Vaginal candidiasis prevalence, associated factors, and antifungal susceptibility patterns among pregnant women attending antenatal care at bule hora university teaching hospital, Southern Ethiopia | BMC Pregnancy and Childbirth

Study area and period

This hospital-based cross-sectional study was conducted at Bule Hora University Teaching Hospital from May to August 2023. The hospital is located in Bule Hora town and is one of the towns of the Oromia Regional state in the southern part of Ethiopia. Bule Hora is the capital of the West Guji zone located 467 km from Addis Ababa in the south and is a teaching hospital that provides health services to more than 1.5 million inhabitants in the zone. The hospital’s antenatal care (ANC) clinic provides routine antenatal screening services for more than 20 pregnant women per day. This town is bordered on the south by the Borena zone, on the west by the Southern Nations, Nationalities, and Peoples Region, on the north by the Abaya River, which separates it from the SNNPR, and on the east by the Guji zone.

Study design and population

A hospital-based cross-sectional study design was used among pregnant women who attended antenatal care (ANC) at the Bule Hora University Teaching Hospital from May to August 2023. All pregnant women who were randomly selected and attended the antenatal care (ANC) during the data collection period, and met the selection criteria, were included in the study.

Inclusion and exclusion criteria

All randomly selected pregnant women attending Bule Hora University Teaching Hospital ANC aged between 18 and 49 were included in the study, while the pregnant women who had either on/taken intra-vaginally or systemic antifungal therapy within the last 2 or 3 weeks were excluded.

Sample size determination

The sample size for this study was determined using the single population proportion formula. When the proportion of vaginal candidiasis (p = 25%) was taken from a previous study conducted at Debra Markos Referral Hospital [23], with the assumption of precision or degree of error 0.05, the confidence interval was 95% and the non-response rate was assumed to be 10%.

Adding 10% of the non-respondent rate, the final sample size was 317 pregnant women. Where n = Sample size, Z = value corresponding to 95% level of significance = 1.96, P = proportion of prevalence vaginal candidiasis in pregnant women = 25%, d = marginal error assumed to be 5%.

Sampling technique

Monthly estimated ANC attendant in the Bule Hora University teaching hospital, which was calculated from the antenatal care followers from registration. Then, the total sample size of pregnant women is proportional to their population size based on the previously registered ANC followers. Pregnant women attending ANC at the time of data collection were carefully chosen using a systematic random sampling method. To decide the interval (k), the estimated total number of attendants of ANC 1135 is divided into an allocated sample size of the study, that is, K = N/ n = 1,135/ 317 = 3.58 ≈ 4.

where, N = total ANC attendants, n = required sample size, K = sampling interval.

So, all fourth pregnant women attending ANC were included in the sample until the total sample size for this study was obtained in the hospital. Based on the client’s sequences, which were used as a sampling frame using systematic random sampling. The process was continued throughout data collection until the required sample size was achieved.

Data collection instruments and procedures

Three Medical laboratory technicians and two midwives were recruited for data collection. Training was provided for the data collector and supervisor. The sociodemographic information, clinical factors, and behavioral factors data were collected using structured questionnaires. The vaginal swab was collected from each participant using a sterile cotton swab moistened with physiological saline and then inserted and rotated gently to pick up the swab. In case an appropriate vaginal swab could not be obtained, alternatively, the participant was informed to collect a first voided urine sample through contamination control using a labeled screw-capped universal container [24].

Identification of candida species

Microscopy examination and culture procedures

The specimens were collected from each pregnant woman and immediately transported to the Bule Hora University Microbiology Laboratory. Smears were prepared from vaginal swabs or urine sediments, stained with Gram’s stain, and examined under a microscope using 10x and 40x objectives. The Gram stain revealed Gram-positive yeast-like budding cells, which were then cultured on Sabouraud Dextrose Agar (SDA) containing 2% chloramphenicol. The inoculated plate was incubated at 37 °C and examined after 24 h for cream-colored pastry colonies and budding yeast cells suggestive of Candida species. The SDA isolates were inoculated in Candida selective agar medium (Chromogenic Candida Differential Agar – TM 1977, New Delhi, India) using an inoculating needle and incubated at 37 °C for 72 h to ensure detection of mixed cultures by colon colors such as C. albicans (light green), C. tropicalis (blue to metallic blue), C. glabrata (cream to white), and C. krusie (purple-pink). The method is based on the differential release of chromogenic breakdown products from various substrates by Candida species after differential exoenzyme activity. It served as the presumptive identification of C. albicans, C. tropicalis, C. glabrata, and C. krusie [25].

Germ tube test (GTT)

A colony was transferred from the agar slant using a sterile wire loop and then inoculated into a germ tube test to determine the presence of germ tubes. A germ tube test was performed by mixing 3–4 colonies in 0.5 ml of human serum, incubated at 37 ° C for 2–4 h, and examined under a microscope using 10x and 40x objectives for germ tubes. The presence of Candida albicans was confirmed by observation of a short, slender, tube-shaped growth structure (pseudohyphae) [26, 27].

Antifungal susceptibility test

Antifungal susceptibility testing for all Candida isolates was performed using a modified disc diffusion method following the 2018 Clinical Laboratory Standards Institute (CLSI) guidelines. The test was carried out by adding 2% glucose and 0.5 µg/mL methylene blue dye to Mueller-Hinton agar. The suspension, prepared with normal saline and four identical colonies, was incubated overnight on Mueller-Hinton agar and then compared to the 0.5 McFarland standard. A cotton swab soaked in the fungal suspension streaked the modified Mueller-Hinton medium. Antifungal discs, including amphotericin B 100 µg, clotrimazole 10 µg, fluconazole 10 µg, itraconazole 10 µg, miconazole, and ketoconazole 30 µg, were placed on the agar using a disc dispenser. The plates were then incubated at 37 ° C for 24 h. Subsequently, the inhibition zones (zone diameters) were measured and interpreted according to CLSI guidelines [28].

Operational definition

Risk behavior

refers to the frequent use of antibiotics and certain types of contraceptives, which can increase the risk of Candida infection [29].

Pregnancy-related factor

Refers to the gestational period, which means the time from the beginning of pregnancy to the birth time of the subject that exposed them to the risk of Candida infection [30].

Clinical factors

Refers to post-medical diseases such as chronic diseases, such as diabetes mellitus, HIV, and previous episodes of candidiasis [31].

Data quality control

Data collectors received three days of training. The questionnaire was initially prepared in English, then translated into Afan Oromo, and subsequently retranslated into English to maintain the consistency of the questions. 5% of the questionnaires underwent pre-testing at the Bule Hora Health Center. Culture media were prepared following the manufacturer’s instructions, and sterility was confirmed by incubating 35% of the batch at 37 °C overnight to check for any microbial growth. Culture medium exhibited growth was discarded and replaced with a new sterile batch. The standard strain of C. albicans (American Type Culture Collection (ATCC 10231)) was used for quality control.

Data processing and statistical analysis

Data was entered using EPi data version 4.6 and exported to the SPSS Version-25 Statistical Package for analysis. Descriptive statistics, numbers, frequency, percentages, tables, and odds ratio (OR) were used to describe the findings. In addition, bivariate and multivariate logistic regression was used to assess the association between dependent and independent variables. A P-value less than 0.25 at 95% CI during the bivariate analysis was further calculated using multivariate logistic regression to avoid the effect of confounding. Finally, a p-value < 0.05 at 95% CI was considered statistically significant.