Bacterial contamination upon the opening of injection needles

Opening methods

Two injection needle product opening methods were employed: the “peel-apart method” (Fig. 1a) where the adhesive surface of the mount for packaging is peeled off, and the “push-off top method” (Fig. 1b) where the needle hub is pressed against the mount to break it [5].

Bacterial strains and preparation of bacterial solutions

The methicillin-susceptible Staphylococcus aureus strain of the American Type Culture Collection (ATCC) 29213 was used [6]. ATCC 29213 was provided by the Kitasato University-Laboratory of Infection Control and Research Center. A bacterial solution was cultured for 10 h with shaking and was diluted with physiological saline to an absorbance of 0.3 using an absorption spectrometer at 578 nm [7]. The concentration of the bacterial suspension was 10⁸ colony-forming units/ml (CFUs/ml) before the experiment. This solution was diluted with physiological saline to six different concentrations (10⁸, 10⁷, 10⁶, 10⁵, 10⁴, and 10³ CFUs/ml).

Experimental contamination of mounts

A total of 240 injection needles, including 120 each adherently packaged with a paper-mount and transparent plastic blister (18G: Terumo Co., Ltd., Tokyo, Japan) or with a plastic (combination of polystyrene and polyethylene terephthalate) mount and transparent plastic blister (18G: NIPRO Co., Ltd., Osaka, Japan) were used. The injection needle was taken out of the box just before the initiation of experiments and was stored on a clean bench after disinfection. Injection needles were classified into two groups according to the opening methods: the peel-apart and push-off top methods (60 needles each). Experiments were conducted separately for 10 needles each at six different concentrations of the bacterial suspension. To assess the risk of needle contamination by various quantitative concentrations under clinical settings, 10 μl of each of the bacterial suspensions (10⁸, 10⁷, 10⁶, 10⁵, 10⁴, and 10³ CFUs/ml) was applied to the part near the needle hub’s opening at the mount of an unopened injection needle product using a pipette tip on a clean bench (shaded parts in Fig. 1a, b). Using the peel-apart method, the bacterial suspension was applied to the gripped part of the mount (shaded parts in Fig. 1a). Using the push-off method, the bacterial suspensions were applied to the part potentially touching the mount when removing the needle (shaded parts in Fig. 1b).

Injection needles were then opened using the peel-apart or push-off top method with disinfected gloves (Fig. 1a, b). On a clean bench, half of the needles were opened as soon as the bacterial suspensions had been applied (wetness group). The other half were dried using the filtering airflow of the clean bench at room temperature. One hour later, the dry state of suspensions applied was confirmed visually and needles were then opened on the clean bench (dryness group). Injection needles were taken out to examine the degree of contamination in each part of the needle hub.

To examine the degree of contamination on each site of the needle hub (Fig. 2), all lateral surfaces (Fig. 2a) were placed on agar medium and rotated to be brought into contact with the medium. The bottom surface of the needle hub (Fig. 2b) was then pressed against the agar medium. To examine contamination in the inner lumen (Fig. 2c), a 1-ml syringe containing 0.1 ml of saline was connected to the needle to discharge all saline onto the agar medium.

The agar medium was incubated at 37 °C for 30 h for colony counting. Brain heart infusion agar (Becton, Dickinson, and Company, USA) was used as the agar medium.

Emulated clinical contamination (hand and saliva contamination of mounts)

Based on the rare occurrence of needle hub contamination in the previous experiment using the peel-apart method, various conditions (i.e., dry or wet hands, saliva contamination) were examined to evaluate the clinical risk of needle hub contamination using the push-off top method. Five anesthesiologists were included in the present study. This investigation was conducted in accordance with the current Declaration of Helsinki. The authors’ own samples were collected, and patients and volunteers were not included. All samples were anonymized after collection for the impossibility to identify the specific individual. A total of 150 injection needles, including 75 each in the paper-mount group and plastic-mount group, were used.

Anesthesiologists rubbed dry/wet hands on the paper or plastic-mounts without gloves. To simulate wet hands, 10 μl of autoclaved physiological saline was applied to dry hands using a micropipette. To simulate a hand contaminated with a patient’s saliva, gloved fingers licked by anesthesiologists were applied to each paper and plastic-mount.

Five saliva samples were obtained from each of the five anesthesiologists and were quantitatively cultured.

All injection needle products with clinically contaminated mounts were opened on a clean bench in the same manner as described for examination of the experimental contamination of mounts.

Statistical analysis

The numbers of bacteria on the lateral surfaces, bottom surfaces, and total surface (sum of the lateral surface, bottom surface, and inner lumen) of needle hubs were compared between the opening methods, dryness/wetness of bacterial solution, and mount materials using the Cochran-Mantel-Haenszel test considering the concentration of S. aureus as a stratum. By comparing the dryness/wetness of bacterial solution and mount materials, only data obtained using the push-off top method was used because only one needle hub was contaminated in the peel-apart method. The trend test for the concentration of S. aureus-contamination relationship was performed using the push-off top method data with the Jonckheere test.

The number of bacteria in the inner lumen of a needle hub was classified into contaminated (≥ 1 colony) or uncontaminated (no colony), and this binary response was compared between opening methods, the dryness/wetness of the bacterial solution, and mount materials using Fisher’s exact test without considering the concentration of S. aureus. A trend test for the S. aureus concentration-contamination relationship in the number of bacteria in the inner lumen was not performed because of only five hubs were contaminated. Fisher’s exact test was instead applied to compare S. aureus concentrations.

Regarding emulated clinical contamination data, the number of bacteria was compared between mounts using the Cochran-Mantel Haenszel test considering the anesthesiologist and wet/saliva hands as strata, excluding dry hand data because all were zero. The numbers of bacteria on the lateral surfaces, bottom surfaces, inner lumens, and total surface of needle hubs were compared between dry/wet/saliva hands using the Cochran-Mantel-Haenszel test considering anesthesiologist as a stratum. The mount was not included into stratum because a large p value was obtained for the mount comparison. Pairwise comparisons were also performed. The family-wise error rate was controlled using the closed testing procedure; first, data was compared between dry, wet, and saliva hands with a significance level of 5% and the testing procedure was stopped if not significant. Second, pairwise comparisons were performed with a significance level of 5% for each test if the first step was significant. A p value of < 0.05 was considered to be significant. Data were analyzed using SAS version 9.4 (SAS Institute, Cay, North Carolina, USA).

Opening methods

The lateral and bottom surfaces of needle hubs were contaminated significantly more by the push-off top method than by the peel-apart method (Figs. 3a, b). However, contamination of the inner lumen did not significantly increase (Fig. 3c).

S. aureus concentrations

Using the push-off top method, contamination of the needle hub increased with the concentration of S. aureus applied to the opening portions. Contamination of the needle hub was rare at a concentration of ≤ 10⁴ CFUs/ml. The number of contaminated needle hubs was 5% (1 out of 20 needles) at a concentration of 10⁴ CFUs/ml, and 0% (0 out of 20 needles) at a concentration of 10³ CFUs/ml.

Wet/dry

Contamination of the needle hub was significantly greater in the wet than in the dry opening portions (Fig. 4a).

Paper/plastic

No significant differences were noted in the needle hub contamination between mount materials (paper and plastic) (Fig. 4b).

Emulated clinical contaminations (hand and saliva contamination of mounts)

No significant differences were observed in the needle hub contamination between the dry and wet hand contamination groups (Fig. 5a–c). The lateral and bottom surfaces of the needle hub were contaminated significantly more in the saliva contamination group than in the dry and wet hand groups (Fig. 5a, b). However, contamination of the inner lumen did not significantly increase (Fig. 5c). No significant differences were observed in the emulated clinical contamination of the needle hub between mount materials (paper and plastic) (Fig. 5d). The mean bacterial concentration of the saliva of five anesthesiologists was 2.36 × 10⁷ CFUs/ml (ranging between 2.2 × 10⁶ and 6.1 × 10⁷ CFUs/ml).