2.1 Study design
This study was conducted on medical students from diverse clinical years and different rotations in the faculty of medicine and health sciences at An-Najah National University in the West Bank, Palestine, which the diaphragm and bell of the stethoscope were selected for sampling and evaluation for microbial contamination rate. Statistical analysis was conducted to determine the distribution of microbial level and the participants were sampled without any prior intimation.
The research utilized a cross-sectional study design to collect data from a diverse population sample in a defined point of time; the study was conducted within a university research laboratory (Central research lab) over a period of five months, starting from August 2022 until February 2023. We confirm that all methods were performed in accordance with the relevant guidelines and regulations according to our university regulation and policies.
A descriptive study based on laboratory was conducted on medical student at An-Najah National University from three hospitals; hospital A (32° 13’ 32” N, 35° 14’ 30” E), hospital B (32° 13’ 22” N, 35° 15’ 45” E), and hospital C (32° 14’ 23” N, 35° 14’ 45” E). Every participant in the study completed a questionnaire; the questionnaire addresses their daily use and practice in hospitals and medical practice. It includes questions about their gender, clinical year and stethoscope ownership. Other questions assessed the frequency of disinfection, the cognizance and understanding of infection transmission, and cleansing practices. A special section asks about their training in hospitals, the availability of disinfectant solutions, the quality of teaching and reminders of disinfection, and who they examine with their stethoscopes (patient or other students). Lastly some questions about their personal status such as medical conditions, hands hygiene and nails. Their answers evaluated in order to know the awareness about the stethoscope handling, disinfectant use and adherence to the infection control practices.
2.2 Bacterial count and identification of Staphylococci spp
A sterile cotton swab wet in a sterile normal saline, then its tip used to swab samples from the entire diaphragm of the stethoscope, and then put in 1ml sterile normal saline Eppendorf tube. Then, 0.1ml cultivated by spreading method on Tryptic Soy Agar media (TSA) for each sample in triplicates, and then the culture plates incubated at 37°C for 48 hours. After incubation, colony counting was measured and the mean was credence (22, 23).
Using different microbiological identification approaches, including morphological and biochemical features, identification was done, all isolates were subjected to Gram stain. After 48 hours of incubation of TSA, colonies that ferment mannitol (yellow colonies) suspected S. aureus, and colonies that cannot ferment mannitol suspected CoNS (22, 23). Investigation of Staphylococcal contamination is targeted since we believed that Staphylococcus was primarily responsible for the contamination of skin and surfaces, a belief that was validated by prior investigations. Because we desire to keep patients safe from cross-infection as stethoscope is in direct contact with skin, yellow colonies were cultivated consequently by spreading method on Mannitol Soy Agar (MSA) for each sample in triplicates, and then the culture plates were incubated at 37°C for 48 hours. After incubation, colony counting was counted and the mean was credence (22, 23).
For Gram-positive cocci, isolates were sub cultured on blood agar and subjected to catalase test; positive reactions are considered S. aureus and CoNS. For positive results further identification is done with coagulase test to confirm the Staphylococcus spp, according to the response to coagulase, we will discern between S. aureus and CoNS. Those who are positive (mannitol fermenter-yellow) are considered S. aureus, and negative ones are considered CoNS (22, 23).
After antibiotics use (section 2.3), we used API Staph (bio Mérieux, Lyon, France) according to the manufacturer procedure to differentiate between the different types of staphylococcus bacteria. Novobiocin sensitivity disc was used to differentiate S. Saprophyticus (less than 16mm consider resistance and positive S. Saprophyticus) (24).
Although Staphylococcus was focused, other types of bacteria were not neglected. For gram negative bacteria MacConkey agar was used, and any cultivated transparent colony (lactose nonfermenter) was tested with oxidase test for the detection of Pseudomonas spp (oxidase positive). And for those colonies that revealed oxidase negative test, Glucose fermentation test was used, the colonies that revealed a negative result were considered Actinobacter Baumannii; nevertheless none of the results disclosed the existence of gram-negative nosocomial bacteria (Data not shown) (22, 25).
2.3 Antibiotics Sensitivity Test
Antibiotic sensitivity test was done on Staphylococcus spp colonies that grew on MSA. This test was performed by the Kirby–Bauer disc diffusion method on Mueller-Hinton (MH) agar according to Weinstein and Lewis (26). Using Antibiotic-classes from (Oxoid United Kingdom): Cefoxitin (FOX), Vancomycin (VA), Oxacillin (OX) (J01CF04) for examination of MRSA and VRSA according to Weinstein and Lewis (CLSI 2018) (26). Also, Gentamicin (CN) (D06AX07), Erythromycin (E) (J01FA01), Trimethoprim (SXT) (J01EE01), Clindamycin (DA) (J01FF01), Levofloxacin (LEV) (J01MA12), antibiotics were performed for sensitivity pattern, by using 0.5 McFarland estimated by the densitometer (26).
2.4 Statistical analysis
Statistical Package for Social Science (SPSS) software version 25 was used for analyzing. Descriptive statistics were used to summarize different variables and presented in the form of texts and tables, Categorical variables were described as frequencies and proportions, and continuous variables tested by Spearman’s correlation test due to the data being not normally distributed. Also, we used Pearson Chi-Square and Fisher’s Exact Test to examine the comparison between categorical variables. We used Kruskal One-Way ANOVA test to compare between variables with more than 3 groups and due to the data not being normally distributed, and being appropriate for comparing means across many groups.
The study formulated the following hypotheses to evaluate the contamination rates and students’ awareness:
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Null Hypothesis (H0): There is no stethoscope contamination, and high awareness of disinfection among medical students does exist.
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Alternative Hypothesis (Ha): There is stethoscope contamination, and high awareness of disinfection among medical students does not exist.
Stethoscope Contamination rate was calculated using the following equation: (Cfu/ml) / radus2 x 3.14 of the stethoscope’s diaphragm, with contamination rate of (2.5 cm2) or more was conceded as an acceptable contamination rate.[23] The tests were conducted to determine whether there were significant differences in contamination rates among different variables. The tests produced an F-statistic and associated p-value. To determine statistical significance, the resultant p-value from the tests was compared to a pre-set significance level (e.g., α = 0.05). If the p-value is less than the significance level, the null hypothesis is refuted due to strong evidence against it, indicating a significant difference in contamination rates between the variables and vice versa.