Evaluation of a Liquid Chromatography-Tandem Mass Spectrometry Assay for Second-line Tuberculosis Drug Concentrations in Small Hair Samples

Abstract Background Treatment monitoring of multidrug-resistant (MDR) and extensively drug-resistant tuberculosis (XDR-TB) in resource-limited settings is challenging. We developed a multi-analyte assay for eleven anti-TB drugs in small hair samples as an objective metric of drug exposure. Methods Small hair samples were collected from participants at various timepoints during directly-observed MDR/XDR-TB treatment at an inpatient tertiary referral facility in South Africa (DR-TB cohort). We assessed an LC-MS/MS index panel assay including isoniazid, ethambutol, pyrazinamide, levofloxacin, moxifloxacin, ethionamide, prothionamide, linezolid, clofazimine, pretomanid, and bedaquiline against a reference standard of inpatient treatment records. Because treatment regimens prior to hospitalization were not available, we also analyzed specificity (for all drugs except isoniazid) using an external cohort of HIV-positive patients treated for latent TB infection with daily isoniazid (HIV/LTBI cohort) in Uganda. Results Among the 57 DR-TB patients (58% with pre-XDR/XDR-TB; 70% HIV-positive) contributing analyzable hair samples, the sensitivity of the investigational assay was 94% or higher for all drugs except ethionamide (58.5%, 95% confidence interval [CI], 40.7-99.9). Assay specificity was low across all tested analytes within the DR-TB cohort; conversely, assay specificity was 100% for all drugs in the HIV/LTBI cohort. Conclusions We developed an 11-drug panel assay to quantitatively ascertain drug exposure within second- and third-line DR-TB treatment regimens. Because hair concentrations reflect long-term exposure, multiple successive regimens commonly employed in DR-TB treatment may result in apparent false-positive qualitative and falsely elevated quantitative hair drug levels when prior treatment histories are not known.