VirologyDetection of human enterovirus and human parechovirus (HPeV) genotypes from clinical stool samples: polymerase chain reaction and direct molecular typing, culture characteristics, and serotyping
Introduction
Human enteroviruses (HEVs) and parechoviruses (HPeVs) belong to Picornaviridae family of small, nonenveloped RNA viruses. HEVs were originally subdivided into poliovirus (PV, 3 serotypes), coxsackievirus A (CAV, 23 serotypes), coxsackievirus B (CBV, 6 serotypes), and echovirus (28 serotypes), and newer HEVs were numerically classified as enterovirus (EV) 68–102 (Minor et al., 1995, Oberste et al., 2004, Oberste et al., 2005, Oberste et al., 2007). Based on phylogenetic analysis, HEVs are now classified into 4 species: HEV-A, HEV-B, HEV-C (containing PV), and HEV-D (Hyypiä et al., 1997, King et al., 2000).
HPeVs were originally classified within the Enterovirus genus as echovirus 22 and 23. However, based on genetic data, they were recently assigned a separate genus of Parechovirus, which also includes Ljungan virus (Niklasson et al., 1999). Currently, 14 HPeV genotypes are known.
HEVs and HPeVs are associated with a wide array of clinical manifestations ranging from mild respiratory or gastrointestinal symptoms, hand–foot and mouth disease, myocarditis, neonatal sepsis, and infections of the central nervous system (Abzug, 2004, Benschop et al., 2008a, Stanway et al., 2000). HEVs and HPeVs have a seasonal distribution that can vary between the genera as well as between the types (Benschop et al., 2006, Khetsuriani et al., 2006, Roth et al., 2007, van der Sanden et al., 2008), and different types can cocirculate depending on the year and place.
HEVs and HPeVs are widespread and are commonly detected by routine diagnostic procedures (Benschop et al., 2008a). The classic method for diagnosis of infection with HEVs or HPeVs has been virus isolation by cell culture from stool samples, throat swabs, cerebral spinal fluid (CSF), and blood. The standard cell culture for isolation of HEV involves at least 3 cell lines, usually including monkey kidney cells and human fibroblasts, followed by neutralization with a panel of specific antibodies to determine the serotype (Kapsenberg, 1988, Rotbart and Romero, 1995, Terletskaia-Ladwig et al., 2008, van Doornum et al., 2007). Standard culture conditions for HPeVs are not well defined. The cytopathologic effect (CPE) induced by HPeV is comparable to that induced by HEV and with classic serotyping for HEV; HPeV1 and 2 can be identified as well. However, the standard cell lines used for isolation of HEV will not support growth of all HPeV types, and the antibodies used to identify HPeV types other than types 1 and 2 are not available.
Many laboratories have introduced polymerase chain reaction (PCR) as the method of choice for detection of viral pathogens in CSF (Espy et al., 2006, Wolthers et al., 2008). PCRs for HEV diagnosis generally target the 5′UTR, which is highly conserved and therefore suitable to detect all HEV serotypes (Beld et al., 2004, Iturriza-Gomara et al., 2006, van Doornum et al., 2007). Because the nucleotide sequences of the HPeVs are divergent from the HEVs, pan-EV reverse transcriptase PCR fails to detect HPeVs (Benschop et al., 2006, Hyypiä et al., 1992). Therefore, specific 5′UTR PCRs have been developed to sensitively detect HPeV infections in CSF as well as in stool samples, throat swabs, or blood (Benschop et al., 2008b, Harvala et al., 2008, Nix et al., 2008, Noordhoek et al., 2008). However, the 5′UTR is not discriminative enough for typing (Oberste et al., 1998). It has been shown that typing by sequencing part of the VP1 region is in good agreement with serotyping by neutralization because type-specific antibodies are also directed against the VP1 region (Oberste et al., 1999b). Typing methods may also involve sequencing of the capsid genes, VP4 and/ or VP2. However, sequencing of these regions was found to be less discriminatory to define the different types (Casas et al., 2001, Oberste et al., 2003). In addition, molecular typing of the different HEV/HPeV types can be done directly from the sample by sequencing part of the capsid region (Benschop et al., 2008c, Harvala et al., 2008, Nix et al., 2006).
Despite the availability of molecular techniques for identification and typing of HEVs and HPeVs, many laboratories still rely on cell culture and serotyping. Therefore, specific HEV and HPeV types will be missed or underdiagnosed. However, many laboratories are not familiar with molecular typing techniques. Furthermore, validation of their performance in a clinical–diagnostic setting is lacking.
We performed an analysis in which we detected HEV/HPeV by PCR in stool samples from hospitalized patients and compared this with HEV and HPeV detected by our cell culture system, thereby establishing the efficacy of different cell lines to support virus growth. Furthermore, we compared the yield of genotyping directly from stool with serotyping by neutralization in the optimized cell culture system.
Section snippets
Clinical samples
Stool samples were obtained in 2007 and 2008 from patients of different age groups and sent to the Laboratory of Clinical Virology for viral diagnostics. Stool samples were suspended in 2% broth (Oxoid, Drongen, Belgium) and stored at −80 °C.
Viral culture and serotyping
Viral culture was performed by cocultivation of patient material with tertiary monkey kidney cells (tMK), Vero cells, human embryonic lung fibroblasts (HEL), human colon carcinoma cells (HT29), rhabdomyosarcoma cells (RD), and human lung fibroblasts (A549).
Epidemiology of patients positive for HEV and/or HPeV by PCR
Over 2007 and 2008, we tested 1174 patients for HEV and/or HPeV. Because several patients had more than one sample tested, 1460 samples were analyzed in total by HEV PCR and HPeV PCR. We found 117 samples from 105 patients positive for HEV (8.9%) and 114 samples from 101 patients positive for HPeV (8.6%). Fifteen patients (17 samples, 1.2%) had a double infection with HPeV and HEV (Table 1). In total, 248 samples from 221 patients (18.8%) were identified as positive for HEV and/or HPeV. Taking
Discussion
Here, we describe the value of direct stool screening by PCR and genotyping to detect HEV and HPeV genotypes.
By PCR, we identified 18.8% of the patients tested positive for HEV and/or HPeV. From the 248 samples found positive, we could isolate a virus from 107 samples. The ability to culture an HPeV- or HEV-positive sample correlated with the Ct value in PCR (Iturriza-Gomara et al., 2006, Shoja et al., 2007a, van Doornum et al., 2007).
Thus, detection by PCR is more sensitive than cell culture
Acknowledgments
This study was financed by the Netherlands Organization for Health Research and Development's Clinical Fellowship and the Department of Medical Microbiology.
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