Abstract
ObjectiveTo examine the diagnostic performance of real-time reverse transcription (RT)-polymerase chain reaction (PCR) assays for Zika virus detection.MethodsWe compared seven published real-time RT–PCR assays and two new assays that we have developed. To determine the analytical sensitivity of each assay, we constructed a synthetic universal control ribonucleic acid (uncRNA) containing all of the assays’ target regions on one RNA strand and spiked human blood or urine with known quantities of African or Asian Zika virus strains. Viral loads in 33 samples from Zika virus-infected patients were determined by using one of the new assays.FindingsOligonucleotides of the published real-time RT–PCR assays, showed up to 10 potential mismatches with the Asian lineage causing the current outbreak, compared with 0 to 4 mismatches for the new assays. The 95% lower detection limit of the seven most sensitive assays ranged from 2.1 to 12.1 uncRNA copies/reaction. Two assays had lower sensitivities of 17.0 and 1373.3 uncRNA copies/reaction and showed a similar sensitivity when using spiked samples. The mean viral loads in samples from Zika virus-infected patients were 5 × 104 RNA copies/mL of blood and 2 × 104 RNA copies/mL of urine.ConclusionWe provide reagents and updated protocols for Zika virus detection suitable for the current outbreak strains. Some published assays might be unsuitable for Zika virus detection, due to the limited sensitivity and potential incompatibility with some strains. Viral concentrations in the clinical samples were close to the technical detection limit, suggesting that the use of insensitive assays will cause false-negative results.
Highlights
The Zika virus is a mosquito-borne flavivirus with an approximately 11 kilobase ribonucleic acid (RNA) genome.[1]
All assay-specific in vitro transcripts (IVT) and the universal control ribonucleic acid (uncRNA) allowed comparable quantification of Zika virus RNA with a mean twofold deviation of results, suggesting the ability to use these controls to generate comparable results even when different real-time reverse transcription (RT)–polymerase chain reaction (PCR) assays are used in different laboratories
The two marker assays showed no detection of Zika virus RNA even upon using RNA from high-titred cell culture isolates
Summary
The Zika virus is a mosquito-borne flavivirus with an approximately 11 kilobase ribonucleic acid (RNA) genome.[1]. Reliable detection of the Zika virus in infected people is key to understanding the epidemiology, the pathogenesis and alternative transmission routes of the virus, such as sexual intercourse and blood transfusions.[12] in areas where the Zika virus is co-circulating with dengue and chikungunya viruses, physicians cannot reliably diagnose the Zika virus infection by clinical presentation, because the viruses cause similar symptoms. Using serology for Zika virus diagnostics can be challenging because of the cross-reactivity of antibodies elicited by other endemic flaviviruses – such as dengue, yellow fever, St Louis encephalitis and West Nile viruses.[3,13,14] Molecular detection of viral nucleic acid using real-time reverse transcription (RT)-polymerase chain reaction (PCR) assay is a highly reliable diagnostic method during acute infection. There are six widely used real-time RT–PCR assays for Zika virus detection.[7,13,15,16] The Pan American Health
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