Fifteen laboratories from 7 Brazilian states participated in this study; these laboratories are spread across ≈2,500 km longitude, including the areas most affected during Brazil’s Zika virus outbreak (1). Participants were university laboratories, hospital laboratories, federal research institutes supporting public health services, and a diagnostic testing company. We provided EQA panels to all laboratories. Each panel comprised 12 lyophilized samples containing inactivated full virus spiked into human plasma tested negative for arboviruses beforehand. The panel consisted of 4 Zika virus–positive specimens of 10³–10⁶ RNA copies/mL to assess sensitivity and determine viral load. Zika virus–negative specimens to assess specificity comprised dengue virus serotypes 2 and 4, Japanese encephalitis virus, St. Louis encephalitis virus, West Nile virus, yellow fever virus, and chikungunya virus at ≈10⁵ 50% tissue culture infective dose/mL each and a negative plasma specimen (Table 1). Moreover, each panel included the international World Health Organization (WHO) Zika virus standard for quantification (7). However, the WHO standard has limited availability. Importation of the WHO standard may be restricted by countries that perceive heat-inactivated materials that derive from live virus as potentially infectious. Therefore, we designed and acquired a Zika virus armored RNA (Asuragen, Austin, TX, USA). The Zika virus armored RNA is a synthetic RNA covering the target sites of 9 Zika virus–specific real-time RT-PCRs as described previously (3), encapsulated into bacteriophage proteins. This highly stable, noninfectious, pseudoviral particle can be used as a universal control for the covered assays, shipped without biosafety concerns, and used as a control for both nucleic acid preparation and RT-PCR.

We asked all laboratories to conduct molecular Zika virus diagnostics as routinely done with clinical samples and to quantify Zika virus–positive specimens using both standards. All but 1 laboratory used the same real-time RT-PCR protocol developed by Lanciotti et al. (2), highlighting the wide dissemination of this assay in Brazil and suggesting comparability of test results within this study (Table 1). We found no significant difference between samples containing comparable quantities of the Asian and the African Zika virus lineage, suggesting suitability of the protocols for both lineages (p = 0.313 by Fisher exact test).

EQA results varied among laboratories. Of 15 laboratories, 4 (27%) reported correct results for all samples. Five (33%) reported 1 or 2 false-negative results from samples with low Zika virus concentrations (Table 1; Figure 1, panel A). EQA participants correctly tested only the 2 samples containing the highest Zika virus concentrations of 8.1 × 10⁵ and 7.0 × 10³ copies/mL (exact test of goodness-of-fit p = 1.00 and p = 0.14, respectively). This finding suggests a potential lack of sensitivity that may be problematic given that viral loads of 10³–10⁴ copies/mL are commonly observed in Zika virus–infected patients (3).

Six (40%) laboratories reported >3 false results, including at least 2 false-positive detections of Zika virus–negative specimens. No heterologous flavivirus was particularly affected by false-positive detection, suggesting that false-positive results did not result from unspecific binding of assay oligonucleotides (Table 1). Instead, false-positive results hint at the possibility of laboratory contamination potentially resulting from virus isolation attempts or PCR amplicons generated during prior Zika virus experimentation.

EQA performance varied according to the way viral RNA was prepared. The 8 laboratories conducting Zika virus detection using automated platforms performed generally superior (n = 8; Youden index, 0.661) compared with the 7 laboratories conducting manual RNA extraction (Youden index, 0.446) (Table 2). This finding might indicate an increased risk for contamination during manual RNA preparation. However, automated RNA preparation also might represent a proxy for more affluent settings of those laboratories.

As previously reported (3), RNA extraction critically influences the clinical lower limit of detection (LOD). Although all participants used highly sensitive real-time RT-PCRs, clinical LODs varied considerably because of different RNA extraction protocols (Figure 1, panel B). Lack of detection of low-concentration EQA samples is thus not surprising because even a small decrease in sensitivity readily causes clinical LODs above the concentration of the lowest EQA panel specimen (Figure 1, panel C). This finding highlights that optimized RNA extraction protocols are crucial for sensitive Zika virus diagnostics.

Quantification of Zika virus loads did not differ significantly between use of the armored RNA and the WHO Zika virus standard, with only 0.76 log₁₀ median deviation between results (p = 0.429 by Wilcoxon signed rank test). This observation suggests usability of the armored RNA for Zika virus quantification in tropical regions. Irrespective of the standard, viral load determinations among laboratories were comparable with 0.12–0.88 log₁₀ median deviations of viral load estimates among laboratories for individual Zika virus specimens. However, we also observed drastic deviations of up to 6 orders of magnitude (Figure 2), suggesting that caution must be taken upon comparing viral load determinations as markers for severe Zika virus disease (8,9) among different laboratories.

Some laboratories in Brazil showed suboptimal sensitivity and specificity of Zika virus diagnostic testing. However, these laboratories performed comparably to those in Europe (6). Neither sensitivity nor specificity differed significantly between laboratories in Brazil compared with those in Europe (p = 0.767 and p = 0.324, respectively, by Fisher exact test). Similarly, the proportion of perfectly performing laboratories in this EQA (27%) was comparable with previous EQAs of flavivirus molecular diagnostics, including yellow fever virus (18%), dengue virus (24%), and West Nile virus (27%) (10–12). Flavivirus molecular diagnostics are thus generally challenging and benefit greatly from controls, such as those provided in this EQA. This study underscores the need to combine RT-PCR and serologic testing in Zika virus diagnostic testing, despite their inherent limitations (3).

Independently of the challenges of Zika virus molecular detection, because of taxation and distributor margins, RT-PCR reagents in Latin America are usually 100%–200% more expensive than in affluent countries (13). Limited resources and relatively higher costs potentially force laboratories in Brazil to seek inferior, more affordable solutions. Enhanced access of laboratories in tropical regions to state-of-the-art reagents is thus an unresolved key component of outbreak response. Further EQAs in Brazil should involve state laboratories that carry a large proportion of Zika virus testing within the public health care system. Unfortunately, the state laboratories we contacted for this EQA could not participate because of limited resources.

Finally, lack of sensitivity directly affects estimates of the absolute risk for Zika virus–induced congenital disease upon maternal infection during pregnancy (14). False-positive results potentially have dramatic consequences for patients, as illustrated by a >90% increase in illegal abortion requests in Latin America during the 2016 Zika virus epidemic (15). Our results emphasize the need for continuous quality assessments of Zika virus diagnostic testing globally.