Abstract
Objectives
We aimed to identify the prevalence, bacterial isolates, antimicrobial susceptibility profile, and factors associated with asymptomatic bacteriuria (ASB) in patients with type 2 diabetes mellitus (T2DM) in rural southwestern Nigeria.
Methods
We performed a hospital-based cross-sectional study of patients with T2DM and ASB. Demographic and clinical data were collected using questionnaires. Urine samples were cultured using standard laboratory procedures, and bacterial colonies were isolated and antimicrobial sensitivity was performed using the disc diffusion technique. Relationships between variables were assessed using adjusted odds ratios (AORs) and 95% confidence intervals (CIs).
Results
Of the 280 participants, 73 (26.1%) had ASB (95% CI: 20.9%–31.2%). The most commonly identified isolate was E. coli (45/73; 61.7%), 100.0% of which were sensitive to cefuroxime but resistant to ciprofloxacin. Female sex (AOR, 6.132; 95% CI: 2.327–16.157), living below the poverty line (AOR, 2.066; 95% CI: 1.059–4.029), uncontrolled blood glucose (AOR, 2.097; 95% CI: 1.000–4.404), and a history of indwelling urethral catheterization (AOR, 14.521; 95% CI: 4.914–42.908) were associated with ASB.
Conclusion
The findings suggest that cefuroxime should be used as an empirical treatment, pending urine culture and sensitivity, and that efforts should be made to prevent ASB in rural southwestern Nigeria.
Keywords
Introduction
Diabetes mellitus is a metabolic disease that is characterized by high blood glucose concentrations. 1 Type 2 diabetes mellitus (T2DM) develops when the level of insulin secretion is insufficient to overcome the inability of target cells to utilize glucose normally, which is referred to as insulin resistance.1,2 Globally, in 2021, 536.6 million adults (10.5%) had received diagnoses of T2DM, and this total is predicted to rise to 783.2 million by 2045. 3 In sub-Saharan Africa (SSA), including Nigeria, there has been an increase in the prevalence of T2DM, despite an increase in people’s knowledge of the disease. This is likely the result of insufficient resources to address the challenges of its clinical diagnosis and treatment.2,4
One complication of T2DM is the high risk of asymptomatic bacteriuria (ASB), but this is underdiagnosed and undertreated. 4 People with T2DM are at a four-fold higher risk of ASB than those without,4,5 and the majority of previous studies have shown that untreated ASB progresses to symptomatic urinary tract infection (UTI), which is an important cause of morbidity in patients with diabetes mellitus.5–7 There is a close association between the retention of urine, owing to poor bladder function, and greater bacterial growth. 4 This rapid bacterial growth occurs in patients with T2DM in the presence of poor glycemic control, which results in poor blood supply and abnormal cell-mediated immunity. 8 Genetic variants, such as the C1QA gene polymorphism, as well as immunocompromise, are linked to the development of ASB in patients with T2DM. 9
There are regional variations in the prevalence of ASB in patients with T2DM, including in southwestern Nigeria, where its prevalence has been reported to be 16% 10 and 26.6%, 11 although higher prevalences of 32% and 38.3% have been reported in India and southwestern Cameroon, respectively.7,12 A number of factors, including age, the duration of diabetes, and poor socioeconomic status have been suggested to contribute to these variations.8,13,14 Furthermore, comorbidities such as human immunodeficiency virus (HIV)/acquired immune deficiency syndrome (AIDS), obesity, chronic renal disease, urinary tract defects, and a history of UTI have been reported previously.6,8,15
Previous studies performed in SSA have shown that Escherichia coli (E.coli), Staphylococcus aureus (S. aureus), and Klebsiella species are the most common bacterial isolates from urine samples in patients with T2DM.6–8 However, with the exception of those for pregnant women and for patients about to undergo urological procedures, there are no standard guidelines in Nigeria for the management of ASB in patients with T2DM.6,16 Therefore, early diagnosis, the identification of the etiological agent, and appropriate treatment could help to reduce the incidence of the complications associated with ASB in such patients. Even though an association between poor glycemic control and the development of ASB in patients with T2DM has been shown in several previous studies,4,8,13,14 the relationship between glycated hemoglobin (HbA1c) level and ASB has not been thoroughly studied in rural southwestern Nigeria. This is important because, to the best of our knowledge, most of the Nigerian population lives in a rural environment. Therefore, in this study, we aimed to determine the prevalence of ASB and evaluate the relationships of ASB with sociodemographic parameters, co-morbid conditions, and urinary tract-related factors in patients with T2DM who were attending a tertiary hospital in rural southwestern Nigeria.
Materials and methods
Study setting
The study was conducted between October and December 2022 at the family medicine clinic of a tertiary institution that is located in a rural community of southwestern Nigeria. This community had a population of 219,000, according to the 2006 national census, and an annual growth in the population of 3.2%. 17 This center serves as a referral center for the two primary health centers in the area, and for patients attending private and other government-run hospitals in the state.
Study design and sample
We performed a health facility-based cross-sectional study of patients with T2DM, defined using the American Diabetes Association criteria. 18 The inclusion criteria were the provision of informed consent and a diagnosis of T2DM. The exclusion criteria were urethral catheterization, the presence of signs or symptoms suggestive of UTI, and the use of antibiotics during the 2 weeks prior to the start of the study.
Sample size determination
The sample size was calculated using the formula 19 z2pq/d2, where ‘p’ is the prevalence (21.0%) of ASB calculated during a study previously conducted in a developing country, 8 z is the type 1 error (5%), and d is the margin of error. Using z = 1.96, p = 0.21, q = 1−p, and d = 0.05, N = (1.96 ×1.96 × 0.21 × 0.79)/(0.05)2 and N = 0.63732/0.0025, equal to 255. An estimated attrition rate of 10% was then added, such that the minimum required sample size was calculated to be 280.
Selection of participants
We used systematic sampling to select the study participants from the family medicine clinic register for 2021. A mean of 10 patients with T2DM were seen daily, which yielded a total of 650 (sample frame) over a period of 13 weeks that the study was conducted. Using the formula K (sampling interval) = sample frame (650) divided by the required sample size (280), K was calculated to be approximately 2. To select the first participant of the day, a simple random sampling technique (balloting) was used. A ballot box containing two pieces of paper, one labeled ‘NO’ and the other labeled ‘YES’, was presented to the first two patients on the list on their arrival at the clinic, and the patient who picked the ‘YES’-labeled piece of paper became the first participant for the day. Thereafter, every third patient who gave their informed consent to participate was selected by systematic sampling, until the calculated sample size was reached. To identify the selected participants and prevent their re-sampling on subsequent days, a tag was attached to the notes for each selected participant.
Data collection methods
A standardized, interviewer-administered questionnaire was used to record the participants’ sociodemographic characteristics (age in years, sex, marital status, educational level, occupation, and monthly income), the time elapsed since their diagnosis of diabetes, any history of catheterization, and any previous history of UTI. Furthermore, any history of co-morbid conditions, such as hypertension, obesity, chronic renal disease, and HIV/AIDS was also identified from their clinical records.
Assessment of glycated hemoglobin (HbA1c)
Approximately 4 mL of venous blood was collected from each participant into fluoride- and EDTA-coated tubes and sent to the hospital laboratory, where the HbA1c level was measured immunochemically on glycated hemoglobin (HbA1c) was measured immunochemically on a DCA 2000 HbA1c auto analyzer (Bayer AG, Leverkusen, Germany), using kits supplied by Boehringer Mannheim (Ingelheim am Rhein, Germany) 20 . Commercially prepared standards and control samples were used to ensure the accuracy of the test results. The glycemic status of the participants was categorized as good if their HbA1c was ≤7.0% (≤53 mmol/mol) and poor if it was >7.0% (>53 mmol/mol). 1
Clean-catch midstream urine sample collection
The participants were instructed regarding the method of collection of approximately 20 mL of midstream urine using the “clean catch” method in a wide-mouthed screw-capped universal urine container. The participant’s identification number, date, and time of collection were written on the outside of each container. The collected samples were kept in a cold box and sent to the medical microbiology laboratory of the study center for processing within 45 minutes of collection.
Bacterial isolation through urine culture
Bacterial isolation was performed in accordance with national detection guidelines. Culture and sensitivity were performed using the standard wire-loop method. Briefly, a loop-full of well-mixed urine was streak-inoculated onto cystine/lactose/electrolyte-deficient, MacConkey, and Chocolate agar plates, which were incubated aerobically at 37°C for 24 hours. A diagnosis of ASB was made in female participants if there were >105 cfu/mL in two consecutive urine cultures and in male participants if there was a single urine culture that contained ≥105 cfu/mL. Urinary pathogens were characterized using microbiological and biochemical tests and their identities confirmed using the API®20E identification system (BioMerieux SA, Marcy I’Etoile, France).
Antibiotic susceptibility testing
Antibiotic susceptibility testing was performed using the Kirby–Bauer Disc diffusion test and 0.5 McFarland standard turbidity of the inocula, and the Muller–Hinton agar inhibition zone diameters were measured. 21 The following antibiotic discs (Oxoid, Ltd., Basingstoke, UK), which included representatives of antimicrobials for the treatment of methicillin-resistant S. aureus (MRSA) infections and the cephalosporin group (cefuroxime (30 µg), clindamycin (2 µg), ciprofloxacin (5 µg), amoxicillin-clavulanate (20/10 µg), fosfomycin (200 µg), nitrofurantoin (300 µg), and erythromycin (15 µg)) were used. The selections for testing were performed and the results were interpreted by two medical microbiologists in the hospital, according to the Clinical Laboratory Standards Institute Protocols. 22 A senior medical microbiologist cross-checked the results and ensured that quality control was maintained.
Definitions of terms
ASB was defined as the presence of ≥105 cfu/mL actively multiplying bacteria in the urine, in the absence of any obvious signs or symptom of UTI. 4 T2DM was defined using documented treatment with antidiabetic medication or a blood glucose concentration >7.0 mmol/L. 1 The presence of concomitant chronic diseases was recorded. 1
Ethical considerations
The Ethics and Research Committee of the Federal Teaching Hospital, Ido Ekiti, Nigeria approved the study (approval number ERC/2022/09/28/851A). The methods and objectives of the study were carefully explained to each patient individually, and all were thoroughly informed about the risks and advantages of the procedures. Written informed consent for treatment was obtained from each of the participants (a signature or thumbprint was provided) before they participated in the study, and they were enrolled if willing to participate. The participants were free to refuse to participate or to discontinue their participation at any time without losing any healthcare, and there were no additional benefits for those who participated. For participants who could not read or write, the questionnaire was translated from English into their local language by an independent interpreter who also served as their legal representative. The responses were subsequently translated into English to maintain their consistency. All the recorded information was kept anonymous, and patient confidentiality and privacy were ensured throughout the study. The study was administered at no cost to the respondents. The reporting of the study conforms to the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) statement. 23 All the patient data have been de-identified in the manuscript. Our methodology may be reproduced by fellow researchers if they desire.
Treatment of participants with significant ASB
The participants who were diagnosed with significant ASB were referred to a physician for further evaluation. Only those who had two or more co-morbidities were treated prophylactically to prevent the complications of ASB. 24
Statistical analysis
The data collected were checked, cleaned, and entered into EPI Info Version 7.0 (Informer Technologies, Inc., Los Angeles, CA, USA, then exported to SPSS version 22.0 (IBM Corp., Armonk, NY, USA) for analysis. The prevalence of ASB was calculated as by dividing the number of positive cultures by the total sample size, and is reported as a percentage. The chi-square test was used to evaluate the relationships of the sociodemographic parameters, co-morbid conditions, and glycemic control of the participants with ASB. All the factors found to be significantly associated with ASB in the bivariate analysis (p < 0.05) were included in a stepwise multivariate logistic regression model to identify factors that are independently associated with ASB, and results are presented as adjusted odds ratios (aORs) with corresponding 95% confidence intervals (CIs). A p-value <0.05 was regarded as indicating statistical significance.
Results
A total of 280 participants were studied. Their mean age was 54.5 ± 11.4 years (range: 36 to 82 years). There were more women (175/280; 62.5%) than men (105/280; 37.5%). The majority of the participants were married (196/280; 70.0%) and their most common occupation was farming (118/280; 42.1%) (Table 1).
Sociodemographic data for the participants.
Of the participants, 167/280 (59.6%) had their blood glucose concentrations under control and 73/280 (26.1%) had ASB (95% CI: 20.9% to 31.2%). For the 73 participants that had ASB, E. coli was the commonest organism isolated (in 45/73; 61.7%) (Table 2). All of the organisms isolated were 100% sensitive to cefuroxime but fully resistant to ciprofloxacin (Table 3).
Clinical parameters of the participants.
Antibiotic sensitivity pattern for the bacterial isolates obtained from the participants.
CEF, cefuroxime; CD, clindamycin; CPF, ciprofloxacin; ETM, erythromycin; AM-CL, amoxicillin-clavulanate; FFM, fosfomycin; NIT, nitrofurantoin.
We found statistically significant associations of ASB with the age of the participants (χ2 = 26.715, P < 0.001), their sex (χ2 = 5.545, P = 0.019), and their income (χ2 = 4.652, P = 0.031) (Table 4). There were also statistically significant associations of ASB with the level of glycemia of the participants (χ2 = 11.573, P = 0.001), their HIV/AIDS status (χ2 = 7.951, P = 0.005), the presence or absence of a history of indwelling urethral catheterization (χ2 = 43.366, P = 0.001), the presence or absence of a history of UTI (χ2 = 36.354, P < 0.001), and the period of time elapsed since a diagnosis of T2DM had been made (χ2 = 7.415, P = 0.006) (Table 5).
Relationships between sociodemographic parameters and ASB in the participants (n = 280).
ASB, asymptomatic bacteriuria. Poverty line: USD 2.15/day.
Relationships of co-morbidities and urinary tract-related factors with ASB in the participants (n = 280).
ASB, asymptomatic bacteriuria; HbA1c, glycated hemoglobin; HIV/AIDS, human immunodeficiency virus/acquired immune deficiency syndrome; DM, diabetes mellitus; UTI, urinary tract infection.
After adjustment for possible confounders, female participants were 6.132 times more likely to have ASB (95% CI: 2.327 to 16.157), participants who lived below the poverty line were 2.066 times more likely (95% CI: 1.059 to 4.029), those with poor glucose control were 2.097 times more likely (95% CI: 1.000 to 4.404), and those with a history of indwelling urethral catheterization were 14.521 times more likely (95% CI: 4.914 to 42.908) (Table 6).
Results of the multivariate regression analysis to identify factors associated with ASB in the participants.
AOR, adjusted odds ratio; ASB, asymptomatic bacteriuria; CI, confidence interval; DM, diabetes mellitus; HbA1c, glycated hemoglobin; OR, odds ratio.
Discussion
The mean age (54.5 ± 11.4 years) of the participants was comparable to the mean age of the participants in the study by Banerjee et al., probably because of similar sociodemographic factors and study design. 25 However, it was lower than that of the participants in the studies by Asghar et al., 26 Odetoyin et al., 10 and Matthiopoulou et al, 6 and higher than that of the participants in the studies by Venketesen et al. 7 and Bissong et al. 12 These differences may be results of differences in sample size, study location, or the age group of the participants.
The prevalence of ASB in the study sample was 26.1%, which is comparable with the prevalences reported for southwestern 11 and southern Nigeria. 27 These figures are similar because of the similar geographical locations and study design. However, other authors have reported higher4,12 or lower (in Nigeria, 11 Greece, 6 and India 8 ) prevalences. These variations may be the results of variations in sociodemographics, geographical factors, and/or the screening methods used.4,28 The high susceptibility of patients with diabetes to ASB is not yet fully understood; however, previous studies have identified defective polymorphonuclear leucocyte function and poor blood supply as contributing factors.4,8
The most frequently isolated organism in the present study was E.coli (61.7%), followed by group B Streptococcus pneumonia (S. pneumonia) (16.4%), S. aureus (12.3%), and Klebsiella pneumonia (K. pneumonia) (9.6%). The prevalence of E.coli in this study is comparable with those identified in previous studies, in which E. coli was also the most commonly isolated organism.4,6,29 The explanation for the high prevalence of E. coli infection in patients with diabetes is that it can readily initiate infection by binding to glycoconjugate receptors on the epithelial cells in the human urinary tract. 7 However, the previous studies showed variations in the prevalences of infections with the other organisms. Aghemwenhio et al. found that K. pneumonia was the second most frequently isolated organism, followed by S. aureus, 29 and Venkatesan et al. found that K. pneumonia was the second most frequently isolated organism, followed by Enterococcus faecalis, S. aureus, Pseudomonas, Proteus, and Candida spp. 7 In contrast to the findings of the present study, Bissong et al. found that coagulase-negative staphylococci were the predominant organisms, followed by Klebsiella, Candida, and Serratia spp. 12 Finally, Alebiosu et al. found that Klebsiella pneumonia was the most prevalent organism, followed by E.coli. 11 These differences may be the results of variations in the sociodemographic characteristics, personal hygiene, and/or co-morbidities of the participants. Nevertheless, the results of the present study should assist clinicians to instill awareness of the high incidence of ASB and how best to prevent such infections in patients with diabetes in rural settings.
In the present study, the antibiotic sensitivity of the various pathogens was tested, and we found that cefuroxime was the most effective antibiotic, with all of the 73 isolates being sensitive to it. Therefore, cefuroxime could be considered to be the ideal first-line drug for the treatment of ASB in patients with diabetes. In contrast, Bharti et al. 8 found that the majority of organisms isolated were resistant to ceftriaxone. We also found that nearly all of the organisms isolated were resistant to ciprofloxacin, similar to the results of a study by Bharti et al., 8 in which the majority of the organisms isolated were resistant to this antibiotic.
Interestingly, the multivariate logistic regression showed that female sex is associated with ASB in patients with T2DM. This finding is confirmatory of those of several previous studies, in which female patients were found to be at higher risk of ASB than male patients.7,12,14 The higher prevalence of ASB in women may be the result of the lower pH and altered microbiota of the vagina in patients with diabetes14,28 or the relatively easy access to the female urethra for enteric bacteria because of the close proximity of the vulva and anus. 14
Previous studies have demonstrated associations between socioeconomic factors, and particularly the level of poverty, and ASB in patients with T2DM.4,30 The findings of the present study are consistent, because we found that participants who were living below the poverty line were at a higher risk of ASB. 30 These findings imply that more rapid interventions are required to manage this condition in patients with diabetes in low-income countries.
In the present study, we found a significant relationship between the outcome of urine culture and HbA1c level: patients with poor glycemic control were more than twice as likely to have ASB. Gurjar et al. 4 and Matthiopoulou et al. 6 also showed that patients with poor glycemic control were at a higher risk of ASB. Furthermore, in the study by Jha et al., all the cases of ASB were identified in patients with hyperglycemia. 31 Hyperglycemia results in glucose spilling over into the urine, which favors bacterial growth.4,21,32 However, a study by Raoofi et al. 13 showed no association between high plasma glucose concentration and the risk of ASB in patients with diabetes. This disparity may be the result of variations in plasma glucose concentration, the lifestyles of the participants, and/or their compliance with therapy using oral hypoglycemic agents.
We also found that patients with a previous history of urethral catheterization were at a higher risk of ASB, which is consistent with the findings of several previous studies.33,34 Urethral catheters are indwelling foreign bodies that can serve as means of access for microorganisms, and previous studies have shown that 5% of patients with chronic medical conditions require urinary catheterization.32,33 The association between ASB and long-term indwelling catheterization during the treatment of chronic medical conditions is an important public health concern, owing to the proliferation of multidrug-resistant bacteria. The etiology of ASB in patients with long-term indwelling catheters involves the acquisition of a biofilm on their surfaces that promotes bacterial growth and the eventual migration of the bacteria into the bladder. 35 The extent of the bacterial colonization depends on the length of time that the catheter is in situ: a previous study showed that for each day an indwelling catheter is kept in situ, the risk of bacterial colonization increases by 3% to 7%. 34 However, in this study, the participants had a previous history of urethral catheterization, but the impact of the duration of catheterization was not assessed. Therefore, studies of the relationship between the duration of catheterization and ASB should be performed to better understand the importance of ASB for patients with diabetes who have a history of indwelling catheter placement.
The present study had several limitations. First, it was cross-sectional in nature, and therefore inferences regarding causality cannot be made. Second, it was conducted in a hospital setting and involved a relatively small number of participants, and therefore the findings may not be representative of the wider population of patients with diabetes. However, it provides additional information regarding the burden of, and the risk factors associated with, ASB in patients with T2DM in rural southwestern Nigeria.
In conclusion, we have shown that the prevalence of ASB in patients with T2DM in rural southwestern Nigeria is 26.1% and that E. coli is the most commonly isolated organism. Furthermore, ASB is more common in women, in patients living below the poverty line, in those with poor glycemic control, and in those with a history of urethral catheterization. The results of the study suggest that cefuroxime should be administered empirically as a treatment, pending the outcome of urine culture and sensitivity testing. The data imply the need to raise awareness and improve the prevention of ASB in rural southwestern Nigeria. Indeed, there may be need for routine screening for ASB in patients with T2DM, to facilitate its early diagnosis and treatment and to reduce the risk of the associated complications.
Footnotes
Acknowledgements
The authors would like to thank the nurses and resident physicians of the family medicine clinic and the management of FETHI, where the study was conducted, as well as the participants in the study.
Author contributions
AOI: Conceptualization, methodology, formal analysis, data acquisition, processing, coding, literature review, interpretation of findings, writing—review and editing.
ISB: Data processing, formal analysis, data analysis, supervision, review of the manuscript for intellectual content.
OAA: Literature review, methodology, formal analysis, data analysis, interpretation of findings.
KMO: Literature review, data curation, formal analysis, interpretation of findings, writing—review and editing.
GOA: Data acquisition, methodology, review of the manuscript for intellectual content.
KRA: Literature review, data analysis, formal analysis, interpretation of findings.
OOO: Literature review, data curation, formal analysis, interpretation of findings, writing—review and editing.
OOS: Data curation, analysis, methodology, writing—review and editing.
AKA: Literature review, methodology, data analysis, writing—review and editing.
Declaration of conflicting interest
The authors declare that there is no conflict of interest.
Funding
This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.
