Evaluation of a Rapid Immunochromatographic Assay and Two Enzyme-Linked Immunosorbent Assays for Detection of IgM-Class Antibodies to Zika Virus.
ABSTRACT: There are currently five serologic assays available for detection of anti-Zika virus (ZIKV) IgM-class antibodies with U.S. Food and Drug Administration emergency use authorization. Among these are the Chembio DPP Zika IgM system (DPP Zika ICA; Chembio, Medford, NY), a rapid immunochromatographic assay (ICA), and the InBios ZIKV Detect 2.0 IgM antibody capture enzyme-linked immunosorbent assay (ZIKV 2.0 MAC-ELISA; InBios international, Inc., Seattle, WA), which has replaced the original InBios ZIKV Detect MAC-ELISA. We evaluated performance of these three serologic assays using 72 specimens characterized by plaque reduction neutralization testing (PRNT) for the presence or absence of neutralizing antibodies (NAbs) to ZIKV, dengue virus (DENV), and West Nile virus (WNV). The InBios ZIKV 2.0 MAC-ELISA was "presumptive Zika positive" in all 15 PRNT-confirmed ZIKV samples, while the Chembio DPP Zika ICA was nonreactive in three (20%) and the InBios ZIKV MAC-ELISA was negative in four (27%). The Chembio DPP Zika ICA and InBios ZIKV 2.0 MAC-ELISA showed >95% specificity in 22 ZIKV/DENV-seronegative specimens and in 13 samples positive for NAbs to non-ZIKV flaviviruses. Comparatively, the InBios ZIKV MAC-ELISA was "presumptive" or "possible Zika positive" in 8 of 12 WNV or DENV PRNT-positive samples and in 12 of 22 PRNT-seronegative sera. Our findings suggest that replacement of the InBios ZIKV MAC-ELISA with the InBios ZIKV 2.0 MAC-ELISA will lead to fewer samples requiring PRNT, minimizing unnecessary anxiety among patients ultimately determined to be seronegative for ZIKV and DENV by PRNT and alleviating some of the testing burden on laboratories performing PRNT.
Project description:The recent outbreak of Zika virus (ZIKV) in the Americas has challenged diagnostic laboratory testing strategies. At the Wadsworth Center, ZIKV serological testing was performed for over 10,000 specimens, using a combination of an enzyme-linked immunosorbent assay (ELISA) for IgM antibodies (Abs) to ZIKV, a polyvalent microsphere immunoassay (MIA) to detect Abs broadly reactive with flaviviruses, and a plaque reduction neutralization test (PRNT) for further testing. Overall, 42% of patients showed serological evidence of flavivirus infection (primarily past dengue virus [DENV] infection), while 7% possessed IgM Abs to ZIKV and/or DENV. ZIKV IgM Abs typically arose within 3 to 4 days, with only one instance of duration beyond 100 days after reported symptoms. PRNT analysis of 826 IgM-positive specimens showed 7% positive neutralization to ZIKV alone, 9% to DENV alone, and 85% to both ZIKV and DENV. Thus, the extensive Ab cross-reactivity among flaviviruses significantly reduced the value of performing PRNT analysis, especially when a traditional paired serum algorithm with viral neutralization titering was used. Nevertheless, the finding of a negative ZIKV result by PRNT was invaluable for reassuring both physicians and patients. The MIA detected both IgM and IgG, which enabled us to identify patients who presented without IgM anti-ZIKV Abs but still had ZIKV-specific neutralizing Abs. On the basis of these results, a new algorithm, which included an IgM Ab capture (MAC)-ELISA to detect recent infection, a flavivirus MIA to identify patients no longer producing IgM, and a single-dilution PRNT for ZIKV exclusion and occasional discrimination of ZIKV and DENV, was implemented.
Project description:With the emerging Zika virus (ZIKV) epidemic, serologic diagnosis relies on a labor-intensive IgM antibody capture enzyme-linked immunosorbent assay (MAC-ELISA) and confirmation by a plaque reduction neutralization test (PRNT). To streamline serologic testing, several commercial assays have been developed. Our aim was to compare the commercial Euroimmun anti-ZIKV IgM and IgG assays to the reference MAC-ELISA and PRNT currently in use. Serum specimens submitted to Public Health Ontario Laboratory, Canada, were tested for IgM and IgG using the Euroimmun assays and the results were compared with those from MAC-ELISA. The PRNT was performed on positive or equivocal specimens using either MAC-ELISA or Euroimmun assays, MAC-ELISA-inconclusive specimens, and a convenience sample of specimens negative by both assays (cohort 1). Another set of specimens selected on the basis of PRNT results was subsequently tested by the Euroimmun assays (cohort 2). MAC-ELISA was positive, equivocal, negative, and inconclusive in 57/223, 15/223, 147/223, and 4/223 specimens, respectively. Among the 76 specimens that were MAC-ELISA positive, equivocal, or inconclusive, 30 (39.5%) were Euroimmun IgM and/or IgG positive or equivocal. Among the 147 MAC-ELISA-negative specimens, 136 (92.5%) were Euroimmun IgM and IgG negative. The sensitivity of the combined Euroimmun IgM/IgG against the PRNT was 83% (cohort 1) and 92% (cohort 2), whereas the specificity was 81% (cohort 1) and 65% (cohort 2). The combined Euroimmun IgM/IgG showed good specificity (92.5%) but suboptimal sensitivity (39.5%) compared with that of the MAC-ELISA. However, the sensitivity of the combined Euroimmun IgM/IgG against the PRNT was significantly higher (83 to 92%). More studies are needed before commercial assays are implemented for routine ZIKV serologic diagnosis.
Project description:Zika virus (ZIKV) is an emerging arbovirus belonging to the genus flavivirus that comprises other important public health viruses, such as dengue (DENV) and yellow fever (YFV). In general, ZIKV infection is a self-limiting disease, however cases of Guillain-Barré syndrome and congenital brain abnormalities in newborn infants have been reported. Diagnosing ZIKV infection remains a challenge, as viral RNA detection is only applicable until a few days after the onset of symptoms. After that, serological tests must be applied, and, as expected, high cross-reactivity between ZIKV and other flavivirus serology is observed. Plaque reduction neutralization test (PRNT) is indicated to confirm positive samples for being more specific, however it is laborious intensive and time consuming, representing a major bottleneck for patient diagnosis. To overcome this limitation, we developed a high-throughput image-based fluorescent neutralization test for ZIKV infection by serological detection. Using 226 human specimens, we showed that the new test presented higher throughput than traditional PRNT, maintaining the correlation between results. Furthermore, when tested with dengue virus samples, it showed 50.53% less cross reactivity than MAC-ELISA. This fluorescent neutralization test could be used for clinical diagnosis confirmation of ZIKV infection, as well as for vaccine clinical trials and seroprevalence studies.
Project description:Diagnostic testing for Zika virus (ZIKV) or dengue virus (DENV) infection can be accomplished by a nucleic acid detection method; however, a negative result does not exclude infection due to the low virus titer during infection depending on the timing of sample collection. Therefore, a ZIKV- or DENV-specific serological assay is essential for the accurate diagnosis of patients and to mitigate potential severe health outcomes. A retrospective study design with dual approaches of collecting human serum samples for testing was developed. All serum samples were extensively evaluated by using both noninfectious wild-type (wt) virus-like particles (VLPs) and soluble nonstructural protein 1 (NS1) in the standard immunoglobulin M (IgM) antibody-capture enzyme-linked immunosorbent assay (MAC-ELISA). Both ZIKV-derived wt-VLP- and NS1-MAC-ELISAs were found to have similar sensitivities for detecting anti-premembrane/envelope and NS1 antibodies from ZIKV-infected patient sera, although lower cross-reactivity to DENV2/3-NS1 was observed. Furthermore, group cross-reactive (GR)-antibody-ablated homologous fusion peptide-mutated (FP)-VLPs consistently showed higher positive-to-negative values than homologous wt-VLPs. Therefore, we used DENV-2/3 and ZIKV FP-VLPs to develop a novel, serological algorithm for differentiating ZIKV from DENV infection. Overall, the sensitivity and specificity of the FP-VLP-MAC-ELISA and the NS1-MAC-ELISA were each higher than 80%, with no statistical significance. The accuracy can reach up to 95% with the combination of FP-VLP and NS1 assays. In comparison to current guidelines using neutralization tests to measure ZIKV antibody, this approach can facilitate laboratory screening for ZIKV infection, especially in regions where DENV infection is endemic and capacity for neutralization testing does not exist.
Project description:BACKGROUND:Dengue, chikungunya and Zika viruses (DENV, CHIKV and ZIKV) are transmitted in sylvatic transmission cycles between non-human primates and forest (sylvan) mosquitoes in Africa and Asia. It remains unclear if sylvatic cycles exist or could establish themselves elsewhere and contribute to the epidemiology of these diseases. The Caribbean island of St. Kitts has a large African green monkey (AGM) (Chlorocebus aethiops sabaeus) population and is therefore ideally suited to investigate sylvatic cycles. METHODS:We tested 858 AGM sera by ELISA and PRNT for virus-specific antibodies and collected and identified 9704 potential arbovirus vector mosquitoes. Mosquitoes were homogenized in 513 pools for testing by viral isolation in cell culture and by multiplex RT-qPCR after RNA extraction to detect the presence of DENV, CHIKV and ZIKVs. DNA was extracted from 122 visibly blood-fed individual mosquitoes and a polymorphic region of the hydroxymethylbilane synthase gene (HMBS) was amplified by PCR to determine if mosquitoes had fed on AGMs or humans. RESULTS:All of the AGMs were negative for DENV, CHIKV or ZIKV antibodies. However, one AGM did have evidence of an undifferentiated Flavivirus infection. Similarly, DENV, CHIKV and ZIKV were not detected in any of the mosquito pools by PCR or culture. AGMs were not the source of any of the mosquito blood meals. CONCLUSION:Sylvatic cycles involving AGMs and DENV, CHIKV and ZIKV do not currently exist on St. Kitts.
Project description:Zika virus (ZIKV) serological diagnostics are compromised in areas where dengue viruses (DENV) co-circulate because of their high levels of protein sequence homology. Here, we describe the characterization of a Zika blockade-of-binding ELISA (Zika BOB) and a Zika microneutralization assay (Zika MN) for the detection of ZIKV nonstructural protein 1 (NS1)-specific antibodies and ZIKV neutralizing antibodies, respectively. Zika BOB and Zika MN cutoffs were established as 10 and 100 endpoint titers, respectively, using samples collected pre- and post-virologically confirmed ZIKV infection from subjects living in DENV-endemic areas. Specificity of the assays was equally high, whereas sensitivity of Zika BOB was lower than that of Zika MN, especially in samples collected > 6 months post-infection. Immunosurveillance analysis, using combined results from both Zika BOB and Zika MN, carried out also in DENV-endemic regions in Colombia, Honduras, Mexico, and Puerto Rico before (2013-2014) and after (2017-2018) ZIKV introduction in the Americas suggests unapparent ZIKV seroprevalence rates ranged from 25% to 80% over the specified period of time in the regions investigated.
Project description:Zika virus (ZIKV) infections have been reported from all over Thailand, but the number of reported cases remains low, suggesting a degree of immune protection against ZIKV infection. To address this possibility, the presence of ZIKV neutralizing antibodies was determined in serum from 135 healthy Thai adults with a plaque reduction neutralization test (PRNT), and a number of samples were subsequently analyzed for the presence of neutralizing antibodies to dengue virus (DENV) and Japanese encephalitis virus (JEV). Results showed that 70.4% (PRNT50???10), 55.6 (PRNT50???20) or 22.2% (PRNT90???20) of the samples showed neutralizing antibodies to ZIKV. Detailed analysis showed no association between the presence of neutralizing antibodies to other flaviviruses (DENV, JEV) and the presence of ZIKV neutralizing antibodies. These results suggest that the level of ZIKV neutralizing antibodies in the Thai population is enough to dampen the transmission of the virus in Thailand.
Project description:Imported cases of infections due to Dengue (DENV) and Chikungunya (CHIKV) viruses and, more recently, Zika virus (ZIKV) are commonly reported among travelers returning from endemic regions. In areas where potentially competent vectors are present, the risk of autochthonous transmission of these vector-borne pathogens is relatively high. Laboratory surveillance is crucial to rapidly detect imported cases in order to reduce the risk of transmission. This study describes the laboratory activity performed by the National Reference Laboratory for Arboviruses (NRLA) at the Italian National Institute of Health in the period from July 2014 to October 2015.Samples from 180 patients visited/hospitalized with a suspected DENV/CHIKV/ZIKV infection were sent to the NRLA from several Italian Hospitals and from Regional Reference Laboratories for Arboviruses, in agreement with the National Plan on human surveillance of vector-borne diseases. Both serological (ELISA IgM test and Plaque Reduction Neutralization Test-PRNT) and molecular assays (Real Time PCR tests, RT-PCR plus nested PCR and sequencing of positive samples) were performed.DENV infection was the most frequently diagnosed (80 confirmed/probable cases), and all four genotypes were detected. However, an increase in imported CHIKV cases (41 confirmed/probable cases) was observed, along with the detection of the first ZIKV cases (4 confirmed cases), as a consequence of the recent spread of both CHIKV and ZIKV in the Americas.Main diagnostic issues highlighted in our study are sensitivity limitations of molecular tests, and the importance of PRNT to confirm serological results for differential diagnosis of Arboviruses. The continuous evaluation of diagnostic strategy, and the implementation of laboratories networks involved in surveillance activities is essential to ensure correct diagnosis, and to improve the preparedness for a rapid and proper identification of viral threats.
Project description:BACKGROUND:Zika virus (ZIKV) was first isolated in Uganda in 1947. In Brazil, the first reported case of ZIKV infection was in May 2015. Additionally, dengue (DENV) is endemic and there has been a recent outbreak of chikungunya (CHIKV). Since the clinical manifestations of different arboviral infections (AI) can be similar, definitive diagnosis requires laboratory testing. OBJECTIVES:To determine the prevalence of ZIKV, DENV, and CHIKV infections in a Brazilian cohort of HIV-infected pregnant women, to assess clinical/immunological characteristics and pregnancy outcomes of women with evidence of recent AI. STUDY DESIGN:Laboratory diagnosis of ZIKV, DENV and CHIKV infections utilized serological assays, RT-PCR and PRNT. The tests were performed at the first visit, 34-36 weeks of gestation and at any time if a woman had symptoms suggestive of AI. Mann-Whitney tests were used for comparison of medians, Chi-square or Fisher's to compare proportions; p< 0.05 was considered statistically significant. Poisson regression was used to analyze risk factors for central nervous system (CNS) malformations in the infant according to maternal symptomatology. RESULTS:Of 219 HIV-infected pregnant women enrolled, 92% were DENV IgG+; 47(22%) had laboratory evidence of recent AI. Of these, 34 (72%) were ZIKV+, nine (19%) CHIKV+, and two (4%) DENV+. Symptoms consistent with AI were observed in 23 (10%) women, of whom 10 (43%) were ZIKV+, eight (35%) CHIKV+. No CNS abnormalities were observed among infants of DENV+ or CHIKV+ women; four infants with CNS abnormalities were born to ZIKV+ women (three symptomatic). Infants born to ZIKV+ women had a higher risk of CNS malformations if the mother was symptomatic (RR = 7.20), albeit not statistically significant (p = 0.066). CONCLUSIONS:Among HIV-infected pregnant women with laboratory evidence of a recent AI, 72% were ZIKV-infected. In this cohort, CNS malformations occurred among infants born to both symptomatic and asymptomatic pregnant women with Zika infection.
Project description:Zika virus (ZIKV) is a mosquito-borne flavivirus that is responsible for recent explosive epidemics in the Americas. Notably, ZIKV infection during pregnancy has been found to cause congenital birth defects, including microcephaly, and ZIKV has been associated with Guillain-Barré syndrome in adults. Diagnosis and surveillance of Zika in the Americas have been challenging due to similar clinical manifestations and extensive antibody cross-reactivity with endemic flaviviral diseases, such as dengue. We evaluated four serological and two reverse transcription-PCR (RT-PCR) methods in acute-phase (mean day, 1.8), early-convalescent-phase (mean day, 16.7), and late-convalescent-phase (mean, ~7 months) samples from the same individuals in a long-term pediatric cohort study in Nicaragua. Well-characterized samples from 301 cases of Zika, dengue, or non-Zika, nondengue febrile illnesses were tested. Compared to a composite reference, an in-house IgM antibody capture enzyme-linked immunosorbent assay (MAC-ELISA) and the NIAID-Biodefense and Emerging Infections (BEI) MAC-ELISA measuring IgM yielded sensitivities of 94.5% and 70.1% and specificities of 85.6% and 82.8%, respectively. The NS1 blockade-of-binding ELISA measuring anti-ZIKV NS1 antibody levels yielded sensitivities of 85.0% and 96.5% and specificities of 91.4% and 92.6% at early and late convalescence, respectively. An inhibition ELISA detecting total anti-ZIKV antibodies had sensitivity and specificity values of 68.3% and 58.3% for diagnosis and 94.0% and 98.6% for measuring annual infection incidence. Finally, the ZCD and Trioplex real-time RT-PCR assays detecting Zika, chikungunya, and dengue viruses both yielded a sensitivity of 96.1% and specificity of 100%. Together, these assays resolve the urgent need for diagnostic and surveillance tools for countries affected by Zika virus infections.