Utilizing Moist or Dry Swabs for the Sampling of Nasal MRSA Carriers? An In Vivo and In Vitro Study

This study investigates the quantitative bacterial recovery of Methicillin-resistant Staphylococcus aureus (MRSA) in nasal screenings by utilizing dry or moistened swabs within an in vivo and an in vitro experimental setting. 135 nasal MRSA carriers were each swabbed in one nostril with a dry and in the other one with a moistened rayon swab. Quantitative bacterial recovery was measured by standard viable count techniques. Furthermore, an anatomically correct artificial nose model was inoculated with a numerically defined suspension of MRSA and swabbed with dry and moistened rayon, polyurethane-foam and nylon-flocked swabs to test these different settings and swab-materials under identical laboratory conditions. In vivo, quantities of MRSA per nostril in carriers varied between <101 and >107 colony forming units, with a median of 2.15x104 CFU. However, no statistically significant differences could be detected for the recovery of MRSA quantities when swabbing nasal carriers with moist or dry rayon swabs. In vitro testing confirmed the in vivo data for swabs with rayon, polyurethane and nylon-flocked tips, since pre-moistening of swabs did not significantly affect the quantities of retrieved bacteria. Therefore, pre-moistening of swabs prior to nasal MRSA sampling provides no advantage in terms of recovering greater bacterial quantities and therefore can be omitted. In addition, this situation can be mimicked in an in vitro model, thereby providing a useful basis for future in vitro testings of new swab types or target organisms for screening approaches.

Study design

For in vivo experiments, subjects were swabbed in one nostril with a dry and in the other one with a moistened rayon swab. Recovered MRSA quantities were assessed. In total, n = 135 participants (males, n = 78; females, n = 57) were recruited from the tertiary hospital of the University Medicine Rostock. Inclusion criteria were (i) a positive result in a routineously performed nasal MRSA screening diagnosed by the accredited diagnostic laboratory of University Medicine Rostock; (ii) no ongoing decolonization procedure; (iii) no antibiotic treatment. To avoid bias due to sample collection order, for instance by always targeting the left nostril with a dry and the right nostril with a moistened swab, the collection mode was altered after swabbing a series of ten patients.

For in vitro experiments, artificial, autoclave resistant, silicone nose models, based on an imprint of a human nose [29], were inoculated with a numerically defined amount of MRSA and subsequently swabbed. Four different swab types were tested. Nose models were swabbed in one nostril with a dry and in the other one with a moistened swab. For each swab type and swabbing scenario n = 10 swabs were tested. Recovered MRSA quantities were assessed.

Swabs

1. Rayon: Nerbe Plus, Winsen/Luhe, Germany, Invasive sterile collection swab, ref. 09-812-8051
2. Polyurethane cellular foam: MWE medical wire, Corsham Wiltshire England, Sigma Dry Swab Tubed, ∑-Swab, ref. MW941
3. Nylon flocked fiber: Copan, Brescia, Italy, FLOQSwabs, regular, ref 552C
4. Rayon: MWE medical wire, Corsham Wiltshire England, Tubed Sterile Dryswab, ref. MW102

Swabbing of patients was exclusively performed with Nerbe Plus (Invasive sterile collection swab), swabbing of the artificial nose model was executed with all four swab types.

Bacterial culture techniques for in vitro experiments

Staphylococcus aureus MRSA strain (ST22-MRSA-IV, Barnim epidemic strain) and Staphylococcus epidermidis strain (DSMZ 1798) were separately propagated at 37°C in brain-heart-infusion (BHI) medium as overnight standing cultures in ambient air. Early stationary phase cells were harvested, washed in phosphate-buffered saline (PBS; NaCl (137 mmol/l), KCl (2.7 mmol/l), Na2HPO4 x 2 H2O (10 mmol/l), KH2PO4 (2.0 mmol/l)) at pH 7.4 and resuspended in BHI + 20% glycerol. Aliquots were stored at -80°C for up to 3 months. Each stock concentration was determined by viable cell count of 3 representative tubes according to standard techniques.

Inoculation of the nose models

Nose models were prepared at the day of usage. Autoclaved, sterile nose models were inoculated with a suspension of MRSA and Staphylococcus epidermidis strains following a protocol as described elsewhere [20]. Briefly, amounts of 2x104 colony forming units (CFU) MRSA (adjusted to the median bacterial quantities recovered from patients) and 1x105 CFU Staphylococcus epidermidis (to simulate nasal flora) per nostril were applied at defined locations in each nasal vestibule, distributed in two 10 μl droplets of bacterial suspension. Inoculated nose models were dried for one hour at room temperature.

Inoculation dose was controlled by plating serial dilutions of the bacterial suspension onto Columbia agar supplemented with 5% sheep blood (BD, Heidelberg, Germany) followed by cultivation at 37°C under ambient atmosphere for 48 h and CFU counting.

Sample collection techniques

Subjects and nose models were swabbed according to a protocol as described elsewhere [20]. Briefly, for both situations one swab was used per nostril. Swabs were either used in dry state directly from the sterile sleeve or pre-moistened by dipping the swab tip into a sterile 15 ml Round Bottom Tube (Greiner Bio-One, Frickenhausen, Germany, Ref. 164161) containing 1 ml of 0.85% NaCl solution, followed by thoroughly squeezing at the tube wall to discard surplus liquid before usage. The nasal vestibule was swabbed by applying gentle pressure. The swab was rotated while circulating in the nasal vestibule for approximately 5 seconds.

After swabbing the nostrils, moist swabs were placed in the corresponding tube used to moisten the swab. In the case of dry swabs, these were set down into a fresh sterile 15 ml Round Bottom Tube containing 1 ml of sterile 0.85% NaCl solution.

Sterile Nasal Swab Nylon

Quantification of bacteria

All swabs were subjected to microbiological analysis immediately after swabbing the noses or the nose models. The swab-NaCl-combination was vortexed for 5 seconds. CFU were determined by plating 100 μl of 1:10 serial dilutions onto Columbia agar supplemented with 5% sheep blood (BD, Heidelberg, Germany), resulting in a detection threshold of 10 CFU. Bacterial amounts below this limit could not be detected and swabs were regarded as MRSA negative. In parallel, a chromogenic MRSA medium (chromID MRSA, bioMérieux, Marcy l’Etoile, France) was inoculated with 100 μl of the undiluted suspension. Agar plates were subsequently cultured at 37°C under ambient atmosphere for 48 h. CFU were then counted by macroscopic inspection. Staphylococcus aureus was identified by β-hemolysis and colony color (golden yellow on Columbia agar, green color on chromID MRSA agar), if necessary by agglutination assay (Slidex Staph Plus, bioMérieux, Marcy l’Etoile, France), or by matrix-assisted laser-desorption-ionization time-of-flight mass spectrometry (MALDI-TOF-MS) using a Shimadzu “AXIMA Assurance” MALDI-TOF mass spectrometer (Shimadzu Germany Ltd., Duisburg, Germany) as described elsewhere [31].

Statistical analysis

Data were analyzed using nonparametric Wilcoxon-Mann-Whitney U-test. All p-values resulted from two-tailed statistical testing. p-values of <0.05 were considered to be statistically significant. Raw data of in vivo and in vitro experiments are provided in the supplementary information (S1 Table, S2 Table).

Ethics statement

All clinical investigation has been conducted according to the principles expressed in the Declaration of Helsinki. The study was approved by the institutional review board of University Medicine Rostock (Ethikkommission an der Medizinischen Fakultät der Universität Rostock; approval no: A 2012–0079) in line with national and ICH-GCP guidelines. All patients provided written informed consent.

Recovered MRSA quantities from patients utilizing dry or moist rayon swabs

Patients were swabbed with dry or moist rayon swabs. The median of the recovered bacterial quantities obtained with dry nasal swabs was log10 CFU = 4,10 (1.25x104 CFU), with moistened swabs log10 CFU = 4,48 (median = 3.05x104 CFU). These differences were not statistically significant (p = 0.17) (Fig 1).

 

Fig 1

Recovered MRSA quantities from patients utilizing dry or moistened rayon swabs.

Patients were swabbed in the nostrils utilizing either dry or moist rayon swabs. CFU = colony forming units. Data result from n = 135 patients. p-value = 0.17 (Wilcoxon-Mann-Whitney U-test).

Recovered MRSA quantities from the artificial nose model utilizing dry or moist rayon, polyurethane-foam and nylon-flocked swabs

Inoculated nose models were tested with dry or moistened swabs of the indicated swab-types. Bacterial quantities were assessed. Within the same swab-type, no statistically significant differences could be detected between dry or moist sample collection (p = 0.62 (Nerbe plus, rayon); 0.90 (MWE, polyurethane); 0.72 (Copan, nylon-flocked); 0.96 (MWE, rayon)) (Fig 2).

 

Fig 2

Recovered MRSA quantities from the artificial nose model utilizing dry or moist rayon, polyurethane foam and nylon-flocked swabs.

Nose models were swabbed utilizing either dry or moistened rayon, polyurethane foam or nylon-flocked swabs. Data results from n = 10 swabs for each setting. p-value (comparing dry and moist swabs within one swab-type) = 0.62 (Nerbe plus, rayon); 0.90 (MWE, polyurethane); 0.72 (Copan, nylon-flocked); 0.96 (MWE, rayon) (Wilcoxon-Mann-Whitney U-test).

Statistically significant differences could be detected when comparing recovered bacterial quantities between different swab-types. From MWE polyurethane, Copan nylon-flocked and MWE rayon swabs statistically significant greater quantities of MRSA could be recovered in comparison to Nerbe plus rayon swabs in the dry (p<0.0001; p<0.0001; p = 0.0014, respectively) as well as in the moist (p<0.0001; p<0.0001; p<0.0001, respectively) setting.

Swabs with tips made of PU-foam and nylon-flocked fibers performed best in terms of recovering the greatest bacterial quantities. Performance between these swabs types was similar with no statistically significant differences in both swabbing settings (p>0.05).

MWE rayon swabs recovered less bacterial quantities as compared to the PU foam or nylon-flocked swabs, but these differences were not statistically significant (p>0.05).

Nasal MRSA burden in patients

For this analysis, data from both the dry and moist setting were used, i.e. about half the results per nostril were obtained by sample collection with either dry or moistened swabs. Quantities of MRSA per nostril were clustered in log10 steps. Bacterial quantities ranged between <101 and >107 CFU (Table 1).

First, several patients that were initially tested as MRSA-positive and were therefore included in this study, now presented themselves as MRSA-negative (see also next subchapter). Second, based upon the documented MRSA-quantities, the MRSA-positive patient population segregated into two major groups, one with less than 1,000 MRSA CFU per nostril and another even larger one with 10,000 to 1,000,000 MRSA CFU per nostril.

Sensitivity of dry or moist rayon swabs for the detection of MRSA

All patients were initially screened positive for carriage of MRSA in the routine MRSA screening program of the hospital. The study population was therefore stated as 100% MRSA positive. Under this aspect, sensitivity of consecutively executed sample collection with dry or moist nasal swabs was qualitatively analyzed. Both sampling methods displayed similar sensitivities. By sampling with dry rayon swabs 79.1% of the initially positive tested patients were MRSA positive, with moistened rayon swabs 79.4%, respectively.