Search

Introduction: Isoniazid (INH) is one of the most effective and potent first-line anti-tubercular drug. INH is also effectively administered as a preventative monotherapy and has been shown to significantly reduce TB incidence. INH is primarily metabolised to acetyl-isoniazid (AcINH) in the liver. AcINH is mainly excreted in urine presenting as a target for monitoring adherence to INH therapy. Objective: The study aimed to develop and fully validate a bioanalytical method using liquid chromatography-tandem mass spectrometry for the quantification of INH and AcINH in human urine. Methods: The samples were prepared using solid phase extraction, with the internal standards isoniazid-d4 and acetyl-isoniazid-d4 being used. The extracts were chromatographed on an Atlantis T3 analytical column with an isocratic mobile phase. For detection, a AB Sciex™ API 5500 triple quadrupole mass spectrometer was used at unit resolution in the multiple reaction monitoring mode, following positive electrospray ionization. Results: The analytical method demonstrated sufficient sensitivity, as indicated by average signal-to-noise ratios of 7.07 and 6.23 at the lower limit of quantification for INH and AcINH, respectively. Validation was performed over three consecutive batches, demonstrating accuracy, precision, and overall robustness based on peak area ratios within the analytical range of 0.234–30.0 µg/mL for both INH and AcINH. All required validation experiments were assessed and met the acceptance criteria guidelines of the US Food and Drug Administration and European Medicines Agency. The validated method was utilized to measure concentrations of AcINH in urine as a means of assessing adherence to the intake of isoniazid in order to prevent TB infection during a phase III open-label multicenter trial. Conclusion: A bioanalytical method was developed and fully validated for quantifying isoniazid (INH) and acetyl-isoniazid (AcINH) in 100 µL of human urine.

Abstract Mycobacterial pathogens present a significant challenge to disease control efforts globally due to their inherent resistance to multiple antibiotics. The rise of drug-resistant strains of Mycobacterium tuberculosis has prompted an urgent need for innovative therapeutic solutions. One promising way to discover new tuberculosis drugs is by utilizing natural products from the vast biochemical space. Multidisciplinary methods can used to harness the bioactivity of these natural products. This study aimed to evaluate the antimycobacterial efficacy of functional crude extracts from bacteria isolated from gold mine tailings in South Africa. Bacterial strains were identified using 16S rRNA sequencing. The crude extracts obtained from the bacteria were tested against Mycobacterium tuberculosis H37Rv, Mycobacterium smegmatis mc2155, and Mycobacterium aurum A+. Untargeted HPLC-qTOF and molecular networking were used to identify the functional constituents present in extracts that exhibited inhibitory activity. A virtual screening workflow (VSW) was used to filter compounds that were strong binders to Mycobacterium tuberculosis Pks13 and PknG. The ligands returned from the VSW were subjected to optimization using density functional theory (DFT) at M06-2X/6-311++ (d,p) level of theory and basis set implemented in Gaussian16 Rev.C01. The optimized ligands were re-docked against Mycobacterium tuberculosis Pks13 and PknG. Molecular dynamics simulation and molecular mechanics generalized born surface area were used to evaluate the stability of the protein–ligand complexes formed by the identified hits. The hit that showed promising binding characteristics was virtually modified through multiple synthetic routes using reaction-driven enumeration. Three bacterial isolates showed significant activity against the two strains of Mycobacterium, while only two, Bacillus subtilis and Bacillus licheniformis, exhibited activity against both Mycobacterium tuberculosis H37Rv, Mycobacterium smegmatis mc2155, and Mycobacterium aurum A+. The tentatively identified compounds from the bacterial crude extracts belonged to various classes of natural compounds associated with antimicrobial activity. Two compounds, cyclo-(L-Pro-4-OH-L-Leu) and vazabitide A, showed strong binding against PknG and Pks13, with pre-MD MM-GBSA values of − 42.8 kcal/mol and − 47.6 kcal/mol, respectively. The DFT-optimized compounds exhibited the same docking scores as the ligands optimized using the OPSL-4 force field. After modifying vazabitide A, its affinity to the Pks13 binding site increased to − 85.8 kcal/mol, as revealed by the post-MD MM-GBSA analysis. This study highlights the potential of bacteria isolates from gold mine tailings as a source of new scaffolds for designing and optimizing anti-Mycobacterium agents. These agents synthesized in-silico can be further tested in-vitro to evaluate their efficacy.

Objective: To develop and validate an assay for the analysis of bedaquiline and its M2 metabolite in human breast milk. Methods: The analytes were extracted using solid phase extraction following protein precipitation. Quantification was performed with liquid chromatography coupled with tandem mass spectrometry. Chromatographic separation was achieved using gradient chromatography on a Poroshell 120 SB-C18 analytical column at 40 °C, with a flow rate of 350 µL/minute and a total run time of eight minutes. An AB Sciex 3000 mass spectrometer with electrospray ionization in the positive mode was used for detection, employing multiple reaction monitoring scan mode. Bedaquiline-d6 and M2-d3-13C were used as internal standards. Results: Calibrations curves for bedaquiline and M2 exhibited quadratic (weighted 1/x concentration) regressions over the respective concentration ranges of 0.0780 to 5.00 µg/mL and 0.0312 to 2.00 µg/mL. Inter- and intra-day validation accuracies ranged between 96.7 % and 103.5 % for bedaquiline, and 104.2 % to 106.5 % for M2, with a coefficient of variation below 9.2 % for both compounds. Conclusion: The developed assay demonstrated selectivity and robustness, enabling differentiation between bedaquiline and M2 within the context of endogenous compounds from six separate lots of breast milk samples. Successful application was observed in the analysis of breast milk samples sourced from patients treated for multidrug-resistant tuberculosis within a clinical study setting.

Background Early mortality among hospitalized HIV-associated tuberculosis (TB/HIV) patients is high despite treatment. The pharmacokinetics of rifampicin, isoniazid, and pyrazinamide were investigated in hospitalized TB/HIV patients and a cohort of outpatients with TB (with or without HIV) to determine whether drug exposures differed between groups. Methods Standard first-line TB treatment was given daily as per national guidelines, which consisted of oral 4-drug fixed-dose combination tablets containing 150 mg rifampicin, 75 mg isoniazid, 400 mg pyrazinamide, and 275 mg ethambutol. Plasma samples were drawn on the 3rd day of treatment over eight hours post-dose. Rifampicin, isoniazid, and pyrazinamide in plasma were quantified and NONMEM® was used to analyze the data. Results Data from 60 hospitalized patients (11 of whom died within 12 weeks of starting treatment) and 48 outpatients were available. Median (range) weight and age were 56 (35 - 88) kg, and 37 (19 - 77) years, respectively. Bioavailability and clearance of the three drugs were similar between TB/HIV hospitalized and TB outpatients. However, rifampicin’s absorption was slower in hospitalized patients than in outpatients; mean absorption time was 49.9% and 154% more in hospitalized survivors and hospitalized deaths, respectively, than in outpatients. Higher levels of conjugated bilirubin correlated with lower rifampicin clearance. Isoniazid’s clearance estimates were 25.5 L/h for fast metabolizers and 9.76 L/h for slow metabolizers. Pyrazinamide’s clearance was more variable among hospitalized patients. The variability in clearance among patients was 1.70 and 3.56 times more for hospitalized survivors and hospitalized deaths, respectively, than outpatients. Conclusions We showed that the pharmacokinetics of first-line TB drugs are not substantially different between hospitalized TB/HIV patients and TB (with or without HIV) outpatients. Hospitalized patients do not seem to be underexposed compared to their outpatient counterparts, as well as hospitalized patients who survived vs who died within 12 weeks of hospitalization.

Efforts to develop new artemisinin triple combination therapies effective against artemisinin-tolerant strains of Plasmodium falciparum based on rational combinations comprising artemisone or other amino-artemisinins, a redox active drug and a third drug with a different mode of action have now been extended to evaluation of three potential redox partners. These are the diethyl analogue AD01 of methylene blue (MB), the benzo [α]phenoxazine PhX6, and the thiosemicarbazone DpNEt. IC50 values in vitro against CQ-sensitive and resistant P. falciparum strains ranged from 11.9 nM for AD01–41.8 nM for PhX6. PhX6 possessed the most favourable pharmacokinetic (PK) profile: intrinsic clearance rate CLint was 21.47 ± 1.76 mL/min/kg, bioavailability was 60% and half-life was 7.96 h. AD01 presented weaker, but manageable pharmacokinetic properties with a rapid CLint of 74.41 ± 6.68 mL/min/kg leading to a half-life of 2.51 ± 0.07 h and bioavailability of 15%. DpNEt exhibited a half-life of 1.12 h and bioavailability of 8%, data which discourage its further examination, despite a low CLint of 10.20 mL/min/kg and a high Cmax of 6.32 µM. Efficacies of AD01 and PhX6 were enhanced synergistically when each was paired with artemisone against asexual blood stages of P. falciparum NF54 in vitro. The favourable pharmacokinetics of PhX6 indicate this is the best partner among the compounds examined thus far for artemisone. Future work will focus on extending the drug combination studies to artemiside in vitro, and conducting efficacy studies in vivo for artemisone with each of PhX6 and the related benzo[α]phenoxazine SSJ-183.

Abstract Tenofovir disoproxil fumarate (TDF) and tenofovir alafenamide (TAF) are prodrugs of the nucleotide analogue tenofovir, which acts intracellularly to inhibit HIV replication. Whereas TDF converts to tenofovir in plasma and may cause kidney and bone toxicity, TAF mostly converts to tenofovir intracellularly, so it can be administered at lower doses. TAF leads to lower tenofovir plasma concentrations and lower toxicity, but there are limited data on its use in Africa. We used data from 41 South African adults living with HIV from the ADVANCE trial and described, with a joint model, the population pharmacokinetics of tenofovir given as TAF or TDF. The TDF was modeled to appear in plasma as tenofovir with a simple first‐order process. Instead, two parallel pathways were used for a TAF dose: an estimated 32.4% quickly appeared as tenofovir into the systemic circulation with first‐order absorption, whereas the rest was sequestered intracellularly and released into the systemic circulation as tenofovir slowly. Once in plasma (from either TAF or TDF), tenofovir disposition followed two‐compartment kinetics and had a clearance of 44.7 L/h (40.2–49.5), for a typical 70‐kg individual. This semimechanistic model describes the population pharmacokinetics of tenofovir when dosed as either TDF or TAF in an African population living with HIV and can be used as a tool for exposure prediction in patients, and to simulate alternative regimes to inform further clinical trials.

Background: Poor medication adherence leads to poor health outcomes and increased healthcare costs among patients with heart failure (HF). This study aimed to objectively assess medication adherence by measuring carvedilol and enalaprilat plasma concentrations among patients with HF. Methods: The present sub-study of the Safety, Tolerability, and Efficacy of Rapid Optimization, helped by NT-proBNP testing, of Heart Failure therapies (STRONG-HF) study involved adult patients with acute HF admitted in two Mozambican and two Nigerian hospitals who were not optimally treated with oral enalapril and carvedilol. Patients in the high-intensity arm of the STRONG-HF study, and those not meeting the biomarker criteria for persistent congestion, were included in the “frequent visit” (FV) arm. In the FV arm, blood for bioanalysis of plasma enalaprilat or/and carvedilol was drawn at the 2,6,12th week post-discharge. Patients in the usual care arm of STRONG-HF were included in the “standard visit” (SV) arm, which followed the usual local practice with blood sampling in week 12. Results: The study involved 113 (79 FV and 34 SV) participants with a mean age of 48.6 years and a mean left ventricular (LV) ejection fraction of 33.1%. Theenalaprilat below the lower level of quantification (LLOQ) was documented in 7.7%, 11.9%, and 15.6% of participants in FV during the 2,6 and 12th weeks. Carvedilol concentration below LLOQ was documented in 37%, 30%, and 44.4% of participants in the FV arm during the 2,6 and 12th weeks, respectively. For the SV arm, enalaprilat and carvedilol concentrations below LLOQ in the twelfth week were documented in 37.3% and 42.9% of patients, respectively. Conclusion: Up to a third of patients using enalapril and carvedilol did not take any medication during the 12 weeks of follow-up. Non adherence was more common in patients who had less follow up, emphasizing the importance of close follow up to adherence. No adherence was also more common in medications know to have more side effects such as carvedilol.

Tuberculous meningitis treatment outcomes are poor and alternative regimens are under investigation. Reliable methods to measure drug concentrations in cerebrospinal fluid are required to evaluate distribution into the cerebrospinal fluid. A simple and quick method was developed and validated to analyse linezolid in human cerebrospinal fluid. Samples were prepared by protein precipitation followed by isocratic liquid chromatography and tandem mass spectrometry. The run time was 3.5 min. Accuracy and precision were assessed in three independent validation batches with a calibration range of 0.100–20.0 μg/mL. The method was used to analyse cerebrospinal fluid samples from patients with tuberculous meningitis enrolled in a clinical trial. Potentially infective patient samples could be decontaminated using Nanosep® nylon and Costar® nylon filter tubes under biosafety level 3 conditions before analysis. The filtration process did not significantly affect the quantification of linezolid. Linezolid concentration in cerebrospinal fluid obtained from tuberculous meningitis patients ranged from 0.197 μg/mL to 15.0 μg/mL. The ratio between average CSF and plasma linezolid concentrations varied with time, reaching a maximum of 0.9 at 6 h after dosing.

Introduction: Adherence to medication is an important determinant of outcomes in chronic diseases like heart failure. Drug assays provide objective adherence biomarkers. Dried blood spots (DBS) are appealing samples for drug assays due to less demanding transportation and storage requirements. Objectives: To analytically validate a LC-MS/MS method for the simultaneous quantification of carvedilol, enalaprilat, and perindoprilat in DBS and evaluate the feasibility of using the method as an adherence determining assay. To validate the assay further clinically by establishing correlation and agreement between plasma and DBS samples from a pharmacokinetic pilot study. Methods: The method was validated over a concentration range of 1.00–200 ng/mL according to FDA guidelines. Adherence tracking ability of the assay was evaluated using a pharmacokinetic pilot study. Correlation and agreement were evaluated through Deming regression and Bland-Altman analysis, respectively. Results: Accuracy, precision, selectivity, and sensitivity were proven with complete and reproducible extraction recovery at all concentrations tested. Stability of the analytes in the matrix and throughout sample processing was proven. The full range of concentrations of the pharmacokinetic pilot study could be quantified for enalaprilat, but not for carvedilol and perindoprilat. The difference between the observed and calculated plasma concentrations was less than 20 % of their mean for >67 % of samples for all analytes. Conclusions: The assay is suitable as a screening tool for carvedilol and perindoprilat, while suitable as an adherence determining assay for enalaprilat. Equivalence between observed and predicted plasma concentrations proves DBS and plasma concentrations can be used interchangeably.

Objectives: Critically ill patients with tuberculosis (TB) face a high mortality risk and require effective treatment. There is a paucity of data on rifampicin pharmacokinetics, the impact of continuous enteral feeding on drug absorption, and the potential of therapeutic drug monitoring (TDM) to optimize drug exposure in these patients. Methods: We performed a sequential pharmacokinetic study to determine the impact of feeding and TDM with rifampicin dose escalation in critically ill patients with TB. Noncompartmental pharmacokinetic analysis was performed. Results: Among 20 critically ill patients (40% were HIV-infected), median rifampicin Cmax (maximum serum concentration) in the fasted and fed states were 5.1 µg/ml versus 3.3 µg/ml, respectively (P

Objectives: There is inconsistent evidence on the relationship between pesticide exposure and childhood respiratory outcomes in non-agricultural settings. This study investigated the association between organophosphate (OP) pesticide exposure and asthma-related outcomes in children residing in four informal settlements.Methods: The study was a longitudinal study of 590 schoolchildren, with a 12 months follow-up period. A standardised questionnaire adopted from the International Study of Asthma and Allergies in Childhood was administered to caregivers for child’s respiratory symptoms and household characteristics. Spirometry and fractional-exhaled nitric oxide, including a phadiatop test (atopy status) and urinary dialkyl phosphate (DAP) metabolites were measured at baseline and follow-up. DAP metabolites included diethylphosphate (DEP) and dimethyl phosphate (DMP) measured at baseline and follow-up and dimethylthiophosphate (DMTP) measured only at baseline.Results: The mean ages of schoolchildren were 9.9 ± 0.91 years and the overal incidence proportions of new doctor diagnosed asthma was 2.2%. No consistent patterns of increased risk of asthma outcomes with increasing DAP concentrations was found in multivariate analysis.Conclusion: Future studies with longer follow-up periods and repeated OP biomonitoring are recommended.

Background: Efavirenz (EFV) is associated with neuropsychiatric symptoms. Severe neurotoxicity has been reported but the clinical phenotype and risk factors are poorly defined. Objectives: To characterise clinical presentations, risk factors and outcomes to help clinicians recognise severe neurotoxicity earlier. Method: The authors retrospectively identified adults with supratherapeutic EFV concentrations ( 4 mg/L) obtained during routine clinical care in Cape Town, South Africa. Clinical and laboratory data at the time of EFV quantification were extracted from medical records. Logistic regression was performed to identify associations with neuropsychiatric symptoms, and with severe neurotoxicity. Results: Eighty one patients were included; 62 with neuropsychiatric manifestations (most frequently ataxia [n = 20] and psychomotor slowing [n = 24]); and 19 with hepatotoxicity. Overall, 28 (34.6%) were male, 49 (60.5%) had concomitant isoniazid exposure, and median EFV concentration was 12.1 mg/L (interquartile range [IQR]: 6.6–20.0). Neuropsychiatric symptoms were associated with longer duration of EFV therapy, adjusted odds ratio (aOR) 1.3/180-day increment (95% confidence interval [CI]: 1.0–1.7); higher EFV concentrations, aOR 1.2/1 mg/L increase (95% CI: 1.0–1.4) and isoniazid exposure, aOR 8.2 (95% CI: 2.5–26.7). Severe neuropsychiatric symptoms occurred in 47 (75%) patients at a median of 5.9 months (IQR: 2.1–40.8) after EFV initiation. Severe symptoms odds were 1.2-fold higher (95% CI: 1.1–1.4) per 1 mg/L increase in EFV concentration. Symptoms resolved completely within 1 month in 25 (76%) patients with severe neurotoxicity who discontinued EFV. Conclusion: A concentration–effect relationship for severe neurotoxicity exists, which occurred late and resolved in most patients after EFV discontinuation. Contribution: The authors highlighted clinical heterogeneity and morbidity of EFV-associated neurotoxicity.

Pulmonary tuberculosis is an inflammatory disease associated with an elevated cortisol/cortisone ratio at the site of infection and an array of cytokine changes. Tuberculous pericarditis is a less common but more lethal form of tuberculosis and has a similar inflammatory process in the pericardium. As the pericardium is largely inaccessible, the effect of tuberculous pericarditis on pericardial glucocorticoids is largely unknown. We wished to describe pericardial cortisolcortisone ratio in relation to plasma and saliva cortisol/cortisone ratios and the associated changes in cytokine concentrations. The median (interquartile range) of plasma, pericardial, and saliva cortisol concentration was 443 (379–532), 303 (257–384), and 20 (10–32) nmol/L, respectively, whereas the median (interquartile range) of plasma, pericardial, and saliva cortisone concentrations was 49 (35–57), 15.0 (0.0–21.7), and 37 (25–55) nmol/L, respectively. The cortisol/cortisone ratio was highest in pericardium with median (interquartile range) of 20 (13–445), followed by plasma of 9.1 (7.4–12.1) and saliva of 0.4 (0.3–0.8). The elevated cortisol/cortisone ratio was associated with elevated pericardial, interferon gamma, tumor necrosis factor–alpha, interleukin-6, interleukin-8, and induced protein 10. Administration of a single dose of 120 mg of prednisolone was associated with the suppression of pericardial cortisol and cortisone within 24 h of administration. The cortisol/cortisone ratio was highest at the site of infection, in this case, the pericardium. The elevated ratio was associated with a differential cytokine response. The observed pericardial cortisol suppression suggests that 120 mg of prednisolone was sufficient to evoke an immunomodulatory effect in the pericardium.

Background: Dolutegravir, a component of the preferred first-line antiretroviral therapy regimen, has been associated with increased weight gain. South Africa has a high prevalence of obesity, especially among women. Understanding dolutegravir exposure in patients with obesity is important for dose optimisation. Objectives: We compared the pharmacokinetic parameters of dolutegravir in Southern African adults living with HIV with and without obesity. Method: Blood samples were collected at various time points over a 24 h-period for dolutegravir assays. Non-compartmental analysis was conducted and geometric mean ratios (GMRs), with 90% confidence intervals (CIs), were generated to compare dolutegravir pharmacokinetic parameters between the groups. Regression analyses to assess predictors of dolutegravir exposure were done. Results: Forty participants were enrolled, 26 were women and 10 had obesity. Dolutegravir area under the concentration-time curve to 24-h and the maximum concentrations were not statistically significantly lower in participants with obesity: GMR 0.91 (90% CI: 0.71–1.16) and GMR 0.86 (90% CI: 0.68–1.07), respectively. In a multivariate linear regression analysis adjusting for age, gender, body mass index, creatinine clearance and randomisation arm (tenofovir alafenamide or tenofovir disoproxil fumarate), a unit increase in body mass index was associated with 1.2% lower dolutegravir area under the concentration-time curve to 24-h (P = 0.035). Conclusion: Dolutegravir exposure was marginally lower in participants with obesity, but this is not clinically significant. Our findings suggest that there is no need to dose adjust dolutegravir in people with obesity.

Abstract Introduction Daily oral pre‐exposure prophylaxis (PrEP) can reduce HIV acquisition. However, prevention effectiveness requires daily adherence prior to and during periods of sexual activity. Little is known about pharmacologic measures of PrEP adherence during pregnancy and postpartum and the factors related to optimal adherence during periods of sexual activity in this population. Methods Between August 2019 and October 2021, we enrolled pregnant women without HIV at their first antenatal care visit followed‐up through 12 months postpartum. Eligible women ≥16 years old received HIV prevention counselling and were offered oral PrEP (TDF‐FTC). We quantified tenofovir‐diphosphate (TFV‐DP) in dried blood spots in women who reported taking PrEP in the past 30 days (at quarterly follow‐up visits). We used regression models with generalized estimating equations to evaluate correlates of TFV‐DP (any vs. none, and ≥2 vs. 5/month vs. no sex or

Background. Early mortality among hospitalized HIV-associated tuberculosis (TB/HIV) patients is high despite treatment. The pharmacokinetics of rifampicin, isoniazid, and pyrazinamide were investigated in hospitalized TB/HIV patients and a cohort of outpatients with TB (with or without HIV) to determine whether drug exposures differed between groups. Methods. Standard first-line TB treatment was given daily as per national guidelines, which consisted of oral 4-drug fixed-dose combination tablets containing 150 mg rifampicin, 75 mg isoniazid, 400 mg pyrazinamide, and 275 mg ethambutol. Plasma samples were drawn on the 3rd day of treatment over eight hours post-dose. Rifampicin, isoniazid, and pyrazinamide in plasma were quantified and NONMEM® was used to analyze the data. Results. Data from 60 hospitalized patients (11 of whom died within 12 weeks of starting treatment) and 48 outpatients were available. Median (range) weight and age were 56 (35 - 88) kg, and 37 (19 - 77) years, respectively. Bioavailability and clearance of the three drugs were similar between TB/HIV hospitalized and TB outpatients. However, rifampicin’s absorption was slower in hospitalized patients than in outpatients; mean absorption time was 49.9% and 154% more in hospitalized survivors and hospitalized deaths, respectively, than in outpatients. Higher levels of conjugated bilirubin correlated with lower rifampicin clearance. Isoniazid’s clearance estimates were 25.5 L/h for fast metabolizers and 9.76 L/h for slow metabolizers. Pyrazinamide’s clearance was more variable among hospitalized patients. The variability in clearance among patients was 1.70 and 3.56 times more for hospitalized survivors and hospitalized deaths, respectively, than outpatients. Conclusion. We showed that the pharmacokinetics of first-line TB drugs are not substantially different between hospitalized TB/HIV patients and TB (with or without HIV) outpatients. Hospitalized patients do not seem to be underexposed compared to their outpatient counterparts, as well as hospitalized patients who survived vs who died within 12 weeks of hospitalization.

The emergence of Plasmodium falciparum (Pf) parasite strains tolerant of the artemisinin component and resistant to the other drug component in artemisinin combination therapies (ACTs) used for treatment now markedly complicates malaria control. Thus, development of new combination therapies are urgently required. For the non-artemisinin component, the quinolone ester decoquinate (DQ) that possesses potent activities against blood stage Pf and acts on a distinct target, namely the Pf cytochrome bc1 complex, was first considered. However, DQ has poor drug properties including high lipophilicity and exceedingly poor aqueous solubility (0.06 μg/ml), rendering it difficult to administer. Thus, DQ was chemically modified to provide the secondary amide derivative RMB005 and the quinoline O-carbamate derivatives RMB059 and RMB060. The last possesses sub-nanomolar activities against multidrug resistant blood stages of Pf, and P. berghei sporozoite liver stages. Here we present the results of ADME analyses in vitro and pharmacokinetic analyses using C57BL/6 mice. The amide RMB005 had a maximum mean whole blood concentration of 0.49 ± 0.02 µM following oral administration; however, the area under the curve (AUC), elimination half-life (t1/2) and bioavailability (BA) were not significantly better than those of DQ. Surprisingly, the quinoline O-carbamates which can be recrystallized without decomposition were rapidly converted into DQ in human plasma and blood samples. The maximum concentrations of DQ reached after oral administration of RMB059 and RMB060 were 0.23 ± 0.05 and 0.11 ± 0.01 µM, the DQ elimination half-lives were 4.79 ± 1.66 and 4.66 ± 1.16 h, and the DQ clearance were 19.40 ± 3.14 and 21.50 ± 3.38 respectively. Under these assay conditions, the BA of DQ could not be calculated Overall although RMB059 and -060 are labile in physiological medium with respect to the DQ parent, the potential to apply these as prodrugs is apparent from the current data coupled with their ease of preparation.

High-throughput metabolomics techniques are a useful tool to understand many disease conditions including cardiovascular disease such as valvular heart disease(s) (VHD). VHD involves damage to heart valves, mostly presenting as stenosis, regurgitation or prolapse and can be classified into degenerative, rheumatic, congenital, or prosthetic valve disease. Gaps remain in our understanding of the pathogenesis of the common VHD. It is now fitting to place into perspective the contribution of metabolomics in the mechanism of development, diagnosis, and prognosis of VHD. A structured search for metabolomics studies centred on human VHD was undertaken. Biomarkers associated with the pathogenesis of bicuspid aortic valve disease, mitral valve disease, rheumatic heart disease, and degenerative aortic valve stenosis are reviewed and discussed. In addition, metabolic biomarkers reported to prognosticate patient outcomes of post-valve repair or replacement are highlighted. Finally, we also review the pitfalls and limitations to consider when designing metabolomics studies, especially from a clinician’s viewpoint. In the future, reliable and simple metabolic biomarker(s) may supplement the existing diagnostic tools in the early diagnosis of VHD.

Novel antitubercular compounds are urgently needed to combat drug-resistant Mycobacterium tuberculosis (Mtb). Filamentous actinobacteria have historically been an excellent source of antitubercular drugs. Despite this, drug discovery from these microorganisms has fallen out of favour due to the continual rediscovery of known compounds. To increase the chance of discovering novel antibiotics, biodiverse and rare strains should be prioritised. Subsequently, active samples need to be dereplicated as early as possible to focus efforts on truly novel compounds. In this study, 42 South African filamentous actinobacteria were screened for antimycobacterial activity using the agar overlay method against the Mtb indicator Mycolicibacterium aurum under six different nutrient growth conditions. Known compounds were subsequently identified through extraction and high-resolution mass spectrometric analysis of the zones of growth inhibition produced by active strains. This allowed the dereplication of 15 hits from six strains that were found to be producing puromycin, actinomycin D and valinomycin. The remaining active strains were grown in liquid cultures, extracted and submitted for screening against Mtb in vitro. Actinomadura napierensis B60T was the most active sample and was selected for bioassay-guided purification. This resulted in the identification of tetromadurin, a known compound, but which we show for the first time to have potent antitubercular activity, with the MIC90s within the range of 73.7–151.6 nM against M. tuberculosis H37RvT in vitro under different test conditions. This shows that South African actinobacteria are a good source of novel antitubercular compounds and warrant further screening. It is also revealed that active hits can be dereplicated by HPLC-MS/MS analysis of the zones of growth inhibition produced by the agar overlay technique.

Background: Cycloserine, or its structural analogue terizidone, has been associated with neuropsychiatric toxicity (psychosis, depression, and neuropathy). Prospective clinical data on the incidence of and risk factors for neuropsychiatric toxicity in TB patients treated with cycloserine are limited. Methods: A prospective evaluation of neuropsychiatric toxicity was performed using validated screening tools in patients with multidrug-resistant tuberculosis treated with terizidone. Cox proportional hazard modelling was performed to explore the effects of clinical variables and measures of cycloserine pharmacokinetics in plasma. Results: A total 144 participants were recruited: 86 were male and 58 were female; their median age was 35.7 years and 91 (63%) were HIV-infected. Fifty-five (38%) participants developed at least one neuropsychiatric event (30 cases per 100 person-months): 50 (35%) neuropathy, 14 (10%) depression, and 11 (8%) psychosis. Neuropathy was independently associated with cycloserine clearance ((adjusted hazard ratio 0.34 (aHR), P = 0.03)) and high-dose pyridoxine (200 mg vs 150 mg daily, aHR: 2.79, P = 0.01). Conclusions: A high incidence of early neuropsychiatric toxicity was observed in this cohort of patients treated with terizidone. Cycloserine clearance and higher doses of pyridoxine are associated with incident or worsening peripheral neuropathy.

Abstract Background Accurate measurement of anti-malarial drug concentrations in therapeutic efficacy studies is essential to distinguish between inadequate drug exposure and anti-malarial drug resistance, and to inform optimal anti-malarial dosing in key target population groups. Methods A sensitive and selective LC–MS/MS method was developed and validated for the simultaneous determination of amodiaquine and its active metabolite, desethylamodiaquine, and used to describe their pharmacokinetic parameters in Ghanaian patients with uncomplicated falciparum malaria treated with the fixed-dose combination, artesunate-amodiaquine. Results The day-28 genotype-adjusted adequate clinical and parasitological response rate in 308 patients studied was > 97% by both intention-to-treat and per-protocol analysis. After excluding 64 patients with quantifiable amodiaquine concentrations pre-treatment and 17 with too few quantifiable concentrations, the pharmacokinetic analysis included 227 patients (9 infants, 127 aged 1–4 years, 91 aged ≥ 5 years). Increased median day-3 amodiaquine concentrations were associated with a lower risk of treatment failure [HR 0.87 (95% CI 0.78–0.98), p = 0.021]. Amodiaquine exposure (median AUC0-∞) was significantly higher in infants (4201 ng h/mL) and children aged 1–5 years (1994 ng h/mL) compared to older children and adults (875 ng h/mL, p = 0.001), even though infants received a lower mg/kg amodiaquine dose (median 25.3 versus 33.8 mg/kg in older patients). Desethylamodiaquine AUC0-∞ was not significantly associated with age. No significant safety concerns were identified. Conclusions Efficacy of artesunate-amodiaquine at currently recommended dosage regimens was high across all age groups. Reassuringly, amodiaquine and desethylamodiaquine exposure was not reduced in underweight-for-age young children or those with high parasitaemia, two of the most vulnerable target populations. A larger pharmacokinetic study with close monitoring of safety, including full blood counts and liver function tests, is needed to confirm the higher amodiaquine exposure in infants, understand any safety implications and assess whether dose optimization in this vulnerable, understudied population is needed.

Despite extensive global efforts, sub-Saharan Africa remains disproportionately affected by the HIV epidemic. This generalized epidemic can be seen in Lesotho which in 2014 the HIV prevalence rate of those aged 15-49 years was 24.6%, with and incidence of 1.9 new infections per 100-person-year exposures. To better understand the impact of Lesotho's national HIV response and significant predictors associated with HIV infection, the Lesotho Population-based HIV Impact Assessment was conducted. This survey provided a nationally representative sample of individuals aged 15-59 years old in which participants were tested for HIV and given an individual questionnaire that included socio-demographic and behavioral risk questions. The association of factors between survey questions and HIV incident was assessed using logistic regression. Multivariate logistic regression models for men and women were constructed for each outcome using variables known to be or plausibly associated with recent or chronic infection. Overall annualized incidence among people aged 15-49 was 1.19% (95% CI 0.73-1.65) per year. The overall prevalence of HIV was 25.6% with women having significantly higher prevalence. Multiple variables, including decreased wealth status, lower education levels, marital status, condom use at first sex, and circumcision (men only) were identified as being significantly associated with HIV infection for both men and women. In combination with improving the awareness of HIV status, an increased focus is needed on AGYW and men 35-49 years old to prevent new infections. HIV education and prevention programs should focus heavily on younger age groups prior to and soon after sexual debut to prevent HIV transmission. The findings of the survey showed significant room for improvement in increasing awareness of HIV status and reinforcing the need for continued HIV prevention and treatment efforts in Lesotho to prevent new infections.

Given the high prevalence of multidrug-resistant (MDR)-TB in high HIV burden settings, it is important to identify potential drug-drug interactions between MDR-TB treatment and widely used nucleoside reverse transcriptase inhibitors (NRTIs) in HIV-positive children. Population pharmacokinetic models were developed for lamivudine (n = 54) and abacavir (n = 50) in 54 HIV-positive children established on NRTIs; 27 with MDR-TB (combinations of high-dose isoniazid, pyrazinamide, ethambutol, ethionamide, terizidone, fluoroquinolones, and amikacin), and 27 controls without TB. Two-compartment models with first-order elimination and transit compartment absorption described both lamivudine and abacavir pharmacokinetics, respectively. Allometric scaling with body weight adjusted for the effect of body size. Clearance was predicted to reach half its mature value ∼2 (lamivudine) and ∼3 (abacavir) months after birth, with completion of maturation for both drugs at ∼2 years. No significant difference was found in key pharmacokinetic parameters of lamivudine and abacavir when co-administered with routine drugs used for MDR-TB in HIV-positive children.

ABSTRACT The therapeutic repertoire for tuberculosis (TB) remains limited despite the existence of many TB drugs that are highly active in in vitro models and possess clinical utility. Underlying the lack of efficacy in vivo is the inability of TB drugs to penetrate microenvironments inhabited by the causative agent, Mycobacterium tuberculosis, including host alveolar macrophages. Here, we determined the ability of the phenoxazine PhX1 previously shown to be active against M. tuberculosis in vitro to differentially penetrate murine compartments, including plasma, epithelial lining fluid, and isolated epithelial lining fluid cells. We also investigated the extent of permeation into uninfected and M. tuberculosis-infected human macrophage-like Tamm-Horsfall protein 1 (THP-1) cells directly and by comparing to results obtained in vitro in synergy assays. Our data indicate that PhX1 (4,750 ± 127.2 ng/ml) penetrates more effectively into THP-1 cells than do the clinically used anti-TB agents, rifampin (3,050 ± 62.9 ng/ml), moxifloxacin (3,374 ± 48.7 ng/ml), bedaquiline (4,410 ± 190.9 ng/ml), and linezolid (770 ± 14.1 ng/ml). Compound efficacy in infected cells correlated with intracellular accumulation, reinforcing the perceived importance of intracellular penetration as a key drug property. Moreover, we detected synergies deriving from redox-stimulatory combinations of PhX1 or clofazimine with the novel prenylated amino-artemisinin WHN296. Finally, we used compound synergies to elucidate the relationship between compound intracellular accumulation and efficacy, with PhX1/WHN296 synergy levels shown to predict drug efficacy. Collectively, our data support the utility of the applied assays in identifying in vitro active compounds with the potential for clinical development. IMPORTANCE This study addresses the development of novel therapeutic compounds for the eventual treatment of drug-resistant tuberculosis. Tuberculosis continues to progress, with cases of Mycobacterium tuberculosis (M. tuberculosis) resistance to first-line medications increasing. We assess new combinations of drugs with both oxidant and redox properties coupled with a third partner drug, with the focus here being on the potentiation of M. tuberculosis-active combinations of compounds in the intracellular macrophage environment. Thus, we determined the ability of the phenoxazine PhX1, previously shown to be active against M. tuberculosis in vitro, to differentially penetrate murine compartments, including plasma, epithelial lining fluid, and isolated epithelial lining fluid cells. In addition, the extent of permeation into human macrophage-like THP-1 cells and H37Rv-infected THP-1 cells was measured via mass spectrometry and compared to in vitro two-dimensional synergy and subsequent intracellular efficacy. Collectively, our data indicate that development of new drugs will be facilitated using the methods described herein.

BackgroundPre-exposure prophylaxis (PrEP) is highly effective and an important prevention tool for African adolescent girls and young women (AGYW), but adherence and persistence are challenging. PrEP adherence support strategies for African AGYW were studied in an implementation study.Methods and findingsHIV Prevention Trials Network (HPTN) 082 was conducted in Cape Town, Johannesburg (South Africa) and Harare (Zimbabwe) from October 2016 to October 2018 to evaluate PrEP uptake, persistence, and the effect of drug level feedback on adherence. Sexually active HIV-negative women ages 16-25 were offered PrEP and followed for 12 months; women who accepted PrEP were randomized to standard adherence support (counseling, 2-way SMS, and adherence clubs) or enhanced adherence support with adherence feedback from intracellular tenofovir-diphosphate (TFV-DP) levels in dried blood spots (DBS). PrEP uptake, persistence through 12 months (no PrEP hold or missed visits), and adherence were assessed. The primary outcome was high adherence (TFV-DP ≥700 fmol/punch) at 6 months, compared by study arm. Of 451 women enrolled, median age was 21 years, and 39% had curable sexually transmitted infections (STIs). Most (95%) started PrEP, of whom 55% had uninterrupted PrEP refills through 12 months. Of those with DBS, 84% had detectable TFV-DP levels at month 3, 57% at month 6, and 31% at month 12. At 6 months, 36/179 (21%) of AGYW in the enhanced arm had high adherence and 40/184 (22%) in the standard adherence support arm (adjusted odds ratio [OR] of 0.92; 95% confidence interval [CI] 0.55, 1.34; p = 0.76). Four women acquired HIV (incidence 1.0/100 person-years), with low or undetectable TFV-DP levels at or prior to seroconversion, and none of whom had tenofovir or emtricitabine resistance mutations. The study had limited power to detect a modest effect of drug level feedback on adherence, and there was limited awareness of PrEP at the time the study was conducted.ConclusionsIn this study, PrEP initiation was high, over half of study participants persisted with PrEP through month 12, and the majority of young African women had detectable TFV-DP levels through month 6 with one-fifth having high adherence. Drug level feedback in the first 3 months of PrEP use did not increase the proportion with high adherence at month 6. HIV incidence was 1% in this cohort with 39% prevalence of curable STIs and moderate PrEP adherence. Strategies to support PrEP use and less adherence-dependent formulations are needed for this population.Trial registrationClinicalTrials.gov NCT02732730.

Background: Blood-cerebrospinal fluid (CSF) barrier transporters affect the influx and efflux of drugs. The antiretrovirals tenofovir and emtricitabine may be substrates of blood-brain barrier (BBB) and blood-CSF barrier transporters, but data are limited regarding the pharmacogenetics and pharmacokinetics of their central nervous system (CNS) penetration. Objectives: We investigated genetic polymorphisms associated with CSF disposition of tenofovir and emtricitabine. Method: We collected paired plasma and CSF samples from 47 HIV-positive black South African adults who were virologically suppressed on efavirenz, tenofovir and emtricitabine. We considered 1846 single-nucleotide polymorphisms from seven relevant transporter genes (ABCC5, ABCG2, ABCB1, SLCO2B1, SCLO1A2, SLCO1B1 and ABCC4) and 782 met a linkage disequilibrium (LD)-pruning threshold. Results: The geometric mean (95% confidence interval [CI]) values for tenofovir and emtricitabine CSF-to-plasma concentration ratios were 0.023 (0.021–0.026) and 0.528 (0.460–0.605), respectively. In linear regression models, the lowest p-value for association with the tenofovir CSF-to-plasma ratio was ABCB1 rs1989830 (p = 1.2 × 10−3) and for emtricitabine, it was ABCC5 rs11921035 (p = 1.4 × 10−3). None withstood correction for multiple testing. Conclusion: No genetic polymorphisms were associated with plasma, CSF concentrations or CSF-to-plasma ratios for either tenofovir or emtricitabine.

Abstract Background To reduce onward falciparum malaria transmission, the World Health Organization recommends adding single low-dose (SLD) primaquine to artemisinin-based combination treatment in low transmission areas. However, uptake of this recommendation has been relatively slow given concerns about whether individual risks justify potential community benefit. This study was undertaken to generate comprehensive local data on the risk–benefit profile of SLD primaquine deployment in a pre-elimination area in South Africa. Methods This randomized, controlled open-label trial investigated adding a single low primaquine dose on day 3 to standard artemether–lumefantrine treatment for uncomplicated falciparum malaria. Efficacy, safety and tolerability of artemether–lumefantrine and primaquine treatment were assessed on days 3, 7, 14, 28 and 42. Lumefantrine concentrations were assayed from dried blood spot samples collected on day 7. Results Of 217 patients screened, 166 were enrolled with 140 randomized on day 3, 70 to each study arm (primaquine and no primaquine). No gametocytes were detected by either microscopy or PCR in any of the follow-up samples collected after randomization on day 3, precluding assessment of primaquine efficacy. Prevalence of the CYP2D6*4, CYP2D6*10 and CYP2D6*17 mutant alleles was low with allelic frequencies of 0.02, 0.11 and 0.16, respectively; none had the CYP2D6*4/*4 variant associated with null activity. Among 172 RDT-positive patients G6PD-genotyped, 24 (14%) carried the G6PD deficient (A−) variant. Median haemoglobin concentrations were similar between treatment arms throughout follow-up. A third of participants had a haemoglobin drop > 2 g/dL; this was not associated with primaquine treatment but may be associated with G6PD genotype [52.9% (9/17) with A− genotype vs. 31% (36/116) with other genotypes (p = 0.075)]. Day 7 lumefantrine concentrations and the number and nature of adverse events were similar between study arms; only one serious adverse event occurred (renal impairment in the no primaquine arm). The artemether–lumefantrine PCR-corrected adequate clinical and parasitological response rate was 100%, with only one re-infection found among the 128 patients who completed 42-day follow-up. Conclusions Safety, tolerability, CYP2D6 and G6PD variant data from this study support the deployment of the WHO-recommended SLD primaquine without G6PD testing to advance malaria elimination in South African districts with low-intensity residual transmission. Trial registration Pan African Clinical Trial Registry, PACTR201611001859416. Registered 11 November 2016, https://pactr.samrc.ac.za/TrialDisplay.aspx?TrialID=1859

Background: In patients with tuberculous pericarditis [TBP] adjunctive prednisolone reduces the incidence of constrictive pericarditis. It is unknown whether prednisolone permeates adequately into pericardial fluid. Drug measurements in pericardial fluid require invasive procedures, and thus less invasive methods are needed to perform full pharmacokinetic characterization of prednisolone in large numbers of patients. We sought to evaluate the relationship between prednisolone concentrations in pericardial fluid, plasma, and saliva. Methods: Plasma, pericardial fluid, and saliva samples were collected at 7 time points from TBP patients randomized to 120 mg prednisolone or placebo. Compartmental pharmacokinetic parameters, peak concentration [Cmax], and 0–24 h area under the concentration-time curve [AUC0–24] were identified in plasma, saliva and pericardial fluid. Results: There were five patients each in the prednisolone and placebo groups. Prednisolone concentrations were best described using a one compartment model. The absorption half-life into plasma was 1 h, while that into pericardial fluid was 9.4 h, which led to a median time-to-maximum concentration in plasma of 2.0 h versus 5.0 h in pericardial fluid [p = 0.048]. The concentration-time profiles in pericardial fluid versus plasma exhibited system hysteresis. The pericardial fluid-to-plasma Cmax peak concentration ratio was 0.28 (p = 0.032), while the AUC0–24 ratio was 0.793. The concentration-time profiles in saliva had a similar shape to those in plasma, but the saliva-to-plasma Cmax was 0.59 [p = 0.032]. Conclusion: The prednisolone AUC0–24 achieved in pericardial fluid approximates that in plasma, but the Cmax is low due to delayed absorption. Saliva can be used as surrogate sampling site for pericardial fluid prednisolone.

Tuberculosis (TB), the leading cause of death due to an infectious agent, requires prolonged and costly drug treatments. With the rise in incidence of MDR and XDR TB, newer more efficacious treatments which are better able to permeate into the deeper recesses of the human lung where bacteria reside are urgently required. To this end, two new promising drug candidates, the decoquinate derivative RMB041 and the phenoxazine PhX1, were assessed for their abilities to permeate into specific murine organs. In particular, PhX1 permeation into the lungs and heart was notably efficient, as reflected in the high relative AUC values of 9669 ± 120.2 min/nmol/mg and 12450 ± 45.2 min/nmol/mg for lung and heart tissue, respectively. However, neither compound maintained a free concentration in the lung which exceeded the compound’s respective MIC90 values, indicating the importance of correcting for organ specific binding.

We have demonstrated previously that amino-artemisinins including artemiside and artemisone in which an amino group replaces the oxygen-bearing substituents attached to C-10 of the current clinical artemisinin derivatives dihydroartemisinin (DHA), artemether and artesunate, display potent activities in vitro against the asexual blood stages of Plasmodium falciparum (Pf). In particular, the compounds are active against late blood stage Pf gametocytes, and are strongly synergistic in combination with the redox active drug methylene blue. In order to fortify the eventual selection of optimum amino-artemisinins for development into new triple combination therapies also active against artemisinin-resistant Pf mutants, we have prepared new amino-artemisinins based on the easily accessible and inexpensive DHA-piperazine. The latter was converted into alkyl- and aryl sulfonamides, ureas and amides. These derivatives were screened together with the comparator drugs DHA and the hitherto most active amino-artemisinins artemiside and artemisone against asexual and sexual blood stages of Pf and liver stage P. berghei (Pb) sporozoites. Several of the new amino-artemisinins bearing aryl-urea and -amide groups are potently active against both asexual, and late blood stage gametocytes (IC50 0.4-1.0 nM). Although the activities are superior to those of artemiside (IC50 1.5 nM) and artemisone (IC50 42.4 nM), the latter are more active against the liver stage Pb sporozoites (IC50 artemisone 28 nM). In addition, early results indicate these compounds tend not to display reduced susceptibility against parasites bearing the Pf Kelch 13 propeller domain C580Y mutation characteristic of artemisinin-resistant Pf. Thus, the advent of the amino-artemisinins including artemiside and artemisone will enable the development of new combination therapies that by virtue of the amino-artemisinin component itself will possess intrinsic transmission-blocking capabilities and may be effective against artemisinin resistant falciparum malaria.

Abstract Background The Angolan government recommends three artemisinin-based combinations for the treatment of uncomplicated Plasmodium falciparum malaria: artemether–lumefantrine (AL), artesunate–amodiaquine (ASAQ), and dihydroartemisinin–piperaquine (DP). Due to the threat of emerging anti-malarial drug resistance, it is important to periodically monitor the efficacy of artemisinin-based combination therapy (ACT). This study evaluated these medications’ therapeutic efficacy in Benguela, Lunda Sul, and Zaire Provinces. Methods Enrollment occurred between March and July 2017. Study participants were children with P. falciparum monoinfection from each provincial capital. Participants received a 3-day course of a quality-assured artemisinin-based combination and were monitored for 28 (AL and ASAQ arms) or 42 days (DP arm). Each ACT was assessed in two provinces. The primary study endpoints were: (1) follow-up without complications and (2) failure to respond to treatment or development of recurrent P. falciparum infection. Parasites from each patient experiencing recurrent infection were genotyped to differentiate new infection from recrudescence of persistent parasitaemia. These parasites were also analysed for molecular markers associated with ACT resistance. Results Of 608 children enrolled in the study, 540 (89%) reached a primary study endpoint. Parasitaemia was cleared within 3 days of medication administration in all participants, and no early treatment failures were observed. After exclusion of reinfections, the corrected efficacy of AL was 96% (91–100%, 95% confidence interval) in Zaire and 97% (93–100%) in Lunda Sul. The corrected efficacy of ASAQ was 100% (97–100%) in Benguela and 93% (88–99%) in Zaire. The corrected efficacy of DP was 100% (96–100%) in Benguela and 100% in Lunda Sul. No mutations associated with artemisinin resistance were identified in the pfk13 gene in the 38 cases of recurrent P. falciparum infection. All 33 treatment failures in the AL and ASAQ arms carried pfmdr1 or pfcrt mutations associated with lumefantrine and amodiaquine resistance, respectively, on day of failure. Conclusions AL, ASAQ, and DP continue to be efficacious against P. falciparum malaria in these provinces of Angola. Rapid parasite clearance and the absence of genetic evidence of artemisinin resistance are consistent with full susceptibility to artemisinin derivatives. Periodic monitoring of in vivo drug efficacy remains a priority routine activity for Angola.

Abstract Background The Joint United Nations Programme on HIV/AIDS (UNAIDS) has developed an ambitious strategy to end the AIDS epidemic. After eight years of antiretroviral therapy (ART) program we assessed progress towards the UNAIDS 90–90-90 targets in Mbongolwane and Eshowe, KwaZulu-Natal, South Africa. Methods We conducted a cross-sectional household-based community survey using a two-stage stratified cluster probability sampling strategy. Persons aged 15–59 years were eligible. We used face-to-face interviewer-administered questionnaires to collect information on history of HIV testing and care. Rapid HIV testing was performed on site and venous blood specimens collected from HIV-positive participants for antiretroviral drug presence test, CD4 count and viral load. At the time of the survey the CD4 threshold for ART initiation was 350 cells/μL. We calculated progression towards the 90–90-90 UNAIDS targets by estimating three proportions: HIV positive individuals who knew their status (first 90), those diagnosed who were on ART (second 90), and those on ART who were virally suppressed (third 90). Results We included 5649/6688 (84.5%) individuals. Median age was 26 years (IQR: 19–40), 62.3% were women. HIV prevalence was 25.2% (95% CI: 23.6–26.9): 30.9% (95% CI: 29.0–32.9) in women; 15.9% (95% CI: 14.0–18.0) in men. Overall progress towards the 90–90-90 targets was as follows: 76.4% (95% CI: 74.1–78.6) knew their status, 69.9% (95% CI: 67.0–72.7) of those who knew their status were on ART and 93.1% (95% CI: 91.0–94.8) of those on ART were virally suppressed. By sex, progress towards the 90–90-90 targets was: 79%–71%–93% among women; and 68%–68%–92% among men (p-values of women and men comparisons were

With drug resistance threatening our first line antimalarial treatments, novel chemotherapeutics need to be developed. Ionophores have garnered interest as novel antimalarials due to their theorized ability to target unique systems found in the Plasmodium-infected erythrocyte. In this study, during the bioassay-guided fractionation of the crude extract of Streptomyces strain PR3, a group of cyclodepsipeptides, including valinomycin, and a novel class of cyclic ethers were identified and elucidated. Further study revealed that the ethers were cyclic polypropylene glycol (cPPG) oligomers that had leached into the bacterial culture from an extraction resin. Molecular dynamics analysis suggests that these ethers are able to bind cations such as K+, NH4+ and Na+. Combination studies using the fixed ratio isobologram method revealed that the cPPGs synergistically improved the antiplasmodial activity of valinomycin and reduced its cytotoxicity in vitro. The IC50 of valinomycin against P. falciparum NF54 improved by 4–5-fold when valinomycin was combined with the cPPGs. Precisely, it was improved from 3.75 ± 0.77 ng/mL to 0.90 ± 0.2 ng/mL and 0.75 ± 0.08 ng/mL when dosed in the fixed ratios of 3:2 and 2:3 of valinomycin to cPPGs, respectively. Each fixed ratio combination displayed cytotoxicity (IC50) against the Chinese Hamster Ovary cell line of 57–65 µg/mL, which was lower than that of valinomycin (12.4 µg/mL). These results indicate that combinations with these novel ethers may be useful in repurposing valinomycin into a suitable and effective antimalarial.

Because of the need to replace the current clinical artemisinins in artemisinin combination therapies, we are evaluating fitness of amino-artemisinins for this purpose. These include the thiomorpholine derivative artemiside obtained in one scalable synthetic step from dihydroartemisinin (DHA) and the derived sulfone artemisone. We have recently shown that artemiside undergoes facile metabolism via the sulfoxide artemisox into artemisone and thence into the unsaturated metabolite M1; DHA is not a metabolite. Artemisox and M1 are now found to be approximately equipotent with artemiside and artemisone in vitro against asexual P. falciparum (Pf) blood stage parasites (IC50 1.5–2.6 nM). Against Pf NF54 blood stage gametocytes, artemisox is potently active (IC50 18.9 nM early-stage, 2.7 nM late-stage), although against the late-stage gametocytes, activity is expressed, like other amino-artemisinins, at a prolonged incubation time of 72 h. Comparative drug metabolism and pharmacokinetic (DMPK) properties were assessed via po and iv administration of artemiside, artemisox, and artemisone in a murine model. Following oral administration, the composite Cmax value of artemiside plus its metabolites artemisox and artemisone formed in vivo is some 2.6-fold higher than that attained following administration of artemisone alone. Given that efficacy of short half-life rapidly-acting antimalarial drugs such as the artemisinins is associated with Cmax, it is apparent that artemiside will be more active than artemisone in vivo, due to additive effects of the metabolites. As is evident from earlier data, artemiside indeed possesses appreciably greater efficacy in vivo against murine malaria. Overall, the higher exposure levels of active drug following administration of artemiside coupled with its synthetic accessibility indicate it is much the preferred drug for incorporation into rational new artemisinin combination therapies.

BackgroundLinezolid is increasingly important for multidrug-resistant tuberculosis (MDR-TB) treatment. However, among children with MDR-TB, there are no linezolid pharmacokinetic data, and its adverse effects have not yet been prospectively described. We characterised the pharmacokinetics, safety, and optimal dose of linezolid in children treated for MDR-TB.Methods and findingsChildren routinely treated for MDR-TB in 2 observational studies (2011-2015, 2016-2018) conducted at a single site in Cape Town, South Africa, underwent intensive pharmacokinetic sampling after either a single dose or multiple doses of linezolid (at steady state). Linezolid pharmacokinetic parameters, and their relationships with covariates of interest, were described using nonlinear mixed-effects modelling. Children receiving long-term linezolid as a component of their routine treatment had regular clinical and laboratory monitoring. Adverse events were assessed for severity and attribution to linezolid. The final population pharmacokinetic model was used to derive optimal weight-banded doses resulting in exposures in children approximating those in adults receiving once-daily linezolid 600 mg. Forty-eight children were included (mean age 5.9 years; range 0.6 to 15.3); 31 received a single dose of linezolid, and 17 received multiple doses. The final pharmacokinetic model consisted of a one-compartment model characterised by clearance (CL) and volume (V) parameters that included allometric scaling to account for weight; no other evaluated covariates contributed to the model. Linezolid exposures in this population were higher compared to exposures in adults who had received a 600 mg once-daily dose. Consequently simulated, weight-banded once-daily optimal doses for children were lower than those currently used for most weight bands. Ten of 17 children who were followed long term had a linezolid-related adverse event, including 5 with a grade 3 or 4 event, all anaemia. Adverse events resulted in linezolid dose reductions in 4, temporary interruptions in 5, and permanent discontinuation in 4 children. Limitations of the study include the lack of very young children (none below 6 months of age), the limited number who were HIV infected, and the modest number of children contributing to long-term safety data.ConclusionsLinezolid-related adverse effects were frequent and occasionally severe. Careful linezolid safety monitoring is required. Compared to doses currently used in children in many settings for MDR-TB treatment, lower doses may approximate current adult target exposures, might result in fewer adverse events, and should therefore be evaluated.

Background: Antiretroviral concentrations in hair provide a longer window of drug detection and are useful for measuring longer-term drug exposure. Efavirenz is an important component of first-line treatment in resource-limited settings, but its concentrations in hair have not been well studied. Methods: This study is a supplementary to a randomised controlled trial of an adherence intervention using an electronic adherence measuring device. Hair and plasma samples were collected from human immunodeficiency virus-positive patients in Cape Town, South Africa. Previously validated liquid chromatography tandem mass spectrometry methods were used to measure efavirenz concentrations in the collected hair and plasma samples. CYP2B6 genotyping of participants was also performed. Data analysis was performed using descriptive and comparative statistics as well as regression modelling. Results: Hair samples were collected from 59% of patients enrolled in the parent study. Results indicated that hair efavirenz concentrations were significantly influenced by participants’ CYP2B6metaboliser status. Median efavirenz concentrations for extensive, intermediate and slow metaboliser genotypes were 3.54 ng/mg, 5.11 ng/mg and 10.66 ng/mg, respectively. A strong correlation was observed between the efavirenz concentrations measured in hair and plasma samples (Spearman’s correlation coefficients, 0.672–0.741, p < 0.0001). No relationship between hair efavirenz concentrations and virological failure or adherence measured using an electronic adherence was shown. Conclusion: The results from this study provide further insight into the potential of using hair as a matrix for measuring antiretroviral concentrations. However, challenges experienced in collecting hair samples suggest that this adherence measure may have limited utility in an African population.

Tuberculosis (TB) is currently the leading cause of mortality due to an infectious disease, despite the existence of multiple effective first-line and second-line drugs. The current anti-TB regimen requires a prolonged treatment period of around 6 months and is only efficacious against drug-sensitive strains of Mycobacterium tuberculosis (Mtb). With a rise in cases of multi-drug resistant and extensively drug resistant strains of Mtb, newer treatments comprising compounds with novel mechanisms of action are required. Although decoquinate (DQ) is inactive against Mtb, its derivatives are of interest to anti-TB drug discovery because of their potential to permeate the mycobacterial cell wall, Mtb-infected macrophages, and granulomatous lesions by passive diffusion. The compounds also display mechanisms of action which are unlike those of currently used quinolones, potentially displaying activity against new targets. Three such derivatives bearing an alkyl group at N-1 and an amide group at C-3 (RMB 041, -043, and -073) displayed potent in vitro activities against Mtb H37Rv (90% minimum inhibitory concentrations, MIC90 = 1.61, 4.18, and 1.88 μM, respectively) and high selectivity indices (10–25). In this study, we evaluated the drug-like properties (in vitro microsomal stability, microsomal/plasma protein binding, kinetic solubility, lipophilicity, and passive permeability) and pharmacokinetic (PK) parameters of these compounds after intravenous and oral administration to male C57BL/6 mice. The compounds showed markedly improved kinetic solubilities compared to that of the parental DQ and were metabolically stable in vitro. The maximum concentrations reached after oral administration were 5.4 ± 0.40, 5.6 ± 1.40, and 2.0 ± 0.03 μM; elimination half-lives were 23.4 ± 2.50, 6.2 ± 0.80, and 11.6 ± 1.30 h; and bioavailabilities were 21.4 ± 1.0, 22.1 ± 2.2, and 5.9 ± 1.3 for RMB041, -043, and -073, respectively. These compounds therefore display promising drug-like properties, and their PK/toxicity profiles (including long half-lives both in vitro and in vivo) support their potential as candidates for further investigation in animal models of Mtb infection.

An amendment to this paper has been published and can be accessed via the original article.

Patients receiving anti-retroviral drug treatment are sometimes simultaneously taking herbal remedies, which may result in pharmacokinetic herb-drug interactions. This study aimed to determine if pharmacokinetic interactions exist between selected commercially available herbal products (i.e., Linctagon Forte®, Viral Choice® and Canova®) and indinavir in terms of in vitro transport and metabolism. Bi-directional transport of indinavir was evaluated across Caco-2 cell monolayers in the presence and absence of the selected herbal products and verapamil (positive control). Metabolism of indinavir was determined in LS180 cells in the presence and absence of the selected herbal products as well as ketoconazole (positive control). The secretory transport of indinavir increased in a concentration dependent way in the presence of Linctagon Forte® and Viral Choice® when compared to that of indinavir alone. Canova® only slightly affected the efflux of indinavir compared to that of the control group. There was a pronounced inhibition of the metabolism of indinavir in LS180 cells over the entire concentration range for all the herbal products investigated in this study. These in vitro pharmacokinetic interactions indicate the selected herbal products may affect indinavir’s bioavailability, but the clinical significance needs to be confirmed with in vivo studies before final conclusions can be made.

Pericardial tuberculosis (TB) is associated with high therapy failure and high mortality rates. Antibiotics have to penetrate to site of infection at sufficient non-protein bound concentrations, and then enter bacteria to inhibit intracellular biochemical processes. The antibiotic concentrations achieved in pericardial fluid in TB pericarditis have never been measured before. We recruited two cohorts of patients with TB pericarditis, and left a pigtail catheter in-situ for serial drug concentration measurements over 24 h. Altogether, 704 drug concentrations were comodeled for pharmacokinetic analyses. The drug concentrations achieved in pericardial fluid were compared to the minimum inhibitory concentrations (MICs) of clinical Mycobacterium tuberculosis isolates. The total rifampicin concentration pericardial-to-serum ratios in 16 paired samples were 0.19 ± 0.33. The protein concentrations of the pericardial fluid in TB pericarditis were observed to be as high as in plasma. The non-protein bound rifampicin concentrations in pericardial fluid were 4-fold lower than rifampicin MICs in the pilot study, and the peak concentration was 0.125 versus 0.208 mg/L in the second (p = 0.001). The rifampicin clearance from pericardial fluid was 9.45 L/h versus 7.82 L/h in plasma (p = 0.002). Ethambutol peak concentrations had a pericardial-to-plasma ratio of 0.55 ± 0.22; free ethambutol peak concentrations were 2.30-lower than MICs (p

Exposure to lower-than-therapeutic levels of anti-tuberculosis drugs is likely to cause selection of resistant strains of Mycobacterium tuberculosis and treatment failure. The first-line anti-tuberculosis (TB) regimen consists of rifampicin, isoniazid, pyrazinamide, and ethambutol, and correct management reduces risk of TB relapse and development of drug resistance. In this study we aimed to investigate the effect of standard of care plus nutritional supplementation versus standard care on the pharmacokinetics of isoniazid, pyrazinamide and ethambutol among sputum smear positive TB patients with and without HIV. In a clinical trial in 100 Tanzanian TB patients, with or without HIV infection, drug concentrations were determined at 1 week and 2 months post initiation of anti-TB medication. Data was analysed using population pharmacokinetic modelling. The effect of body size was described using allometric scaling, and the effects of nutritional supplementation, HIV, age, sex, CD4+ count, weight-adjusted dose, NAT2 genotype, and time on TB treatment were investigated. The kinetics of all drugs was well characterised using first-order elimination and transit compartment absorption, with isoniazid and ethambutol described by two-compartment disposition models, and pyrazinamide by a one-compartment model. Patients with a slow NAT2 genotype had higher isoniazid exposure and a lower estimate of oral clearance (15.5 L/h) than rapid/intermediate NAT2 genotype (26.1 L/h). Pyrazinamide clearance had an estimated typical value of 3.32 L/h, and it was found to increase with time on treatment, with a 16.3% increase after the first 2 months of anti-TB treatment. The typical clearance of ethambutol was estimated to be 40.7 L/h, and was found to decrease with age, at a rate of 1.41% per year. Neither HIV status nor nutritional supplementations were found to affect the pharmacokinetics of these drugs in our cohort of patients.

Insight into the mechanisms of intestinal transport and metabolism of aspalathin will provide important information for dose optimisation, in particular for studies using mouse models. Aspalathin transportation across the intestinal barrier (Caco-2 monolayer) tested at 1–150 µM had an apparent rate of permeability (Papp) typical of poorly absorbed compounds (1.73 × 10−6 cm/s). Major glucose transporters, sodium glucose linked transporter 1 (SGLT1) and glucose transporter 2 (GLUT2), and efflux protein (P-glycoprotein, PgP) (1.84 × 10−6 cm/s; efflux ratio: 1.1) were excluded as primary transporters, since the Papp of aspalathin was not affected by the presence of specific inhibitors. The Papp of aspalathin was also not affected by constituents of aspalathin-enriched rooibos extracts, but was affected by high glucose concentration (20.5 mM), which decreased the Papp value to 2.9 × 10−7 cm/s. Aspalathin metabolites (sulphated, glucuronidated and methylated) were found in mouse urine, but not in blood, following an oral dose of 50 mg/kg body weight of the pure compound. Sulphates were the predominant metabolites. These findings suggest that aspalathin is absorbed and metabolised in mice to mostly sulphate conjugates detected in urine. Mechanistically, we showed that aspalathin is not actively transported by the glucose transporters, but presumably passes the monolayer paracellularly.