Detection of rhinovirus and enterovirus in upper respiratory tract samples using a multiplex nested PCR

Billaud, Geneviève; Peny, Stéphanie; Legay, Vincent; Lina, Bruno; Valette, Martine

doi:10.1016/s0166-0934(03)00038-7

Cited by 23 publications

(17 citation statements)

References 22 publications

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“…A sterile nylon brush was placed on the nasal, pharyngeal mucosa and in the external acoustic meatus, and removed gently without anesthesia. Every brush was immediately placed in a closed container that contained viral transport medium, placed on ice, and sent immediately to the laboratory where every medium with suspended cells was centrifuged for 15 min and inoculated in four 667 ADHESION MOLECULES AND CYTOKINES IN URTI cell lines (American Type Culture Collection, Manassas, VA, U.S.A.) in tube cultures while remaining aliquot was frozen at Ϫ70°C until tested by PCR human lung carcinoma cells (A549), HeLa Ohio cells, Madin-Darby kidney cells, and LLC-MK2 cells, and for adenovirus, rhinovirus, influenza virus, and parainfluenza viruses 1, 2 and 3 cell cultures and virus cytopathic effects were performed as described by Billaud et al (15).…”

Section: Methodsmentioning

confidence: 99%

Soluble Adhesion Molecules and Cytokines in Children Affected by Recurrent Infections of the Upper Respiratory Tract

et al. 2004

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The objective of this study was to compare plasma levels of soluble adhesion molecules and Th1-Th2 type cytokines in 44 children with frequently recurrent respiratory infections (FRRI) of upper airways, defined as having nine or more episodes per year, and in 34 children without recurrence; all subjects were followed-up for 12 mo. The viral etiology was determined by cultures from nasal, pharyngeal, and ear secretions, using PCR and immunofluorescence. Plasma levels of five soluble adhesion molecules (E-selectin, P-selectin, L-selectin, intercellular adhesion molecule-1, and vascular cell adhesion molecule-1) and interferon (IFN)-␥, IL-12, IL-4, and IL-10 were measured in patients and in 15 healthy controls using sandwich ELISA. During acute phase, all patients showed significant increase in plasma levels of soluble adhesion molecules; during the followup, the levels were greater in children with FRRI. A difference of cytokine profile was demonstrated between the patients with and without FRRI: an increased IL-4 and IL-10 release with decreased levels of IFN-␥ and IL-12 suggested a skewing into Th2-type response, in patients with FRRI. This pattern persisted during the follow-up. In patients without recurrence, an increased IFN-␥ and IL-12 release, together with decreased levels of IL-4 and IL-10, showed a skewing into Th1-type responses; in the follow-up these cytokines reached normal values. In conclusion, the abnormal levels of all examined parameters in children with FRRI may reflect the persistence of an inflammatory microenvironment in the airways and an activation of the immune system that may contribute to the frequently recurrence of the respiratory disease. Infections of the URTI are the most common cause of viral morbidity in children. They represent the most frequent managed problem in general pediatric practice (1) and are responsible for more than one third of school absences (2). Many different immunologic problems can concur to the development of respiratory infections (3). Disorders of the aspecific humoral and the specific cellular defense are the principal cause of inflammation in the upper respiratory diseases (4). The recruitment and activation of inflammatory cells at the site of infection involve the expression of leukocyte and vascular adhesion molecules and the generation of pro-inflammatory cytokines, which contribute to the injury of lung epithelial and endothelial barriers (5). An important role in the early transient adhesion phases of leukocytes is mediated by the selectin family, which includes three distinct carbohydrate receptors expressed by either endothelial cells (E-selectin), leukocytes (L-selectin), or platelets and endothelial cells (P-selectin) (6). ICAM-1 and VCAM-1 are membrane glycoproteins, belonging to the immunoglobulin superfamily; ICAM-1 are widely expressed on respiratory epithelial cells, monocytes, macrophages, dendritic cells, and, together with VCAM-1, on vascular endothelial cells (7,8). These adhesion molecules, after a proteolytic cleavage, are found as so...

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Section: Methodsmentioning

confidence: 99%

Soluble Adhesion Molecules and Cytokines in Children Affected by Recurrent Infections of the Upper Respiratory Tract

et al. 2004

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“…These assays typically target the 5Ј noncoding region (5ЈNCR) of the viral genome that contains highly conserved sequences suitable for molecular assay development. However, most of these assays require postamplification processing of the amplicon by gel electrophoresis, probe hybridization, sequencing, or restriction analysis to confirm and differentiate HRVs from HEVs (1,3,4,5,14,24,31,34,41). More recently, real-time RT-PCR assays have been described for HRVs/HEVs (8,9,25,38,44) that offer potentially rapid, sensitive, and quantitative results and that are less prone to amplicon contamination.…”

mentioning

confidence: 99%

Real-Time Reverse Transcription-PCR Assay for Comprehensive Detection of Human Rhinoviruses

Lu¹,

Holloway

Dare³

et al. 2008

J Clin Microbiol

257

229

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Human rhinoviruses (HRVs) are important contributors to respiratory disease, but their healthcare burden remains unclear, primarily because of the lack of sensitive, accurate, and convenient means of determining their causal role. To address this, we developed and clinically validated the sensitivity and specificity of a real-time reverse transcription-PCR (RT-PCR) assay targeting the viral 5 noncoding region defined by sequences obtained from all 100 currently recognized HRV prototype strains and 85 recently circulating field isolates. The assay successfully amplified all HRVs tested and could reproducibly detect 50 HRV RNA transcript copies, with a dynamic range of over 7 logs. In contrast, a quantified RNA transcript of human enterovirus 68 (HEV68) that showed the greatest sequence homology to the HRV primers and probe set was not detected below a concentration of 5 ؋ 10 5 copies per reaction. Nucleic acid extracts of 111 coded respiratory specimens that were culture positive for HRV or HEV were tested with the HRV real-time RT-PCR assay and by two independent laboratories that used different in-house HRV/HEV RT-PCR assays. Eighty-seven HRVculture-positive specimens were correctly identified by the real-time RT-PCR assay, and 4 of the 24 HEVpositive samples were positive for HRV. HRV-specific sequences subsequently were identified in these four specimens, suggesting HRV/HEV coinfection in these patients. The assay was successfully applied in an investigation of a coincidental outbreak of HRV respiratory illness among laboratory staff.

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“…In this context, we tested three different protocols, including one that differentiated HRV form HEV by nested-PCR (2), in samples that were discordant when tested with two other protocols (17, 20). …”

Section: Discussionmentioning

confidence: 99%

“…The first step detects picornaviruses (EV2 and EV3 primers), and the second step distinguishes human rhinovirus from human enterovirus with specific primers (CCRV3/CCRV4 to HRV and EV3/EVNC1 to HEV) (2). The amplified products were detected by 2% agarose gel electrophoresis to identify a 93 base pair (bp) fragment for rhinovirus or a 154 bp fragment for enterovirus.…”

Section: Methodsmentioning

confidence: 99%

“…These assays provide high sensitivity, but because the gene sequences are similar between rhinovirus and other enterovirus species, the assays also necessitate an additional step to differentiate the two. Examples of other methods to detect these viruses include hybridization with specific probes (4, 17), nested-PCR with specific HRV primers (1), and duplex nested-PCR with enterovirus and rhinovirus primers that distinguish between enterovirus- and rhinovirus-positive samples in only one reaction (2). Moreover, sequencing can distinguish these viruses following their detection (14).…”

Section: Introductionmentioning

confidence: 99%

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Rhinovirus detection using different PCR-based strategies

Silva¹,

Watanabe²,

Carraro³

et al. 2012

Braz. J. Microbiol.

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Human rhinoviruses (HRVs) are the major cause of the common cold. HRVs were recently reclassified into the Enterovirus genus (HEV) in the Picornaviridae family. HRVs and other members of the HEV genus share many common features, including sense RNA genomes and partial nucleotide sequence identity. The aim of this study was to evaluate different HRV detection strategies. Samples from adults with acute respiratory infection (n = 291) who were treated in Sao Paulo Hospital (2001-2003) were tested using three assays. The first assay detected picornaviruses by RT-PCR and hybridization, the second detected rhinoviruses using RT-PCR/sequencing, and the third differentiated HRV from HEV using duplex semi-nested-RT-PCR. Analysis of the results obtained from the first two strategies revealed 83% concordance. Discordant samples were then evaluated by the third protocol, and 82% were negative. The picornavirus detection protocol was more sensitive but less specific than the rhinovirus detection protocols. The semi-nested protocol utilized in the present study was less sensitive and was not useful in differentiating HRV from HEV. Sequencing assays examining different genes would address the best strategy of confirming rhinovirus and enterovirus infections.

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Detection of rhinovirus and enterovirus in upper respiratory tract samples using a multiplex nested PCR

Cited by 23 publications

References 22 publications

Soluble Adhesion Molecules and Cytokines in Children Affected by Recurrent Infections of the Upper Respiratory Tract

Soluble Adhesion Molecules and Cytokines in Children Affected by Recurrent Infections of the Upper Respiratory Tract

Real-Time Reverse Transcription-PCR Assay for Comprehensive Detection of Human Rhinoviruses

Rhinovirus detection using different PCR-based strategies

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