Analysis of unsuccessful tests and the effect of prolonged clinical sample preprocessing in the GeneXpert MTB/RIF assay.

BACKGROUND: The GeneXpert MTB/RIF (Xpert) assay is a widely used technology for detecting Mycobacterium tuberculosis (MTB) in clinical samples. However, the study on the failure of the Xpert assay during routine implementation and its potential solutions is limited. METHODS: We retrospectively analyzed the records of unsuccessful tests in the Xpert and the GeneXpert MTB/RIF Ultra (Ultra) assays between April 2017 and April 2021 at the Shanghai Public Health Clinical Center. To further investigate the effect of prolonged preprocessing on clinical sputum, an additional 120 sputum samples were collected for Xpert testing after 15 min, 3 h, and 6 h preprocessing. The analysis was performed by SPSS version 19.0 software. RESULTS: A total of 11,314 test records were analyzed, of which 268 (2.37%) had unsuccessful test results. Among these, 221 (1.95%) were reported as "Error", 43 (0.38%) as "Invalid", and 4 (0.04%) as "No result". The most common clinical specimen for Xpert tests was sputum, accounting for 114 (2.17%) unsuccessful tests. The failure rate of urine specimens was lower than that of sputum (OR = 0.12, 95% CI: 0.02-0.88, χ2 = 6.22, p = 0.021). In contrast, the failure rate of stool specimens was approximately twice as high as that of sputum (OR = 1.93, 95% CI: 1.09-3.40, χ2 = 5.35, p = 0.014). In the prolonged preprocessing experiment, 102 cases (85%) yielded consistent results in Xpert tests. Furthermore, 7 cases (5.83%) detected an increase in MTB load, 8 cases (6.67%) detected a decrease in MTB load, and 3 cases (2.5%) yielded incongruent results in MTB and rifampicin resistance detection. CONCLUSIONS: The primary cause of unsuccessful tests in the Xpert assay was reported as "Error". Despite varying failure rates depending on the samples, the Xpert assay can be applied to extrapulmonary samples. For paucibacillary specimens, retesting the remaining preprocessed mixture should be carefully considered.

Identification of gene targets that potentiate the action of rifampicin on Mycobacterium bovis BCG.

Tuberculosis (TB) caused by bacteria of the Mycobacterium tuberculosis complex remains one of the most important infectious diseases of mankind. Rifampicin is a first line drug used in multi-drug treatment of TB, however, the necessary duration of treatment with these drugs is long and development of resistance is an increasing impediment to treatment programmes. As a result, there is a requirement for research and development of new TB drugs, which can form the basis of new drug combinations, either due to their own anti-mycobacterial activity or by augmenting the activity of existing drugs such as rifampicin. This study describes a TnSeq analysis to identify mutants with enhanced sensitivity to sub-minimum inhibitory concentrations (MIC) of rifampicin. The rifampicin-sensitive mutants were disrupted in genes of a variety of functions and the majority fitted into three thematic groups: firstly, genes that were involved in DNA/RNA metabolism, secondly, genes involved in sensing and regulating mycobacterial cellular systems, and thirdly, genes involved in the synthesis and maintenance of the cell wall. Selection at two concentrations of rifampicin (1/250 and 1/62 MIC) demonstrated a dose response for mutants with statistically significant sensitivity to rifampicin. The dataset reveals mechanisms of how mycobacteria are innately tolerant to and initiate an adaptive response to rifampicin; providing putative targets for the development of adjunctive therapies that potentiate the action of rifampicin.

A multi-AS-PCR-coupled CRISPR/Cas12a assay for the detection of ten single-base mutations.

Single-nucleotide polymorphism (SNP) detection is critical for diagnosing diseases, and the development of rapid and accurate diagnostic tools is essential for treatment and prevention. Allele-specific polymerase chain reaction (AS-PCR) is widely used for detecting SNPs with multiplexing capabilities, while CRISPR-based technologies provide high sensitivity and specificity in targeting mutation sites through specific guide RNAs (gRNAs). In this study, we have integrated the high sensitivity and specificity of CRISPR technology with the multiplexing capabilities of AS-PCR, achieving the simultaneous detection of ten single-base mutations. As for Multi-AS-PCR, our research identified that competitive inhibition of primers targeting the same loci, coupled with divergent amplification efficiencies of these primers, could result in diminished amplification efficiency. Consequently, we adjusted and optimized primer combinations and ratios to enhance the amplification efficacy of Multi-AS-PCR. Finally, we successfully developed a novel nested Multi-AS-PCR-Cas12a method for multiplex SNPs detection. To evaluate the clinical utility of this method in a real-world setting, we applied it to diagnose rifampicin-resistant tuberculosis (TB). The limit of detection (LoD) for the nested Multi-AS-PCR-Cas12a was 102 aM, achieving sensitivity, specificity, positive predictive value, and negative predictive value of 100 %, 93.33 %, 90.00 %, and 100 %, respectively, compared to sequencing. In summary, by employing an innovative design that incorporates a universal reverse primer alongside ten distinct forward allele-specific primers, the nested Multi-AS-PCR-Cas12a technique facilitates the parallel detection of ten rpoB gene SNPs. This method also holds broad potential for the detection of drug-resistant gene mutations in infectious diseases and tumors, as well as for the screening of specific genetic disorders.

[Evaluation of molecular diagnosis of tuberculosis and resistance to rifampicin with GeneXpert® MTB/RIF in Algeria]. / Évaluation du GeneXpert® MTB/RIF dans le diagnostic moléculaire de la tuberculose et de la résistance à la rifampicine en Algérie.

Objective: 1) To evaluate the contribution of the GeneXpert® MTB/RIF (GX) test in the diagnosis of pulmonary and extra-pulmonary tuberculosis compared to culture. 2) To compare the rifampicin results resistance obtained by GX with the phenotypic sensitivity test. Materials and methods: Retrospective study carried out over a period of five years, from May 2017 to June 2022 at the microbiology laboratory of the Central army Hospital Mohamed Seghir Nekkache, Algiers (Algeria). The pulmonary and extrapulmonary clinical specimens were collected, cultivated, tested by GX PCR and direct examination by Ziehl-Neelsen staining. The study of sensitivity to antituberculosis drugs was performed according to the proportion method on liquid medium Bactec MGIT 960 (or on solid medium Lowenstein-Jensen at the Algerian Pasteur Institute). Results: 310 samples were included in the final analysis of the study, of which 156 were of pulmonary origin and 154 of extrapulmonary origin. Mycobacterium tuberculosis complex (MTBC) was detected in 95 samples from 88 tuberculosis patients (sex ratio 2,03 and middle age 37 years) with 49 cases of pulmonary tuberculosis and 39 cases of extra-pulmonary tuberculosis. For 2 cases, the GX was positive while the culture was negative and for 11 cases, the GX was negative while the culture was positive. Thus, in our study and compared to culture, GX showed an overall sensitivity of 88.2%, a specificity of 98.6%, a positive predictive value (PPV) of 96.4% and a negative predictive value (NPV) of 95.2%. The analysis of the data according to the type of samples, the sensitivity, specificity, PPV and NPV of GX for the pulmonary and extrapulmonary samples were 96.3% vs. 77.0%, 98.0% vs. 99.1%, 96.2% vs. 96.5% and 98.0% vs. 92.7% respectively. The sensitivity of GX for disco-vertebral, lymph node, meningeal and pleural tuberculosis were 100%, 90.0%, 71.4% and 57.1% respectively. The sensitivity of GX for pulmonary tuberculosis compared to microscopy was 96% vs. 68%. The comparison of the results of detection of resistance to rifampicin by GX and by phenotypic methods showed perfect agreement. Discussion and conclusion: A good sensitivity of GX compared to microscopy was revealed. The GX is a useful tool for the diagnosis of pulmonary tuberculosis, especially in smear-negative cases. The sensitivity of GX in extrapulmonary tuberculosis varied depending on the location of the infection. A negative result by GX does not exclude tuberculosis and cases of resistance to RIF detected by GX must be confirmed by phenotypic method.

Dose-dependent induction of CYP3A activity by St. John's wort alone and in combination with rifampin.

The dose dependence of the effect of enzyme inducers and the effect of the combined administration of two inducers that exert their effect via the same induction pathway (pregnane X receptor) have not been well studied. Using oral midazolam microdoses (30 µg), we have investigated CYP3A4 induction by St. John's wort (SJW) in 11 healthy volunteers using low (300 mg/day containing 7.48 mg hyperforin), therapeutic (900 mg/day), and supratherapeutic doses of SJW (1800 mg/day) for 14 days. SJW was then co-administered with rifampin (600 mg/day) for a further 7 days to evaluate the effect of the combined administration of two inducers. In addition, intravenous midazolam microdoses (10 µg) were administered before SJW, at SJW 1800 mg/day, and during administration of the two inducers to assess the hepatic contribution to total induction (semi-simultaneous administration). Administration of SJW increased oral midazolam clearance 1.96-fold (300 mg/day), 3.86-fold (900 mg/day), and 5.62-fold (1800 mg/day), and 17.5-fold after the addition of rifampin. Concurrently, the clearance of intravenous midazolam increased 2.05-fold (1800 mg/day) and 2.93-fold (SJW + rifampin). These results show that rifampin significantly enhances the induction of the highest SJW doses both hepatically and overall and suggest that these metabolic effects occur predominantly in the gut. These findings also suggest that in drug interactions involving strong and moderate enzyme inducers, the perpetrator effects of the strong inducer are decisive for the interaction.

Analysis of epidemiological characteristics of extrapulmonary tuberculosis from South-Central China.

Objectives: This study aimed to investigate the epidemiological and drug resistance (DR) characteristics of extrapulmonary tuberculosis (EPTB) in South-Central China. Methods: EPTB inpatients who were culture-positive for Mycobacterium tuberculosis were retrospectively included in a study at a provincial TB hospital in Hunan, a province in South-Central China, from January 2013 to December 2021. Demographic, clinical, and drug susceptibility data were retrieved from TB treatment records. Descriptive statistical methods and a Chi-squared test were used to analyze the epidemiological and DR characteristics of EPTB patients. A logistic regression model was used to explore the risk factors of rifampicin-resistant/multidrug-resistant (RR/MDR)-EPTB. Results: A total of 1,324 cases were included. The majority of EPTB patients were in the age range of 20-29 years, were predominantly men (male-to-female ratio: 2.03), and were farmers (65.63%). Most EPTB cases were found in 2013 and 2017 from 2013 to 2021. The most prevalent subtypes of EPTB were lymphatic TB (29.83%, 395/1,324), multiple EPTB (20.85%, 276/1,324), and musculoskeletal TB (14.65%, 194/1,324). Musculoskeletal TB and genitourinary TB predominantly presented as exclusive EPTB forms, while lymphatic TB and pharyngeal/laryngeal TB often co-occurred with pulmonary TB (PTB). Drug susceptibility testing results showed that total DR rates (resistance to any of RFP, isoniazid [INH], streptomycin [STR], and/or ethambutol [EMB]) and RR/MDR rates in EPTB were 25.23% and 12.39%, respectively. Musculoskeletal TB exhibited the highest rates of total DR (31.40%), INH resistance (28.90%), STR resistance (20.10%), EMB resistance (6.20%), MDR (13.90%), and poly-DR (6.70%). The multivariable logistic regression model showed that patients aged from 20 to 59 years (compared to those aged 10 years), workers (compared to retirees), and EPTB patients from the south and west of Hunan (compared to those from the east of Hunan) were at an increased risk of developing RR/MDR EPTB (all OR values > 1). Conclusion: Our study provided a detailed account of the epidemiological and DR characteristics of EPTB in Hunan province, China. The significant DR rates, particularly in musculoskeletal TB cases, highlight the need for timely diagnosis, effective drug susceptibility testing, and the development of more effective treatment regimens for EPTB, especially targeting musculoskeletal TB treatments.

[A New Method for Determination of Rifampicin and Isoniazid Resistance in Mycobacterium tuberculosis complex Isolates: Capillary Tube Method]. / Mycobacterium tuberculosis Kompleks Izolatlarinda Rifampisin ve Izoniyazid Direncinin Belirlenmesi için Yeni Bir Yöntem: Kapiller Tüp Yöntemi.

Tuberculosis continues to be an important public health problem worldwide. Culture methods are still considered the gold standard in the diagnosis of tuberculosis and the determination of drug resistance. The most important limitation of these methods is their long turnaround time. Commercial culture systems developed to shorten the duration are emerging as an economic problem, especially for developing countries. Therefore, cheap, fast, easy to apply and objectively evaluable tests are needed. In this study, in addition to culture-based methods for determining RIF and INH resistance in Mycobacterium tuberculosis complex isolates, it was aimed to develop the capillary tube method to accelerate the evaluation process. The study included 27 RIF-resistant, 36 RIF -sensitive, 30 INH-resistant, and 33 INH-sensitive isolates obtained from the mycobacteriology laboratory culture collection, for which susceptibility testing to firstline drugs were previously performed using the BACTEC MGIT 960 system (BD, USA) and were stored. H37Rv standard strain and an external quality control strain (IDT3) with known RIF and INH resistance were used as quality control isolates in the study. As a new testing method, the capillary tube method for detecting rifampicin and isoniazid resistance was compared to the standard BACTEC MGIT 960 system. In the determination of RIF and INH resistance, the sensitivity of the capillary tube method compared to the reference method was determined as 85% and 80%, respectively; however, the specificity values (25% and 45.5%, respectively) for both drugs were found to be low in the studies. The time to detect resistance with the capillary tube method varied between 4-9 days. Capillary tube method, which was developed especially for the rapid identification and treatment of multidrug-resistant isolates, is promising in that it detects resistant strains in a short time with a relatively high sensitivity, although its specificity is very low. It is thought that it would be beneficial to continue the study with a larger number of samples and even improve the method with studies conducted directly from clinical samples.

Missed rifampicin and isoniazid resistance by commercial molecular assays.

Drug-resistant tuberculosis (TB) has poor outcomes unless resistance is detected early, ideally by commercially available molecular tests. We present a case of occult multidrug-resistant TB where both rifampicin and isoniazid resistance were missed by molecular testing and were only identified by phenotypic testing.

EasyNAT MTC Assay: a Rapid Method to Detect Mycobacterium Tuberculosis in Respiratory Specimens.

BACKGROUND: This study aimed to effectively evaluate the diagnostic performance of the EasyNAT Mycobacterium tuberculosis complex (MTC) assay for tuberculosis (TB) detection from sputum. METHODS: The retrospectively analyzed data was collected from September 1, 2021, to November 1, 2023, in our hospital. RESULTS: Forty EasyNAT-positive sputum specimens were simultaneously detected using the GeneXpert MTB/ rifampicin (RIF) assay. The concordance rate between the EasyNAT and GeneXpert MTB/RIF assays was 100%. CONCLUSIONS: Because of the complexity of detecting RIF resistance data information, the rapid EasyNAT system used in conjunction with GeneXpert might be a better choice for the detection of TB in hospitals.

Assembly-Induced Membrane Selectivity of Artificial Model Peptides through Entropy-Enthalpy Competition.

Peptide design and drug development offer a promising solution for combating serious diseases or infections. In this study, using an AI-human negotiation approach, we have designed a class of minimal model peptides against tuberculosis (TB), among which K7W6 exhibits potent efficacy attributed to its assembly-induced function. Comprising lysine and tryptophan with an amphiphilic α-helical structure, the K7W6 sequence exhibits robust activity against various infectious bacteria causing TB (including clinically isolated and drug-resistant strains) both in vitro and in vivo. Moreover, it synergistically enhances the effectiveness of the first-line antibiotic rifampicin while displaying low potential for inducing drug resistance and minimal toxicity toward mammalian cells. Biophysical experiments and simulations elucidate that K7W6's exceptional performance can be ascribed to its highly selective and efficient membrane permeabilization activity induced by its distinctive self-assembly behavior. Additionally, these assemblies regulate the interplay between enthalpy and entropy during K7W6-membrane interaction, leading to the peptide's two-step mechanism of membrane interaction. These findings provide valuable insights into rational design principles for developing advanced peptide-based drugs while uncovering the functional role played by assembly.

Isoniazid resistance in Rifampicin sensitive pulmonary tuberculosis in children and adolescents.

BACKGROUND: Isoniazid (INH) and Rifampicin (RIF) are two crucial drugs used in antitubercular therapy. INH is known for its potent bactericidal effects and has a relatively higher prevalence of resistance compared to RIF. However, RIF resistance has been the subject of more extensive research. On the other hand, Ethambutol (EMB) and Streptomycin (STR) resistance have not been thoroughly studied, particularly in the context of children and adolescents. To address this knowledge gap, a study was designed to investigate the resistance patterns of INH, EMB, and STR in RIF-sensitive pulmonary tuberculosis (PTB) cases among children and adolescents. METHODS: Seventy-five newly diagnosed RIF sensitive PTB cases up to 18 years of age were enrolled. Retreatment cases were excluded. Sputum/gastric aspirate sample of these patients were sent for culture in Mycobacterium Growth Indicator Tube (MGIT) followed by drug susceptibility testing and Line Probe Assay. RESULTS: INH, EMB and STR resistance among RIF sensitive PTB cases was found to be 5.7%, 0% and 0.7% respectively. RIF resistance detected by CBNAAT was found to be 8.4%. CONCLUSION: Detection of INH resistance is as important as detecting RIF resistance as prevalence of INH resistance in RIF sensitive PTB among children and adolescents up to 18 years is around 6%.

In-silico transcriptome analysis of antibiotic-treated Mycobacterium tuberculosis identifies novel antibiotic resistance factors.

The emergence of drug resistant Mycobacterium tuberculosis strains increases the burden on the treatment of tuberculosis. In this study, through in-silico transcriptome analysis of drug-treated M. tuberculosis samples, novel drug targets for the treatment of drug resistance in tuberculosis were identified. Gene expression datasets of tuberculosis patients samples treated with different antibiotics (Isoniazid, Rifampicin, Pyrazinamide, Bedaquiline and Linezolid) were considered in this study. DESeq2 was used to identify the differentially regulated genes. Novel genes which were up-regulated during antibiotic treatment were identified which could be antibiotic resistance factors. Further, to understand the resistance mechanism of the novel genes, we performed gene ontology and gene network analysis for the differentially up-regulated genes. Thus, the in-silico transcriptome analysis paves way for a deeper understanding of the antibiotic resistance in M. tuberculosis.

Mutational analysis in drug resistant Mycobacterium tuberculosis in Western Uttar Pradesh.

BACKGROUND: Multi-drug resistant tuberculosis (MDR-TB) results in treatment failure and poor clinical outcomes. This study was carried out with the aim to determine the pattern of drug resistance against Mycobacterium tuberculosis towards first line ATT (anti-tubercular treatment) in sputum smear-positive patients using Line Probe Assay (LPA). METHODS: A cross sectional prospective study was carried out in a tertiary care Hospital of Meerut. A total of 898 sputum samples (on spot and early morning) collected from 449 suspected pulmonary tuberculosis patients as per RNTCP guidelines were screened by microscopy. Decontamination was done by N-acetyl-l-cysteine and sodium hydroxide. Then smear positive samples were subjected to 1st line drug susceptibility testing (DST) using LPA GenoType® MTBDRplus (HAIN Life Science) assay, a molecular method which allows rapid detection of Rifampicin (Rif) and Isoniazid (INH) resistance. RESULTS: The overall burden of MDR TB in this geographical area was 7.9 %. Mono-resistance with Rif alone was around 2.8 %. However, the mono-resistance with INH (inhA gene) and INH (katG gene) was 2.8 % and 1.1 % respectively. Drug resistance of Rif was due to mutations in rpoB gene while resistances to INH were more commonly due to mutation in inhA gene followed by katG gene. TB was more commonly seen in the age group of 30-59 years (43.8 %) and predominantly in males. CONCLUSION: Tuberculosis positivity rate is high in Western Uttar Pradesh. Burden of MDR TB in Western Uttar Pradesh was similar to National data. Line probe assay can be used as a primary method to diagnose multi drug resistant TB as done in present study which can help in earlier initiation of correct therapy.

Potential of Pan-Tuberculosis Treatment to Drive Emergence of Novel Resistance.

New tuberculosis (TB) drugs with little existing antimicrobial resistance enable a pan-TB treatment regimen, intended for universal use without prior drug-susceptibility testing. However, widespread use of such a regimen could contribute to an increasing prevalence of antimicrobial resistance, potentially rendering the pan-TB regimen ineffective or driving clinically problematic patterns of resistance. We developed a model of multiple sequential TB patient cohorts to compare treatment outcomes between continued use of current standards of care (guided by rifampin-susceptibility testing) and a hypothetical pan-TB approach. A pan-TB regimen that met current target profiles was likely to initially outperform the standard of care; however, a rising prevalence of transmitted resistance to component drugs could make underperformance likely among subsequent cohorts. Although the pan-TB approach led to an increased prevalence of resistance to novel drugs, it was unlikely to cause accumulation of concurrent resistance to novel drugs and current first-line drugs.

Reassessment of the genetic basis of natural rifampin resistance in the genus Rickettsia.

Rickettsia, a genus of obligate intracellular bacteria, includes species that cause significant human diseases. This study challenges previous claims that the Leucine-973 residue in the RNA polymerase beta subunit is the primary determinant of rifampin resistance in Rickettsia. We investigated a previously untested Rickettsia species, R. lusitaniae, from the Transitional group and found it susceptible to rifampin, despite possessing the Leu-973 residue. Interestingly, we observed the conservation of this residue in several rifampin-susceptible species across most Rickettsia phylogenetic groups. Comparative genomics revealed potential alternative resistance mechanisms, including additional amino acid variants that could hinder rifampin binding and genes that could facilitate rifampin detoxification through efflux pumps. Importantly, the evolutionary history of Rickettsia genomes indicates that the emergence of natural rifampin resistance is phylogenetically constrained within the genus, originating from ancient genetic features shared among a unique set of closely related Rickettsia species. Phylogenetic patterns appear to be the most reliable predictors of natural rifampin resistance, which is confined to a distinct monophyletic subclade known as Massiliae. The distinctive features of the RNA polymerase beta subunit in certain untested Rickettsia species suggest that R. raoultii, R. amblyommatis, R. gravesii, and R. kotlanii may also be naturally rifampin-resistant species.

Cryo-EM structure of orf virus scaffolding protein orfv075.

Capsid-like poxvirus scaffold proteins self-assemble into semi-regular lattice that govern the formation of spherical immature virus particles. The scaffolding is a critical step in virus morphogenesis as exemplified by the drug rifampicin that impairs the recruitment of scaffold onto the viral membrane in vaccinia virus (VACV). Here we report cryo-electron microscopy structure of scaffolding protein Orfv075 of orf virus (ORFV) that causes smallpox-like diseases in sheep, goats and occasionally humans via zoonotic infection. We demonstrate that the regions that are involved in intertrimeric interactions for scaffold assembly are largely conserved in comparison to its VACV orthologue protein D13 whose intermediate assembly structures have been previously characterized. By contrast, less conserved regions are located away from these interfaces, indicating both viruses share similar assembly mechanisms. We also show that the phenylalanine-rich binding site of rifampicin in D13 is conserved in Orfv075, and molecular docking simulation confirms similar binding modes. Our study provides structural basis of scaffolding protein as a target for anti-poxvirus treatment across wide range of poxvirus genera.

Pharmacokinetics of anti-Mycobacterium avium-intracellulare disease drugs in silkworms.

Pulmonary Mycobacterium avium-intracellulare complex (MAC) disease is a typical non-tuberculous mycobacterial infection. The incidence of pulmonary MAC is increasing worldwide. This study aimed to clarify the pharmacokinetic parameters of anti-pulmonary MAC disease drugs in silkworms. The pharmacokinetic parameters investigated included maximum concentration, area under the concentration-time curve, total clearance, and volume of distribution at steady-state. In addition, protein-binding rates, fat body transferability, and drug-drug interactions were examined. Antibiotic concentrations were measured using a validated high-performance liquid chromatography-mass spectrometry method. Among the antibiotics investigated, amikacin was not eliminated from silkworms during the 48-h observation period. In contrast, dose-proportional pharmacokinetics were observed in silkworms for all antibiotics tested, except for amikacin. Protein-binding rates in hemolymph for clarithromycin, azithromycin, rifampicin, ethambutol, and amikacin were 39.6 ± 3.0%, 39.5 ± 4.3%, 76.3 ± 3.2%, 20.9 ± 4.2%, and 73.1 ± 4.7%, respectively (mean ± standard deviation). The distribution of antibiotics in the fat bodies of silkworms was related to drug lipophilicity. No drug-drug interactions were observed in the silkworms. The pharmacokinetics of these drugs in silkworms differed significantly from those in humans. Therefore, while it is challenging to predict the pharmacokinetics of these drugs in humans based on silkworm data, the silkworm infection model has facilitated a comprehensive assessment of the relationship between antibiotic exposure and efficacy.

A Microtiter Plate Assay at Acidic pH to Identify Potentiators that Enhance Pyrazinamide Activity Against Mycobacterium tuberculosis.

Pyrazinamide (PZA) is a key component of chemotherapy for the treatment of drug-susceptible tuberculosis (TB) and is likely to continue to be included in new drug combinations. Potentiation of PZA could be used to reduce the emergence of resistance, shorten treatment times, and lead to a reduction in the quantity of PZA consumed by patients, thereby reducing the toxic effects. Acidified medium is required for the activity of PZA against Mycobacterium tuberculosis. In vitro assessments of pyrazinamide activity are often avoided because of the lack of standardization, which has led to a lack of effective in vitro tools for assessing and/or enhancing PZA activity.We have developed and optimized a novel, robust, and reproducible, microtiter plate assay, that centers around acidity levels that are low enough for PZA activity. The assay can be applied to the evaluation of novel compounds for the identification of potentiators that enhance PZA activity. In this assay, potentiation of PZA is demonstrated to be statistically significant with the addition of rifampicin (RIF), which can, therefore, be used as a positive control. Conversely, norfloxacin demonstrates no potentiating activity with PZA and can be used as a negative control. The method, and the associated considerations, described here, can be adapted in the search for potentiators of other antimicrobials.

Rifabutin central nervous system concentrations in a rabbit model of tuberculous meningitis.

Tuberculous meningitis (TBM) has a high mortality, possibly due to suboptimal therapy. Drug exposure data of antituberculosis agents in the central nervous system (CNS) are required to develop more effective regimens. Rifabutin is a rifamycin equivalently potent to rifampin in human pulmonary tuberculosis. Here, we show that human-equivalent doses of rifabutin achieved potentially therapeutic exposure in relevant CNS tissues in a rabbit model of TBM, supporting further evaluation in clinical trials.

A Novel 3D Printed Model of Infected Human Hair Follicles to Demonstrate Targeted Delivery of Nanoantibiotics.

Hair follicle-penetrating nanoparticles offer a promising avenue for targeted antibiotic delivery, especially in challenging infections like acne inversa or folliculitis decalvans. However, demonstrating their efficacy with existing preclinical models remains difficult. This study presents an innovative approach using a 3D in vitro organ culture system with human hair follicles to investigate the hypothesis that antibiotic nanocarriers may reach bacteria within the follicular cleft more effectively than free drugs. Living human hair follicles were transplanted into a collagen matrix within a 3D printed polymer scaffold to replicate the follicle's microenvironment. Hair growth kinetics over 7 days resembled those of simple floating cultures. In the 3D model, fluorescent nanoparticles exhibited some penetration into the follicle, not observed in floating cultures. Staphylococcus aureus bacteria displayed similar distribution profiles postinfection of follicles. While rifampicin-loaded lipid nanocapsules were as effective as free rifampicin in floating cultures, only nanoencapsulated rifampicin achieved the same reduction of CFU/mL in the 3D model. This underscores the hair follicle microenvironment's critical role in limiting conventional antibiotic treatment efficacy. By mimicking this microenvironment, the 3D model demonstrates the advantage of topically administered nanocarriers for targeted antibiotic therapy against follicular infections.