The safety of pyrazinamide in pediatric drug-sensitive tuberculosis treatment: A real-world study.

Model-informed precision pyrazinamide dosing: The establishment of a population pharmacokinetic model repository for clinical decision support.

Antibacterial drug mechanisms have traditionally been examined through a drug-pathogen lens, with limited attention to host influences on drug activity. However, growing evidence suggests that the host environment is crucial for antibacterial efficacy. Pyrazinamide (PZA), a key component of modern tuberculosis therapy, exemplifies this complexity, exhibiting potent in vivo activity despite its inability to reduce Mycobacterium tuberculosis viability in standard in vitro culture. Here, using macrophage and murine infection models, we identify a critical role for CD4+ T cell-dependent cell-mediated immunity in PZA's antitubercular action. Using MHC class II knockout mice, we demonstrate that CD4 T-cell help is essential for PZA efficacy. While interferon gamma (IFN-γ) is required for PZA-mediated clearance of M. tuberculosis at extrapulmonary sites, bacterial reduction in the lungs occurs, independent of IFN-γ signaling. We show that PZA leverages cell-mediated immunity in part through activation of the oxidative burst. Our findings underscore the need to incorporate host factors into antibacterial drug evaluation and highlight potential avenues for host-directed therapies and adjunctive antibiotics in first- and second-line tuberculosis treatment.

Tuberculosis (TB), an infectious disease caused by Mycobacterium tuberculosis (M. tuberculosis), remains a major public health concern, particularly because of the increasing incidence of drug-resistant TB. In Republic of Korea, research on genes related to TB resistance is limited; therefore, in this study, we identified resistance-associated mutations in TB clinical isolates from Republic of Korea. We evaluated and compared phenotypic drug susceptibility testing (pDST) and genotypic drug susceptibility testing (gDST) using whole-genome sequencing (WGS) and targeted sequencing in 75 clinical M. tuberculosis isolates collected in Republic of Korea between 2005 and 2009. Specifically, we analyzed mutations associated with resistance against isoniazid (INH), rifampicin (RIF), moxifloxacin (MFX), pyrazinamide (PZA), pretomanid (PMD), delamanid (DLM), linezolid (LZD), and bedaquiline (BDQ) and compared them with those in the 2023 WHO mutation catalog. We detected resistance-associated mutations in 98.7% of INH- and RIF-resistant isolates, with a high degree of concordance between the pDST and gDST results for most drugs. However, PZA results were discrepant for 16 isolates. Our findings highlight the potential of WGS and targeted sequencing as powerful tools for diagnosing TB drug resistance and emphasize the need for further validation before their routine implementation in clinical settings.

Bovine tuberculosis (bTB) has a certain incidence on farms in China, but little is known about its prevalence and the pathogenic Mycobacterium species. With the increasing application of whole-genome sequencing (WGS), it is now possible to investigate the genomic diversity, lineage distribution, and drug resistance profiles of M. bovis at high resolution. In this study, we sequenced 29 Mycobacterium tuberculosis complex (MTBC) isolates from two farms in China and incorporated genomic data from 79 publicly available M. bovis isolates from cattle farms in China for a comprehensive genomic analysis. The majority of the 106 M. bovis isolates (63.2%, 67/106) belong to lineage La1.4. The dataset was screened for isolates of two known clonal complexes, Eu1 and Eu3, corresponding to La1.8.1 and La1.2, respectively. In silico spoligotyping revealed nine previously identified profiles, and the most common spoligotype in this study is SB1903. All isolates have the H57D mutation in the pncA gene associated with pyrazinamide (PZA) resistance. 16.0% (17/106) of isolates have resistance mutations to first- or second-line drugs other than PZA, with one isolate classified as pre-XDR-TB. The distribution of cluster strains suggests the possibility of recent transmission and outbreak in the two farms. It is noted that two M. tuberculosis isolates from tissue samples had detectable drug resistance mutations. The results of this study provide the basis for in-depth analysis of genomic population characteristics and drug resistance of M. bovis in China. It should be noted that close contact between humans and cattle increases the risk of M. tuberculosis transmission from humans to cattle in rural areas or developing countries with low levels of human-animal separation.

Genomic insights into pyrazinamide and fluoroquinolone resistance in multidrug-resistant tuberculosis in Khyber Pakhtunkhwa, Pakistan.

Background: Tuberculosis (TB) is still one of the most important causes of morbidity and mortality worldwide. The problem multiplies whenever there is multidrug-resistant TB (MDR-TB), due to Mycobacterium tuberculosis susceptibility to both Rifampicin and Isoniazid. Pyrazinamide (PZA) is often added to TB regimens if there is susceptibility; however, one adverse reaction to it is that it causes hyperuricemia. This study was conducted to investigate hyperuricemia induced by PZA among MDR-TB patients attending the Abu Ajja Teaching Hospital in the State of Khartoum, Sudan. Methods: A retrospective cross-sectional study was carried out among 84 MDR-TB patients who received treatment in the hospital’s MDR-TB ward during 2020-2021. Serum uric acid measurements (pre-PZA baseline values and months 1-5 post-PZA treatment initiation) were obtained by spectrophotometry. The data was analyzed with paired t-test to compare each group to the baseline values. A p-value < 0.05 was taken to be statistically significant. Results: Among the study population, 67.9% were male, and 48.8% were aged between 18 and 30 years. The majority (89.3%) were Sudanese, and 58.3% were classified as underweight. The mean baseline serum uric acid level prior to PZA therapy was 4.47 ± 1.17 mg/dL. Following the initiation of PZA, a progressive and statistically significant increase in serum uric acid was observed across all subsequent months: 8.67 ± 2.39 mg/dL (p < 0.001) in month 1, 8.94 ± 2.07 mg/dL (p < 0.001) in month 2, 9.04 ± 1.89 mg/dL (p < 0.001) in month 3, and 9.56 ± 1.85 mg/dL (p < 0.001) in month 4. By month 5, the mean serum uric acid level further increased to 9.69 ± 1.96 mg/dL (p < 0.001) compared with baseline values. Conclusion: In this group of patients with MDR-TB undergoing PZA-containing regimens, there was a statistically significant increase in the levels of uric acid in the blood during the initial five months of treatment. These results highlight the need to take uric acid levels into consideration during TB treatment regimens that include PZA, even in resource-limited centers.

Oral antituberculosis drugs (OAT) used repeatedly can negatively impact the liver. Natural compounds with high antioxidant potential could be used as hepatoprotective agents. This study aims to determine the hepatoprotective activity of the combination ethanolic leaf extract of Syzygium polyanthum (SPEE) and Moringa oleifera (MOEE) in rats administered with OAT. The hepatoprotective activity of the combination of SPE and MOE was determined by calculating the SGOT and SGPT levels of rats administered rifampicin (RMP), pyrazinamide (PZA), and isoniazid (INH) for 14 days. An experimental study using eight groups: standard control (CMCM-Na), negative control treatment with OAT (RMP 100 mg/kg BW; PZA: 252 mg/kg BW INH 50 mg/kg BW), positive control (OAT + Curcumin 100 mg/kg BW), OAT+SPEE 150 mg/kg BW group, OAT+MOEE 400 mg/kg BW group, and 3 group OAT + combination SPEE and MOEE (25%: 75%; 50%:50%; 75%:25%). SGPT & SGOT levels were determined using spectrophotometry with analytical methods using specific SGPT & SGOT reagent kits at 340 nm. After 14 days of OAT administration, SGOT levels increased by 1.3-fold, and SGPT levels increased by 1.8-fold compared to baseline levels. This considerable increase is still considered mild hepatotoxicity. The combination of SPEE: MOEE (25%:75%) was most effective in decreasing SGOT levels by 23.5%, or 1.3-fold, whereas the combination treatment of SPEE: MOEE (75%; 25%) was most effective in reducing SGPT levels by 51.56%, or 2-fold. The ratio value of the decrease in SGOT and SGPT levels from single SPEE and MOEE and the combination of both (p>0.05). The combination treatment of SPEE and MOEE in rats administered OAT showed an additive effect and could evolve as a hepatoprotective substance.

Pyrazinamide (PZA), an FDA-approved antibiotic, was investigated for potential use as a hyperpolarized MRI contrast agent. PZA was readily hyperpolarized via parahydrogen-based NMR Signal Amplification By Reversible Exchange (SABRE) at ∼5.5 mT, allowing 1H polarization and relaxation dynamics to be characterized. More importantly, performing SABRE hyperpolarization in the microtesla regime (i.e., SABRE in SHield Enables Alignment Transfer to Heteronuclei, SABRE-SHEATH) allowed direct detection of enhanced 15N NMR and corresponding polarization/relaxation dynamics to be characterized in this system for the first time, despite the low natural abundance of 15N. Initial experiments detected at 9.4 T following SABRE-SHEATH at 0.2 μT permitted observation of a single 15N resonance at 332.7 ppm (tentatively assigned to the ring N site meta to the amide group, based on the 1H enhancement pattern, suggesting the less sterically hindered N site dominates this substrate's interaction with the Ir-based SABRE catalyst). Direct SABRE-SHEATH 15N hyperpolarization using a more optimized setup (with respect to mixing field, temperature, p-H2 flow rate, etc.) that is directly coupled to a 1.4 T benchtop NMR system resulted in improved detection sensitivity, including 15N enhancements of >140,000-fold for the primary 15N resonance (corresponding to a polarization of ∼7% with substrate concentration of over 100 mM). This effort also yielded a 15N T1 measurement for PZA of over 2 min at 1.4 T. Finally, the potential utility of hyperpolarized PZA as a contrast agent was also demonstrated via quantitative 1H MRI studies performed using a low-field (64 mT) portable "point-of-care" clinical scanner.

CwlM, identified as an N-acetylmuramoyl-l-alanine amidase, plays crucial roles in the synthesis and remodeling of peptidoglycan in mycobacteria. This protein also appears to participate in both drug susceptibility and tolerance mechanisms within these organisms. In our study, we employed CRISPR interference (CRISPRi) to deplete CwlM in Mycobacterium smegmatis (M. smegmatis) and examined the resulting effects on the susceptibility of mycobacteria to first-line anti-tuberculosis drugs, including isoniazid (INH), rifampicin (RIF), pyrazinamide (PZA), and ethambutol (EMB), as well as the β-lactams cefoxitin and imipenem. Our findings revealed that CwlM depletion increased the susceptibility of the bacterium to RIF, EMB, cefoxitin, and imipenem, while tolerance was heightened against INH and PZA. The enhanced antibiotic susceptibility can primarily be attributed to increased permeability of the bacterial cell wall. Conversely, the observed tolerance to INH might be ascribed to elevated expression of the amidase known as hydrazidase along with its LuxR-type regulator. Furthermore, several genes associated with peptidoglycan synthesis appeared to correlate with increased expression levels of either hydrazidase or its LuxR-type regulator. Collectively, these findings indicate that CwlM depletion significantly influences the susceptibility of M. smegmatis towards certain anti-tuberculosis drugs and may be implicated in drug susceptibility and tolerance mechanisms in M. smegmatis.

Pyrazinamide (PZA) is a cornerstone drug in tuberculosis (TB) treatment with a strong bactericidal activity in vivo on both actively and non-replicating bacterial subpopulations. Yet the precise mode of action of its active form, pyrazinoic acid (HPOA), remains unclear.In this study, we comprehensively explore and challenge the two major and conflicted models of PZA mode of action. The pH-dependent model, where the drug is mostly effective at acidic pH by acidifying Mycobacterium tuberculosis (Mtb) cytosol, and the PanD-dependent model where PZA’s active form targets the aspartate decarboxylase PanD, therefore depleting pantothenate and subsequently coenzyme A (CoA) levels regardless of the surrounding pH.By combining standard antimicrobial susceptibility testing at various pH with fluorescence-based live recording of Mtb intrabacterial pH, we demonstrate that PZA kills Mtb by decreasing intrabacterial pH, independently of pantothenate levels. Comparative studies between a prototrophic Mtb strain and a pantothenate auxotrophic mutant lacking the panCD locus confirmed that PZA bactericidal activity is primarily driven by pH and its ability to acidify Mtb cytosol, independently of the aspartate decarboxylase PanD. Bio-electrophysiology experiments revealed that acidic pH promotes the conversion of the pyrazinoate anion POA− into HPOA which in turn acts as conventional weak acid that facilitates membrane permeation and cytosolic acidification. Finally, using custom-based culture media, we demonstrate that PZA displays heterogeneous efficacy according to the media composition, therefore proposing a revisited biological model that might explain the discrepancies around PZA’s unique mode of action.Overall, this work constitutes the first comprehensive side-by-side investigation of the two models and univocally supports a pH-dependent mechanism of action underlying PZA sterilizing activity, providing new insights for the development of more effective PZA-like drugs.

Antibacterial drug mechanisms have traditionally been examined through a drug-pathogen lens, often overlooking the host’s role in shaping drug activity. However, growing evidence suggests that the host environment is crucial for antibacterial efficacy. Pyrazinamide (PZA), a key component of modern tuberculosis therapy, exemplifies this complexity—exhibiting potent in vivo activity despite its inability to reduce Mycobacterium tuberculosis viability in standard in vitro culture. Here, using macrophage and murine infection models, we identify a critical role for host cell-mediated immunity in PZA’s antitubercular action. Through the use of MHC II knockout mice, we demonstrate that CD4 T cell help is essential for PZA efficacy. Notably, while IFN-γ is required for PZA-mediated clearance of M. tuberculosis at extrapulmonary sites, bacterial reduction in the lungs occurs independently of IFN-γ signaling. Additionally, we show that PZA leverages cell-mediated immunity in part through activation of the oxidative burst. Our findings underscore the need to incorporate host factors into antibacterial drug evaluation and highlight potential avenues for host-directed therapies and adjunctive antibiotics in first- and second-line tuberculosis treatment.

Mitigating unfavourable treatment outcomes and acquired rifampicin resistance in isoniazid-resistant tuberculosis: the role of fluoroquinolone.

ObjectiveMultidrug-resistant tuberculosis (MDR-TB) remains a major public health challenge in China. Hainan, China’s largest tropical island, possesses distinct socio-geographical features. However, the drug resistance patterns and molecular epidemiology of MDR-TB in this region have not been fully elucidated. This study aimed to assess the correlation between drug resistance genotypes and phenotypic resistance levels in multidrug-resistant Mycobacterium tuberculosis (MDR-MTB) strains collected from Hainan Island, using whole-genome sequencing (WGS) and phenotypic drug susceptibility testing (DST).MethodsMDR-MTB strains isolated from patients on Hainan Island (2019–2021) were analyzed. Minimum inhibitory concentrations (MIC) for 15 anti-TB drugs were determined by broth microdilution (BMD). Whole-genome sequencing (WGS) was performed using Illumina NovaSeq 6000. Genotypic resistance was predicted via TB-Profiler, and correlations between resistance mutations and MIC levels were assessed.ResultsA total of 209 MDR-MTB strains were analyzed. Strains of lineage 2.2 exhibited significantly higher resistance to ethambutol (EMB) compared to non-lineage 2 strains (P < 0.05). The sensitivity of WGS in predicting resistance to first-line drugs isoniazid (INH), rifampicin (RIF), EMB, and pyrazinamide (PZA) was 94.7%, 99.0%, 96.5%, and 80.8%, respectively. However, specificity for EMB and PZA was lower at 60.2% and 79.4%. WGS also demonstrated high sensitivity and specificity (> 95%) for second-line injectable aminoglycosides (amikacin [AMK], capreomycin [CPM], and kanamycin [KM]), but sensitivity for other second-line drugs except for fluoroquinolone drug moxifloxacin (MOX, 94.4%) was below 80.0%. Notably, mutations in katG_S315T, rpoB_S450L, and gyrA_D94G were strongly associated with high-level resistance, while mutations in fabG1, ahpC, embA promoters, and gyrA at codon 90 were linked to low-level resistance.ConclusionsThis study quantitatively demonstrates the relationship between specific drug resistance genotypes and resistance levels. It is the first to characterize the regional resistance spectrum of MDR-MTB strains on Hainan Island. These findings offer a novel foundation for MIC-based dose adjustment and the optimization of treatment strategies in this region. Trial registrationMR-46–23-020530. Date of registration:2023–07-03.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12879-025-11312-8.

A five-step plan is presented to propose an HPLC method with the aid of the fishbone diagram to balance efficiency and sustainability. The five steps include method development, method optimization, method validation, greenness assessment, and sustainability profiling. A sensitive, accurate, robust, and rapid HPLC method has been developed for simultaneous determination and separation of anti-tubercular medications: rifampicin (RIF), isoniazid (INH), and pyrazinamide (PYR) in a fixed dose combination (FDC). The separation was attained by using a stationary phase Kromasil C18 column (250mm, 4.6,5 µm), accompanied by a gradient mode of the mobile phase in which the first solution was made up by using acetonitrile and phosphate buffer in a ratio of 4:96 v/v, and the second solution consisted of phosphate buffer and acetonitrile in ratio 45:55 v/v. The method was optimized by applying a fractional factorial design (22), where the flow rate was set at 1.5mL/min, the injection volume was 10 µL, and the detection was performed at 238nm at a temperature of 35°C. The retention time for INH, PYR, and RIF were 3.82, 5.36, and 8.72min (±0.02), respectively. The method was validated per the ICH guidelines, and the proposed method was found to be accurate, robust, precise, and selective. A design of experiment was established to assess the robustness of the study, where the obtained results proved that the developed method is robust. Two greenness tools, namely moGAPI and AGREE, were used to evaluate the method's greenness. In addition, the method was subjected to sustainability profiling using the EVG framework, where the suggested method was compared to other reported methods. The proposed method was found to be more sustainable and green, with a low negative impact on the environment.

Pyrazinamide (PZA) is a cornerstone of first-line antitubercular drug therapy and is unique in its ability to kill nongrowing populations of Mycobacterium tuberculosis through disruption of coenzyme A (CoA) metabolism. Unlike other drugs, PZA action is conditional and requires potentiation by host-relevant environmental stressors, such as low pH and nutrient limitation. Despite its pivotal role in tuberculosis therapy, the durability of this crucial drug is challenged by the emergent spread of drug resistance. To advance drug discovery efforts, we characterized the activity of a more potent PZA analog, morphazinamide (MZA). Here, we demonstrate that like PZA, MZA acts in part through impairment of CoA metabolism. Unexpectedly, we find that, in contrast to PZA, MZA does not require potentiation and maintains bactericidal activity against PZA-resistant strains due to an additional mechanism involving aldehyde release. Further, we find that the principal mechanism for resistance to the aldehyde component is through promoter mutations that increase expression of the mycothiol oxidoreductase MscR. Our findings reveal a dual-action synergistic mechanism of MZA that results in a faster kill rate and a higher barrier to resistance. These observations provide new insights for the discovery of improved therapeutic approaches for addressing the growing problem of drug-resistant tuberculosis.

Pyrazinamide (PZA) is a key component of tuberculosis treatment, with drug resistance (PZAR) primarily related to pncA mutations. However, discordance between phenotypic resistance and conventional pncA-based molecular diagnostics challenges diagnostic accuracy. This study investigates discrepancies between phenotypic and genotypic resistance profiles among Mycobacterium tuberculosis (Mtb) clinical isolates. Fifty-three Mtb isolates from Guangzhou Chest Hospital were tested for PZA resistance using the BACTEC MGIT 960 system and PZase activity assay. Thirty-one phenotypically PZAR strains were genetically assessed by Sanger sequencing of PZAR-associated customary genes. Five pncA-wild-type PZAR strains were investigated through whole-genome sequencing. ClpC1P1P2 activity was evaluated by proteolytic degradation assay. Notably, 26/31 of the PZAR strains harbored mutations in pncA and/or its upstream region, aligning PZase activity and phenotypic profiles. However, five PZAR strains lacked pncA mutations. The WGS of five discordant strains revealed four novel mutations (Gly58Ser, Val63Ala, Ala567Val, and Pro796Leu) across ClpC1 domains. Incorporating clpC1 mutations improved molecular diagnostic sensitivity and accuracy from 48.3% and 69.8% (pncA alone) to 100%. This is the first report from southern China that identifies novel clpC1 mutations in wild-type pncA PZAR Mtb isolates. Our findings underscore the limitations of pncA-targeted diagnostics and support the integration of WGS and clpC1 analysis in molecular diagnostics to prevent false-negative diagnoses and improve clinical outcomes.

Flexible and dispersible paediatric oral formulations produced via extrusion spheronisation for the treatment of tuberculosis.

Selective crystallization of pyrazinamide polymorphs in supramolecular gels: Synergistic selectivity by mimetic gelator and solvent.

Background/Objectives: Isoniazid (INH) and pyrazinamide (PZA) are first-line drugs used to treat tuberculosis (TB), but their use is generally contraindicated in patients with porphyria, a group of metabolic disorders caused by defects in the heme biosynthetic pathway. To investigate the basis for these contraindications, we compared the effects of INH and PZA on the heme biosynthetic pathway in mouse liver. Method: We investigated the hepatic expression and activity of the key enzymes involved in the heme biosynthetic pathway, including aminolevulinic acid synthase 1 (Alas1) and ferrochelatase (Fech). Additionally, we employed a metabolomic approach to analyze liver and fecal samples from the mice treated with INH or PZA. Result: We found that INH, but not PZA, significantly upregulated the expression and activity of Alas1, the rate-limiting enzyme in heme biosynthesis, while concurrently downregulating Fech, which converts protoporphyrin IX (PPIX) to heme. These changes resulted in the accumulation of the toxic intermediate aminolevulinic acid (ALA) and PPIX in the liver of INH-treated mice. In contrast, PZA had no measurable effect on the expression or function of Alas1 or Fech. Conclusions: These findings provide mechanistic insight into INH-induced porphyria exacerbation and suggest that PZA may not carry the same risk, challenging its current contraindication.