Conventional fluorescent-antibody (FA) methods were compared to real-time PCR assays for detection of respiratory syncytial virus (RSV), influenza virus type A (FluA), parainfluenza virus types 1, 2, and 3 (PIV1, PIV2, and PIV3), human metapneumovirus (MPV), and adenovirus (AdV) in 1,138 specimens from children with respiratory illnesses collected over a 1-year period. At least one virus was detected in 436 (38.3%) specimens by FA and in 608 (53.4%) specimens by PCR (P < 0.001). Specimen quality was inadequate for FA in 52 (4.6%) specimens; 13 of these (25%) were positive by PCR. In contrast, 18 (1.6%) specimens could not be analyzed by PCR; 1 of these was positive by FA. The number of specimens positive only by PCR among specimens positive by PCR and/or FA was 18 (7.0%) of 257 for RSV, 18 (13.4%) of 134 for FluA, 25 (64.1%) of 39 for PIV1, 8 (88.9%) of 9 for PIV2, 17 (30.1%) of 55 for PIV3, and 101 (76.5%) of 132 for AdV. MPV was detected in 6.6% of all specimens and in 9.5% of the 702 specimens negative by FA. The mean number of virus copies per milliliter in specimens positive by both PCR and FA was significantly higher, at 6.7 ؋ 10 7 , than that in specimens positive only by PCR, at 4.1 ؋ 10 4 (P < 0.001). The PCR assays were significantly more sensitive than FA assays for detecting respiratory viruses, especially parainfluenza virus and adenovirus. Use of real-time PCR to identify viral respiratory pathogens in children will lead to improved diagnosis of respiratory illness.Accurate detection of respiratory viruses is important to guide antiviral therapy, prevent nosocomial spread, provide surveillance, and in some cases, decrease hospital costs and lengths of stay (1, 2, 11, 21). By using standard laboratory methods, such as staining with fluorescent antibodies (FA) and isolation by culture, viruses have been detected in 13 to 45% of children with symptoms of respiratory illness (3,8,12,22,28). Disadvantages of FA include requiring multiple reagents which may vary in sensitivity, potential variability in technical reading, and the need for an adequate number of cells to examine each specimen. Several studies have shown that PCR methods appear to be more sensitive than FA and culture for the diagnosis of acute respiratory virus infections (8,22,23,24,26,28). PCR is less affected by specimen quality and transport and provides an objective interpretation of results. Real-time PCR technology, which combines nucleic acid amplification with amplicon detection, provides results more quickly than conventional PCR, has in some cases shown improved sensitivity compared to conventional PCR, and provides a uniform platform for quantifying both single and multiple pathogens in a single sample (4,7,18).In this study, separate quantitative real-time reverse transcription (RT)-PCR assays were used to detect six RNA viruses, including respiratory syncytial virus (RSV), influenza virus type A (FluA), parainfluenza virus types 1, 2, and 3 (PIV1, PIV2, and PIV3), and human metapneumovirus (MPV). A quantitative real-time PCR a...

The incidence of respiratory virus infection after hematopoietic cell transplantation (HCT) has probably been underestimated with conventional testing methods in symptomatic patients. This prospective study assessed viral infection episodes by testing weekly respiratory samples collected from HCT recipients, with and without symptoms reported by questionnaire, for 100 days after HCT. Samples were tested by culture and direct fluorescent antibody testing for respiratory syncytial virus (RSV), parainfluenza virus (PIV), and influenza A and B, and by quantitative reverse transcription–polymerase chain reaction for RSV, PIV, influenza A and B, and metapneumovirus (MPV). Of 122 patients, 30 (25%) had 32 infection episodes caused by RSV (5), PIV (17), MPV (6), influenza (3), RSV, or influenza (1). PIV, with a cumulative incidence estimate of 17.9%, was the only virus for which asymptomatic infection was detected. Lower virus copy number in patients with no or one symptom compared with 2 or more symptoms was found for all viruses in all patients (P < .001), with PIV infection having a similar virus-specific comparison (P = .004). Subclinical infection with PIV may help explain why infection-control programs that emphasize symptoms are effective against RSV and influenza but often not against PIV.

Host and transplant related factors appear to determine the risk of progression to LRD more than viral factors. Dysfunctional cell-mediated immunity appears to be important in the pathogenesis of progressive RSV disease after HCT. A characterization of RSV-specific T-cell immunity is warranted.

Reproducible quantitative assays to detect viral nucleic acids have proven useful in defining disease progression and following response to therapy in a wide variety of viral infections. We describe the development of a quantitative assay to detect hepatitis B virus (HBV) DNA using real-time fluorescent-probe polymerase chain reaction (PCR) (TaqMan). The assay is highly reproducible, highly automated, and much more sensitive than the currently used branchedchain DNA (bDNA) assay for HBV. The quantitative PCR assay accurately detected samples ranging from 10 to 10 9 copies of HBV DNA per milliliter. Of 157 serum samples submitted for HBV quantitation, 119 were positive by TaqMan PCR versus only 55 by bDNA (P < .001). All 55 bDNApositives were positive by TaqMan. Of the 77 samples with detectable HBV-DNA titers below 3.75 ؋ 10 5 copies by TaqMan, only 13 were detected by bDNA. We tested 119 patients negative for all HBV serologic markers, and all tested negative in the TaqMan assay. Hepatitis B virus (HBV) infects approximately 400 million persons worldwide. HBV can cause pathology ranging from self-limited illness to chronic hepatitis, cirrhosis, and hepatocellular carcinoma. 1 The progression of HBV disease is usually monitored by serologic assays. The branched-chain DNA assay (bDNA) (Chiron, Emeryville, CA) can be used to accurately determine viral load. However, the sensitivity of the bDNA test is limited to a range of approximately 6 ϫ 10 5 to 5 ϫ 10 9 HBV-DNA copies/mL. 2,3 This level of detection is far above what most authorities would consider optimal for suppression of HBV replication during therapy or for reducing transmission of HBV via sexual, bloodborne, or maternal-fetal routes. 4 Advances in antiviral therapy (lamivudine) and liver transplantation have also brought about a need for a more sensitive assay for HBV viral load. 5 We and others have used quantitative "real-time" fluorescent-based polymerase chain reaction (PCR) systems to detect nucleic acid from other viruses in plasma and to measure response to antiviral therapy. 6,7 In this article, we describe a new TaqMan-based quantitative PCR 8,9 for the detection of HBV DNA in serum, and compare this assay with the established serologic and bDNA tests for monitoring HBV infection.PCR primers (forward primer HBV1F: 5Ј CCG TCT GTG CCT TCT CAT CTG; reverse primer HBV1R: 5Ј AGT CCA AGA GTY CTC TTA TGY AAG ACC TT) and fluorescent probe (HBV1TAQ: 5Ј CCG TGT GCA CTT CGC TTC ACC TCT GC) were designed against a conserved region of the HBV genome overlapping the genes encoding the X-protein and DNA polymerase, defining an amplicon corresponding to bases 1549 to 1653 of the HBV genome. TaqMan PCR was performed as previously described. 6 To define the linear range of the assay, we used serially diluted bacterial plasmids encoding a wild subtype of HBV (ATCC #39629). The TaqMan PCR assay reproducibly quantitated bacterial plasmid samples ranging between 10 1 to 10 6 copies per reaction (Fig. 1). The standard deviation for each curve was less than 1% of each ...

Background-Sensitive detection of respiratory viruses is important for early diagnosis of infection in patients following hematopoietic cell transplantation (HCT). To evaluate the relative sensitivity of respiratory virus detection in specimens from HCT recipients, we compared the results of conventional and quantitative molecular methods.

Human metapneumovirus (HMPV), a common respiratory virus, can cause severe disease in pre- and post-hematopoietic cell transplant (HCT) recipients. We conducted a retrospective cohort analysis in HCT patients with HMPV (N=23) or respiratory syncytial virus (RSV, N=23) detected in bronchoalveolar lavage (BAL) samples by reverse-transcription PCR between 2006 and 2011 to determine disease characteristics and factors associated with outcome. Mortality rates at 100 days were 43% for both HMPV and RSV lower respiratory tract disease (LRTD). Steroid therapy, oxygen requirement > 2L or mechanical ventilation and bone marrow as cell source were significant risk factors for overall and virus-related mortality in multivariable models while the virus type was not. The presence of centrilobular/nodular radiographic infiltrates was a possible protective factor for mechanical ventilation. Thus, HMPV LRTD is associated with high mortality in HCT recipients. Earlier detection in combination with new antiviral therapy is needed to reduce mortality among HCT recipients.

The FilmArray respiratory virus panel detects 15 viral agents in respiratory specimens using polymerase chain reaction. We performed FilmArray respiratory viral testing in a core laboratory at a regional children's hospital that provides service 24 hours a day 7 days a week. The average and median turnaround time were 1.6 and 1.4 hours, respectively, in contrast to 7 and 6.5 hours documented 1 year previously at an on-site reference laboratory using a direct fluorescence assay (DFA) that detected 8 viral agents. During the study period, rhinovirus was detected in 20% and coronavirus in 6% of samples using FilmArray; these viruses would not have been detected with DFA. We followed 97 patients with influenza A or influenza B who received care at the emergency department (ED). Overall, 79 patients (81%) were given oseltamivir in a timely manner defined as receiving the drug in the ED, a prescription in the ED, or a prescription within 3 hours of ED discharge. Our results demonstrate that molecular technology can be successfully deployed in a nonspecialty, high-volume, multidisciplinary core laboratory.

Currently licensed enzyme immunoassays give inaccurate or misleading results about the correct herpes simplex virus infecting subtype.