Diagnosing rotavirus A associated IID: Using ELISA to identify a cut-off for real time RT-PCR

Phillips, Gemma; Lopman, Ben; Tam, Clarence C.; Iturriza‐Gómara, Miren; Brown, David; Gray, Jim

doi:10.1016/j.jcv.2008.12.001

Cited by 74 publications

(69 citation statements)

References 18 publications

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“…Phillips and colleagues (40) suggested that asymptomatic shedding was more likely to be associated with lower fecal viral loads, as assessed by higher C T values (Ͼ31) in real-time RT-PCR assays. Application of a similar approach to rotavirus testing yielded a good correlation between detection of rotavirus by antigen detection and symptomatic disease (41). In the current study, the norovirus sandwich ELISA identified all NoVs present in fecal samples at higher concentrations (C T values of Ͻ31), while only 1 of 17 samples with C T values of Ͼ31 were positive.…”

Section: Discussionmentioning

confidence: 49%

Characterization of Cross-Reactive Norovirus-Specific Monoclonal Antibodies

Kou¹,

Crawford²,

Ajami³

et al. 2014

Clin. Vaccine Immunol.

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Section: Discussionmentioning

confidence: 49%

Characterization of Cross-Reactive Norovirus-Specific Monoclonal Antibodies

Kou¹,

Crawford²,

Ajami³

et al. 2014

Clin. Vaccine Immunol.

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“…These data are in agreement with previous studies, such as that by Kang et al, who reported that C T values for rotavirus were associated with the severity of disease as assessed by the Vesikari severity score (7). Philips et al proposed a C T cutoff value for rotavirus real-time PCR results which is between 25 and 28 (16). Although C T values will change depending on the PCR assay used, a similar cutoff C T value of 28 based on our data of samples in which rotavirus was found (by any method) would result in a clinical sensitivity and specificity of 80% and 80%, respectively, in comparison to a specificity and sensitivity of RAT of 60% and 78%, respectively.…”

Section: Discussionmentioning

confidence: 99%

Replacing Traditional Diagnostics of Fecal Viral Pathogens by a Comprehensive Panel of Real-Time PCRs

et al. 2011

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Molecular DNA-based diagnostics are increasingly being used for diagnosis of viral infections. For enteric viruses, PCR assays have also been developed. The aims of this study were to compile and evaluate a comprehensive panel of PCR assays for diagnosis of viruses causing diarrheal disease and to evaluate its use in a largely pediatric population in a 750-bed university medical center. The PCR panel was designed to include assays for detection of adenovirus, astrovirus, enterovirus, norovirus, parechovirus, rotavirus, and sapovirus. The results of the PCR panel were evaluated in relation to conventional viral diagnostics consisting of viral culture and/or rotavirus and adenovirus rapid antigen tests on samples that were taken for routine diagnostics. Comparing conventional with PCR-based testing, the number of viruses detected increased dramatically from 25 to 106 when PCR assays were used. This increase was due mainly to detection of previously undetected viruses, i.e., astrovirus, norovirus, and sapovirus. In 24% of the samples, norovirus was detected. Also, the lower detection limit of PCR-based adenovirus, enterovirus, parechovirus, and rotavirus diagnostics further increased the detection rate. By focusing on samples from patients with complaints of gastroenteritis, detection of a causative agent was increased from 49% by conventional tests to 97% by molecular diagnostics. However, many samples containing low viral loads were found in patients with complaints other than intestinal complaints. In conclusion, the proposed comprehensive PCR panel with appropriate cutoff values can be used for sensitive, rapid, and clinically relevant diagnosis of gastrointestinal viruses.

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“…A broad quantification range is useful since the concentrations of RV can vary manyfold between different specimens, e.g., when sampling is performed at different time points after the onset of illness. Quantification can also enable the use of cutoff values in order to distinguish between clinical and subclinical infections, as suggested by Phillips et al (30). Also, if the assay is to be applied on environmental samples to measure RV concentration, e.g., in wastewater, a broad quantification range is valuable.…”

Section: Discussionmentioning

confidence: 99%

“…A PCR assay for direct genogrouping has not, to our knowledge, been reported previously. The VP6 protein is also the primary target for RV diagnosis using ELISA (12,18,30). There is a limited number of RV real-time PCR assays reported, many targeting the VP6 gene, such as the SYBR green assay (31) and the TaqMan assay (22).…”

mentioning

confidence: 99%

Novel Light-Upon-Extension Real-Time PCR Assay for Simultaneous Detection, Quantification, and Genogrouping of Group A Rotavirus

et al. 2010

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We have developed a light-upon-extension (LUX) real-time PCR assay for detection, quantification, and genogrouping of group A rotavirus (RV), the most common cause of acute gastroenteritis in children. The LUX system uses a fluorophore attached to one primer and having a self-quenching hairpin structure, making it cost-effective and specific. We designed genogroup-specific primers having different fluorophores, making it possible to differentiate between the two main genogroups of human group A RVs. The assay was applied on clinical stool specimens from Sweden and Central America (n ‫؍‬ 196) and compared to immunological and conventional PCR assays. The genogrouping ability was further validated against a subset of clinical specimens, which had been genogrouped using monoclonal antibodies. Our real-time PCR assay detected and quantified all positive specimens (n ‫؍‬ 145) and exhibited higher sensitivity than immunological assays and conventional PCR. The assay exhibited a wide dynamic range, detecting from 5 to >10 7 genes per PCR, resulting in a theoretical lower detection limit of <10,000 viruses per gram of stool. No cross-reaction was observed with specimens containing norovirus, sapovirus, astrovirus, or adenovirus. In total, 22 (15%) of the positive clinical specimens were identified as genogroup I, 122 (84%) were identified as genogroup II, and 1 specimen was found to contain a mix of both genogroups. All genogroup I-positive specimens were associated with capsid glycoprotein 2 (G2). No significant difference in viral load was found between genogroups or geographic region. The detection and quantification, combined with the genogrouping ability, make this assay a valuable tool both for diagnostics and for molecular epidemiological investigations.

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Diagnosing rotavirus A associated IID: Using ELISA to identify a cut-off for real time RT-PCR

Abstract: Gray (2009)

Cited by 74 publications

References 18 publications

Characterization of Cross-Reactive Norovirus-Specific Monoclonal Antibodies

Characterization of Cross-Reactive Norovirus-Specific Monoclonal Antibodies

Replacing Traditional Diagnostics of Fecal Viral Pathogens by a Comprehensive Panel of Real-Time PCRs

Novel Light-Upon-Extension Real-Time PCR Assay for Simultaneous Detection, Quantification, and Genogrouping of Group A Rotavirus

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