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Objectives Globally, tuberculosis (TB) incidence and mortality are declining; however, low detection of drug-resistant disease threatens to reverse current progress toward global TB control. Multiple, rapid molecular diagnostic tests have recently been developed to detect genetic mutations in Mycobacterium tuberculosis (Mtb) known to confer anti-TB drug resistance. Their utility, though, depends on the frequency and distribution of the resistance-associated mutations in the pathogen population. Therefore, this review aimed to assess the prevalence of the gene mutations associated with rifampicin (RIF) and isoniazid (INH) resistant Mtb in Ethiopia. Methods Using PRISMA guidelines, we searched the literature on PubMed/MEDLINE, Web of Science, Scopus, and Cochrane library databases. Data analysis was conducted in STATA 11. Results In total, 909 (95.8%) of 949 INH resistant Mtb isolates had detectable gene mutations: 95.8% in katG315 and 5.9% in the inhA-promoter region. The meta-analysis resulting an estimated prevalence of katGMUT1(S315T1) was 89.2% (95%CI:81.94-96.43%), while a pooled prevalence of inhAMUT1 (C15T) was 77.5% (95%CI:57.84-97.13%). Besides, 769 (90.8%) of 847 RIF resistant strains had detectable rpoB gene mutations, and the meta-analysis resulting in a pooled prevalence of rpoBMUT3 (S531L) was 74.2% (95%CI: 66.39-82.00%). Conclusions RIF-resistant Mtb isolates were spread widely, particularly those harboring S531L mutations. Similarly, INH-resistant Mtb strains with S315T1 and C15T mutations were common. Tracking S531L, S315T1, and C15T mutations among RIF and INH resistant isolates, respectively, would be diagnostically and epidemiologically valuable. Rapid diagnosis of RIF and INH-resistant Mtb in TB patients would expedite alteration of treatment regimens, and proper timely infection control interventions could reduce the risk of progression and transmission of multidrug-resistant TB (MDR-TB).

Clinical specimens unfit for laboratory processing represent missed opportunities for diagnosing tuberculosis. Poor-quality cough specimens (n=61) from presumptive tuberculosis cases were cultured and GeneXpert MTB/RIF (Xpert) successfully performed on samples transferred by flocked swab into PrimeStore molecular transport medium (PS-MTM). Mycobacterium tuberculosis was grown in culture from 13 (21.3 %) and Xpert reported 15 (24.2 %) positive, of which 10 concordant. RT-PCR of PS-MTM samples showed enhanced sensitivity; three positives were missed by Xpert, five by culture and three more detected for a total of 21 positives (34.4 %).

Objectives To assess the performance of an innovative method of transporting sputum to centralised facilities for molecular detection of Mycobacterium tuberculosis: using a swab to inoculate sputum in a transport medium, PrimeStore® Molecular Transport Medium (PS-MTM). Methods Two sputum specimens were obtained from suspected patients with tuberculosis (TB) at rural healthcare facilities in South Africa. A swab was taken from each specimen and placed into PS-MTM, prior to it being processed by either liquid culture or Xpert MTB/RIF assay (Xpert). Results A total of 141 patients (including 47 with laboratory-confirmed TB) were included in this analysis. M. tuberculosis was detected at 29% by culture and 29% by Xpert, whereas 31% tested positive by IS6110 real-time PCR of PS-MTM from the culture and 36% from the Xpert-paired specimen. Concordance between the method under evaluation with culture was 82% (McNemar, P = 0.55) and 84% (McNemar, P = 0.05) for Xpert. Stratified by culture result, the detection rate by IS6110 real-time PCR of PS-MTM was similar to Xpert for patients with positive culture (P = 0.32), but significantly higher if culture was negative (P = 0.008). Conclusions These results suggest that swab collection of sputum into PS-MTM for transport is a promising method for diagnosis of TB in rural healthcare settings, thereby potentially improving the options available for molecular diagnosis of TB in countries incapable of applying decentralised high-tech molecular testing.

The technical limitations of common tests used for detecting pyrazinamide (PZA) resistance in Mycobacterium tuberculosis isolates pose challenges for comprehensive and accurate descriptions of drug resistance in patients with multidrug-resistant tuberculosis (MDR-TB). In this study, a 606-bp fragment (comprising the pncA coding region plus the promoter) was sequenced using Ion Torrent next-generation sequencing (NGS) to detect associated PZA resistance mutations in 88 recultured MDR-TB isolates from an archived series collected in 2001. These 88 isolates were previously Sanger sequenced, with 55 (61%) designated as carrying the wild-type pncA gene and 33 (37%) showing mutations. PZA susceptibility of the isolates was also determined using the Bactec 460 TB system and the Wayne test. In this study, isolates were recultured and susceptibility testing was performed in Bactec 960 MGIT. Concordance between NGS and MGIT results was 93% ( n = 88), and concordance values between the Bactec 460, the Wayne test, or pncA gene Sanger sequencing and NGS results were 82% ( n = 88), 83% ( n = 88), and 89% ( n = 88), respectively. NGS confirmed the majority of pncA mutations detected by Sanger sequencing but revealed several new and mixed-strain mutations that resolved discordancy in other phenotypic results. Importantly, in 53% (18/34) of these isolates, pncA mutations were located in the 151 to 360 region and warrant further exploration. In these isolates, with their known resistance to rifampin, NGS of pncA improved PZA resistance detection sensitivity to 97% and specificity to 94% using NGS as the gold standard and helped to resolve discordant results from conventional methodologies.

Patients with impaired immunity often have rapid progression of tuberculosis (TB) which can lead to highly lethalBALB/c mice were immunized with ethanol-killed MTB (EK-MTB) and MABs were produced and screened by ELISA for binding to killed and liveTwo MABs (GG9 and JG7) bound to killed and live SMEG and MTB (susceptible and resistant), and promoted OPKA with live MTB. MAB JG7 significantly enhanced OPKA of MTB. Both MABs significantly enhanced clearance of killed MTB from murine blood at 4 and 24 h as measured by qPCR. These opsonic MABs bound to PGN, a major cell wall constituent.Anti-MTB MABs that promote bactericidal phagocytic activity of MTB and enhance clearance of killed MTB from the blood, may offer an immunotherapeutic approach for treatment of MTB bacteremia or sepsis, and augment treatment of multi-drug resistant (MDR) or extensively drug resistant (XDR) TB.

Multidrug-resistant tuberculosis (MDR-TB) threatens global TB control. The lengthy treatment includes one of the injectable drugs kanamycin, amikacin, and capreomycin, usually for the first 6 months. These drugs have potentially serious toxicities, and when given as intramuscular injections, dosing can be painful. Advances in particulate drug delivery have led to the formulation of capreomycin as the first antituberculosis drug available as a microparticle dry powder for inhalation and clinical study. Delivery by aerosol may result in successful treatment with lower doses. Here we report a phase I, single-dose, dose-escalating study aimed at demonstrating safety and tolerability in healthy subjects and measuring pharmacokinetic (PK) parameters. Twenty healthy adults ( n = 5 per group) were recruited to self-administer a single dose of inhaled dry powder capreomycin (25-mg, 75-mg, 150-mg, or 300-mg nominal dose) using a simple, handheld delivery device. Inhalations were well tolerated by all subjects. The most common adverse event was mild to moderate transient cough, in five subjects. There were no changes in lung function, audiometry, or laboratory parameters. Capreomycin was rapidly absorbed after inhalation. Systemic concentrations were detected in each dose group within 20 min. Peak and mean plasma concentrations of capreomycin were dose proportional. Serum concentrations exceeded 2 μg/ml (MIC for Mycobacterium tuberculosis ) following the highest dose; the half-life ( t 1/2 ) was 4.8 ± 1.0 h. A novel inhaled microparticle dry powder formulation of capreomycin was well tolerated. A single 300-mg dose rapidly achieved serum drug concentrations above the MIC for Mycobacterium tuberculosis , suggesting the potential of inhaled therapy as part of an MDR-TB treatment regimen.

Item does not contain fulltext OBJECTIVES: Extensive but fragmented data from existing studies were used to describe the drug-drug interaction between rifabutin and HIV PIs and predict doses achieving recommended therapeutic exposure for rifabutin in patients with HIV-associated TB, with concurrently administered PIs. METHODS: Individual-level data from 13 published studies were pooled and a population analysis approach was used to develop a pharmacokinetic model for rifabutin, its main active metabolite 25-O-desacetyl rifabutin (des-rifabutin) and drug-drug interaction with PIs in healthy volunteers and patients who had HIV and TB (TB/HIV). RESULTS: Key parameters of rifabutin affected by drug-drug interaction in TB/HIV were clearance to routes other than des-rifabutin (reduced by 76%-100%), formation of the metabolite (increased by 224% in patients), volume of distribution (increased by 606%) and distribution to the peripheral compartment (reduced by 47%). For des-rifabutin, clearance was reduced by 35%-76% and volume of distribution increased by 67%-240% in TB/HIV. These changes resulted in overall increased exposure to rifabutin in TB/HIV patients by 210% because of the effects of PIs and 280% with ritonavir-boosted PIs. CONCLUSIONS: Given together with non-boosted or ritonavir-boosted PIs, rifabutin at 150 mg once daily results in similar or higher exposure compared with rifabutin at 300 mg once daily without concomitant PIs and may achieve peak concentrations within an acceptable therapeutic range. Although 300 mg of rifabutin every 3 days with boosted PI achieves an average equivalent exposure, intermittent doses of rifamycins are not supported by current guidelines.

A new efficacious tuberculosis (TB) vaccine has the potential to dramatically assist control efforts for the global TB epidemic. Good progress has been made with the clinical development of new TB vaccine candidates with twelve being actively tested in clinical trials. However, there are many challenges that need to be addressed before a new vaccine is licensed for public use. The diversity of risk in populations needs to be factored into clinical development plans, specific but feasible clinical endpoints need to be agreed upon, and TB vaccines need to be effective in both uninfected and infected populations. An achievable efficacy target needs to be set while standardisation of trial outcomes and critical choices based on the vaccine development pipeline need to be made. Alternative routes of vaccine administration should be thoroughly explored, sufficient adequately prepared trial sites for performing TB vaccine assessments are required and creative use of study designs should be used to expedite progress towards licensure while at the same time containing costs. Lastly, there needs to be sufficient funding to support TB vaccine development. These challenges can be met through commitment by all role-players within the TB vaccine arena and with support from external stakeholders.

Rifapentine and its primary metabolite, 25-desacetyl rifapentine, are active against mycobacterium tuberculosis. The objectives of this study were to describe the population pharmacokinetics of rifapentine and 25-desacetyl rifapentine in fasting and fed states. Thirty-five male healthy volunteers were enrolled in an open-label, randomized, sequential, five-way crossover study. Participants received a single 900-mg dose of rifapentine after meals with high fat (meal A), bulk and low fat (meal B), bulk and high fat (meal C), high fluid and low fat (meal D), or 200 ml of water (meal E). Venous blood samples were collected over 72 h after each rifapentine dose, and plasma was analyzed for rifapentine and 25-desacetyl rifapentine using high-performance liquid chromatography. Pharmacokinetic data were analyzed by nonlinear mixed-effect modeling using NONMEM. Compared with the fasting state, meal A had the greatest effect on rifapentine oral bioavailability, increasing it by 86%. Meals B, C, and D resulted in 33%, 46%, and 49% increases in rifapentine oral bioavailability, respectively. Similar trends were observed for 25-desacetyl rifapentine. As meal behavior has a substantial impact on rifapentine exposure, it should be considered in the evaluation of optimal dosing approaches.

Objectives: Pharmacokinetic drug-drug interactions were investigated between the fluoroquinolone gatifloxacin and a fixed dose combination (FDC) of rifampicin, isoniazid and pyrazinamide. Patients and methods: The single-dose pharmacokinetics of the four drugs was evaluated in an open-label three-way cross-over study amongst 22 healthy volunteers following administration of gatifloxacin, the FDC or the two products together. Results: Modest but potentially important drug-drug interactions affecting gatifloxacin and rifampicin concentrations were detected. The elimination rate of gatifloxacin was reduced such that the AUC from Oh to infinity was increased with a geometric mean ratio (GMR) [90% confidence interval (Cl)] of 1.14 (1.10, 1.18). Conversely, the AUC from Oh to infinity for rifampicin was reduced (GMR: 0.81, 90% Cl: 0.81, 0.96) when rifampicin, isoniazid and pyrazinamide were given together with gatifloxacin. Conclusions: Studies in patients including pharmacokinetic evaluation at steady state, efficacy and toxicity are required to determine the importance of the interactions for use of the combination of gatifloxacin, rifampicin, isoniazid and pyrazinamide in the treatment of tuberculosis.

Comparison of the early bactericidal activity (EBA) of rifapentine and its pharmacokinetics with those of rifampin to determine the cause of poor clinical response and regrowth between doses, leading to rifamycin monoresistance at relapse.Determination of the dose size of rifapentine that gives sufficient drug exposure to prevent regrowth.EBA study over initial 5 days of treatment of 123 patients, half at Durban and half at Cape Town, who received single rifapentine doses of 300, 600, 900, or 1,200 mg rifapentine or five daily doses of 150, 300, or 600 mg rifampin, with a pharmacokinetic study on 58 patients measuring standard parameters for each dose size of rifamycin and their desacetyl metabolites.The EBAs for both rifamycins were similar, with a linear relationship to log dose at lower doses and a curvilinear response at higher doses giving a plateau at 1,136 mg rifapentine. The area under the concentration-time curve (AUC) divided by the minimal inhibitory concentration (MIC) agreed well for both rifamycins on the assumption that the only free 2% of free rifapentine and the 14% of free rifampin after plasma binding were active in the lesions.Only the free proportions of the rifamycins were active in lesions. From consideration of the pulse size and the duration of the postantibiotic lag, a 1,200-mg dose of rifapentine seemed necessary to improve response and to prevent regrowth between doses, and hence rifamycin monoresistance.

Inhalable clofazimine-containing dry powder microparticles (CFM-DPI) and native clofazimine (CFM) were evaluated for activity against Mycobacterium tuberculosis in human monocyte-derived macrophage cultures and in mice infected with a low-dose aerosol. Both formulations resulted in 99% killing at 2.5 μg/ml in vitro . In mice, 480 μg and 720 μg CFM-DPI inhaled twice per week over 4 weeks reduced numbers of CFU in the lung by as much as log 10 2.6; 500 μg oral CFM achieved a log 10 0.7 reduction.

Health services in Africa are being overburdened by a continuous increase of cases of tuberculosis (TB), largely resulting from the large pool of infected individuals becoming co-infected with HIV. To help deal with the situation, TB treatment schedules need to be shorter and simpler, with minimal contact between the patient and the service provider required, if the problems of non-compliance and of ineffective service provision are to be overcome. Various drugs not marketed for use in the treatment of TB are currently under investigation for their potential roles in the simplification or shortening of treatment schedules. These mainly include the long-acting rifamycins and the fluoroquinolones. Furthermore, new drug development is focused on an understanding of the host-pathogen interaction leading to infection, latency and disease. Of these, latency is least understood. The use of molecular diversity and combinatorial chemistry, proteomics, and the use of the whole genome to discover drug targets are expected to produce new lead compounds for turning into drugs to treat active, latent and multi-drug-resistant TB more effectively in the foreseeable future.

Inhaled therapies offer a unique approach to the treatment of tuberculosis (TB) using a relevant target organ system as a route of administration. The number of research reports on this topic has been increasing exponentially in the last decade but studies of clinical efficacy have been rare in recent times. The challenge is to take many research findings and translate them into a strategy for product development. Dry powder inhalers are the dominant drug product under consideration by those interested in the inhaled therapy for TB. A range of factors including candidate drug, formulation, device selection, drug product testing for proof of concept, and preclinical and clinical purposes all demand different considerations. The following review is intended to raise awareness of a growing body of evidence, suggesting that inhaled therapy for TB is possible and desirable. In addition, it is intended to outline key elements of the product-development activity for this particular application that has not been discussed elsewhere in the literature. Hopefully, this will encourage those with development expertise to seriously contemplate the steps required to bring such products forward.

BACKGROUND: "TBDx" is an innovative smear microscopy system that automatically loads slides onto a microscope, focuses and digitally captures images and then classifies smears as positive or negative using computerised algorithms. OBJECTIVES: To determine the diagnostic accuracy of TBDx, using culture as the gold standard, and compare this to a microscopist's diagnostic performance. METHODS: This study is nested within a cross-sectional study of tuberculosis suspects from South African gold mines. All tuberculosis suspects had one sputum sample collected, which was decontaminated prior to smear microscopy, liquid culture and organism identification. All slides were auramine-stained and then read by both a research microscopist and by TBDx using fluorescence microscopes, classifying slides based on the WHO classification standard of 100 fields of view (FoV) at 400× magnification. RESULTS: Of 981 specimens, 269 were culture positive for Mycobacterium tuberculosis (27.4%). TBDx had higher sensitivity than the microscopist (75.8% versus 52.8%, respectively), but markedly lower specificity (43.5% versus 98.6%, respectively). TBDx classified 520/981 smears (53.0%) as scanty positive. Hence, a proposed hybrid software/human approach that combined TBDx examination of all smears with microscopist re-examination of TBDx scanty smears was explored by replacing the "positive" result of slides with 1-9 AFB detected on TBDx with the microscopist's original reading. Compared to using the microscopist's original results for all 981 slides, this hybrid approach resulted in equivalent specificity, a slight reduction in sensitivity from 52.8% to 49.4% (difference of 3.3%; 95% confidence interval: 0.2%, 6.5%), and a reduction in the number of slides to be read by the microscopist by 47.0%. DISCUSSION: Compared to a research microscopist, the hybrid software/human approach had similar specificity and positive predictive value, but sensitivity requires further improvement. Automated microscopy has the potential to substantially reduce the number of slides read by microscopists.

The lungs have received attention as a portal for drug delivery in tuberculosis (TB) from researchers addressing diverse objectives. These include: (a) targeting alveolar macrophages that harbour TB bacilli; (b) maintaining high drug concentrations in lung tissue; (c) systemic delivery of potent or second-line anti-TB agents; and (d) delivering agents that may change the host-pathogen dialectic. Formulation design considerations for each of the above objectives differ in slight, but important ways. As distinct from vaccine delivery formulations, inhalations intended for drug delivery are presumed to require chronic and repeated administration of larger amounts of material. This review seeks to summarize the consensus on the ways and means available or under development, to deliver different anti-TB agents as aerosols for inhalation. These agents include drugs in current clinical use, singly or in combination, experimental chemical entities, siRNA against host molecules, and finally, drugs in clinical use for unrelated pharmacological action, as modifiers of the host-pathogen dialectic. The pharmacokinetics of drug bioavailability in the lung, the blood and other tissues following lung deposition of inhaled therapies are also addressed. Finally, considerations on efficacy studies of drugs administered through aerosol delivery are discussed.

The global emergence of multidrug-resistant and extensively drug-resistant TB has refocused attention on preventing acquired resistance. This article reviews basic science, pharmacology and public policy to understand the contribution of these factors to acquired rifamycin resistance (ARR), a critical factor in multidrug resistance. Directly observed therapy short course (DOTS) effectiveness requires that each drug's effects persist equally throughout the dosing interval. Although rifampin and isoniazid have similar pharmacokinetics, the postantibiotic effect of rifampin is several times greater than that of isoniazid. As a result, rifampin's effects may persist unopposed when standard multidrug regimens are administered at intervals longer than 24 h. ARR may occur in this setting when outgrowth is not otherwise contained by the host immune response. Most countries do not provide weekend therapy under DOTS. Limiting TB therapy to weekdays may promote the emergence of acquired drug resistance in patients with advanced AIDS and TB. A large, simple trial to examine this question is both warranted and feasible.

The last word on Mycobacterium vaccae and its potential role in tuberculosis control has not been spoken. What is the way forward? Properly designed clinical trials have not shown efficacy. On the other hand some studies suggest activity for clinical radiological and laboratory endpoints. Future trials must incorporate dose-ranging with the best available correlate of protection--whole-blood production of interferon-to provide a rationale for selection of the dose and schedule for administration. (excerpt)

SummaryThe ultimate goal of vaccine development is licensure of a safe and efficacious product that has a well-defined manufacturing process resulting in a high quality product. In general, clinical development and regulatory approval occurs in a linear, sequential manner: Phase 1 – safety, immunogenicity; Phase 2 – immunogenicity, safety, dose ranging and preliminary efficacy; Phase 3 – definitive efficacy, safety, lot consistency; and, following regulatory approval, Phase 4 – post-marketing safety and effectiveness. For candidate TB vaccines, where correlates of protection are not yet identified, phase 2 and 3 efficacy of disease prevention trials are, by necessity, very large. Each trial would span 2–5 years, with full licensure expected only after 1 or even 2 decades of development. Given the urgent unmet need for a new TB vaccine, a satellite discussion was held at the International African Vaccinology Conference in Cape Town, South Africa in November 2012, to explore the possibility of expediting licensure by use of an “adaptive licensure” process, based on a risk/benefit assessment that is specific to regional needs informed by epidemiology. This may be appropriate for diseases such as TB, where high rates of morbidity, mortality, particularly in high disease burden countries, impose an urgent need for disease prevention. The discussion focused on two contexts: licensure within the South African regulatory environment – a high burden country where TB vaccine efficacy trials are on-going, and licensure by the United States FDA --a well-resourced regulatory agency where approval could facilitate global licensure of a novel TB vaccine.