Background
Methods/design
Aim
Setting
Study design
Specific aim 1
Study population
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18–69 years of age
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Willing and able to provide informed consent.
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Pregnant (all women with reproductive potential will undergo urine pregnancy testing)
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Known congenital kidney disease or autoimmune disease (e.g., systemic lupus erythematosus) that can cause albuminuria
Study procedures
HIV-negative adults
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Participant will undergo point-of-care (POC) HIV rapid testing (via finger prick)
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If their POC HIV rapid test is positive, the Determine HIV-1/2 assay kit (Abbott Laboratories) with UniGold test (Trinity Biotech) will be used as the confirmatory test. If needed, the HIV-1/2 STAT-PAK Dipstick assay (Chembio Diagnostic Systems will be used as tiebreaker.
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If their confirmatory HIV testing is positive, they are not eligible to continue in the study but will be informed of their status and will receive appropriate counseling by study staff, all of whom have IRB training and extensive HIV counseling experience. They will be referred for care per existing national standards. If the HIV test is negative, participants will be asked to complete a brief risk assessment, asking, “Have you engaged in any high risk (i.e., unprotected intercourse with a person with known HIV or unknown HIV status, etc.) one or more times within the prior 4–6 weeks?” (NOTE: those who are currently healthcare workers will also be asked if they have had any needlestick and/or mucous membrane exposures from someone with known HIV-positive or unknown HIV status within the prior 4–6 weeks). Based on responses to the risk assessment question, subjects deemed high-risk (answering yes to any question), will undergo viral load (VL) testing.
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ALL consenting subjects with a negative POC HIV rapid test at their screening visit, regardless of their responses to the above risk assessment question, will be asked to provide urine specimens at the study clinic (visits 1 and 2) for uACR measurement. They will also have a blood specimen obtained for genetic testing (at visit 2; see Table 1).
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When VL results are available (in ~ 2–3 weeks), participants will be called back to receive results and counseling to reduce transmission risk or for referral to care if HIV-positive. Screened high-risk individuals having a detectable VL (> 50 copies/mL) are not eligible for the study and will be provided counseling and referral to care, as detailed above.
Visit 1 | Visit 2 (4–8 weeks after Visit 1) |
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– Screening, informed consent – HIV testing (HIV-negative participants only) – Blood pressure measurement – Diabetes screeninga – Enrollment (if meet eligibility criteria) – Participant to provide urine specimen at study clinic on day of visit (for 1st uACR and for urine albumin-to-protein ratio (uAPR) (if HIV-positive)) | – Participant to provide urine specimen at study clinic on day of visit (for 2nd uACR) – Specimen collection (~ 6 tubes; ~ 30 mL total) i) Genotyping (Two DBS cards) ii) Serum creatinine (3 mL gold top tube) iii) HBVb and HCV testingc (Two 3.5 mL gold top tubes) iv) CMV testingd (One 3.5 mL gold top tube) v) Additional specimens to store (Two urine aliquots: Two 3 mL EDTA tubes; toenail/hair samples from consenting patients) |
Visit 1 | |
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-Screening, informed consent -Blood pressure measurement -Diabetes screeninga -Enrollment (if meet eligibility criteria) -Participant to provide urine specimen at study clinic on day of visit (for uACR and uAPR) | -Participant to provide urine specimen at study clinic on day of visit (for one-time uACR and uAPR) Specimen collection (~ 3 tubes; ~ 15 mLs total) i) Serum creatinine (3 mL gold top tube) ii) HBVb and HCV testingc (leverage existing stored plasma aliquots) iii) CMV testingd (leverage existing stored plasma aliquots) iv) Additional specimens to store (Two urine aliquots: Two 3 mL EDTA tubes; toenail/hair samples from consenting patients) |
Data collection and privacy considerations
Study measures
Urine albumin-to-creatinine ratio (uACR)
Urine albumin-to-protein ratio (uAPR)
Kidney function
Hepatitis B and C infection status
Cytomegalovirus (CMV)
Hypertension
Study outcomes
Laboratory investigations
Albuminuria and proteinuria measurement
Genetics specimen collection and genotyping
Statistical considerations
Sample size
Number of HIV-negative participants (N = 3000 HIV-positive participants) | Detectable proportion with micro-/macroalbuminuria among HIV-negative controls (RR = relative risk) | |
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90% power | 80% power | |
300 | 0.306 (RR = 1.31) | 0.319 (RR = 1.25) |
500 | 0.325 (RR = 1.23) | 0.335 (RR = 1.19) |
750 | 0.336 (RR = 1.19) | 0.345 (RR = 1.16) |
1000 | 0.343 (RR = 1.17) | 0.350 (RR = 1.14) |
Number of HIV-negative participants (n = 3000 HIV-positive participants) | Detectable difference between mean uACR in HIV-positive and HIV-negative participants (standard deviations) | |
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90% power | 80% power | |
300 | 0.196 | 0.170 |
500 | 0.157 | 0.135 |
750 | 0.132 | 0.114 |
1000 | 0.118 | 0.102 |
Number of HIV-negative participants | Albuminuria prevalence for HIV-negative participants without hypertension | Albuminuria prevalence for HIV-negative participants with hypertension | Relative Risk (HIV-negative participants) |
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750 | 0.10 | 0.27 | 2.7 |
750 | 0.15 | 0.36 | 2.4 |
750 | 0.20 | 0.44 | 2.2 |
750 | 0.25 | 0.50 | 2.0 |
Power calculations for interaction effects
Statistical analysis plan
Genetic analysis plans
Specific aim 2
Study population
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Participants with normoalbuminuria (uACR < 30 mg/g) who complete Aim 1 are eligible for Aim 2 (Fig. 1).
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Participants that completed Aim 1 of the separate, ongoing R3 study [13, 14] having normoalbuminuria (uACR < 30 mg/g) based on the average of two previously obtained uACR measurements (4–8 weeks apart) and re-confirmed via an additional uACR measurement obtained at time of screening for this study are also eligible.
Exclusion criteria
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Did not complete all scheduled Aim 1 visits
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Pregnancy (all women with reproductive potential will undergo urine pregnancy testing)
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Diabetes mellitus (documented diagnosis or random glucose ≥ 6.1 mmol/L [110 mg/dL])
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Known congenital kidney disease and/or autoimmune disease that can cause albuminuria
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Actively taking ACEi or ARBs, which may suppress albuminuria
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Regular use of NSAIDs or traditional medications
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HIV-negative but use TDF as pre-exposure prophylaxis or HBV treatment
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Comorbid condition with life expectancy < 3 years
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Planning to relocate within three years
Study procedures
Study measures
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Demographics: age, sex, self-reported ethnicity (Hausa/Fulani, Igbo, Yoruba, Other (Specify)
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Education: not literate, primary, secondary, tertiary (university or other college).
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Occupation: employment status (and if employed; details to be obtained)
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HIV transmission risk factors: heterosexual activity, homosexual activity, transfusion, other, unknown
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Date of HIV diagnosis (PWH only): date of first positive HIV test
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Date of ART initiation (PWH only): defined as three or more active antiretrovirals from two or more drug classes
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Dates of AIDS-defining illnesses (PWH only): defined per CDC list of AIDS-defining illnesses
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Dates of comorbid disease diagnoses: infections including TB (pulmonary and extrapulmonary), viral infections (HBV, HCV [Aim 1], Lassa fever, COVID-19, etc.), AIDS-defining illnesses; NCDs including hypertension, diabetes, sickle cell trait or disease, dyslipidemia, cardiovascular disease, cancer, liver disease, lung disease, depression, and bone mineral density abnormalities (including non-traumatic fractures).
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Antiretroviral medication start/stop dates (PWH only): from clinical record and pharmacy data (evaluating current exposure as well as prior/current TDF, protease inhibitor use to determine cumulative exposure time).
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Current medications: all medications taken at least once daily for ≥ 30 days, including traditional medications.
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Laboratory HIV data: all CD4 cell count and viral load measurements (collected per standard of care). SARS-CoV-2 testing will be performed per existing standard of care and results of all COVID-19 screening tests will be captured in the patients’ study medical record).
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Substance use: The Alcohol, Smoking and Substance Involvement Screening Test (ASSIST) is a cross-cultural screening of recent and lifetime substance use [90].
Procedure / Visit (month (mo)) | Enrollment | 6 mo | 12 mo | 24 mo | 30 mo |
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Seated blood pressure check | X | X | X | X | X |
Diabetes mellitus screening (perform fasting blood glucose if any signs/symptoms suggestive of underlying diabetes) | X* | X | X | X | X |
uACRa (No urine protein testing necessary for enrolled Aim 2 HIV-positive adults with normoalbuminuria as all HIV-positive ART-treated adults from Aim 1 will have one uAPR measurement) (We will have stored urine at 3 timepoints to go back and perform uAPR testing in patients with incident microalbuminuria) | X# (We will use Aim 1 uACR as participants baseline value) | X | X | ||
Serum creatinine (SCr)b | X (We will use Aim 1 SCr as their baseline value) | X | X | ||
Parasite screening | |||||
Complete blood count (CBC) with differentialc | X | X | X | X | X |
Urine dipstick (spun for all patients with hematuria followed by urine microscopy (S. haematobium) | X | X | X | X | X |
Malaria testing (rapid diagnostic test) at point-of-care (POC) using fingerprick specimen | X | X | X | X | X |
Filariasis POC testing for Wuchereria bancroftid | X | X | |||
Onchocerciasis testing (bilateral skin snips)e | X | X | |||
Stool for ova and parasite (stool O&P)f | X | X | X | X | X |
Daytime thick blood smear for Loa loa (10 AM–2 PM) | X | X | |||
Strongyloides IgG serology (plasma)g | X | X | |||
Tuberculosis screeningh | X | X | X | X | X |
Inflammatory biomarkers (urine and plasma)i | X | X | |||
CD4 cell count | X | X | |||
Viral load (plasma HIV RNA) | X | X | |||
Social and Behavioral data | X | X | X | ||
Additional tubes for storagej | X | X | X |
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Vital signs: temperature, respiratory rate, heart rate, blood pressure (Seated BP checked every study visit using a validated Omron series 5 upper arm digital BP device).
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Anthropometrics: height, weight, waist circumference
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Urine albumin testing, uACR measurements: Albuminuria will be assessed at each visit and categorized as defined in Aim 1. Subjects with incident macroalbuminuria will be referred to a clinician for evaluation and to a nephrologist for additional testing/management, per standard of care.
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Urine albumin-to-protein [uAPR] ratios (HIV-positive participants): will be assessed to screen for possible TDF-mediated tubular insult as a contributing cause of microalbuminuria. Subjects with uAPR < 40% will be classified as tubular albuminuria and those with uAPR ≥ 40% will be classified as glomerular albuminuria.
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Diabetes screening: All potentially eligible Aim 2 participants will undergo fasting/random blood glucose testing to evaluate for diabetes (and if ≥ 6.1 mmol/L (110 mg/dL), then patient may have diabetes, is ineligible, and is referred to medical provider/clinic for evaluation. Once enrolled, during each study visit, participants will be screened for symptoms of diabetes. In those with symptoms, we will perform fasting/random blood glucose testing. if ≥ 6.1 mmol/L (110 mg/dL), they will be immediately referred to a specialist for further evaluation.
Co-infection screening
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Mycobacterium tuberculosis (TB): Sputum specimens for acid fast bacilli (AFB) microscopy and/or Gene Xpert testing in patients having a cough of ≥ 2 weeks in duration, weight loss, and/or night sweats). Mycobacterial blood cultures, lymph node aspirates, body fluid (i.e., thoracentesis) and imaging studies will be obtained per standard of care when evaluating for suspected extrapulmonary TB.
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Parasitic infections (see Table 6) (The results obtained from enrollment filariasis (W. bancrofti, O. volvulus, and L. loa) screening will determine the need for repeat testing at the 30-month visit (i.e., if prevalence is extremely low, we will not re-test at the 30-month visit).
Adverse clinical outcomes will be assessed at all study visits by trained study research staff
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Hospitalizations: We will ask all participants if they have been hospitalized in the previous 6 months. If they answer affirmatively, we will ask the reason for hospitalization and approximate dates.
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Death: Ascertained from medical records or from a contact identified by the participant during consent.
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Inflammation will be assessed from plasma and urine specimens collected, as detailed in Table 6. Plasma inflammatory biomarker assay testing will be performed in duplicate at the DMPI Biomarker Core Facility using the Mesoscale Discovery Platform. Based on the literature, we chose a panel of plasma and urine biomarkers [92‐101] that will help us elucidate the processed by which chronic inflammation contributes to albuminuria. Urine inflammatory/tubular injury biomarker assay testing will be performed at the Bonventre laboratory (Brigham and Women’s Hospital Biomarker Laboratory, Boston, MA, USA). Some are novel biomarkers whose role in clinical research is being defined.
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Plasma inflammatory markers: highly sensitive C-reactive protein (hsCRP), IL-6, soluble TNF-receptor 1 (TNFR-1), soluble TNF-receptor 2 (TNFR-2), IL-1β, and TNF (with biomarker levels of sCD14, fibrinogen, and possibly additional plasma biomarker levels to be ascertained pending additional funding/approved sub-studies).
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Urine inflammatory/tubular injury biomarkers: NGAL, KIM-1, and MCP-1 (with biomarker levels of IL-18, interferon-gamma inducible protein-10 (IP-10), and possibly additional urine biomarker levels to be ascertained pending additional funding/approved sub-studies).
Study outcomes
Statistical considerations
Sample size
HIV-positive adults, N | HIV-negative adults, N | 3-year incidence of albuminuria for HIV-positive adults | 3-year incidence of albuminuria, HIV-negative adults, 80% power to detect | Relative Risk |
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1000 | 500 | 5% | 2.2% | 2.28 |
1000 | 500 | 10% | 5.9% | 1.70 |
1000 | 500 | 15% | 10.0% | 1.51 |