Optimization of a combined human parechovirus-enterovirus real-time reverse transcription-PCR assay and evaluation of a new parechovirus 3-specific assay for cerebrospinal fluid specimen testing

Abstract

Human parechoviruses (HPeVs), particularly type 3 (HPeV3), are known central nervous system (CNS) pathogens, causing serious infections in infants similar to those caused by enteroviruses (EVs). The primary aim of this study was to combine and validate HPeV and EV real-time reverse transcription-PCR (RT-PCR) detection assays with the best available RT-PCR reagents and conditions for parallel detection of HPeV and EV on a single platform. The secondary aim was to develop and validate a newly developed HPeV3-specific real-time RT-PCR assay. Five commercially available RT-PCR kits were evaluated with the pan-HPeV and EV assays in one-step and two-step RT-PCRs. Two-step RT-PCR with the AgPath ID RT-PCR (AGP) kit performed best for both pan-HPeV and EV assays. The pan-HPeV-specific assay performed best with the AGP kit in a one-step RT-PCR. Frozen aliquots of 145 (for HPeV, n = 70; for EV, n = 75) previously characterized cerebrospinal fluid (CSF) specimens were tested by EV-, pan-HPeV-, and HPeV3-specific (HPeV specimens only) assays. The pan-HPeV and EV assays demonstrated 100% analytical sensitivity and specificity compared to historic results, while the HPeV3-specific assay demonstrated 97% sensitivity and 100% specificity. We propose a real-time pan-HPeV, EV two-step RT-PCR algorithm for simultaneous detection of HPeV and EV from CSF specimens on a single platform. The HPeV3-specific one-step RT-PCR assay can be used as a rapid and cost-effective assay to detect and identify HPeV3 in pan-HPeV RT-PCR assay-positive CSF specimens.

Fig 1

Performance of five commercial RT-PCR kits in one-step and two-step reactions with pan-HPeV and EV assays. The control strains, a clinical isolate of HPeV3 (A) and echovirus 9 (B) (initial titer, 1 × 10^7.2 50% tissue culture infective doses/ml), were 10-fold serially diluted using negative CSF specimens. RT-PCRs were tested in duplicate with enzymes from the AGP, QTP, QTRTP, and SSIII kits and in triplicate with the enzyme from ABI. All kits and protocols included 45 cycles of amplification.

Fig 2

Comparison of C_T values of EV, pan-HPeV, and HPeV3 assays with CSF clinical specimens tested in one-step and two-step RT-PCR reactions. (A) Two-step EV assay with the AGP kit enzyme versus one-step Cepheid enterovirus ASR (year 2008; n = 25); (B) two-step EV assay with the AGP kit enzyme versus one-step Argene enterovirus RUO assay (year 2009; n = 25); (C) two-step versus one-step EV assay with the AGP kit enzyme (year 2012; n = 30); (D) two-step versus one-step pan-HPeV assay with the AGP kit enzyme and historic two-step pan-HPeV assay with the ABI enzyme; (E) two-step versus one-step HPeV3 assay with the AGP kit enzyme and historic two-step HPeV3 assay with the ABI enzyme.