Comparison of a rapid culture method combining an immunoperoxidase test and a group specific anti-VP1 monoclonal antibody with conventional virus isolation techniques for routine detection of enteroviruses in stools

Bourlet, Thomas; Gharbi, Jawhar; Omar, Shabir; Aouni, Mahjoub; Pozzetto, Bruno

doi:10.1002/(sici)1096-9071(199803)54:3<204::aid-jmv11>3.0.co;2-h

Cited by 21 publications

(14 citation statements)

References 20 publications

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“…Immunohistochemical staining with enterovirus-specific antibody was positive in the islets and in the endothelia of some blood vessels, while the exocrine pancreas was negative. This positivity was observed using the same VP1-specific commercial antibody (clone 5-D8/1; DakoCytomation) as that used in previous studies indicating the presence of enterovirus proteins in the islets [21,22]. Expression of enterovirus proteins in the islets was confirmed using another enterovirus-specific antibody.…”

Section: Discussionsupporting

confidence: 72%

“…Although this inhouse antibody was made by immunising rabbits with purified echovirus 11, it also detects other enterovirus serotypes in formalin-fixed cells (S. Tauriainen, unpublished observations). In addition, the VP1-specific clone 5- D8/1 is known to bind to several enterovirus serotypes [21]. Therefore, on the basis of the current data, we cannot define the precise serotype of the virus infecting the pancreatic islets of this child.…”

Section: Discussionmentioning

confidence: 85%

“…The enterovirus-specific antibody (clone 5-D8/1) has been previously used in studies showing enteroviruses in the pancreas of diabetic patients and in the heart tissue of patients with dilated cardiomyopathy [21,22,24]. Even though its specificity has been demonstrated in these studies, it has also been shown that antibodies against enterovirus VP1 protein may cross-react with heat shock proteins, especially HSP60 [27].…”

Section: Discussionmentioning

confidence: 99%

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Analysis of pancreas tissue in a child positive for islet cell antibodies

et al. 2008

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Aims/hypothesis Type 1 diabetes is caused by an immunemediated process, reflected by the appearance of autoantibodies against pancreatic islets in the peripheral circulation. Detection of multiple autoantibodies predicts the development of diabetes, while positivity for a single autoantibody is a poor prognostic marker. The present study assesses whether positivity for a single autoantibody correlates with pathological changes in the pancreas. Methods We studied post mortem pancreatic tissue of a child who repeatedly tested positive for islet cell antibodies (ICA) in serial measurements. Paraffin sections were stained with antibodies specific for insulin, glucagon, somatostatin, interferon alpha, CD3, CD68, cyclooxyge- Diabetologia (2008) nase-2 (COX-2), beta-2-microglobulin, coxsackie B and adenovirus receptor (CAR), natural killer and dendritic cells. Apoptosis was detected using Fas-specific antibody and TUNEL assay. Enterovirus was searched for using immunohistochemistry and in situ hybridisation, as well as enterovirus-specific RT-PCR from serum samples. Results The structure of the pancreas did not differ from normal. The number of beta cells was not reduced and no signs of insulitis were observed. Beta-2-microglobulin and CAR were strongly produced in the islets, but not in the exocrine pancreas. Enterovirus protein was detected selectively in the islets by two enterovirus-specific antibodies, but viral RNA was not found. Conclusions/interpretation These observations suggest that positivity for ICA alone, even when lasting for more than 1 year, is not associated with inflammatory changes in the islets. However, it is most likely that the pancreatic islets were infected by an enterovirus in this child.

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Section: Discussionsupporting

confidence: 72%

Section: Discussionmentioning

confidence: 85%

Section: Discussionmentioning

confidence: 99%

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Analysis of pancreas tissue in a child positive for islet cell antibodies

et al. 2008

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“…Although SV assays reduce the turnaround time for diagnosis, there are major disadvantages, including tedious manual preparation and washing of cover slips. A more convenient rapid cell culture technique using microwell cell cultures and immunoperoxidase staining with 5-D8/1 mAb has been described by Bourlet et al (1998). According to literature published up until now, this staining technique for detection of enteroviruses is not widely used (Table 1).…”

Section: Introductionmentioning

confidence: 99%

A convenient rapid culture assay for the detection of enteroviruses in clinical samples: comparison with conventional cell culture and RT-PCR

Terletskaia-Ladwig¹,

Meier²,

Hahn³

et al. 2008

Journal of Medical Microbiology

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A convenient rapid culture assay (RCA) for the detection of enteroviruses was evaluated against RT-PCR using 576 stool and 102 cerebrospinal fluid (CSF) samples. One hundred and ninety stool samples were also tested by conventional cell culture (CCC). The RCA used immunoperoxidase staining of cell culture plates with a blend of monoclonal antibodies (mAbs) against enterovirus VP1 on the second and sixth days after inoculation. This blend was composed of 5D8/1 (Dako) and four 'in-house' mAbs. CCC was performed using fluorescence staining with the Enterovirus Screening Set (Chemicon International) for culture confirmation. Detection of enteroviruses by the RCA was more successful in colonic carcinoma (CaCo-2) and rhabdomyosarcoma (RD) cells than in human embryonic lung fibroblasts, HEp2 and A549 cells. The performance of CCC in RD cells was hindered by rapid cell degeneration and non-specific staining of cells during culture confirmation. The sensitivity of the RCA compared to RT-PCR in stool samples was found to be 71 % (115/161) on the second day and 87 % (140/161) on the sixth day. The sensitivity of the RCA in CSF samples was 38 % (22/58) after 2 days and 59 % (34/58) after 6 days. The specificity of the RCA was 100 %. All CCC-positive samples were positive by the RCA. CCC required 3-14 days for virus recovery. In conclusion, the RCA has the same sensitivity as CCC, significantly shortens the time required for the detection of enteroviruses, and prevents pitfalls associated with using RD cells for CCC. For diagnosis of aseptic meningitis in CSF samples, RT-PCR should be performed.

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“…The need to perform subpassages on toxic cultures and/or the performance of culture confirmation may add an additional 24 or more h to test turnaround time. In one study with stool isolates, for example, a mean time to isolation and confirmation of 11.5 Ϯ 5 days was reported (1).…”

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confidence: 99%

Detection of Precytopathic Effect of Enteroviruses in Clinical Specimens by Centrifugation-Enhanced Antigen Detection

et al. 2001

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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.During the summer season, enteroviruses are responsible for the majority of viral diseases among pediatric and adult patients. Approximately 10 to 15 million symptomatic infections due to enteroviruses occur each year, resulting in a variety of disease syndromes (9). A rapid laboratory diagnosis of an enterovirus infection is important in patient care and management (e.g., decisions about antibiotic use and length of hospital stay). The significance of rapid enterovirus diagnostics is further underscored by recent progress in enterovirus drug research (8).Enteroviruses in general grow rapidly in cell culture. Most clinical enterovirus strains are isolated within 4 to 5 days of conventional culture inoculation (S. M. Lipson, unpublished observations). The need to perform subpassages on toxic cultures and/or the performance of culture confirmation may add an additional 24 or more h to test turnaround time. In one study with stool isolates, for example, a mean time to isolation and confirmation of 11.5 Ϯ 5 days was reported (1).Molecular diagnostics, both "automated" and nonautomated, have been introduced t...

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Comparison of a rapid culture method combining an immunoperoxidase test and a group specific anti-VP1 monoclonal antibody with conventional virus isolation techniques for routine detection of enteroviruses in stools

Cited by 21 publications

References 20 publications

Analysis of pancreas tissue in a child positive for islet cell antibodies

Analysis of pancreas tissue in a child positive for islet cell antibodies

A convenient rapid culture assay for the detection of enteroviruses in clinical samples: comparison with conventional cell culture and RT-PCR

Detection of Precytopathic Effect of Enteroviruses in Clinical Specimens by Centrifugation-Enhanced Antigen Detection

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