Development and Validation of a UPLC-MS/MS Method for Therapeutic Drug Monitoring, Pharmacokinetic and Stability Studies of First-Line Antituberculosis Drugs in Urine.

Tuberculosis (TB) remains one of the leading global causes of mortality. Several methods have been established to detect anti-TB agents in human plasma and serum. However, there is a notable absence of studies analyzing TB drugs in urine. Thus, our objective was to validate a method for quantifying first-line anti-TB agents: isoniazid (INH), pyrazinamide (PZA), ethambutol (ETH), and rifampicin (RIF), along with its metabolite 25-desacetylrifampicin, and degradation products: rifampicin quinone and 3-formyl-rifampicin in 10 µL of urine. Chromatographic separation was achieved using a Kinetex Polar C18 analytical column with gradient elution (5 mM ammonium acetate and acetonitrile with 0.1% formic acid). Mass spectrometry detection was carried out using a triple-quadrupole tandem mass spectrometer operating in positive ion mode. The lower limit of quantification (LLOQ) was 0.5 µg/mL for INH, PZA, ETH, and RIF, and 0.1 µg/mL for RIF's metabolites and degradation products. The method was validated following FDA guidance criteria and successfully applied to the analysis of the studied compounds in urine of TB patients. Additionally, we conducted a stability study of the anti-TB agents under various pH and temperature conditions to mimic the urine collection process in different settings (peripheral clinics or central laboratories).

Simplified urine-based method to detect rifampin underexposure in adults with tuberculosis: a prospective diagnostic accuracy study.

Variable pharmacokinetics of rifampin in tuberculosis (TB) treatment can lead to poor outcomes. Urine spectrophotometry is simpler and more accessible than recommended serum-based drug monitoring, but its optimal efficacy in predicting serum rifampin underexposure in adults with TB remains uncertain. Adult TB patients in New Jersey and Virginia receiving rifampin-containing regimens were enrolled. Serum and urine samples were collected over 24 h. Rifampin serum concentrations were measured using validated liquid chromatography-tandem mass spectrometry, and total exposure (area under the concentration-time curve) over 24 h (AUC0-24) was determined through noncompartmental analysis. The Sunahara method was used to extract total rifamycins, and rifampin urine excretion was measured by spectrophotometry. An analysis of 58 eligible participants, including 15 (26%) with type 2 diabetes mellitus, demonstrated that urine spectrophotometry accurately identified subtarget rifampin AUC0-24 at 0-4, 0-8, and 0-24 h. The area under the receiver operator characteristic curve (AUC ROC) values were 0.80 (95% CI 0.67-0.90), 0.84 (95% CI 0.72-0.94), and 0.83 (95% CI 0.72-0.93), respectively. These values were comparable to the AUC ROC of 2 h serum concentrations commonly used for therapeutic monitoring (0.82 [95% CI 0.71-0.92], P = 0.6). Diabetes status did not significantly affect the AUC ROCs for urine in predicting subtarget rifampin serum exposure (P = 0.67-0.92). Spectrophotometric measurement of urine rifampin excretion within the first 4 or 8 h after dosing is a simple and cost-effective test that accurately predicts rifampin underexposure. This test provides critical information for optimizing tuberculosis treatment outcomes by facilitating appropriate dose adjustments.

New approach to rifampicin stability and first-line anti-tubercular drug pharmacokinetics by UPLC-MS/MS.

Successful tuberculosis (TB) therapy requires achieving sufficient exposure to multiple drugs. Limited stability of several first-line anti-TB drugs might compromise reliable therapeutic drug monitoring (TDM). We developed and validated a sensitive and selective UPLC-MS/MS method for simultaneous quantification of isoniazid (INH), pyrazinamide (PZA), rifampicin (RIF), its metabolite 25-desacetylrifampicin and degradation products: rifampicin quinone and 3-formyl-rifampicin. Analysis was completed from a very small plasma volume (20 µL) using only protein precipitation with methanol. Chromatographic separation was achieved on a Kinetex Polar C18 column (2.6 µm; 150 × 3 mm) with a mobile phase consisting of 5 mM ammonium acetate and acetonitrile, both containing 0.1 % formic acid, in gradient elution. The analytes were detected using a positive ionization mode by multiple reaction monitoring. The LLOQ for RIF and its degradation products was 0.1 µg/mL, 0.05 µg/mL for INH, and 0.2 µg/mL for PZA. The method was validated based on the FDA guidance. The application of the method was confirmed in the analysis of RIF, INH, and PZA, as well as RIF metabolism/degradation products in plasma samples of patients with TB. Based on the detailed stability study of the analyzed compounds at various storage conditions, we proposed recommendations for handling the plasma and serum samples in TDM and other pharmacokinetic studies.

Rifampin urinary excretion to predict serum targets in children with tuberculosis: a prospective diagnostic accuracy study.

OBJECTIVE: Pharmacokinetic variability drives tuberculosis (TB) treatment outcomes but measurement of serum drug concentrations for personalised dosing is inaccessible for children in TB-endemic settings. We compared rifampin urine excretion for prediction of a serum target associated with treatment outcome. DESIGN: Prospective diagnostic accuracy study. SETTING: Inpatient wards and outpatient clinics, northern Tanzania. PATIENTS: Children aged 4-17 years were consecutively recruited on initiation of WHO-approved treatment regimens. INTERVENTIONS: Samples were collected after directly observed therapy at least 2 weeks after initiation in the intensive phase: serum at pre-dose and 1, 2 and 6 hours post-dose, later analysed by liquid chromatography-tandem mass spectrometry for calculation of rifampin total exposure or area under the concentration time curve (AUC0-24); urine at post-dose intervals of 0-4, 4-8 and 8-24 hours, with rifampin excretion amount measured onsite by spectrophotometry. MAIN OUTCOME MEASURES: Receiver operating characteristic (ROC) curve for percentage of rifampin dose excreted in urine measured by spectrophotometry to predict serum rifampin AUC0-24 target of 31.7 mg*hour/L. RESULTS: 89 children, 52 (58%) female, with median age of 9.1 years, had both serum and urine collection. Only 59 (66%) reached the serum AUC0-24 target, reflected by a range of urine excretion patterns. Area under the ROC curve for percentage of rifampin dose excreted in urine over 24 hours predicting serum AUC0-24 target was 69.3% (95% CI 56.7% to 81.8%), p=0.007. CONCLUSIONS: Urine spectrophotometry correlated with a clinically relevant serum target for rifampin, representing a step toward personalised dosing for children in TB-endemic settings.

Duration of Symptoms Prior to Pediatric and Adolescent Tuberculosis Diagnosis and Its Impact on Schooling in Tanzania: A Mixed Methods Study.

At least a third of tuberculosis (TB) cases remain undiagnosed, disproportionately so in children and adolescents, which is hampering global elimination goals. Prolonged symptom duration presents a high-risk scenario for childhood TB in endemic areas, but the prolonged period of symptoms and its impact on educational attainment are rarely documented. Using a mixed method approach, we aimed to quantify the duration of respiratory symptoms and describe their impact on education among children from a rural area of Tanzania. We used data from a prospectively enrolled cohort of children and adolescents aged 4-17 years in rural Tanzania at the start of active TB treatment. We report on the cohort's baseline characteristics and explore the correlation between duration of symptoms and other variables. In-depth qualitative interviews were designed on the basis of a grounded theory approach to explore the impact of TB on educational attainment among school-aged children. In this cohort, children and adolescents diagnosed with TB experienced symptoms for a median of 85 days (interquartile range: 30, 231 days) prior to treatment initiation. In addition, 56 participants (65%) had a TB exposure in the household. Of the 16 families with school-aged children who were interviewed, 15 (94%) reported a significant negative impact of TB on the schooling of their children. Children in this cohort experienced a long duration of TB symptoms; the extent of illness impacted absenteeism at school. Screening initiatives for households affected by TB may lead to a shortened duration of symptoms and may minimize the impact on school attendance.

Alternative Methods for Therapeutic Drug Monitoring and Dose Adjustment of Tuberculosis Treatment in Clinical Settings: A Systematic Review.

BACKGROUND AND OBJECTIVE: Quantifying exposure to drugs for personalized dose adjustment is of critical importance in patients with tuberculosis who may be at risk of treatment failure or toxicity due to individual variability in pharmacokinetics. Traditionally, serum or plasma samples have been used for drug monitoring, which only poses collection and logistical challenges in high-tuberculosis burden/low-resourced areas. Less invasive and lower cost tests using alternative biomatrices other than serum or plasma may improve the feasibility of therapeutic drug monitoring. METHODS: A systematic review was conducted to include studies reporting anti-tuberculosis drug concentration measurements in dried blood spots, urine, saliva, and hair. Reports were screened to include study design, population, analytical methods, relevant pharmacokinetic parameters, and risk of bias. RESULTS: A total of 75 reports encompassing all four biomatrices were included. Dried blood spots reduced the sample volume requirement and cut shipping costs whereas simpler laboratory methods to test the presence of drug in urine can allow point-of-care testing in high-burden settings. Minimal pre-processing requirements with saliva samples may further increase acceptability for laboratory staff. Multi-analyte panels have been tested in hair with the capacity to test a wide range of drugs and some of their metabolites. CONCLUSIONS: Reported data were mostly from small-scale studies and alternative biomatrices need to be qualified in large and diverse populations for the demonstration of feasibility in operational settings. High-quality interventional studies will improve the uptake of alternative biomatrices in guidelines and accelerate implementation in programmatic tuberculosis treatment.

Population Pharmacokinetics of Levofloxacin and Moxifloxacin, and the Probability of Target Attainment in Ethiopian Patients with Multidrug-Resistant Tuberculosis.

Objective: This study aimed to explore the population pharmacokinetic modeling (PopPK) of levofloxacin (LFX) and moxifloxacin (MXF), as well as the percent probability of target attainment (PTA) as defined by the ratio of the area under the plasma concentration-time curve over 24 h and the in vitro minimum inhibitory concentration (AUC0-24/MIC) in Ethiopian multidrug resistant tuberculosis (MDR-TB) patients. Methods: Steady state-plasma concentration of the drugs in MDR-TB patients were determined using optimized liquid chromatography-tandem mass spectrometry. PopPK and simulations were run at various doses, and pharmacokinetic parameters were estimated. The effect of covariates on the PK parameters and PTA for maximum mycobacterial kill and resistance prevention was also investigated. Results: LFX and MXF both fit in a one-compartment model with adjustments. Serum-creatinine (Cr) influenced (p = 0.01) the clearance of LFX, whereas body mass index (BMI) influenced (p = 0.01) the apparent volume of distribution (V) of MXF. The PTA for LFX maximal mycobacterial kill at the critical MIC of 0.5 mg/L with the simulated 750 mg, 1000 mg, and 1500 mg doses were 29%, 62%, and 95%, respectively, whereas the PTA for resistance prevention at 1500 mg was only 4.8%, with none of the lower doses achieving this target. At the critical MIC of 0.25 mg/L, there was no change in the PTA for maximum bacterial kill when the MXF dose was increased (600 mg, 800 mg, and 1000 mg), but the PTA for resistance prevention was improved. Conclusion: The standard doses of LFX and MXF may not provide adequate drug exposure. PopPK of LFX is more predictable for maximum mycobacterial kill, whereas MXF's resistance prevention target increases with dose. Cr and BMI are likely important covariates for dose optimization in Ethiopian patients.

Therapeutic Drug Monitoring of Anti-infective Drugs: Implementation Strategies for 3 Different Scenarios.

BACKGROUND: Therapeutic drug monitoring (TDM) supports personalized treatment. For successful implementation, TDM must have a turnaround time suited to the clinical needs of patients and their health care settings. Here, the authors share their views of how a TDM strategy can be tailored to specific settings and patient groups. METHODS: The authors selected distinct scenarios for TDM: high-risk, complex, and/or critically ill patient population; outpatients; and settings with limited laboratory resources. In addition to the TDM scenario approach, they explored potential issues with the legal framework governing dose escalation. RESULTS: The most important issues identified in the different scenarios are that critically ill patients require rapid turnaround time, outpatients require an easy sampling procedure for the sample matrix and sample collection times, settings with limited laboratory resources necessitate setting-specific analytic techniques, and all scenarios warrant a legal framework to capture the use of escalated dosages, ideally with the use of trackable dosing software. CONCLUSIONS: To benefit patients, TDM strategies need to be tailored to the intended population. Strategies can be adapted for rapid turnaround time for critically ill patients, convenient sampling for outpatients, and feasibility for those in settings with limited laboratory resources.

Population Pharmacokinetics and Significant Under-Dosing of Anti-Tuberculosis Medications in People with HIV and Critical Illness.

Critical illness from tuberculosis (TB) bloodstream infection results in a high case fatality rate for people living with human immunodeficiency virus (HIV). Critical illness can lead to altered pharmacokinetics and suboptimal drug exposures. We enrolled adults living with HIV and hospitalized with sepsis, with and without meningitis, in Mbarara, Uganda that were starting first-line anti-TB therapy. Serum was collected two weeks after enrollment at 1-, 2-, 4-, and 6-h post-dose and drug concentrations quantified by validated LC-MS/MS methods. Non-compartmental analyses were used to determine total drug exposure, and population pharmacokinetic modeling and simulations were performed to determine optimal dosages. Eighty-one participants were enrolled. Forty-nine completed pharmacokinetic testing: 18 (22%) died prior to testing, 13 (16%) were lost to follow-up and one had incomplete testing. Isoniazid had the lowest serum attainment, with only 4.1% achieving a target exposure over 24 h (AUC0-24) of 52 mg·h/L despite appropriate weight-based dosing. Simulations to reach target AUC0-24 found necessary doses of rifampin of 1800 mg, pyrazinamide of 2500-3000 mg, and for isoniazid 900 mg or higher. Given the high case fatality ratio of TB-related critical illness in this population, an early higher dose anti-TB therapy should be trialed.

Levofloxacin pharmacokinetics in saliva as measured by a mobile microvolume UV spectrophotometer among people treated for rifampicin-resistant TB in Tanzania.

BACKGROUND: Early detection and correction of low fluoroquinolone exposure may improve treatment of MDR-TB. OBJECTIVES: To explore a recently developed portable, battery-powered, UV spectrophotometer for measuring levofloxacin in saliva of people treated for MDR-TB. METHODS: Patients treated with levofloxacin as part of a regimen for MDR-TB in Northern Tanzania had serum and saliva collected concurrently at 1 and 4 h after 2 weeks of observed levofloxacin administration. Saliva levofloxacin concentrations were quantified in the field via spectrophotometry, while serum was analysed at a regional laboratory using HPLC. A Bayesian population pharmacokinetics model was used to estimate the area under the concentration-time curve (AUC0-24). Subtarget exposures of levofloxacin were defined by serum AUC0-24 <80 mg·h/L. The study was registered at Clinicaltrials.gov with clinical trial identifier NCT04124055. RESULTS: Among 45 patients, 11 (25.6%) were women and 16 (37.2%) were living with HIV. Median AUC0-24 in serum was 140 (IQR = 102.4-179.09) mg·h/L and median AUC0-24 in saliva was 97.10 (IQR = 74.80-121.10) mg·h/L. A positive linear correlation was observed with serum and saliva AUC0-24, and a receiver operating characteristic curve constructed to detect serum AUC0-24 below 80 mg·h/L demonstrated excellent prediction [AUC 0.80 (95% CI = 0.62-0.94)]. Utilizing a saliva AUC0-24 cut-off of 91.6 mg·h/L, the assay was 88.9% sensitive and 69.4% specific in detecting subtarget serum AUC0-24 values, including identifying eight of nine patients below target. CONCLUSIONS: Portable UV spectrophotometry as a point-of-care screen for subtarget levofloxacin exposure was feasible. Use for triage to other investigation or personalized dosing strategy should be tested in a randomized study.

Determination of Rifampin Concentrations by Urine Colorimetry and Mobile Phone Readout for Personalized Dosing in Tuberculosis Treatment.

BACKGROUND: Individual pharmacokinetic variability is a driver of poor tuberculosis (TB) treatment outcomes. We developed a method for measurement of rifampin concentrations by urine colorimetry and a mobile phone photographic application to predict clinically important serum rifampin pharmacokinetic measurements in children treated for TB. METHODS: Among spiked urine samples, colorimetric assay performance was tested with conventional spectrophotometric and the mobile phone/light box methods under various environmental and biologic conditions. Urine rifampin absorbance (Abs) was then determined from timed specimens from children treated for TB in Tanzania, and compared to serum pharmacokinetic measurements collected throughout the dosing interval. RESULTS: Both the mobile phone/light box and spectrophotometry demonstrated excellent correlation across a wide range of urine rifampin concentrations (7.8-1000 mg/L) in intra- and interday trials, 24-hour exposure to ambient light or darkness, and varying urinalysis profiles (all r ≥ 0.98). In 12 Tanzanian children, the urine mobile phone/light box measurement and serum peak concentration (Cmax) were significantly correlated (P = .004). Using a Cmax target of 8 mg/L, the area under the receiver operating characteristic curve was 80.1% (range, 47.2%-100%). A urine mobile phone/light box threshold of 50 Abs correctly classified all patients (n = 6) with serum measurements below target. CONCLUSIONS: The urine colorimetry with mobile phone/light box assay accurately measured rifampin absorbance in varying environmental and biological conditions that may be observed clinically. Among children treated for TB, the assay was sensitive for detection of low rifampin serum concentrations. Future work will identify the optimal timing for urine collection, and operationalize use in TB-endemic settings.

Urine colorimetry for levofloxacin pharmacokinetics and personalized dosing in people with drug-resistant tuberculosis.

Background: Levofloxacin is a preferred drug for multidrug-resistant (MDR)-tuberculosis (TB) with bactericidal activity that correlates with the pharmacokinetic exposures of serum peak concentration (Cmax) and total area under the concentration time curve (AUC0-24). Pharmacokinetic exposures can be measured to personalize dosing to reach targets, but this practice requires venepuncture, chromatographic or mass spectrometry equipment, and technical expertise. We sought to demonstrate the accuracy of using urine colorimetry as a more feasible estimation of levofloxacin exposure. Method: A colorimetric method using bromocresol green was tested on spiked urine samples with levofloxacin measured using a spectrophotometer. This method was tested in urine samples of healthy volunteers given one 750 mg dose of levofloxacin with urine collected at 0-4 h, 4-8 h, and 8-24 h intervals, and concomitant serum samples were collected and analyzed by high-performance liquid chromatography. Validation of this assay was done in a cohort of people living with human immunodeficiency virus (PLWH), initiating a levofloxacin containing MDR-TB regimen. Results: Urine colorimetry was reproducible in spiked samples and the calibration was curve linear for levofloxacin concentrations ranging from 7.8 µg/ml to 250 µg/ml, with r = 0.98. In healthy volunteers, correlation between urine absorbance values and serum AUC0-24 was highest in urine collected between 4 and 8 h (r = 0.91, P = 0.01), yet in PLWH, urine collected between 0 and 4 h had highest correlation (r = 0.66, P = 0.05). The area under the receiver operating characteristics curve was >0.8 in the derivation, as well as the validation cohort for the urine absorbance values identifying people with total serum exposure below target. Conclusion: Urine colorimetry was highly sensitive in predicting target serum concentrations. Colorimetric methods to determine levofloxacin in urine may improve the feasibility of therapeutic drug monitoring and personalized dose adjustment in TB endemic settings.

Outpatient Versus Inpatient Surgical Fixation of Isolated Ankle Fractures: An Analysis of 90-Day Complications, Readmissions, and Costs.

Despite increasing interest toward managing isolated ankle fractures in an outpatient setting, evidence of its safety remains largely limited. The 2007 to 2014 Humana Administrative Claims database was queried to identify patients undergoing open reduction internal fixation for unimalleolar, bimalleolar, or trimalleolar isolated closed ankle fractures. Two cohorts (outpatient versus inpatient) were then matched on the basis of age, sex, race, region, fracture type (uni-/bi-/trimalleolar) and Elixhauser Comorbidity Index to control for selection bias. Multivariate regression analyses were performed to report independent impact of outpatient-treated ankle fracture surgery on 90-day complications, readmission, and emergency department visit rates. Independent-samples t test was used to compare global 90-day costs between cohorts. A total of 5317 inpatient-treated and 6941 outpatient-treated closed ankle fractures were included in the final cohort. After matching and multivariate analyses, patients with outpatient ankle fractures, compared with patients with inpatient ankle fractures, had statistically lower rates of pneumonia (2.3% versus 4.0%; p < .001), myocardial infarction (0.9% versus 1.8%; p = .005), acute renal failure (2.2% versus 5.3%; p < .001), urinary tract infections (7.4% versus 12.3%; p < .001), and pressure ulcers (0.9% versus 2.0%; p = .001). Outpatient ankle fractures also had lower rates of 90-day readmissions (9.7% versus 14.1%; p < .001) and emergency department visits (13.8% versus 16.2%; p = .028). Last, overall 90-day costs for outpatient ankle fractures were nearly $9000 lower than costs for inpatient ankle fractures ($12,923 versus $21,866; p < .001). Based on our findings, it appears that outpatient treatment of ankle fractures can be deemed safe and feasible in a select cohort of patients.