Analytical profile index

Blood, lung aspirate, cerebrospinal fluid (CSF), pleural fluid, and other microbiological samples were processed at the MRC Basse Field Station using standard methods [23 (link)]. Aerobic and anaerobic blood cultures were taken for each adult patient, with 5 mL of blood added to each bottle. Children had 2–3 mL added to a single pediatric blood culture bottle. Bottles were weighed before and after blood was added to ensure adequate filling, with weights reported to clinicians. Biochemical tests, including the Analytical Profile Index (bioMérieux, UK), and serological tests were used to confirm suspected pathogens. Latex agglutination tests were used on all CSF samples to identify Streptococcus pneumoniae, Neisseria meningitidis, and group B streptococci. Results were aggregated with culture-positive cases. Pneumococcal isolates were serotyped at the MRC Fajara laboratory using a latex agglutination assay employing factor and group-specific antisera (Statens Serum Institut, Copenhagen, Denmark) and polymerase chain reaction [24 (link)]. The laboratories in Basse and Fajara submitted to external quality assurance throughout the study (UK National External Quality Assessment Service, World Health Organization [WHO] reference laboratory in Denmark, and the Royal Australasian College of Pathologists).

At each study visit, sterile swabs (BactiSwab, Remel, Lenexa, KS) were used to obtain samples from 5 anatomical sites (ie, perirectal area, groin, nares, oropharynx, and dominant hand), as well as any wounds, indwelling urinary catheters, or feeding tubes. All patients provided consent. Swabs from the hand were enriched overnight in brain–heart infusion broth at 36°C to improve culture sensitivity, considering lower bacterial burden from hands compared to anatomical sites. All swabs were plated on MacConkey agar. Genus and species of colonies were identified using bioMerieux analytical profile index (API, bioMerieux, Marcy l’Etoile, France). Antimicrobial susceptibility testing by disk diffusion was performed according to Clinical and Laboratory Standards Institute (CLSI) guidelines (M100-S23). Gram-negative bacilli were considered antimicrobial resistant (R-GNB) if nonsusceptible to ciprofloxacin, ceftazidime, or imipenem for all species except Proteus mirabilis. P. mirabilis isolates were considered R-GNB if they were nonsusceptible to ciprofloxacin, ceftazidime, or meropenem.

Positive blood cultures were sub-cultured on sheep-blood, MacConkey, chocolate, and Sabouraud Dextrose agar (SDA) plates. Standard microbiological methods such as conventional biochemical tests and analytical profile index (API) (bioMérieux SA, Marcy l’Etoile, France) were used for identifying bacterial isolates. Fungal isolates that grew on SDA were Gram stained, and for yeast cells germ-tube test was performed. Final identification of both bacterial and fungal isolates was done with MALDI-TOF MS using the Microflex LT instrument and MALDI Biotyper 3.1 software (Bruker Daltonics, Bremen, Germany).

All CSF samples were processed in the microbiology laboratory according to the standard operating procedure [20 ]. Briefly, CSF samples were inoculated on sheep blood agar, chocolate agar, and MacConkey agar. Bacterial identification was performed by analytical profile index (API) (Biomerieux) [20 ]. Antibiotic susceptibility was determined by the Kirby-Bauer disc diffusion method and minimum inhibitory concentration (MIC) determination according to the Clinical and Laboratory Standards Institute (CLSI) guidelines [13 ] The laboratory deployed antibacterial testing of the drugs per the CLSI criteria for each bacterium and the laboratory's availability of antibiotic discs for the given years. Agents administered by oral routes only, first and second-generation cephalosporins and cephamycins, doripenem, ertapenem, imipenem and lefamulin, clindamycin, macrolides, tetracyclines, fluoroquinolones were excluded for the CSF isolates as per CLSI recommendation [13 ]. Based on a review of clinical practice in PINS throughout the study, the following antibiotics were tested, amikacin, gentamicin, cotrimoxazole (trimethoprim-sulphamethoxazole), ceftriaxone, ceftazidime, cefoperazone, cefotaxime, cefepime, piperacillin, ampicillin, amoxicillin-clavulanic acid, piperacillin-tazobactam, ampicillin-sulbactam, meropenem, oxacillin, penicillin, vancomycin, and linezolid.