Exploring of pyrazinamidase recombinant activity from PZA-sensitive and resistant Mycobacterium tuberculosis expressed in Escherichia coli BL21 (DE3).

The mutations of pncA gene encoding pyrazinamidase/PZase in Mycobacterium tuberculosis are often associated with pyrazinamide/PZA resistance. The H and R1 isolates showed significant phenotypic differences to PZA. The H isolate was PZA sensitive, but R1 was PZA resistant up to 100 ug/ml. The paper reports the pncA profile for both isolates and the activity of their protein expressed in Escherichia coli BL21(DE3). The 0.6 kb of each pncA genes have been subcloned successfully into the 5.4 kb pET30a vector and formed the pET30a-pncA recombinant with a size of 6.0 kb. The pncAR1 profile exhibited base mutations, but not for pncAH against to pncA from the PZA-sensitive M. tuberculosis H37RV published in Genbank ID: 888260. Three mutations were found in pncAR1, ie T41C, G419A, and A535G that subsequently changed amino acids of Cys14Arg, Arg140His and Ser179Gly in its protein level. The mutant PZase R1 that expressed as a 21 kDa protein in E. coli Bl21(DE3) lost 32% of its performance in activating PZA drug to pyrazinoic acid/POA compared to the wild-type PZase H. The mutation in the pncAR1 gene that followed by the decreasing of its PZase activity underlies the emergence of pyrazinamide resistance in the clinical isolate. Structural studies for the R1 mutant PZase protein should be further developed to reveal more precise drug resistance mechanisms and design more effective TB drugs.

Evaluation and Docking Study of Pyrazine Containing 1, 3, 4-Oxadiazoles Clubbed with Substituted Azetidin-2-one: A New Class of Potential Antimicrobial and Antitubercular.

BACKGROUND: Tuberculosis (TB) caused by Mycobacterium tuberculosis is one of the main killers of people all over the world. The major hurdles with existing therapy are the lengthy regimen and appearance of multi drug resistant (MDR) and extensively drug resistant (XDR) strains of M.tuberculosis. AIMS: The present work was aimed to synthesize and determine antitubercular and antimicrobial potential of some novel 3-chloro-4-aryl-1-[4-(5-pyrazin-2-yl[1,3,4]oxadiazole-2-ylmethoxy)-phenyl]-azetidin-2-one derivatives 7: (A: -H: ) from pyrazinoic acid as precursor, which is a well-established antitubercular agent. Here we report the synthesis of a new class of heterocyclic molecules in which pyrazine, 1, 3, 4-oxadiazole and azetidinone moieties were present in one frame work. METHODS: Pyrazinoic acid (1: ) was esterified first (2: ) followed by amination to produce hydrazide (3: ) which was refluxed with POCl3 to obtain 2-chloromethyl-5pyrazino-1, 3, 4-oxadiazole (4: ). This was then further reacted with 4-amino phenol to obtain 4-[5-pyrazino-1, 3, 4-oxadiazol-2-yl-methoxy]-phenyl amine (5: ) which on condensation with various aromatic aldehydes afforded a series Schiff's bases 6(A-H): . Dehydrative annulations of 6(A-H): in the presence of chloroacetyl chloride and triethylamine yielded 3-chloro-4-aryl-1-[4-(5-pyrazin-2-yl-[1, 3, 4]oxadiazole-2-ylmethoxy)-phenyl]-azetidin-2-one derivatives 7(A-H): . Antibacterial, antifungal and antitubercular potential of all the synthesized compounds were assessed. Docking study was performed using the software VLife Engine tools of Vlifemds 4.6 on the protein lumazine synthase of M. tuberculosis (PDB entry code 2C92). RESULTS: The present studies demonstrated that synthesized oxadiazole derivatives have good antimicrobial activity against the various microorganisms. Among the synthesized derivative, 7B: and 7G: were found to be prominent compounds which have potential antibacterial, antifungal and antitubercular activity (with MIC 3.12 µg/ml and high dock score ranging from -59.0 to -54.0) against Mycobacterium tuberculosis. CONCLUSIONS: Derivatives 7B: and 7G: would be effective lead candidates for tuberculosis therapy.

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

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

Introducing molecular testing of pyrazinamide susceptibility improves multidrug-resistant tuberculosis treatment outcomes: a prospective cohort study.

The current treatment for multidrug-resistant tuberculosis (MDR-TB) takes a lengthy period of 18-24 months and has a poor cure rate of 50-60%. A multicenter, prospective cohort study was conducted to assess the role of testing for molecular susceptibility to pyrazinamide (PZA) in optimising treatment for MDR-TB.We assigned 76 patients to an optimised molecular susceptibility group and 159 patients to a regular treatment group where PZA susceptibility was not determined. Of these patients, 152 were matched after propensity score matching (76 in the optimised group and 76 in the regular group). Treatment success rate was measured in the propensity-matched cohort as the primary outcome.Patients in the optimised group achieved a higher treatment success rate than those in the regular group (76.3% versus 55.3%, p=0.006). Of 51 patients with isolates that were susceptible to PZA and who were receiving a 12-month regimen, 42 (82.4%) were treated successfully. The optimised group showed faster culture conversion than the regular group (p=0.024). After exclusion of pre-extensively drug-resistant TB (pre-XDR-TB), the treatment outcome in the optimised group was still better than the regular group (83.1% versus 62.1%, p=0.009).Introducing molecular susceptibility testing for PZA improved the treatment outcomes for MDR-TB without the use of new drugs. Introducing PZA for patients with PZA-susceptible (PZA-S) MDR-TB allows the current regimen to be shortened to 12 months with comparable success rates to the World Health Organization (WHO) recommended shorter regimen.

Optimized Background Regimen for Treatment of Active Tuberculosis with the Next-Generation Benzothiazinone Macozinone (PBTZ169).

The efficacy of the standardized four-drug regimen (comprising isoniazid, rifampin, pyrazinamide, and ethambutol) for the treatment of tuberculosis (TB) is menaced by the emergence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) strains of Mycobacterium tuberculosis Intensive efforts have been made to develop new antibiotics or to repurpose old drugs, and several of these are currently being evaluated in clinical trials for their antitubercular activity. Among the new candidate drugs is macozinone (MCZ), the piperazine-containing benzothiazinone PBTZ169, which is currently being evaluated in phase I/II clinical trials. Here, we determined the in vitro and in vivo activity of MCZ in combination with a range of anti-TB drugs in order to design a new regimen against active TB. Two-drug combinations with MCZ were tested against M. tuberculosis using checkerboard and CFU enumeration after drug exposure assays. MCZ was observed to have no interactions with all first- and second-line anti-TB drugs. At the MIC of each drug, MCZ with either bedaquiline (BDQ), clofazimine (CLO), delamanid (DMD), or sutezolid (STZ) reduced the bacterial burden by 2 logs compared to that achieved with the drugs alone, indicating synergism. MCZ also displayed synergism with clomiphene (CLM), a potential inhibitor of the undecaprenyl pyrophosphate synthase (UppS) in mycobacteria. For all the other drugs tested in combination with MCZ, no synergistic activity was observed. Neither antagonism nor increased cytotoxicity was found for most combinations, suggesting that MCZ could be added to different TB treatment regimens without any significant adverse effects.

Characterizing the Effects of Glutathione as an Immunoadjuvant in the Treatment of Tuberculosis.

Mycobacterium tuberculosis is the etiological agent that is responsible for causing tuberculosis (TB), which continues to affect millions of people worldwide, and the rate of resistance of M. tuberculosis to antibiotics is ever increasing. We tested the synergistic effects of N-acetyl cysteine (NAC; the precursor molecule for the synthesis of glutathione [GSH]) and individual first-line antibiotics typically given for the treatment of TB, such as isoniazid (INH), rifampin (RIF), ethambutol (EMB), and pyrazinamide (PZA), to improve the ability of macrophages to control intracellular M. tuberculosis infection. GSH, a pleiotropic antioxidant molecule, has previously been shown to display both antimycobacterial and immune-enhancing effects. Our results indicate that there was not only an increase in beneficial immunomodulatory effects but also a greater reduction in the intracellular viability of M. tuberculosis when macrophages were treated with the combination of antibiotics (INH, RIF, EMB, or PZA) and NAC.

Evaluation of a novel line probe assay to detect resistance to pyrazinamide, a key drug used for tuberculosis treatment.

OBJECTIVES: The development of rapid molecular diagnostic assays for pyrazinamide (PZA) resistance is considered technically challenging as mutations are highly diverse, scattered along the full length of the pncA gene and not all are associated with PZA resistance. We evaluated the performance of the novel Genoscholar PZA-TB II line probe assay (PZA-LPA2; NIPRO Corporation, Japan). METHODS: To evaluate the applicability of the PZA-LPA2 in clinical settings, we compared the performance of the PZA-LPA2 to a composite reference standard pncA Sanger and Illumina sequencing plus phenotypic susceptibility testing on a panel of 87 Mycobacterium tuberculosis isolates from World Health Organization (WHO) drug resistance surveys, harbouring mutations previously classified as associated or not associated with resistance according to data from peer-reviewed literature. In addition, the PZA-LPA2 was challenged against a selection of isolates with lineage-specific and non-resistance-associated mutations, for which the frequency among clinical isolates is unknown, and tested directly on 59 sputum extracts. RESULTS: For the survey isolates, the PZA-LPA2 reached an overall agreement with the composite reference of 97.6% (80/82) or 94.3% (82/87) excluding or including heteroresistance, respectively. The PZA-LPA2 failed on 8.5% (5/59) of clinical samples; among valid results, 100% (14/14) sensitivity and 100% (7/7) specificity was reached relative to pncA Sanger sequencing. CONCLUSIONS: The PZA-LPA2 represents a valid and rapid alternative for indirect PZA susceptibility testing. Preliminary findings on clinical samples show promise for direct testing. Further studies are needed to assess the clinical risk of missing heteroresistance and falsely detecting lineage-specific, silent and nonassociated mutations.

Ureidopyrazine Derivatives: Synthesis and Biological Evaluation as Anti-Infectives and Abiotic Elicitors.

Tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb) has become a frequently deadly infection due to increasing antimicrobial resistance. This serious issue has driven efforts worldwide to discover new drugs effective against Mtb. One research area is the synthesis and evaluation of pyrazinamide derivatives as potential anti-TB drugs. In this paper we report the synthesis and biological evaluations of a series of ureidopyrazines. Compounds were synthesized by reacting alkyl/aryl isocyanates with aminopyrazine or with propyl 5-aminopyrazine-2-carboxylate. Reactions were performed in pressurized vials using a CEM Discover microwave reactor with a focused field. Purity and chemical structures of products were assessed, and the final compounds were tested in vitro for their antimycobacterial, antibacterial, and antifungal activities. Propyl 5-(3-phenylureido)pyrazine-2-carboxylate (compound 4, MICMtb = 1.56 µg/mL, 5.19 µM) and propyl 5-(3-(4-methoxyphenyl)ureido)pyrazine-2-carboxylate (compound 6, MICMtb = 6.25 µg/mL, 18.91 µM) had high antimycobacterial activity against Mtb H37Rv with no in vitro cytotoxicity on HepG2 cell line. Therefore 4 and 6 are suitable for further structural modifications that might improve their biological activity and physicochemical properties. Based on the structural similarity to 1-(2-chloropyridin-4-yl)-3-phenylurea, a known plant growth regulator, two selected compounds were evaluated for similar activity as abiotic elicitors.

Concentration-Dependent Antagonism and Culture Conversion in Pulmonary Tuberculosis.

BACKGROUND: There is scant evidence to support target drug exposures for optimal tuberculosis outcomes. We therefore assessed whether pharmacokinetic/pharmacodynamic (PK/PD) parameters could predict 2-month culture conversion. METHODS: One hundred patients with pulmonary tuberculosis (65% human immunodeficiency virus coinfected) were intensively sampled to determine rifampicin, isoniazid, and pyrazinamide plasma concentrations after 7-8 weeks of therapy, and PK parameters determined using nonlinear mixed-effects models. Detailed clinical data and sputum for culture were collected at baseline, 2 months, and 5-6 months. Minimum inhibitory concentrations (MICs) were determined on baseline isolates. Multivariate logistic regression and the assumption-free multivariate adaptive regression splines (MARS) were used to identify clinical and PK/PD predictors of 2-month culture conversion. Potential PK/PD predictors included 0- to 24-hour area under the curve (AUC0-24), maximum concentration (Cmax), AUC0-24/MIC, Cmax/MIC, and percentage of time that concentrations persisted above the MIC (%TMIC). RESULTS: Twenty-six percent of patients had Cmax of rifampicin <8 mg/L, pyrazinamide <35 mg/L, and isoniazid <3 mg/L. No relationship was found between PK exposures and 2-month culture conversion using multivariate logistic regression after adjusting for MIC. However, MARS identified negative interactions between isoniazid Cmax and rifampicin Cmax/MIC ratio on 2-month culture conversion. If isoniazid Cmax was <4.6 mg/L and rifampicin Cmax/MIC <28, the isoniazid concentration had an antagonistic effect on culture conversion. For patients with isoniazid Cmax >4.6 mg/L, higher isoniazid exposures were associated with improved rates of culture conversion. CONCLUSIONS: PK/PD analyses using MARS identified isoniazid Cmax and rifampicin Cmax/MIC thresholds below which there is concentration-dependent antagonism that reduces 2-month sputum culture conversion.

Optimizing treatment outcome of first-line anti-tuberculosis drugs: the role of therapeutic drug monitoring.

INTRODUCTION: Tuberculosis (TB) remains one of the world's deadliest communicable diseases. Although cure rates of the standard four-drug (rifampicin, isoniazid, pyrazinamide, ethambutol) treatment schedule can be as high as 95-98 % under clinical trial conditions, success rates may be much lower in less well resourced countries. Unsuccessful treatment with these first-line anti-TB drugs may lead to the development of multidrug resistant and extensively drug resistant TB. The intrinsic interindividual variability in the pharmacokinetics (PK) of the first-line anti-TB drugs is further exacerbated by co-morbidities such as HIV infection and diabetes. METHODS: Therapeutic drug monitoring has been proposed in an attempt to optimize treatment outcome and reduce the development of drug resistance. Several studies have shown that maximum plasma concentrations (C max), especially of rifampicin and isoniazid, are well below the proposed target C max concentrations in a substantial fraction of patients being treated with the standard four-drug treatment schedule, even though treatment's success rate in these studies was typically at least 85 %. DISCUSSION: The proposed target C max concentrations are based on the concentrations of these agents achieved in healthy volunteers and patients receiving the standard doses. Estimation of C max based on one or two sampling times may not have the necessary accuracy since absorption rate, especially for rifampicin, may be highly variable. In addition, minimum inhibitory concentration (MIC) variability should be taken into account to set clinically meaningful susceptibility breakpoints. Clearly, there is a need to better define the key target PK and pharmacodynamic (PD) parameters for therapeutic drug monitoring (TDM) of the first-line anti-TB drugs to be efficacious, C max (or area under the curve (AUC)) and C max/MIC (or AUC/MIC). CONCLUSION: Although TDM of first-line anti-TB drugs has been successfully used in a limited number of specialized centers to improve treatment outcome in slow responders, a better characterization of the target PK and/or PK/PD parameters is in our opinion necessary to make it cost-effective.

Crystal structures of the pyrazinamide-p-aminobenzoic acid (1/1) cocrystal and the transamidation reaction product 4-(pyrazine-2-carboxamido)benzoic acid in the molten state.

The synthesis of pharmaceutical cocrystals is a strategy to enhance the performance of active pharmaceutical ingredients (APIs) without affecting their therapeutic efficiency. The 1:1 pharmaceutical cocrystal of the antituberculosis drug pyrazinamide (PZA) and the cocrystal former p-aminobenzoic acid (p-ABA), C7H7NO2·C5H5N3O, (1), was synthesized successfully and characterized by relevant solid-state characterization methods. The cocrystal crystallizes in the monoclinic space group P21/n containing one molecule of each component. Both molecules associate via intermolecular O-H···O and N-H···O hydrogen bonds [O···O = 2.6102 (15) Šand O-H···O = 168.3 (19)°; N···O = 2.9259 (18) Šand N-H···O = 167.7 (16)°] to generate a dimeric acid-amide synthon. Neighbouring dimers are linked centrosymmetrically through N-H···O interactions [N···O = 3.1201 (18) Šand N-H···O = 136.9 (14)°] to form a tetrameric assembly supplemented by C-H···N interactions [C···N = 3.5277 (19) Šand C-H···N = 147°]. Linking of these tetrameric assemblies through N-H···O [N···O = 3.3026 (19) Šand N-H···O = 143.1 (17)°], N-H···N [N···N = 3.221 (2) Šand N-H···N = 177.9 (17)°] and C-H···O [C···O = 3.5354 (18) Šand C-H···O = 152°] interactions creates the two-dimensional packing. Recrystallization of the cocrystals from the molten state revealed the formation of 4-(pyrazine-2-carboxamido)benzoic acid, C12H9N3O3, (2), through a transamidation reaction between PZA and p-ABA. Carboxamide (2) crystallizes in the triclinic space group P1̅ with one molecule in the asymmetric unit. Molecules of (2) form a centrosymmetric dimeric homosynthon through an acid-acid O-H···O hydrogen bond [O···O = 2.666 (3) Šand O-H···O = 178 (4)°]. Neighbouring assemblies are connected centrosymmetrically via a C-H···N interaction [C···N = 3.365 (3) Šand C-H···N = 142°] engaging the pyrazine groups to generate a linear chain. Adjacent chains are connected loosely via C-H···O interactions [C···O = 3.212 (3) Šand C-H···O = 149°] to generate a two-dimensional sheet structure. Closely associated two-dimensional sheets in both compounds are stacked via aromatic π-stacking interactions engaging the pyrazine and benzene rings to create a three-dimensional multi-stack structure.

Pantothenate and pantetheine antagonize the antitubercular activity of pyrazinamide.

Pyrazinamide (PZA) is a first-line tuberculosis drug that inhibits the growth of Mycobacterium tuberculosis via an as yet undefined mechanism. An M. tuberculosis laboratory strain that was auxotrophic for pantothenate was found to be insensitive to PZA and to the active form, pyrazinoic acid (POA). To determine whether this phenotype was strain or condition specific, the effect of pantothenate supplementation on PZA activity was assessed using prototrophic strains of M. tuberculosis. It was found that pantothenate and other ß-alanine-containing metabolites abolished PZA and POA susceptibility, suggesting that POA might selectively target pantothenate synthesis. However, when the pantothenate-auxotrophic strain was cultivated using a subantagonistic concentration of pantetheine in lieu of pantothenate, susceptibility to PZA and POA was restored. In addition, we found that ß-alanine could not antagonize PZA and POA activity against the pantothenate-auxotrophic strain, indicating that the antagonism is specific to pantothenate. Moreover, pantothenate-mediated antagonism was observed for structurally related compounds, including n-propyl pyrazinoate, 5-chloropyrazinamide, and nicotinamide, but not for nicotinic acid or isoniazid. Taken together, these data demonstrate that while pantothenate can interfere with the action of PZA, pantothenate synthesis is not directly targeted by PZA. Our findings suggest that targeting of pantothenate synthesis has the potential to enhance PZA efficacy and possibly to restore PZA susceptibility in isolates with panD-linked resistance.

Evaluation of a biphasic media assay for pyrazinamide drug susceptibility testing of Mycobacterium tuberculosis.

OBJECTIVES: Pyrazinamide is a key first-line tuberculosis drug. Reliable drug susceptibility testing (DST) data are of clinical importance, but in vitro testing is challenging since the activity of pyrazinamide is pH sensitive. The BACTEC MGIT 960 is considered the principal reference technique, but Wayne's test is an alternative, although it may be difficult to interpret. A further alternative is the use of a biphasic media assay (BMA). The objective of this work was to evaluate the BMA against the MGIT method and with screening of pncA gene mutations. METHODS: Twenty strains were inoculated in tubes containing 2 mL of Löwenstein-Jensen (LJ) medium and 2 mL of semi-solid Kirchner medium with a critical concentration of 66 mg/L pyrazinamide at a pH of 5.2 or 5.5, incubated for 2 weeks and visually read. The results obtained were compared with MGIT 960 and DNA sequencing. RESULTS: Results were obtained in duplicate for 19 strains. One strain failed to grow on two occasions and only one result was available. Reproducibility was 95%. Eleven of the 19 strains were susceptible to pyrazinamide, whereas 7 were resistant. One strain was susceptible initially and pyrazinamide resistant on repeat testing. At pH 5.5, two strains reported as susceptible at pH 5.2 gave resistant results. CONCLUSIONS: The BMA might serve as a reliable low-cost DST alternative for pyrazinamide, particularly in laboratories using locally made solid media for DST. Its major drawback is the time to result. A reliable and affordable test method for the detection of pyrazinamide resistance is needed, especially in settings where multidrug-resistant tuberculosis is increasing. Proficiency testing should be routinely introduced wherever pyrazinamide DST is performed.

The pyrazinamide susceptibility breakpoint above which combination therapy fails.

OBJECTIVES: To identify the pyrazinamide MIC above which standard combination therapy fails. METHODS: MICs of pyrazinamide were determined for Mycobacterium tuberculosis isolates, cultured from 58 patients in a previous randomized clinical trial in Cape Town, South Africa. The MICs were determined using BACTEC MGIT 960 for isolates that were collected before standard treatment with isoniazid, rifampicin, pyrazinamide and ethambutol commenced. Weekly sputum collections were subsequently made for 8 weeks in order to culture M. tuberculosis in Middlebrook broth medium. Classification and regression tree (CART) analysis was utilized to identify the pyrazinamide MIC predictive of sputum culture results at the end of pyrazinamide therapy. The machine learning-derived susceptibility breakpoints were then confirmed using standard association statistics that took into account confounders of 2 month sputum conversion. RESULTS: The pyrazinamide MIC range was 12.5 to >100 mg/L for the isolates prior to therapy. The epidemiological 95% cut-off value was >100 mg/L. The 2 month sputum conversion rate in liquid cultures was 26% by stringent criteria and 48% by less stringent criteria. CART analysis identified an MIC breakpoint of 50 mg/L, above which patients had poor sputum conversion rates. The relative risk of poor sputum conversion was 1.5 (95% CI: 1.2-1.8) for an MIC >50 mg/L compared with an MIC ≤ 50 mg/L. CONCLUSIONS: We propose a pyrazinamide susceptibility breakpoint of 50 mg/L for clinical decision making and for development of rapid susceptibility assays. This breakpoint is identical to that identified using computer-aided simulations of hollow fibre system output.

Pyrazinamide resistance, Mycobacterium tuberculosis lineage and treatment outcomes in San Francisco, California.

BACKGROUND: Pyrazinamide (PZA) is a first line agent for the treatment of active tuberculosis. PZA is also considered a potent companion drug for newer regimens under development. There are limited data on the demographic, clinical, and pathogen characteristics of PZA resistant tuberculosis. METHODS: Using a retrospective cohort study design, we evaluated all PZA resistant M. tuberculosis (M.tb) and M. bovis cases reported in San Francisco from 1991 to 2011. Demographic, clinical, and molecular data were analyzed. M.tb lineage was determined for all PZA resistant strains and compared to PZA susceptible strains. RESULTS: PZA resistance was identified in 1.8% (50 of 2,842) of mycobacterial isolates tested, corresponding to a case rate of 0.3 per 100,000 in the population. Monoresistant PZA infection was associated with the Hispanic population ([OR], 6.3; 95% [CI], 1.97-20.16) and 48% of cases were due to M. bovis. Infection with monoresistant PZA was also associated with extrapulmonary disease ([OR], 6.0; 95% [CI], 2.70-13.26). There was no statistically significant difference between treatment failure and mortality rates in patients infected with PZA monoresistance compared to pansusceptible controls (4% vs. 8%, p = 0.51), or those with PZA and MDR resistance (PZA-MDR) compared to MDR controls (18% vs. 29%, p = 0.40). PZA resistance was not associated with M.tb lineage. CONCLUSIONS: Across two decades of comprehensive epidemiologic data on tuberculosis in San Francisco County, PZA resistance was uncommon. PZA resistance caused predominantly extrapulmonary disease and was more common in Hispanics compared to other ethnicities, with nearly half the cases attributed to M. bovis. No association was found between PZA monoresistance and M.tb lineage. Treatment outcomes were not adversely influenced by the presence of PZA resistance.

Alkylamino derivatives of pyrazinamide: synthesis and antimycobacterial evaluation.

A series of pyrazinamide derivatives with alkylamino substitution was designed, synthesized and tested for their ability to inhibit the growth of selected mycobacterial, bacterial and fungal strains. The target structures were prepared from the corresponding 5-chloro (1) or 6-chloropyrazine-2-carboxamide (2) by nucleophilic substitution of chlorine by various non-aromatic amines (alkylamines). To determine the influence of alkyl substitution, corresponding amino derivatives (1a, 2a) and compounds with phenylalkylamino substitution were prepared. Some of the compounds exerted antimycobacterial activity against Mycobacterium tuberculosis H37Rv significantly better than standard pyrazinamide and corresponding starting compounds (1 and 2). Basic structure-activity relationships are presented. Only weak antibacterial and no antifungal activity was detected.

Enhancement of in vitro activity of tuberculosis drugs by addition of thioridazine is not reflected by improved in vivo therapeutic efficacy.

OBJECTIVES: Assessment of the activity of thioridazine towards Mycobacterium tuberculosis (Mtb), in vitro and in vivo as a single drug and in combination with tuberculosis (TB) drugs. METHODS: The in vitro activity of thioridazine as single drug or in combination with TB drugs was assessed in terms of MIC and by use of the time-kill kinetics assay. Various Mtb strains among which the Beijing genotype strain BE-1585 were included. In vivo, mice with TB induced by BE-1585 were treated with a TB drug regimen with thioridazine during 13 weeks. Therapeutic efficacy was assessed by the change in mycobacterial load in the lung, spleen and liver during treatment and 13 weeks post-treatment. RESULTS: In vitro, thioridazine showed a concentration-dependent and time-dependent bactericidal activity towards both actively-replicating and slowly-replicating Mtb. Thioridazine at high concentrations could enhance the activity of isoniazid and rifampicin, and in case of isoniazid resulted in elimination of mycobacteria and prevention of isoniazid-resistant mutants. Thioridazine had no added value in combination with moxifloxacin or amikacin. In mice with TB, thioridazine was poorly tolerated, limiting the maximum tolerated dose (MTD). The addition of thioridazine at the MTD to an isoniazid-rifampicin-pyrazinamide regimen for 13 weeks did not result in enhanced therapeutic efficacy. CONCLUSIONS: Thioridazine is bactericidal towards Mtb in vitro, irrespective the mycobacterial growth rate and results in enhanced activity of the standard regimen. The in vitro activity of thioridazine in potentiating isoniazid and rifampicin is not reflected by improved therapeutic efficacy in a murine TB-model.

Thioridazine pharmacokinetic-pharmacodynamic parameters "Wobble" during treatment of tuberculosis: a theoretical basis for shorter-duration curative monotherapy with congeners.

Phenothiazines are being repurposed for treatment of tuberculosis. We examined time-kill curves of thioridazine and first-line drugs against log-growth-phase and semidormant bacilli under acidic conditions and nonreplicating persistent Mycobacterium tuberculosis. While both the potency and the efficacy of first-line drugs declined dramatically as M. tuberculosis replication rates decreased, those of thioridazine improved. The mutation prevalence to 3 times the thioridazine MIC was <1 × 10(-11), better than for ≥2 first-line drugs combined. Hollow fiber system studies revealed that the relationship between sterilizing effect and pharmacodynamic indices (PDI) was characterized by an r(2) of 0.88 for peak/MIC, an r(2) of 0.47 for the area under the concentration-time curve (AUC) to MIC, and an r(2) of 0.14 for the cumulative percentage of a 24-h period that the drug concentration exceeds the MIC under steady-state pharmacokinetic conditions (%TMIC) at the end of the first week. However, the PDI linked to effect "wobbled" as the duration of therapy increased, so that by the fourth week the r(2) was 0.88 for AUC/MIC, 0.78 for %TMIC, and 0.72 for peak/MIC. This "wobble" has implications on general pharmacokinetic/pharmacodynamic theory, whereby efficacy is linked to only one of the three PDIs in deterministic models. The potency changed 8.9-fold from the first to the fourth weeks. The non-protein-bound AUC/MIC associated with maximal kill at the end of therapy was 50.53 (protein binding = 99.5%). This thioridazine exposure was calculated to extinguish all three M. tuberculosis metabolic populations in human lungs in only 42.9 days of monotherapy. However, this concentration exceeds the 2- to 8-mg/liter thioridazine concentration in serum known to be lethal to humans. Therefore, the way forward for phenothiazine monotherapy that also reduces therapy duration is via synthesis of less toxic congeners.

Impact of tuberculosis treatment on CD4 cell count, HIV RNA, and p24 antigen in patients with HIV and tuberculosis.

OBJECTIVES: To describe HIV RNA levels during tuberculosis (TB) infection in patients co-infected with TB and HIV. Moreover, to examine the p24 antigen profile during TB treatment. METHODS: We examined the changes in CD4 cell count, HIV RNA, and p24 levels during anti-tuberculous therapy in a group of TB/HIV-1 co-infected and HIV-untreated patients from Guinea-Bissau. RESULTS: A total of 365 TB patients were enrolled, of whom 76 were co-infected with HIV-1 and 19 were dually infected with HIV-1 + HIV-2. No significant changes in CD4, HIV RNA, or p24 levels were found during 8 months of TB treatment. HIV RNA levels correlated well with p24 (Spearman's R(2)=0.52, p<0.00001) and both markers were strong predictors of mortality. Initial HIV RNA levels correlated with a clinical TB severity index--the TBscore (Spearman's R(2)=0.23, p=0.02)--and the TBscore decreased dramatically during TB treatment although HIV RNA levels remained unchanged. CONCLUSION: We found no significant changes in CD4, HIV RNA, or p24 antigen levels during 8 months of TB treatment among TB/HIV co-infected individuals, who did not receive antiretroviral treatment. The markers were unaffected by a strong improvement in TBscore and all three markers showed predictive capacity for mortality risk.