scholarly journals A systematic evaluation of IgM and IgG antibody assay accuracy in diagnosing acute Zika Virus infection in Brazil; lessons relevant to emerging infections

2021 ◽  
Author(s):  
Raquel Medialdea-Carrera ◽  
Flavia Levy ◽  
Priscila Castanha ◽  
Patricia Carvalho de Sequeira ◽  
Patricia Brasil ◽  
...  
Keyword(s):  
Symptom Onset ◽  
Zika Virus ◽  
Target Population ◽  
Systematic Evaluation ◽  
Emerging Infections ◽  
Emerging Viruses ◽  
Serum Samples ◽  
Igg Antibody ◽  
Igm Elisa ◽  
Lower Accuracy

Accurate diagnostics underpin effective public health responses to emerging viruses. For viruses, such as Zika virus (ZIKV), where the viremia clears quickly, antibody-based (IgM or IgG) diagnostics are recommended for patients who present seven days after symptom onset. However, cross-reactive antibody responses can complicate test interpretation among populations where closely related viruses circulate. We examined the accuracy (proportion of samples correctly categorized as Zika-positive or negative) for antibody-based diagnostics among Brazilian residents (Rio de Janeiro) during the ZIKV outbreak. Four ZIKV ELISAs (IgM and IgG Euroimmun, IgM Novagnost and CDC MAC), two dengue ELISAs (IgM and IgG Panbio), and the ZIKV plaque reduction neutralization test (PRNT) were evaluated. Positive samples were ZIKV PCR confirmed clinical cases collected in 2015-2016 (n=169); Negative samples (n=236) were collected before ZIKV was present in Brazil (≤2013). Among serum samples collected ≥7 days from symptom onset, PRNT exhibited the highest accuracy (93.7%), followed by the Euroimmun IgG ELISA (77.9%). All IgM assays exhibited lower accuracy (<75%). IgG was detected more consistently than IgM among ZIKV cases using Euroimmun ELISAs (68% versus 22%). Anti-DENV IgM ELISA was positive in 41.1% of confirmed ZIKV samples tested. The Euroimmun IgG assay, although misdiagnosing 22% of samples, provided the most accurate ELISA. Anti-ZIKV IgG was detected more reliably than IgM among ZIKV patients, suggesting a secondary antibody response to assay antigens following ZIKV infection. Antibody ELISAs need careful evaluation in their target population to optimise use and minimise misdiagnosis, prior to widespread deployment, particularly where related viruses co-circulate.

scholarly journals A systematic evaluation of IgM and IgG antibody assay accuracy in diagnosing acute Zika Virus infection in Brazil; lessons relevant to emerging infections

2020 ◽  
Author(s):  
Raquel Medialdea-Carrera ◽  
Flavia Levy ◽  
Priscila Castanha ◽  
Patricia Carvalho de Sequeira ◽  
Patricia Brasil ◽  
...  
Keyword(s):  
Symptom Onset ◽  
Zika Virus ◽  
Target Population ◽  
Systematic Evaluation ◽  
Emerging Infections ◽  
Emerging Viruses ◽  
Serum Samples ◽  
Igg Antibody ◽  
Igm Elisa ◽  
Lower Accuracy

AbstractAccurate diagnostics underpin effective public health responses to emerging viruses. For viruses, such as Zika virus (ZIKV), where the viremia clears quickly, antibody-based (IgM or IgG) diagnostics are recommended for patients who present seven days after symptom onset. However, cross-reactive antibody responses can complicate test interpretation among populations where closely related viruses circulate.We examined the accuracy (proportion of samples correctly categorized as Zika-positive or negative) for antibody-based diagnostics among Brazilian residents (Rio de Janeiro) during the ZIKV outbreak. Four ZIKV ELISAs (IgM and IgG Euroimmun, IgM Novagnost and CDC MAC), two dengue ELISAs (IgM and IgG Panbio), and the ZIKV plaque reduction neutralization test (PRNT) were evaluated. Positive samples were ZIKV PCR confirmed clinical cases collected in 2015-2016 (n=169); Negative samples (n=236) were collected before ZIKV was present in Brazil (≤2013).Among serum samples collected ≥7 days from symptom onset, PRNT exhibited the highest accuracy (93.7%), followed by the Euroimmun IgG ELISA (77.9%). All IgM assays exhibited lower accuracy (<74%). IgG was detected more consistently than IgM among ZIKV cases using Euroimmun ELISAs (68% versus 22%). Anti-DENV IgM ELISA was positive in 41.1% of confirmed ZIKV samples tested.The Euroimmun IgG assay, although misdiagnosing 22% of samples, provided the most accurate ELISA. Anti-ZIKV IgG was detected more reliably than IgM among ZIKV patients, suggesting a secondary antibody response to assay antigens following ZIKV infection. Antibody ELISAs need careful evaluation in their target population to optimise use and minimise misdiagnosis, prior to widespread deployment, particularly where related viruses co-circulate.

scholarly journals Zika Virus Serologic Diagnosis by NS1 ELISA in Curacao

2017 ◽  
Vol 4 (suppl_1) ◽  
pp. S302-S303 ◽  
Author(s):  
Samantha Manuel ◽  
Liane Virginia-Cova ◽  
Loubiela Joseph ◽  
Chris Roggeveen ◽  
Radjin Steingrover
Keyword(s):  
Zika Virus ◽  
Cross Reactivity ◽  
Pregnant Female ◽  
Serum Samples ◽  
Zikv Infection ◽  
Igm Elisa ◽  
Pcr Diagnosis ◽  
Two Samples ◽  
Negative Controls

Abstract Background Zika virus (ZIKV) was introduced in the Caribbean island of Curacao in January 2016. A commercially available ZIKV IgM and IgG ELISA was evaluated on patients that were PCR-positive for ZIKV. Methods ZIKV infection was established by PCR in urine samples. Samples from PCR-positive patients were selected for validation of a ZIKV NS1 IgG and IgM ELISA. Patients with a follow-up sample ≥ 2 weeks after initial presentation were used to assess the sensitivity of the assay. Samples of 15 historical controls with serological evidence of Dengue, Chikungunya or an unrelated viral infection were included to establish specificity and cross-reactivity. Results Fourteen patients with positive ZIKV PCR diagnosis had repeated serum samples drawn ≥ 2 weeks after the initial sample. The combined results of these repeated IgM and IgG tests resulted in a sensitivity of 92%. One pregnant female showed no presence of IgG or IgM in any of the two samples. Testing of the panel of historical ZIKV-negative controls resulted in a specificity of 100% in both the quantitative and semi-quantitative setting of the ELISA. One patient with known high-titers of antibodies against Chikungunya virus in the respective panel displayed borderline reactive results for ZIKV IgG in both quantitative and semi-quantitative setting of the assay. Conclusion In this PCR-positive ZIKV cohort of patients, the newly available ZIKV NS1 ELISA displayed excellent performance characteristics. Cross-reactivity was indicated for Chikungunya in one case. No cross-reactivity was found for Dengue virus infection. One pregnant female showed no signs of developing anti-ZIKV IgM or IgG in this study. In the light of intrauterine pathogenesis, the lack of development of maternal IgG during ZIKV infection is a concern. Disclosures All authors: No reported disclosures.

scholarly journals Enhanced Immune Responses and Protective Immunity to Zika Virus Induced by a DNA Vaccine Encoding a Chimeric NS1 Fused With Type 1 Herpes Virus gD Protein

2020 ◽  
Vol 2 ◽  
Author(s):  
Lennon Ramos Pereira ◽  
Rúbens Prince dos Santos Alves ◽  
Natiely Silva Sales ◽  
Robert Andreata-Santos ◽  
Aléxia Adrianne Venceslau-Carvalho ◽  
...  
Keyword(s):  
Immune Responses ◽  
Zika Virus ◽  
Mammalian Cells ◽  
Hek293 Cells ◽  
Target Antigen ◽  
Structural Protein ◽  
Serum Samples ◽  
Igg Antibody ◽  
Simplex Virus ◽  
Cellular Immune

Zika virus (ZIKV) is a globally-distributed flavivirus transmitted to humans by Aedes mosquitoes, usually causing mild symptoms that may evolve to severe conditions, including neurological alterations, such as neonatal microcephaly and Guillain-Barré syndrome. Due to the absence of specific and effective preventive methods, we designed a new subunit vaccine based on a DNA vector (pgDNS1-ZIKV) encoding the non-structural protein 1 (NS1) genetically fused to the Herpes Simplex Virus (HSV) glycoprotein D (gD) protein. Recombinant plasmids were replicated in Escherichia coli and the expression of the target protein was confirmed in transfected HEK293 cells. C57BL/6 and AB6 (IFNAR1–/–) mice were i.m. immunized by electroporation in order to evaluate pgDNS1-ZIKV immunogenicity. After two doses, high NS1-specific IgG antibody titers were measured in serum samples collected from pgDNS1-ZIKV-immunized mice. The NS1-specific antibodies were capable to bind the native protein expressed in infected mammalian cells. Immunization with pgDNS1-ZIKV increased both humoral and cellular immune responses regarding mice immunized with a ZIKV NS1 encoding vaccine. Immunization with pgDNS1-ZIKV reduced viremia and morbidity scores leading to enhanced survival of immunodeficient AB6 mice challenged with a lethal virus load. These results give support to the use of ZIKV NS1 as a target antigen and further demonstrate the relevant adjuvant effects of HSV-1 gD.

scholarly journals Seroprevalence of anti-SARS-CoV-2 antibodies in Japanese COVID-19 patients

PLoS ONE ◽  
2021 ◽  
Vol 16 (4) ◽  
pp. e0249449
Author(s):  
Makoto Hiki ◽  
Yoko Tabe ◽  
Tomohiko Ai ◽  
Yuya Matsue ◽  
Norihiro Harada ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Symptom Onset ◽  
Detection Rate ◽  
Clinical Manifestations ◽  
Rapid Test ◽  
Igg Antibodies ◽  
Antibody Assay ◽  
Serum Samples ◽  
Igg Antibody ◽  
Chemiluminescent Microparticle Immunoassay

Objectives To determine the seroprevalence of anti-SARS-CoV-2 IgG and IgM antibodies in symptomatic Japanese COVID-19 patients. Methods Serum samples (n = 114) from 34 COVID-19 patients with mild to critical clinical manifestations were examined. The presence and titers of IgG antibody for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) were determined by a chemiluminescent microparticle immunoassay (CMIA) using Alinity i SARS-CoV-2 IgG and by an immunochromatographic (IC) IgM/IgG antibody assay using the Anti-SARS-CoV-2 Rapid Test. Results IgG was detected by the CMIA in 40%, 88%, and 100% of samples collected within 1 week, 1–2 weeks, and 2 weeks after symptom onset in severe and critical cases, and 0%, 38%, and 100% in mild/moderate cases, respectively. In severe and critical cases, the positive IgG detection rate with the IC assay was 60% within one week and 63% between one and two weeks. In mild/moderate cases, the positive IgG rate was 17% within one week and 63% between one and two weeks; IgM was positive in 80% and 75% of severe and critical cases, and 42% and 88% of mild/moderate cases, respectively. On the CMIA, no anti-SARS-CoV-2 IgG antibodies were detected in COVID-19 outpatients with mild symptoms within 10 days from onset, whereas 50% of samples from severe inpatients were IgG-positive in the same period. The IC assay detected higher IgM positivity earlier from symptom onset in severe and critical cases than in mild/moderate cases. Conclusions A serologic anti-SARS-CoV-2 antibody analysis can complement PCR for diagnosing COVID-19 14 days after symptom onset.

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

2021 ◽  
Author(s):  
Kanittha Sirikajornpan ◽  
Piyarat Suntarattiwong ◽  
Detchvijitr Suwanpakdee ◽  
Sutchana Tabprasit ◽  
Darunee Buddhari ◽  
...  
Keyword(s):  
Zika Virus ◽  
High Sensitivity ◽  
Cross Reactivity ◽  
Energy Information Administration ◽  
Elisa Assay ◽  
Serum Samples ◽  
Igm Elisa ◽  
Assay Performance ◽  
Detection And Diagnosis ◽  
Commercial Kits

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 US Energy Information Administration units 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.

scholarly journals Immunoglobulin-like Domain of HsFcμR as a Capture Molecule for Detection of Crimean-Congo Hemorrhagic Fever Virus- and Zika Virus-Specific IgM Antibodies

2019 ◽  
Vol 65 (3) ◽  
pp. 451-461 ◽  
Author(s):  
Anne Rackow ◽  
Christa Ehmen ◽  
Ronald von Possel ◽  
Raquel Medialdea-Carrera ◽  
David Brown ◽  
...  
Keyword(s):  
Zika Virus ◽  
Specific Binding ◽  
Hemorrhagic Fever ◽  
Fever Virus ◽  
Size Exclusion ◽  
Animal Origin ◽  
Serum Samples ◽  
Crimean Congo Hemorrhagic Fever ◽  
Hemorrhagic Fever Virus ◽  
Igm Antibodies

Abstract BACKGROUND The cellular surface molecule HsTOSO/FAIM3/HsFcμR has been identified as an IgM-specific Fc receptor expressed on lymphocytes. Here, we show that its extracellular immunoglobulin-like domain (HsFcμR-Igl) specifically binds to IgM/antigen immune complexes (ICs) and exploit this property for the development of novel detection systems for IgM antibodies directed against Crimean-Congo hemorrhagic fever virus (CCHFV) and Zika virus (ZIKV). METHODS His-tagged HsFcμR-Igl was expressed in Escherichia coli and purified by affinity chromatography, oxidative refolding, and size-exclusion chromatography. Specific binding of HsFcμR-Igl to IgM/antigen ICs was confirmed, and 2 prototypic ELISAs for the detection of anti-CCHFV and anti-ZIKV IgM antibodies were developed. Thereby, patient sera and virus-specific recombinant antigens directly labeled with horseradish peroxidase (HRP) were coincubated on HsFcμR-Igl-coated ELISA plates. Bound ICs were quantified by measuring turnover of a chromogenic HRP substrate. RESULTS Assay validation was performed using paired serum samples from 15 Kosovar patients with a PCR-confirmed CCHFV infection and 28 Brazilian patients with a PCR-confirmed ZIKV infection, along with a panel of a priori CCHFV/ZIKV-IgM-negative serum samples. Both ELISAs were highly reproducible. Sensitivity and specificity were comparable with or even exceeded in-house gold standard testing and commercial kits. Furthermore, latex beads coated with HsFcμR-Igl aggregated upon coincubation with an IgM-positive serum and HRP-labeled antigen but not with either component alone, revealing a potential for use of HsFcμR-Igl as a capture molecule in aggregation-based rapid tests. CONCLUSIONS Recombinant HsFcμR-Igl is a versatile capture molecule for IgM/antigen ICs of human and animal origin and can be applied for the development of both plate- and bead-based serological tests.

scholarly journals A Label and Probe-Free Zika Virus Immunosensor Prussian Blue@carbon Nanotube-Based for Amperometric Detection of the NS2B Protein

Biosensors ◽  
2021 ◽  
Vol 11 (5) ◽  
pp. 157
Author(s):  
Bárbara V. M. Silva ◽  
Marli T. Cordeiro ◽  
Marco A. B. Rodrigues ◽  
Ernesto T. A. Marques ◽  
Rosa F. Dutra
Keyword(s):  
Carbon Nanotube ◽  
Prussian Blue ◽  
Zika Virus ◽  
Point Of Care ◽  
Amperometric Detection ◽  
Cross Reactivity ◽  
Structural Protein ◽  
Serum Samples ◽  
Polypyrrole Composite ◽  
Congenital Disabilities

Zika virus (ZIKV) is a mosquito-borne infection, predominant in tropical and subtropical regions causing international concern due to the ZIKV disease having been associated with congenital disabilities, especially microcephaly and other congenital abnormalities in the fetus and newborns. Development of strategies that minimize the devastating impact by monitoring and preventing ZIKV transmission through sexual intercourse, especially in pregnant women, since no vaccine is yet available for the prevention or treatment, is critically important. ZIKV infection is generally asymptomatic and cross-reactivity with dengue virus (DENV) is a global concern. An innovative screen-printed electrode (SPE) was developed for amperometric detection of the non-structural protein (NS2B) of ZIKV by exploring the intrinsic redox catalytic activity of Prussian blue (PB), incorporated into a carbon nanotube–polypyrrole composite. Thus, this immunosensor has the advantage of electrochemical detection without adding any redox-probe solution (probe-less detection), allowing a point-of-care diagnosis. It was responsive to serum samples of only ZIKV positive patients and non-responsive to negative ZIKV patients, even if the sample was DENV positive, indicating a possible differential diagnosis between them by NS2B. All samples used here were confirmed by CDC protocols, and immunosensor responses were also checked in the supernatant of C6/36 and in Vero cell cultures infected with ZIKV.

scholarly journals THU0296 PROTEIN PROFILING IN INDIVIDUALS BEFORE ONSET OF ANCA-ASSOCIATED VASCULITIS

2020 ◽  
Vol 79 (Suppl 1) ◽  
pp. 376.2-376
Author(s):  
E. Berglin ◽  
A. Esberg ◽  
J. Dahlqvist ◽  
J. Sjöwall ◽  
A. Lundquist ◽  
...  
Keyword(s):  
Immune System ◽  
Growth Factor ◽  
Random Forest ◽  
Symptom Onset ◽  
Partial Least Square ◽  
Protein Profiling ◽  
Serum Samples ◽  
Anca Associated Vasculitis ◽  
Significant Difference ◽  
Novel Biomarkers

Background:Etiology and pathogenesis of ANCA-associated vasculitis (AAV) is multifactorial and understanding of the processes leading from a healthy immune system to autoimmunity and on to debut of symptoms in AAV is rudimentary.Objectives:To identify inflammatory proteins related to the early processes preceding AAV development, and potential novel biomarkers, using large-scale protein analysesMethods:The Swedish National Patient Register of in-patient carevand the Swedish Cause of Death Register with discharge diagnosis from ICD-9 and-10 for AAV were co-analysed with the registers of 4 different blood biobanks to identify AAV individuals with available samples predating onset of symptom. Of the pre-AAV cases 86 (36 male, 50 female; mean age (SD); 51.9 (16.9) years) were identified with at least one plasma or serum sample (28 plasma, and 100 serum) pre-dating symptom onset (mean (SD); -4.3 (3.1) years), and 14 had 2-3 samples. Serum and plasma control samples matched for sex, age and sampling date were identified (n=198; 82 male, 116 female; mean age (SD); 51.9±15.9 years). The samples were analysed for levels of 92 proteins using proximity extension assay (OLINK inflammation panel, SciLifeLab, Uppsala, Sweden). Data were analysed using routine statistical methods, random forest and Partial Least square-discriminant analysis (PLS-DA).Results:As previously described for the assay significant difference between plasma and serum samples were observed both in pre-AAV individuals and controls. In pre-AAV plasma samples significantly increased concentrations of interleukin (IL)-2, chemokine ligand (CCL)-4, fibroblast growth factor (FGF)21, IL-4 and CCL20 were found closer to symptom onset, (<5 years) than later (> 5 years) and compared with controls. In serum tumor necrosis factor receptor superfamily member (TNFRSF)9, CXCL9, osteoprotegerin and vascular endothelial growth factor-A were significantly increased <5 years before onset vs. later (>5 years) and compared with controls. PLS-DA score scattered plot separated the pre-AAV individuals from healthy controls (R2=0.26), with significantly increased levels of CCL23, CXCL5, and matrix metalloproteinases-1 (MMP-1),transforming growth factor-ß, orosomucoid, en-rage (S100A12) and IL-7 and decreased FGF-19 level in serum. Binary logistic regression analyses comparing tertiles for these proteins confirmed significantly increased odds ratios for disease development of CCL23, CXCL5 and MMP-1. The findings were confirmed in random forest analysis where these factors were among the 20 most discriminatory factors between pre-symptomatic AAV and controls.Conclusion:In serum samples collected years before symptom onset of AAV, proteins involved in immune system activation were increased, suggesting that the inflammatory process is initiated long before clinical manifestations of the disease appear. These findings propose the elevated proteins as novel biomarkers for disease progression.References:[1]Watts et al. Ann Rheum Dis 2007;66:222-22Acknowledgments:Vasculitis Foundation, USADisclosure of Interests:Ewa Berglin: None declared, Anders Esberg: None declared, Johanna Dahlqvist: None declared, Johanna Sjöwall: None declared, Anders Lundquist: None declared, Kristina Lejon: None declared, Ingegerd Johansson: None declared, Aladdin J Mohammad Speakers bureau: lecture fees from Roche and Elli Lilly Sweden, PI (GiACTA study), Solbritt Rantapää Dahlqvist: None declared

scholarly journals Fludarabine Inhibits Infection of Zika Virus, SFTS Phlebovirus, and Enterovirus A71

Viruses ◽  
2021 ◽  
Vol 13 (5) ◽  
pp. 774
Author(s):  
Chengfeng Gao ◽  
Chunxia Wen ◽  
Zhifeng Li ◽  
Shuhan Lin ◽  
Shu Gao ◽  
...  
Keyword(s):  
Broad Spectrum ◽  
Zika Virus ◽  
Therapeutic Agent ◽  
Viral Infections ◽  
Rna Virus ◽  
Emerging Viruses ◽  
Enterovirus A71 ◽  
Ic50 Values ◽  
High Doses ◽  
Severe Fever

Viral infections are one of the leading causes in human mortality and disease. Broad-spectrum antiviral drugs are a powerful weapon against new and re-emerging viruses. However, viral resistance to existing broad-spectrum antivirals remains a challenge, which demands development of new broad-spectrum therapeutics. In this report, we showed that fludarabine, a fluorinated purine analogue, effectively inhibited infection of RNA viruses, including Zika virus, Severe fever with thrombocytopenia syndrome virus, and Enterovirus A71, with all IC50 values below 1 μM in Vero, BHK21, U251 MG, and HMC3 cells. We observed that fludarabine has shown cytotoxicity to these cells only at high doses indicating it could be safe for future clinical use if approved. In conclusion, this study suggests that fludarabine could be developed as a potential broad-spectrum anti-RNA virus therapeutic agent.

scholarly journals Low seroprevalence rates of Zika virus in Kuala Lumpur, Malaysia

2019 ◽  
Vol 113 (11) ◽  
pp. 678-684 ◽  
Author(s):  
I-Ching Sam ◽  
Magelda Montoya ◽  
Chong Long Chua ◽  
Yoke Fun Chan ◽  
Andrew Pastor ◽  
...  
Keyword(s):  
Zika Virus ◽  
Large Scale ◽  
Binding Assay ◽  
Seroprevalence Rate ◽  
Protective Effects ◽  
Kuala Lumpur ◽  
Serum Samples ◽  
Denv Serotypes ◽  
Population Immunity ◽  
Low Incidence

Abstract Background Zika virus (ZIKV) is believed to be endemic in Southeast Asia. However, there have been few Zika cases reported to date in Malaysia, which could be due to high pre-existing levels of population immunity. Methods To determine Zika virus (ZIKV) seroprevalence in Kuala Lumpur, Malaysia, 1085 serum samples from 2012, 2014–2015 and 2017 were screened for anti-ZIKV antibodies using a ZIKV NS1 blockade-of-binding assay. Reactive samples were confirmed using neutralization assays against ZIKV and the four dengue virus (DENV) serotypes. A sample was possible ZIKV seropositive with a ZIKV 50% neutralization (NT50) titre ≥20. A sample was probable ZIKV seropositive if, in addition, all DENV NT50 titres were &lt;20 or the ZIKV NT50 titre was &gt;4-fold greater than the highest DENV NT50 titre. Results We found low rates of possible ZIKV seropositivity (3.3% [95% confidence interval {CI} 2.4 to 4.6]) and probable ZIKV seropositivity (0.6% [95% CI 0.3 to 1.4]). Possible ZIKV seropositivity was independently associated with increasing age (odds ratio [OR] 1.04 [95% CI 1.02 to 1.06], p&lt;0.0001) and male gender (OR 3.5 [95% CI 1.5 to 8.6], p=0.005). Conclusions The low ZIKV seroprevalence rate, a proxy for population immunity, does not explain the low incidence of Zika in dengue-hyperendemic Kuala Lumpur. Other factors, such as the possible protective effects of pre-existing flavivirus antibodies or reduced transmission by local mosquito vectors, should be explored. Kuala Lumpur is at high risk of a large-scale Zika epidemic.

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