Anne Cent

Comparison of Real-Time PCR Assays with Fluorescent-Antibody Assays for Diagnosis of Respiratory Virus Infections in Children

ABSTRACT 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 × 107, than that in specimens positive only by PCR, at 4.1 × 104 (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.

Comparison of conventional and molecular detection of respiratory viruses in hematopoietic cell transplant recipients

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.

Six Rapid Tests for Direct Detection of Clostridium difficile and Its Toxins in Fecal Samples Compared with the Fibroblast Cytotoxicity Assay

ABSTRACT Clostridium difficile is one of the most frequent causes of nosocomial gastrointestinal disease. Risk factors include prior antibiotic therapy, bowel surgery, and the immunocompromised state. Direct fecal analysis for C. difficile toxin B by tissue culture cytotoxin B assay (CBA), while only 60 to 85% sensitive overall, is a common laboratory method. We have used 1,003 consecutive, nonduplicate fecal samples to compare six commercially available immunoassays (IA) for C. difficile detection with CBA: Prima System Clostridium difficile Tox A and VIDAS Clostridium difficile Tox A II, which detect C. difficile toxin A; Premier Cytoclone A/B and Techlab Clostridium difficile Tox A/B, which detect toxins A and B; and ImmunoCard Clostridium difficile and Triage Micro C. difficile panels, which detect toxin A and a species-specific antigen. For all tests, Triage antigen was most sensitive (89.1%; negative predictive value [NPV] = 98.7%) while ImmunoCard was most specific (99.7%; positive predictive value [PPV] = 95.0%). For toxin tests only, Prima System had the highest sensitivity (82.2%; NPV = 98.0%) while ImmunoCard had the highest specificity (99.7%; PPV = 95.0%). Hematopoietic stem cell transplant (HSCT) patients contributed 44.7% of all samples tested, and no significant differences in sensitivity or specificity were noted between HSCT and non-HSCT patients. IAs, while not as sensitive as direct fecal CBA, produce reasonable predictive values, especially when both antigen and toxin are detected. They also offer significant advantages over CBA in terms of turnaround time and ease of use.

Implementation of FilmArray respiratory viral panel in a core laboratory improves testing turnaround time and patient care

Abstract 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.

Respiratory Syncytial Virus in Hematopoietic Cell Transplant Recipients: Factors Determining Progression to Lower Respiratory Tract Disease

Abstract Background. Respiratory syncytial virus (RSV) lower respiratory tract disease (LRD) is a life-threatening complication in hematopoietic cell transplant (HCT) recipients. Lymphopenia has been associated with an increased risk of progression from upper respiratory tract infection (URI) to LRD. Methods. This study retrospectively analyzed the significance of lymphocyte engraftment dynamics, lung function, smoking history, corticosteroids, antiviral treatment, viral subtypes, and RSV-specific neutralizing antibodies for the progression to LRD in 181 HCT recipients with RSV URI. Results. In multivariable models, smoking history, conditioning with high-dose total body irradiation, and an absolute lymphocyte count (ALC) ≤100/mm3 at the time of URI onset were significantly associated with disease progression. No progression occurred in patients with ALCs of >1000/mm3 at URI onset. Lymphocyte engraftment dynamics were similar in progressors and nonprogressors. Pre- and posttransplant donor and posttransplant recipient RSV subtype-specific neutralizing antibody levels, RSV viral subtypes, and corticosteroids also were not significantly associated with LRD progression. Conclusions. 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.

Use of a glycoprotein G-based type-specific assay to detect antibodies to herpes simplex virus type 2 among persons attending sexually transmitted disease clinics.

Background Most genital herpes simplex virus type 2 (HSV-2) infections are unrecognized, thus, strategies to reduce the sexual transmission of HSV-2 are partly dependent on serologic screening. Goal To define performance characteristics of the Gull/Meridian glycoprotein G-based HSV-2 enzyme-linked immunosorbent assay among sexually transmitted disease clinic attendees and correlates of test acceptance. Study Design The cross-sectional study was conducted during two periods. Serologic testing was offered at a US

Development of multiplexed real-time quantitative polymerase chain reaction assay for detecting human adenoviruses ☆ ☆☆

15 charge during the first period and at no charge during the second period. Sera were tested by a type-specific glycoprotein G enzyme-linked immunosorbent assay and Western blot analysis, with the latter test used as the reference standard. Results Acceptance of HSV-2 testing was associated with free testing (odds ratio, 7.5; 95% CI, 6.0–9.9), older age, and white race. Sensitivity of the HSV-2 assay was 80.5% and specificity was 98.5%. The HSV-2 positive and negative predictive values were 95.8% (95% CI, 91.6–98.0%) and 92.2% (95% CI, 89.6–94.2%), respectively. Antibodies to HSV-2 were detected in 25.9% of 606 persons with no history of genital herpes. Conclusion Acceptance of HSV-2 serologic testing was cost sensitive. In this high-prevalence population, the positive predictive value of the enzyme-linked immunosorbent assay was sufficient to warrant its use without a confirmatory test. This assay could be useful in the screening of sexually active adults to detect unrecognized HSV-2 infection.

Bloodborne Viral Pathogen Contamination in the Era of Laboratory Automation

Adenoviruses (AdVs) have been associated with a wide variety of human disease and are increasingly recognized as viral pathogens that can cause significant morbidity and mortality in immunocompromised patients. Early detection of AdV DNA in plasma and sterile fluids has been shown to be useful for identifying patients at risk for invasive AdV disease. Because of the large number of existing Adv types, few real-time quantitative AdV polymerase chain reaction (PCR) assays published effectively cover all AdV types. We designed a series of AdV PCR primers and probes and empirically multiplexed them into 2 separate real-time PCR assays to quantitatively detect all 49 serotypes of human AdV (types 1-49) available from American Type Culture Collection. We then subsequently multiplexed all the primers and probes into 1 reaction. The sensitivity of these assays was determined to be less than 10 copies per reaction (500 copies/mL plasma). In a retrospective evaluation, we detected all 84 clinical AdV isolates isolated in cell culture from patients undergoing hematopoietic stem cell transplantation between 1981 and 1987. Prospective analysis of 46 consecutive clinical samples submitted for AdV testing showed greater sensitivity and equal specificity of the AdV PCR than viral culture. This real-time PCR assay allows rapid, sensitive, and specific quantification of all currently defined AdVs into either 2 or 1 multiplex assay for clinical samples.

Myeloablation-associated deletion of ORF4 in a human coronavirus 229E infection

BACKGROUND The CDC states that laboratory testing for persons under investigation for Ebola virus disease can be safely performed using automated laboratory instruments by adhering to bloodborne pathogen practices. We therefore sought to investigate the levels of viral contamination of a total laboratory automation (TLA) system to guide risk mitigation strategies for handling infectious agents. METHODS Environmental swabs followed by PCR for hepatitis B (HBV) and hepatitis C (HCV) viruses were taken from a chemistry TLA system during routine clinical use and after running a small number of high-titer HCV samples. Control experiments were performed to ensure the recovery of DNA and RNA viruses by swabs from a representative nonporous surface. RESULTS Of 79 baseline swabs for nucleic acids performed on the TLA system, 10 were positive for HBV and 8 for HCV. Viral nucleic acid was consistently detected from swabs taken from the distal inside surface of the decapper discharge chute, with areas adjacent to the decapper instrument and the centrifuge rotor also positive for HBV or HCV nucleic acid. Contamination was occasionally detected on exposed surfaces in areas without protective barriers between samples and personnel. After running known HCV-positive samples, at least one additional site of contamination was detected on an exposed area of the line. CONCLUSIONS A low level of viral contamination of automated clinical laboratory equipment occurs in clinical use. Given the risks associated with highly infectious agents, there is a need for risk-mitigation procedures when handling all samples.

Respiratory syncytial virus in hematopoietic cell transplant recipients

We describe metagenomic next-generation sequencing (mNGS) of a human coronavirus 229E from a patient with AML and persistent upper respiratory symptoms, who underwent hematopoietic cell transplantation (HCT). mNGS revealed a 548-nucleotide deletion, which comprised the near entirety of the ORF4 gene, and no minor allele variants were detected to suggest a mixed infection. As part of her pre-HCT conditioning regimen, the patient received myeloablative treatment with cyclophosphamide and 12 Gy total body irradiation. Iterative sequencing and RT-PCR confirmation of four respiratory samples over the 4-week peritransplant period revealed that the pre-conditioning strain contained an intact ORF4 gene, while the deletion strain appeared just after conditioning and persisted over a 2.5-week period. This sequence represents one of the largest genomic deletions detected in a human RNA virus and describes large-scale viral mutation associated with myeloablation for HCT.