This was a multicentre cross-sectional study conducted from October 2019 to March 2020 in the Deido Health District. Laboratories that carry out culture and antimicrobial susceptibility testing (AST) within the Deido Health District were identified and assessed to determine their capacity as well as the quality of results from microbiological investigations using the Modified WHO Antimicrobial Resistance Surveillance Questionnaire 16 and Stepwise Laboratory Quality Improvement Process Towards Accreditation (‎SLIPTA) Checklist 19.

The laboratory staff working on the microbiology bench were trained on how to collect information using a structured questionnaire incorporated in tablet phones. Information on antimicrobial susceptibility of various isolates was collected using tablet phones in which the study questionnaires had been incorporated.

Participants included in this study were individuals of all ages and sex who visited any of the three (Deido District Hospital, St Padre Pio Hospital and Daniel Muna Memorial Clinic) main hospitals in the Deido Health District for culture and sensitivity tests between October 2019 and March 2020.

Antimicrobial susceptibility of the isolates was determined using the disc diffusion technique on Mueller Hinton agar for bacteria isolates and Sabouraud dextrose agar for fungi isolates as described in the guidelines of the Clinical and Laboratory Standard Institute (CLSI) and the European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints2021

After inoculating the isolates and placing the antimicrobial discs, the plates were incubated for 24h against Staphylococcus isolates and 16–18h for other isolates. The diameters of the zones of complete inhibition (as qualified with the eye) were measured, the diameter of the antimicrobial disk was also measured. The measured diameter was compared to the critical values of each antimicrobial disc to determine whether the target isolate was sensitive, resistant or intermediate. Mindful of the difficulty in obtaining commercial control strains in the country to be used for daily routine, the laboratories had established in house controls that are derived from commercial controls. Control tests were performed with Staphylococcus aureus and E. coli in house derived strains.

For mycoplasma susceptibility testing, the specimen was inoculated into the Mycoplasma Susceptibility kit (Autobio Diagnostics Co.,Ltd., Zhengzhou, China) within an hour as described by the manufacturer's guidelines. If there is a growth, Urealpasmaurealyticum and Mycoplasma hominis metabolizes urea and arginine, respectively. This changes the colour of the culture medium (from yellow to red). The kit was incubated at 37˚C for 48 h after which susceptibility or resistance bacterial were determined with the aid guidelines of Clinical and Laboratory Standards Institute.

Data on the laboratory capacity and quality of culture and antimicrobial susceptibility testing was collected with the modified WHO Antimicrobial Resistance Surveillance Questionnaire and the SLIPTA ‎ Checklist. A structured questionnaire was used to collect demographic information, patient history concerning the usage of antimicrobials. Furthermore, the questionnaire was also designed to capture information on the type of specimen that was cultured, the isolate, and the drugs to which the microbe was resistant as well as the turnaround time for culture and antimicrobial susceptibility testing. Since the questionnaire was incorporated into a tablet phone, an email address was created which was used for weekly backup of the data set.

Data was collected with a questionnaire designed using Epi Info Data software. The data set was exported from Epi Info to excel spreadsheet. Missing variables or discrepancies in data were corrected from the medical records of patient. The data was then exported and analyzed using SPSS version 20 (IBM, Chicago, IL). Descriptive statistics such as the number of microbes isolated, type of specimen cultured were expressed as proportions. The overall resistance of the isolates to a class of antimicrobial agents was calculated as median resistance and inter quarter range. The resistance rate of a specific group of the isolate to the antimicrobial agent was also calculated as median resistance. Comparison of the proportion of antimicrobial resistance between groups was assessed with the chi-square test and the threshold for statistical significance set at p < 0.05.

Ethical approval was obtained from the Faculty of Health Sciences Institutional review board of the University of Buea (N0: 2019/941-01/UB.SG.IRB.FHS). Administrative authorization was obtained from the Littoral Regional Delegation of Public Health and the Deido Health District. Written consent was obtained from the participants after the purpose of the study was orally explained to them. Since the questionnaire was incorporated into the tablet phones, passwords were given for each tablet to avoid unauthorized accessto the database

From the audit results using WHO Antimicrobial Resistance Surveillance and SLIPTA checklist, the methods in use for culture, identification, and antimicrobial susceptibility testing in the laboratories were kit-based (only for mycoplasma) and conventional methods. With respect to personnel, the laboratories were headed by qualified microbiologists and Quality Assurance Officers were present in all the laboratories. The laboratories reported not to be using control strains but they had established in-house quality control isolates. All the laboratories had a backup system for power. The laboratories were using either the “CLSI or the EUCAST” guide for the interpretation of AST.

On the other hand, gaps identified which cut across the three laboratories included; insufficient standard operating procedures, inadequate records on personnel training and competency assessment, lack of safety equipment such as biosafety cabinet, stock out, non-participation in external quality assurance programs, and audits were not regularly performed. The study findings also showed that there are no structures in place to oversee AMR activities in the facilities and at district levels, hence non-utilization of laboratory AST data to inform authorities. Another gap that cut across these laboratories was the non-usage of quality indicators to measure performance.

A total of 1797 samples were cultured in the three laboratories during the study period among which, 424 (23.6%) were female samples. The samples were collected from individuals aged 1 to 94 years, with a mean age of 19.84 years (SD 14.9). The majority of the participants 1199 (66.7) were within the age group 20 – 40 years. Most of the specimens cultured were, urine 731(40.7%) follow by vagina smear 642(35.7%) while the CSF was 2(0.1%). It took the laboratories 3 to 7 days to give out culture and susceptibility testing results. It is important to note that 1164(64.7%) of results were given within 3 working days while 16(0.9%) took more than 5 days (Table 1).

Out of the 1797 samples cultured, 437(24.3) had at least one isolate. A total of 15 different isolates were identified with Candida albicans being the most frequent 178 (40.7%), mainly isolated from virginal smears followed by Escherichia coli 80 (18.3%) isolated predominantly from urine cultures. Only one Neisseria gonorrhoeae was isolated from a urethra smear during the study period. Overall the greatest number of isolates was from virginal smears 198 (45.3%) followed by urine 116 (26.5%) and stool 81(18.5%) (Table 2).

Among the cephalosporins, cefixime showed 100% resistance to Staphylococcus aureus and CoNSwhile cefuroxime showed no resistance to these two groups of bacterial. Among penicillins, ampicillin showed a resistance rate of at least 50% to all gram-positive isolates while cloxacillin (9.1%), amoxicillin-clavulanic acid (33.3%) piperacillin (21.4%) and amoxicillin (11.1%) were the resistance to Staphylococcus aureus, CoNS, Enterococcus spp and Streptococcus respectively. Among the Beta-lactam, Staphylococcus aureus and Enterococcusspp showed 100% resistance to oxacillin. With respect to the Quinolones, Staphylococcus aureus, Enterococcus spp, and Streptococcus showed 100% resistance to nalidixic acid while perfloxacin was the least resistant to Staphylococcus aureus, and Streptococcus with a resistance of 11.1% and 28.6% respectively. Streptococcus also had 100% resistance to ciprofloxacin. Most of the tetracyclines showed 100% resistance.

Generally, among the 10 classes of drugs tested against the gram-positive bacteria in the study, only clarithromycin (a quinolone) and rifampicin (an antimycobacterial) showed resistance rate with a significant difference across the different category of gram-positive bacteria ( p-value 0.044 and 0.0001 respectively) (Table 5)

Escherichia coli showed high resistance of above 70% to ampicillin, amoxicillin-clavulanic acid, oxacillin, nalidixic acid, vancomycin, and trimethoprim while it was least resistant to netilmicin (4.2%) followed by ceftriaxone and perfloxacin 11.4% and 11.8% respectively. Klebsiella spp was less resistant to netilmicin (5.9%) and pefloxacin (16.7%) while it showed resistance above 70% to cefuroxime, cefotaxime, amoxicillin-clavulanic acid, oxacillin and rifampicin.

Pseudomonas aeruginosa showed high resistance of above 70% to cefuroxime, cefotaxime, cefixime, piperacillin, cloxacillin, amoxicillin-clavulanic acid, oxacillin, ofloxacin, azithromycin, antifolate and chloramphenicol, whereas, its lowest resistance wastto ceftriaxone (20.0%) while it showed no resistance to ampicillin, pefloxacin and amikacin (Table 6).

Serratia liquefaciens resistance to ceftazidime, amoxicillin-clavulanic acid, oxacillin, ofloxacin, nalidixic acid, perfloxacin, trimethoprim, and rifampicin was above 70% while it was not resistant to aztreonam, levofloxacin, gentamicin, amikacin, and nitrofurantoin. Citrobacter showed 100% resistance to trimethoprim, oxacillin, nalidixic acid, and perfloxacin. (Table 6).

For the resistance of Ureaplasmaurealyticum and Mycoplasma hominis to the various antimicrobials, no statistical significance difference was noted. Ureaplasmaurealyticum showed no resistance to oxacillin, perfloxacin, erythromycin, vancomycin, and nitrofurantoin while Mycoplasma hominis showed no resistance to only ciprofloxacin. Ureaplasmaurealyticum highest resistance was to Clarithromycin (63.6%) while Mycoplasma hominis showed 100% resistance to trimethoprim (Table 7).

Among the antifungal agents, the highest resistance of Candida albicans was observed against Amphotericin B (88.8%). Fluconazole, ketoconazole, and nystatin showed resistance above 50%. Candida albicans showed the least resistance to Miconazole (9.8%)

The antimicrobial resistance situation in the Deido Health District is preoccupying as is the case with other developing countries. Apart from aminoglycosides, pathogens showed an antibiotic class median resistance over 25% of the various antimicrobial agents. Despite the importance of testing in the fight against AMR, the laboratory turnaround time remains long and the laboratories are under-equipped and the quality of AST is seriously affected.

This study has shown there is a need to develop a coordinated national approach by Cameroon's ministry of public health to fight AMR with much priority on antimicrobial susceptibility testing. Scaling-up AST testing will, therefore, require strengthening the microbiology units of laboratory systems and ensuring the use of laboratory data for clinical decision-making.

The authors express sincere gratitude to the Faculty of Science University of Buea, Cameroon, the directors and laboratory staff of the hospitals involved, all the study participants and the littoral Regional Delegation of Public Health

This study was funded by the corresponding author and also received support from the faculty of Science University of Buea, Cameroon. However, the results and conclusions made in this publication are made by the authors and may not represent the official position of the faculty of Science University of Buea, Cameroon 

AMR: antimicrobial resistance

AST: antimicrobial susceptibility testing

CLSI: Clinical and Laboratory Standard Institute

CSF: Cerebrospinal fluid

EUCAST: European Committee on Antimicrobial Susceptibility Testing

IRB: Institutional review board

SLIPTA: Stepwise Laboratory Quality Improvement Process towards Accreditation

US: Urethral Smear

VS: Vagina Smear

WHO: World Health Organization

All relevant data are included in this manuscript

This study protocol was reviewed and approved by the Faculty of Health Sciences Institutional Review Board (IRB) of the University of Buea, Cameroon (N0: 2019/941-01/UB.SG.IRB.FHS).