This was a cross sectional study in which pregnant women registered for ANC within the months of August to November 2017 in the major Integrated Health Centers of the Buea Health District and gave signed informed consent were recruited in this study. Data on the symptoms of UTIs reported, demographic characteristics, medical history and information on the risk factors tested were obtained using a questionnaire. Urine samples were collected for urine dipstick analysis and culture for the identification of bacteria. The validity of dipstick test in the screening of UTIs was calculated and antimicrobial susceptibility testing was carried out on the isolates by the disc diffusion method.
This study was carried out in the Buea Health District, located in Buea, the capital city of South West Region of Cameroon. It is situated on latitude 4o.1538’ North, longitude 9o.2337’ East and 959 m above sea level. Buea Health District is one of the four health districts in Fako Division. With regards to its health structure, the Buea Health District is made up of 7 health areas. It has a total population of 153,827 as of 2015 . The Integrated Health Centers (IHC) sampled included; Mile 16 IHC, Muea Divisional Hospital, Buea road IHC, Bova IHC, Tole IHC and Molyko IHC.
Sample size estimation and study population
The sample size was calculated as previously described for cross sectional studies . Following this method, a minimum sample size of 284 participants was obtained assuming a prevalence of 23.5% bacteriuria reported in the same area .
All pregnant women attending ANC in the major Integrated Health Centers in all Health Areas of the Buea Health District were included in this study. Each participant signed an informed consent form. Assent was sought from participants < 21years old and consent from their parents/guardian. Pregnant women who were on antibiotics or had taken any within the previous 2 weeks, as well as those who refused to give consent, were excluded from the study.
Sterile urine samples were collected from each participant for urinalysis and urine culture. The women were given instructions on how to collect a clean catch mid-stream sterile urine sample. They were asked to clean the labiae and area around the urethra with sterile water and urine collected with the labiae held apart. They were to pass out urine into the toilet pot for a few seconds, then collect a portion of the mid-stream urine before emptying their bladder into the toilet pot. About 3 mL of the specimen was poured into another wide-mouth container and used for urine dipstick analysis and microscopy. The rest of the sample was kept in a cold box and transported to the Faculty of Science Clinical Diagnostic Laboratory, University of Buea within 3 hours of sample collection for culture. Capillary blood from a finger prick was also collected for the determination of haemoglobin concentration using a haemoglobinometer (Mission HB haemoglobinometer, Italy).
Macroscopy and dipstick analysis
The color and consistency of freshly collected urine samples were recorded. A Combii 11 dipstick (URIT Medical Electronic Co-LTD) was used for urinalysis. The strip contained the following analytes; ascorbic acid, specific gravity, pH, ketone, glucose, urobilinogen, bilirubin, nitrite, leucocytes and proteins. The reagent strip pad was completely immersed in fresh urine and observed for color changes. These were read against the color chart on the labeled test strip container and the results recorded.
The sensitivity and specificity of nitrite and leucocyte esterase of the urine dipstick analyses were calculated using formulae previously described .
Sensitivity = sum of participants who were positive to both culture and dipstick
Total number of participants positive to culture
Specificity= sum of participants who were negative to both culture and dipstick
Total number of participants’ negative to culture
Considering the two parameters simultaneously, the combined sensitivity and specificity were calculated thus;
Net (combined) sensitivity = (sensitivity of leucocytes) + (sensitivity of nitrites) – (sensitivity of leucocytes x sensitivity of nitrite)
Net (combined) specificity = specificity of leucocytes x specificity of nitrites.
Urine culture and biochemical testing
Cysteine Lactose Electrolyte Deficient (CLED) agar (Liofilchemsrl. Roseto d. Abruzzi TE-Italy), was prepared according to manufacturer’s instructions and stored in the refrigerator at 2-8oC. Prior to inoculation, the pre-casted culture media were removed and placed on a clean bench to warm to room temperature. Ten microliters (10 μL) of each sample was streaked on the media and incubated at 37oC for 18- 24 hours, after which the number of colonies on each plate was recorded. Only samples with bacterial count ≥105 colony forming unit (CFU) per mL of urine were considered positive. Colony morphology on CLED was recorded. Pure cultures were prepared by sub-culturing on nutrient agar. Smears of isolates were prepared and Gram stained . The Coagulase and Catalase tests were performed on gram positive isolates while for the gram negatives, glucose and lactose breakdown were tested on KIA medium as well as indole test and API20E used for species identification.
Antibiotic susceptibility testing
Antimicrobial susceptibility of pure isolates was tested by the disc diffusion method on Mueller-Hinton agar. The antibiotics (Cypress Diagnostics, Italy) used included: gentamycin (20 μg), ciprofloxacin (5 μg), trimethoprim (1.25 μg) and sulphamethoxazole (23.75 μg), kanamycin (30 μg), chloramphenicol (5 μg), tetracycline (30 μg), ceftriaxone (30 μg), cefatoxime (30 μg) and ampicillin (10 μg).The plates with the discs were incubated in an inverted position at 37°C overnight. Diameters of zones of inhibition were measured using a ruler to the nearest mm and compared with recommended CLSI M100-S12 Standards ([13) and the organisms reported as susceptible, intermediate, or resistant to the antimicrobial agents.
Data was entered into a prepared template on Microsoft Excel 2010 and later exported to the Statistical Package for Social Sciences (SPSS) for Windows Version 18.0 (Chicago, IL, USA) and EPI Info 7.0 (CDC, USA) for analyses. Relationships between categorical variables were assessed using either the Chi Square test or the Fischer Exact test. A bivariate analysis was performed to investigate the level of association of the risk factors (exposure) to the outcome variable (Culture positivity). Ninety-five percent (95%) confidence intervals and Odds ratios (OR) were reported for key parameters under investigation to quantitate associations. OR>1with the null value-1 lying outside the 95% confidence interval were considered as positive associations whereas OR< 1 were consider as negative association. Statistical significance was set at p value ≤0.05.
Ethical clearance was granted by the Faculty of Health Sciences Institutional Review Board (FHS-IRB), University of Buea. Administrative clearances were obtained from the South West Regional Delegation of Public Health and the Buea District Health Service. Permission was obtained from the management of the participating health facilities. Every participant indicated their willingness to participate in the study by signing an informed consent form. Participants < 21 years gave assent and their parents/guardians gave informed consent. Participants were free to withdraw at any stage of the research without any sanctions. All experiments were performed in accordance with the relevant guidelines and regulations. Laboratory results of urine analysis and culture were sent to the respective health centers in sealed envelopes for delivery to the participants by the Chief of Centers. Personal information of all participants was kept confidential and electronic files were pass-word protected and accessible only to authorised research group members.