C - reactive protein and urinary tract infection due to Gram-negative bacteria in a pediatric population at a tertiary hospital, Mwanza, Tanzania

Introduction Gram-negative bacteria are the major cause of urinary tract infections (UTI) in children. There is limited data on UTI systemic response as measured using C-reactive protein (CRP). Here, we report the association of CRP and UTI among children attending the Bugando Medical Centre, Mwanza, Tanzania. Methods A cross-sectional study was conducted between May and July 2017. Urine and blood were collected and processed within an hour of collection. Data were analyzed using STATA version 13. Results Of 250 enrolled children, 76(30.4%) had significant bacteriuria with 56(22.4%, 95%CI; 11.5–33.3) having gram-negative bacteria infection. There was dual growth of gram-negative bacteria in 3 patients. Escherichia coli (32.2%, 19/59) was the most frequently pathogen detected. A total of 88/250(35.2%) children had positive CRP on qualitative assay. By multinomial logistic regression, positive CRP (RRR=4.02, 95%CI: 2.1–7.7, P<0.001) and age ≤ 2years (RRR=2.4, 95%CI: 1.23–4.73, P<0.01) significantly predicted the presence of significant bacteriuria due to gram-negative enteric bacteria. Conclusion C-reactive protein was significantly positive among children with UTI due to gram-negative bacteria and those with fever. In children with age ≤ 2 years, positive CRP indicates UTI due to gram-negative enteric bacteria.


Background
Urinary tract infection (UTI) have been noted as the commonest cause of bacterial infection among children under five years of age in low-income countries [1][2][3] . It is documented that 91% of bacteria causing UTI in chil-dren are gram negative bacteria [2][3][4] . This observation is of public health concern due to the increased antimicrobial resistance associated with gram negative bacteria. In the city of Mwanza, Tanzania, it was reported that more than 37% of gram negative bacteria causing UTI in children were producing extended spectrum beta lactamases (ESBL) [1][2][3] . In addition, ESBL producing gram negative bacteria had co-resistance to trimethoprim/sulfamethoxazole, fluoroquinolones and aminoglycosides [5][6][7][8] , which further complicates the management of infection caused by these pathogens. In children, if UTI is not properly and adequately treated can lead to complications like renal scaring 9,10 , the leading cause of end-stage renal disease in this population 1 . Therefore, a rapid test to indicate the presence of pathogens is highly needed especially in the place where there are no culture services.
The recommended diagnosis of UTI is quantitative urine culture, nevertheless this technique is expensive, take long time and not commonly available in many health facilities in resource limited settings. The C-reactive protein(CRP) often becomes elevated within few hours after tissue injury or the start of an infection 11,12 . In healthy individuals, CRP is normally present in a very low concentration of less than 6 mg/l 11,13 . Elevated CRP has been used as the predictor of inflammations among patients with infections including neonatal sepsis 14 , fungal infections 15 and pelvic inflammatory diseases 16 . Previous studies done in Europe and India reported a significant high CRP among patients with UTI than those without UTI [17][18][19] . Increase in CRP has commonly being detected in UTI caused by Escherichia coli, Proteus spp., Klebsiella pneumoniae, Staphylococcus aureus and others 17 . In addition, CRP had been suggested as the marker for the treatment progress 20,21 . However, the association of the CRP and UTI caused by gram negative bacteria among children with signs and symptoms of UTI has never being documented in many low-income settings. Therefore, this study was done to determine the association of C-reactive protein and UTI caused by gram negative bacteria among children attending the Bugando Medical Centre. The qualitative CRP assay is inexpensive and can be adopted in many settings to guide appropriate antibiotic treatment in patients with suspected bacterial infections including UTI.

Methodology
This cross-sectional hospital based study was conducted from May to July 2017 at pediatric out patients' clinic and pediatric wards of the Bugando Medical Centre (BMC). BMC is a tertiary and teaching hospital with a bed capacity of 1000. BMC serve about 13 million people from six regions. This study included children with presumptive diagnosis of UTI admitted or attending pediatric clinic at BMC. In this study children with either fever, painful micturition or pus in urine were presumptively diagnosed with UTI. To reduce CRP false positive results as indicated in the manufacturer guidelines; children with hepatitis and those who were HIV positive were excluded.

Data and sample collection
A clean catch method for obtaining a midstream urine was used to collect urine sample from children above 2 years of age 22,23 . For children below two years of age and in all children who have not been pre-trained on toileting supra-pubic aspiration was aseptically done 3,24 . Urine samples were collected in clean sterile container (HiMedia Laboratories. Pvt. Ltd, India) and transported to the microbiology laboratory for processing within an hour of sample collection. Standard quantitative urine culture was done on Cysteine lactose electrolyte deficient (CLED), MacConkey and blood agar plates (Oxoid, UK) 3 . Plates were aerobically incubated at 37 0 c for 18-24 hours. The presence of at least 10 5 CFU/ml for midstream urine and any growth for supra-pubic urine was defined as significant bacteriuria. Bacterial specie identifications were done using in house biochemical test 22,25 . Antibiotic susceptibility test was performed using Kirby-Bauer disc diffusion method following the guidelines laid down by the Clinical Laboratory Standard Institute(CLSI) 26 . E. coli ATCC 25922 and Staphylococcus aureus ATCC 25923 were used for quality control. Furthermore, 2 ML of venous blood was collected and placed in to the plain vacutainer tube (BD Vacutainer, Nairobi Kenya). Qualitative C-reactive protein assay was performed following manufacturer instructions (Diagnostics Euromedi Equip UK). Presence of agglutinations similar with a positive control was considered as positive C-reactive protein (i.e greater than 6mg/dl).

Data management and analysis
Data entry was done using Microsoft excel 2007 cleaned and transferred to STATA version 13(College station, Texas) for analysis. Categorical variables were presented as proportions while continuous variables were summarized using median (Inter quartile range). Chi square or Fisher's exact were used to establish statistical differences in proportions while logistic regression analysis was used to establish strength of association between CRP and the age, sex, gram-negative culture results, diarrhea and fever. Multivariate logistic was performed for all factors with P less than 0.2 controlled by age and sex to establish independent predictors of elevated CRP levels. Furthermore, multinomial logistic regression analysis was done using gram reaction (no significant bacteriuria, gram positive significant bacteriuria and gram negative significant bacteriuria) as the outcome. Statistical significance was set at African Health Sciences Vol 19 Issue 4, December, 2019 95% confidence interval with a p value of less than 0.05 as significant.

Demographic Characteristics of Study population
A total of 250 children were enrolled in the study, their median age (IQR) was 3(1-4.5) years. The slightly majority of the study participants were male 142(56.8%) and 128(51.2%) had fever (Table 1). Failure to gain weight was observed in 6(2.4%) children while 29 (11.6%) had diarrhoea. The isolated gram negative bacteria were highly resistant to ampicillin (94%) and amoxicillin/clavulanic acid (82%).
Regarding the resistance to third generation cephalosporins, 39% and 33% of enteric gram negative isolates were resistant to ceftriaxone and ceftazidime, respectively (Figure 1). All gram negative bacteria detected were 100% sensitive to meropenem.
African Health Sciences Vol 19 Issue 4, December, 2019 Positive CRP was significantly more in children with UTI due to gram negative bacteria than in children with gram positive UTI (30%) and those with no significant bacteriuria (28.2%), P<0.001, Table 3.   (Table 3). By multinomial logistic regression analysis was done, no bacteria was used as base and other outcomes being gram positive significant bacteriuria and gram negative significant bacteriuria, factors found to independently predict gram negative significant bacteriuria were positive CRP (RRR=4.02, 95%CI: 2.1-7.7, P<0.001) and age ≤ 2years(RRR=2.4, 95%CI: 1.23-4.73, P<0.01). Fever was not subjected to multinomial logistic regression due to its strong collinearity with positive CRP.

Discussion
The prevalence of significant bacteriuria due to gram negative enteric bacteria among children with presumptive diagnosis of UTI in the current study was 22.4%. This is similar to the results from the previous study conducted 6 years ago in the same settings which observed the prevalence of enteric gram negative bacteria UTI to be 20.3% 1,2 . The current results indicate that the contribution of gram negative in causing UTI at BMC has remained relative the same. Nevertheless, the observed prevalence of gram negative enteric bacteria causing UTI in children is slightly lower than 29.3% and 39.7% reported from Kenya and Uganda 27,28 , respectively. The ob-served difference could be explained by the differences in study population, a significant proportion of children in studies from Kenya and Uganda had co-morbidities such as malnutrition and HIV. Co-morbidities have been found to pedispose to infections such as UTI 1,27 . In the current study out of six children who failed to gain weight only 1 had significant bacteriuria, the number is too low for any statistical test therefore no conclusion could be made. Escherichia coli and Klebsiella spp., have been reported in previous studies [29][30][31] to contribute more than three quarter of the bacteria causing UTI in children of all ages. This was confirmed in the present study whereby these were predominant pathogens causing UTI. This can partly be explained by the fact that Escherichia coli and Klebsiella spp. are among the normal flora in the gastrointestinal tract (GIT), hence can easily cause UTI due to the close proximity of GIT and urinary tract.
As it was reported previous by other studies in the same settings [1][2][3]5 , bacteria detected in the current study were highly resistant to ampicillin and amoxicillin/clavulanic acid. This could be explained by the fact that these antimicrobial agents are inappropriately used to treat respiratory infections among children in Tanzania, hence increase the chance of pathogens to develop resistance against them 3 . Similar findings were reported elsewhere 32,33 which necessity the need of increasing effort in promoting ap-propriate antimicrobial use through antimicrobial stewardship programmes. Early detection of bacterial infection is crucial for the proper management of patients to reduce the associated morbidity and mortality. In the current study about one third of the enrolled children had positive CRP (i.e elevated CRP). CRP has been found to be a more sensitive maker in predicting gram negative bacterial infections than gram positive bacterial infections in children 14 . This has been confirmed in the current study whereby patient with gram negative bacterial infections had 3.54 times higher odds of having positive CRP results with positive CRP being an independent predictor of presence of gram negative significant bacteriuria. These findings are similar to previous studies reported in Turkey, England, Iran and US among children with UTI 20,29,34,35 .
As it was reported previously from other studies 13,36 ; this study has confirmed that, fever strongly predicts positive CRP results. This may be due to presence of pro-inflammatory cytokines like interleukin 1 and interleukin 6 that signal the liver to produce high concentration of CRP 37 . Therefore, in the place with limited diagnostic facilities positive qualitative CRP assay in children with fever and other symptoms and signs of UTI can be used to predict the possible group of pathogens which is important for early appropriate antibiotic treatment. Early appropriate initiation of the right antibiotics has been found to reduce morbidity such as renal scarring and mortality associated with UTI.

Limitation
Failure to do serial dilution of CRP and possibility of having other undiagnosed conditions might affect the result of this study.

Conclusion and recommendations
Qualitative CRP assay is mostly likely to be positive in children with fever and can predict the significant bacteriuria of gram negative bacteria especially in young children.
In place with limited culture services, clinicians should start appropriate treatment for gram negative bacteria in young children(≤ 2years) with presumptive diagnosis of UTI and positive qualitative CRP assay in order to reduce UTI associated morbidity and mortality. Further studies to evaluate the effectiveness of CRP in monitoring treat-ment of UTI and establish cutoff value of quantitative CRP in the diagnosis of gram negative enteric bacteria UTI are warranted.

Declaration Ethical approval and consent to participate
The protocol for conducting the study was approved by the Joint Catholic University of Health and Allied Sciences/Bugando Medical Centre (CUHAS/BMC) research ethics and review committee (CREC) with certificate no: CREC/274/2017. Consent to participate was obtained from guardians/parents.

Consent for publication
Not applicable.