RECOVERY OF RESPIRATORY SYNCYTIAL VIRUS FROM STETHOSCOPES BY CONVENTIONAL VIRAL CULTURE AND POLYMERASE CHAIN REACTION (1999)

Respiratory syncytial virus (RSV) is a common respiratory pathogen causing upper and lower respiratory tract infections, especially in children. It is responsible for 25 to 50% of pediatric hospital admissions for pneumonia and bronchiolitis. RSV is also recognized as a major cause of nosocomial respiratory infections and is associated with significant morbidity in high risk patients, such as those with underlying cardiopulmonary disease (bronchopulmonary dysplasia or congenital heart disease), prematurity and immune compromise.1 RSV is transmitted by direct inoculation of the ocular and nasal mucosa with infected secretions, or by contact with objects contaminated by droplets containing RSV. It is known that RSV can be recovered from inanimate hard surfaces such as countertops, for up to 6 h, and that it remains infectious if transferred onto the hands of those who touch these surfaces.2, 3 Several infection control strategies have been designed to deal with the problem of nosocomial viral spread, each with varying success.4-8 None has specifically evaluated the role of stethoscopes. Because stethoscopes are frequently in direct contact with patients, especially those with respiratory infections, it is reasonable to expect that they could become contaminated and then serve as a vehicle for infection of the caregiver and other patients. Casual observation suggests that stethoscopes are infrequently cleaned between patients. We wished to evaluate the potential role of stethoscopes in RSV transmission. To assess this we examined the ability of RSV to survive in dried secretions on stethoscopes after deliberate contamination. We also wished to determine whether a simple wipe with an alcohol swab was effective in eliminating infectious virus from the stethoscope. Methods. Fresh secretions were obtained by routine nasopharyngeal aspiration from patients at the Montreal Children's Hospital who were known to be infected with RSV. Samples consisting of 200 μl of the secretions from an infected patient were used to inoculate the diaphragms of nine stethoscopes and were allowed to dry. The stethoscopes were left at room temperature on the bench top during this time. For the purposes of sampling, the diaphragm of each stethoscope was divided in half with a wax pencil line to provide two samples per stethoscope. One stethoscope served as a control. The diaphragms of the stethoscopes were sampled initially, and then every hour for 8 h by the technique of Hall et al.2 Basically 200 μl of minimal essential medium were applied on the area to be cultured and then drawn back into a pipette. The sample was then diluted in another 200 μl of viral transport medium. The sample was then divided, and 200 μl were inoculated into HEp-2 cells for viral culture. Cultures were examined three times per week for cytopathic effect (CPE) and were kept for a minimum of 16 days, with blind passage at 9 days. CPE suggestive of RSV was confirmed by immunofluorescent staining with an RSV monoclonal antibody. All cultures that did not show CPE were screened by RSV monoclonal immunofluorescent staining before discard. All cultures were examined by a qualified and experienced virology technician. The remaining 200 μl of sample were examined by PCR. Sample (100 μl) was digested with proteinase K (2 mg/ml) at 37°C for 10 min. The digest was extracted with acid phenolchloroform, and nucleic acids were recovered by ethanol precipitation. Samples were then resuspended in 10 μl of diethyl pyrocarbonate-treated water. Reverse transcription-PCR was carried out in 2 steps using superscript II RNase H− reverse transcriptase and Taq DNA polymerase (Life Technologies, Gaithersburg, MD). Primer sequences were obtained from Paton et al.9 Oligonucleotides were synthesized using a Pharmacia Gene Assembler Plus. The forward priming sequence was 5′-TTAACCAGCAAAGTGTTAGA-3′, and the reverse primer sequence was 5′-TTTGTTATAGGCATATCATTG-3′. These primers defined a 243-base pair segment of the RSV genome encoding the F1 subunit of the fusion protein conserved between subgroup A and subgroup B strains. PCR was done with the use of 35 cycles consisting of 1 min of denaturation at 94°C, followed by 2 min of annealing at 37°C, followed by 3 min of elongation at 72°C. The amplified product was detected by electrophoresing 16-μl aliquots through 2% agarose-Tris-buffered EDTA gels in the presence of ethidium bromide. To evaluate the sensitivity of the PCR protocol, a titered supernatant of stock RSV was serially diluted in 10-fold dilutions and tested until 10−8. In the second experiment three stethoscopes were each inoculated with RSV-containing secretions as above and allowed to dry. Each stethoscope was sampled immediately after the specimen had dried. The three stethoscopes were then wiped with a single commercial prepackaged alcohol swab. After the alcohol had dried, each stethoscope was again sampled for viral culture and PCR as described previously. Results. Fresh secretions on the control stethoscope were confirmed to be positive for RSV by culture and PCR. Control cultures of Hep-2 cells were negative for RSV by culture and PCR. In two trials RSV could be cultured from the diaphragm of the stethoscopes for 6 h after the samples had dried. When PCR was used RSV was identified for up to 8 h after the samples had dried. No sampling was done beyond 8 h. Three stethoscopes were wiped with alcohol swabs after being contaminated with secretions that had been allowed to dry. RSV could not be recovered by viral culture from stethoscopes wiped with alcohol but was easily recovered from controls. RSV was detected by PCR on one of three stethoscopes that had been wiped with alcohol. PCR signal was present up to a dilution of 10−3 of a known RSV-positive isolate. Thus it was able to detect 3 TCID50 of the virus. Discussion. Respiratory syncytial virus is able to survive for as long as 6 h in dried secretions on the diaphragms of stethoscopes. Detection by PCR was more sensitive, but whether this represents viable infectious virus at 8 h is unknown. Wiping the diaphragm with alcohol was an effective way of disinfecting the contaminated surface. Although viral DNA was detected by PCR in one of the three cleaned stethoscopes, the negative tissue culture tests suggest there was not a large amount of infectious virus present. It has been shown previously by Hall et al.2 that RSV can survive for varying lengths of time on inanimate surfaces and that these secretions remain infectious. Similarly stethoscopes could represent an important fomite in the transmission of RSV in the hospital setting. In instances when stethoscopes are used repeatedly without being cleaned, the risk of transmission to patients and caregivers could be significant. Infection control strategies aimed at controlling nosocomial RSV infection should emphasize the need for proper cleaning of stethoscopes and other equipment that comes in contact with patients, especially when gross contamination occurs. The practice of caregivers using the same stethoscope on multiple patients should be reconsidered, especially in high risk areas such as intensive care units or during RSV epidemics. Stethoscopes used in patients with RSV disease should be considered contaminated until cleaned. Tom Blydt-Hansen, M.D. Kanta Subbarao, M.D. Patrick Quennec, R.T. Jane McDonald, M.D. Departments of Pediatrics (TBH, KS, JM) and Microbiology (PQ, JM); Montreal Children's Hospital; McGill University; Montreal, Quebec, Canada