High-Dose Isoniazid Lacks EARLY Bactericidal Activity Against Isoniazid-resistant Tuberculosis Mediated by katG Mutations: A Randomized, Phase 2 Clinical Trial.

RATIONALE: Observational studies suggest that high-dose isoniazid may be efficacious in treating multidrug-resistant tuberculosis (MDR-TB). However, its activity against Mycobacterium tuberculosis (M.tb) with katG mutations (which typically confer high-level resistance) is not established. OBJECTIVE: To characterize early bactericidal activity (EBA) of high-dose isoniazid in patients with tuberculosis caused by katG-mutated M.tb. METHODS: A5312 was a Phase 2A randomized, open-label trial. Participants with tuberculosis caused by katG-mutated M.tb were randomized to receive 15 or 20 mg/kg isoniazid daily for 7 days. Daily sputum samples were collected for quantitative culture. Intensive PK sampling was performed on day 6. Data were pooled across all A5312 participants for analysis (drug-sensitive, inhA-mutated, and katG-mutated M.tb). EBA was determined using nonlinear mixed-effects modelling. RESULTS: Of 80 treated participants, 21 had katG-mutated M.tb. Isoniazid PK was best described by a two-compartment model with an effect of NAT2 acetylator phenotype on clearance. Model-derived Cmax and AUC in the 15 and 20 mg/kg groups were 15.0 and 22.1 mg/L and 57.6 and 76.8 mg∙h/L, respectively. Isoniazid bacterial kill was described using an effect compartment and a sigmoidal Emax relationship. Isoniazid potency against katG-mutated M.tb was approximately 10-fold lower than against inhA-mutated M.tb. The highest dose (20 mg/kg) did not demonstrate measurable EBA, except in a subset of slow NAT2 acetylators (who experienced the highest concentrations). There were no grade 3 or higher drug-related adverse events. CONCLUSIONS: This study found negligible bactericidal activity of high-dose isoniazid (15-20 mg/kg) in the majority of participants with tuberculosis caused by katG-mutated M.tb. Clinical trial registration available at www. CLINICALTRIALS: gov, ID: NCT01936831.

Mycobacteriophages to Treat Tuberculosis: Dream or Delusion?

Rates of antimicrobial resistance are increasing globally while the pipeline of new antibiotics is drying up, putting patients with disease caused by drug-resistant bacteria at increased risk of complications and death. The growing costs for diagnosis and management of drug resistance threaten tuberculosis control where the disease is endemic and resources limited. Bacteriophages are viruses that attack bacteria. Phage preparations served as anti-infective agents long before antibiotics were discovered. Though small in size, phages are the most abundant and diverse biological entity on earth. Phages have co-evolved with their hosts and possess all the tools needed to infect and kill bacteria, independent of drug resistance. Modern biotechnology has improved our understanding of the biology of phages and their possible uses. Phage preparations are available to treat meat, fruit, vegetables, and dairy products against parasites or to prevent contamination with human pathogens, such as Listeria monocytogenes, Escherichia coli, or Staphylococcus aureus. Such phage-treated products are considered fit for human consumption. A number of recent case reports describe in great detail the successful treatment of highly drug-resistant infections with individualized phage preparations. Formal clinical trials with standardized products are slowly emerging. With its highly conserved genome and relative paucity of natural phage defence mechanisms Mycobacterium tuberculosis appears to be a suitable target for phage treatment. A phage cocktail with diverse and strictly lytic phages that kill all lineages of M. tuberculosis, and can be propagated on Mycobacterium smegmatis, has been assembled and is available for the evaluation of optimal dosage and suitable routes of administration for tuberculosis in humans. Phage treatment can be expected to be safe and active on extracellular organisms, but phage penetration to intracellular and granulomatous environments as well as synergistic effects with antibiotics are important questions to address during further evaluation.

A Semimechanistic Model of the Bactericidal Activity of High-Dose Isoniazid against Multidrug-Resistant Tuberculosis: Results from a Randomized Clinical Trial.

Rationale: There is accumulating evidence that higher-than-standard doses of isoniazid are effective against low-to-intermediate-level isoniazid-resistant strains of Mycobacterium tuberculosis, but the optimal dose remains unknown. Objectives: To characterize the association between isoniazid pharmacokinetics (standard or high dose) and early bactericidal activity against M. tuberculosis (drug sensitive and inhA mutated) and N-acetyltransferase 2 status. Methods: ACTG (AIDS Clinical Trial Group) A5312/INHindsight is a 7-day early bactericidal activity study with isoniazid at a normal dose (5 mg/kg) for patients with drug-sensitive bacteria and 5, 10, and 15 mg/kg doses for patients with inhA mutants. Participants with pulmonary tuberculosis received daily isoniazid monotherapy and collected sputum daily. Colony-forming units (cfu) on solid culture and time to positivity in liquid culture were jointly analyzed using nonlinear mixed-effects modeling. Measurements and Main Results: Fifty-nine adults were included in this analysis. A decline in sputum cfu was described by a one-compartment model, whereas an exponential bacterial growth model was used to interpret time-to-positivity data. The model found that bacterial kill is modulated by isoniazid concentration using an effect compartment and a sigmoidal Emax relationship (a model linking the drug concentration to the observed effect). The model predicted lower potency but similar maximum kill of isoniazid against inhA-mutated compared with drug-sensitive isolates. Based on simulations from the pharmacokinetics-pharmacodynamics model, to achieve a drop in bacterial load comparable to 5 mg/kg against drug-sensitive tuberculosis, 10- and 15-mg/kg doses are necessary against inhA-mutated isolates in slow and intermediate N-acetyltransferase 2 acetylators, respectively. Fast acetylators underperformed even at 15 mg/kg. Conclusions: Dosing of isoniazid based on N-acetyltransferase 2 acetylator status may help patients attain effective exposures against inhA-mutated isolates. Clinical trial registered with www.clinicaltrials.gov (NCT01936831).

Increased bactericidal activity but dose-limiting intolerability at 50†mg·kg-1 rifampicin.

BACKGROUND: Accumulating data indicate that higher rifampicin doses are more effective and shorten tuberculosis (TB) treatment duration. This study evaluated the safety, tolerability, pharmacokinetics, and 7- and 14-day early bactericidal activity (EBA) of increasing doses of rifampicin. Here we report the results of the final cohorts of PanACEA HIGHRIF1, a dose escalation study in treatment-naive adult smear-positive patients with TB. METHODS: Patients received, in consecutive cohorts, 40 or 50 mg·kg-1 rifampicin once daily in monotherapy (day 1-7), supplemented with standard dose isoniazid, pyrazinamide and ethambutol between days 8 and 14. RESULTS: In the 40 mg·kg-1 cohort (n=15), 13 patients experienced a total of 36 adverse events during monotherapy, resulting in one treatment discontinuation. In the 50 mg·kg-1 cohort (n=17), all patients experienced adverse events during monotherapy, 93 in total; 11 patients withdrew or stopped study medication. Adverse events were mostly mild/moderate and tolerability rather than safety related, i.e. gastrointestinal disorders, pruritis, hyperbilirubinaemia and jaundice. There was a more than proportional increase in the rifampicin geometric mean area under the plasma concentration-time curve from time 0 to 12 h (AUC0-24 h) for 50 mg·kg-1 compared with 40 mg·kg-1; 571 (range 320-995) versus 387 (range 201-847) mg·L-1·h, while peak exposures saw proportional increases. Protein-unbound exposure after 50 mg·kg-1 (11% (range 8-17%)) was comparable with lower rifampicin doses. Rifampicin exposures and bilirubin concentrations were correlated (Spearman's ρ=0.670 on day 3, p<0.001). EBA increased considerably with dose, with the highest seen after 50 mg·kg-1: 14-day EBA -0.427 (95% CI -0.500- -0.355) log10CFU·mL-1·day-1. CONCLUSION: Although associated with an increased bactericidal effect, the 50 mg·kg-1 dose was not well tolerated. Rifampicin at 40 mg·kg-1 was well tolerated and therefore selected for evaluation in a phase IIc treatment-shortening trial.

Early Bactericidal Activity of Different Isoniazid Doses for Drug-Resistant Tuberculosis (INHindsight): A Randomized, Open-Label Clinical Trial.

Rationale: High-dose isoniazid is recommended in short-course regimens for multidrug-resistant tuberculosis (TB). The optimal dose of isoniazid and its individual contribution to efficacy against TB strains with inhA or katG mutations are unknown.Objectives: To define the optimal dose of isoniazid for patients with isoniazid-resistant TB mediated by inhA mutations.Methods: AIDS Clinical Trials Group A5312 is a phase 2A, open-label trial in which individuals with smear-positive pulmonary TB with isoniazid resistance mediated by an inhA mutation were randomized to receive isoniazid 5, 10, or 15 mg/kg daily for 7 days (inhA group), and control subjects with drug-sensitive TB received the standard dose (5 mg/kg/d). Overnight sputum cultures were collected daily. The 7-day early bactericidal activity (EBA) of isoniazid was estimated as the average daily change in log10 cfu on solid media (EBAcfu0-7) or as time to positivity (TTP) in liquid media in hours (EBATTP0-7) using nonlinear mixed-effects models.Measurements and Main Results: Fifty-nine participants (88% with cavitary disease, 20% HIV-positive, 16 with isoniazid-sensitive TB, and 43 with isoniazid-monoresistant or multidrug-resistant TB) were enrolled at one site in South Africa. The mean EBAcfu0-7 at doses of 5, 10, and 15 mg/kg in the inhA group was 0.07, 0.17, and 0.22 log10 cfu/ml/d, respectively, and 0.16 log10 cfu/ml/d in control subjects. EBATTP0-7 patterns were similar. There were no drug-related grade ≥3 adverse events.Conclusions: Isoniazid 10-15 mg/kg daily had activity against TB strains with inhA mutations similar to that of 5 mg/kg against drug-sensitive strains. The activity of high-dose isoniazid against strains with katG mutations will be explored next.Clinical trial registered with www.clinicaltrials.gov (NCT01936831).

Fourteen-Day Bactericidal Activity, Safety, and Pharmacokinetics of Linezolid in Adults with Drug-Sensitive Pulmonary Tuberculosis.

Linezolid is increasingly used for the treatment of tuberculosis resistant to first-line agents, but the most effective dosing strategy is yet unknown. From November 2014 to November 2016, we randomized 114 drug-sensitive treatment-naive pulmonary tuberculosis patients from Cape Town, South Africa, to one of six 14-day treatment arms containing linezolid at 300 mg once daily (QD), 300 mg twice daily (BD), 600 mg QD, 600 mg BD, 1,200 mg QD, 1,200 mg three times per week (TIW), or a combination of isoniazid, rifampin, pyrazinamide, and ethambutol. Sixteen-hour sputum samples were collected overnight, and bactericidal activity was characterized by the daily percentage change in time to positivity (TTP) and the daily rate of change in log10(CFU). We also assessed the safety and pharmacokinetics of the study treatments. We found that bactericidal activity increased with increasing doses of linezolid. Based on the daily percentage change in TTP, activity was highest for 1,200 mg QD (4.5%; 95% Bayesian confidence interval [BCI], 3.3 to 5.6), followed by 600 mg BD (4.1%; BCI, 2.5 to 5.7), 600 mg QD (4.1%; BCI, 2.9 to 5.3), 300 mg BD (3.3%; BCI, 1.9 to 4.7), 300 mg QD (2.3%; BCI, 1.1 to 3.5), and 1,200 mg TIW (2.2%; BCI, 1.1 to 3.3). Similar results were seen with bactericidal activity characterized by the daily rate of change in CFU count. Antimycobacterial activity correlated positively with plasma drug exposure and percentage time over MIC. There were no unexpected adverse events. All linezolid doses showed bactericidal activity. For the same total daily dose, once-daily dosing proved to be at least as effective as a divided twice-daily dose. An intermittent dosing regimen, with 1,200 mg given three times weekly, showed the least activity. (This study has been registered at ClinicalTrials.gov under identifier NCT02279875.).

Large unstained cells: a potentially valuable parameter in the assessment of immune activation levels in HIV infection.

INTRODUCTION: Chronic immune activation is associated with the accelerated progression of HIV to AIDS; however, affordable markers reflecting this have not yet been determined. The percentage of large unstained cells (%LUCs) is a differential count parameter measured by certain routine hematology analyzers and reflects activated lymphocytes and peroxidase-negative cells. We hypothesized that the %LUCs would be increased in HIV infection and would correlate with markers of immune activation [i.e. CD38 expression on CD8+ T cells (%CD38onCD8) and lipopolysaccharide-binding protein (LBP)] and CD4 counts. METHODS: In this cross-sectional study, 78 HIV-infected, antiretroviral therapy-naïve adults and 52 uninfected controls were recruited. %CD38onCD8 and CD4 counts were determined by flow cytometry, LBP levels were assessed by immunoassay, and the %LUCs was tested on a Siemens ADVIA 2120. RESULTS: Significant differences were found between the HIV-infected and control groups for %LUCs (95% CI 2.3-2.7 vs. 1.8-2.2, respectively; p = 0.001), as well as for %CD38onCD8, LBP, and CD4 counts. Furthermore, %LUCs correlated directly with %CD38onCD8 and LBP and inversely with CD4 counts. CONCLUSION: The %LUCs was significantly increased in this untreated, asymptomatic, HIV-infected group and correlated with markers of immune activation and CD4 counts. Therefore, the %LUCs may be of value in identifying HIV-infected patients at risk of accelerated disease progression.

Serum adenosine deaminase and total immunoglobulin G correlate with markers of immune activation and inversely with CD4 counts in asymptomatic, treatment-naive HIV infection.

PURPOSE: HIV-infection is characterized by aberrant immune activation and ongoing inflammation. Markers of inflammation are now recognized to have prognostic value for adverse events, independent of viral loads and CD4 counts. This study aimed to delineate a panel of affordable markers of immune activation in untreated HIV-infection that may have an impact on the management of HIV in resource-limited settings. METHODS: This was a cross-sectional study of 86 untreated newly diagnosed HIV-infected patients and 54 matched controls attending a voluntary testing clinic in Cape Town, South Africa. Serum levels of adenosine deaminase (ADA), total immunoglobulin G (IgG), soluble CD14 and lipopolysaccharide-binding protein (LBP) were measured and correlated with CD4 counts, viral loads and expression of CD38 on CD8+ T cells. RESULTS: ADA, IgG and LBP were all significantly increased in the HIV infected group (p < 0.0001) compared with uninfected controls. Soluble CD14 was also significantly increased (p = 0.0187). Furthermore, all these parameters correlated inversely with CD4 counts (r = -0.481 p < 0.0001; r = -0.561; p < 0.0001; r = -0.387 p = 0.0007 and r = -0.254 p = 0.0240, respectively). Only ADA correlated with viral load (r = 0.260 p = 0.0172). Importantly, ADA, IgG and LBP correlated directly with %CD38 on CD8+ T cells (r = 0.369 p < 0.0001; r = 0.284 p = 0.001; r = 0.408 p = 0.0006, respectively). CONCLUSION: Affordable parameters such as serum ADA and IgG correlated significantly with immune activation levels and markers of disease progression in untreated HIV-infection and therefore may add value to the management of these patients in resource-limited settings.

The Population Pharmacokinetics of Meropenem in Adult Patients With Rifampicin-Sensitive Pulmonary Tuberculosis.

Background: Meropenem is being investigated for repurposing as an anti-tuberculosis drug. This study aimed to develop a meropenem population pharmacokinetics model in patients with pulmonary tuberculosis and identify covariates explaining inter-individual variability. Methods: Patients were randomized to one of four treatment groups: meropenem 2 g three times daily plus oral rifampicin 20 mg/kg once daily, meropenem 2 g three times daily, meropenem 1 g three times daily, and meropenem 3 g once daily. Meropenem was administered by intravenous infusion over 0.5-1 h. All patients also received oral amoxicillin/clavulanate together with each meropenem dose, and treatments continued daily for 14 days. Intensive plasma pharmacokinetics sampling over 8 h was conducted on the 14th day of the study. Nonlinear mixed-effects modeling was used for data analysis. The best model was chosen based on likelihood metrics, goodness-of-fit plots, and parsimony. Covariates were tested stepwise. Results: A total of 404 concentration measurements from 49 patients were included in the analysis. A two-compartment model parameterized with clearance (CL), inter-compartmental clearance (Q), and central (V1) and peripheral (V2) volumes of distribution fitted the data well. Typical values of CL, Q, V1, and V2 were 11.8 L/h, 3.26 L/h, 14.2 L, and 3.12 L, respectively. The relative standard errors of the parameter estimates ranged from 3.8 to 35.4%. The covariate relations included in the final model were creatinine clearance on CL and allometric scaling with body weight on all disposition parameters. An effect of age on CL as previously reported could not be identified. Conclusion: A two-compartment model described meropenem population pharmacokinetics in patients with pulmonary tuberculosis well. Covariates found to improve model fit were creatinine clearance and body weight but not rifampicin treatment. The final model will be used for an integrated pharmacokinetics/pharmacodynamics analysis linking meropenem exposure to early bactericidal activity.

Fourteen-day PET/CT imaging to monitor drug combination activity in treated individuals with tuberculosis.

Early bactericidal activity studies monitor daily sputum bacterial counts in individuals with tuberculosis (TB) for 14 days during experimental drug treatment. The rate of change in sputum bacterial load over time provides an informative, but imperfect, estimate of drug activity and is considered a critical step in development of new TB drugs. In this clinical study, 160 participants with TB received isoniazid, pyrazinamide, or rifampicin, components of first-line chemotherapy, and moxifloxacin individually and in combination. In addition to standard bacterial enumeration in sputum, participants underwent 2-deoxy-2-[18F]fluoro-d-glucose positron emission tomography and computerized tomography ([18F]FDG-PET/CT) at the beginning and end of the 14-day drug treatment. Quantitating radiological responses to drug treatment provided comparative single and combination drug activity measures across lung lesion types that correlated more closely with established clinical outcomes when combined with sputum enumeration compared to sputum enumeration alone. Rifampicin and rifampicin-containing drug combinations were most effective in reducing both lung lesion volume measured by CT imaging and lesion-associated inflammation measured by PET imaging. Moxifloxacin was not superior to rifampicin in any measure by PET/CT imaging, consistent with its performance in recent phase 3 clinical trials. PET/CT imaging revealed synergy between isoniazid and pyrazinamide and demonstrated that the activity of pyrazinamide was limited to lung lesion, showing the highest FDG uptake during the first 2 weeks of drug treatment. [18F]FDG-PET/CT imaging may be useful for measuring the activity of single drugs and drug combinations during evaluation of potential new TB drug regimens before phase 3 trials.

Laboratory test result interpretation for primary care doctors in South Africa.

BACKGROUND: Challenges and uncertainties with test result interpretation can lead to diagnostic errors. Primary care doctors are at a higher risk than specialists of making these errors, due to the range in complexity and severity of conditions that they encounter. OBJECTIVES: This study aimed to investigate the challenges that primary care doctors face with test result interpretation, and to identify potential countermeasures to address these. METHODS: A survey was sent out to 7800 primary care doctors in South Africa. Questionnaire themes included doctors' uncertainty with interpreting test results, mechanisms used to overcome this uncertainty, challenges with appropriate result interpretation, and perceived solutions for interpreting results. RESULTS: Of the 552 responses received, the prevalence of challenges with result interpretation was estimated in an average of 17% of diagnostic encounters. The most commonly-reported challenges were not receiving test results in a timely manner (51% of respondents) and previous results not being easily available (37%). When faced with diagnostic uncertainty, 84% of respondents would either follow-up and reassess the patient or discuss the case with a specialist, and 67% would contact a laboratory professional. The most useful test utilisation enablers were found to be: interpretive comments (78% of respondents), published guidelines (74%), and a dedicated laboratory phone line (72%). CONCLUSION: Primary care doctors acknowledge uncertainty with test result interpretation. Potential countermeasures include the addition of patient-specific interpretive comments, the availability of guidelines or algorithms, and a dedicated laboratory phone line. The benefit of enhanced test result interpretation would reduce diagnostic error rates.

The use of the full blood count and differential parameters to assess immune activation levels in asymptomatic, untreated HIV infection.

BACKGROUND: A feature of HIV/AIDS is chronic immune activation, which results in a number of complications including inflammation-related disorders and blood cytopaenias. Immune activation status is not routinely tested in HIV infection. However, the full blood count (FBC) is a commonly performed test. OBJECTIVE: We hypothesised that FBC parameters would be significantly different in HIV-infected v. -uninfected individuals, and that some of these parameters would correlate with markers of immune activation (i.e. percentage CD38 expression on CD8(+) T cells (%CD38onCD8)) and disease progression (i.e. CD4(+) counts) in HIV infection. METHODS: This was a cross-sectional study with 83 HIV-infected adults who were antiretroviral therapy-naive and clinically well, and 51 HIV-uninfected adults. The %CD38onCD8 and CD4(+) counts were determined by flow cytometry and the FBC was performed on a Siemens ADVIA 2120 system. FBC parameters investigated were total white cell count (WCC), haemoglobin (Hb) concentration, platelet count, absolute neutrophil count, absolute lymphocyte count, and percentage of large unstained cells (%LUCs). RESULTS: Significant differences were found between the HIV-infected and -uninfected groups for total WCC, Hb, neutrophil count, lymphocyte count and %LUCs. The mean ± standard deviation (SD) for the total WCC (5.3±1.3 v. 6.9±2.2; p≤0.001) and the %LUCs (2.5±0.9 v. 2.0±0.9; p=0.001) both showed correlations with CD4(+) counts and %CD38onCD8. CONCLUSION: The total WCC and %LUCs showed significant differences in HIV-infected individuals and correlated with markers of immune activation and disease progression. This suggests the potential use of these parameters as markers of immune activation in HIV infection.

A Six Sigma approach to the rate and clinical effect of registration errors in a laboratory.

BACKGROUND: Laboratory errors made during the pre-analytical phase can have an impact on clinical care. Quality management tools such as Six Sigma may help improve error rates. AIM: To use elements of a Six Sigma model to establish the error rate of test registration onto the laboratory information system (LIS), and to deduce the potential clinical impact of these errors. METHODS: In this retrospective study, test request forms were compared with the tests registered onto the LIS, and all errors were noted before being rectified. The error rate was calculated. The corresponding patient records were then examined to determine the actual outcome, and to deduce the potential clinical impact of the registration errors. RESULTS: Of the 47 543 tests requested, 72 errors were noted, resulting in an error rate of 0.151%, equating to a sigma score of 4.46. The patient records reviewed indicated that these errors could, in various ways, have impacted on clinical care. CONCLUSION: This study highlights the clinical effect of errors made during the pre-analytical phase of the laboratory testing process. Reduction of errors may be achieved through implementation of a Six Sigma programme.