|Home | About | Journals | Submit | Contact Us | Français|
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.
Human parechoviruses (HPeVs) were previously classified in the Enterovirus genus but on the basis of distinct genetic and biological properties have been reclassified into a new genus, Parechovirus, within the family Picornaviridae (1). To date, 16 HPeV types have been identified. However, only HPeV type 1 (HPeV1) to HPeV6 and their clinical associations have been monitored in parts of the United States, Europe, and Japan. Among the 6 HPeV types, HPeV1 has been the most prevalent type in these geographic areas and is primarily associated with asymptomatic or mild respiratory and gastrointestinal infection and less frequently with central nervous system (CNS) disease (2, 3). Among all HPeV types, HPeV3 is the most common type recovered from cerebrospinal fluid (CSF) (4, 5). HPeV3 causes sepsis-like illness in young infants (4–7) and severe CNS disease, such as meningitis, encephalitis, and white matter abnormalities with long-term sequelae (4, 5, 7–9). Recently, association of HPeV3 with sudden unexplained infant deaths was reported in the United States (10).
HPeV is difficult to culture, and Vero cells, the optimal cell line, are not commonly used in clinical laboratories in the United States. Moreover, culture is time-consuming, laborious, and less sensitive than molecular approaches for CSF pathogen detection. Today, nucleic acid amplification tests are accepted for rapid and accurate diagnosis of CSF viral pathogens and help in specifying appropriate and cost-effective treatments (11–15). A few real-time reverse transcription-PCR (RT-PCR) assays have been developed for detection of HPeV in different matrices (16–19). In general, the assays for detecting CSF pathogens warrant careful investigation of their performance characteristics, particularly the sensitivity, because of the severity of illnesses associated with CNS disease, and the presence of low viral loads in CSF is not uncommon. Several studies have shown that the choice of master mix reagents and PCR conditions can influence the sensitivity and consistency of results for real-time PCR assays (20–22). As rapid real-time PCR assays have become routine in diagnostic laboratories, there has been an ever increasing number of commercially available reagent systems to support these methods. In anticipation of improving our diagnostic capabilities, this study evaluated a pan-HPeV assay and an EV assay previously described in the literature (14, 16) with five commercially available kits in one-step and two-step RT-PCR formats. It would be beneficial if both HPeV and EV assays were combined in a single run for simultaneous detection of HPeV and EV, considering the similar clinical presentations caused by both infections.
A new HPeV3 genotype-specific real-time RT-PCR assay based on VP1 gene sequences was developed for rapid and cost-effective detection of HPeV3 in pan-HPeV assay-positive CSF specimens. The aims of this study were (i) to evaluate commercial real-time RT-PCR kits in one-step and two-step protocols with the pan-HPeV and EV assays, (ii) to validate pan-HPeV and EV real-time PCR assays in the same run for parallel detection of both HPeV and EV from CSF specimens, and (iii) to develop and validate a HPeV3-specific real-time RT-PCR for HPeV3 identification in pan-HPeV assay-positive CSF specimens.
Pan-HPeV (16) and EV (14) real-time RT-PCR assays were selected for clinical testing. Five commercial real-time RT-PCR reagent kits were evaluated to determine the best-performing master mix in one-step and two-step reactions (Table 1). For two-step RT-PCR, TaqMan reverse transcription reagents (Applied Biosystems [ABI], Foster City, CA) were used to generate cDNA as described earlier (4). An HPeV3 clinical isolate and an echovirus 9 (E9) strain from the American Type Culture Collection (ATCC; VR-39) were spiked in negative CSF specimens and 10-fold serial dilutions were tested by pan-HPeV and EV assays in one-step and two-step reactions to compare sensitivity. A bacteriophage MS2 internal control (for both extraction and amplification) was included in the EV assay. Lyophilized MS2 obtained from ATCC (15597-B1) was resuspended with 1 ml of viral transport medium (VTM) and diluted serially 10-fold, using the VTM to yield a threshold cycle (CT) value in the range of 30 to 33. Nucleic acid was extracted from 180 μl of clinical specimen plus 20 μl of MS2 internal control using a NucliSENS easyMAG automated extraction system and eluted in 55 μl of elution buffer (bioMérieux Inc., Durham, NC). The primers and probe sequences for the pan-HPeV, EV, and MS2 internal control and the HPeV3 real-time RT-PCR assays are given in Table 2.
Pan-HPeV and EV real-time RT-PCRs were performed on a 7500 Fast real-time PCR system (Applied Biosystems) in 25-μl reaction mixtures containing 5 μl of template RNA (one-step RT-PCR) or 5 μl of cDNA (equivalent to about 2 μl of RNA in a two-step RT-PCR) with optimized concentrations of the respective primers and probes (Table 2).
Pan-HPeV and EV real-time RT-PCR assays were tested in a single-target format (HPeV or EV only) and dual-target format (HPeV plus MS2 or EV plus MS2) to identify the ideal target (HPeV or EV) that could be detected without competitive inhibition due to the MS2 internal control. The HPeV3 strain (ZeptoMetrix Corporation, Buffalo, NY) diluted from 1 × 102.6 to 1 × 100.06 median (50%) cell culture infectious doses (CCID50s)/ml and the ATCC E9 strain diluted from 1 × 101.2 to 1 × 100.002 CCID50s/ml were used to determine the limit of detection of the pan-HPeV and EV assay. Six replicates of each dilution of test organism with MS2 added were extracted and tested, using two sets of master mixes; one set each was with or without MS2-specific primers and probe.
For clinical validation of the pan-HPeV and EV two-step RT-PCR assays, the AgPath ID RT-PCR (AGP) kit was selected on the basis of its superior performance.
The ability of the two-step pan-HPeV assay to detect six HPeV types was analyzed using ATCC cultures of HPeV1 (VR-52) and HPeV2 (VR-53), HPeV3 (ZeptoMetrix), and CDC culture isolates of HPeV4, -5, and -6. The limit of detection (LoD) was analyzed by testing six replicates of the commercial HPeV3 strain using 5 μl of template RNA or 5 μl of cDNA (2 μl of equivalent RNA), which was extracted from 200 μl of viral cultures at a level of 1 × 102.6 to 1 × 100.06 CCID50s/ml. Similarly, LoDs for other HPeV types, types 1, 2, and 4 to 6 (obtained from CDC), were determined after making serial dilutions. The analytical specificity of the pan-HPeV assay reagents was tested against 13 enterovirus strains and other viruses obtained from ATCC: coxsackievirus types A9 (VR-186), B1 (VR-1032), B2 (VR-29), B3 (VR-30), B4 (VR-184), and B5 (VR-185); echovirus types 3 (VR-1040), 4 (VR-1041), 5 (VR-1043), 6 (VR-36), 7 (VR-37), 9 (VR-39), and 11 (VR-41); rhinovirus types 39 (ATCC VR-340), 8 (ATCC VR-488), 13 (ATCC VR-1123), 26 (ATCC VR-1136), and 27 (ATCC VR-1137); herpes simplex virus 1 (HSV-1) (ATCC VR-539) and HSV-2 (ATCC VR-540); human herpesvirus 6 (HHV-6; ATCC VR-1480); and laboratory-grown clinical isolates of cytomegalovirus (CMV) and Epstein-Barr virus (EBV). Reproducibility was analyzed utilizing HPeV3 (ZeptoMetrix) diluted to 1 × 101.6 CCID50s/ml. Reactions were run in triplicate on three different days, and fresh nucleic acid was extracted for each run. A total of 70 frozen aliquots of CSF specimens that were previously characterized to be HPeV positive (n = 33) and negative (n = 37) were analyzed by both one-step and two-step real-time pan-HPeV RT-PCRs with the AGP kit, and the results were compared. Further, the two-step RT-PCR results obtained with the AGP kit were compared with the historic results of the two-step RT-PCR that used the TaqMan Universal PCR master mix (ABI).
The limit of detection of the two-step EV assay was determined by testing six replicates of 10-fold dilutions of a culture of an ATCC strain of E9 at 1 × 101.2 to 1 × 100.002 CCID50s/ml. Twelve other enterovirus strains, coxsackievirus types A9 and B1 to B5 and echovirus types 3 to 7 and 11, were serially diluted and tested to determine the LoD of the two-step EV RT-PCR assay. These 13 EV types were reported to be highly prevalent types in the United States during the period from 1970 to 2005 (3). The analytical specificity of this assay was assessed by testing HPeV types 1 and 2, five rhinovirus strains, HSV-1 and -2, HHV-6, CMV, and EBV, as listed above. The reproducibility was tested by using the E9 standard at 1 × 100.2 CCID50/ml as described above. EV-positive (n = 41; 12 each from 2008 and 2009 and 17 from 2010) and EV-negative (n = 34; 13 each from 2008 and 2009 and 8 from 2010) CSF specimens that were previously characterized by validated EV assays in clinical use from 2008 to 2010 (the Cepheid assay in 2008, the Argene assay in 2009, and the laboratory-developed test [LDT] in 2010) were tested by both one-step and two-step real-time EV RT-PCRs with the AGP kit.
The primers and probe specific for HPeV3 were designed on the basis of the complete VP1 capsid gene sequences available in GenBank. The VP1 sequences of HPeV3, other HPeV types, enteroviruses, and rhinoviruses were analyzed using Lasergene software (version 8.1; DNAStar Inc., Madison, WI) to identify the HPeV3-specific consensus sequence. The primers and probe specific for HPeV3 were designed using Primer Express software (version 3.0; Applied Biosystems). HPeV3-specific RT-PCR was validated with a one-step RT-PCR protocol, using the AGP kit. HPeV3 culture fluid (ZeptoMetrix) was tested at 1 × 102.6 to 1 × 100.06 CCID50s/ml in six replicates to determine the LoD. The analytical specificity of the HPeV3-specific reagents was tested against other HPeV types (types 1, 2, and 4 to 16), enteroviruses, rhinoviruses, and the other viruses listed above. Reproducibility was analyzed in a manner similar to that used for the pan-HPeV assay with HPeV3 (ZeptoMetrix) culture fluid at 1 × 101.6 CCID50s/ml. The HPeV3-specific RT-PCR assay was validated with the same nucleic acid extracts from CSF specimens (33 HPeV-positive and 37 HPeV-negative specimens) used for pan-HPeV assay evaluation. All HPeV-positive specimens were sequenced and typed as HPeV3 in our earlier study (4) before evaluation with the HPeV3-specific RT-PCR. The results of the HPeV3-specific one-step and two-step RT-PCRs with the AGP kit and the historic results with the two-step ABI kits were compared.
Overall, the AGP kit was the best, compared to the other kits, in both the one-step and two-step RT-PCR formats of pan-HPeV and EV assays (Fig. 1). Although the AGP and QuantiTect Probe RT-PCR (QTRTP) kits detected HPeV3 at the 10−6 dilution, the QuantiTect Probe PCR (QTP) kit showed a higher CT, with an average difference of 1.5 ± 0.2 cycles, suggesting a lower amplification efficiency. The LoD for the SuperScript III one-step RT-PCR system (SSIII) was 1 log dilution higher (10−5 dilution) than that for the enzymes from the other kits, indicating lower sensitivity. In the two-step RT-PCR, the AGP kit detected HPeV3 at a 10−7 dilution, which is 1 log dilution lower than that for the SSIII, QTP, and ABI kits, indicating a better efficiency of the AGP kit than the other three kits. In addition, the AGP kit showed a lower CT, with an average difference of 2.6 ± 0.3 cycles, suggesting a higher amplification efficiency (Fig. 1).
For the EV assay, all the three one-step RT-PCR kits showed a similar LoD of 100.2 CCID50/ml, although Invitrogen's SSIII kit yielded a 1-log higher CT than the AGP and QTRTP kits. In the two-step RT-PCR, the AGP and QTP kits detected E9 at a level of 100.02 CCID50/ml, while the other two kits, SSIII and ABI, detected only 100.2 CCID50/ml (Fig. 1). The two-step EV assay with the AGP and QTP kits demonstrated the highest sensitivity when its sensitivity was compared to that of the one-step protocol (with the AGP, SSIII, and QTRTP kits) or the two-step protocol (with SSIII or ABI). Among the best two-step EV assays (AGP or QTP assay), the AGP assay demonstrated a lower CT (0.8 ± 0.4 cycles), indicating better amplification efficiency.
Clinical CSF samples positive for HPeV and EV were tested, using the AGP kit in one-step and two-step RT-PCR protocols, and the CT values were compared. The two-step pan-HPeV assay with the AGP kit performed better than the one-step AGP protocol, as the latter assay failed to detect 10 (33%) HPeV-positive samples. Further, the two-step pan-HPeV with the AGP kit yielded lower CT values for positive samples, with a mean difference of 2.9 (range, 0.9 to 6.1) compared to the CT values of the one-step assay (Fig. 2D). Similarly, there was a mean CT difference of 1.8 (range, 0.1 to 4.9) between the AGP and ABI kits in two-step reactions in favor of the AGP assay (Fig. 2D).
The results of the two-step EV assay with the AGP kit were compared with historic results obtained from the use of the Cepheid enterovirus analyte-specific reagent (ASR) for 2008 samples, the Argene research-use-only (RUO) assay for 2009 samples, and a one-step LDT using the AGP kit for 2010 samples. The results demonstrated the superior performance of the AGP kit in the two-step RT-PCR, with lower CT values for the AGP kit than the Cepheid one-step enterovirus ASR (mean CT difference, 2.8 [range, 1.3 to 4.6]; Fig. 2A) and the one-step LDT using the AGP kit (mean CT difference, 2.53 [range, 0.6 to 5.5]; Fig. 2C). The two-step AGP kit results were comparable to those for the Argene one-step enterovirus RUO assay (Fig. 2B).
Detection of E9 was not inhibited when tested with the MS2 internal control. The LoD was 1 × 100.02 CCID50/ml with a 100% detection rate (n = 6/6) in both the single- and dual-target formats (Table 3). In contrast, HPeV detection was inhibited when the dual-target assay for HPeV and MS2 was tested. The LoD was 1 × 101.6 CCID50s/ml for HPeV and MS2 (n = 6/6), whereas the LoD was 1 × 100.6 TCID50/ml with HPeV only (n = 6/6) (Table 3).
The analytical performance characteristics of the pan-HPeV and EV two-step protocol with the AGP kit was determined using cDNA synthesized with TaqMan reverse transcription reagents obtained from Applied Biosystems.
The pan-HPeV two-step assay with the AGP kit demonstrated 100% analytical sensitivity in detecting HPeV-positive (n = 33/33) and -negative (n = 37/37) CSF specimens (see Table 5). The pan-HPeV RT-PCR reagents detected other HPeV types (types 1 to 6) but did not detect any of the rhinoviruses, enteroviruses, or other CNS viral pathogens. The LoDs for HPeV types are given in Tables 3 and and4.4. All six replicates of HPeV3 were detected at a level of 1 × 101.6 CCID50s/ml. Excellent reproducibility was observed when the HPeV3 test strain was repeatedly tested at 1 × 101.6 CCID50s/ml, with an interrun variability of 1 cycle (coefficient of variation [CV], 2.8%) and an intrarun variability of 0.5 cycles (CV, 1.3%).
Of the 75 CSF specimens tested, the two-step EV assay with the AGP kit demonstrated a good overall sensitivity of 100% and a specificity of 100% with EV-positive (n = 41/41) and -negative (n = 34/34) samples compared with the historic results of the Cepheid enterovirus ASR, Argene enterovirus RUO, and one-step LDT assays (Table 5). The MS2 internal control was detected in all samples (100%; n = 75). The CT value for the MS2 internal control ranged from 29.8 to 33.2 cycles, with a median CT of 31.4 cycles. The LoD of the two-step EV assay was analyzed by testing serial dilutions of 13 EV strains. All six replicates of E9 were detected at a level of 1 × 100.02 CCID50/ml (Table 3). The LoDs for 12 other common EV serotypes were also the same as the LoD for E9, but a 5-log-unit reduction in sensitivity was observed with E6 (ATCC VR-36; Table 4). No cross-reactivity was observed with any other viruses, including rhinoviruses, tested in this study. The reproducibility analysis with E9 at 1 × 100.2 CCID50/ml (1 log unit above the LoD) showed an interrun variability of 0.6 cycle with a CV of 1.7% and an intrarun variability of 0.3 cycle with a CV of 0.9%.
The results of the HPeV3-specific one-step RT-PCR with the AGP kit were compared to those of our previous pan-HPeV RT-PCR (using a two-step ABI kit) and sequencing results to determine the sensitivity. The HPeV3 assay correctly identified most positive (32/33) and all negative (37/37) specimens, with an analytical sensitivity of 97% and a specificity of 100% (Table 5). The LoD of this assay was 1 × 101.6 CCID50s/ml. The HPeV3-specific assay did not detect any other HPeV type (types 1, 2, and 4 to 16) and did not amplify any other CSF-associated viruses tested. With the HPeV3 control, the assay was highly reproducible, with interrun and intrarun CT variabilities of 0.4 cycle and interrun and intrarun CVs of 1.1% and 0.8%, respectively.
EV and HPeV, particularly HPeV3, are often detected in the cerebrospinal fluid of neonates and young children less than 6 months old with serious CNS disease (4, 5, 7–9). The recent increase in the incidence of HPeV-associated CNS infections with clinical syndromes similar to those caused by EV warrants testing for HPeV along with EV for hospitalized children aged <6 months. It has been reported that HPeV molecular methods increased the CSF viral detection rate by 31% in a Dutch clinical center (5) and by 63% in 2007 and 122% in 2009 in our experience (4, 24) in cases of neonatal sepsis-like illness and meningitis in young children.
No Food and Drug Administration (FDA)-approved assays for HPeV testing are available in the United States. Although several HPeV PCR methods have been published (16–19), the assays were not properly validated either with sufficient numbers of clinical specimens or with controls for clinical testing. Hence, using a good collection of characterized CSF samples, we evaluated the assays previously reported by Benschop and coworkers for HPeV (16) and by Verstrepen and coworkers for EV (14) under both one-step and two-step reaction conditions with a few amplification kits, some of which became available only recently. Identifying the most suitable kit for real-time PCR applications among several commercially available reagent kits is important, as the reagents are known to impact the assay performance characteristics (20, 21, 25). Originally, both assays utilized the ABI enzyme in a two-step reaction (HPeV) or one-step reaction (EV). By substituting the AGP kit enzyme in both assays and optimizing the assays for a two-step RT-PCR assay, we demonstrated a 1-log-unit lower detection for HPeV and a 2-log-unit lower detection for EV (Fig. 1). Similarly, Stephens and coworkers found the AGP kit enzyme to be the most sensitive in amplification of Ebola virus RNA in a study that compared five commercial real-time RT-PCR master mixes (25). Our previous HPeV surveillance studies (2006 to 2010) showed that the two-step RT-PCR yielded CT values of >35 in 51.8% (n = 28/54) of samples in the year 2007 (4) and 33.3% (22/66) of samples in the year 2009 (24). Hence, a sensitive assay is needed to test clinical specimens with a low abundance of HPeV RNA. Among 66 samples positive by HPeV assay in the year 2009, only 55 were detected as positive by a one-step RT-PCR assay, while 65 were repeatedly positive by a two-step HPeV assay (unpublished data). In contrast, the one-step RT-PCR performed better than the two-step RT-PCR for the HPeV3-specific assay. It is possible that the gene-specific primers used in the one-step kit may have been more efficient at generating higher copy numbers of VP1 cDNA than the random hexamers in the two-step kit, as reported earlier (26).
In this study, we designed the pan-HPeV and EV assays to run in two separate reaction vials at the same time with the same nucleic acid extract. Then, the HPeV3-specific assay can follow with the same nucleic acid extract, if needed, to identify the HPeV3 genotype. An MS2 internal control was tested with the EV well only and was not tested with HPeV because of the competitive inhibition of HPeV when MS2 was coamplified. This algorithm would appropriately control for inhibitors in the sample. On the contrary, since the pan-HPeV assay or HPeV3-specific assay lacks an internal control, using them alone will not control for nonspecific inhibitors of PCR amplification.
The primer and probe sequences of the EV assay were predicted to detect all known EV serotypes; however, cross-reactivity with some rhinoviruses has been reported in an earlier publication (11). The analysis indicated that the assay should detect all EVs with good sensitivity except for some strains of E6, where a C-to-U difference at the 3′ end of the forward primer was observed (11). Our validation study agreed with the published data, as the assay with our E6 strain (ATCC VR-36; D'Amori) had a 5-log-unit reduced sensitivity. The HPeV3-genotype specific assay developed in this study can be used for rapid identification of HPeV3 from pan-HPeV-positive CSF samples but may not be suitable for diagnostic testing of CSF specimens because non-HPeV3 types are not detected by this assay. However, our earlier HPeV sequencing study showed that the viruses in 52/53 (98.1%) HPeV-positive CSF samples tested were HPeV3 (4). In the current study, one sequence-confirmed HPeV3-positive sample was negative by the HPeV3-specific assay, and this may have been due to sequence differences in the primer/probe binding region. Analysis of all available complete HPeV3 VP1 sequences at CDC (n = 39) suggests that the assay would fail to amplify one HPeV3 strain, detected in Bangladesh, which has a 5/7-bp mismatch at the 5′ end of the TaqMan probe. Other HPeV3 strains from Bangladesh, Japan, Europe, and South America conformed to the HPeV3-specific primer/probe set. The HPeV3-specific real-time RT-PCR assay is less expensive and less labor-intensive than sequencing for typing pan-HPeV-positive CSF samples and can provide a better understanding of the disease burden associated with HPeV3 infection.
In summary, the EV and HPeV two-step RT-PCR assay using the AGP kit is optimized for parallel detection of both EV (EV with the MS2 internal control) and HPeV in two separate reaction vials in a single test run. The one-step HPeV3-specific RT-PCR assay can be followed to rapidly identify HPeV3 in pan-HPeV-positive CSF specimens for epidemiological purposes.
Published ahead of print 21 November 2012