Pharmacokinetic-Pharmacodynamic Determinants of Clinical Outcomes for Rifampin-Resistant Tuberculosis: A Multisite Prospective Cohort Study.

BACKGROUND: Rifampin-resistant and/or multidrug-resistant tuberculosis (RR/MDR-TB) treatment requires multiple drugs, and outcomes remain suboptimal. Some drugs are associated with improved outcome. It is unknown whether particular pharmacokinetic-pharmacodynamic relationships predict outcome. METHODS: Adults with pulmonary RR/MDR-TB in Tanzania, Bangladesh, and the Russian Federation receiving local regimens were enrolled from June 2016 to July 2018. Serum was collected after 2, 4, and 8 weeks for each drug's area under the concentration-time curve over 24 hours (AUC0-24). Quantitative susceptibility of the M. tuberculosis isolate was measured by minimum inhibitory concentrations (MICs). Individual drug AUC0-24/MIC targets were assessed by adjusted odds ratios (ORs) for favorable treatment outcome, and hazard ratios (HRs) for time to sputum culture conversion. K-means clustering algorithm separated the cohort of the most common multidrug regimen into 4 clusters by AUC0-24/MIC exposures. RESULTS: Among 290 patients, 62 (21%) experienced treatment failure, including 30 deaths. Moxifloxacin AUC0-24/MIC target of 58 was associated with favorable treatment outcome (OR, 3.75; 95% confidence interval, 1.21-11.56; P = .022); levofloxacin AUC0-24/MIC of 118.3, clofazimine AUC0-24/MIC of 50.5, and pyrazinamide AUC0-24 of 379 mg × h/L were associated with faster culture conversion (HR >1.0, P < .05). Other individual drug exposures were not predictive. Clustering by AUC0-24/MIC revealed that those with the lowest multidrug exposures had the slowest culture conversion. CONCLUSIONS: Amidst multidrug regimens for RR/MDR-TB, serum pharmacokinetics and M. tuberculosis MICs were variable, yet defined parameters to certain drugs-fluoroquinolones, pyrazinamide, clofazimine-were predictive and should be optimized to improve clinical outcome. CLINICAL TRIALS REGISTRATION: NCT03559582.

Gridlock from diagnosis to treatment of multidrug resistant tuberculosis (MDR-TB) in Tanzania: patients' perspectives from a focus group discussion.

BACKGROUND: Molecular diagnostics have revolutionized the diagnosis of multidrug resistant tuberculosis (MDR-TB). Yet in Tanzania we found delay in diagnosis with more than 70% of MDR-TB patients having a history of several previous treatment courses for TB signaling prior opportunities for diagnosis. We aimed to explore patients' viewpoints and experiences with personal and socio-behavioral obstacles from MDR-TB diagnosis to treatment in an attempt to understand these prior findings. METHODS: The study was conducted in December 2016 with MDR-TB patients admitted at Kibong'oto Infectious Diseases Hospital. A qualitative approach deploying focus group discussions (FGDs) was used to gather information. Groups were sex aggregated to allow free interaction and to gauge gender specific issues in the social and behavioral contexts. The FGDs explored pathways and factors in the service delivery that may have contributed in the delay in accessing MDR-TB diagnostics and/or treatment. Collected data were coded, categorized and thematically interpreted. RESULTS: Forty MDR-TB patients participated in six FGDs. Challenges and barriers contributing to the delay in accessing MDR-TB diagnosis to treatment were as follows: 1) Participants had a different understanding of MDR-TB that led to seeking services outside the conventional health system; 2) Socio-economic adversity made health-seeking behavior difficult and often unproductive; 3) In the health system, challenges included inadequacy of MDR-TB diagnostic centers, lack of knowledge on behalf of health care providers to consider MDR-TB and order appropriate diagnostics; 4) The specimen referral system for early diagnosis of MDR-TB was inefficient. Non-adherence of TB patients to first-line anti-TB drugs prior to MDR-TB diagnosis, given the multitude of barriers discussed, was coupled with both intentional and unintentional non-adherence of health care providers to international standards of TB care. CONCLUSION: Patient-centered strategies bridging communities and the health system are urgently required for optimum MDR-TB control in Tanzania.

Model-Based Relationship between the Molecular Bacterial Load Assay and Time to Positivity in Liquid Culture.

The molecular bacterial load (MBL) assay is a new tuberculosis biomarker which provides results in ∼4 hours. The relationship between MBL and time-to-positivity (TTP) has not been thoroughly studied, and predictive models do not exist. We aimed to develop a model for MBL and identify the MBL-TTP relationship in patients. The model was developed on data from 105 tuberculosis patients from Malawi, Mozambique, and Tanzania with joint MBL and TTP observations quantified from patient sputum collected for 12 weeks. MBL was quantified using PCR of mycobacterial RNA and TTP using the mycobacterial growth indicator tube (MGIT) 960 system. Treatment consisted of isoniazid, pyrazinamide, and ethambutol in standard doses together with rifampin 10 or 35 mg/kg of body weight. The developed MBL-TTP model included several linked submodels, a component describing decline of bacterial load in sputum, another component describing growth in liquid culture, and a hazard model translating bacterial growth into a TTP signal. Additional components for contaminated and negative TTP samples were included. Visual predictive checks performed using the developed model gave good description of the observed data. The model predicted greater total sample loss for TTP than MBL due to contamination and negative samples. The model detected an increase in bacterial killing for 35 versus 10 mg/kg rifampin (P = 0.002). In conclusion, a combined model for MBL and TTP was developed that described the MBL-TTP relationship. The full MBL-TTP model or each submodel was used separately. Second, the model can be used to predict biomarker response for MBL given TTP data or vice versa in historical or future trials.

Using WGS to identify antibiotic resistance genes and predict antimicrobial resistance phenotypes in MDR Acinetobacter baumannii in Tanzania.

BACKGROUND: Reliable phenotypic antimicrobial susceptibility testing can be a challenge in clinical settings in low- and middle-income countries. WGS is a promising approach to enhance current capabilities. AIM: To study diversity and resistance determinants and to predict and compare resistance patterns from WGS data of Acinetobacter baumannii with phenotypic results from classical microbiological testing at a tertiary care hospital in Tanzania. METHODS AND RESULTS: MLST using Pasteur/Oxford schemes yielded eight different STs from each scheme. Of the eight, two STs were identified to be global clones 1 (n = 4) and 2 (n = 1) as per the Pasteur scheme. Resistance testing using classical microbiology determined between 50% and 92.9% resistance across all drugs. Percentage agreement between phenotypic and genotypic prediction of resistance ranged between 57.1% and 100%, with coefficient of agreement (κ) between 0.05 and 1. Seven isolates harboured mutations at significant loci (S81L in gyrA and S84L in parC). A number of novel plasmids were detected, including pKCRI-309C-1 (219000 bp) carrying 10 resistance genes, pKCRI-43-1 (34935 bp) carrying two resistance genes and pKCRI-49-1 (11681 bp) and pKCRI-28-1 (29606 bp), each carrying three resistance genes. New ampC alleles detected included ampC-69, ampC-70 and ampC-71. Global clone 1 and 2 isolates were found to harbour ISAba1 directly upstream of the ampC gene. Finally, SNP-based phylogenetic analysis of the A. baumannii isolates revealed closely related isolates in three clusters. CONCLUSIONS: The validity of the use of WGS in the prediction of phenotypic resistance can be appreciated, but at this stage is not sufficient for it to replace conventional antimicrobial susceptibility testing in our setting.

Effect of diabetes mellitus on TB drug concentrations in Tanzanian patients.

BACKGROUND: Diabetes mellitus (DM) is associated with poor TB treatment outcome. Previous studies examining the effect of DM on TB drug concentrations yielded conflicting results. No studies have been conducted to date in an African population. OBJECTIVES: To compare exposure to TB drugs in Tanzanian TB patients with and without DM. PATIENTS AND METHODS: A prospective pharmacokinetic study was performed among 20 diabetic and 20 non-diabetic Tanzanian TB patients during the intensive phase of TB treatment. Plasma pharmacokinetic parameters of isoniazid, rifampicin, pyrazinamide and ethambutol were compared using an independent-sample t-test on log-transformed data. Multiple linear regression analysis was performed to assess the effects of DM, gender, age, weight, HIV status and acetylator status on exposure to TB drugs. RESULTS: A trend was shown for 25% lower total exposure (AUC0-24) to rifampicin among diabetics versus non-diabetics (29.9 versus 39.9 mg·h/L, P=0.052). The AUC0-24 and peak concentration (Cmax) of isoniazid were also lower in diabetic TB patients (5.4 versus 10.6 mg·h/L, P=0.015 and 1.6 versus 2.8 mg/L, P=0.013). Pyrazinamide AUC0-24 and Cmax values were non-significantly lower among diabetics (P=0.08 and 0.09). In multivariate analyses, DM remained an independent predictor of exposure to isoniazid and rifampicin, next to acetylator status for isoniazid. CONCLUSIONS: There is a need for individualized dosing of isoniazid and rifampicin based on plasma concentration measurements (therapeutic drug monitoring) and for clinical trials on higher doses of these TB drugs in patients with TB and DM.

Underestimated pyrazinamide resistance may compromise outcomes of pyrazinamide containing regimens for treatment of drug susceptible and multi-drug-resistant tuberculosis in Tanzania.

BACKGROUND: Tuberculosis (TB) is the leading cause of death from an infectious disease and the roll-out of rapid molecular diagnostics for rifampin resistance has resulted in a steady rise in the number of patients with multidrug-resistant (MDR)-TB referred for treatment. Pyrazinamide is used in susceptible TB treatment for 6 months when used in combination with rifampin, isoniazid and ethambutol and is an important companion drug in novel MDR-TB trials. This study was undertaken to determine the prevalence of pyrazinamide resistance by either phenotypic or pncA testing among patients admitted to a referral hospital in Tanzania for drug-susceptible and MDR-TB treatment. METHODS: Surveillance sputa were sent among subjects beginning TB therapy at the national MDR-TB referral hospital during a 6 month period in 2013-2014. Mycobacterial cultures of pretreatment sputa were performed at the Kilimanjaro Clinical Research Institute (KCRI) in the BACTEC mycobacterial growth indicator tubes (MGIT) 960 system. Speciation of M. tuberculosis complex was confirmed by MTBc assay. Isolates were sub-cultured on to Lowenstein-Jensen (LJ) slants. Phenotypic resistance to pyrazinamide was performed in the MGIT system while a real-time PCR with High Resolution Melt (HRM) technique was used to determine mutation in the pncA gene from the same pure subculture. Sputa were then collected monthly to determine the time to culture negativity. Final treatment outcome was determined. RESULTS: Ninety-one M. tuberculosis isolates from individual patients were available for analysis of which 30 (32.9%) had MDR-TB, the mean (±SD) age was 33 ± 10 years, and the majority 23 (76.7%) were males. Of the 30 MDR-TB patients, 15(50%) had isolates with pyrazinamide resistance by conventional MGIT testing. This proportion expectedly exceeded the number with pyrazinamide resistance in the 61 patients without MDR-TB, 13 (21.3%) (p = 0.008). Six (20%) of MDR-TB patients had a poor outcome including treatment failure. Among patients with treatment failure, 5 (83%) had pyrazinamide resistance compared to only 10 (41.6%) with treatment success (p = 0.08). Two patients died, and both had isolates with pyrazinamide resistance. No other pretreatment characteristic was associated with treatment outcome. CONCLUSION: Pyrazinamide susceptibility appears to be important in clinical outcomes for MDR-TB patients, and susceptibility testing appears to be a critical adjunct to TB care. The high proportion of PZA resistance in non-MDR TB cases calls for further local investigation.

Gridlock from diagnosis to treatment of multidrug-resistant tuberculosis in Tanzania: low accessibility of molecular diagnostic services and lack of healthcare worker empowerment in 28 districts of 5 high burden TB regions with mixed methods evaluation.

BACKGROUND: Multidrug-resistant tuberculosis (MDR-TB) outcomes are adversely impacted by delay in diagnosis and treatment. METHODS: Mixed qualitative and quantitative approaches were utilized to identify healthcare system related barriers to implementation of molecular diagnostics for MDR-TB. Randomly sampled districts from the 5 highest TB burden regions were enrolled during the 4th quarter of 2016. District TB & Leprosy Coordinators (DTLCs), and District AIDS Coordinators (DACs) were interviewed, along with staff from all laboratories within the selected districts where molecular diagnostics tests for MDR-TB were performed. Furthermore, the 2015 registers were audited for all drug-susceptible but retreatment TB cases and TB collaborative practices in HIV clinics, as these patients were in principal targeted for drug susceptibility testing by rapid molecular diagnostics. RESULTS: Twenty-eight TB districts from the 5 regions had 399 patients reviewed for retreatment with a drug-susceptible regimen. Only 160 (40%) had specimens collected for drug-susceptibility testing, and of those specimens only 120 (75%) had results communicated back to the clinic. MDR-TB was diagnosed in 16 (13.3%) of the 120 specimens but only 12 total patients were ultimately referred for treatment. Furthermore, among the HIV/AIDS clinics served in 2015, the median number of clients with TB diagnosis was 92 cases [IQR 32-157] yet only 2 people living with HIV were diagnosed with MDR-TB throughout the surveyed districts. Furthermore, the districts generated 53 front-line healthcare workers for interviews. DTLCs with intermediate or no knowledge on the clinical application of XpertMTB/RIF were 3 (11%), and 10 (39%), and DACs with intermediate or no knowledge were 0 (0%) and 2 (8%) respectively (p = 0.02). Additionally, 11 (100%) of the laboratories surveyed had only the 4-module XpertMTB/RIF equipment. The median time that XpertMTB/RIF was not functional in the 12 months prior to the investigation was 2 months (IQR 1-4). CONCLUSIONS: Underutilization of molecular diagnostics in high-risk groups was a function of a lack of front-line healthcare workforce empowerment and training, and a lack of equipment access, which likely contributed to the observed delay in MDR-TB diagnosis in Tanzania.

The Potential for Treatment Shortening With Higher Rifampicin Doses: Relating Drug Exposure to Treatment Response in Patients With Pulmonary Tuberculosis.

Background: Tuberculosis remains a huge public health problem and the prolonged treatment duration obstructs effective tuberculosis control. Higher rifampicin doses have been associated with better bactericidal activity, but optimal dosing is uncertain. This analysis aimed to characterize the relationship between rifampicin plasma exposure and treatment response over 6 months in a recent study investigating the potential for treatment shortening with high-dose rifampicin. Methods: Data were analyzed from 336 patients with pulmonary tuberculosis (97 with pharmacokinetic data) treated with rifampicin doses of 10, 20, or 35 mg/kg. The response measure was time to stable sputum culture conversion (TSCC). We derived individual exposure metrics with a previously developed population pharmacokinetic model of rifampicin. TSCC was modeled using a parametric time-to-event approach, and a sequential exposure-response analysis was performed. Results: Higher rifampicin exposures increased the probability of early culture conversion. No maximal limit of the effect was detected within the observed range. The expected proportion of patients with stable culture conversion on liquid medium at week 8 was predicted to increase from 39% (95% confidence interval, 37%-41%) to 55% (49%-61%), with the rifampicin area under the curve increasing from 20 to 175 mg/L·h (representative for 10 and 35 mg/kg, respectively). Other predictors of TSCC were baseline bacterial load, proportion of culture results unavailable, and substitution of ethambutol for either moxifloxacin or SQ109. Conclusions: Increasing rifampicin exposure shortened TSCC, and the effect did not plateau, indicating that doses >35 mg/kg could be yet more effective. Optimizing rifampicin dosage while preventing toxicity is a clinical priority.

Molecular epidemiology of virulence and antimicrobial resistance determinants in Klebsiella pneumoniae from hospitalised patients in Kilimanjaro, Tanzania.

This study aimed to use whole-genome sequencing to determine virulence and antimicrobial resistance genes in K. pneumoniae isolated from patients in a tertiary care hospital in Kilimanjaro. K. pneumoniae isolates from patients attending Kilimanjaro Christian Medical Centre between August 2013 and August 2015 were fully genome-sequenced and analysed locally. Sequence analysis was done for identification of virulence and AMR genes. Plasmid and multi-locus sequence typing and capsular or capsular (K) typing were performed and phylogeny was done to ascertain K. pneumoniae relatedness. Stata 13 (College Station, TX, 77845, USA) was used to determine Cohen's kappa coefficient of agreement between the phenotypically tested and sequence-predicted resistance. A total of 16 (47.1%) sequence types (STs) and 10 (29.4%) K types were identified in 30 (88.2%) and 17 (50.0%) of all analysed isolates, respectively. K. pneumoniae ST17 were 6 (17.6%). The commonest determinants were blaCTX-M-15 in 16 (47.1%) isolates, blaSHV in 30 (88.2%), blaOXA-1 in 8 (23.5%) and blaTEM-1 in 18 (52.9%) isolates. Resistance genes for aminoglycosides were detected in 21 (61.8%) isolates, fluoroquinolones in 13 (38.2%) and quinolones 34 (100%). Ceftazidime and ceftriaxone showed the strongest agreement between phenotype- and sequence-based resistance results: 93.8%, kappa = 0.87 and p = 0.0002. Yersiniabactin determinant was detected in 12 (35.3%) of K. pneumoniae. The proportion of AMR and virulence determinants detected in K. pneumoniae is alarming. WGS-based diagnostic approach has showed promising potentials in clinical microbiology, hospital outbreak source tracing virulence and AMR detection at KCMC.

A new trial design to accelerate tuberculosis drug development: the Phase IIC Selection Trial with Extended Post-treatment follow-up (STEP).

BACKGROUND: The standard 6-month four-drug regimen for the treatment of drug-sensitive tuberculosis has remained unchanged for decades and is inadequate to control the epidemic. Shorter, simpler regimens are urgently needed to defeat what is now the world's greatest infectious disease killer. METHODS: We describe the Phase IIC Selection Trial with Extended Post-treatment follow-up (STEP) as a novel hybrid phase II/III trial design to accelerate regimen development. In the Phase IIC STEP trial, the experimental regimen is given for the duration for which it will be studied in phase III (presently 3 or 4 months) and patients are followed for clinical outcomes of treatment failure and relapse for a total of 12 months from randomisation. Operating characteristics of the trial design are explored assuming a classical frequentist framework as well as a Bayesian framework with flat and sceptical priors. A simulation study is conducted using data from the RIFAQUIN phase III trial to illustrate how such a design could be used in practice. RESULTS: With 80 patients per arm, and two (2.5 %) unfavourable outcomes in the STEP trial, there is a probability of 0.99 that the proportion of unfavourable outcomes in a potential phase III trial would be less than 12 % and a probability of 0.91 that the proportion of unfavourable outcomes would be less than 8 %. With six (7.5 %) unfavourable outcomes, there is a probability of 0.82 that the proportion of unfavourable outcomes in a potential phase III trial would be less than 12 % and a probability of 0.41 that it would be less than 8 %. Simulations using data from the RIFAQUIN trial show that a STEP trial with 80 patients per arm would have correctly shown that the Inferior Regimen should not proceed to phase III and would have had a high chance (0.88) of either showing that the Successful Regimen could proceed to phase III or that it might require further optimisation. CONCLUSIONS: Collection of definitive clinical outcome data in a relatively small number of participants over only 12 months provides valuable information about the likelihood of success in a future phase III trial. We strongly believe that the STEP trial design described herein is an important tool that would allow for more informed decision-making and accelerate regimen development.

Feasibility of Real Time Medication Monitoring Among HIV Infected and TB Patients in a Resource-Limited Setting.

HIV infected and tuberculosis (TB) patients need high levels of treatment adherence to achieve optimal treatment outcomes. We conducted a pilot-study on real time medication monitoring (RTMM) in a resource-limited setting. We enrolled five HIV infected and five TB patients from Kilimanjaro, Tanzania. They took their medication using RTMM. When the device was not opened on time, patients received a reminder SMS. After 3 months, we interviewed patients. Six patients (60 %) reached adherence of >95 %. Nine-hundred-twenty-two of 1104 intakes (84 %) were on time. Five-hundred reminders (45 %) were sent, of which 202 (40 %) were incorrect, because of an unstable mobile network. Nine patients found the device helpful and nine mentioned it keeps medication safe. Six patients reported that the size was too big. Five patients mentioned they received incorrect reminders. The device is considered useful in Kilimanjaro. Optimization of the device should consider network connectivity and the size of the device.

Sensititre MycoTB plate compared to Bactec MGIT 960 for first- and second-line antituberculosis drug susceptibility testing in Tanzania: a call to operationalize MICs.

MIC testing for Mycobacterium tuberculosis is now commercially available. Drug susceptibility testing by the MycoTB MIC plate has not been directly compared to that by the Bactec MGIT 960. We describe a case of extensively drug-resistant tuberculosis (XDR-TB) in Tanzania where initial MIC testing may have prevented acquired resistance. From testing on archived isolates, the accuracy with the MycoTB plate was >90% for important first- and second-line drugs compared to that with the MGIT 960, and clinically useful quantitative interpretation was also provided.

Pharmacological interactions between rifampicin and antiretroviral drugs: challenges and research priorities for resource-limited settings.

Coadministration of antituberculosis and antiretroviral therapy is often inevitable in high-burden countries where tuberculosis (TB) is the most common opportunistic infection associated with HIV/AIDS. Concurrent use of rifampicin and many antiretroviral drugs is complicated by pharmacokinetic drug-drug interactions. Rifampicin is a very potent enzyme inducer, which can result in subtherapeutic antiretroviral drug concentrations. In addition, TB drugs and antiretroviral drugs have additive (pharmacodynamic) interactions as reflected in overlapping adverse effect profiles. This review provides an overview of the pharmacological interactions between rifampicin-based TB treatment and antiretroviral drugs in adults living in resource-limited settings. Major progress has been made to evaluate the interactions between TB drugs and antiretroviral therapy; however, burning questions remain concerning nevirapine and efavirenz effectiveness during rifampicin-based TB treatment, treatment options for TB-HIV-coinfected patients with nonnucleoside reverse transcriptase inhibitor resistance or intolerance, and exact treatment or dosing schedules for vulnerable patients including children and pregnant women. The current research priorities can be addressed by maximizing the use of already existing data, creating new data by conducting clinical trials and prospective observational studies and to engage a lobby to make currently unavailable drugs available to those most in need.

Plasma drug activity in patients on treatment for multidrug-resistant tuberculosis.

Little is known about plasma drug concentrations relative to quantitative susceptibility in patients with multidrug-resistant tuberculosis (MDR-TB). We previously described a TB drug activity (TDA) assay that determines the ratio of the time to detection of plasma-cocultured Mycobacterium tuberculosis versus control growth in a Bactec MGIT system. Here, we assess the activity of individual drugs in a typical MDR-TB regimen using the TDA assay. We also examined the relationship of the TDA to the drug concentration at 2 h (C2) and the MICs among adults on a MDR-TB regimen in Tanzania. These parameters were also compared to the treatment outcome of sputum culture conversion. Individually, moxifloxacin yielded superior TDA results versus ofloxacin, and only moxifloxacin and amikacin yielded TDAs equivalent to a -2-log killing. In the 25 patients enrolled on a regimen of kanamycin, levofloxacin, ethionamide, pyrazinamide, and cycloserine, the C2 values were found to be below the expected range for levofloxacin in 13 (52%) and kanamycin in 10 (40%). Three subjects with the lowest TDA result (<1.5, a finding indicative of poor killing) had significantly lower kanamycin C2/MIC ratios than subjects with a TDA of ≥1.5 (9.8 ± 8.7 versus 27.0 ± 19.1; P = 0.04). The mean TDAs were 2.52 ± 0.76 in subjects converting to negative in ≤2 months and 1.88 ± 0.57 in subjects converting to negative in >2 months (P = 0.08). In Tanzania, MDR-TB drug concentrations were frequently low, and a wide concentration/MIC range was observed that affected plasma drug activity ex vivo. An opportunity exists for pharmacokinetic optimization in current MDR-TB regimens, which may improve treatment response.

A randomized controlled trial of standard versus intensified tuberculosis diagnostics on treatment decisions by physicians in Northern Tanzania.

BACKGROUND: Routine tuberculosis culture remains unavailable in many high-burden areas, including Tanzania. This study sought to determine the impact of providing mycobacterial culture results over standard of care [unconcentrated acid-fast (AFB) smears] on management of persons with suspected tuberculosis. METHODS: Adults and children with suspected tuberculosis were randomized to standard (direct AFB smear only) or intensified (concentrated AFB smear and tuberculosis culture) diagnostics and followed for 8 weeks. The primary endpoint was appropriate treatment (i.e. antituberculosis therapy for those with tuberculosis, no antituberculous therapy for those without tuberculosis). RESULTS: Seventy participants were randomized to standard (n = 37, 53%) or intensive (n = 33, 47%) diagnostics. At 8 weeks, 100% (n = 22) of participants in follow up randomized to intensive diagnostics were receiving appropriate care, vs. 22 (88%) of 25 participants randomized to standard diagnostics (p = 0.14). Overall, 18 (26%) participants died; antituberculosis therapy was associated with lower mortality (9% who received antiuberculosis treatment died vs. 26% who did not, p = 0.04). CONCLUSIONS: Under field conditions in a high burden setting, the impact of intensified diagnostics was blunted by high early mortality. Enhanced availability of rapid diagnostics must be linked to earlier access to care for outcomes to improve.

Pharmacokinetics of first-line tuberculosis drugs in Tanzanian patients.

East Africa has a high tuberculosis (TB) incidence and mortality, yet there are very limited data on exposure to TB drugs in patients from this region. We therefore determined the pharmacokinetic characteristics of first-line TB drugs in Tanzanian patients using intensive pharmacokinetic sampling. In 20 adult TB patients, plasma concentrations were determined just before and at 1, 2, 3, 4, 6, 8, 10, and 24 h after observed drug intake with food to estimate the areas under the curve from 0 to 24 h (AUC0-24) and peak plasma concentrations (Cmax) of isoniazid, rifampin, pyrazinamide, and ethambutol. Acetylator status for isoniazid was assessed phenotypically using the isoniazid elimination half-life and the acetylisoniazid/isoniazid metabolic ratio at 3 h postdose. The geometric mean AUC0-24s were as follows: isoniazid, 11.0 h · mg/liter; rifampin, 39.9 h · mg/liter; pyrazinamide, 344 h · mg/liter; and ethambutol, 20.2 h · mg/liter. The Cmax was below the reference range for isoniazid in 10/19 patients and for rifampin in 7/20 patients. In none of the patients were the Cmaxs for pyrazinamide and ethambutol below the reference range. Elimination half-life and metabolic ratio of isoniazid gave discordant phenotyping results in only 2/19 patients. A substantial proportion of patients had an isoniazid and/or rifampin Cmax below the reference range. Intake of TB drugs with food may partly explain these low drug levels, but such a drug intake reflects common practice. The finding of low TB drug concentrations is concerning because low concentrations have been associated with worse treatment outcome in several other studies.

Application of quantitative second-line drug susceptibility testing at a multidrug-resistant tuberculosis hospital in Tanzania.

BACKGROUND: Lack of rapid and reliable susceptibility testing for second-line drugs used in the treatment of multidrug-resistant tuberculosis (MDR-TB) may limit treatment success. METHODS: Mycobacterium tuberculosis isolates from patients referred to Kibong'oto National TB Hospital in Tanzania for second-line TB treatment underwent confirmatory speciation and susceptibility testing. Minimum inhibitory concentration (MIC) testing on MYCOTB Sensititre plates was performed for all drugs available in the second-line formulary. We chose to categorize isolates as borderline susceptible if the MIC was at or one dilution lower than the resistance breakpoint. M. tuberculosis DNA was sequenced for resistance mutations in rpoB (rifampin), inhA (isoniazid, ethionamide), katG (isoniazid), embB (ethambutol), gyrA (fluoroquinolones), rrs (amikacin, kanamycin, capreomycin), eis (kanamycin) and pncA (pyrazinamide). RESULTS: Of 22 isolates from patients referred for second-line TB treatment, 13 (59%) were MDR-TB and the remainder had other resistance patterns. MIC testing identified 3 (14%) isolates resistant to ethionamide and another 8 (36%) with borderline susceptibility. No isolate had ofloxacin resistance, but 10 (45%) were borderline susceptible. Amikacin was fully susceptible in 15 (68%) compared to only 11 (50%) for kanamycin. Resistance mutations were absent in gyrA, rrs or eis for all 13 isolates available for sequencing, but pncA mutation resultant in amino acid change or stop codon was present in 6 (46%). Ten (77%) of MDR-TB patients had at least one medication that could have logically been modified based on these results (median 2; maximum 4). The most common modifications were a change from ethioniamide to para-aminosalicylic acid, and the use of higher dose levofloxacin. CONCLUSIONS: In Tanzania, quantitative second-line susceptibility testing could inform and alter MDR-TB management independent of drug-resistance mutations. Further operational studies are warranted.

Prevalence and risk factors for infection of bovine tuberculosis in indigenous cattle in the Serengeti ecosystem, Tanzania.

BACKGROUND: Bovine tuberculosis (bTB) is a chronic debilitating disease and is a cause of morbidity and mortality in livestock, wildlife and humans. This study estimated the prevalence and risk factors associated with bovine tuberculosis transmission in indigenous cattle at the human-animal interface in the Serengeti ecosystem of Tanzania. RESULTS: A total of 1,103 indigenous cattle from 32 herds were investigated for the presence of bTB using the Single Intradermal Comparative Tuberculin Test. Epidemiological data on herd structure, management and grazing system were also collected.The apparent individual animal prevalence of tuberculin reactors was 2.4% (95% confidence interval (CI), 1.7 - 3.5%), whereas the true prevalence was 0.6% CI, 0.6 - 0.7% as indicated by a reaction to avian tuberculin purified protein derivatives (PPD) which is more than 4 mm greater than the reaction to avian tuberculin PPD. The results showed that 10.6% (117/1,103) showed non-specific reactions (atypical mycobacterium). The herd prevalence of 50% (16/32) was found. Tuberculin skin test results were found to be significantly associated with age, location, size of the household and animal tested. Of 108 respondents, 70 (64.8%) individuals had not heard about bovine tuberculosis at all. Thirty five percent (38/108) of respondents at least were aware of bTB. About 60% (23/38) of respondents who were aware of bTB had some knowledge on how bTB is spread. Eighty one percent (87/108) of respondents were not aware of the presence of bTB in wildlife. There is regular contact between cattle and wild animals due to sharing of grazing land and water sources, with 99% (107/108) of households grazing cattle in communal pastures. CONCLUSION: The study has demonstrated a high reported interaction of livestock with wildlife and poor knowledge of most cattle owners concerning bTB and its transmission pathways among people, livestock and wildlife. Although the overall proportion of animals with bTB is relatively low, herd prevalence is 50% and prevalence within herds varied considerably. Thus there is a possibility of cross transmission of bTB at wildlife-livestock interface areas that necessitates use of genetic strain typing methods to characterize them accurately.

Protocol for a feasibility randomized controlled trial to evaluate the efficacy, safety and tolerability of N-acetylcysteine in reducing adverse drug reactions among adults treated for multidrug-resistant tuberculosis in Tanzania.

BACKGROUND: Adverse drug reactions (ADRs) frequently occur in patients using second-line anti-tuberculosis medicine for treatment of multidrug resistant tuberculosis (MDR-TB). ADRs contribute to treatment interruptions which can compromise treatment response and risk acquired drug resistance to critical newer drugs such as bedaquiline, while severe ADRs carry considerable morbidity and mortality. N-acetylcysteine (NAC) has shown promise in reducing ADRs for medications related to TB in case series or randomized controlled trials in other medical conditions, yet evidence is lacking in MDR-TB patients. TB endemic settings have limited capacity to conduct clinical trials. We designed a proof-of-concept clinical trial primarily to explore the preliminary evidence on the protective effect of NAC among people treated for MDR-TB with second-line anti-TB medications. METHODS: This is a proof-of-concept randomized open label clinical trial with 3 treatment arms including a control arm, an interventional arm of NAC 900 mg daily, and an interventional arm of NAC 900 mg twice-daily administered during the intensive phase of MDR-TB treatment. Patients initiating MDR-TB treatment will be enrolled at Kibong'oto National Center of Excellence for MDR-TB in the Kilimanjaro region of Tanzania. The minimum anticipated sample size is 66; with 22 participants in each arm. ADR monitoring will be performed at baseline and daily follow-up over 24 weeks including blood and urine specimen collection for hepatic and renal function and electrolyte abnormalities, and electrocardiogram. Sputum will be collected at baseline and monthly thereafter and cultured for mycobacteria as well as assayed for other molecular targets of Mycobacterium tuberculosis. Adverse drug events will be analysed over time using mixed effect models. Mean differences between arms in change of the ADRs from baseline (with 95% confidence intervals) will be derived from the fitted model. DISCUSSION: Given that NAC promotes synthesis of glutathione, an intracellular antioxidant that combats the impact of oxidative stress, it may protect against medication induced oxidative damage in organs such as liver, pancreas, kidney, and cells of the immune system. This randomized controlled trial will determine if NAC leads to fewer ADRs, and if this protection is dose dependent. Fewer ADRs among patients treated with MDR-TB may significantly improve treatment outcomes for multidrug regimens that necessitate prolonged treatment durations. Conduct of this trial will set the needed infrastructure for clinical trials. TRIAL REGISTRATION: PACTR202007736854169 Registered 03 July 2020.

Effect of seven anti-tuberculosis treatment regimens on sputum microbiome: a retrospective analysis of the HIGHRIF study 2 and PanACEA MAMS-TB clinical trials.

BACKGROUND: Respiratory tract microbiota has been described as the gatekeeper for respiratory health. We aimed to assess the impact of standard-of-care and experimental anti-tuberculosis treatment regimens on the respiratory microbiome and implications for treatment outcomes. METHODS: In this retrospective study, we analysed the sputum microbiome of participants with tuberculosis treated with six experimental regimens versus standard-of-care who were part of the HIGHRIF study 2 (NCT00760149) and PanACEA MAMS-TB (NCT01785186) clinical trials across a 3-month treatment follow-up period. Samples were from participants in Mbeya, Kilimanjaro, Bagamoyo, and Dar es Salaam, Tanzania. Experimental regimens were composed of different combinations of rifampicin (R), isoniazid (H), pyrazinamide (Z), ethambutol (E), moxifloxacin (M), and a new drug, SQ109 (Q). Reverse transcription was used to create complementary DNA for each participant's total sputum RNA and the V3-V4 region of the 16S rRNA gene was sequenced using the Illumina metagenomic technique. Qiime was used to analyse the amplicon sequence variants and estimate alpha diversity. Descriptive statistics were applied to assess differences in alpha diversity pre-treatment and post-treatment initiation and the effect of each treatment regimen. FINDINGS: Sequence data were obtained from 397 pre-treatment and post-treatment samples taken between Sept 26, 2008, and June 30, 2015, across seven treatment regimens. Pre-treatment microbiome (206 genera) was dominated by Firmicutes (2860 [44%] of 6500 amplicon sequence variants [ASVs]) at the phylum level and Streptococcus (2340 [36%] ASVs) at the genus level. Two regimens had a significant depressing effect on the microbiome after 2 weeks of treatment, HR20mg/kgZM (Shannon diversity index p=0·0041) and HR35mg/kgZE (p=0·027). Gram-negative bacteria were the most sensitive to bactericidal activity of treatment with the highest number of species suppressed being under the moxifloxacin regimen. By week 12 after treatment initiation, microbiomes had recovered to pre-treatment level except for the HR35mg/kgZE regimen and for genus Mycobacterium, which did not show recovery across all regimens. Tuberculosis culture conversion to negative by week 8 of treatment was associated with clearance of genus Neisseria, with a 98% reduction of the pre-treatment level. INTERPRETATION: HR20mg/kgZM was effective against tuberculosis without limiting microbiome recovery, which implies a shorter efficacious anti-tuberculosis regimen with improved treatment outcomes might be achieved without harming the commensal microbiota. FUNDING: European and Developing Countries Clinical Trials Partnership and German Ministry of Education and Research.