Surveillance of Childhood Influenza Virus Infection: What Is the Best Diagnostic Method To Use for Archival Samples?

Abstract

Despite the clinical importance of influenza virus in pediatric respiratory infections, the optimal set of diagnostic tests to use when conducting studies using archival samples is not clear. In this study, we compared diagnostic tests for influenza virus in 75 children younger than 5 years of age who presented with symptomatic respiratory infection during one of four influenza seasons, had negative viral cultures for other respiratory pathogens, and had both an archival nasal aspirate obtained at the time of illness and serology spanning that influenza season. For all eligible children, we compared the results of viral culture performed at the time of collection with serology and PCR of archival nasal aspirates. Using real-time viral culture as the "gold standard," the test characteristics of PCR of archival nasal aspirates (sensitivity, 82%; specificity, 100%) and serology (sensitivity, 82%; specificity, 87%) were similar. The relatively low sensitivity of PCR of archival nasal samples in this study compared to that of PCR of fresh samples in a previous study suggests that RNA degradation occurred despite storage of the specimens at ؊70°C. RNA degradation would also explain why only 11 (52%) of 21 archival nasal samples that had positive influenza virus cultures at the time of collection had positive repeat cultures in the summer of 2000. Thus, in archival specimens stored at ؊70°C, PCR was more sensitive than viral culture. However, testing of fresh specimens had the highest yield in this study. Studies of optimal methods for specimen storage are needed.Influenza virus is an important cause of febrile respiratory infections in children. Large population-based studies using administrative databases indicate that rates of hospitalization, antibiotic use, and outpatient visits for children consistently increase when influenza virus is circulating (8,10). Prospective studies of children in inpatient and outpatient settings using viral culture alone or in combination with serology indicate that a high proportion of respiratory illness during the winter is caused by influenza virus (1,3,5,6,11,21). However, estimates of the medical care burden of influenza virus in children have been uncertain, due in part to the lack of large population-based studies that have used state-of-the-art viral diagnostic tests. In fact, the optimal set of diagnostic tests to detect influenza virus infections is debatable, particularly when retrospective epidemiologic studies are conducted using archival samples.The Centers for Disease Control and Prevention (CDC) recently established a new population-based surveillance network, the New Vaccine Surveillance Network (NVSN), to determine the burden of viral respiratory illnesses in hospitalized children younger than 5 years of age. In a previous NVSN study, the results of a rapid diagnostic test for influenza virus were compared to those of viral culture and/or PCR (13). In that study a fully validated and Clinical Laboratory Improvement Amendments-approved PCR of freshly collected samples...

Relevance of Influenza A Virus Detection by PCR, Shell Vial Assay, and Tube Cell Culture to Rapid Reporting Procedures

“…In a previous evaluation of 557 respiratory tract specimens in our laboratory, we detected influenza A virus in 92 specimens (16.5%) by PCR, 49 specimens (8.8%) by R-Mix, and 24 specimens (4.3%) by the Binax NOW Flu A rapid enzyme immunoassay test (3). Thus, PCR yielded 88% and 283% increases in sensitivity compared to R-Mix shell vial cell culture and Binax NOW antigen detection, respectively, in agreement with other publications (1,4,6,8,10,11). The negative predictive value (available within hours) of the influenza A PCR assay (99.9%) also made this assay superior to culture techniques.…”

Evaluation of a Multiplexed PCR Assay for Detection of Respiratory Viral Pathogens in a Public Health Laboratory Setting

“…This is because molecular testing requires only the agent's genetic fingerprint, and assay development cycles are short (1 to 4 weeks). Recent data even suggest that when properly developed, molecular testing for viral agents can be more sensitive and more specific than viral culture and/or immunoassays (16,19,41). The molecular tests typically target the anticipated conserved regions of one or a few viral genomes (1,47).…”

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

“…PCR is expected to have an increased sensitivity of detection relative to culture because PCR detects short segments of viral RNA from viable as well as nonviable viruses; however, the reported rates of increased PCR detection over culture differ widely in different studies. Differences in the recovery rates of viable influenza viruses are frequently attributed to less than optimal specimen collection, storage, and transport of samples, RNA degradation during laboratory storage at 4°C and Ϫ20°C, and multiple freeze-thaws of specimens before testing (22). Notably, FluA detection by TM assays was nearly double that detected by culture when the samples were collected and sent through the mail at room temperature or at 4°C and were in transit between 1 and 4 days (41,47).…”

“…In the second year, two different PCR methods, designated primary and secondary PCRs, were used for the detection of each virus in the ethanol-fixed specimens. Because PCR-based methods have been shown to be more sensitive than culture for the detection of many viruses, a specimen with positive results for two different PCR targets was considered a "molecular true positive"-a second gold standard-for the comparison of assay performances (22,37,38,41,47,48). Results were calculated based on defining a true-positive specimen by culture or two molecular tests.…”