.0 IgM Capture ELISA in two reference laboratories compared to the original ZIKV Detect™ IgM Capture ELISA

ZIKV Detect™ 2.0 IgM Capture ELISA (InBios International, Seattle, WA) recently replaced the ZIKV Detect™ IgM Capture ELISA and a number of significant changes have been made to the original version. This study compares data generated from the ZIKV Detect™ 2.0 IgM Capture ELISA, to data generated using the original version of the kit. The same sample sets were used in this comparison, and reference test results for these samples were used to assess sensitivity, specificity, accuracy and concordance of results across two laboratories. Average sensitivity increased from 90.4% to 92.5% with the updated kit where the increase was not statistically different, and specificity increased from 79.5% to 97.4%, a statistically-significant difference. Accuracy of the ZIKV Detect™ 2.0 IgM Capture ELISA was 89% compared to 63.9% for the original version of the kit, and agreement across the laboratories increased from 79.5% to 97.4%. With secondary dengue virus infections, specificity increased from 9.3% to 82.6% with the updated kit, primarily due to the change in interpretation criteria that no longer includes “Possible Zika positive.”

Performance evaluation of the Diasorin LIAISON® XL Zika capture IgM CLIA test

Because Zika virus (ZIKV) can cause serious birth defects and is involved in cases of Guillain-Barré syndrome, the ZIKV outbreak in the American continent in 2015 resulted in an enormous need for ZIKV diagnostic tools. We evaluated the LIAISON® XL Zika Capture IgM test on 106 samples from patients, mainly travelers, with a confirmed or probable ZIKV infection. Sensitivity between 0 and 84 days after onset of symptoms was 92.5%. Specificity was evaluated on a panel of 56 samples known to cause possible cross-reactions. Cross-reaction with DENV antibodies was limited (10.5%) but false-positive results occurred in samples from patients with malaria, CMV and EBV infections.

Establishment of an immunofluorescence assay to detect IgM antibodies to Nipah virus using HeLa cells expressing recombinant nucleoprotein

A novel indirect fluorescent antibody test (IFAT) for detection of IgM against Nipah virus (NiV) was developed using HeLa 229 cells expressing recombinant NiV nucleocapsid protein (NiV-N). The NiV IFAT was evaluated using three panels of sera: a) experimentally produced sera from NiV-N-immunized/pre-immunized macaques, b) post-infection human sera associated with a Nipah disease outbreak in the Philippines in 2014, and c) human sera from a non-exposed Malaysian population. Immunized macaque sera showed a characteristic granular staining pattern of the NiV-N expressed antigen in HeLa 229 cells, which was readily distinguished from negative-binding results of the pre-immunized macaque sera. The IgM antibody titers in sequential serum samples (n = 7) obtained from three Nipah patients correlated well with previously published results using conventional IgM capture ELISA and SNT serology. The 90 human serum samples from unexposed persons were unreactive by IFAT. The IFAT utilizing NiV-N-expressing HeLa 229 cells to detect IgM antibody in an early stage of NiV infection is an effective approach, which could be utilized readily in local laboratories to complement other capabilities in NiV-affected countries.

Comparative study of chikungunya Virus-Like Particles and Pseudotyped-Particles used for serological detection of specific immunoglobulin M

The incidence of chikungunya virus (CHIKV) infection has increased dramatically in recent decades. Effective diagnostic methods must be available to optimize patient management. IgM-capture Enzyme-Linked Immunosorbent Assay (MAC-ELISA) is routinely used for the detection of specific CHIKV IgM. This method requires inactivated CHIKV viral lysate (VL). The use of viral bioparticles such as Virus-Like Particles (VLPs) and Pseudotyped-Particles (PPs) could represent an alternative to VL.Bioparticles performances were established by MAC-ELISA; physico-chemical characterizations were performed by field-flow fractionation (HF5) and confirmed by electron microscopy.Non-purified PPs give a detection signal higher than for VL. Results suggested that the signal difference observed in MAC-ELISA was probably due to the intrinsic antigenic properties of particles.The use of CHIKV bioparticles such as VLPs and PPs represents an attractive alternative to VL. Compared to VL and VLPs, non-purified PPs have proven to be more powerful antigens for specific IgM capture.

Establishment of a comprehensive and high throughput serological algorithm for Zika virus diagnostic testing

The previous serological algorithm for Zika virus (ZIKV) comprised screening by anti-ZIKV IgM capture ELISA (MAC-ELISA) for samples collected within 3 months postexposure or onset (MPEO). Samples positive by MAC-ELISA and samples collected beyond 3 MPEO were tested by the confirmatory plaque reduction neutralization test (PRNT), which proved laborious and time-consuming during the 2015 outbreak. Thus, we evaluated several ZIKV ELISAs to establish an anti-IgM and anti-IgG combination for use as a screening tool for all samples prior to PRNT confirmation. The MAC-ELISA or InBios-M in combination with the Euroimmun-G demonstrated sensitivities of 99.1% and 97.2%, respectively, and nonflavivirus specificity of 96.0%. Their cross-reactivities were 71.4% and 50.0%, respectively, for sera positive for Dengue virus antibodies. Due to near-perfect interrater agreement with PRNT and excellent detection of samples collected beyond 3 MPEO, these combinations were recommended as a screening protocol in a new high-throughput algorithm with special considerations for ZIKV diagnostics.

Cytokine Signature Associated with Disease Severity in Dengue.

Dengue is the most rapidly spreading viral disease transmitted by the bite of infected Aedes mosquitos. The pathogenesis of dengue is still unclear; although host immune responses and virus serotypes have been proposed to contribute to disease severity. In this study, we examined the circulating dengue virus (DENV) and measured plasma levels of inflammatory mediators. Ninety-eight patients during a dengue outbreak in eastern India in 2016 were included in the study. The presence of DENV was demonstrated by detecting NS1 antigen; IgM capture ELISA and serotypes were discriminated by type-specific RT-PCR and/or sequencing. Plasma samples were assayed for 41-plex cytokine/chemokines using multiplex Luminex assay. Eighty-five (87%) samples were positive by NS1/IgM capture ELISA/RT-PCR. All four serotypes of DENV were detected in this outbreak, with DENV-2 as the predominant type, seen in 55% of cases. Mixed infections were seen in 39% of subjects. Among the host inflammatory biomarkers, GM-CSF, IFN-γ, IL-10, IL-15, IL-8, MCP-1, IL-6, MIP-1β, and TNF-α levels were significantly increased in dengue with and without warning signs, in severe dengue patients in comparison to healthy controls. Four cytokines IFN-γ, GM-CSF, IL-10, and MIP-1β correlated significantly with disease severity and could serve as potential predictor for disease severity. Information on the host biomarkers and the dengue serotype may help guide in optimizing effective intervention strategies.

Geographical distribution of primary & secondary dengue cases in India - 2017: A cross-sectional multicentric study.

Background & objectives:Dengue virus infection is endemic in India with all the four serotypes of dengue virus in circulation. This study was aimed to determine the geographic distribution of the primary and secondary dengue cases in India.Methods:A multicentre cross-sectional study was conducted at Department of Health Research / Indian Council of Medical Research (DHR)/(ICMR) viral research and diagnostic laboratories (VRDLs) and selected ICMR institutes located in India. Only laboratory-confirmed dengue cases with date of onset of illness less than or equal to seven days were included between September and October 2017. Dengue NS1 antigen ELISA and anti-dengue IgM capture ELISA were used to diagnose dengue cases while anti-dengue IgG capture ELISA was used for identifying the secondary dengue cases.Results:Of the 1372 dengue cases, 897 (65%) were classified as primary dengue and 475 (35%) as secondary dengue cases. However, the proportion varied widely geographically, with Theni, Tamil Nadu; Tirupati, Andhra Pradesh and Udupi-Manipal, Karnataka reporting more than 65 per cent secondary dengue cases while Srinagar, Jammu and Kashmir reporting as low as 10 per cent of the same. The median age of primary dengue cases was 25 yr [interquartile range (IQR 17-35] while that of secondary dengue cases was 23 yr (IQR 13.5-34). Secondary dengue was around 50 per cent among the children belonging to the age group 6-10 yr while it ranged between 20-43 per cent among other age groups.Interpretation & conclusions:Our findings showed a wide geographical variation in the distribution of primary and secondary dengue cases in India. It would prove beneficial to include primary and secondary dengue differentiation protocol in the national dengue surveillance programme.

Methods to discriminate primary from secondary dengue during acute symptomatic infection

BackgroundDengue virus infection results in a broad spectrum of clinical outcomes, ranging from asymptomatic infection through to severe dengue. Although prior infection with another viral serotype, i.e. secondary dengue, is known to be an important factor influencing disease severity, current methods to determine primary versus secondary immune status during the acute illness do not consider the rapidly evolving immune response, and their accuracy has rarely been evaluated against an independent gold standard.MethodsTwo hundred and ninety-three confirmed dengue patients were classified as experiencing primary, secondary or indeterminate infections using plaque reduction neutralisation tests performed 6 months after resolution of the acute illness. We developed and validated regression models to differentiate primary from secondary dengue on multiple acute illness days, using Panbio Indirect IgG and in-house capture IgG and IgM ELISA measurements performed on over 1000 serial samples obtained during acute illness.ResultsCut-offs derived for the various parameters demonstrated progressive change (positively or negatively) by day of illness. Using these time varying cut-offs it was possible to determine whether an infection was primary or secondary on single specimens, with acceptable performance. The model using Panbio Indirect IgG responses and including an interaction with illness day showed the best performance throughout, although with some decline in performance later in infection. Models based on in-house capture IgG levels, and the IgM/IgG ratio, also performed well, though conversely performance improved later in infection.ConclusionsFor all assays, the best fitting models estimated a different cut-off value for different days of illness, confirming how rapidly the immune response changes during acute dengue. The optimal choice of assay will vary depending on circumstance. Although the Panbio Indirect IgG model performs best early on, the IgM/IgG capture ratio may be preferred later in the illness course.

Viral etiology and clinico-epidemiological profile of clinically suspected acute viral encephalitis

Background: Acute viral encephalitis is a medical emergency which could lead to considerable morbidity and mortality. The spectrum of brain involvement, with varying severity, and the outcome of the disease depend on the causative virus. Geography is a major determinant for encephalitis caused by vector-borne viruses. This retrospective study will focus on Flaviviral etiology, diagnosis, geography and clinical profile of acute viral encephalitis in Sri Lanka. Methods & Materials: This study included 381clinically Japanese Encephalitis(JE) suspected acute encephalitis patients’ samples from December 2016 through February 2017. The CSF samples received from both children and adults across the country were first tested for JE using JE IgM Capture Enzyme Linked Immunosorbent (ELISA) assay (NIV Pune). The positive samples for JE IgM were tested with the Anti Dengue Ig M capture ELISA (SD Korea) assay and to rule-out dengue, with the dengue real time typing PCR assay. Patients were diagnosed with dengue if they were positive for dengue RNA. Only detectable JE IgM was judged to have JE infection, while detectable JE IgM and dengue IgM, but not dengue RNA were identified as having Flavivirus infection. The clinico-epidemiological profiles sent along with the samples were analyzed on diagnosed cases. Results: A total of 381 CSF samples were tested and 23(6%) were positive for JE IgM. Of the JE positives, 08(35%) were positive for dengue IgM and only 04(17%) positive for dengue RNA. JE only was positive in 11(48%) patients. Dengue and JE serology both positive in 8(35%) while 4(17%) were confirmed with dengue RNA. All cases experienced fever. Altered consciousness and seizures observed in 91%(21/23) of patients. It was noted that 63%(7/11) of JE was reported from the Sabaragamuwa Province and young adults and elderly were affected most. Conclusion: JE and dengue were identified as contributing viral etiology for acute viral encephalitis. Even though dengue is not primarily neurotropic, the study convinced the importance of considering dengue encephalitis in appropriate situations. Findings appreciated common clinical profile of acute encephalitis to intervene timely. Data explained the geography of high JE activity and the most affected age group, to strengthen the preventive/control measures.

Evidence for histidine-rich protein 2 immune complex formation in symptomatic patients in Southern Zambia

BackgroundRapid diagnostic tests based on histidine-rich protein 2 (HRP2) detection are the primary tools used to detect Plasmodium falciparum malaria infections. Recent conflicting reports call into question whether α-HRP2 antibodies are present in human host circulation and if resulting immune complexes could interfere with HRP2 detection on malaria RDTs. This study sought to determine the prevalence of immune-complexed HRP2 in a low-transmission region of Southern Zambia.MethodsAn ELISA was used to quantify HRP2 in patient sample DBS extracts before and after heat-based immune complex dissociation. A pull-down assay reliant on proteins A, G, and L was developed and applied for IgG and IgM capture and subsequent immunoprecipitation of any HRP2 present in immune complexed form. A total of 104 patient samples were evaluated using both methods.ResultsImmune-complexed HRP2 was detectable in 17% (18/104) of all samples evaluated and 70% (16/23) of HRP2-positive samples. A majority of the patients with samples containing immune-complexed HRP2 had P. falciparum infections (11/18) and were also positive for free HRP2 (16/18). For 72% (13/18) of patients with immune-complexed HRP2, less than 10% of the total HRP2 present was in immune-complexed form. For the remaining samples, a large proportion (≥ 20%) of total HRP2 was complexed with α-HRP2 antibodies.ConclusionsEndogenous α-HRP2 antibodies form immune complexes with HRP2 in the symptomatic patient population of a low-transmission area in rural Southern Zambia. For the majority of patients, the percentage of HRP2 in immune complexes is low and does not affect HRP2-based malaria diagnosis. However, for some patients, a significant portion of the total HRP2 was in immune-complexed form. Future studies investigating the prevalence and proportion of immune-complexed HRP2 in asymptomatic individuals with low HRP2 levels will be required to assess whether α-HRP2 antibodies affect HRP2 detection for this portion of the transmission reservoir.

Diagnosis of a Suspected Rift Valley Fever Outbreak Using Capture IgM ELISA in the Sudan

Diagnosis of a Suspected Rift Valley Fever Outbreak Using Capture IgM ELISA in the Sudan

A COMPREHENSIVE STUDY ON EBOLA (EBOV) VIRUS: A THREAT TO HUMAN EXISTENCE

Ebola hemorrhagic disease is a severe, an acute, often fatal disease in humans and non-humans which is caused by infection with a virus of family: Filoviridae, genus: Ebola virus. The incubation period of ebola virus disease (EVD) varies from 2-21 days, with an observed average of 8 to 10 days by following introduction of Ebola virus in the human population through animal-to-human transmission, person-to-person transmission by direct contact body fluids/secretions of infected persons. The most common symptoms include sudden onset of fever, intense weakness, muscle pain, headache and sore throat, vomiting, diarrhea, rash, impaired kidney and liver function, and at advanced stage both internal and external bleeding. The virus is transmitted by contact with body fluids of infected humans or animals is primarily responsible for the virus outbreak. Fruit bats are considered as the natural reservoirs of the virus. The most general assays used for antibody detection are direct IgG and IgMÂ ELISAs and IgM capture ELISA. An IgM or rising IgG titer (four-fold) contributes to strong presumptive diagnosis. Currently neither a licensed vaccine nor an approved treatment is available for human use. In this review, the Ebola virus: life cycle and pathogenicity in humans, diagnosis, pharmacotherapy and their prevention is summarized. Keywords: Filoviridae, Ebola, Outbreak, Transmission, Symptoms, Hemorrhagic fever.

Neuroimaging evaluation of pattern of brain involvement in Japanese encephalitis and other viral encephalitis in paediatric age group

Introduction: Japanese encephalitis virus, a flavivirus is a significant cause of arboviral encephalitis worldwide. The virus is transmitted to humans via the bite of infected Culex mosquitoes especially C. tritaeniorhynchus, they prefer to bite outdoors and are extremely active and are extremely active in the evening and night. Neuroimaging reveals by MRI and CT scan of patients of Japanese encephalitis shows characteristic lesions particularly in the thalami, substantia nigra, basal ganglia, cerebral cortex, cerebellum, brainstem and white matter.Material & Method: A total of 50 patients divided in 25 anti JE antibody positive patients and another 25 in anti JE antibody negative group. A cross sectional observational analytical study was performed in the time period of January 2015 to August 2016, the patients was studied using IgM capture ELISA and 1.5T MRI machine.Result Analysis: The results were analysed using chi square test and p values. It was found that gray matter structures were the most commonly affected, the thalamus, basal ganglia but white matter lesions were also noted. The thalamus was the most commonly involved structure, followed by the brainstem and cerebral cortex. Haemorrhagic lesions were noted in the cortex, midbrain and cerebellum.Conclusion: Involvement of the thalamus showed the most significant difference between JE and non JE patients with 64% of the JE patients showed thalamic involvement. No other part of the brain showed such significant difference.

Diagnosis of dengue fever: roles of different laboratory test methods

Background: Dengue fever is currently the most important arthropod borne viral disease. Since occurrence of dengue infections has been an epidemic in many parts of India and complications like DHF and DSS are increasing, while at the same time the diagnosis is challenging, particularly the laboratory diagnosis is confusing, this study was conducted to evaluate the different laboratory test methods and to compare their respective efficacy, timing, advantages and disadvantages.Methods: This study was done in the Department of Microbiology in collaboration with the Department of Medicine and Pediatrics in two tertiary care medical colleges and hospitals in eastern India. Blood samples from 319 patients with clinical features suggestive of Dengue fever were included in this study. Laboratory investigations were done which included immunological assays that were performed using commercially available kits - SD dengue duo NS1Ag + Ab combo rapid test, NS1 Ag capture ELISA, IgM capture ELISA, IgG capture ELISA test for dengue and other routine tests -full blood cell count, coagulation tests, routine biochemical and lipid profile were also done. Ethical considerations were taken care of and statistical evaluations were done.Results: An increased detection of IgM antibody (46.15%) was seen in the early febrile period (1-5 days) as compared to the mid-febrile period (6-10 days), and late febrile period (6-10 days) when it is 6.89%. IgG antibody is much less in early febrile period (4.16%). Compared to mid-febrile period (24.13%), and late febrile period (62.5%). IgM antibodies were detected in 44.5% of the samples, IgG antibodies were detected in 43.5% of the samples, Rapid test was positive in 36.9% and NS1AG ELISA was detected in 43.5% of the samples in the study.Conclusions: It can be inferred from our study that for detection of dengue in the early febrile period (1-5 days), estimation of dengue-specific serum IgM is the most sensitive antibody detection method.