Julie D. Fox

Effect of Influenza Vaccination of Children on Infection Rates in Hutterite Communities: A Randomized Trial

CONTEXT Children and adolescents appear to play an important role in the transmission of influenza. Selectively vaccinating youngsters against influenza may interrupt virus transmission and protect those not immunized. OBJECTIVE To assess whether vaccinating children and adolescents with inactivated influenza vaccine could prevent influenza in other community members. DESIGN, SETTING, AND PARTICIPANTS A cluster randomized trial involving 947 Canadian children and adolescents aged 36 months to 15 years who received study vaccine and 2326 community members who did not receive the study vaccine in 49 Hutterite colonies in Alberta, Saskatchewan, and Manitoba. Follow-up began December 28, 2008, and ended June 23, 2009. INTERVENTION Children were randomly assigned according to community and in a blinded manner to receive standard dosing of either inactivated trivalent influenza vaccine or hepatitis A vaccine, which was used as a control. MAIN OUTCOME MEASURES Confirmed influenza A and B infection using a real-time reverse transcriptase polymerase chain reaction (RT-PCR) assay and by measuring serum hemagglutination inhibition titers. RESULTS The mean rate of study vaccine coverage among eligible participants was 83% (range, 53%-100%) for the influenza vaccine colonies and 79% (range, 50%-100%) for the hepatitis A vaccine colonies. Among nonrecipients, 39 of 1271 (3.1%) in the influenza vaccine colonies and 80 of 1055 (7.6%) in the hepatitis A vaccine colonies had influenza illness confirmed by RT-PCR, for a protective effectiveness of 61% (95% confidence interval [CI], 8%-83%; P = .03). Among all study participants (those who were and those who were not vaccinated), 80 of 1773 (4.5%) in the influenza vaccine colonies and 159 of 1500 (10.6%) in the hepatitis A vaccine colonies had influenza illness confirmed by RT-PCR for an overall protective effectiveness of 59% (95% CI, 5%-82%; P = .04). No serious vaccine adverse events were observed. CONCLUSION Immunizing children and adolescents with inactivated influenza vaccine significantly protected unimmunized residents of rural communities against influenza. TRIAL REGISTRATION clinicaltrials.gov Identifier: NCT00877396.

Viral Infection in Adults Hospitalized With Community-Acquired Pneumonia: Prevalence, Pathogens, and Presentation

Background The potential role of respiratory viruses in the natural history of community-acquired pneumonia (CAP) in adults has not been well described since the advent of nucleic amplification tests (NATs). Methods From 2004 to 2006, adults with CAP who were admitted to five hospitals were prospectively enrolled in the study, and clinical data, cultures, serology, and nasopharyngeal swabs were obtained. NATs from swabs were tested for influenza, human metapneumovirus (hMPV), respiratory syncytial virus (RSV), rhinovirus, parainfluenza virus 1–4, coronaviruses (OC43, 229E, and NL63), and adenovirus. Results A total of 193 patients were included; the median age was 71 years, 51% of patients were male, and 47% of patients had severe CAP. Overall, 75 patients (39%) had a pathogen identified. Of these pathogens, 29 were viruses (15%), 38 were bacteria (20%), 8 were mixed (4%), and the rest were “unknown.” Influenza (n = 7), hMPV (n = 7), and RSV (n = 5) accounted for most viral infections; other infections included rhinovirus (n = 4), parainfluenza (n = 3), coronavirus (n = 4), and adenovirus (n = 2). Streptococcus pneumoniae was the most common bacterial infection (37%). Compared with bacterial infection, patients with viral infection were older (76 vs 64 years, respectively; p = 0.01), were more likely to have cardiac disease (66% vs 32%, respectively; p = 0.006), and were more frail (eg, 48% with limited ambulation vs 21% of bacterial infections; p = 0.02). There were few clinically meaningful differences in presentation and no differences in outcomes according to the presence or absence of viral infection. Conclusions Viral infections are common in adults with pneumonia. Easily transmissible viruses such as influenza, hMPV, and RSV were the most common, raising concerns about infection control. Routine testing for respiratory viruses may be warranted for adults who have been hospitalized with pneumonia.

Interlaboratory Comparison of Cytomegalovirus Viral Load Assays

To assess interlaboratory variability in qualitative and quantitative cytomegalovirus (CMV) viral load (VL) testing, we distributed a panel of samples to 33 laboratories in the USA, Canada and Europe who performed testing using commercial reagents (n = 17) or laboratory‐developed assays (n = 18). The panel included two negatives, seven samples constructed from purified CMV nucleocapsids in plasma (2.0–6.0 log10 copies/mL) and three clinical plasma samples. Interlaboratory variation was observed in both actual (range, 2.0–4.0 log10 copies/mL) and self‐reported lower limits of detection (range, 1.0–4.0 log10 copies/mL). Variation observed in reported results for individual samples ranged from 2.0 log10 (minimum) to 4.3 log10 (maximum). Variation was greatest at low VLs. Assuming ± 0.5 log10 relative to the expected result represents an acceptable result, 57.6% of results fell within this range. Use of commercially available reagents and procedures was associated with less variability compared with laboratory‐developed assays. Interlaboratory variability on replicate samples was significantly greater than intralaboratory variability (p < 0.0001). The significant interlaboratory variability in CMV VL observed may be impacting patient care and limiting interinstitutional comparisons. The creation of an international reference standard for CMV VL assay calibration would be an important step in quality improvement of this laboratory tool.

Comparison of the Luminex xTAG Respiratory Viral Panel with In-House Nucleic Acid Amplification Tests for Diagnosis of Respiratory Virus Infections

ABSTRACT Detection of respiratory viruses using sensitive real-time nucleic acid amplification tests (NATs) is invaluable for patient and outbreak management. However, the wide range of potential respiratory virus pathogens makes testing using individual real-time NATs expensive and laborious. The objective of this study was to compare the detection of respiratory virus targets using the Luminex xTAG respiratory viral panel (RVP) assay with individual real-time NATs used at the Provincial Laboratory of Public Health, Calgary, Alberta, Canada. The study included 1,530 specimens submitted for diagnosis of respiratory infections from December 2006 to May 2007. Direct-fluorescent-antigen-positive nasopharyngeal samples were excluded from this study. A total of 690 and 643 positives were detected by RVP and in-house NATs, respectively. Kappa correlation between in-house NATs and RVP for all targets ranged from 0.721 to 1.000. The majority of specimens missed by in-house NATs (96.7%) were positive for picornaviruses. Samples missed by RVP were mainly positive for adenovirus (51.7%) or respiratory syncytial virus (27.5%) by in-house NATs and in general had low viral loads. RVP allows for multiplex detection of 20 (and differentiation between 19) respiratory virus targets with considerable time and cost savings compared with alternative NATs. Although this first version of the RVP assay has lower sensitivity than in-house NATs for detection of adenovirus, it has good sensitivity for other targets. The identification of picornaviruses and coronaviruses and concurrent typing of influenza A virus by RVP, which are not currently included in our diagnostic testing algorithm, will improve our diagnosis of respiratory tract infections.

Interlaboratory comparison of epstein-barr virus viral load assays.

To assess interlaboratory variability in qualitative and quantitative Epstein‐Barr virus (EBV) viral load (VL) testing, we distributed a panel of samples to 28 laboratories in the USA, Canada and Europe who performed testing using commercially available reagents (n = 12) or laboratory‐developed assays (n = 18). The panel included two negatives, seven constructed samples using Namalwa and Molt‐3 cell lines diluted in plasma (1.30–5.30 log10 copies/mL) and three clinical plasma samples. Significant interlaboratory variation was observed for both actual (range 1.30–4.30 log10 copies/mL) and self‐reported (range, 1.70–3.30 log10 copies/mL) lower limits of detection. The variation observed in reported results on individual samples ranged from 2.28 log10 (minimum) to 4.14 log10 (maximum). Variation was independent of dynamic range and use of commercial versus laboratory‐developed assays. Overall, only 47.0% of all results fell within acceptable standards of variation: defined as the expected result ± 0.50 log10. Interlaboratory variability on replicate samples was significantly greater than intralaboratory variability (p < 0.0001). Kinetics of change in VL appears more relevant than absolute values and clinicians should understand the uncertainty associated with absolute VL values at their institutions. The creation of an international reference standard for EBV VL assay calibration would be an initial important step in quality improvement of this laboratory tool.

Development and Evaluation of a Real-Time Nucleic Acid Sequence Based Amplification Assay for Rapid Detection of Influenza A

The development and introduction of effective treatment for influenza A in the form of neuraminidase inhibitors have made the rapid diagnosis of infection important especially in high‐risk populations. The aim of this study was to develop a real‐time nucleic acid sequenced based amplification (NASBA) using a molecular beacon that could detect a wide range of influenza A subtypes and strains in a single reaction by targeting a conserved region of the influenza genome, and to evaluate its sensitivity and specificity against traditional laboratory techniques on a range of clinical samples usefulness during the 2003/2004 influenza season. The results demonstrated the assay to be highly sensitive and specific, detecting <0.1 TCID50 of virus stock. Three hundred eighty‐nine clinical samples were tested in total from two patient groups. Overall, the real‐time NASBA assay detected 64% (66/103) more influenza positive samples than cell culture and direct immunofluorescence (IF) and, therefore, was shown to be more sensitive in detecting influenza A in a wide range of respiratory samples than traditional methods. In conclusion, the real‐time influenza A assay demonstrated clinical usefulness in both hospital and community populations. J. Med. Virol. 74:619–628, 2004.

Design and Validation of Real-Time Reverse Transcription-PCR Assays for Detection of Pandemic (H1N1) 2009 Virus

ABSTRACT Tracking novel influenza viruses which have the potential to cause pandemics, such as the pandemic (H1N1) 2009 virus, is a public health priority. Pandemic (H1N1) 2009 virus was first identified in Mexico in April 2009 and spread worldwide over a short period of time. Well-validated diagnostic tools that are rapid, sensitive, and specific for the detection and tracking of this virus are needed. Three real-time reverse transcription PCR (RT-PCR) assays for the amplification and detection of pandemic (H1N1) 2009 virus were developed, and their performance characteristics were compared with those of other published diagnostic assays. Thirty-nine samples confirmed to be positive for pandemic (H1N1) 2009 virus from Alberta, Canada, and six additional samples that were positive for influenza A virus but that were not typeable by using published seasonal influenza H1/H3 virus assays were available for this validation. Amplification and direct sequencing of the products was considered the “gold standard” for case identification. The new assays were sensitive and able to reproducibly detect virus in a 10−6 dilution of 4 × 106 50% tissue culture infective doses/ml when 5 μl was used as the template. They showed 100% specificity and did not cross-react with other respiratory viruses or seasonal influenza A virus subtypes. The coefficient of variation in crossing cycle threshold values for the detection of different template concentrations of pandemic (H1N1) 2009 virus was ≤3.13%, showing good reproducibility. The assays had a wide dynamic range for the detection of pandemic (H1N1) 2009 virus and utilized testing platforms appropriate for high diagnostic throughput with rapid turnaround times. We developed and validated these real-time PCR procedures with the goal that they will be useful for diagnosis and surveillance of pandemic (H1N1) 2009 virus. These findings will contribute to the informed management of this novel virus.

Detection of a broad range of human adenoviruses in respiratory tract samples using a sensitive multiplex real‐time PCR assay

Human adenoviruses (hAdVs) are associated with acute respiratory tract infections in pediatric populations and have been identified as a cause of outbreaks in institutional settings. Rapid diagnosis of hAdV infection is critical for appropriate and timely management. This study reports the design and validation of a sensitive and specific multiplex real‐time PCR for the detection of a broad range of hAdV serotypes in respiratory samples. The assay targets the conserved region of the hAdV hexon gene and utilizes hydrolysis probes for the detection of amplified products. The assay was evaluated using retrospectively (n = 864) and prospectively (n = 11,451) collected samples from November 2005 to July 2006. Seasonality studies and analysis of outbreaks was conducted over a 2‐year period from January 2005 to December 2006 (n = 33,067 samples). The assay gave a hAdV positive rate of 7.1% (n = 811) for specimens tested prospectively and was able to detect a broad range of hAdV serotypes with good sensitivity and specificity. A high rate of co‐infection was noted (21.7%). Adenovirus infections were more prevalent in the young with a median age of 24 months for positive patients. Sequence analysis of hAdV positives showed that serotype 7 was the most prevalent followed by serotypes 2 and 3. Association of hAdVs with respiratory outbreaks was low at 2.3% (6 of 266 outbreaks tested) and no seasonal variation was observed for hAdV infections during the 2‐year study period. This assay can improve the detection of hAdVs in respiratory samples and can be used to provide valuable epidemiological information. J. Med. Virol. 80:856–865, 2008.

A Commutable Cytomegalovirus Calibrator Is Required to Improve the Agreement of Viral Load Values between Laboratories

BACKGROUND Viral load testing for cytomegalovirus (CMV) is an important diagnostic tool for the management of transplant recipients and immunocompromised individuals; however, inconsistency among laboratories in quantitative measurements of viral load limits interinstitutional comparisons. These inconsistencies stem from the lack of assays cleared by the US Food and Drug Administration, the absence of international standards, the wide variety of CMV-extraction and -detection methods, and differences in materials used for calibration. A critical component of standardization is the use of calibrators that are traceable and commutable. METHODS Bland-Altman plots and prediction ellipses were used to test the commutability of 2 CMV calibrators for 2 different quantification methods. RESULTS Tests with 2 methods showed 1 calibrator to be commutable and the other to be noncommutable. The results for the commutable calibrator were within the 95% prediction interval of the clinical samples in the Bland-Altman plot and within the 95% prediction ellipse for a simulated commutable calibrator, whereas the results for the noncommutable calibrator were not within these prediction intervals. When used to calibrate patient results, only the commutable calibrator, the OptiQuant CMV(tc) Calibration Panel, significantly improved the comparability of viral loads for the 2 different measurement methods. CONCLUSIONS This study demonstrates that an important goal in the effort to improve healthcare for patients with CMV-related disease is the establishment of traceable and commutable reference materials, including both calibrators and controls. .

Nucleic acid amplification tests for detection of respiratory viruses

Abstract Nucleic acid amplification tests (NATs) are increasingly being used for diagnosis of respiratory virus infections. The most familiar formats use DNA or RNA target amplification methods for enhanced sensitivity above culture and antigen-based procedures. Although gel and plate-hybridisation methods are still utilised for analysis of amplified products, detection using “real-time” methods which do not require handling of amplified products are favoured in many laboratories. Assays based on nucleic acid amplification and detection can be designed against a broad range of respiratory viruses and have been particularly useful for detection of recently identified viruses such as human metapneumovirus and coronaviruses NL63 and HKU1. However, the wide range of potential pathogens which can cause similar respiratory symptomology and disease makes application of individual diagnostic assays based on detection of DNA and RNA both complex and expensive. One way to resolve this potential problem is to undertake multiplexed nucleic acid amplification reactions with analysis of amplified products by suspension microarray. The Respiratory Virus Panel (RVP) from Luminex Molecular Diagnostics is one example of such an approach which could be made available to diagnostic and public health laboratories for broad spectrum respiratory virus detection.