Enteroviruses In Clinical Specimens Research Articles

Molecular strategy for the direct detection and identification of human enteroviruses in clinical specimens associated with hand, foot and mouth disease.

BackgroundDiseases caused by human enteroviruses (EVs) are a major global public health problem. Thus, the effective diagnosis of all human EVs infections and the monitoring of epidemiological and ecological dynamic changes are urgently needed.MethodsBased on two comprehensive virological surveillance systems of hand, foot and mouth disease (HFMD), real-time PCR and nested RT-PCR (RT-snPCR) methods based on the enteroviral VP1, VP4-VP2 and VP4 regions were designed to directly detect all human EVs serotypes in clinical specimens.ResultsThe results showed that the proposed serotyping strategy exhibit very high diagnostic efficiency (Study 1: 99.9%; Study 2: 89.5%), and the variance between the study was due to inclusion of the specific Coxsackie virus A6 (CVA6) real-time RT-PCR and VP4 RT-snPCR in Study 1 but not Study 2. Furthermore, only throat swabs were collected and analyzed in Study 2, whereas in Study 1, if a specific EV serotype was not identified in the primary stool sample, other sample types (rectal swab and throat swab) were further tested where available. During the study period from 2013 to 2018, CVA6 became one of the main HFMD causative agents, whereas the level of enterovirus A71 (EV-A71) declined in 2017.ConclusionThe findings of this study demonstrate the appropriate application of PCR methods and the combination of biological sample types that are useful for etiological studies and propose a molecular strategy for the direct detection of human EVs in clinical specimens associated with HFMD.

Preponderance of enterovirus C in RD-L20B-cell-culture-negative stool samples from children diagnosed with acute flaccid paralysis in Nigeria.

Recently, a reverse transcriptase semi-nested polymerase chain reaction (RT-snPCR) assay was recommended by the WHO for direct detection of enteroviruses in clinical specimens. In this study, we use this assay and a modification thereof to screen acute flaccid paralysis (AFP) samples that had previously tested negative for enteroviruses by the RD-L20B algorithm. Thirty paired stool suspensions collected in 2015 as part of the national AFP surveillance program in different states of Nigeria were analyzed in this study. The samples had previously tested negative for enteroviruses in the polio laboratory in accordance with the WHO-recommended RD-L20B-cell-culture-based algorithm. Two samples that had previously been found to contain enteroviruses were included as positive controls. All samples were subjected to RNA extraction, the RT-snPCR assay and a modified version of the RT-snPCR. All amplicons were sequenced, and enteroviruses were identified using the enterovirus genotyping tool and phylogenetic analysis. Amplicons were recovered from the two controls and 50% (15/30) of the samples screened. Fourteen were successfully typed, of which, 7.1% (1/14), 21.4% (3/14), 64.3% (9/14) and 7.1% (1/14) were enterovirus (EV) -A, EV-B, EV-C and a mixture of EV-B and C (EV-C99 and E25), respectively. The two controls were identified as EV-C99 and coxsackievirus (CV) -A1, both of which belong to the species Enterovirus C. In one sample, poliovirus serotype 2 was detected and found to have the VP1 ILE143 variation and was therefore identified as a vaccine strain. The results of this study showed that significant proportion of enterovirus infections (including some with Sabin PV2) are being missed by the RD-L20B-cell-culture-based algorithm, thus highlighting the value of the RT-snPCR assay and its modifications. The circulation and preponderance of EV-C in Nigeria was also confirmed.

Simultaneous Detection and Differentiation of Human Rhino- and Enteroviruses in Clinical Specimens by Real-Time PCR with Locked Nucleic Acid Probes

Human rhinoviruses (HRVs) and human enteroviruses (HEVs) are significant respiratory pathogens. While HRV infections are restricted to the respiratory tract, HEV infections may spread to secondary target organs. The method of choice for sensitive specific detection of these viruses is reverse transcription (RT)-PCR with primers targeting the conserved 5' noncoding region of the viral RNA. On the other hand, sequence similarities between HRVs and HEVs complicate their differential detection. In this study, we describe the use of locked nucleic acid (LNA) analogues in short double-dye probes which contained only two selectively HRV- or HEV-specific bases. The double-stranded DNA dye BOXTO (4-[6-(benzoxazole-2-yl-(3-methyl-)-2,3-dihydro-(benzo-1,3-thiazole)-2-methylidene)]-1-methyl-quinolinium chloride) was used with the LNA probes in a tricolor real-time PCR assay to allow specific detection of HRVs (probes labeled with 6-carboxyfluorescein [FAM] [green]) and HEVs (Cy5 [red]) with additional melting curve analysis (BOXTO [yellow]). The functionality of the probes was validated in PCR and RT-PCR assays using plasmids containing viral cDNA, quantified viral RNA transcripts, cultivated rhino- and enterovirus prototypes, and clinical specimens. Of 100 HRV and 63 HEV prototypes, the probes correctly identified all HEVs except one that produced only a BOXTO signal. Among 118 clinical specimens with sequencing results, concordant results were obtained for 116 specimens. Two specimens were reactive with both probes, but sequencing yielded only a single virus. Real-time PCR with LNA probes allowed sensitive group-specific identification of HRVs and HEVs and would enable relative copy number determination. The assay is suitable for rapid and accurate differential detection of HRVs and HEVs in a diagnostic laboratory setting.

Comparing Molecular Methods for Early Detection and Serotyping of Enteroviruses in Throat Swabs of Pediatric Patients

BackgroundEnteroviruses include over 100 serotypes and usually cause self-limited infections with non-specific symptoms in children, with the exceptions of polioviruses and enterovirus 71 which frequently cause neurologic complications. Therefore, early detection and serotyping of enteroviruses are critical in clinical management and disease surveillance. Traditional methods for detection and serotyping of enteroviruses are virus isolation and immunofluorescence assay, which are time-consuming. In this study, we compare virus isolation and two molecular tests for detection and serotyping of enteroviruses in clinical samples.MethodsOne hundred and ten throat swabs were collected from pediatric outpatients with enterovirus-like illnesses (hand-foot-mouth disease, herpangina, and non-specific febrile illness). Virus isolation was conducted using multiple cell lines and isolated viruses were serotyped using immunofluorescent assay. In the molecular tests, a semi-nested RT-PCR and a novel CODEHOP platform were used to detect the 5′UTR and VP1 genes of enteroviruses, respectively. Amplified nucleotides were sequenced and genotyped.ResultsAmong the 110 cases, 39(35%), 52(47%), and 46(42%) were tested positive with these three tests, respectively. Using the consensus results of these three tests as the gold standard, agreement of the VP1 CODEHOP test was 96%, which is higher than those of the virus isolation (89%) and the 5′-UTR test (88%). The VP1 CODEHOP test also has the best performance on serotyping confirmed with serum neutralization tests.ConclusionsThe VP1 CODEHOP test performed well for detection and serotyping of enteroviruses in clinical specimens and could reduce unnecessary hospitalization cares during enterovirus seasons.

Comparative evaluation of Taqman real-time PCR and semi-nested VP1 PCR for detection of enteroviruses in clinical specimens

Background Molecular diagnostic tests to detect enterovirus in clinical specimens usually target highly conserved sites in the 5′-non-translated region, allowing detection of all members of the genus. The sequences in the 5′-NTR do not correlate with serotype, but PCR and sequencing of the VP1 region can be used for typing; this system has largely replaced traditional antigenic typing. Objective To investigate the relative performance of two common enterovirus assays. Study design We compared the relative sensitivities of Taqman ® real-time RT-PCR (rRT-PCR) and a VP1 semi-nested PCR (RT-snPCR) assay in which sequencing the VP1 amplicon also provides typing information. Results There was 89% concordance between the two methods among the 371 clinical specimens tested (74 positive in both assays and 257 negative in both assays). Twenty-seven rRT-PCR-negative/VP1-positive specimens were confirmed positive by sequencing; 13 specimens were rRT-PCR-positive/VP1-negative. Conclusions The results suggest that either assay can produce satisfactory results, but that using both assays in parallel should provide the highest sensitivity for clinical diagnostic testing.

Multicenter evaluation of the ENTEROVIRUS R-gene™ real-time RT-PCR assay for the detection of enteroviruses in clinical specimens

Background The rapid molecular diagnosis of enteroviral meningitis has been shown important for an adequate management of the patients. Objectives A new CE-marked real-time RT-PCR assay (ENTEROVIRUS R-gene™, Argene) was evaluated in two university hospital virology laboratories. Study design Reactivity, analytical sensitivity and specificity were evaluated using 54 prototype and 173 clinical human enterovirus (HEV) strains, a 12-sample HEV proficiency panel, and 30 non-HEV microorganisms. The clinical performance of the ENTEROVIRUS R-gene™ assay was evaluated by testing 197 cerebrospinal fluid (CSF) and 103 respiratory specimens, comparatively to the routinely used diagnostic techniques. Results Sixty-four out of the 65 HEV serotypes tested were detected. The analytical sensitivity ranged between 10 −2.64 and 10 2.39 TCID 50/50 μl. Cross-reactivity was observed with four human rhinoviruses. On 59 CSF specimens analyzed prospectively, the results of the ENTEROVIRUS R-gene™ assay showed a 94.8% concordance with those of the Smart enterovirus (EV) assay (Cepheid). On 138 CSF specimens tested retrospectively, the results of the ENTEROVIRUS R-gene™ assay showed a 97.1% concordance with those of either the GeneXpert EV assay (Cepheid) or the in-house RT-PCR HEV assays used at the time of specimen collection. On 103 respiratory specimens, the concordance between the results of the ENTEROVIRUS R-gene™ assay and those of the routine RT-PCRs or viral culture was 90.2% and 96.1% before and after retest, respectively. Conclusions The new test was found able to detect a large panel of enterovirus serotypes; it was sensitive when used on clinical specimens; and, easy and rapid to perform on a routine basis.

OP6-1 Multicenter evaluation of the ENTEROVIRUS R-gene™ real time RT-PCR assay for the detection of enteroviruses in clinical specimens

OP6-1 Multicenter evaluation of the ENTEROVIRUS R-gene™ real time RT-PCR assay for the detection of enteroviruses in clinical specimens

Establishing evidence for enterovirus infection in chronic disease.

Viruses have long been considered among potential environmental triggers of type 1 diabetes mellitus. Epidemiologic and seroprevalence studies have associated enterovirus infection with development of prediabetic autoimmunity and with the onset of clinical diabetes. Enterovirus infection has also been temporally correlated with disease onset by virus isolation or by detection of viral genome by reverse transcription-polymerase chain reaction (RT-PCR). For the large-scale prospective studies that are required to firmly establish a causal relationship between enterovirus infection and development of prediabetic autoimmunity or progression from autoimmunity to clinical diabetes, sensitive RT-PCR methods must be used to detect virus prior to the onset of diabetic symptoms. We have developed an RT-seminested PCR protocol to detect enteroviruses in clinical specimens. This method is approximately 10,000-fold more sensitive than conventional, single-amplification PCR. Further, we have developed molecular methods to rapidly and reliably identify enterovirus serotype, bypassing the cumbersome and often problematic neutralization test. The molecular serotyping approach will be valuable in examining the relationships between particular virus serotypes or genotypes and specific diseases.

Detection of precytopathic effect of enteroviruses in clinical specimens by centrifugation-enhanced antigen detection.

Rapid enterovirus detection is important for decisions about antibiotic administration and length of hospital stay. The efficacy of rapid antigen detection-cell culture amplification (Ag-CCA) was evaluated with monoclonal antibodies (MAbs) 5-D8/1 (DAKO) and Pan-Enterovirus clone 2E11 (Chemicon) with 10 poliovirus, echovirus, and coxsackievirus type A and B stock isolates and College of American Pathologists check samples. By using Ag-CCA technology, MAb 2E11 was more sensitive than 5-D8/1 at detecting a greater number of stock isolates at or past tube (cytopathic effect [CPE]) culture (TC) end points. The efficacy of Ag-CCA in the clinical setting was subsequently confirmed with 273 consecutively freshly collected nasopharyngeal aspirate or swab specimens, rectal swab, and cerebrospinal fluid specimens during the 1999 enterovirus season. All specimens were tested by Ag-CCA in parallel with rhesus monkey kidney (RhMk), MRC-5, and A549 conventional TCs. Approximately 60% of field specimens were additionally tested with Hep-2 and HNK conventional TCs. Sixty-two percent of the clinical specimens tested were Ag-CCA positive after 48 h. Among 51 isolates, the mean time to CPE or culture confirmation was 5.5 days (range, 2 to 18 days). After 48 h, Ag-CCA achieved sensitivity, specificity, and positive and negative predictive values of 62, 100, 100, and 93%, respectively. During the same period, TC-CPE displayed test parameters of 12, 100, 100, and 85%, respectively. After 5 days, the sensitivity and specificity of Ag-CCA increased to 92 and 98%, respectively. Within the same period, isolation attained sensitivity and specificity of 52 and 100%, respectively. Although Ag-CCA displayed slightly reduced sensitivity and reduced specificity compared with conventional cell culture after 14 days, the markedly superior 48-h enterovirus Ag-CCA detection rate supports incorporation of this assay into the routine clinical setting.

Variations in the recognition of echovirus type 11 strains by a group-specific anti-VP1 monoclonal antibody.

Mab 5-D8/1 is a monoclonal antibody (Mab) that was shown to be directed towards a conserved epitope of the capsid protein VP1 among the genus enterovirus. The use of this Mab for the routine detection of enteroviruses in clinical specimens led to the observation that several strains of echovirus type 11 (EV-11) could not be detected on spontaneously detached cells from 26-h cultures using a two-step immunofluorescence (IF) assay. Conversely, these strains were detected positive with the same Mab when tested on adherent or trypsinizated cells. The aim of this study was to understand the misrecognition of some strains of EV-11 by this Mab. IF tests at different times of the viral cycle brought evidence that the detection of a variant strain of EV-11 decreased rapidly with time, becoming undetectable 26 h post-infection, since the reference strain remained positive up to 46 h post-infection. The infective titres of the variant strains were shown to be high in comparison with those of well-recognised strains. Sequencing the Mab binding epitope confirmed that the variant strains exhibited no antigenic shift. These results suggest that the poor recognition of some strains of EV-11 by Mab 5-D8/1 is due to a rapid decrease of the expression of the binding epitope in the cell, maybe in relation with the high lytic power of these strains. From a practical point of view, our data indicate that a negative result when Mab 5-D8/1 is used for enterovirus typing must be interpreted cautiously with highly replicative strains and that detached cells should not be used for enterovirus identification under these circumstances.

Specific detection of echoviruses 22 and 23 in cell culture supernatants by RT-PCR.

Reverse transcription-polymerase chain reaction (RT-PCR) methods are available for the rapid detection of enteroviruses in clinical specimens or virus isolates. Pan-enterovirus PCR primers, however, fail to amplify echovirus (E) type 22 or 23 because of their extreme sequence divergence from the other enteroviruses. We have developed an RT-PCR method to detect specifically E22 and E23 RNA directly in tissue culture supernatants without a viral RNA purification step. The E22/E23 primers successfully amplified 20 of 20 clinical isolates of E22 and 4 of 4 E23 isolates representing viruses isolated in 15 states over a 19-year period, as well as E22 and E23 prototype strains isolated in the 1950s. The primers did not amplify any of the other 64 enterovirus prototype strains.

Identification of enteroviruses in clinical specimens by competitive PCR followed by genetic typing using sequence analysis.

A sensitive method based on competitive PCR was developed to detect and quantitate enteroviral RNA in clinical specimens, with special emphasis on controlling contamination and the presence of potential inhibitory factors in the specimens. Oligonucleotide primers from the conserved parts of the 5' untranslated and VP2 capsid protein-coding regions were selected to differentiate between enteroviruses and rhinoviruses on the basis of the length of the cDNA amplicons. RNA transcribed from a truncated cDNA copy of the echovirus 11 genome was used as an internal control for the reverse transcription reaction and PCR. This allowed simple differentiation of the control and viral PCR products from each other by agarose gel electrophoresis and nonradioactive quantitation of the viral RNA in the clinical specimens. By direct sequencing of the PCR products and subsequent computer analysis of the data, potential laboratory contaminations could be monitored and enteroviruses from clinical samples could be grouped into four distinct clusters, thus enabling genetic typing of the viruses. The described method can be applied to the diagnosis and epidemiology of enteroviral infections.

A combination of four cell types for rapid detection of enteroviruses in clinical specimens.

Isolation in cell culture is currently the most sensitive and reliable way to demonstrate enterovirus (EV) in clinical specimens. During July-October 1982 and 1983, we studied the impact of adding two new cell lines, Buffalo green monkey kidney (BGM) and human rhabdomyosarcoma (RD), to the more traditional cell combination used for EV isolation, human embryonic lung (HEL) and primary cynomolgus monkey kidney (CMK) cells; 2,558 specimens were studied: 632 fecal, 677 respiratory, 537 CSF, and 712 blood. An EV was isolated from 417 (16%); of these, 77 (18%) were positive only in BGM or RD; 35% (146/417) of the specimens were positive in BGM, RD, or both, at least one day earlier than in the traditional cells. BGM cells were helpful in isolation of group B coxsackieviruses (CB): 99% of 121 positive specimens were detected in BGM vs 73% in CMK and 23% in HEL; 72% of the CB isolates were detected by day 2 in BGM vs 48% in CMK and 0% in HEL. RD cells were helpful in the isolation of echoviruses: 59% of the 189 positive specimens were detected in RD vs 67% in HEL and 58% in CMK. RD was the only positive cell type in 28/189 (15%) positive specimens; 31% of the echovirus isolates were detected by day 2 in RD, vs 20% in HEL and 19% in CMK. Using the cell types described, we provided the clinician with results in 42% of the EV-positive specimens by day 2 after inoculation and in 61% by day 4.