Relación del control interno con los derechos de los accionistas

The commercial IgG and IgM ZIKV NS1 ELISAs and the MAC ELISA were the first commercial diagnostic assays approved by the National Health Surveillance Agency of Brazil (ANVISA).

The IgG NS1 Euroimmun assay showed the highest accuracy with 72.5% and the highest NPV with 68.6%. The poor specificity observed with the IgG NS1 Euroimmun assay indicates that this assay provides 22.4% of false positive results in flavivirus exposed populations.

Moreover, users must be aware, 19.6% of the ZIKV positive samples collected between 6 to 14 days (median) post-illness onset were IgG negative. Missed positive diagnosis of ZIKV cases measured with the IgG NS1 assay increased to 65% when samples were collected acutely between 0 to 5 days post-illness onset. The commercial IgM µ-capture assay showed the highest PPV of 86.9 and could potentially be used as a rule-in method for recent ZIKV infection for patients presenting after acute phase.

When I started this research work, there was no published validation study available that had evaluated serological assays. To date (as of July 2018), there are few published validation studies of commercial serological assays using well-characterised samples, but none from Brazil, the country that was impacted by ZIKV the most. Moreover, none of the published studies use serum collections from before the 2015-2016 epidemic, which was when ZIKV spread widely across the Americas.

My findings evaluating these assays in a highly flavivirus exposed population contrast with recently published studies conducted using traveller populations which reported much higher sensitivity (of over 90%) for the commercial IgM and IgG Euroimmun (NS1) assays

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[106, 136, 159]. Their evaluation using mainly European travellers’ samples also found a high specificity. However, it has also been reported that the specificity of the IgM NS1 assay may be low among malaria positive subjects [137]. Surprisingly, it was also reported a very high specificity for the IgG NS1 assay of 90% among Canadian travellers [14] which is much higher to the specificity levels that I obtain of 77.6%, with the test showing even lower specificity, in my study, for subjects previously infected with DENV-2.

A recently published multicentre study conducted in North America by Basile et al. (2018) includes evaluations performed in three different laboratories, reported sensitivity and specificity for the IgM NS1 assay of 53.1% and 97.0% (respectively) [160]. In the results presented in this chapter, similar levels of specificity (96.4) were observed while a much lower sensitivity of 22% (which is 31% less). This study also found much lower sensitivity for the IgG NS1 Euroimmun assay of 34.4% compared to 67.9%. However, they report specificity values of 94.2%, which are much higher than the specificity values that the found in this study at 77.6%. This evidence demonstrates the relevance of testing in populations with previous flavivirus exposure. All these suggest that among DENV immune populations (such as Brazil), the IgG NS1 assay has a much higher sensitivity because the specificity is also much lower due to cross-reactivity with antibodies such as DENV or yellow fever. This also reinforces the claim that the IgM and IgG NS1 Euroimmun assays have poor performance among the Brazilian population and its use for routine diagnostics should be revised.

The IgM µ-capture ELISA which has recently been approved by the FDA and became the recommended method by ANVISA (Brazil) in 2017, is currently being used across the Americas. However, appropriate validation with samples from South America is needed. To date, only two publications have evaluated this assay both with samples from North America, which reported low specificity values ranging from 66% (study conducted in Canada) to 97.6% (study conducted in the USA) [14] [160].

The CDC recommends the use of an IgM antibody capture enzyme-linked immunosorbent assay (MAC ELISA), which is based on the CDC FDA-emergency-use-authorization protocol.

Nevertheless, it has recently recognised the existence of multiple limitations on the use of this assay and related serologic assays for ZIKV diagnosis due to potential false-positive and false-negative test results [161]. Differences among the assays accuracies observed are probably linked to differences between travellers and the Brazilian population [162].

Moreover, recent results now in Nicaragua and the USA have suggested this assay has low specificity (around 82 to 85%) and full evaluations are needed [163, 164]. Based on this

Evaluation of diagnostic assays for Zika virus Chapter 3

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results, I believe it is not appropriate to use exclusively specimens from travellers for validation of serological assays for flaviviral diagnostics.

The cut-off levels established for this commercial assays were optimised mainly using samples from travellers. For example, the cut-offs for the IgM and IgG NS1 Euroimmun ELISAs were established with testing of 29 ZIKV positive samples and over 600 non ZIKV controls from German blood donors or travellers (as reported in their manual). Similarly, the IgM μ-capture was validated using a panel of samples from the US and North America. I suggest the performance of optimizations for the South American population and recommend adjusting the cut-off assay values to flavivirus-exposed populations in order to improve assay accuracy.

Nevertheless, this is a compromise and the overall performance of the commercial assays is still poor. I believe that my findings reinforce the limited capacity for these assays to discriminate between infections caused by closely related flaviviruses.

Cross-reaction seems to be one of the most challenging problems around the diagnosis of flaviviral infections. Recent studies have suggested that the epitopes in the E region may not be as specific as the NS1 region of the flaviviruses [165]. In these results I observed a very high degree of false positives with the MAC ELISA assay. Specificity improved as the cut-off was raised, but sensitivity was then lowered. As expected, the in-house MAC ELISA assay required several additional steps when compared to the commercial ELISAs with over five hours required distributed in over at least two days, compared to around three to four hours for the commercial assays. Moreover, the CDC MAC-ELISA needs site-specific optimization of the dilutions used for some of the reagents leading to potential variability among sites.

Regarding the key features of the kit and taking into account the assay length and number of samples that can be processed per plate, both the IgM and IgG Euroimmun NS1 assays have shown to be the easiest to perform and most high throughput methods among the methods evaluated in this chapter.

Analysis of the performance of the assays demonstrated that the four of them performed quite poorly. Serological testing remains the primary method for diagnosis of infections following acute phase, even though the cross-reactive nature of antibodies elicited during flavivirus infections. Therefore, I tried to better understand the host immunological response following ZIKV infections in order to identify the factors influencing in the poor performance of the commercial assays.

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Investigations of the biological diversity of the ZIKV antibody response