Study design
We conducted a cross-sectional study recruiting pregnant women attending routine antenatal care (ANC) in rural Burkina Faso. Urine samples routinely obtained for dipstick analysis (glucose and protein) were semi-quantitatively cultured by dipslide technique to assess for asymptomatic bacteriuria (ASB). For the purpose of this study, isolates growing in counts of ≥ 104 colony forming units (CFU/ml) belonging to the Enterobacterales species and Enterococcus faecalis were considered as "significant growth". Antibiotic susceptibility testing was done for E. coli isolates. AMR profiles were compared to E. coli isolates obtained from blood culture surveillance studies performed in the same district and the same laboratory. Women were asked about recent antibiotic use prior to sampling and urine samples were screened for antibiotic residues.
Study site, period and participants, routine antenatal care
The study was conducted from October 2016 to September 2018 at the Clinical Research Unit of Nanoro (CRUN) in the Center-West Region of Burkina Faso. Samples were obtained in 9 health centers within the Health and Demographic Surveillance System (HDSS) of CRUN, at 11–38 km from CRUN (Fig. 1). The HDSS monitors changes in a total population of 60,000 persons distributed over 24 villages [9]. Routine ANC is provided at the health centers and is organized in morning hours between 7 and 12 a.m. A total of 4 ANC visits are recommended during each pregnancy [10] and comprise collection of demographic data (age, week of pregnancy and Gestation Parity Abortion score [GPA score]) and uranalysis for glucose and protein by dipstick test.
Study intervention: urine culture and collection of demographic and clinical data
Informed consent was sought from all women attending routine ANC by the ANC nurse. After written consent was obtained, a study nurse provided a sterile cup and instructions on how to obtain a clean midstream urine sample. Apart from routinely collected ANC data, information on antibiotic use in the past 2 weeks, and signs and symptoms of urinary tract infection were collected. For semi-quantitative culture, dipslide devices (Uricult MC/CLED, International Medical Products, Brussels, Belgium and Servocults, Meus S.R.L, Padova, Italy) were used. The dipslide consisted of cysteine-, lactose, and electrolyte-deficient (CLED) agar on one side and MacConkey agar on the other side. Inoculation was done by the study nurse: dipslides were fully submerged in the urine sample, alternatively, a sterile pipette was used to inoculate both agar slides. The dipslide was then placed straight-up on a piece of absorbent paper to allow excess urine to leak off. Subsequently, the urine was tested for presence of glucose and protein using a dipstick analysis (UroColor strips [Standard Diagnostics, Gyeonggi-do, Republic of Korea] or Urine-10 strips [Cypress Diagnostics, Hulshout, Belgium]). Leukocyturia was quantified according to the manufacturers’ instruction as negative (−), + (25–74 cells/µL), ++ (75–499 cells/µL), or +++ (500 or more cells/µL). The urine dipslides and the left-over urine samples were stored in the fridge (2–8 °C). Transport to the laboratory was done in a light protected box by motorcycle, within 24 h after collection, at room temperature.
Semi-quantitative culture, bacterial identification and antibiotic susceptibility testing
Upon reception at the laboratory of CRUN, dipslides were incubated for 16–24 h at 35 °C. Grown cultures were assessed for colony counts by comparing the number of colonies to the figure provided in the product's instructions for use. Bacterial isolates were identified using standardized biochemical techniques and API (bioMérieux, Marcy l’Etoile, France) in case of doubtful test reactions. The isolates were subsequently stored in Tryptic Soy Agar (CM0131, Oxoid Ltd). All bacteria growing in counts of ≥ 104 colony forming units/ml (CFU/ml) except for non-fermentative Gram-negative bacteria and bacteria considered as contaminants [Bacillus spp. and coagulase-negative staphylococci (CNS)] were shipped to the Institute of Tropical Medicine in Antwerp (Belgium) for confirmation of identification by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) technology (Bruker MALDI Biotyper, Bruker, Billerica, MA, US) at the University Hospital of Leuven (Belgium). Isolates identified as E. coli were processed for AST by disk diffusion (Neo-Sensitabs, Rosco Diagnostica A/S, Taastrup, Denmark) according to Clinical and Laboratory Standards Institute (CLSI) guidelines [11]. Combination disk tests (Neo-Sensitabs, Rosco Diagnostica A/S, Taastrup, Denmark) were performed to assess the production of Extended Spectrum ß-lactamases (ESBL) [11]. Neo-Sensitabs disk contents were as follows: nitrofurantoin 300 µg, fosfomycin 200 µg, ampicillin 10 µg, cotrimoxazole (trimethoprim 1.25 µg + sulfamethoxazole 23.75 µg), ciprofloxacin 5 µg, gentamicin 10 µg, ceftriaxone 30 µg, ceftazidime 30 µg, ceftriaxone-clavulanic acid 30 + 10 µg and ceftazidime-clavulanic acid 30 + 10 µg.
Antibiotic residue testing
Urine samples were tested for the presence of antibiotic residues as part of the work-up at the laboratory of CRUN. For each urine sample, a Mueller–Hinton plate was inoculated with 0.5 McFarland saline solutions of Bacillus spizizenii (ATCC 6633). An absorbent paper disk was saturated with urine and placed on the Mueller–Hinton agar. Plates were incubated at 35 °C for 18–24 h. The appearance of an inhibition zone around the urine disk was considered positive for the presence of residue antibiotics [12].
Comparison with clinical isolates
For comparison, AST results from clinical isolates of urine and blood cultures collected under different study protocols in the Nanoro district hospital and processed at CRUN during the same period were used. Two studies assessed the proportion and differentiation of malaria and bacteremia in the Nanoro district hospital [13, 14], a third study assessed the incidence and reservoir of non-typhoidal Salmonella bloodstream infection [15, 16] and finally, several isolates were obtained from a blood culture surveillance study for follow-up of antimicrobial resistance (unpublished data).
Definitions
For the purpose for this study, single-organism cultures with counts of ≥ 104 CFU/ml belonging to the Enterobacterales species or Enterococcus faecalis were considered as "significant growth" and the isolates were considered as “pathogens”. Isolates obtained from febrile patients are referred to as “clinical samples”. Asymptomatic bacteriuria was defined as the presence of actively multiplying bacteria in the urinary tract in patients that have no obvious symptoms of urinary tract infection (UTI) [17]. Skin- or environmental bacteria (CNS, Bacillus spp.), non-fermentative Gram-negative bacteria and bacteria growing as mixed flora (≥ 2 different isolates) were considered as contaminants [12]. In case a culture grew with mixed isolates including Enterobacterales, the latter were also subcultured for the purpose of antibiotic susceptibility testing (AST). Staphylococcus aureus isolates were not considered for antibiotic susceptibility testing.
Multi-drug resistance (MDR) for Enterobacterales was defined as resistance to the three principal oral antibiotic categories for urinary tract infection (penicillins, cotrimoxazole and fluoroquinolones). The number of parities of each participant was categorized as in nullipara (never given birth), primipara (given birth once), multipara (≥ 2 births) or grand multipara (≥ 5 births) [18].
Sample size, data registration and statistical analysis
In line with the CLSI M39 [19], a minimum number of 30 E. coli isolates was targeted for separate antibiotic susceptibility reporting. Assuming a prevalence of 5–10% asymptomatic bacteriuria with 10% contamination rate and E. coli being 40% of retrieved isolates, 6000 women were targeted. Data were recorded in a coded database (Microsoft Excel, Redmond, US). Differences in proportions were compared using as appropriate a Mann–Whitney-u test, a Kruskall Wallis test or a Chi-square test. For smaller sample sizes (value in one of the cells ≤ 5), the Fischer exact test was used. A p value of 0.05 was considered as statistically significant. Reporting of the methods and results was done according to the STROBE guidelines for cross-sectional studies [20].
Ethics
The study was approved by the national ethics committee of Burkina Faso (Comité d’Ethique pour la Recherche en Santé (Reference No. 2015-7-96 July 1st, 2015), the institutional review board of ITM, Antwerp (Reference 1008/15 from December 15th, 2015) and the ethics committee of the University Hospital of Antwerp (Reference 15/51/563, January 4th, 2016). Written informed consent was obtained before participation in the study. A screening log with reasons for refusal was completed at each health center included in the study. If ASB was diagnosed, laboratory staff of the study site communicated the recovery of clinically significant isolates and their AST results to the study investigator, who informed the ANC nurse or the clinician responsible of the ANC. Participants were treated according to national treatment guidelines.