Study design and period

A cross-sectional study was conducted, from November 2021 to August 2022 to include all participants selected during follow-up monitoring visits at baseline, 1, 2, 4, 12, 16 and 24 weeks of treatment as indicated in Ethiopian National consolidated guideline for ART ¹¹.

Study setting

The study was conducted at the Asella Referral and Teaching University Hospital (ARTH), approximately 175 km south of Addis Ababa, the capital city of Ethiopia. Located in Arsi Zone, Oromia Regional State, the hospital has 260 inpatient beds and serves as a referral center for 3.5 million inhabitants. About 3700 registered PLWHA receive regular care at the hospital’s specialized anti-retroviral therapy (ART) follow-up clinic.

Sample size and sampling technic

A total of 400 participants were enrolled after estimating the minimum required sample size using single population formula at a 95% confidence level, 5% margin of error and a S. aureus colonization rate of 61.7%, using a similar study from Ethiopia ¹², and a 10% non-response rate. The registration book served as a sampling frame for the selection of participants. The first participant was selected from the first 10 ART registered users by the lottery method. Using k = 9, systematic sampling was used to choose the remaining 399 individuals. Participants had to be at least eighteen years old, intellectually and physically competent of making decisions, able to answer questions, and have written, signed consent in order to be eligible for this study. Accordingly, every chosen participant was contacted, ask for their consent and then enrolled in the study.

Sociodemographic and clinical data collection

A semi-structured questionnaire was used to interview consenting participants in order to collect data on their sociodemographic, hospitalization history and disease condition. Clinical information from the participants’ medical records, including as clinical conditions, CPT data, CD4 + cell count, and HAART regimen data, was extracted using case report forms (CRF).

Nasal swab collection and transportation

As described previously, one nasal swab was collected from each participant using moistened sterile cotton swabs ¹³. All the collected swabs were put into Amie’s transport medium and transported to the Hirsch Institute of Tropical Medicine (HITM) laboratory located in Asella, Ethiopia. Safety and sterile conditions were maintained according to the study’s standard operating procedures (SOPs).

Culture identification of S. aureus

The specimens were immediately inoculated in the HITM laboratory under aseptic conditions using 5% sheep blood agar (SBA) plates first, then Oxoid Mannitol Salt Agar (MSA) plates. Then, SBA plates were incubated in a 5% CO₂-enriched atmosphere at 37 °C. MSA plates were aerobically incubated at 37 °C as described previously ¹⁴. In accordance with the manufacturer’s instructions, the coagulase test was conducted using CoaguStaph™, a tube coagulase test ¹⁵.

Plates were examined after 18–24 h and 48 h of incubation for the presence of growth and characteristic colonies ¹⁴. Gram-positive cocci bacteria that were grown as colonies of golden yellow color on MSA plates and beta hemolytic bacteria on SBA plates ¹⁶ and that tested positive for the enzymes catalase and coagulase were presumptively identified as S. aureus. The rest of colorless/white, pink or red colonies on MSA and/or blood agar, catalase-positive, coagulase-negative Gram-positive cocci were presumptively identified as coagulase negative staphylococci (CoNS) ¹⁷.

Antimicrobial susceptibility test

A standardized suspension matched with 0.5 McFarland was prepared from all isolated strains of S. aureus. Mueller Hinton Agar (MHA) was inoculated with the suspension. In accordance with the European Committee on Antimicrobial Susceptibility Testing (EUCAST), the modified Kirby-Bauer disc diffusion method was used¹⁸. The antimicrobial discs used were selected considering locally available antibiotics, EUCAST and CLSI guidelines. Accordingly, the isolated S. aureus were tested against amikacin (AK) (30 µg), ciprofloxacin (CIP) (5 µg), cefoxitin (FOX) (30 µg), clindamycin (CD) (2 µg), co-trimoxazole (25 µg) (trimethoprim/sulfamethoxazole (TMP-SMX) (1.25/23.75 µg), erythromycin (E) (15 µg), gentamicin (GEN) (10 µg), penicillin G (PG) (30 µg) and tetracycline (TET) (30 µg) antibiotics ¹⁹. The diameters of zones of inhibitions were measured and the results were interpreted by the EUCAST guideline, as S-susceptible, standard dosing regimen (high likelihood of therapeutic success using standard dosing regimen of the antimicrobial), I-susceptible, increased exposure (high likelihood of therapeutic success by increasing dosing regimen or its concentration at the site of infection) and R-resistant (high likelihood of therapeutic failure even when there is increased exposure ¹⁸. In this study, all susceptible isolates with increased exposure were considered resistant. Isolates that showed non-susceptibility to at least one agent in three or more tested antimicrobial categories were considered as multidrug resistant (MDR) isolates ²⁰.

Methicillin-resistant S. aureus (MRSA) identification

Screening for methicillin resistance in S. aureus isolates were done on MHA, by using cefoxitin (FOX) (30 µg) antibiotic disc. Those S. aureus that grow in a zone of inhibition size < 22 mm around cefoxitin disc were presumptively considered as MRSA ¹⁸.

Co-trimoxazole-resistant S. aureus (COTRSA) identification

Screening for co-trimoxazole resistance S. aureus was done by TMP-SMX (25 µg) (trimethoprim/sulfamethoxazole) (1.25/23.75 µg) antibiotic disc on MHA. All isolates with zone of diameter < 14 mm for SXT disc, were considered as co-trimoxazole-resistant S. aureus (COTRSA) according to the EUCAST recommendation ¹⁸.

Data quality control and management

Data collectors received instruction on how to conduct questionnaires and gather high-quality data. The questionnaire was pretested for comprehensiveness, efficacy, reliability and validity. Laboratory standard operating procedures (SOPs) were strictly adhered during specimen collection, labeling, storage and transportation. By incubating 5% of each batch of the produced media at 37 °C for 24 h, the sterility of the culture media was guaranteed. To evaluate the effectiveness of the media and antibiotic discs, S. aureus standard strain ATCC 25923 was employed as a control organism¹⁸. Furthermore, known MRSA clinical isolates from Asella Hospital were used to assure the quality of Methicillin-resistance testing. Using monitoring log books, the proper operation of the autoclave, incubator, refrigerator, chemicals, and microscope was routinely verified. Media preparations, antimicrobial resistance testing and internal quality controls of all the laboratory procedures were carried out in accordance to the manufacturer’s recommendations and specifications.

Statistical analysis

Data were exported to IBM’s statistical package for social sciences (SPSS) version 25 after being entered into Epidata V3.1. Participants’ clinical and demographic traits in relation to isolated microorganisms were compiled using descriptive statistics like frequency and percentage. The association between sociodemographic and clinical variables with co-trimoxazole-resistance S. aureus was evaluated using Chi-squared test at a cut off value of p < 0.05.

Ethical considerations

Under reference number PM23/326/2021, ethical clearance was acquired from the IRB at St. Paul’s Hospital Millennium Medical College (SPHMMC). All participants also provided written informed consent. In order to ensure the confidentiality of their information and guarantee that the data would only be used for research, all participants received comprehensive and detailed information on the method, risk and benefit, and purpose. Participants’ identities are kept anonymous, and the research report does not use any other personal information. All study procedures were carried out in accordance with the Helsinki declaration and other local and international research ethics guidelines.

Ethics approval and consent to participate

The study was approved by the Institutional Review Board of St. Paul’s Hospital Millennium Medical College (SPHMMC) with reference number PM23/326/2021.

Consent for publication

Not applicable—This manuscript does not contain any individual person’s data.