A total of 177 respiratory viruses were isolated in tube culture from 613 specimens tested from 22 March through 18 July 2009 (Table ). Influenza A viruses accounted for 145 of these isolates. Other viruses included parainfluenza virus (22 isolates), adenovirus (7 isolates), respiratory syncytial virus (2 isolates), and influenza B virus (1 isolate). The peak influenza A virus activity occurred during the weeks ending on 30 May 2009 through 20 June 2009.
Respiratory viruses isolated in tube culture during weeks ending 28 March through 18 July 2009a
Both the number of respiratory specimens tested and the number of viruses isolated far exceeded what is normally seen during that time of year. By comparison, a total of 156 specimens tested during the same time period in 2007 and 146 specimens tested during the same time period in 2008 only yielded five and seven viral isolates, respectively.
A variety of symptoms were identified in the 145 patients harboring influenza A virus. The most prevalent symptom was fever (temperature, 102.7 ± 0.32°F), which occurred over a mean duration of 2.9 ± 0.61 days prior to examination. Other reported symptoms included cough (n = 134 patients), URI (i.e., rhinorrhea, congestion, and sore throat; n = 127), nausea/vomiting (n = 59), myalgia (n = 55), headache (n = 52), bronchitis (n = 45), diarrhea (n = 39), and pleurisy (n = 23). A majority of patients (n = 98) reported either exposure to a person known to be influenza A virus positive or recent travel outside the United States.
The age distribution of the patients harboring respiratory viruses is found in Table . A majority of influenza A virus infections (38/145) occurred in those 13 to 21 years of age, whereas only 2 patients were older than 64 years of age. Influenza A virus infection was significantly greater in the 13- to 21-year-old age group than in those in the age groups under 1 year, 42 to 51 years, 52 to 64 years, and 65 years and older (P < 0.05). Over the previous two influenza seasons, a majority of infected patients were found to be over 64 years of age.
Age distribution of patients culture positive for respiratory viruses between 22 March and 18 July 2009a
Parainfluenza virus infection was observed across all age groups. In the previous five respiratory virus seasons, parainfluenza virus was exclusively isolated from children 5 years old or younger. Adenovirus and RSV were isolated from pediatric patients 3 years of age or younger.
By using traditional tube culture as the “gold standard,” we were able to evaluate other rapid diagnostic assays for influenza A virus utilized in the laboratory (Table ). Rapid shell vial culture (R-mix) was able to generically identify the presence of common respiratory viruses by approximately 1 day postinoculation. The sensitivity and specificity were 98.6% and 100%, respectively, for those patients infected with influenza A virus. The R-mix shell vials demonstrated 100% sensitivity and 100% specificity for the other respiratory viruses.
Evaluation of a chromatographic immunoassay, R-mix shell vial culture, and tube culture methods for identification and isolation of influenza A virus in respiratory specimens
Reporting of the rapid chromatographic immunoassay result for influenza A and B viruses occurred within 1 h of specimen collection. The immunoassay failed to detect influenza A virus in 43 of 145 patients, yielding a sensitivity of 70.3%. The specificity was found to be 100%.
For 50 patients, the DFA assay was added to the testing regimen. A total of 15 patients were positive for influenza A virus by the tube culture assay (Table ). Compared to the results of the tube culture assay, sensitivities of 66.7, 100, and 80% were obtained by the chromatographic immunoassay, shell vial culture, and DFA assay methods, respectively. The specificities of all assays were 100%.
Evaluation of assay performance and nasopharyngeal swab specimen adequacy with 50 respiratory specimens examined for influenza A virus
Estimates of cell numbers on the DFA assay slides yielded 39 cases in which more than 100 cells were observed and only 3 cases in which less than 10 cells were found. Ample cell numbers (>100 cells) were available in those specimens for which the chromatographic immunoassay or the DFA assay yielded false-negative results. Differentiation between squamous and columnar epithelial cells was not done in the present study.
Of 45 specimens forwarded to the Wadsworth Center laboratory for testing by RT-PCR, 21 were found to be positive for the novel influenza A (H1N1) virus and 2 were found to be positive for seasonal influenza A (H3N2) virus. The specimens containing seasonal influenza A (H3N2) virus were collected in early May of 2009, whereas specimens positive for the novel influenza A (H1N1) virus strain were collected from mid-May into July 2009. The culture results were identical to the findings of RT-PCR for these 45 specimens. In addition, in three patient specimens, tube culture isolated 3 adenoviruses that were not identified by RT-PCR. The chromatographic immunoassay yielded seven false-negative results (70% sensitivity) compared with the results of RT-PCR.
The mean time intervals from specimen collection to reporting of the results were found to be 5.12 ± 42 days for culture and 5.69 ± 0.37 for RT-PCR, demonstrating statistically nonsignificant differences (P = 0.33). Further analysis of the RT-PCR procedures demonstrated a mean time interval of 1.62 ± 0.21 days from specimen receipt at the Wadsworth Center laboratory to reporting of the results. In contrast, it took approximately 4.1 days to evaluate the need for molecular testing and for specimen processing and transport to the laboratory to occur.
A number of potential shortages resulted from the increased testing demands placed on the laboratory. We were able to substitute culture maintenance medium, which regularly undergoes monthly quality control testing, for viral transport medium. Aliquots of maintenance medium were placed in sterile conical centrifuge tubes, packaged along with sterile swabs, and labeled with the maintenance medium lot number and an expiration date set before the next scheduled quality control testing. The shortage of transporting supplies was handled by obtaining suitable boxes and preparing the necessary shipping labels.
Increased personnel needs arose. Nighttime staffing of the microbiology laboratory was utilized during “off hours” in the Virology Laboratory to accommodate rapid immunoassay testing on a 24-h-a-day, 7-day-a-week schedule. In addition, approximately 12% increased technical time (12 to 15 h of overtime per week) was needed to process and package the specimens, transport the materials for outside testing, etc.