External Quality Assessment (EQA) of Molecular Detection of Zika Virus: Value of the 1st World Health Organization International Standard

LETTER

Charrel et al. have carried out an external quality assessment (EQA) study for European expert laboratories performing Zika virus (ZIKV) molecular tests, which emphasized the lack of standardization and assay sensitivity for some participating laboratories (1). However, the study omitted a key element: assays and samples traceable to internationally agreed-upon reference materials.

The World Health Organization (WHO) declaration that the microcephaly-associated ZIKV outbreak constituted a public health emergency of international concern facilitated the development, assessment, and validation of ZIKV diagnostic assays (2). These activities included the establishment of the 1st WHO international standard (IS) for ZIKV for molecular assays in October 2016 (3). The IS strain (Paul-Ehrlich-Institut code number 11468/16 [4]) is lyophilized French Polynesian ZIKV strain PF13/251013-18, which is heat inactivated, fully sequenced, and used at a concentration of 25,000,000 international units (IU)/vial (3, 5). The WHO IS strain was used directly in two EQA studies in 2016 and 2017 coordinated by QCMD, an ISO 17043:2010 (6)-accredited EQA organization. In 2016, of the 106 participating laboratories (116 data sets), all were able to detect the ZIKV IS strain diluted to a concentration of 5.7 and 4.7 log₁₀ IU/ml. In 2017, of the 108 participating laboratories (114 data sets), 99.1% could detect the ZIKV IS strain diluted to a concentration of 4.7 log₁₀ IU/ml, and 96.5% were able to detect a lower dilution of 3.7 log₁₀ IU/ml. Inclusion of an African strain in both studies (MR766, supplied by the Robert Koch-Institut, in a heat-inactivated and gamma-irradiated form) revealed that a nominal concentration of 4.7 log₁₀ IU/ml was detected by 97.4% of laboratories in both EQAs; 3.7 log₁₀ IU/ml was detected by 95.7% and 95.6% of laboratories in 2016 and 2017, respectively. A sample with 2.7 log₁₀ IU/ml was detected by 93.1% and 89.5% of laboratories in 2016 and 2017, respectively. The false-positive rates were 2.6% and 3.5% for true-negative samples and for samples containing a mixture of flaviviruses (West Nile, yellow fever, and dengue viruses), and rates of 5.2% and 1.8% were observed in 2016 and 2017, respectively. In 2016, a Chikungunya virus sample gave a false-positive rate of 3.4%. Review of these two EQAs show that overall performance in participating laboratories for the molecular detection of ZIKV was at an acceptable level, although specificity and sensitivity with lower viral loads remain a challenge.

In the EQA described by Charrel et al. (1), the study materials were evaluated using an in-house assay, calibrated using in vitro-transcribed ZIKV RNA. However, without traceability to the WHO IS, it is not possible to compare the results between the different EQA programs and understand acceptable levels of assay sensitivity. WHO ISs play an important role in standardizing diagnostic assays, and expression of viral loads in a common reporting unit (i.e., the international unit) enables comparison of results between different assays and laboratories (7). The continued use of the IS will help ensure the traceability of EQA materials and allow comparability of laboratory performances between EQA programs to help resolve the lack of assay standardization.