| Abstract |
Ιntroduction: Diabetes mellitus (DM) is the most common endocrinologic disease, and has an increasing prevalence that has risen from 108 million cases in 1980 to more than 460 million cases in 2019 worldwide. Based on these trends, it is estimated that almost 700 million people will have diabetes by the year 2045, according to the International Diabetes Federation. Patients with diabetes are more prone to developing infections, and this may be due to multiple mechanisms involving abnormalities in innate and adaptive immunity, chemotaxis and phagocyte function. Asymptomatic bacteriuria (ASB) is defined as the presence of a single species of bacteria at specified quantitative counts ≥105 cfu/ml (≥108 cfu/l) in a voided urine specimen without signs or symptoms attributable to urinary tract infection (UTI). The prevalence of ASB in diabetics has been reported to be three fold higher than in patients without DM. Presence of ASB in diabetics has been correlated with several clinical, laboratory and genetic factors, as well. Single nucleotide polymorphisms (SNPs) of genes relating to innate immunity have been linked with the occurrence of ASB in patients with diabetes. C1q gene encodes for the initial molecule of the classical complement pathway and certain polymorphisms of C1q increase the risk of bacterial infections as well as other diseases. Moreover, several SNPs of Toll-like receptor 4 (TLR4) gene affect the genetic susceptibility to infections. The aim of this study was to identify independent risk factors associated with ASB and the responsible pathogenic microorganisms of ASB, as well as to investigate the association of C1q rs292001 (G>A) and TLR4 rs4986790 (Asp299Gly) SNPs with ASB in patients with Type 2 diabetes mellitus (T2D).
Patients & methods: This is a prospective case-control study performed from 2012 to 2019 at the University Hospital and the General Hospital of Heraklion Venizeleio, Heraklion, Crete, Greece. In the clinical part of this study 437 Cretan adult male and female patients with T2D attending the diabetes and hypertension outpatient clinics at both hospitals, were enrolled. Data regarding age; gender; body weight; height; Body mass index (BMI); findings from physical examination; family history of diabetes; duration of diabetes; glycosylated hemoglobin A1c (HbA1c); past medical history; previous antimicrobial use (within three months) or UTI within the last 12 months; recurrent UTIs (rUTIs); recent sexual contact; results of urine culture; laboratory data of blood and urine samples were collected. Quantitative urine cultures were performed by standard techniques using Columbia blood and MacConkey agar plates (BioMérieux, Marcy l’ Etoile, France). Bacterial species were identified by the use of standard biochemical methods and the Vitek 2 automated system (BioMérieux SA). Antimicrobial susceptibility testing was performed using the Vitek 2 system. Statistical analysis was based on Fisher’s exact test, Student’s t-test and Mann–Whitney U-test according to the variable’s type and all statistics were calculated using GraphPad Prism 6.0 (GraphPad Software, Inc., San Diego, CA). In the genetic analysis, the study population consisted of 76 patients with T2D and ASB (cases) and 76 patients with T2D without ASB (controls). Age and gender were well balanced between the study groups. Genomic DNA extraction was performed by using PureLinkTM Genomic DNA Mini Kit (Invitrogen, Waltham, Massachusetts, USA), according to the manufacturer’s protocol, and the extracted DNA was stored at −20oC until analyzed. C1q rs292001 and TLR4 rs4986790 SNPs were analyzed by polymerase chain reaction (PCR) amplification followed by restriction fragment length polymorphisms (RFLPs) assays. Genotypes were scored blindly and analysis of all ambiguous samples was repeated. PCR analysis of the C1q rs292001 SNP was performed using the upstream primer 5’-GTC CAA AGC AGA CCA GAA GGA TCA CAT AGA CAT TTA-3’ and the downstream primer 5’-GGC ACT TGG GAA AGT GTC AG-3’ (Invitrogen, ThermoScientific LSG, Waltham, Massachusetts, USA) to generate a 197-bp PCR fragment region of the intron 2 of C1q gene (located on chromosome 1). Regarding the TLR4 rs4986790 SNP, a fragment of 249 bp was amplified by PCR using the upstream primer 5′-GATTAGCATACTTAGACTACTACCTCCATG-3′ and the downstream primer 3′-GATCAACTTCTGAAAAAGCATTCCCAC-5′. Genotyping of the samples of both the C1q rs292001 SNP and the TLR4 rs4986790 SNP was performed by restriction analysis. RFLPs products of both polymorphisms were analyzed through electrophoresis on agarose gel and ethidium bromide fluorescence in reference to a molecular weight marker, and visualized using a UV-light trans-illuminator apparatus. Statistical analysis was performed using GraphPad Prism statistical program (GraphPad Prism 9 Software, San Diego, CA). The TLR4 and C1q variants under investigation were evaluated for deviation from Hardy-Weinberg equilibrium using chi-squared statistical test to compare observed and expected genotype frequencies in patients and control groups. The chi-squared test, with one or two degrees of freedom, or Fisher’s exact test was used to examine differences of genotypes and allele frequencies between patients and controls. Odds ratios (OR) and their 95% confidence intervals (CI) were calculated. A two-tailed p value of less than 0.05 was defined as statistically significant.
Results: In the clinical part of the study, the final study group consisted of 437 adult patients with T2D, of which 267 (61%) were female and 170 (39%) were male with a mean age of 70.5 ± 9.6 years. In 27% female participants and in 9.4% male participants ASB was noted. In total 20.1% presented ASB. Antimicrobial use within the last 3 months, was noted in 6.6%, while 17.8% of all patients had experienced a UTI in the last year before evaluation. Additionally, 2.7% had had recurrent UTIs, and 6.8% had had recent sexual intercourse. The characteristics of are shown in Table 2.
Several statistically significant differences were noted in patients with T2D with and without ASB, as, for example, in terms of age,BMI, duration of T2D, mean HbA1c, prior antimicrobial use, prior UTI, recurrent UTIs,recent sexual contact, serum B12 levels, albuminuria, nitrites and leukocyte esterase in theurine. More specifically, in female diabetic patients, age, duration of diabetes, BMI, HbA1c, recurrent UTIs, sexual intercourse, albuminuria, presence of leucocyte esterase or nitrites, recent use of antimicrobials and a history of UTI,were correlated with ASB. Moreover, women with T2D and ASB exhibited lower levels of vitamin B12 compared to women with T2D but without ASB. Male patients with T2D and ASB had a higher HbA1c and BMI, recurrent UTIs, a history of UTI, prior antimicrobial use and albuminuria, nitrites and leukocyte esterase in their urine. Differences in clinical and laboratoryparameters were noted between male patients with T2D and ASB and male patients withT2D but without ASB. Male patients with T2D and ASB had a higherHbA1c and BMI, recurrent UTIs, a history of UTI, prior antimicrobial use and albuminuria, nitrites, and leukocyte esterase in their urine. No statistically significant relation wasnoted between findings from physical examination, complications of diabetes and ASB inmen and women with T2D.
The results of a comparison between male and female patients with T2D and ASB, showed that only BMI (p = 0.0455) and family history of diabetes (p = 0.0468) were significantly different between the two groups. Female patients with T2D and ASB had a slightly higher BMI compared to male patients with T2D and ASB (33.50 ± 0.5992 vs. 30.73 ± 1.050) and a more frequent family history of T2D (p = 0.0468). Escherichia coli was the most frequently isolated microorganism which was found in more than half of all cases (62.5%). The most commonly isolated microorganisms in the urine samples of diabetic patients with ASB were Gram-negative bacteria which were identified in 80.7% of all cultures obtained from patients with T2D and ASB. Higher levels of HbA1c (OR = 3.921, 95%CI: 1.521-10.109, p<0.001) and a UTI episode within the last 12 months (OR = 13.254, 95%CI: 2.245-78.241, p<0.001), were found to be independently, positively associated with the development of ASB in the multivariate logistic regression analysis. In the same analysis, higher levels of vitamin B12 were found to be independently, negatively associated with the development of ASB (OR = 0.994 per ng/ml, 95%CI: 0.989-0.999, p<0.001). The results of the genetic study showed that the G/G genotype of the C1q rs292001 SNP was more common in T2D patients with ASB than in T2D patients without ASB (41,3% and 32%, respectively) and the observed difference was statistically significant (p=0.048, OR=0.339, 95% CI: 0.120–0.954). However, frequencies of allele G did not exhibit any statistical significant difference between patients with and without ASB (p=0.076, OR=0.638, 95% CI: 0.400–1.018). Regarding TLR4 rs4986790 SNP, neither the A/G genotype (p=1, OR=0.761, 95% CI: 0.164–3.527) nor the minor allele G (p=1, OR=0.766, 95% CI: 0.168–3.485) were found to differ significantly between T2D patients with and without ASB (Table 2). Thus, no association was observed with regard to the TLR4 rs4986790 SNP between cases and controls. Importantly, no deviation from Hardy–Weinberg equilibrium was observed in the distribution of genotypes of the controls either in rs4986790 or rs292001 SNPs at the 0.05 level.
Conclusions: This study identifies the characteristics of patients with T2D and ASB, and describes their differences from diabetic patients without ASB. Furthermore, a multivariate regression analysis identified higher HbA1c and recent UTI to be positively associated and higher B12 levels to be negatively associated with the development of ASB. Moreover, the present study provides the first evidence of an association between the C1q rs292001 SNP and an increased susceptibility for ASB in an adult Cretan population with T2D, suggesting that this SNP may be a risk factor for ASB in patients with T2D.
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