Vol. 20 (2) Nov. 2011 Respiratory Virus Test Protocol 2011-12: DFA vs. PCR

Direct immunofluorescence (DFA): Since 2000, direct immunofluorescence (DFA) has been the mainstay of respiratory virus diagnosis at YNHH. DFA is done continuously during operating hours and takes about 2 hours to complete. It performs well when compared to culture, except for adenovirus. For tests and viruses detected, see Appendix on page 2. Respiratory Virus PCR Panel: In the 2010-2011 season, a PCR panel for respiratory viruses was introduced for hospitalized patients. Clinicians can order DFA and/or PCR. Culture is reserved for lower respiratory tract samples. The table below shows the sensitivity of DFA compared to PCR last season when both tests were performed on the same sample.

Sensitivity of DFA compared to PCR on a Subset of Inpatients in 2010-11 Season Viruses tested by DFA and PCR

Total positive

PCR positive (%)

DFA positive (%)a Adult Pediatric

RSV A, B 101 99/101 (99%)b 39/61 (64%) 35/40 (88%) Influenza A 154 154/154 (100%) 70/134 (52%) 15/20 (75%) Influenza B 32 32/32 (100%) 9/17 (53%) 12/15 (80%) Parainfluenza 1,2,3 33 31/33 (94%)b 14/23 (61%) 7/10 (70%) Adenovirus 27 27/27 (100%) 1 /4 (25%) 5/23 (22%) hMPV 22 22/22 (100%) 8/15 (53%) 5/7 (71%) Total Positive 369 365/369 (99%) 141/254 (56%) 79/115 (69%)

a, Both DFA-negative and DFA-inadequate samples are included b, PCR negative/ DFA positive c, Note 68.8% of samples were on adults, who shed lower titers of virus than children

Respiratory Virus DFA vs PCR: The results from last season show that the sensitivity of DFA was significantly less than PCR in samples tested by both methods, averaging 56% in adult and 69% in pediatric inpatients. Thus for infection control and patient management in hospitalized patients, PCR is the preferred test to due its greater sensitivity. For our 2011-2012 respiratory season test protocol, we will add a second PCR run for RSV and influenza late in the day on weekdays and limit DFA testing in the evenings.

Respiratory Virus Test Protocol for 2011-12 Winter Respiratory Virus Season PCR Panels DFA Weekdays During peak season, RSV, Influenza A and B

PCRs will be performed twice a day HMPV, adenovirus and rhinovirus PCRs will be continue to be done once a day

During peak season, DFA will be performed from 7 AM to 8 PM

After 8 PM, DFA will be done only for intensive care units or admissions via the ED; samples from other locations will be held until the morning

Weekends PCR panels will be run once a day, at midday DFA will be performed until 11 PM with results reported by 12:30- 1:00 AM.

Subytping of influenza A: Due to greatly increased test volumes and lack of clinical impact, routine subtyping of Influenza A positives will not be performed in 2011-12 season. For special requests, including fatal cases, please contact the laboratory.

References Landry ML, Ferguson D. SimulFluor Respiratory Screen for rapid detection of multiple respiratory viruses in clinical specimens by

immunofluorescence staining. J Clin Microbiol 38:708-711, 2000. Landry ML, Cohen S, Ferguson D. Prospective study of human metapneumovirus detection in clinical samples using Light DiagnosticsTM

direct immunofluorescence reagent and real-time PCR. J Clin Microbiol 46:1098-1100, 2008. Landry ML, Cohen S, Ferguson D. Real-time PCR compared to BINAX NOW and cytospin-immunofluorescence for detection of influenza in

hospitalized patients. J Clin Virol 43:148-151, 2008. Landry ML and Ferguson D. Cytospin-enhanced immunofluorescence and impact of sample quality on diagnosis of novel swine-origin (H1N1)

influenza virus. J Clin Microbiol 48:957-959, 2010. Appendix of Respiratory Virus Tests Offered at YNHH Virology Laboratory:

Routine Respiratory Virus Tests Offered at YNHH Test Viruses detected Time to result Respiratory virus screen DFA RSV, Flu A, Flu B, Paraflu 1,2,3, Adenovirus 2 hrs, when lab open

Human metapneumovirus DFA hMPV 2 hrs, when lab open

Respiratory Virus PCR Panel All viruses above plus Rhinovirus 6-30 hrs

Viral Culture*

(reserved for lower resp. tract samples)

RSV, Flu A, Flu B, Paraflu 1,2,3, adenovirus, rhinovirus,

enterovirus, parechovirus, CMV, HSV, VZV

1-21 days

*In most cases PCR is more sensitive than culture, but culture includes some viruses not in the PCR panel. NOTE: The PCR tests used at YNHH Clinical Virology Laboratory are not commercial kits, but are set up and validated in the Clinical Virology Laboratory. Initially, no FDA approved commercial kits were available. In the past few years, some PCR kits have been FDA approved but do not yet perform as well as our laboratory developed tests. In a recent evaluation in our lab, the best commercial influenza PCR test missed 13% of influenza A and 20% of influenza B positives detected by the CDC assay. The sources of our PCR tests are listed below. Laboratory Developed PCR Tests used at YNHH

Respiratory virus Origin of YNHH Virology Laboratory PCR Protocol Influenza A Centers for Disease Control protocol; WHO website

Influenza B Centers for Disease Control protocol; WHO website

hMPV Centers for Disease Control protocol; Landry et al J Clin Microbiol 46:1098-1100, 2003.

Rhinovirus Centers for Disease Control protocol; Lu X, et al, J Clin Microbiol 46:533-539, 2008.

Adenovirus Heim et al, J Med Virol 70:228-239, 2003 (also used at CDC)

RSV A, B Van Elden LJR et al, J Clin Microbiol, 41:4378-81, 2003; Erratum 43:4308, 2005

Parainfluenza 1,2,3 R. Hodinka, Children’s Hospital of Philadelphia, personal communication

For Questions: Call Marie Landry, M.D., 688-3475, or David Ferguson, Laboratory Manager, 688-3524.