Over 15 million children are exposed to tuberculosis (TB) each year.1 For young children, risk of TB is ≈20% within 2 years of exposure.2 Isoniazid (INH) preventative therapy (IPT) reduces pediatric TB risk by 60%.3 However, efficacy depends on adherence, often reported as low in programmatic settings. INH metabolites are detectable in urine, providing a noninvasive objective means of adherence assessment.
We performed a biomarker-based assessment of infant IPT adherence comparing caregiver-reported adherence to INH metabolite testing using a urine dipstick in a TB prevention trial of HIV-exposed uninfected Kenyan infants.
In the parent trial, HIV-exposed uninfected infants 6 weeks of age without known TB exposure were randomized to 12 months daily INH (≈10 mg/kg) versus no INH to evaluate INH efficacy to prevent primary Mycobacterium tuberculosis infection.4 Follow-up visits were at 10 and 14 weeks and at 6, 9 and 12 months of age, with final study visit at 12 months postrandomization at ≈14 months of age. Standardized adherence questionnaires were administered to caregivers of infants randomized to INH.
Urine was collected using a pediatric collection bag. Dipsticks designed at the Lutz laboratory (University of Washington Department of Bioengineering) used a modified Arkansas method based on a colorimetric change, which occurs when INH metabolite isonicotinic acid reacts with cyanogen chloride and barbituric acid (see text, Figure, Supplemental Digital Content 1; https://links.lww.com/INF/E151 and 2; https://links.lww.com/INF/E152).5 INH metabolite is typically detectable in urine up to 24–30 hours after ingestion. A positive test was defined as any color change and was considered negative if no color change occurred. Cost to make each strip is <1 USD. A subset of tests were compared with the commercially available IsoScreen (≈8 USD per test; GFC Diagnostics Limited, Oxfordshire, United Kingdom) with reported 95%–99% sensitivity within 24 hours and 85% within 48 hours of INH ingestion, with 98% specificity in evaluations of adults.6,7
We compared caregiver-reported adherence measures to assay results and evaluated correlates of a positive INH dipstick using generalized linear models with a log-binomial link and clustered by participant. Agreement to IsoScreen was assessed and concordance measured using kappa (κ) statistics and sensitivity and specificity with IsoScreen as reference. Time between caregiver-reported INH dose and urine collection was compared using Mann-Whitney U test. Statistical significance was evaluated at α = 0.05 and regression estimates reported with 95% confidence intervals.
Caregivers provided written informed consent. Study procedures were approved by University of Washington Institutional Review Board and Kenyatta National Hospital/University of Nairobi Ethics and Research Committee. The parent trial is registered at ClinicalTrials.gov (NCT02613169).
In the parent trial, 150 HIV-exposed infants were randomized to INH; 145 received at least one dose. Ninety-seven of 145 infants (67%) had 155 urine tests performed (Fig. 1A and B). Among 97 infants with urine results, median enrollment age was 6.3 weeks (interquartile range [IQR], 6.0–6.4) and 56 (57.7%) were male (see Table, Supplemental Digital Content 3; https://links.lww.com/INF/E153). On enrollment, all mothers had received antiretroviral therapy and 83 (93.3%) had HIV viral load <1000 copies/ml. Seventy-three mothers (75.3%) had ever received programmatic IPT (26 [37.0%] current use), 10 (10.3%) reported TB history and 43 (44.3%) initiated secondary education.
Among 155 INH metabolite assays performed, 77 (49.7%) were positive (see Fig. 1C; see Table, Supplemental Digital Content 4; https://links.lww.com/INF/E154). Overall, median caregiver-reported time since last INH dose was 15.1 hours (IQR, 5.0–17.1) with 134 (86.5%) taken <24 hours since urine testing (see Table, Supplemental Digital Content 3; https://links.lww.com/INF/E153). Dipstick results by caregiver-reported adherence measures by visit are shown in Table, Supplemental Digital Content 5; https://links.lww.com/INF/E155. Median caregiver-reported time since last INH dose was 14.5 hours (IQR, 4.6–16.3) among infants with a positive urine INH assay versus 15.5 hours (IQR, 6.3–17.5) (P = 0.04) for those with a negative result. Urine tests were positive in ≈50% of infants with caregiver-reported optimal INH use (≥90% pills taken since last visit; 76/149), INH taken <24 hours (69/134), or no missed doses past 3 days (72/136) (Fig. 1C). Positive urine INH test was associated with increased infant weight-for-age Z score (relative risk [RR], 1.3 [95% CI, 1.1–1.6; P = 0.002]), maternal secondary education (RR, 1.5 [95% CI, 1.1–2.2; P = 0.02]), maternal HIV viral load <1000 copies/ml (RR, 2.1 [95% CI, 1.1–4.0]; P = 0.02]) and no missed doses past 3 days (RR, 2.4 [95% CI, 1.0–5.6]; P = 0.05) (see Table, Supplemental Digital Content 3; https://links.lww.com/INF/E153). There was a trend for caregiver-reported last dose <24 hours (vs >48 hours; RR, 4.9 [95% CI, 0.8–28.9]; P = 0.08) and positive urine INH result. Infant sex, age at visit, time since enrollment and maternal history of TB or IPT were not associated with biomarker-confirmed adherence.
Fifteen urine samples were tested concurrently with the commercial IsoScreen test. Among 8 IsoScreen positive samples, 7 were positive by the in-house dipstick (sensitivity 87.5%); among 7 negative IsoScreen tests, 6 in-house dipsticks were negative (specificity 85.7%) (see Table, Supplemental Digital Content 6; https://links.lww.com/INF/E156). Overall agreement was 86.7% (kappa 0.73, P = 0.002). Similar to the whole cohort, IsoScreen results were positive in ≈50%–60% of children with caregiver-reported optimal INH use, INH taken in past 24 hours, or no missed doses past 3 days (see Table, Supplemental Digital Content 7; https://links.lww.com/INF/E157).
In our study, urine biomarker assessment suggested overreported infant INH adherence in a primary TB infection prevention trial in Kenya. A low-cost urine dipstick assay performed reasonably well compared with a more expensive commercially available test. Although the assay did not strongly correlate with caregiver report of >90% adherence between study visits, there was a significant 2.4-fold increased likelihood of positive tests among infants who reportedly missed no doses in past 3 days and a trend for >4-fold increased likelihood of positive test if last dose was received within past 24 hours. Association of maternal secondary education and HIV viral suppression with infant INH adherence suggests maternal education and success in their own medication use predicts infant adherence.
This is one of the first studies to evaluate TB prevention adherence using a biomarker approach in young infants, specifically HIV-exposed infants, a population with high risk of TB exposure and disease.8 The large proportion of infants with negative biomarker tests despite caregiver-reported adherence (≈50%) may be due to assay performance, infants not receiving doses, social desirability bias to report adherence, or infant drug metabolism characteristics. Previous urine INH biomarker evaluations have focused primarily in adults with few pediatric assessments, with nonadherence ranging from 23% to 35%.5,9–15 In a Gambian evaluation among child contacts of adults with smear-positive TB (median age 2.3 years) with IsoScreen, 77% completed a 6-month course of IPT with “good” adherence (consuming >80% of pills); 85% of which had a positive urine test.13 This higher biomarker-assessed adherence compared with our study could be due to parental motivation related to prophylaxis indication (primary prevention vs. known contact), younger age or mode of IPT delivery and urine testing (IPT pickup and urine collection during study visit versus monthly home delivery and urine collection). In our pilot testing, the urine dipstick performed well using positive controls in the laboratory with good diagnostic performance compared with the commercial test using clinical samples, with performance relative to commercial testing similar to previous reports.9 Previous evaluation of commercial IsoScreen reported 95% sensitivity within 24 hours of INH dose taken under direct supervision, with 98% specificity12; it is possible the discrepant positive urine dipstick detected INH missed by the commercial test. In studies evaluating test performance at different time points, sensitivity of urine INH assays were lower at 24 versus 12 hours in South African adults (77% vs 93%, respectively)16 and at 24 versus 4 hours in South African children with HIV (median age 7.7 years; 78% vs 94%, respectively).11 Median time between reported dose and urine testing was 15 hours in our study. Metabolism differences, including acetylator status, are an unlikely explanation for low test positivity in our study; pharmacokinetic data for INH in South African HIV-exposed infants demonstrated >98% of infants achieved therapeutic levels at similar doses, regardless of N-acetyltransferase 2 enzyme (NAT2) genotype.17
Our study had limitations. Despite urine collection bags placed at each visit, many infants did not produce urine, limiting samples for biomarker testing. This could potentially be addressed by home urine collection. Urine INH metabolite testing only assesses recent adherence and may be prone to “white-coat” dosing with administration before clinic visits. Hair analyses are planned, which provide a longer-term objective measure but require significant laboratory infrastructure/expertise and do not provide real-time results. We did not supervise INH dosing or repeat urine testing after administration, which would have allowed direct evaluation at different time points. Study visits were aligned with routine outpatient infant follow-up, and caregivers were encouraged to give infants INH at the same time daily. This study represented initial prototype testing; there are ongoing evaluations of next-generation INH dipsticks in other settings and contexts (South Africa, Argentina and Kenya), including direct comparisons with the commercially available test in children living with HIV receiving IPT and planned evaluation in cohorts receiving combined INH and rifapentine for TB prevention, which will further inform test characteristics.
Point-of-care biomarker monitoring may be useful to assess and motivate infant TB prevention medication adherence. Further evaluation is needed regarding whether the newer generation of assays have improved performance in this population and whether a biomarker-based intervention such as urine adherence testing can improve TB prevention outcomes.
We acknowledge the iTIPS Study Clinic Staff, the Kisumu and Siaya County Directors of Health, health facility staff, UW-Kenya and Kenyatta National Hospital Research and Programs operational staff. We thank the University of Washington Global Center for the Integrated Health of Women, Adolescents, and Children (Global WACh) for comments and insights provided during study design and manuscript development. We appreciate the editorial advice from Adrienne Shapiro and Sarah Iribarren. Most of all, we thank the families who have participated in the study.
S.M.L., G.J.-S., and B.A.R. designed this study. D.L. developed the urine assay under the supervision of N.P. and B.L. G.J.-S. is the principal investigator and protocol chair of the parent study. J.K. is the protocol co-chair and country principal investigator, and E.M.-O. is the Pediatric Clinical TB lead of the parent study. S.M.L. and J.N.E. performed the data analysis with input from G.J.-S. and B.A.R. J.M. and J.N.E. managed study data. S.M.L., D.M., J.K., J.M. and G.J.-S. participated in study implementation. S.M.L. wrote the initial draft of the article. All authors read and approved the article.
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