Zika Virus Diagnosis Research Articles

Strain-Dependent Activity of Zika Virus and Exposure History in Serological Diagnostics.

Zika virus (ZIKV) circulates as two separate lineages, with significant genetic variability between strains. Strain-dependent activity has been reported for dengue virus, herpes simplex virus and influenza. Strain-dependent activity of subject specimens to a virus could be an impediment to serological diagnosis and vaccine development. In order to determine whether ZIKV exhibits strain-dependent activity when exposed to antibodies, we measured the neutralizing properties of polyclonal serum and three monoclonal antibodies (ZKA185, 753(3)C10, and 4G2) against three strains of ZIKV (MR−766, PRVABC59, and R103454). Here, MR−766 was inhibited almost 60% less by ZKA185 than PRVABC59 and R103454 (p = 0.008). ZKA185 enhanced dengue 4 infection up to 50% (p = 0.0058). PRVABC59 was not inhibited by mAb 753(3)C10 while MR−766 and R103453 were inhibited up to 90% (p = 0.04 and 0.036, respectively). Patient serum, regardless of exposure history, neutralized MR−766 ~30%−40% better than PRVABC56 or R103454 (p = 0.005−0.00007). The most troubling finding was the significant neutralization of MR−766 by patients with no ZIKV exposure. We also evaluated ZIKV antibody cross reactivity with various flaviviruses and found that more patients developed cross-reactive antibodies to Japanese encephalitis virus than the dengue viruses. The data here show that serological diagnosis of ZIKV is complicated and that qualitative neutralization assays cannot discriminate between flaviviruses.

Zika Vírus: conhecimentos, sentimentos e rede de apoio social de gestantes

Objetivo: compreender os conhecimentos, os sentimentos e a rede de apoio social de gestantes sob suspeita e diagnóstico positivo de Zika vírus. Método: estudo descritivo, com abordagem qualitativa, realizado com 10 gestantes. Os dados foram coletados no segundo semestre de 2017, por meio de entrevista semiestruturada e analisados pela técnica de análise temática. Resultados: evidenciou-se o déficit de conhecimento sobre a patologia na gestação e os sinais e sintomas apresentados. Sentimentos negativos como medo, desespero e choro estão associados as possibilidades de repercussão fetal frente à suspeita e diagnóstico de microcefalia do bebê. O cuidado multiprofissional, juntamente com a família e as crenças religiosas foram vistos como rede de apoio para o enfrentamento da doença. Considerações finais: o conhecimento, os sentimentos e a rede de apoio são essenciais para se pensar políticas e a própria organização dos serviços de saúde, na perspectiva do enfretamento e do cuidado em saúde.

Zika and women's sexual and reproductive health: Critical first steps to understand the role of gender in the Colombian epidemic.

ObjectivesTo describe the mechanisms of implementation of Zika virus diagnosis, prevention, and management guidelines in Colombia, and to characterize their influence on efforts to defend sexual and reproductive rights.MethodsA qualitative study performed between February and April 2018 in three municipalities in Colombia. We conducted 30 semistructured interviews and five focus groups with key informants who played a role during the epidemic. These included decision‐makers, program coordinators, healthcare providers, pregnant women diagnosed with Zika virus, and members of affected communities.ResultsWe identified barriers to and facilitators for the implementation of the national Zika virus response plan. Barriers included a lack of coordination between vector control efforts and in the realms of sexual and reproductive rights. Facilitators included healthcare providers’ response to the epidemic, the development of technical skills, and the establishment of coordination and referral networks across different institutions.ConclusionA multidimensional approach that considers healthcare services, gender issues, and the environment is crucial. We highlight the epidemic's effects on women's sexual and reproductive rights, mainly related to inequalities in sexual and reproductive health such as the increased risk of sexually transmitted infections experienced by the poorest and most vulnerable women.

A serological point-of-care test for Zika virus detection and infection surveillance using an enzyme-free vial immunosensor with a smartphone

Zika virus (ZIKV) is a mosquito-borne flavivirus (FLAV) that emerged in Brazil in 2015 and has rapidly spread to more than 50 countries worldwide. However, early, accurate, and specific point-of-care (POC) diagnosis of ZIKV is very difficult because most infected patients are asymptomatic or display nonspecific symptoms similar to those of other viral infections, and most of the analysis also requires instruments. Herein, an instrument-free ZIKV POC test using a drop of blood comprising a vial immunosensor, artificial nanozyme platinum/gold core-shell nanoparticles (Pt@Au NPs) as a signal probe, and a smartphone was developed to specifically detect ZIKV without cross-reaction with other FLAVs. A high sensitivity of 1 pg/mL ZIKV, desirable specificity, data storage, and geographic location surveillance were simultaneously achieved for the proposed POC test. Our POC test suitably met the urgent needs of ports of entry, airports, and endemic regions with stressed resources, as well as strict clinical requirements for ZIKV detection.

Loop-Mediated Isothermal Amplification (LAMP) for the Diagnosis of Zika Virus: A Review.

The recent outbreak of Zika virus (ZIKV) in the Americas and its devastating developmental and neurological manifestations has prompted the development of field-based diagnostics that are rapid, reliable, handheld, specific, sensitive, and inexpensive. The gold standard molecular method for lab-based diagnosis of ZIKV, from either patient samples or insect vectors, is reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The method, however, is costly and requires lab-based equipment and expertise, which severely limits its use as a point-of-care (POC) tool in resource-poor settings. Moreover, given the lack of antivirals or approved vaccines for ZIKV infection, a POC diagnostic test is urgently needed for the early detection of new outbreaks and to adequately manage patients. Loop-mediated isothermal amplification (LAMP) is a compelling alternative to RT-qPCR for ZIKV and other arboviruses. This low-cost molecular system can be freeze-dried for distribution and exhibits high specificity, sensitivity, and efficiency. A growing body of evidence suggests that LAMP assays can provide greater accessibility to much-needed diagnostics for ZIKV infections, especially in developing countries where the ZIKV is now endemic. This review summarizes the different LAMP methods that have been developed for the virus and summarizes their features, advantages, and limitations.

Genosensors for differential detection of Zika virus

Zika virus (ZIKV) is considered an emerging infectious disease of high clinical and epidemiological relevance. The epidemiological emergency generated by the virus in Latin America and Southeast Asia in 2014 evidenced the urgent need for rapid and acute diagnostic tools. The current laboratory diagnosis of ZIKV is based on molecular and serological methods. However, molecular tools need expensive and sophisticated equipment and trained personnel; and serological detection may suffer from cross-reactivity. In this context, genosensors offer an attractive alternative for field-ready, early and accurate diagnosis of ZIKV. This work reports on the development of genosensors for the differential detection of ZIKV and its discrimination from dengue (DENV) and chikungunya (CHIKV) homologous arboviruses. We designed specific capture and signal probes by bioinformatics, and prove their specificity to amplify the target genetic material by the polymerase chain reaction (PCR). The designed biotinylated capture and digoxigenin (Dig)-labeled signal probes hybridized the target in a sandwich-type format. An anti-Dig antibody labeled with the horseradish peroxidase (HRP) enzyme allowed for both optical and electrochemical detection. The genosensors detected the ZIKV genetic material in spiked serum, urine, and saliva samples and cDNA from infected patients, discriminating them from the DENV and ZIKV genetic material. The proposed system offers a step forward to the differential diagnosis of the ZIKV, closer to the patient, very promising for diagnosis and surveillance of this rapidly emerging disease.

Serological tests reveal significant cross-reactive human antibody responses to Zika and Dengue viruses in the Mexican population

Zika virus (ZIKV) is a mosquito-borne flavivirus that has caused recent large outbreaks in the Americas. Given its association with severe congenital defects including microcephaly, distinguishing infections caused by ZIKV from those caused by dengue virus (DENV) is of primordial importance. The objectives of this study were to evaluate the recombinant proteins rEIII-ZIKV (Envelope protein domain III) and rNS1ß-leader-ZIKV (non-structural protein 1) for the serological diagnosis of ZIKV in the Mexican population. We also evaluated potential cross-reactivity in commercial enzyme-linked immunosorbent assays (ELISA) based on the ZIKV NS1 and DENV NS1 proteins. rEIII-ZIKV and rNS1ß-leader-ZIKV proteins were tested with sera from 30 PCR-confirmed ZIKV cases, 50 ZIKV-naive, DENV-exposed subjects with no acute febrile disease, (asymptomatic subjects, AS), and 50 ZIKV-naive and DENV naive AS. Commercial ELISA tests were evaluated with sera from 57 ZIKV and 20 DENV PCR-confirmed cases, and 50 ZIKV-naive, DENV-exposed AS. In-house ELISA assays showed that IgM antibody levels against rEIII-ZIKV and rNS1ß-ZIKV were higher in ZIKV naive, DENV-exposed AS than in acutely infected ZIKV individuals. IgG reactivity was highest for rEIII-ZIKV, and indistinguishable between acutely infected ZIKV cases and DENV exposed AS. Positivity for the Euroimmun Zika IgM assay at 7–10 days was considerably higher in DENV-naive ZIKV patients (86%) than in DENV-exposed ZIKV patients (33%), while 39% of the latter had false-negative anti-ZIKV IgG before 7 days of onset. DENV-exposed ZIKV patients presented lower anti-ZIKV IgM and higher IgG responses similar to a secondary dengue response. Forty-four percent of DENV- exposed acute ZIKV patients were DENV IgM positive with the Panbio Dengue assay, and two (15%) of the DENV-naive ZIKV patients presented false DENV IgG conversion. Given the extensive cross-reactivity to both the NS1 and EDIII proteins in current serological methods, the development of sensitive and specific serological tests to distinguish ZIKV from DENV infections is an urgent priority.

One-step pentaplex real-time polymerase chain reaction assay for detection of zika, dengue, chikungunya, West nile viruses and a human housekeeping gene

BackgroundRecent emergence of Zika virus (ZIKV), and the global spread of dengue (DENV), chikungunya (CHIKV) and West Nile viruses (WNV) raised urgent need of accurate and affordable molecular diagnosis of these clinically indistinguishable arboviral infections. ObjectivesWe established a pentaplex real-time reverse transcription PCR (rRT-PCR) assay (CII-ArboViroPlex rRT-PCR) for specific and sensitive detection of the African and American genotypes of ZIKV, all four serotypes of DENV, CHIKV, WNV and a housekeeping gene as internal control in single reaction. Study designSpecific primers and probe sets were designed for ZIKV, DENV, CHIKV, WNV and RNase P (housekeeping gene) and tested for in-vitro transcribed RNA standards, virus cultures, clinical samples positive for ZIKV, DENV, CHIKV and WNV and limit of detection (LOD) were determined for each. Results Using ten-fold serially diluted in-vitro transcribed RNA, CII- ArboViroPlex rRT-PCR assay has LOD of 100 RNA copies/reaction (Rn) for ZIKV in serum or urine, 100 RNA copies/Rn for DENV in serum, and 10 RNA copies/Rn for CHIKV and WNV in serum. LODs from sera spiked with quantitated viral stocks were 2.6 × 102 GEQ/Rn for ZIKV, 2.2 × 101 GEQ/Rn for DENV-1, 9.4 × 100 GEQ/Rn for DENV-2, 2.3 × 102 GEQ/Rn for DENV-3, 1.4 × 103 GEQ/Rn for DENV-4, 2.7 × 102 GEQ/Rn for CHIKV, and 1.05 × 101 GEQ/Rn for WNV. ConclusionsThe CII-ArboViroPlex rRT-PCR assay is a quantitative one-step pentaplex rRT-PCR assay for the molecular detection and differential diagnosis of ZIKV, DENV, CHIKV, WNV and a human housekeeping gene control in a single- PCR reaction.

Implications of the clinical gestational diagnosis of ZIKV infection in the manifestation of symptoms of postpartum depression: a case-control study

BackgroundTraumatic events can trigger postpartum depression. Pregnant women in Brazil today are facing an extremely stressful experience. Thus, the objective here was to analyze the prevalence of symptoms of depression in the immediate postpartum period (IPPD) and associate these symptoms with previous stressful, social, psychological, behavioral, obstetrical, clinical, violent and infectious events, particularly exposure to Zika virus (ZIKV) infection during pregnancy.MethodsThis was a case-control study. The sample comprised 213 puerperal women treated in the maternity ward of a public reference hospital in the Araguaia River Valley of Mato Grosso and Goiás, Brazil. Severe IPPD symptoms were confirmed based on the Edinburgh Postnatal Depression Scale (EPDS). A descriptive statistical analysis was carried out using the Epi Info™ version 7.1.5 suite of software tools and the Statistical Package for Social Sciences (SPSS) version 21.0.ResultsA bivariate analysis revealed a significant association between “severe symptoms of IPPD” and the following variables: “clinical diagnosis of ZIKV during pregnancy” (OR = 13.36; 95% CI = 5.34–33.39); “was separated or divorced in the last year” (OR = 3.58; 95% CI = 1.42–8.99); “suffered an accident in the last year” (OR = 3.32, 95% CI = 1.12–9.82); “suffered emotional violence during pregnancy” (OR = 3.80; 95% CI = 1.81–7.99); “suffered physical violence during pregnancy” (OR = 11.86; 95% CI = 2.07–67.82); “fear of her partner” (OR = 17.90; 95% CI = 3.44–92.99); “dengue fever during pregnancy” (OR = 7.85; 95% CI: 1.66–37.05), and “has a family member diagnosed with mental illness” (OR = 2.54; 95% CI = 1.09–5.93). The multivariate analysis confirmed the association of severe PPD symptoms only with the variables of “clinical diagnosis of ZIKV during pregnancy” (OR = 19.82; 95% CI: 5.35–73.39) and “was separated or divorced in the last year” (OR = 3.92; 95% IC = 1.12–13.63).ConclusionsClinically diagnosed ZIKV during pregnancy may be one of the main events associated with severe IPPD symptoms, showing an almost 20-fold higher chance of occurrence than other factors.

Development of a direct reverse-transcription quantitative PCR (dirRT-qPCR) assay for clinical Zika diagnosis

ObjectiveThe nucleic acid-based polymerase chain reaction (PCR) assay is commonly applied to detect infection with Zika virus (ZIKV). However, the time- and labor-intensive sample pretreatment required to remove inhibitors that cause false-negative results in clinical samples is impractical for use in resource-limited areas. The aim was to develop a direct reverse-transcription quantitative PCR (dirRT-qPCR) assay for ZIKV diagnosis directly from clinical samples. MethodsThe combination of inhibitor-tolerant polymerases, polymerase enhancers, and dirRT-qPCR conditions was optimized for various clinical samples including blood and serum. Sensitivity was evaluated with standard DNA spiked in simulated samples. Specificity was evaluated using clinical specimens of other infections such as dengue virus and chikungunya virus. ResultsHigh specificity and sensitivity were achieved, and the limit of detection (LOD) of the assay was 9.5×101 ZIKV RNA copies/reaction. The on-site clinical diagnosis of ZIKV required a 5μl sample and the diagnosis could be completed within 2h. ConclusionsThis robust dirRT-qPCR assay shows a high potential for point-of-care diagnosis, and the primer–probe combinations can also be extended for other viral detection. It realizes the goal of large-scale on-site screening for viral infections and could be used for early diagnosis and the prevention and control of viral outbreaks.

An update on the aspects of Zika virus infection on male reproductive system.

Zika virus (ZIKV) is mainly transmitted through Aedes mosquito bites, but sexual and post-transfusion transmissions have been reported. During acute infection, ZIKV is detectable in most organs and body fluids including human semen. Although it is not currently epidemic, there is a concern that the virus can still reemerge since the male genital tract might harbor persistent reservoirs that could facilitate viral transmission over extended periods, raising concerns among public health and assisted reproductive technologies(ART) experts and professionals. So far, the consensus is that ZIKV infection in the testes or epididymis might affect sperm development and, consequently, male fertility. Still, diagnostic tests have not yet been adapted to resource-restricted countries. This manuscript provides an updated overview of the cellular and molecular mechanisms of ZIKV infection and reviews data on ZIKV persistence in semen and associated risks to the male reproductive system described in human and animal models studies. We provide an updated summary of the impact of the recent ZIKV outbreak on human-ART, weighing on current recommendations and diagnostic approaches, both available and prospective, with special emphasis on mass spectrometry-based biomarker discovery. In the light of the identified gaps in our accumulated knowledge on the subject, we highlight the importance for couples seeking ART to follow the constantly revised guidelines and the need of specific ZIKV diagnosis tools for semen screening to contain ZIKV virus spread and make ART safer.

A sensitive label-free impedimetric DNA biosensor based on silsesquioxane-functionalized gold nanoparticles for Zika Virus detection

Zika virus (ZIKV) has recently become a global health challenge due to its rapid geographical expansion, since it is associated with serious neurological anomalies such as Guillain-Barré syndrome and microcephaly. Currently, the techniques for ZIKV diagnosis require labor-intensive, expensive and lengthy tests using sophisticated equipment. Moreover, false-positive or false-negative results can occur. In the present work, a DNA biosensor to detect ZIKV in real human serum samples was developed using an oxidized glassy carbon electrode (ox-GCE) modified with silsesquioxane-functionalized gold nanoparticles (AuNPs-SiPy). This nanohybrid was characterized by UV-Vis, FTIR and Raman spectroscopies, DLS, and XRD. The conditions for the immobilization of a ZIKV ssDNA probe on the electrode surface (ox-GCE-[AuNPs-SiPy]) were optimized by univariate and multivariate analysis. The optimized biosensor was characterized by CV, EIS and AFM experiments. The ZIKV target recognition was based on the variation of the charge transfer resistance (ΔRct) of the redox marker ([Fe(CN)6]3-/4-) used and the roughness (Rq) of the electrode surface. The proposed biosensor presented a LOD of 0.82 pmol L-1, with a linear range of 1.0 x10-12 – 1.0 x10-6 mol L-1. Moreover, the reported device showed a suitable stability and satisfactory sensitivity and selectivity to quantify ZIKV in human serum samples, which suggests its promising clinical applications for the early diagnosis of ZIKV-associated pathologies.

Quantum dots-based fluoroimmunoassay for anti-Zika virus IgG antibodies detection

Zika virus (ZIKV) has been declared a public health emergency of international concern. ZIKV has been associated with some neurological disorders, and their long-term effects are not completely understood. The majority of the methods for ZIKV diagnosis are based on the detection of IgM antibodies, which are the first signs of immunological response. However, the detection of IgG antibodies can be an important approach for ZIKV past infection diagnosis, especially for pregnant women, helping the comprehension/treatment of this disease. There has been a growing interest in applying nanoparticles for efficient ZIKV or antibodies detection. Quantum dots (QD) are unique fluorescent semiconductor nanoparticles, highly versatile for biological applications. In the present study, we explored the special QD optical properties to develop an immunofluorescence assay for anti-ZIKV IgG antibodies detection. Anti-IgG antibodies were successfully conjugated with QDs and applied in a fluorescence sensing nanoplatform. After optimization using IgG antibodies, the conjugates were employed to detect anti-ZIKV IgG antibodies in polystyrene microplates sensitized with ZIKV envelope E protein. The nanoplatform was able to detect anti-ZIKV IgG antibodies in a concentration at least 100-fold lower than the amount expected for protein E immune response. Moreover, conjugates were able to detect the antibodies for at least 4 months. Thus, our results showed that this QDs-based fluoroimmunoplatform can be considered practical, simple and promising to detect Zika past infections and/or monitoring immune response in vaccine trials.

2081. Building a Decision Tree with Serial Serology Measurements Improves Classification in a Flavivirus Co-circulation Region

BackgroundRT-PCR (reverse transcriptase polymerase chain reaction) is often considered the “gold standard” for diagnosis of Zika Virus (ZIKV) infection; however, it has been shown to have low sensitivity. A possible remedy is to study ZIKV-specific IgG (ZsIgG) and IgM (ZsIgM) antibodies. However, the in vitro cross-reactivities of Dengue virus (DENV) and ZIKV-specific antibodies are well known, leading to diagnostic difficulties in an area with co-circulation of the two viruses. Our goal was to use Zika and Dengue serologic assays to build a classification model that improves upon the PPV of commercial kits while maintaining sensitivity.MethodsWe conducted a prospective longitudinal study in Southern Mexico where DENV and ZIKV co-circulation occurs (NCT02831699). Patients were included in two cohorts: a cohort of subjects presenting with a febrile rash meeting WHO/PAHO Zika case definition and a household cohort. After signed consent, all subjects enrolled were evaluated on study-visit Days 0, 3 and 7 (for fever rash cohort) and 28. We considered a subject “true positive” for ZIKV or DENV if RT-PCR positive at any time point. The healthy household cohort (with no positive RT-PCR) was considered “true negatives.” We fit a statistical decision tree taking as inputs serial serology measurements and outputting a predicted disease category. Funded in part by the NCI Contract No. HHSN261200800001E. Funded in part by the Mexican Ministry of Health.ResultsAs of March 2018, we have 32 subjects in the Zika PCR+ group, 32 in the Dengue PCR+ group, and 68 in the household group. Our decision tree (Figure 1) achieved PPV of at least 90% on all three disease categories, while maintaining sensitivity above 50%. The highest PPV achieved by the kit manufacturer recommended cutoffs while maintaining a sensitivity of at least 10% on Zika PCR+ subjects is 30/114 (26%), and for Dengue PCR+ subjects is 21/30 (70%).ConclusionUsing serology data in a statistical decision tree improves the PPV exhibited by the kit manufacturer recommendations while still maintaining respectable sensitivity. Physicians in regions with co-circulating flaviviruses should be aware of the pitfalls of using only RT-PCR or using pre-established commercial cutoffs in the serology kits for diagnosis. Disclosures All authors: No reported disclosures.

Identification of peptide based B-cell epitopes in Zika virus NS1

Zika virus (ZIKV), a mosquito-borne flavivirus that has recently emerged globally, poses a major threat to public health. To control this emerging disease, accurate diagnostics are required for monitoring current ZIKV outbreaks. Owing to the high nucleotide sequence similarity and cross-reactivity of ZIKV with other members of the Flaviviridae family, discrimination from other flavivirus infections is often difficult in endemic areas. ZIKV NS1 induces major virus-specific antibodies and is therefore utilized as a serological marker for ZIKV diagnosis. To identify ZIKV specific epitopes for clinical application, 33 NS1 peptides that are 15–30 amino acid in length covering whole NS1 were synthesized and analyzed linear B-cell epitopes with 38 human serum samples (20 ZIKV-positive and 18 ZIKV-negative). As a result of screening, eight epitope regions were identified. In particular, the Z8 and Z14 peptides located in the β-ladder surface region showed higher levels of binding activity in ZIKV-positive sera without cross-reactivity to other flaviviruses. These identified sensitive and specific epitopes provide a tool for design of diagnostics and structure-based vaccine antigens for ZIKV infection.

Zika Virus Liquid Biopsy: A Dendritic Ru(bpy)3 2+-Polymer-Amplified ECL Diagnosis Strategy Using a Drop of Blood.

Zika virus (ZIKV) is a mosquito-borne flavivirus that leads to devastating consequences for fetal development. However, accurate diagnosis of ZIKV is made difficult by the fact that most infected patients are asymptomatic or present with symptoms similar to those of other febrile illnesses. Thus, the development of a simple, accurate, highly sensitive, and reliable method for the biomedical analysis and diagnosis of ZIKV is needed. Herein, a novel ZIKV liquid biopsy system was constructed via a dendritic Ru(bpy)32+-polymer-amplified electro-chemiluminescence (ECL) strategy. This system accomplished amplification-free analysis of ZIKV using a drop of blood, and simultaneously achieved a high sensitivity of 500 copies and superior specificity. This strategy adopted the humoral biomarker as the diagnostic index, which greatly simplified the analysis process, and established a nondestructive detection mode. Furthermore, the performance index for biomedical analysis of clinical ZIKV samples was investigated, and the results indicated that the dendritic Ru(bpy)32+-polymer-amplified ECL strategy reliably responded to ZIKV from the body fluid (blood, saliva, and urine). Hence, this system suitably met the strict clinical requirements for ZIKV detection and thus has the potential to serve as a new paradigm for the biomedical analysis and diagnosis of ZIKV.

The Advancements in the Early Detection of Zika Virus Infection

The Zika Virus (ZIKV) was propelled to international attention during its outbreak from 2015-2016. Interestingly, the most recent outbreak was not ZIKV’s first, although it proved to be the most widespread and impactful, with millions affected in South America, Asia and Africa. Presently no longer considered a global emergency, ZIKV has managed to invoke fear and realization of the susceptibility of the global population to rapidly evolving viruses. In addition, the difficulty of diagnosing the virus demonstrates a deficiency in a rapid, virus specific, and accurate diagnostic tool for the family of flaviviruses that ZIKV belongs to. This paper reviews the approved identification methods along with an analysis of the advantage and disadvantages of each, as well as emerging alternative approaches in ZIKV diagnosis. Common problems with currently utilized methods include slow turnover time, limited throughput, need for further testing to confirm diagnosis, narrow sample compatibility, and cross reactivity to DENV and other similar viruses, Although newer methods discussed in the paper, namely Electrogenerated Chemiluminescence (ECL) and Reporter Virus Neutralization Test (RVNT), show improvement in throughput quantity, speed, and efficiency, it is not certain whether these tests are virus specific and will not react in the presence DENV. The rapidity of diagnosis is important in ensuring timely access to treatment as well as tracking and containing future possible epidemics, Concurrently, virus specificity is equally crucial in ensuring correct diagnosis. Thus, the challenge lies in finding the balance between the two.

Development of multiplex real-time reverse-transcriptase polymerase chain reaction assay for simultaneous detection of Zika, dengue, yellow fever, and chikungunya viruses in a single tube.

Zika virus (ZIKV), dengue virus (DENV), chikungunya virus (CHIKV) and yellow fever virus (YFV) share the same mosquito vectors and have similar clinical manifestations early stage of infection. Therefore, simultaneously differentiating these viruses from each other is necessary. We developed a multiplex real-time reverse-transcriptase polymerase chain reaction (RT-PCR) assay for the differentiation of these four viruses in a single tube. The linear range was established by regression analysis, and the R2 value for each viruswas ≥0.98, and the 95% lower limit of detectionfor each virus was as follows (copies/reaction): ZIKV-Asian, 9; ZIKV-Africa, 15; CHIKV, 11; DENV-1, 19; DENV-2, 13; DENV-3, 24; DENV-4, 36; and YFV, 17. Meanwhile, our multiplex real-time RT-PCR has a good consistency with the commercial singleplex assay. In summary, the developed assay can be effectively used for the diagnosis of ZIKV, DENV, CHIKV, and YFV infections.

Exhaustive TORCH Pathogen Diagnostics Corroborate Zika Virus Etiology of Congenital Malformations in Northeastern Brazil.

The Latin American 2015-2016 Zika virus (ZIKV) outbreak was associated with an increase in microcephaly predominantly in northeastern Brazil. To comparatively investigate infectious causes of congenital malformations, we performed a nested case-control study in 32 mothers of cases of suspected congenital Zika syndrome (CZS) and 160 age-matched controls from Bahia, northeastern Brazil. We collected clinical and imaging data and assessed past exposure to ZIKV, Chikungunya virus (CHIKV), dengue virus, and 8 established TORCH (Toxoplasma gondii, Treponema pallidum, rubella virus, cytomegalovirus, herpes simplex virus 1 [HSV-1] and HSV-2, varicella-zoster virus, parvovirus B19) pathogens using multiple serological tests. Heterogeneous symptoms prevented unequivocal diagnosis of CZS on clinical grounds. Only ZIKV and CHIKV seroprevalence rates differed significantly between cases and controls (93.8% versus 67.8% for ZIKV [Fisher's exact text, P = 0.002] and 20.7% versus 8.2% for CHIKV [χ2, P = 0.039]). High ZIKV seroprevalence rates in cases could not be explained by previous dengue virus infections potentially eliciting cross-reactive antibody responses affecting ZIKV serological tests. In conditional logistic regression analyses, only ZIKV was significantly associated with congenital malformations (P = 0.030; odds ratio, 4.0 [95% confidence interval, 1.1 to 14.1]). Our data support an association between maternal ZIKV exposure and congenital malformations. Parallels between the discrepant ZIKV and CHIKV seroprevalence rates between cases and controls and similar seroprevalence rates between cases and controls for the sexually transmitted T.pallidum and HSV-2 may suggest the occurrence of predominantly vector-borne transmission in our study population. High seroprevalence of TORCH pathogens suggests that exhaustive diagnostics will be necessary in the aftermath of the ZIKV outbreak and provides baseline data for longitudinal studies on ZIKV pathogenesis.IMPORTANCE The Latin American Zika virus (ZIKV) outbreak had a major impact on reproductive health worldwide. The reasons for the massively increased reports of neonatal microcephaly in northeastern Brazil are still unclear. Beyond the technical limitations of laboratory diagnostics, unambiguous diagnosis of ZIKV as the cause of congenital malformations is hampered by similar clinical pictures elicited by other pathogens known as TORCH pathogens. We performed a case-control study comparing mothers of children with congenital malformations to age-matched controls from Salvador, Brazil, one of the areas most extensively affected by the ZIKV outbreak. The ZIKV and Chikungunya virus seroprevalence rates differed significantly, whereas the levels of maternal exposure to TORCH pathogens were similar between cases and controls. Our data support a link between maternal ZIKV infection and congenital malformations and suggest the occurrence of predominantly vector-borne ZIKV transmission in these cases. In addition, some highly prevalent TORCH pathogens may be misinterpreted as representative of ongoing ZIKV activity in the absence of exhaustive diagnostics in northeastern Brazil.

Diagnostic performance of commercial IgM and IgG enzyme-linked immunoassays (ELISAs) for diagnosis of Zika virus infection

BackgroundSerologic detection of Zika virus (ZIKV) infections is challenging because of antigenic similarities among flaviviruses.ObjectiveTo evaluate the sensitivity and specificity of commercial ZIKV IgM and IgG enzyme-linked immunoassay (ELISA) kits.MethodsWe used sera from febrile patients with RT-PCR-confirmed ZIKV infection to determine sensitivity and sera from RT-PCR-confirmed dengue cases and blood donors, both of which were collected before ZIKV epidemics in Brazil (2009–2011 and 2013, respectively) to determine specificity.ResultsThe ZIKV IgM-ELISA positivity among RT-PCR ZIKV confirmed cases was 0.0% (0/14) and 12.5% (1/8) for acute- and convalescent-phase sera, respectively, while its specificity was 100.0% (58/58) and 98.3% (58/59) for acute- and convalescent-phase sera of dengue patients, and 100.0% (23/23) for blood donors. The ZIKV IgG-ELISA sensitivity was 100.0% (6/6) on convalescent-phase sera from RT-PCR confirmed ZIKV patients, while its specificity was 27.3% (15/55) on convalescent-phase sera from dengue patients and 45.0% (9/20) on blood donors’ sera. The ZIKV IgG-ELISA specificity among dengue confirmed cases was much greater among patients with primary dengue (92.3%; 12/13), compared to secondary dengue (7.1%; 3/42).ConclusionsIn a setting of endemic dengue transmission, the ZIKV IgM-ELISA had high specificity, but poor sensitivity. In contrast, the ZIKV IgG-ELISA showed low specificity, particularly for patients previously exposed to dengue infections. This suggests that this ZIKV IgM-ELISA is not useful in confirming a diagnosis of ZIKV infection in suspected patients, whereas the IgG-ELISA is more suitable for ZIKV diagnosis among travelers, who reside in areas free of flavivirus transmission, rather than for serosurveys in dengue-endemic areas.