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3 Chapter 3: Evaluating and optimizing diagnostic assays to improve antibody detection of Zika Virus and understanding immune responses to Zika virus

3.1 Overview Chapter 3

When I first arrived Brazil in March 2016 and held our first few meetings with the team of experts in the Zika Reference Laboratory in Rio de Janeiro (IOC, Fiocruz, Brazil), I found that one of the most urgent needs was to evaluate and improve the serological diagnostics for ZIKV. Alarm had spread across the population following the sensationalist messages expressed in the local newspapers and TV. As a result of this, a large number of individuals, including pregnant woman, were presenting to their physician and health centres wanting to know if they had suffered Zika. Due to the mild nature of this illness many were presenting a few days following symptom onset. Moreover, in many neurological cases, with a suspected association with ZIKV, symptoms began over a week following the presentation of the acute rash-fever. I realised serological diagnostic assays were key to the public health response in this outbreak and this became my priority. At that point, there was no published evidence on the evaluation of these methods and only a few assays were commercially available. Therefore, I set as my first objective, to evaluate the accuracy of the serological methods used to diagnose ZIKV infections following acute phase of infection.

Then, as a second objective, I sought to further investigate if newer methods could help to resolve the ongoing challenge of providing accurate diagnostics for ZIKV infections.

Thus, I aimed to optimize and evaluate another two different ELISA assays used to detect ZIKV antibodies and a neutralization assay, the plaque reduction neutralization tests (PRNT).

Additionally, I also investigated the use of assays with different formats including a lateral flow assay for use as a Rapid Diagnostic Test (RDT) and two immunofluorescence assays.

3.2 Introduction

Zika virus (ZIKV) diagnostics underpin all public health responses to the outbreak and accurate results are essential to guide appropriate clinical management of suspected patients, particularly of vulnerable groups such as patients affected by neurological disorders and gestating women. Both false negative and false positive diagnosis may trigger

Evaluation of diagnostic assays for Zika virus Chapter 3

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catastrophic consequences, especially amongst pregnant women. As a demonstration of this, during the explosion of the ZIKV epidemic, it has been suggested that the rate of illegal abortions increased, with enormous health risks that this entails for pregnant woman [127].

During the ZIKV outbreak, governments from countries such as Colombia, Jamaica and Ecuador advised women to avoid becoming pregnant for a period of months to over a year.

This is impractical and neglectful advice because around 40% of pregnancies worldwide are unplanned [128]. Abortion is also illegal or highly restricted in many countries affected by Zika virus, leaving vulnerable women with few options. This led to an increase in illegal abortions in Latin America, with severe risks for maternal health [127].

In 2016, both the WHO and the American Centres for Disease Control and Prevention (CDC) issued laboratory diagnostic algorithms which recommend serological testing for patients presenting a few days after symptom onset when molecular detection of ZIKV was reported to become less sensitive [129, 130]. However, accurate detection of ZIKV antibodies can be very challenging. We still have limited understanding of the antibody responses to ZIKV in different populations. This is a particular concern in Latin America where extensive co-circulation of other flaviviruses has occurred over the last 30 years [131]. Recent studies have shown that over 50% of pregnant Brazilian women have been reported as anti-DENV Immunoglobulin G (IgG) positive [132]. In my research, I have shown that this percentage of IgG positivity among the population may be higher than 80% (See Chapter 5). Similarly, yellow fever vaccination has been reported to have good coverage in most regions of Brazil, especially after the recent yellow fever outbreaks [133, 134].

In 2016, the WHO emphasised the need for field validation of available serological tests [135]. This prompted prioritisation of the research described in this chapter including search, selection and collection of adequate panels and reagents during my first months in Brazil.

In April 2016, the first validation study evaluating ZIKV commercial assays was published in Eurosurveillance [106, 136, 137]. They reported “high specificity and sensitivity” of the commercial IgM and IgG NS1 ZIKV ELISAs (Euroimmun, Germany) using mainly samples from returned travellers for their evaluation. However, this was a relatively small study that lacked samples collected systematically from flavivirus exposed populations.

In early 2016, the Food and Drug Administration (FDA or USFDA) from the United States Department of Health and Human Services issued an Emergency-Use-Authorization (EUA) protocol for the MAC ELISA assay developed by the CDC. Their emergency use authorization

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protocols are generally intended to “make available diagnostic and therapeutic medical devices to diagnose and respond to public health emergencies” [138].

The CDC declared that the ZIKV MAC –ELISA “is intended for the qualitative detection of Zika virus IgM antibodies in human sera or cerebrospinal fluid (CSF) that is submitted alongside a patient-matched serum specimen, collected from individuals meeting CDC Zika virus clinical criteria (e.g., clinical signs and symptoms associated with Zika virus infection) and/or CDC Zika virus epidemiological criteria (e.g., history of residence in or travel to a geographic region with active Zika virus transmission at the time of travel, or other epidemiologic criteria for which Zika virus testing may be indicated).” Since then, most routine Zika Reference Laboratories in Brazil began to implement this assay.