Evaluation of Altona Diagnostics RealStar Zika Virus Reverse Transcription-PCR Test Kit for Zika Virus PCR Testing

Abstract

With the emerging Zika virus (ZIKV) epidemic, accessible real-time reverse transcription-PCR (rRT-PCR) assays are needed to streamline testing. The commercial Altona Diagnostics RealStar ZIKV rRT-PCR test kit (Altona PCR) has been approved for emergency use authorization by the U.S. FDA. Our aim was to verify the Altona PCR by comparing it to the CDC-designed dual-target ZIKV rRT-PCR reference assay (reference PCR) and describe the demographics of patients tested for ZIKV by rRT-PCR in Ontario, Canada. A large set of clinical specimens was tested for ZIKV by the Altona PCR and the reference PCR. Positive or equivocal specimens underwent PCR and Sanger sequencing targeting the ZIKV NS5 gene. A total of 671 serum specimens were tested by the reference PCR: 58 (8.6%) were positive, 193 (28.8%) were equivocal, and 420 (62.6%) were negative. Ninety percent of the reference PCR-positive patients were tested in the first 5 days after symptom onset. The Altona PCR was performed on 284/671 specimens tested by the reference PCR. The Altona PCR was positive for 53/58 (91%) reference PCR-positive specimens and 16/193 (8%) reference PCR-equivocal specimens; the ZIKV NS5 PCR was positive for all 68 Altona PCR-positive specimens and negative for all 181 Altona PCR-negative specimens that underwent the NS5 PCR. The Altona PCR has very good sensitivity (91%) and specificity (97%) compared to the reference PCR. The Altona PCR can be used for ZIKV diagnostic testing and has less extensive verification requirements than a laboratory-developed test.

Keywords: PCR; Zika virus; diagnostic testing; flavivirus.

FIG 1

Distribution of serum specimens according to ZIKV reference PCR, Altona PCR, and NS5 gene PCR results. ZIKV, Zika virus; reference PCR, 2-step dual-target real-time reverse transcription-PCR, designed by the U.S. Centers for Disease Control and Prevention, targeting the Zika virus envelope (E) and premembrane (prM) genes; NS5, nonstructural protein 5; NSQ, insufficient quantity for testing.

FIG 2

(A) Serum ZIKV reference PCR positivity by day following symptom onset. (B) C_T values of E and prM gene targets by day following symptom onset. ZIKV, Zika virus; reference PCR, 2-step dual-target real-time reverse transcription-PCR, designed by the U.S. Center for Disease Control and Prevention, targeting the Zika virus envelope (E) and premembrane (prM) genes; C_T, cycle threshold. The horizontal bars represent median C_T values.

FIG 3

Distribution of urine specimens according to ZIKV reference PCR, Altona PCR, and NS5 gene PCR results. ZIKV, Zika virus; reference PCR, 2-step dual-target real-time reverse transcription-PCR, designed by the U.S. Centers for Disease Control and Prevention, targeting the Zika virus envelope (E) and premembrane (prM) genes; NS5, nonstructural protein 5; NT, not tested.

FIG 4

ZIKV genome and locations targeted by the ZIKV reference PCR, Altona PCR, and NS5 gene PCR. ZIKV, Zika virus; reference PCR, 2-step dual-target real-time reverse transcription-PCR, designed by the U.S. Centers for Disease Control and Prevention, targeting the Zika virus envelope (E) and premembrane (prM) genes; NS, nonstructural protein gene; C, capsid protein gene. The schematic representation depicts the coding and untranslated regions (UTRs) of the ZIKV genome. The three structural proteins C, prM, and E and the nonstructural (NS) proteins NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5 are specified. The assay targets are indicated above. Briefly, the reference PCR dual-target assay amplifies a 75-bp region spanning the prM-E boundary (Zika1) and a 76-bp portion of the E gene (Zika2), the NS5 PCR targets a 191-bp internal region of the NS5 gene, and the Altona PCR targets the NS1 gene (personal communication with the manufacturer). The viral schematic and genomic locations are based on Zika virus reference strain MR-766 (GenBank accession no. NC_012532.1).