Prevalence and risk factors associated with multi-drug resistant organisms (MDRO) carriage among pediatric patients at the time of admission in a tertiary care hospital of a developing country. A cross-sectional study

We conducted a cross-sectional study including all pediatric patients aged 1–18 years admitted to the pediatric ward at the Aga Khan University Hospital (AKUH), Pakistan, from May to September 2019. Participants were screened for identification of MDRO carriers including MRSA, VRE, CRE and ESBL-producing gram negative bacteria within 72 h of admittance. Patients with a documented history of culture proven MDRO infection or carriage during the previous 12 months of admission, diagnosed cases of primary or secondary immunodeficiency, and those who refused to participate in the study were excluded.

This study was approved by the Ethical Review Committee (ERC) of AKUH (2019–0653-2209). All methods including enrolment of patients, data collection, microbiological and statistical analysis were in accordance with the guidelines and regulations of institutional ERC. The sample size was calculated by OpenEpi. Assuming a rate of MDRO carriers in the general population as 30%, 5% bound on the error, 5% significance level, the maximum sample size was 323. Sample size was also calculated for associated risk factors such as prior use of antibiotics, prior hospitalization, ICU stay etc. by taking frequency of these factors among non-MDRO carriers between 30% and 50%, 80% power, 5% significance level, and ratio of-MDRO carriers to non-MDRO carriers as 1:2, with a prevalence ratio (PR) of 2.00 and 5% attrition, the sample size estimated was at least 323.

Data was collected aftertaking consent from parents or guardians of patients, on demographics, prior use of antibiotics for > 48 h in the last 6 months, history of vaccination in last 6 months, exposure to health care facility regardless of the time of exposure, ICU stay for more than 72 h, and about the prior use of medical devices (urinary catheter, central venous lines etc.) in last 1 year. We cultured the nasal swab for MRSA, and either rectal swab or stool specimen for ESBL Enterobacteriaceae, carbapenem resistant Enterobacteriaceae, vancomycin resistant enterococci, MDR Acinetobacter species, and MDR P. aeruginosa within 72 h of hospitalization for the detection of these pathogens. Patient was identified as MDRO carrier if a (nasal, rectal or stool) was positive, and the patient was without systemic infection caused by these organisms.

For the detection of VRE, rectal swabs/stool specimens were streaked directly onto selective agar plates and incubated aerobically for 24 h. Isolated colonies of enterococci were confirmed as E. faecalis or E. faecium by biochemical test and API strep kit test. These isolates were confirmed as vancomycin resistant by vancomycin disc on SB-Mueller-Hinton agar (SBMHA) plate and isolated on 6mcg/ml vancomycin plate with negative and positive control.

Swabs were also inoculated into a broth medium containing 6 mcg/ml vancomycin (Enterococcosel). Broth with color change from amber to black after 24–48 h incubation at 37 °C was then sub-cultured on blood agar and selective VRE media and incubated overnight at 37 °C. For further confirmation of isolated colonies API strep kit test and vancomycin disc on Mueller Hinton Agar (MHA) media was performed.

For identification of MRSA, nasal swabs plated directly onto sheep blood agar (SBA) and mannitol salt agar (MSA) and incubated aerobically for 24 h in a CO₂ incubator. A Swab was also inoculated into 2.5% NaCl BHI (Brain Heart Infusion) broth and after incubation at 37 °C for 24 h; it is inoculated onto colistin nalidixic acid agar (CNA), MSA, and SBA plates. The plates were incubated at 37 °C for 24 h. Morphologically resembling S. aureus was confirmed by tube coagulase (for secreted extracellular coagulase), DNase agar, and phenolphthalein phosphate agar. Broth or saline suspension of colonies confirmed as S. aureus was prepared and adjusted to achieve turbidity of 0.5 McFarland standards. Sensitivity testing carried out by disc diffusion method against cefoxitin 30 mcg disc susceptibility breakpoints for cefoxitin against S. aureus and was interpreted using Clinical and Laboratory Standards Institute (CLSI) guidelines.

Rectal swabs and stool specimens were inoculated into BHI broth and after overnight incubation at 35 ± 2 °C with ambient air, vortexed, and subcultured 100 μl onto MacConkey and CNA agar plate with vancomycin disc placed on 1st streak of CNA. Followed by overnight incubation, all gram negative bacilli were identified by conventional biochemical identification method that includes oxidase test, triple sugar iron (TSI) agar, Sulphide indole motility (SIM) agar, citrate agar, urea agar, Pseudomonas agar P, and Pseudomonas agar F. Identification of ESBL producing Enterobacteriaceae was performed by disc diffusion method by using combination of clavulanic acid (10 μg) and ceftazidime (30 μg). Both discs were placed on Muller Hinton agar plates which were earlier swabbed by respective culture and incubated for 24 h at 37 °C. More than 5 mm increase in the zone diameter for ceftazidime-clavulanic acid was considered positive ESBL production. Phenotypic disc diffusion method with Imipenem disc (10 μg) on Muller Hinton agar plates and incubated for 24 h at 37 °C were used for detection of carbapenem resistance in Enterobacteriaceae (zone size ≤19 mm), Acinetobacter species (zone size ≤18 mm), and P. aeruginosa (Zone size ≤15 mm). Identification of XDR S. Typhi was done using conventional biochemical methods i.e., TSI, SIM agar, and confirmed with the help of serotyping (BD Difco Salmonella O Antiserum Factor 9) and API 20E (Biomerieux).