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Although urinary tract infection (UTI) resolves with prompt treatment in a majority of children, some children, especially those aged less than 5 years, also develop renal parenchymal scarring (RPS). RPS causes high blood pressure that may lead to severe chronic kidney disease and end-stage renal disease (ESRD). Although the risk of UTI is higher in white children than in black children, it is unknown whether RPS is more common in white children than in black children as data are scarce in this regard. A common genetic predisposition to kidney disease in African Americans and the sub-Saharan African blacks is the possession of apolipoprotein L1 (APOL1). APOL1 risk variants regulate the production of APOL1. APOL1 circulates in the blood, and it is also found in the kidney tissue. While circulating, APOL1 kills the trypanosome parasites; an increased APOL1 in kidney tissues, under the right environmental conditions, can also result in the death of kidney tissue (vascular endothelium, the podocytes, proximal tubules, and arterial cells), which, ultimately, is replaced by fibrous tissue. APOL1 may influence the development of RPS, as evidence affirms that its expression is increased in kidney tissue following UTI caused by bacteria. Thus, UTI may be a putative environmental risk factor responsible for APOL1-induced kidney injury.
The aim of this proposal was to outline a study that seeks to determine if the possession of two copies of either G1 or G2 APOL1 variant increases the risk of having RPS, 6 months following a febrile UTI among Nigerian under-five children.
This case-control association study seeks to determine whether the risk of RPS from febrile UTI is conditional on having 2 APOL1 risk alleles (either G1 or G2). Cases will be children with a confirmed RPS following a febrile UTI. Controls will be age-, gender-, and ethnic-matched children with a febrile UTI but without RPS. Children with vesicoureteral reflux and other congenital anomalies of the urinary tract are to be excluded. Association between predictor variables (ethnicity, APOL1 G1 or G2, and others) and RPS will be tested at bivariate logistic regression analyses. Predictors that attained significance at a
The study is expected to last for 3 years.
The study is contingent on having a platform for undergoing a research-based PhD program in any willing university in Europe or elsewhere. The findings of this study will be used to improve the care of African children who may develop RPS following febrile UTI.
RR1-10.2196/9514
Approximately 7% to 8% of girls and 2% of boys have a urinary tract infection (UTI) during the first 8 years of life [
The clinical features of UTI in childhood are often different from those found in adults and are frequently nonspecific [
Although the pathogenic mechanism of renal scarring following acute pyelonephritis (APN) is not well understood, the combined presence of congenital anomalies (vesicoureteral reflux, VUR and renal hypodysplasia), inflammation seen in pyelonephritis, and genetics may interact to result in scar formation [
These chemokines and cytokines include the interleukin 6, chemokine ligand 2 (CXCL2), interferon β1, CXCL8 (also known as interleukin 8), the interferon regulatory factor 3 (IRF3), interferon-gamma (IFN-γ), tumor necrosis factor (TNF), transforming growth factor beta (TGF-β), and vascular endothelial growth factor (VEGF) [
With the exception of antimicrobial peptides and epithelial cells that have not been well studied, single gene defects or variations in various genes have been shown to affect most of the other innate immune expression and response to invading bacteria [
As observed already, although several significant mutations [
African Americans have a three-fold higher lifetime risk of ESRD as compared with non-Hispanic whites because of a higher incidence of CKD and glomerulonephritis [
Although possession of the APOL1 risk variants in an autosomal recessive manner increases susceptibility to nondiabetic kidney disease, not all people who possess these variants develop kidney disease, which indicates that another factor may initiate progression of kidney disease [
For the following reasons, APOL1 is hypothesized to play a role in the pathogenesis of RPS, as febrile UTI may be a second-hit insult for the APOL1 kidney disease risk variants:
APOL1 is resident in vascular endothelium, podocytes, proximal tubules, and arterial cells [
Two renal transplantation studies suggest that the APOL1 kidney risk allele association is mediated by the gene product isoform that is endogenously expressed within the kidney and not the circulating APOL1 [
APOL1 is a member of the family of BH3-only proteins that interacts with the family of Bcl2 proteins to help regulate their function in autophagy and apoptosis [
Although apoptosis is a beneficial process for the host in lower UTI as it results in exfoliation of the superficial cells of the multilayered epithelium and thus the eradication of the bacteria attached to and invaded into the cells, however, where the epithelium is single-layered and close to the underlying kidney tissue and blood vessels, apoptosis is more likely to be part of a deleterious cycle of tubular atrophy, cytolytic events, and renal scarring [
The expression of APOL1 in human embryonic umbilical vein endothelial cells can be induced by lipopolysaccharide [
APOL1 is involved in innate immunity, which is the primary response of the human host to bacterial invasion of the urinary tract or kidney tissue. It is up-regulated by proinflammatory cytokines gamma interferon and TNFα [
In cell culture, interferon and toll-like receptor agonists increased APOL1 expression by up to 200-fold; in some cases with the appearance of transcripts not detected under basal conditions. PolyI:C, a double-stranded RNA TLR3 agonist, increased APOL1 expression by up-regulating interferon directly or through an interferon-independent, IRF-3-dependent pathway [
The innate immune response to bacterial UTI also involves IRF-3 stimulating pathway, and there may be an increased APOL1 expression [
Little is still known about the roles of APOL1 in kidney disease. Over time, there has been an extension in the spectrum of APOL1-associated kidney diseases, including systemic lupus erythematosus [
This study hypothesizes that APOL1 may be associated with RPS of UTI and that UTI is a trigger that determines RPS in susceptible individuals with APOL1 risk variants.
In addition to the limited availability of DMSA scanner, the issues of cost and exposure to radiation in children have prompted researchers to seek clinical or laboratory predictors of RPS. Most of the studies on the prevalence and clinical predictors of RPS following UTI were conducted in Europe, North America, Australia, and the Middle East and in Asia, with little or no data from sub-Saharan Africa, a region of the world where the APOL1 risk alleles are highly prevalent [
In view of the conflicting reports regarding the predictors of RPS among children with UTI, carrying out a well-designed, highly powered prospective cohort study to determine the risk of acquiring RPS in Nigerian children with a febrile UTI will contribute to the existing data of RPS in children with UTI. The findings of this study will for the first time provide information on the susceptibility factors ethnic sub-Saharan Africans may have for the development of RPS following a febrile UTI.
Africa, and in fact Nigeria, has complex populations and variations in climate, diet, and exposure to infectious diseases, which makes it a good setting for studying diversities in human genetics and phenotypes [
The overarching hypotheses are that RPS is a common complication of a febrile UTI in Nigerian under-fives and that the possession of APOL1 kidney risk variants increases the risk of RPS in these children. The study also proposes that clinical and laboratory predictors of RPS exist among children presenting with a first febrile UTI.
The presence of UTI among Nigerian children aged between >1 month and 60 months, presenting with axillary temperature ≥37.5˚C at the emergency pediatric unit (EPU) shall be determined. Children less than 1 month of age shall be excluded because their clinical presentations are unique. This is because they may present with hypothermia instead of fever [
RPS shall be determined with technetium Tc 99m DMSA renal scintigraphy. However, as studies have demonstrated that many abnormalities seen in DMSA scan done at 2 weeks resolve over time, and that little benefit exists in doing DMSA scan at 2 weeks, DMSA scan will be performed 6 months after the treatment for UTI to confirm or rule out chronic parenchymal scarring [
For DNA samples, blood samples will be obtained from all children with confirmed UTI. We shall collect about 1.5 mL of whole venous blood. DNA will be extracted using labeled collection tubes for blood. All samples will be allocated a unique identifier and will be stored at −4˚C. The blood samples shall be shipped to a reputable diagnostic molecular laboratory in London, United Kingdom. The new
Micturating cystourethrogram (MCUG) shall be performed at 2 weeks of follow-up on children with confirmed UTI who also have abnormal renal and bladder ultrasound (RBUS) features, including hydronephrosis, scarring, high-grade VUR, or obstructive uropathy, in line with the 2011 American Academy of Pediatrics Clinical Practice Guideline that took into consideration the fact that MCUG is an uncomfortable, costly procedure that involves exposure to radiation [
At this time point (2 weeks), the MCUG will be done when the child must have received the full course of antibiotics treatment for the UTI. VUR will be graded into five classes as follows [
The RBUS and the MCUG will be done by a consultant radiologist at the radiological department of the National Hospital, Abuja.
A comprehensive data of the sociodemographic, clinical signs and symptoms, examination findings, and the presumptive diagnoses will be collected on the first encounter for each child after informed consent has been obtained from the parents or caregivers of the children. About 2 mL of blood will be collected for complete blood counts, ESR, CRP, PCT, TNF-α, and IFN-γ. Other routine investigations will be as for the diagnostic work-up toward the presumptive diagnosis. The following factors will be considered for inclusion in the prediction model for RPS: age, sex, ethnicity, measured temperature at the time of diagnosis, duration of fever before presentation, grade of VUR, the organism isolated from culture (
Elevated acute phase reactants will be taken as follows: CRP >40 mg/L [
PCT is produced by the liver and peripheral blood mononuclear cells and modulated by lipopolysaccharides and sepsis-related cytokines. PCT is very specific for bacterial infection and helps to distinguish between viral and bacterial infections, which are particularly useful in children [
Although TNF-α and IFN-γ are both immunomodulating and proinflammatory cytokines, TNF-α secretion by activated macrophages is via bacterial lipopolysaccharide, whereas, IFN-γ is critical for innate and adaptive immunity against viral, some bacterial, and protozoal infections [
This study is important because it is coming at a time when genetic studies have started to explain the differences in the predisposition to kidney diseases in blacks compared with white. The study seeks to unravel for the first time the potential role of APOL1 kidney risk alleles in the evolution of RPS consequent on febrile UTI. The prevention of RPS is the most important goal of all therapies for childhood UTI, and if APOL1 kidney risk variants are shown to be associated with RPS, it will offer less invasive genetic biomarkers for predicting children who will develop RPS following UTI. This approach will reduce the need for urinary tract imaging that is expensive and exposes children to toxic radiation. This study will also offer an opportunity for clinical and translational research of kidney disease, as it will expose the researcher to training in genomic science and genetic studies.
The significance of study is as follows:
Although children with UTI tend to present with fever, it is often difficult on clinical grounds to distinguish UTI from other febrile illness. This makes UTI one of the most often missed diagnoses in the pediatric wards in developing countries [
The long-term complications of febrile UTI (APN) have been previously studied, and they include the risk of RPS, hypertension, preeclampsia, and ESKD that may ultimately require dialysis or renal transplantation [
Some researchers did not find any clinical predictors of RPS following UTI [
RPS following UTI is an important cause of renal morbidity in children. Studies have shown that the intensity of the inflammatory response following infection is related to the risk of RPS. However, genetic variability in this response has not been well studied [
VUR is one of the most common inherited diseases of the genitourinary tract in children. The incidence of primary VUR is 1% in normal infants, whereas it is 30% in infants presenting with UTI [
Presently, there is a paucity of epidemiologic, genomics, and translational studies of kidney disease among Africans. There is inadequate knowledge and exposure to genetic and translational longitudinal studies. The future of predictive, preventive, and individualized medicine in Africa is therefore gloomy but could be remedied by training more clinician researchers. Hopefully, there is a lot to be gained by the researcher in terms of training and exposure in the process of executing this research work.
The researcher’s capacity to conduct this study is presented in
This is a prospective, observational, longitudinal, case-control cohort study involving febrile under-five children presenting at the EPU of the National Hospital, Abuja. The conceptual framework is as depicted in
The National Hospital is located in Abuja, the Federal Capital Territory of the Federal Republic of Nigeria. Abuja is located in the North Central Geo-Political Zone of Nigeria. It occupies a land area of 7753.9 square kilometers [
As of 2016, Abuja’s population is estimated at 6 million persons [
The intention is to enroll a minimum sample size of 500 consecutive febrile under-five children, whose parents or caregivers consent to the study’s objectives. This sample size was expanded from the 260 derived from the Leslie Kish formula [
However, to test the study’s hypotheses, cases will be children with a confirmed RPS following a febrile UTI, and an equal number of controls (age-, gender-, and ethnicity-matched children with a febrile UTI but without RPS) will be targeted from the sample size. The study would aim at having at least 50 cases and 50 controls.
The inclusion criteria are shown in
Four research assistants (RAs) will be employed for the purpose of recruiting the participants for this study. This will comprise 2 medical doctors and 2 nurses. The medical doctors shall be involved in conducting the dipstick urinalysis, automated urinalysis, anthropometrical measurements, and the collection of urine and blood samples from the recruited participants. The nurses will be in charge of obtaining informed consent, the administration of the questionnaire, and the follow-up of the recruited subjects to the radiological department of the National Hospital, Abuja. The RAs will be trained on the concept and objectives of the study and the quality and culture appropriateness of the study’s questionnaire will be discussed with them. The proficiency of the RAs will be verified via role-playing. The study will use brochures, posters, flyers, newsletters, and family engagement to promote recruitment and retention within the study cohort. Parents or caregivers of febrile children coming to the EPU will be approached to be screened for enrollment. Furthermore, the study shall engage the services of a microbiologist, a radiologist, and a radionuclide physician. The microbiologist will be responsible for doing urine culture and interpretation, urine microscopy, and enhanced urinalysis. The radiologist shall be responsible for doing the RBUS and the MCUG. The radionuclide physician shall be responsible for the DMSA scanning for detecting APN and RPS.
Financial compensation will be made available for parents or caregivers who would have to bring their infants back to the National Hospital for MCUG at 14 days and for the DMSA scan at 6 months, following the treatment for the febrile UTI. To this end, the mobile telephone numbers of the consenting parents or caregivers of the children will be obtained for tracking after discharge from the hospital. In the same way, extra efforts will be made to obtain traceable home addresses of the study participants. Attrition would be minimized by ensuring that parents or caregivers fully understand this responsibility or expectation of study’s protocol at the time of signing the consent forms.
Ethical approval for the study shall be obtained from the research and ethics committee of the National Hospital, Abuja and the institutional review board of the university that will be willing to supervise this proposal as a PhD thesis. A written informed consent (see
Consecutive febrile children (≥1 month-59 months of age) presenting at the emergency pediatric unit (EPU) of the National Hospital, Abuja, whose parents or caregivers give consent to the study.
Fever is defined as axillary temperature ≥37.5˚C [
Urinary tract infection (UTI) will be defined as a positive test result for pyuria by either microscopy (≥5 white blood cells per high-power field in uncentrifuged urine specimen) or dipstick test (positive leucocyte esterase test) and a positive growth on culture of at least 50,000 colony-forming unit (CFU) per mL of a single uropathogen in urine specimen obtained by catheterization or greater than 100,000 CFU per mL of a single uropathogen in clean-catch urine specimen or any uropathogen growth in urine obtained suprapubically [
Renal parenchymal scarring (RPS) will be defined as a kidney with a decreased or an absent uptake in one or more areas, or relative function less than 45% on a dimercaptosuccinic acid (DMSA) scan done at 6 months following a confirmed febrile UTI.
Parents or caregivers’ ability to understand and comply with planned study protocols.
Parents or caregivers’ ability to provide informed consent before recruitment into the study.
Parents or caregivers residing in study area and at least within 5 miles radius, for easy follow-up and to reduce the number that may be lost to follow-up.
Febrile under-five children who had taken antibiotics in the preceding 2 weeks.
Children who have been treated for urinary tract infection (UTI) before.
Children with neurological lesion causing bladder dysfunction or those with known stone disease
Children with known congenital abnormalities of the kidney and the urinary tract (CAKUT, ie, vesicoureteral reflux, VUR; cystic kidney diseases; renal dysplasia; renal hypoplasia).
Children with HIV or AIDS and those with sickle cell anemia, as these diseases may confound the finding of renal parenchymal scarring (RPS).
Children with mixed ethnicity defined as having more than one ethnic ancestry in the biological parents and grandparents.
Children who are already part of another ongoing research effort.
Children whose parents or caregivers will refuse to give informed consent for participating in the study.
Children whose parents or caregivers are institutionalized (eg, prisoner, nursing home residents, and prisons).
The first step is to do screening to confirm study eligibility and provide participants with information about the study. A questionnaire (see
The participant will be informed that participation in the study is
The participant will be informed of the
The participant will be informed of
The participant will be informed of any foreseeable
The participant will be informed of any
An outline of safeguards to protect participant
The participant will be informed
The participant will be informed as to whether or not
The participant will be informed that he or she will be notified of any significant changes in the protocol that might affect their willingness to continue in the study.
The informed consent form will be duly signed or thumb-printed and dated by the participant or witness before initiation of any study-related activity.
A well-structured questionnaire (
Sociodemographic data: age as at last birthday, gender, place of residence (urban or rural), ethnicity or tribe, and socioeconomic status of the household
Past medical history: prior use of antibiotics, past history of UTI, family history of recurrent UTI, family or subjects’ history of congenital anomaly of the urogenital tract (ie, VUR), prior history of worm infestations, history of constipation, and history of breastfeeding in the first 6 postnatal months
Symptoms: jaundice, poor feeding, vomiting, diarrhea, irritability, strong smelling urine, abdominal pain, flank or back pain, irritability, dysuria, frequency, dribbling, poor stream, or straining to void
Signs: acutely ill-looking or not, fever (degree and duration before presentation), undernutrition as determined from anthropometric (height or length, weight, midarm circumference, occipitofrontal circumference) measurement, tenderness of the flank or costovertebral angle, suprapubic tenderness, abdominal tenderness, circumcision, signs of irritation on the external genitalia, pinworms, vaginitis, trauma, or sexual abuse
Comorbidity: malaria, sepsis, upper respiratory tract infection or otitis media, pneumonia, NS, viral exanthema, malignancies, etc
The following biological specimen will be collected from the enrolled child:
The urine sample will be collected as per the method relevant to the age of the child. Urine will be studied for culture and sensitivity and antimicrobial activity analysis. Furthermore, the urinalysis will be done by dipstick, urine microscopy, automated urinalysis, and enhanced urinalysis.
About 2 mL of blood will be collected using the sterile procedure for each child and sent for complete blood count, polymorphonuclear cell counts, ESR, CRP, PCT, TNF-α, IFN-γ, HIV, and hemoglobin genotypes.
Other laboratory work, including viral screening (hepatitis B or C), blood culture, stool microscopy, culture and sensitivity, cerebrospinal fluid culture and sensitivity, x-ray investigations, and joint fluid aspirate studies will be according to the diagnostic work-up for the particular child.
Additional 1.5 mL of blood shall be collected for APOL1 DNA analysis for all children with confirmed UTI.
RBUS scan will be done for all children with a confirmed UTI.
Any child with a confirmed febrile UTI as discussed previously will be subjected to MCUG at 14 days follow-up,
All children with a confirmed UTI but without a VUR are expected to have a DMSA scan at 6 months of follow-up for evaluation of chronic RPS. Before this time point (6 months), the RAs will also be in constant contact with the parents or caregivers of discharged children via telephone calls and SMS text messages. The flowchart of the four steps is summarized in
A total of 3.5 mL will be collected from the recruited subjects in step 2. The blood collected will be allowed to clot and centrifuged to produce the serum for biochemical analysis that will include serum TNF-α, IFN-γ, PCT, and CRP. The WBC counts and the differentials (polymorphonuclear cells) will be measured using the automated hematological analyzer (SYSMEX automated hematology analyzer KX-21N, Sysmex Corporation, Kobe, Japan). Serum levels of TNF-α and IFN-γ will be assayed by ELISA (BD Biosciences, United States) according to the manufacturer’s instructions. The TNF-α concentration will be calculated in the test samples on the basis of the curve produced by plotting the optical density values of the known standards (range: 7.5-500 pg/mL) on log-log graph paper.
The level of serum IFN-γ will also be detected with the IFN-γ ELISA kit. The detection limits of the kit for TNF-α and IFN-γ will be 2 and 1 pg/mL, respectively.
PCT will be measured with a quantitative immunoluminometric assay (LUMItest PCT, progressively replaced by PCT sensitive KRYPTOR, both from Brahms Diagnostica, Berlin, Germany), with a maximum interassay variation of approximately 0.3 ng/mL. CRP will be measured using the latex agglutination method and the automated method on Roche Integra 400 with an analytical goal of ±10%.
This study will employ the new “Axiom Genome-Wide Pan-African Array Set” to perform the genome-wide genotyping for variants in the APOL1 gene rather than the limited candidates’ SNPs (ie, rs73885319 and rs60910145 in G1 and rs71785313 in G2). DNA samples will be prepared and brought to a concentration of 100 ng required per array in the
The urine culture will be done within 1 hour of collection, employing the quantitative method as described by Guttmann and Stokes [
The bacterial isolates will be identified based on colony morphology characteristics, Gram stain reaction, and biochemical tests using standard techniques [
When tests on the urine will not be performed within the first hour of urine collection, urine will be stored in the refrigerator (at 4˚C) and will be tested within 4 hours of storage in the refrigerator [
The uncentrifuged urine sample will be used, and the procedure will follow a standard method. The analysis will specifically look for leukocyte esterase, nitrite, hematuria, and proteinuria.
The microscopic analysis will take place after centrifuging of the urine and will follow the standard procedure [
Uncentrifuged urine will be drawn into a Neubauer hemocytometer via capillary action [
Automated urinalysis [
Other investigations, including blood culture, joint aspirates for microbiology study, chest x-ray, stool microscopy culture, and sensitivity will be according to individual appropriateness.
RBUS will be requested for all bacteriologically confirmed cases of UTI while they are still on admission. All children will be examined in a warm room. Mothers will be requested to give water to the children to drink 1 to 1.5 hours before scanning. The abdominopelvic scan will be done using an Ultrasound SDD-3500 Plus, Japan 2005 scan machine with a 3-5 MHz curvilinear transducer. With the patient lying supine, the abdomen will be exposed, and gel will be applied. The transducer will be placed on the abdomen and gently moved laterally to the right and the left flanks from the midabdomen for the visualization of the right and the left kidney, respectively. The transducer will also be moved to the suprapubic region to localize the urinary bladder, especially when adequately distended with urine.
VCUG is after antibiotic therapy and will be done at 2 weeks for cases of confirmed UTI having hydronephrosis, scarring, high-grade VUR, or obstructive uropathy on RBUS. Children will be told to void (for those who can obey a command), after which, a preliminary coned down view of the bladder is taken. With the patient lying supine on the x-ray table and under aseptic technique, a lubricated catheter will be introduced into the bladder, and any residual urine will also be drained. A total of 150 mL of contrast medium (Urografin or Utravist) will be introduced into the urinary bladder until it is adequately distended. When the radiologist is convinced that the child will micturate or when the child shows the urge to micturate, the catheter will then be removed quickly. Spot films will then be taken during micturition. Films of the entire urethra and a full-length view of the abdomen will also be taken to demonstrate any reflux into these organs.
The static renal scintigraphy will be done 2 to 4 hours after DMSA injection at a dose of 1 MBq/kg body weight (minimum 15 MBq). Planar images will be obtained by a high-resolution collimator in one posterior and two oblique projections with 300,000 counts in the posterior view. All data files will be evaluated by the consultant nuclear medicine specialist.
The maximum irradiation dose would be 2 millisieverts (I millisievert from MCUG and 1 millisiervert from DMSA scan [
All specimens will be properly labeled and secured in a specimen bag with an accompanying laboratory request form. All specimens will be secured in a specimen carrier and transported to the study laboratory. All specimens will be delivered to the laboratory no longer than 3 hours after collection. Subject enrollment will occur only during weekdays and between 8.00 AM to 4 PM for logistic reasons. Specimens will be duly processed, and preliminary culture results will be made available to the researcher and the attending physician during the first 48 hours after enrollment and final results within 5 days. All clinical samples will be carefully labeled and coded using freezer-resistant labels. An electronic storage file will be developed to facilitate storage and retrieval of specimens.
2 mL of blood will be allowed to clot for 30 min, centrifuge for 20 min at 2400 rpm, and store at 4˚C and ship with cold packs for TNF-α AND IFN-γ assay.
1.5 mL of blood, stored at 4˚C and ship with cold packs for DNA analysis.
Whenever there is a requirement to store sample at −20˚C or −80˚C, the freezer at the Professor Obaro’s Research Laboratory of the International Foundation Against Infectious Disease in Nigeria (IFAIN) will be used.
A database will be created for logging all stored samples.
All participants will have a study identification to be used on study’s questionnaire and biospecimens. Questionnaires will be kept without patients’ identification information in a secured locked location as per the policies of Institutional Research and Ethics Board per site. All data will be checked for consistency, and outliers will be identified by examining empirical distributions of each outcome All data will be entered into REDCap, a secured online tool.
Statistical analysis will be done with SPSS version 21 (IBM Corp).
For numerical characteristics with symmetrical distribution, means and SD will be used as a measure of dispersion, whereas median and the interquartile range will be used to measure the central tendency for skewed numerical data. For categorical variables, proportions and percentage distribution would be described. Age grouping (≥1 month-2 months, >2 months-12 months, >12 months-24 months, >24 months- 59 months) will be done to take care of the known age-dependent risk of UTI [
The prevalence rates of children who developed RPS will be calculated. Cases shall be children who develop RPS consequent to febrile UTI and who also do not have VUR. Controls shall be children with confirmed UTI but who do not develop RPS. The risk of developing RPS across the age groups (≥1 month-2 months, >2 months-12 months, >12 months-24 months, >24 months-59 months) will be determined. Proportion distribution of RPS in children with UTI will also be done across the various potential risk factors of RPS, including age at diagnosis of UTI, gender, delayed treatment (>6 days of fever), peak of fever, laboratory indices of inflammation (total WBC count, ESR, IFN-γ, TNFα, PCT, and CRP concentration), the extent of renal parenchymal lesions, ethnicity (the major Nigerian ethnic groups of the Yorubas, the Ibos, the Fulanis, the Hausas, and others categorized as Others), and the presence of VUR. Ethnicity of the child will be that of his biological father and mother and the grandparents. Chi-square test of proportions or, in the case of small samples, Fisher exact test will be used to test the association between each of these risk factors and RPS. Predictors that attained significance at
The dependent variable is RPS and the independent variables will be the possession of either the G1 and/or the G2 APOL1 nephropathy risk variants. The possession of either the G1 and/or the G2 APOL1 will also be done among the cases defined by their ethnic groups of Yorubas, Ibos, Fulanis, Hausas, and Others. Ethnicity of the child will be that of his biological father and mother and the grandparents. Chi-square test of proportions or, in the case of small samples, Fisher exact test will be used to test the association between the possession of either the G1 and/or the G2 APOL1 risk variants and RPS. The APOL1 gene alleles associated with RPS will be described. Comparisons will be made between potential predictor variables and RPS using chi-square test of proportions or, in the case of small samples, Fisher exact test. At the least, the following factors will be considered for inclusion in the prediction model: age groups, sex, ethnicity, APOL1 gene allele, measured temperature at the time of diagnosis, duration of fever before presentation, the organism isolated from culture (
The outcome variable will be UTI, and the independent variables will be the various grades (I-V) of VUR. The effect of compounding with other significant risk factors (
Comparisons will be made between clinical predictor variables (ie, age, gender, ethnicity, past medical history, symptoms and signs, and comorbidities) and RPS using chi-square test of proportions or, in the case of small samples, Fisher exact test. In most cases, potential predictors will be dichotomized into either “yes” or “no.” Predictors that attained significance at
All candidate gene analyses will be analyzed. Each of the SNPs to be examined will be analyzed individually with RPS. Logistic regression model will be used for RPS, and the likelihood ratio test for significance will be used. The overall test of genotypic association with two degrees of freedom and any statistical contrasts will be defined by three genetic models: dominant, additive, and recessive models, respectively (each with one degree of freedom), with and without adjustment for covariates. If the test of general association is significant, then three a priori genetic models will be explored, and the best genetic model will be selected without further adjustment for multiple comparisons. Multiple SNPs from each gene will be tested, and Bonferroni correction to account for multiple testing will be done. The effect size for each of the risk haplotypes will be tested using a generalized linear model defined depending on the outcome variable.
The graphical representation of the power to detect association in the 500 minimum sample size populations can be seen in
For the proposed 500 minimum sample size, and assuming frequency of the exposure risk of haplotypes of APOL1 G2 (2 risk alleles) at 15% in the general population [
Statistical analyses of the primary and secondary outcomes of the study.
Outcome | Definition | Variable | Analyses | |
Urinary tract infection (UTI) among febrile children aged 1 month to 5 years | UTI will be defined as a positive test result for pyuria by either microscopy (≥5 white blood cells per high-power field in uncentrifuged urine specimen) or dipstick test (positive leucocyte esterase test) and a positive growth on culture of at least 50,000 colony-forming Unit per mL of a single uropathogen in urine specimen obtained by catheterization or greater than 100,000 CFU per mL of a single uropathogen in clean-catch urine specimen or any uropathogen growth in urine obtained suprapubically | Dichotomous (yes or no) | Prevalence ratios, odds ratio (OR), 95% CI | |
Renal parenchymal scarring among febrile children aged 1 month to 5 years with confirmed UTI | A kidney with decreased or absent uptake in one or more areas or relative function less than 45% on dimercaptosuccinic acid scan | Dichotomous (yes or no) | Prevalence ratio, OR, 95% CI, logistic regression |
Timeline of study
Activity | Year 1 | Year 2 | Year 3 | |
Institutional review board approval | January to June | |||
Hiring and training of research assistants | January to June | |||
Engagement of microbiologist, radiologist, and radionuclide physician | January to June | |||
Project enrollment | July to December | |||
Shipping of samples for apolipoprotein L1 (APOL1) analysis | July to December | |||
Project enrollment | January to December | |||
Shipping of samples for APOL1 analysis | January to December | |||
Project enrollment | January to June | |||
Shipping of samples for APOL1 analysis | January to June | |||
Analysis of data | July to December | |||
Writing reports and manuscript | July to December | |||
Defense of PhD thesis | July to December |
The primary outcome measure is UTI in febrile children aged 1 month to 5 years.
The secondary outcome measure is RPS among the children with confirmed UTI. The study will show for the first time the burden of RPS following a febrile UTI among Nigerian under-five children. It will also highlight if the risk of acquiring RPS is dependent on having APOL1 kidney disease risk variants among the children with confirmed febrile UTI.
Participant recruitment for this case-control cohort study will commence when the researcher gets a placement for a PhD study and after securing adequate funding grants for the study. The study is expected to last for at least 3 years.
This study will employ the new
A major concern is getting funds to execute a study of this nature. However, through application for grants, it is hoped that there would be light at the end of the tunnel.
There is also a concern that there may be an insufficient sample size of children with RPS that would serve as cases. Although this may impact on statistical significance of predictor variables in regression analyses, this limitation will be surmounted by extending the study period until a satisfactory number of 50 is attained.
The pitfall of possible attrition at 2 weeks and 6-month time points will be reduced by ensuring that only parents or caregivers who fully understand the study protocol and are willing to comply with it are recruited into the study in the first place. A modest monetary incentive would also be provided when parents or caregivers bring their children at these two time points. Traceable home addresses and mobile telephone numbers of the participants would be gotten, and close contact would be maintained by periodic telephone calls.
In Nigeria, as in many sub-Saharan African countries, political and economic instability can hamper longitudinal biomedical research via incessant strikes and erratic essential services such as electricity. The engagement of the researcher in other large-scale studies would enable him to have access to the uninterrupted electricity supply already put in place by the IFAIN. Biological specimens requiring refrigeration (including −80˚C freezers) will be kept at the IFAIN laboratory service in Abuja, pending the appropriate time of shipment of such specimens.
All genetic data will be checked for consistency, and outliers will be examined. Genotypic error will be examined by including blind replicates to assess assay reproducibility and the assessment of Hardy-Weinberg (H-W) proportion for each SNP. Reproducibility of the assay using replicate samples will be assessed using the kappa statistic. SNPs showing considerable discordance (kappa ˂.9) will be discarded from subsequent analyses. Allele frequencies for each SNP will be computed and tested for departures from the H-W proportions. SNPs that persist out of H-W proportions after testing for genotyping error will be kept in the analyses as they may provide valuable insight into population ancestry, or signal a genome region for which the study sample is biased or is under selection pressure.
The researcher's capacity to conduct this study.
The conceptual framework of the study.
The informed consent form.
Questionnaire.
The flowchart of the study's four steps.
The power chart to show the strength of association.
angiotensin II
acute pyelonephritis
apolipoprotein L1
colony-forming unit
chronic kidney disease
C-reactive protein
chemokine ligand
dimercaptosuccinic acid
emergency pediatric unit
erythrocyte sedimentation rate
end-stage kidney disease
end-stage renal disease
focal segmental glomerulosclerosis
HIV-associated nephropathy
International Foundation Against Infectious Diseases in Nigeria
interferon-γ
interferon regulatory factor 3
micturating cystourethrogram
nephrotic syndrome
procalcitonin
pattern recognition receptor
research assistant
renal and bladder ultrasound
renal parenchymal scarring
single nucleotide polymorphism
transforming growth factor β
toll-like receptor 4
tumor necrosis factor-alpha
urinary tract infection
vascular endothelial growth factor
vesicoureteral reflux
white blood cell
The author wishes to acknowledge Professors Stephen Obaro (of the University of Nebraska Medical Center, 982162 Nebraska Medical Center, Omaha, Nebraska 68198-2162, United States) and Abdulkareem Airede (of the College of Health Sciences, University of Abuja, Abuja, Nigeria), both of whom encouraged him to put this PhD proposal together.
None declared.