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

Abstract

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.

Determination of the Frequency of Human Bocavirus and Other Respiratory Viruses Among 0-2 Years Age Group Children Diagnosed as Acute Bronchiolitis

“…Hücre kültürü ve direkt immünofloresans yöntemleri, solunum yolu viral enfeksiyonlarının tanısında yaygınlıkla kullanılan geleneksel yöntemler olmakla birlikte, bu metotların duyarlılık ve özgüllüklerinde önemli kısıtlamalar bulunmaktadır 9 . Buna karşın, tek bir örnekten DNA ve RNA viruslarının tespitinde multipleks RT-PCR yöntemi duyarlı, özgül ve hızlı bir metottur 8 .…”

“…Günümüzde polimeraz zincir reaksiyonu (PCR) temelli testler, solunum yolu viruslarının tanımlanmasında, konvansiyonel yöntemlerden (virus izolasyonu ve antijen testleri) daha hızlı ve duyarlı sonuç vermektedir [6][7][8] . Özellikle çok sayıdaki solunum virusunu tek bir örnekte aynı anda tespit edebilen multipleks PCR/mikroarray yöntemlerinin kullanıma girmesi, gerek viral koenfeksiyonların prevalansının belirlenmesinde, gerekse doğru ve etkili antiviral tedaviye karar verilmesinde büyük yarar sağlamıştır 4,[8][9][10] .…”

Novel Neutrophil-Derived Proteins in Bronchoalveolar Lavage Fluid Indicate an Exaggerated Inflammatory Response in Pediatric Cystic Fibrosis Patients

“…We performed total cell and differential cell counts and identified bacterial, fungal, and mycobacterial pathogens from 500 L of BALF by standard methods reported elsewhere (16,17 ). We conducted direct immunofluorescence antigen testing for respiratory viruses on BALF as described (16 ); if results were negative, we performed nucleic acid amplification tests using a multiplex reverse transcriptase-PCR assay (18 ). Final counts for bacterial and fungal pathogens were expressed as CFU per mL of BALF.…”

Evaluation of PCR Testing of Ethanol-Fixed Nasal Swab Specimens as an Augmented Surveillance Strategy for Influenza Virus and Adenovirus Identification

“…The major surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA), are primarily responsible for the antigenic variation seen in influenza viruses and serve as the basis for further subtyping of influenza type A viruses (51). The extreme genetic variability of influenza A viruses makes designing useful, rapid, molecular-based assays challenging (45), but several different approaches have been successfully utilized by targeting the highly conserved RNA gene segment encoding the internally expressed matrix 1 (M1) or nonstructural (NS) protein (16,44). Reverse transcription-PCR (RT-PCR) is effective for initial influenza diagnosis and has greater sensitivity than other available rapid assays (4,5,7,17,36).…”