Evaluation of the Prodesse Hexaplex multiplex PCR assay for direct detection of seven respiratory viruses in clinical specimens

We evaluated the Hexaplex assay (Prodesse, Waukesha, WI) for the detection of 7 respiratory viruses (influenza A and B, parainfluenza 1-3, and respiratory syncytial virus [RSV] A and B). The Hexaplex assay was performed on 300 respiratory samples during the 1999-2000 respiratory virus season. Results of this assay were compared with shell vial cell culture and/or direct fluorescent antibody stain. The overall sensitivity and specificity of the assay were 96.6% and 94.1%, respectively. The respective sensitivity and specificity of the Hexaplex assay for detection of specific virus groups were as follows: influenza A, 98.6% and 97.8%; influenza B, 100% and 100%; and for parainfluenza viruses (1-3), 100% and 99.1%. The assay did not perform as well with patients infected with RSV: sensitivity and specificity were 91.0% and 98.6%, respectively. There are 2 major drawbacks to this assay: it is technically demanding (3-4 hours hands-on time), and it is expensive ($80-$90 direct cost). Nevertheless, because of the excellent sensitivity and specificity, the Hexaplex assay may be valuable in the diagnosis of respiratory viral infections in immunocompromised patients.     

Evaluation of multiple commercial molecular and conventional diagnostic assays for the detection of respiratory viruses in children

This study compares the performance of four commercial multiplex PCR assays (Resplex II Panel v2.0, Seeplex RV15, xTAG RVP and xTAG RVP Fast) and direct fluorescent antibody (DFA) staining and viral isolation. Seven hundred and fifty nasopharyngeal swabs were tested for 17 viral agents. In each assay, the sensitivity and specificity for each target were determined against a composite reference standard. Two hundred and eighty-eight out of 750 (38.4%) specimens were positive by DFA or viral isolation, while an additional 214 (28.5%) were positive by multiplex PCR, for a total positivity rate of 66.9%. Of 502 positive specimens, one virus was detected in 420 specimens (83.7%), two in 77 (15.3%), three in four (0.8%) and four in one case (0.2%). Compared with a composite reference standard, the inter-assay accuracy of the multiplex PCR assays varied, but all were superior to conventional diagnostic methods in detecting a broad range of respiratory viral agents in children. In addition, the sensitivity of two commercial assays, Resplex II Plus PRE and Seeplex Influenza A/B Subtyping, was determined relative to the Astra influenza Screen & Type assay for detection of influenza A viruses, including seasonal influenzas and pandemic H1N1 2009 influenza A virus. Using 75 positive and 55 negative nasopharyngeal swabs for influenza A by the Astra assay, the sensitivity of Seeplex and Resplex was 95.9% and 91.8%, respectively, with a specificity of 100% for both.     

Evaluation of BioStar FLU OIA assay for rapid detection of influenza A and B viruses in respiratory specimens.

BACKGROUND: Demand for the rapid diagnosis of influenza infections has increased with the advent of the availability of neuraminidase antiviral therapy for influenza A and B. Several rapid assays that detect both influenza A and B are now available. OBJECTIVES: In this study we compared the performance of the BioStar FLU OIA assay to Bartels Viral Respiratory Screening and Identification Kit (Bartels Inc., Issaquah, WA), and cell culture. STUDY DESIGN: A total of 145 patient specimens for influenza virus detection submitted in either viral transport medium or in sterile containers were evaluated by the three methods. Specimen types included nasal washings, nasal swabs, sputum, throat swabs, and bronchial alveolar lavage (BAL) fluids. RESULTS: Fifty six positive specimens were identified based on culture and/or DFA. Of these, 30 specimens were positive by the OIA assay for an overall sensitivity of 54%. The OIA assay detected 48% (n = 21) of the 44 culture positive specimens and 81% (n = 29) of the 36 DFA positive specimens. Eighty six of the 89 culture/DFA negative samples were negative by the OIA assay (97% specificity). Analysis of the OIA assay sensitivity from samples submitted in M4 transport medium or in sterile containers revealed that M4 transport medium does not reduce the sensitivity of the OIA assay. Fifteen of the 27 positive samples submitted in M4 transport medium were positive by the OIA assay (56% sensitivity) compared to 15 of 29 positive samples transported in sterile containers (52% sensitivity). Twelve specimens were either culture and/or DFA positive for viruses other than influenza, but negative by the OIA assay, suggesting that there was no cross reactivity of the OIA assay with the other virus types recovered in this study. CONCLUSIONS: The overall excellent specificity of the BioStar FLU OIA allows for treatment of positive patients for influenza, however, a negative result should be confirmed by DFA and culture.     

Evaluation of R-Mix FreshCells in shell vials for detection of respiratory viruses

The performance of a mixture of mink lung and A549 cell lines in shell vials (MSVs) for the detection of respiratory viruses in 159 specimens was evaluated. MSVs, conventional culture, and direct immunofluorescence assay identified 96, 85, and 67% of the influenza A virus-positive specimens, respectively. MSVs provided both a high degree of sensitivity and rapid turnaround times for the detection of influenza A virus.     

Multiplex real-time PCR assay for detection of influenza and human respiratory syncytial viruses

A multiplex real-time PCR assay was developed with a LightCycler instrument for detection of influenza viruses A and B and the human respiratory syncytial virus (HRSV). Detection of each viral product and of an internal control was based on determination of specific melting temperatures by the LightCycler software. The lower limit of detection in the multiplex PCR assay was found to be 50 copies for each viral target. In an evaluation of nasopharyngeal samples collected from hospitalized children (ages, 0 to 3 years) with acute respiratory tract infections during the winter of 2001 to 2002, a viral pathogen was detected by the multiplex PCR test in 139 (66.8%) of 208 cases, including 45 (21.6%) influenza A virus infections, no (0%) influenza B virus infections, 106 (51%) HRSV infections, and 12 (5.8%) coinfections. The multiplex PCR test was compared to rapid antigen detection assays for influenza viruses A and B (Directigen; Becton Dickinson, Sparks, Md.) and HRSV (RSV TestPack; Abbott Laboratories, Abbott Park, Ill.) in 172 and 204 samples, respectively. After resolution of discrepant test results by use of additional PCR assays targeting other viral genes, the sensitivity (Se) and specificity (Sp) of the multiplex PCR assay for influenza A virus were 100 and 97.7% compared to 43.6 and 98.5% for the antigenic test. Similarly, the Se and Sp of the multiplex PCR assay for HRSV were 94.5 and 98.9% compared to 81.6 and 94.7% for the antigenic test. In conclusion, our multiplex real-time PCR assay combines both rapidity and sensitivity for detecting the most important respiratory viral pathogens in children.     

Concurrent detection of other respiratory viruses in children shedding viable human respiratory syncytial virus

Human respiratory syncytial virus (HRSV) is an important cause of respiratory disease. The majority of studies addressing the importance of virus co‐infections to the HRSV‐disease have been based on the detection of HRSV by RT‐PCR, which may not distinguish current replication from prolonged shedding of remnant RNA from previous HRSV infections. To assess whether co‐detections of other common respiratory viruses are associated with increased severity of HRSV illnesses from patients who were shedding viable‐HRSV, nasopharyngeal aspirates from children younger than 5 years who sought medical care for respiratory infections in Ribeirão Preto (Brazil) were tested for HRSV by immunofluorescence, RT‐PCR and virus isolation in cell culture. All samples with viable‐HRSV were tested further by PCR for other respiratory viruses. HRSV‐disease severity was assessed by a clinical score scale. A total of 266 samples from 247 children were collected and 111 (42%) were HRSV‐positive. HRSV was isolated from 70 (63%), and 52 (74%) of them were positive for at least one additional virus. HRSV‐positive diseases were more severe than HRSV‐negative ones, but there was no difference in disease severity between patients with viable‐HRSV and those HRSV‐positives by RT‐PCR. Co‐detection of other viruses did not correlate with increased disease severity. HRSV isolation in cell culture does not seem to be superior to RT‐PCR to distinguish infections associated with HRSV replication in studies of clinical impact of HRSV. A high rate of co‐detection of other respiratory viruses was found in samples with viable‐HRSV, but this was not associated with more severe HRSV infection. J Med. Virol. 85:1852–1859, 2013. © 2013 Wiley Periodicals, Inc.

     

Development of a loop-mediated isothermal amplification assay for the rapid detection of six common respiratory viruses

European Journal of Clinical Microbiology & Infectious Diseases - Due to the highly contagious and spreads quickly of respiratory infectious diseases (ADR), the availability of rapid,...     

Comparison of three techniques for detection of respiratory viruses in nasopharyngeal aspirates from children with lower acute respiratory infections

A comparison of immunofluorescence (IF), enzyme-linked immunosorbent assay (ELISA), and isolation in tissue culture (TC) for detection of respiratory viruses was performed on 496 nasopharyngeal aspirates from children under 5 years of age with lower acute respiratory infections who were receiving attention at three hospitals in Buenos Aires, Argentina. All samples were tested by the three methods for respiratory syncytial virus (RSV), influenza A and B, adenovirus, and parainfluenza 1 and 3. Viral diagnosis was made in 167 samples (33.7%); of these, 124 (74.3%) were isolated in TC, whereas 120 (71.8%) were detected by ELISA and 127 (76%) by IF. RSV was detected in 121 samples, mainly by ELISA and IF. The sensitivity and specificity of each rapid technique as compared with isolation in TC were similar, reaching 98% and 92%, respectively. When ELISA was compared with IF, the sensitivity was 95%, and the specificity was 98%. Adenovirus was detected in 18 patients by TC. For this virus, rapid techniques sensitivity as compared with TC was low (almost 22%). Parainfluenza 3 was readily detected by IF and TC; influenza A, B and parainfluenza 1 were detected in few samples; and tissue culture proved more efficient than rapid techniques. The results indicate that both rapid techniques are good tools for the detection of most respiratory viruses except for adenovirus, for which TC cannot be omitted.     

Penicillinase-based enzyme-linked immunosorbent assay for the detection of plant viruses

A penicillinase (PNC)-based, enzyme-linked immunosorbent assay (ELISA) was standardized to detect maize mosaic virus (MMV) in sorghum leaf extracts, peanut mottle virus (PMV) in pea leaf extracts, and tomato spotted wilt virus (TSWV) in peanut leaf extracts. Rabbit Fc-specific antibodies were conjugated with PNC by a single step glutaraldehyde bridge. Among several indicators tested, bromothymol blue (BTB) was found suitable for measuring PNC activity under simulated conditions. Two reagents, starch-iodine complex (SIC) and a mixed pH indicator, containing bromocresol purple and BTB (2:1) used earlier for the PNC-based ELISA, were compared with BTB for utilization in the PNC-based ELISA. SIC gave a slightly higher virus titre than BTB or the mixed pH indicator, but it often gave nonspecific reactions. Sodium or potassium salts of penicillin-G at 0.5-1.0 mg/ml and BTB at 0.2 mg/ml were found to be suitable as substrate-indicator mixture for PNC-based ELISA. The sensitivity of the PNC system was comparable to those of the alkaline phosphatase (ALP) and horseradish peroxidase (HRP) systems in detecting MMV, PMV, and TSWV. The PNC conjugate could be used at a greater dilution than those of the ALP and HRP conjugates and the BTB substrate mixture was stable for at least 3 weeks at 4 degrees C. Penicillin is readily available in developing countries, and at a substantially lower cost than p-nitrophenyl phosphate, the commonly used substrate for ALP in the plate ELISA. Thus the PNC-based ELISA provides a less expensive means for assaying plant viruses by ELISA.     

Comparison of enzyme-linked immunosorbent assay, indirect immunofluorescence assay, and virus isolation for detection of respiratory viruses in nasopharyngeal secretions.

Nasopharyngeal secretions obtained from 94 children with acute respiratory illness were examined for the presence of respiratory syncytial virus (RSV), adenovirus, and influenza virus type A by virus culturing (virus isolation technique [VIT]), immunofluorescence assay (IFA), and enzyme-linked immunosorbent assay (ELISA). Similar results were obtained in at least two tests for RSV, influenza virus type A, and adenovirus in 92 (97.9%), 88 (93.6%), and 88 (93.6%) cases, respectively. Both rapid virus detection methods showed good specificity for the diagnosis of these virus infections (greater than or equal to 90.7%) and were more sensitive than was VIT for RSV detection. In a more accurate statistical analysis, the indexes of agreement between VIT and ELISA were substantial for RSV (kappa = 0.69; zeta = 5.5; P less than 0.0001), influenza virus type A (kappa = 0.67; zeta = 5.3; P less than 0.0001), and adenovirus (kappa = 0.71; zeta = 6.0; P less than 0.0001), while it was almost perfect for RSV when ELISA was compared with IFA (kappa = 0.88; zeta = 5.7; P less than 0.0001). Although the observed agreement was good in the comparison of these two tests for these three viruses (89%0, the indexes of agreement were moderate in the comparison of IFA and VIT for RSV (K = 0.55; Z = 2.0; P < 0.05), influenza virus type A (K = 0.42; Z = 9.7; P < 0.0001), and adenovirus (K = 0.41; Z = 6.5; P < 0.0001) and of ELISA and IFA for influenza virus type A (K = 0.55; Z = 7.0; P < 0.0001) and adenovirus (K = 0.59; Z = 6.8; P < 0.0001). All of the statistical evaluations demonstrated better agreement between ELISA and VIT for influenza virus type A and adenovirus.     

多重PCR检测儿童呼吸道感染常见病毒

目的 对深圳、粤东地区儿童呼吸道病毒感染进行监测、分析,探讨其流行规律.方法 2006年6月-2008年6月,于汕头大学附属医院、深圳市第四人民医院、揭阳市人民医院采集毛细支气管炎、支气管肺炎、喘息支气管炎等的患儿咽拭子或鼻咽分泌物标本686份,多重PCR进行9种呼吸道病毒检测.结果 在686例标本中病毒阳性362例(52.77%),其中呼吸道感染患儿中呼吸道合胞病毒(RSV)感染占首位,达到31.22%(113/362),其次是鼻病毒(RHV)为16.85%(61/362),最低是流感病毒B型(IVB)占0.83%(3/362),流感病毒A型(IVA)占14.36%(52/362),副流感病毒1型(PIV1)和副流感病毒3型(PIV3)分别占7.73%(28/362)和8.29%(30/362),腺病毒(AdV)达到9.67%(35/362),而人博卡病毒(hBOV)和人偏肺病毒(hMPV)分别占6.08%(22/362)和4.97%(18/362).结论 RSV、RHV及IVA是华南地区儿童呼吸道感染的主要病毒病原,混合感染以RSV、IVA联合其他病毒感染为主,诊治时应根据所感染病原体制定有针对性的措施.     

MultiCode-PLx system for multiplexed detection of seventeen respiratory viruses

The MultiCode-PLx system (EraGen Biosciences, Inc., Madison, WI) for the detection of respiratory viruses uses an expanded genetic alphabet, multiplex PCR chemistry, and microsphere flow cytometry to rapidly detect and specifically identify 17 different respiratory viruses directly in clinical specimens. The MultiCode-PLx system was tested in parallel with direct fluorescent-antibody (DFA) staining and rapid shell vial culture (R-mix cells; Diagnostic Hybrids, Inc. Athens, OH) with 354 respiratory specimens from adult patients that were submitted to the clinical virology laboratory at the Emory University Hospital. Single-target PCRs were performed with retained samples to confirm the positive results obtained with the MultiCode-PLx system for viruses not covered by DFA and R-mix culture (metapneumovirus, coronaviruses [CoV], parainfluenza viruses 4a and 4b, and rhinoviruses) and to resolve any discrepancies between the DFA and R-mix culture and the MultiCode-PLx results for viruses common to both systems. Respiratory viruses were detected in 77 (21.8%) and 116 (32.7%) specimens by DFA and R-mix culture and with the MultiCode-PLx system, respectively. Among the viruses common to both systems, the MultiCode-PLx system detected significantly more influenza A viruses (P = 0.0026). An additional increased diagnostic yield with the MultiCode-PLx system resulted from the detection of human metapneumovirus (HMPV) in 9 specimens, human CoV (HCoV) in 3 specimens, and human rhinovirus (HRV) in 16 specimens. Also, two mixed viral infections were detected by the MultiCode-PLx system (HCoV OC43 and HRV infections and HMPV and HRV infections), but none were detected by DFA and R-mix culture. Single-target PCRs verified the results obtained with the MultiCode-PLx system for 73 of 81 (90.1%) specimens that had discordant results or that were not covered by DFA and R-mix culture. The MultiCode-PLx system provides clinical laboratories with a practical, rapid, and sensitive means for the massively multiplexed molecular detection of common respiratory viruses.     

Rapid multiplex nested PCR for detection of respiratory viruses

Respiratory tract infections can be caused by a heterogeneous group of viruses and bacteria that produce similar clinical presentations. Specific diagnosis therefore relies on laboratory investigation. This study developed and evaluated five groups of multiplex nested PCR assays that could simultaneously detect 21 different respiratory pathogens: influenza A virus (H1N1, H3N2, and H5N1); influenza B virus; parainfluenza virus types 1, 2, 3, 4a, and 4b; respiratory syncytial virus A and B; human rhinoviruses; human enteroviruses; human coronaviruses OC43 and 229E; severe acute respiratory syndrome coronavirus; human metapneumoviruses; Mycoplasma pneumoniae; Chlamydophila pneumoniae; Legionella pneumophila; and adenoviruses (A to F). These multiplex nested PCRs adopted fast PCR technology. The high speed of fast PCR (within 35 min) greatly improved the efficiency of these assays. The results show that these multiplex nested PCR assays are specific and more sensitive (100- to 1,000-fold) than conventional methods. Among the 303 clinical specimens tested, the multiplex nested PCR achieved an overall positive rate of 48.5% (95% confidence interval [CI], 42.9 to 54.1%), which was significantly higher than that of virus isolation (20.1% [95% CI, 15.6 to 24.6%]) and that of direct detection by immunofluorescence assay (13.5% [95% CI, 9.7 to 17.4%]). The improved sensitivity was partly due to the higher sensitivity of multiplex nested PCR than that of conventional methods in detecting cultivatable viruses. Moreover, the ability of the multiplex nested PCR to detect noncultivatable viruses, particularly rhinoviruses, coronavirus OC43, and metapneumoviruses, contributed a major gain (15.6%) in the overall positive rate. In conclusion, rapid multiplex nested PCR assays can improve the diagnostic yield for respiratory infections to allow prompt interventive actions to be taken.     

Comparison of nasopharyngeal aspirate with flocked swab for PCR‐detection of respiratory viruses in children

Fast‐ and high‐throughput molecular workflows require sample matrices to be suitable for automation. Respiratory swabs are better suited for this purpose compared to the more viscous nasopharyngeal aspirates. Samples collected by nasopharyngeal aspiration and nasopharyngeal flocked swab from 81 children were compared for detection and recovery of respiratory viruses. Using real‐time RT‐PCR, no statistically significant differences in virus detection between the two sample types were found, supporting the use of flocked swabs in children aged one month to two years.     

Expression of beta-galactosidase by recombinant respiratory syncytial viruses for microneutralization assay

The beta-galactosidase gene (lacZ) was inserted into a recombinant respiratory syncytial virus (RSV) A2 strain of subgroup A RSV (designated as A-lacZ) and a chimeric RSV that had the G and F surface glycoproteins of A2 replaced by those of the subgroup B RSV 9320 strain (designated as B-lacZ). Both recombinant RSVs, A-lacZ and B-lacZ, grew well in tissue culture and expressed high levels of beta-galactosidase. Using these two beta-galactosidase-expressing recombinant RSVs, a novel microneutralization assay was developed to measure serum anti-RSV neutralizing antibody from subgroup A or subgroup B RSV infection. The assay was carried out in 96-well plates and the unneutralized virus was quantitated by spectrophotometric measurement of the beta-galactosidase enzymatic reaction following incubation of the infected cell lysate with the enzyme substrate, chlorophenol red beta-D-galactopyranoside (CPRG). Adult human sera positive for anti-RSV antibody as shown by Western blot analysis and subgroup A or subgroup B RSV infected monkey sera were examined for the levels of anti-RSV neutralizing antibodies by the microneutralization assay in comparison with the plaque reduction neutralization assay. A higher antibody titer was detected when the neutralization assay was performed with the homologous RSV than the heterologous RSV, indicating that neutralization assay could distinguish antigenic differences between the two RSV subgroups. The microneutralization assay is comparable to the plaque reduction neutralization assay in sensitivity, but it is rapid, less laborious and suitable for screening a large number of samples.     

Simultaneous detection of respiratory viruses in children with acute respiratory infection using two different multiplex reverse transcription-PCR assays

A 4-tube multiplex RT-PCR (mRT-PCR), which showed higher sensitivity over conventional methods, was previously developed for the diagnosis of 14 viral pathogens of the respiratory tract. Herein the mRT-PCR was compared to the commercial Luminex mPCR-microsphere flow cytometry assay (Resplex II) which allows the detection of 12 different viruses. Eleven different viruses were identified in 91 nasopharyngeal swabs of children with acute respiratory infection, influenza A (IAV) and B, respiratory syncytial virus (RSV), human rhinovirus (hRhV), human echovirus, parainfluenza viruses (PIV) 1, 2, 3 and 4, human metapneumovirus (hMPV), and human coronavirus NL63. The results of the two techniques showed 53 and 40 positive patients by the Resplex II assay and mRT-PCR, respectively, with a concordance in 35 positive and 33 negative patients (74.7%). Individual RT-PCR tests were performed to control viruses not simultaneously detected by the two multiplex assays. The major virus misdiagnosed by mRT-PCR was IAV whereas the major viruses misdiagnosed by Resplex II were PIV1, 3 and 4. The mRT-PCR remains a simple, rapid, and specific assay for the specific detection of respiratory viruses, and can be easily implemented with standards in clinical laboratories at a low cost.