Searching for the best real-time RT-PCRs to detect Zika virus infections: the importance of comparing several protocols

Abstract

Clinical manifestations of Zika, dengue, and chikungunya virus infections are very similar, making it difficult to reach a diagnosis based only on clinical grounds. In addition, there is an intense cross-reactivity between antibodies directed to Zika virus and other flaviviruses, and an accurate Zika diagnosis is best achieved by real-time RT-PCR. However, some real-time RT-PCR show better performance than others. To reach the best possible Zika diagnosis, the analytic sensitivity of some probe-based real-time RT-PCR amplifying Zika virus RNA was evaluated in spiked and clinical samples. We evaluated primers and probes to detect Zika virus, which had been published before, and tested sensitivity using serum spiked and patient samples by real-time RT-PCR. When tested against spiked samples, the previously described primers showed different sensitivity, with very similar results when samples from patients (serum and urine) were analyzed. Real-time RT-PCR designed to amplify Zika virus NS1 showed the best analytical sensitivity for all samples.

Recent progresses and remaining challenges for the detection of Zika virus

“…The assay offered specificity and broad coverage for detecting all African and Asian lineages of ZIKV, and could alleviate the problem (could not be used to detect all strains of ZIKV) of earlier real‐time PCR. In addition, the assay also achieved a low LOD for five RNA transcript copies 406 . To improve the diagnosis of ZIKV RNA in the specimens of animal and humans, Chan et al successfully developed a new, highly specific and sensitive real‐time RT‐PCR assay by targeting the 5ʹ‐untranslated region (5ʹ‐UTR) of ZIKV.…”

Dengue Virus and Influenza A Virus Co-Infection in Pregnancy: A Case Report

“…[10]. Additionally, real-time RT-PCR (qRT-PCR) was made for the detection of Zika virus [11], Chikungunya virus [12], influenza B virus and influenza A virus [13]. All the tests were made in duplicate, with a negative control from the extraction and a negative control from the RT-PCR process.…”

“…All the tests were made in duplicate, with a negative control from the extraction and a negative control from the RT-PCR process. Primers used for these experiments were designed and standardized elsewhere [9,10,11,12,13]. Influenza A infection was confirmed in the lung tissue, in two different experiments, with an average Cycle Threshold CT of 35 (cut-off point of 38.5), and the negative controls showed no amplification curve.…”

Parameter estimates of the 2016-2017 Zika outbreak in Costa Rica: An Approximate Bayesian Computation (ABC) Approach

“…In neonates infected with Zika, an increased number of nervous system malformations, in particular microcephaly, were reported [29]. The diagnosis of the virus, is based on clinical examination, however it is very challenging, because of the overlap of symptoms with other arboviral diseases [30], the cross-reaction between antibodies directed to ZIKV and other flaviviruses [31], and the limited reliable techniques to diagnose infection accurately in those who have symptoms [2]. To date, an accurate Zika diagnosis is best achieve by real-time RT-PCR [31] and there are no specific treatments available for ZIKV infections, therefore only symptoms such as pain and fever can be treated [27].…”