Detection of Respiratory Viruses by PCR Assay of Nasopharyngeal Swabs Stored in Skim Milk-Tryptone-Glucose-Glycerol Transport Medium

Abstract

We analyzed 129 paired nasopharyngeal aspirates (stored in viral transport medium [VTM]) and nasopharyngeal swabs (stored in skim milk-tryptone-glucose-glycerol [STGG] bacterial transport and storage medium) using PCRs to detect adenoviruses, influenza virus A or B, and respiratory syncytial virus (RSV). Overall, swabs stored in STGG medium without antimicrobials were found to be an acceptable alternative to aspirates stored in antimicrobial-containing VTM, with PCR agreement of 90.2% (kappa of 0.8).Longitudinal population-based studies of bacterium-virus interactions in the nasopharynx will result in improved understanding of the pathogenesis of respiratory tract infections, a major cause of morbidity and mortality globally (1, 10). Nasopharyngeal aspirates (NPA) or nasopharyngeal swabs (NPS) collected into a viral transport medium (VTM), usually containing antimicrobial agents to prevent bacterial overgrowth, have been considered the optimal specimen for respiratory virus detection (3). However, antimicrobial-containing VTM precludes culture-based detection and characterization of bacterial upper respiratory tract colonizers, necessitating the collection of duplicate samples if both viruses and bacteria are to be studied.We sought to determine whether NPS collected and stored in skim milk-tryptone-glucose-glycerol (STGG) bacterial transport and storage medium could be used to determine the presence of respiratory tract viruses in the upper respiratory tract, thus permitting assessment of bacterium-virus interaction in vivo from a single specimen.We analyzed 129 paired NPA and NPS specimens taken from infants diagnosed with pneumonia during a longitudinal cohort study on the Thailand-Myanmar border (8). NPA were collected in 1 ml of VTM (minimum essential medium with Hanks balanced salt solution [MEM-Hanks] with 0.5% gelatin, amphotericin B, penicillin, and streptomycin; prepared inhouse) and stored at Ϫ80°C until extraction and PCR. Nucleic acid was extracted from these NPA-VTM specimens using spin columns (NucleoSpin RNA virus kit [Macherey-Nagel, Germany] or QIAamp viral RNA minikit [Qiagen, Germany]) following the manufacturer's instructions. Extracts were analyzed by real-time PCR for adenoviruses, influenza viruses, and respiratory syncytial virus (RSV), with a human RNase P PCR to detect the presence of inhibitors as previously described (2, 9). A Rotorgene 6000 real-time PCR thermocycler (Corbett Life Science, Australia) and SuperScript III one-step reverse transcription-PCR (RT-PCR) kit (Invitrogen) were used throughout. The specimens were considered positive if a virus PCR threshold cycle (C T ) value was Ͻ40 with appropriate run control results. Specimens with low positive PCR results (C T values of 35 to 39) were repeated: only if the C T was Ͻ40 in both runs was the virus PCR considered positive. Dacrontipped NPS (Medical Wire & Equipment, United Kingdom) were collected into 1 ml of STGG transport medium (no antimicrobials; prepared in-house) and processed according to the WHO protocol for Streptoc...

Concordance between RT-PCR-based detection of respiratory viruses from nasal swabs collected for viral testing and nasopharyngeal swabs collected for bacterial testing

“…11 Our study, which used samples from young Andean children with acute respiratory illness, complements the prior report and expands it to a larger number of samples including MPV, HRV and PIV3; NS samples, which are commonly obtained; and importantly, by comparing CT values between detection strategies. Notably, even though our NP samples had undergone a previous freeze-thaw cycle, the detection of viruses was remarkably similar between the 2 sampling strategies.…”

Respiratory Viral Detections During Symptomatic and Asymptomatic Periods in Young Andean Children

“…(22, 26-29) Despite different collection methods, prior studies by our group and others have demonstrated a very high agreement (89%-99%) in the detection of common respiratory viruses between NS and NP samples collected in this manner. (16, 30) For samples with virus detections, RT-PCR cycle thresholds (CT) from asymptomatic and ARI periods were considered as surrogates for viral loads, with higher CT indicating lower viral load. CT values of ≤40 were used to define sample positivity.…”

Respiratory viral pathogens associated with lower respiratory tract disease among young children in the highlands of Papua New Guinea

“…18 During the course of the vaccine trial, 1888 pernasal swabs were collected from 319 young children. Swabs were collected into 1 mL of skim milk-tryptoneglucose-glycerin-broth (STGGB) 19 and stored at −80 • C. A limited number of those samples collected between January 2006 and August 2008 were shipped to Perth, Western Australia for viral studies. Those samples included 80 from young children with ALRI and 273 from children with no evidence of ALRI.…”