Evaluation of a quantitative real-time PCR for the detection of respiratory syncytial virus in pulmonary diseases

Borg, Irmgard; Rohde, Gernot; Löseke, Stefan; Bittscheidt, J; Schultze‐Werninghaus, G.; Stephan, Volker; Bufe, Albrecht

doi:10.1183/09031936.03.00088102

Cited by 102 publications

(81 citation statements)

References 41 publications

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“…This analytical sensitivity may be insufficient for reliable detection of microbial pathogens, which can be found at lower clinically relevant loads. For example, infections associated with viral loads as low as 100 viruses per ml of respiratory tract sample may be encountered (1,3). A highly sensitive hybridization on a plastic microarray using DNA capture probes spotted onto microfabricated micropillars has been described recently (19).…”

Section: Discussionmentioning

confidence: 99%

Plastic Polymers for Efficient DNA Microarray Hybridization: Application to Microbiological Diagnostics

et al. 2008

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Fabrication of microarray devices using traditional glass slides is not easily adaptable to integration into microfluidic systems. There is thus a need for the development of polymeric materials showing a high hybridization signal-to-background ratio, enabling sensitive detection of microbial pathogens. We have developed such plastic supports suitable for highly sensitive DNA microarray hybridizations. The proof of concept of this microarray technology was done through the detection of four human respiratory viruses that were amplified and labeled with a fluorescent dye via a sensitive reverse transcriptase PCR (RT-PCR) assay. The performance of the microarray hybridization with plastic supports made of PMMA [poly(methylmethacrylate)]-VSUVT or Zeonor 1060R was compared to that with high-quality glass slide microarrays by using both passive and microfluidic hybridization systems. Specific hybridization signal-to-background ratios comparable to that obtained with high-quality commercial glass slides were achieved with both polymeric substrates. Microarray hybridizations demonstrated an analytical sensitivity equivalent to approximately 100 viral genome copies per RT-PCR, which is at least 100-fold higher than the sensitivities of previously reported DNA hybridizations on plastic supports. Testing of these plastic polymers using a microfluidic microarray hybridization platform also showed results that were comparable to those with glass supports. In conclusion, PMMA-VSUVT and Zeonor 1060R are both suitable for highly sensitive microarray hybridizations.

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Section: Discussionmentioning

confidence: 99%

Plastic Polymers for Efficient DNA Microarray Hybridization: Application to Microbiological Diagnostics

et al. 2008

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“…RSV RNA has been detected in archived lung tissue from infants who died in summer months, raising the question of viral persistence (30). More compelling, two recent studies of patients with COPD reported detectable RSV RNA in 24 to 28% of respiratory samples, regardless of whether samples were collected during illness or stable periods (17,18). In addition, RSV RNA has also been detected by real-time RT-PCR in 70% of a small group of symptom-free smokers (31).…”

Section: Discussionmentioning

confidence: 99%

“…transcriptase-polymerase chain reaction (RT-PCR) from a high percentage (24-28%) of patients with stable COPD as well as from ill patients experiencing acute exacerbations (17,18). RSV detection was associated with elevated markers of inflammation and typically was at low copy number.…”

mentioning

confidence: 99%

Detection of Respiratory Syncytial Virus in Adults with Chronic Obstructive Pulmonary Disease

Falsey

Formica

Hennessey

et al. 2006

Am J Respir Crit Care Med

131

107

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Rationale: Recently, respiratory syncytial virus (RSV) RNA has been identified by reverse transcriptase-polymerase chain reaction (RT-PCR) from a high percentage of patients with stable chronic obstructive pulmonary disease (COPD). These data raise the possibility of persistent low-grade infection in this population, which could have implications in COPD pathogenesis. Objectives: RSV persistence was investigated by testing respiratory secretions from subjects with COPD during illness and at regular intervals over 1 yr. Methods: Nasal and sputum samples from subjects with COPD were tested by one-tube nested RT-PCR for RSV every 2 mo and during respiratory illnesses for 1 yr. Subjects positive for RSV were evaluated weekly until negative in two consecutive samples. Nasal secretions and serum were tested for RSV antibody. A rise of fourfold or greater was defined as evidence of RSV infection. Results: A total of 112 patients were enrolled and the illnesses of 92 patients were evaluated. RSV was detected by RT-PCR in 6/92 (6.5%) illness nasal samples versus 0/685 routine nasal samples and in 5/69 (7.2%) illness sputum samples versus 3 /315 (0.9%) routine. Four additional RSV infections were identified by serum antibody responses. Of the RSV infections 86% were associated with serum or nasal antibody responses and 73% had symptoms of acute respiratory illness. Conclusions: Most RSV infections in patients with COPD are associated with symptomatic respiratory illnesses and measurable immune responses. Our data do not support the concept of RSV persistence in this population. Keywords: chronic obstructive pulmonary disease exacerbation; persistent infection; viral infectionChronic obstructive pulmonary disease (COPD) is a group of disorders characterized by airflow obstruction that can be associated with breathing-related symptoms such as chronic cough, dyspnea, and wheezing (1). It is estimated that approximately 24 million adults in the United States have impaired lung function due to COPD. During 2000, COPD was responsible for 8 million physician office visits, 1.5 million emergency room visits, 726,000 hospitalizations, and 119,000 deaths. Recurrent acute exacerbations of COPD contribute substantially to the morbidity and mortality of this condition and may be due to infections with bacteria, viruses, or both (2-4). In studies of COPD exacerbation, respiratory syncytial virus (RSV) has been identified with variable frequency ranging from 0.8 to 22% depending on the diagnostic methods used (5-16). Recently investigators from the United Kingdom and Germany identified RSV RNA by reverse In our laboratory, we have developed a sensitive and specific real-time, one-tube nested RT-PCR for the diagnosis of RSV in adults (19,20). Because the assay is performed without opening the reaction tube, PCR contamination has been markedly reduced while the sensitivity of a nested assay is retained. Therefore, we sought to explore the question of RSV persistence in patients with COPD by testing upper-and lower-respiratorytract secreti...

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“…Non-quantitative PCR results may also be difficult to interpret, since low numbers of virus that do not signify an infection may be detected, but this problem is largely overcome by real-time PCR. Molecuar methods can also detect several orders of magnitude less than tissue culture methods (Borg et al, 2003).…”

Section: Molecular Methodsmentioning

confidence: 99%

Epidemiology and Diagnosis of Human Respiratory Syncytial Virus Infections

Akhter¹,

Johani²

2011

Human Respiratory Syncytial Virus Infection

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Evaluation of a quantitative real-time PCR for the detection of respiratory syncytial virus in pulmonary diseases

Cited by 102 publications

References 41 publications

Plastic Polymers for Efficient DNA Microarray Hybridization: Application to Microbiological Diagnostics

Plastic Polymers for Efficient DNA Microarray Hybridization: Application to Microbiological Diagnostics

Detection of Respiratory Syncytial Virus in Adults with Chronic Obstructive Pulmonary Disease

Epidemiology and Diagnosis of Human Respiratory Syncytial Virus Infections

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