Comparison of a Multiplex Reverse Transcription-PCR-Enzyme Hybridization Assay with Conventional Viral Culture and Immunofluorescence Techniques for the Detection of Seven Viral Respiratory Pathogens

Liolios, Lisa; Jenney, Adam; Spelman, Denis; Kotsimbos, Tom; Catton, Michael; Wesselingh, Steve Lodewyk

doi:10.1128/jcm.39.8.2779-2783.2001

Cited by 160 publications

(138 citation statements)

References 30 publications

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“…7,12,13,16 Our results illustrated the limitations of virus culture and DFA-based techniques for the detection of viruses in clinical specimens. 8,12,14,36 The failure to detect virus by these techniques (DFA/CA-DFA) might be due to low viral load of or the presence of non-infectious virions in the specimen, which resulted from insufficient specimen or inappropriate transport or storage conditions (ie, specimens were kept for extended period at room temperature or higher). 13 RT-PCR technology is not affected by these limitations because it is dependent on the presence of viral nucleic acid rather than infectious or intact virions.…”

Section: Discussionmentioning

confidence: 99%

“…DFA detection is more rapid but less sensitive than viral culture, and results may be affected by specimen quality (ie, presence of intact, infected cells), virus type, and interpretation of a positive result, which is subjective and requires a great deal of technical skill. 2,[7][8][9][10] DFA is also unable to detect minor variations in amino acid sequence on envelope or capsid proteins. 11 Viral culture is still considered the "gold standard" for respiratory virus detection, but is limited by a prolonged result turnaround time (ie, 2 days to 1 week) and is dependent on stringent specimen transport and storage conditions to preserve the infectivity of the virus.…”

mentioning

confidence: 99%

“…9,[12][13][14][15] Although the combination of both these techniques can provide an increase in the number of positive results, a significant proportion of specimens still remain negative, despite clinical suspicion of viral infection. 8 Several studies have shown that polymerase chain reaction (PCR) amplification can resolve the intrinsic limitations associated with traditional diagnostic techniques by combining increased sensitivity, specificity, and rapid result turnaround time. 16,17 Also, PCR results are not dependent on infectious virus or viable cells.…”

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confidence: 99%

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A Sensitive, Specific, and Cost-Effective Multiplex Reverse Transcriptase-PCR Assay for the Detection of Seven Common Respiratory Viruses in Respiratory Samples

Syrmis¹,

Whiley²,

Thomas³

et al. 2004

The Journal of Molecular Diagnostics

159

153

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Common etiological viral agents of respiratory infections include adenoviruses (ADV), influenza types A and B (Flu A and B), parainfluenza types 1, 2, and 3 (PIV1, 2, 3),and respiratory syncytial virus (RSV). 1-4 These viruses are responsible for a spectrum of acute upper and lower respiratory tract disease. In children, the elderly and other immunocompromised groups, respiratory viruses can cause more serious clinical complications, such as croup, bronchiolitis, and pneumonia, which often require hospitalization. 5,6 Virus isolation by cell culture and direct immunofluorescent antibody assay (DFA) staining with monoclonal antibodies are two of the most commonly used laboratory techniques for detecting respiratory viruses. Both these methods have significant limitations in sensitivity and specificity. DFA detection is more rapid but less sensitive than viral culture, and results may be affected by specimen quality (ie, presence of intact, infected cells), virus type, and interpretation of a positive result, which is subjective and requires a great deal of technical skill. 2,7-10 DFA is also unable to detect minor variations in amino acid sequence on envelope or capsid proteins. 11 Viral culture is still considered the "gold standard" for respiratory virus detection, but is limited by a prolonged result turnaround time (ie, 2 days to 1 week) and is dependent on stringent specimen transport and storage conditions to preserve the infectivity of the virus. 9,[12][13][14][15] Although the combination of both these techniques can provide an increase in the number of positive results, a significant proportion of specimens still remain negative, despite clinical suspicion of viral infection. 8 Several studies have shown that polymerase chain reaction (PCR) amplification can resolve the intrinsic limitations associated with traditional diagnostic techniques by combining increased sensitivity, specificity, and rapid result turnaround time. 16,17 Also, PCR results are not dependent on infectious virus or viable cells. However, Supported by Royal Children's Hospital Foundation grants RA921-006 and I922-034 which were sponsored by the Woolworth's "Care for Kids" campaign.

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Section: Discussionmentioning

confidence: 99%

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confidence: 99%

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confidence: 99%

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A Sensitive, Specific, and Cost-Effective Multiplex Reverse Transcriptase-PCR Assay for the Detection of Seven Common Respiratory Viruses in Respiratory Samples

Syrmis¹,

Whiley²,

Thomas³

et al. 2004

The Journal of Molecular Diagnostics

159

153

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“…With PCR methods, because it is difficult to design compatible multiplex primer sets (3), the maximum number of viruses detectable in a single assay is relatively small (2). Moreover, unambiguous viral identification with degenerate PCR often is complicated by the existence of highly homologous relatives, and discrimination between viral subtypes or genera requires additional labor-intensive procedures such as restriction enzyme analysis, sequencing, or hybridization blotting of the PCR product (4)(5)(6)(7)(8). Perhaps most significantly, even the broadest multiplexing is inherently biased, requiring assumptions that ultimately restrict the possible outcome to the selected candidate viruses.…”

mentioning

confidence: 99%

Microarray-based detection and genotyping of viral pathogens

Wang

Coscoy

Zylberberg

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

694

243

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The detection of viral pathogens is of critical importance in biology, medicine, and agriculture. Unfortunately, existing techniques to screen for a broad spectrum of viruses suffer from severe limitations. To facilitate the comprehensive and unbiased analysis of viral prevalence in a given biological setting, we have developed a genomic strategy for highly parallel viral screening. The cornerstone of this approach is a long oligonucleotide (70-mer) DNA microarray capable of simultaneously detecting hundreds of viruses. Using virally infected cell cultures, we were able to efficiently detect and identify many diverse viruses. Related viral serotypes could be distinguished by the unique pattern of hybridization generated by each virus. Furthermore, by selecting microarray elements derived from highly conserved regions within viral families, individual viruses that were not explicitly represented on the microarray were still detected, raising the possibility that this approach could be used for virus discovery. Finally, by using a random PCR amplification strategy in conjunction with the microarray, we were able to detect multiple viruses in human respiratory specimens without the use of sequence-specific or degenerate primers. This method is versatile and greatly expands the spectrum of detectable viruses in a single assay while simultaneously providing the capability to discriminate among viral subtypes.

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“…There are two subtypes of RSV, serotypes A and B (Liolios et al, 2001). RSV is responsible for approximately 90,000 hospitalisations and 4,500 deaths in children aged six months and younger of the same age group, each year in the US alone.…”

mentioning

confidence: 99%