Perceptions of Research Bronchoscopy in Malawian Adults with Pulmonary Tuberculosis: A Cross-Sectional Study

Bronchoscopy is an established research tool in Malawi, enabling collection of pulmonary samples for immunological, pharmacological, and microbiological studies. It is, however, an invasive clinical procedure that offers no direct benefit to volunteering participants when used in a research capacity alone, and thus informed consent is essential. This study aimed to explore TB patients’ understanding of research bronchoscopy, what would motivate them to participate in research bronchoscopy, and their concerns, in order to inform consenting processes for future clinical studies. We used a qualitative research design. Two focus group discussions were conducted with community members and TB patients to understand their perceptions of bronchoscopy. Transcripts were coded by multiple co-authors and thematic content analysis was used to analyse main findings. We found that Malawian patients with pulmonary TB were willing to participate in a study using research bronchoscopy for health assessment and access to improved healthcare. We identified information of value to potential participants when consenting to that may lessen some of the anxieties expressed by participants. Patient and public involvement is essential to improve informed consent and institutional trust

Genetic determinants of the pharmacokinetic variability of rifampin in Malawian adults with pulmonary tuberculosis

D.J.S. was supported by a Wellcome Trust Clinical PhD Fellowship (086757/Z/08/A to D.J.S.). A.D.M. was supported by a National Institute for Health Research Integrated Clinical Academic Training Fellowship and a Wellcome Trust Clinical PhD Fellowship (105/392/B/14/Z). The Malawi Liverpool Wellcome Trust Clinical Research Programme is supported by a strategic award from the Wellcome Trust.Variable exposure to antituberculosis (TB) drugs, partially driven by genetic factors, may be associated with poor clinical outcomes. Previous studies have suggested an influence of the SLCO1B1 locus on the plasma area under the concentration-time curve (AUC) of rifampin. We evaluated the contribution of single nucleotide polymorphisms (SNPs) in SLCO1B1 and other candidate genes (AADAC and CES-1) to interindividual pharmacokinetic variability in Malawi. A total of 174 adults with pulmonary TB underwent sampling of plasma rifampin concentrations at 2 and 6 h postdose. Data from a prior cohort of 47 intensively sampled, similar patients from the same setting were available to support population pharmacokinetic model development in NONMEM v7.2, using a two-stage strategy to improve information during the absorption phase. In contrast to recent studies in South Africa and Uganda, SNPs in SLCO1B1 did not explain variability in AUC0-∞ of rifampin. No pharmacokinetic associations were identified with AADAC or CES-1 SNPs, which were rare in the Malawian population. Pharmacogenetic determinants of rifampin exposure may vary between African populations. SLCO1B1 and other novel candidate genes, as well as nongenetic sources of interindividual variability, should be further explored in geographically diverse, adequately powered cohorts.Publisher PDFPeer reviewe

Evidence for phage-mediated gene transfer among Pseudomonas aeruginosa strains on the phylloplane

As the use of genetically engineered microorganisms for agricultural tasks becomes more frequent, the ability of bacteria to exchange genetic material in the agricultural setting must be assessed. Transduction (bacterial virus-mediated horizontal gene transfer) is a potentially important mechanism of gene transfer in natural environments. This study investigated the potential of plant leaves to act as surfaces on which transduction can take place among microorganisms. Pseudomonas aeruginosa and its generalized transducing bacteriophage F116 were used as a model system. The application of P. aeruginosa lysogens of F116 to plant leaves resulted in genetic exchange among donor and recipient organisms resident on the same plant. Transduction was also observed when these bacterial strains were inoculated onto adjacent plants and contact was made possible through high-density planting.Peer reviewedMicrobiology and Molecular Genetic

Intrapulmonary Pharmacokinetics of First-line Anti-tuberculosis Drugs in Malawian Patients With Tuberculosis

BACKGROUND: Further work is required to understand the intrapulmonary pharmacokinetics of first-line anti-tuberculosis drugs. This study aimed to describe the plasma and intrapulmonary pharmacokinetics of rifampicin, isoniazid, pyrazinamide, and ethambutol, and explore relationships with clinical treatment outcomes in patients with pulmonary tuberculosis. METHODS: Malawian adults with a first presentation of microbiologically-confirmed pulmonary tuberculosis received standard 6-month first-line therapy. Plasma and intrapulmonary samples were collected 8 and 16 weeks into treatment and drug concentrations measured in plasma, lung/airway epithelial lining fluid, and alveolar cells. Population pharmacokinetic modelling generated estimates of drug exposure (Cmax and AUC) from individual-level post-hoc Bayesian estimates of plasma and intrapulmonary pharmacokinetics. RESULTS: One-hundred-and-fifty-seven patients (58% HIV co-infected) participated. Despite standard weight-based dosing, peak plasma concentrations of first-line drugs were below therapeutic drug monitoring targets. Rifampicin concentrations were low in all three compartments. Isoniazid, pyrazinamide, and ethambutol achieved higher concentrations in epithelial lining fluid and alveolar cells than plasma. Isoniazid and pyrazinamide concentrations were 14.6 (95% CI: 11.2-18.0) and 49.8-fold (95% CI: 34.2-65.3) higher in lining fluid than plasma respectively. Ethambutol concentrations were highest in alveolar cells (alveolar cells:plasma ratio 15.0, 95% CI 11.4-18.6). Plasma or intrapulmonary pharmacokinetics did not predict clinical treatment response. CONCLUSIONS: We report differential drug concentrations between plasma and the lung. While plasma concentrations were below therapeutic monitoring targets, accumulation of drugs at the site of disease may explain the success of the first-line regimen. The low rifampicin concentrations observed in all compartments lend strong support for ongoing clinical trials of high-dose rifampicin regimens

Continuous culture model to examine factors that affect transduction among Pseudomonas aeruginosa strains in freshwater environments

Transduction among Pseudomonas aeruginosa strains was observed in continuous cultures operated under environmentally relevant generation times, cell densities, and phage-to-bacterium ratios, suggesting its importance as a natural mechanism of gene transfer. Transduction was quantified by the transfer of the Tra(sup-) Mob(sup-) plasmid Rms149 from a plasmid-bearing strain to an F116 lysogen that served as both the recipient and source of transducing phages. In control experiments in which transduction was prevented, there was a reduction in the phenotype of the mock transductant over time. However, in experiments in which transduction was permitted, the proportion of transductants in the population increased over time. These data suggest that transduction can maintain a phenotype for an extended period of time in a population from which it would otherwise be lost. Changes in the numbers of transductants were analyzed by a two-part mathematical model, which consisted of terms for the selection of the transductant's phenotype and for the formation of new transductants. Transduction rates ranged from 10(sup-9) to 10(sup-6) per total viable cell count per ml per generation and increased with both the recipient concentration and the phage-to-bacterium ratio. These observations indicate an increased opportunity for transduction to occur when the interacting components are in greater abundance.Peer reviewedMicrobiology and Molecular Genetic

Widespread use of herbal medicines by people living with human immunodeficiency virus and contamination of herbal medicines with antiretrovirals in Nigeria.

Herbal medication use amongst people living with human immunodeficiency virus (PLWH) is widespread and understudied. This study aimed to evaluate the prevalence of herbal medicine use amongst PLWH and possible contamination with antiretrovirals (ARVs). Countrywide collection of herbal samples sold by street vendors in Nigeria for the following indications: human immunodeficiency virus (HIV), acquired immune deficiency syndrome, fever and general weakness. Samples were screened using a validated liquid chromatography-mass spectrometry/mass spectrometry method for the presence of the following ARVs: efavirenz, nevirapine, lopinavir, darunavir, ritonavir, atazanavir, emtricitabine, tenofovir and lamivudine. A survey was conducted among 742 PLWH attending four HIV clinics in Nigeria. Data were collected using a structured questionnaire and analysed using IBM SPSS statistics version 22.0 (IBM Corp., 2013, Armond, NY). Of the 138 herbal medicines sampled, three (2%) contained detectable levels of tenofovir, emtricitabine and/or lamivudine. Additionally, of the 742 PLWH surveyed, 310 (41.8%) reported herbal medicine use. Among the users, 191 (61.6%) started taking herbals after commencing HIV therapy while herbal medicine use preceded ARVs treatment in 119 (38.4%) PLWH. We found herbal use to be widespread among PLWH in Nigeria, with increasing use after commencing ARV. Three herbal preparations were also found to contain detectable levels of ARVs. This is a concern and should be studied widely across the region and countries where herbal medicine use is prevalent and poorly regulated

Perceptions of research bronchoscopy in Malawian adults with pulmonary tuberculosis : a cross-sectional study

Funding: Liverpool Integrated Clinical Academic Training Programme, experience at another institution (ADM); Wellcome Trust Clinical PhD Fellowship 105392/B/14/Z.Bronchoscopy is an established research tool in Malawi, enabling collection of pulmonary samples for immunological, pharmacological, and microbiological studies. It is, however, an invasive clinical procedure that offers no direct benefit to volunteering participants when used in a research capacity alone, and thus informed consent is essential. This study aimed to explore TB patients' understanding of research bronchoscopy, what would motivate them to participate in research bronchoscopy, and their concerns, in order to inform consenting processes for future clinical studies. We used a qualitative research design. Two focus group discussions were conducted with community members and TB patients to understand their perceptions of bronchoscopy. Transcripts were coded by multiple coauthors and thematic content analysis was used to analyse main findings. We found that Malawian patients with pulmonary TB were willing to participate in a study using research bronchoscopy for health assessment and access to improved healthcare. We identified information of value to potential participants when consenting to that may lessen some of the anxieties expressed by participants. Patient and public involvement is essential to improve informed consent and institutional trust.Publisher PDFPeer reviewe

High intrapulmonary rifampicin and isoniazid concentrations are associated with rapid sputum bacillary clearance in patients with pulmonary tuberculosis

This work was supported by a Wellcome Trust Clinical PhD Fellowship [grant number 105392/B/14/Z to A.D.M. and L69AGB to JM]. ELC was supported by Wellcome [200901/Z/16/Z]. The Malawi-Liverpool-Wellcome Clinical Research Programme is supported by a strategic award from the Wellcome Trust [206545/Z/17/Z]. We also acknowledge infrastructural support for bioanalysis from the Liverpool Biomedical Research Centre funded by Liverpool Health Partners.Background Intrapulmonary pharmacokinetics may better explain response to tuberculosis (TB) treatment than plasma pharmacokinetics. We explored these relationships by modelling bacillary clearance in sputum in adult patients on first-line treatment in Malawi. Methods Bacillary elimination rates (BER) were estimated using linear mixed-effects modelling of serial time-to-positivity in mycobacterial growth indicator tubes for sputum collected during the intensive phase of treatment (weeks 0 to 8) for microbiologically confirmed TB. Population pharmacokinetic models used plasma and intrapulmonary drug levels at 8 and 16 weeks. Pharmacokinetic-pharmacodynamic relationships were investigated using individual-level measures of drug exposure (AUC and Cmax) for rifampicin, isoniazid, pyrazinamide, and ethambutol, in plasma, epithelial lining fluid, and alveolar cells as covariates in the bacillary elimination models. Results Among 157 participants (58% HIV co-infected), drug exposure in plasma or alveolar cells was not associated with sputum bacillary clearance. Higher peak concentrations (Cmax) or exposure (AUC) to rifampicin or isoniazid in epithelial lining fluid was associated with more rapid bacillary elimination and shorter time to sputum negativity. More extensive disease on baseline chest radiograph was associated with slower bacillary elimination. Clinical outcome was captured in 133 participants, with 15 (11%) unfavourable outcomes recorded (recurrent TB, failed treatment, or death). No relationship between BER and late clinical outcome was identified. Conclusions Greater intrapulmonary drug exposure to rifampicin or isoniazid in the epithelial lining fluid was associated with more rapid bacillary clearance. Higher doses of rifampicin and isoniazid may result in sustained high intrapulmonary drug exposure, rapid bacillary clearance, shorter treatment duration and better treatment outcomes.Publisher PDFPeer reviewe

Conjugal transfer of R68.45 and FP5 between Pseudomonas aeruginosa strains in a freshwater environment

Recent concern over the release of genetically engineered organisms has resulted in a need for information about the potential for gene transfer in the environment. In this study, the conjugal transfer in Pseudomonas aeruginosa of the plasmids R68.45 and FP5 was demonstrated in the freshwater environment of Fort Loudoun Resevoir, Knoxville, Tenn. When genetically well defined plasmid donor and recipient strains were introduced into test chambers suspended in Fort Loudoun Lake, transfer of both plasmids was observed. Conjugation occurred in both the presence and absence of the natural microbial community. The number of transconjugants recovered was lower when the natural community was present. Transfer of the broad-host-range plasmid R68.45 to organisms other than the introduced recipient was not observed in these chambers but was observed in laboratory simulations when an organism isolated from lakewater was used as the recipient strain. Although the plasmids transferred in laboratory studies were genetically and physically stable, a significant number of transconjugants recovered from the field trials contained deletions and other genetic rearrangements, suggesting that factors which increase gene instability are operating in the environment. The potential for conjugal transfer of genetic material must be considered in evaluating the release of any genetically engineered microorganism into a freshwater environment.Peer reviewedMicrobiology and Molecular Genetic

vitro synergy and enhanced murine brain penetration of saquinavir coadministered with mefloquine

ABSTRACT Highly active antiretroviral therapy has substantially improved prognosis in human immunodeficiency virus (HIV). However, the integration of proviral DNA, development of viral resistance, and lack of permeability of drugs into sanctuary sites (e.g., brain and lymphocyte) are major limitations to current regimens. Previous studies have indicated that the antimalarial drug chloroquine (CQ) has antiviral efficacy and a synergism with HIV protease inhibitors. We have screened a panel of antimalarial compounds for activity against HIV-1 in vitro. A limited efficacy was observed for CQ, mefloquine (MQ), and mepacrine (MC). However, marked synergy was observed between MQ and saquinavir (SQV), but not CQ in U937 cells. Furthermore, enhancement of the antiviral activity of SQV and four other protease inhibitors (PIs) by MQ was observed in MT4 cells, indicating a class specific rather than a drug-specific phenomenon. We demonstrate that these observations are a result of inhibition of multiple drug efflux proteins by MQ and that MQ also displaces SQV from orosomucoid in vitro. Finally, coadministration of MQ and SQV in CD-1 mice dramatically altered the tissue distribution of SQV, resulting in a Ͼ3-fold and Ͼ2-fold increase in the tissue/blood ratio for brain and testis, respectively. This pharmacological enhancement of in vitro antiviral activity of PIs by MQ now warrants further examination in vivo