Rifampicin Resistance Research Articles

The pattern of rpoB gene mutation of Mycobacterium tuberculosis and predictors of rifampicin resistance detected by GeneXpert MTB/RIF assay in Tanzania.

Antimicrobial resistance in Mycobacterium tuberculosis (MTB) poses a significant challenge to tuberculosis (TB) management worldwide. Rifampicin resistance (RR) has been associated with the rpoB gene mutation. No study was conducted in Tanzania to determine the commonest mutation. The inconsistent findings from various studies support the need to determine whether reported mutation patterns are applicable in our setting. We determined the frequency of rpoB gene mutation and factors associated with RR, which were detected using GeneXpert MTB/RIF assay. We conducted a retrospective cross-sectional study involving data from the National Tuberculosis and Leprosy Program database from 2020 to 2022 for cases investigated using GeneXpert MTB/RIF assay. Descriptive analysis was performed to determine the frequency of categorical variables. The chi-square test and logistic regression analysis assessed the relationship between the independent variables and outcome. The 95% confidence interval and a significance level of p<0.05 were used to assess the strength of association. A total of 56,004 participants had a status of MTB and RR, where 38,705/56,004 (69.11%) were males. Probe E mutation (codon 529-533), 89/219 (40.64%) was predominant. Human immunodeficiency virus (HIV)-positive patients had a higher gene mutation, 134/10601 (1.26%) than HIV-negative, 306/45016 (0.68%) (p<0.001). Patients with both pulmonary and extra-pulmonary TB had about four times greater odds of developing rifampicin resistance (AOR 3.88, 95%CI: 1.80-8.32). RR was nearly nine times higher in previously treated patients than new patients (AOR 8.66, 95% CI: 6.97-10.76). HIV-positive individuals had nearly twice the odds of developing RR than HIV-negative individuals (AOR 1.91, 95%CI: 1.51-2.42). The rate of RR was lower compared to other studies in Tanzania, with probe E mutations the most prevalent. Patients with disseminated TB, HIV co-infection and those with prior exposure to anti-TB had more risk of RR. The findings highlight the need to strengthen surveillance of multidrug-resistant TB among high risk patients.

Targeted next-generation sequencing of Mycobacterium tuberculosis from patient samples: lessons learned from high drug-resistant burden clinical settings in Bangladesh

ABSTRACT Lack of appropriate early diagnostic tools for drug-resistant tuberculosis (DR-TB) and their incomplete drug susceptibility testing (DST) profiling is concerning for TB disease control. Existing methods, such as phenotypic DST (pDST), are time-consuming, while Xpert MTB/RIF (Xpert) and line probe assay (LPA) are limited to detecting resistance to few drugs. Targeted next-generation sequencing (tNGS) has been recently approved by WHO as an alternative approach for rapid and comprehensive DST. We aimed to investigate the performance and feasibility of tNGS for detecting DR-TB directly from clinical samples in Bangladesh. pDST, LPA and tNGS were performed among 264 sputum samples, either rifampicin-resistant (RR) or rifampicin-sensitive (RS) TB cases confirmed by Xpert assay. Resistotypes of tNGS were compared with pDST, LPA and composite reference standard (CRS, resistant if either pDST or LPA showed a resistant result). tNGS results revealed higher sensitivities for rifampicin (RIF) (99.3%), isoniazid (INH) (96.3%), fluoroquinolones (FQs) (94.4%), and aminoglycosides (AMGs) (100%) but comparatively lower for ethambutol (76.6%), streptomycin (68.7%), ethionamide (56.0%) and pyrazinamide (50.7%) when compared with pDST. The sensitivities of tNGS for INH, RIF, FQs and AMGs were 93.0%, 96.6%, 90.9%, and 100%, respectively and the specificities ranged from 91.3 to 100% when compared with CRS. This proof of concept study, conducted in a high-burden setting demonstrated that tNGS is a valuable tool for identifying DR-TB directly from the clinical specimens. Its feasibility in our laboratory suggests potential implementation and moving tNGS from research settings into clinical settings.

Identification of Mycobacterium tuberculosis complex species using dual-tube multiplex real-time PCR and determination of their rifampicin and isoniazid resistance profiles: A 5-year study

Identification of Mycobacterium tuberculosis complex species using dual-tube multiplex real-time PCR and determination of their rifampicin and isoniazid resistance profiles: A 5-year study

Diagnostic evaluation of Tru-Nat MTB/Rif test in comparison with microscopy for diagnosis of pulmonary tuberculosis at tertiary care hospital of eastern Uttar Pradesh.

This study evaluated the efficacy of the TrueLab™ Real Time mini-PCR system in providing rapid and accurate diagnostic results for tuberculosis (TB) detection in India. The goal is to improve case detection and accelerate treatment in settings with limited resources. This prospective study was conducted by the Department of Microbiology on 120 patients, age ranging from >=15 years with at least two clinical symptoms of pulmonary TB. Molbio and Universal Cartridge Based Sample Prep were the 2 methods used for processing sputum samples. The diagnosis was based on the MTB Real Time PCR test, which has a detection limit of 100 CFU/mL. Patients under 15 years, samples lacking clinical background, saliva specimens or extra-pulmonary TB cases were excluded from the study. A total of 44.17% samples were positive for TB with maximum positivity in the age group 31-45 years. Positivity rate was found to be higher in females. In 4.17% of cases there was rifampicin resistance, which was significantly high in previously treated cases. Comparison of Truenat with Ziehl-Neelsen and fluorescent method revealed that it was more sensitive and less time consuming. Truenat MTB/RIF is a sensitive detection system for TB with rapid results, which serves as an important tool in the early management of tuberculosis patients and drug-resistant-TB cases.

Prevalence and associated factors of TB and HIV coinfections among adult inmates with presumptive pulmonary TB in a Kenyan prison

BackgroundTuberculosis (TB) is more than ten times higher in prisons compared to the general population, and HIV-infected persons are at increased risk of developing active TB and death. In the World Health Organization (WHO) African region, however, where the TB and HIV coinfections are highest, and prisons rarely factored in national disease surveillance, epidemiological data to inform TB control interventions in correctional facilities is limited. In this study, we assessed the prevalence of TB and HIV coinfections, as well as the factors associated with coinfections in our study setting.MethodsThis was a prospective cross-sectional study among 157 adult (≥ 18 years) prisoners presenting with symptoms of pulmonary TB at Shimo La Tewa Prison, Kenya, between January and June 2023. The study excluded those with a history of anti-TB drugs use or on treatment follow-up and collected demographic and clinical characteristics data using a questionnaire. Sputum samples were collected and processed immediately using Xpert® MTB/RIF assay or stored at 4 °C for three (3) days in case of delay.ResultsThe overall prevalence of TB among inmates with presumptive pulmonary TB was 10.2%, 95% CI 6.37–16.91% (16/157), HIV 19.1%, 95% CI 13.73–25.97% (30/157). All the TB cases were positive for HIV (16/16, 100%), translating to TB/HIV coinfection of 10.2%, 95% CI 6.37–16.91% (16/157), and there was no rifampicin resistance. TB and HIV coinfection cases were found among underweight (100%, 16/16) prisoners. The independent factors associated with TB and HIV coinfections were education level (adjusted OR = 0.17, p = 0.007), smoking history (adjusted OR = 3.01, p = 0.009) and illegal drug use history (adjusted OR = 4.55, p = 0.044).ConclusionWe report a high prevalence of pulmonary TB and HIV coinfections among adult inmates with presumptive pulmonary TB in Kenya, with education level, smoking status, and illegal drug use as the independent factors associated with the coinfection. The authority should take measures to protect HIV-positive prisoners from TB, focusing on education, nutrition, smoking, and illegal drug use.

Burden of drug-resistant tuberculosis by the GeneXpert MTB/RIF assay and its clinico-epidemiological aspects at a tertiary care hospital in West Bengal

Multidrug -resistant tuberculosis (MDRTB) is a serious threat to mankind. India has the highest number of MDRTB cases, although majority remain undiagnosed due to inadequate diagnostic infrastructure, leading to increased community transmission and mortality. This one-year observational retrospective study highlighted the effectiveness of the National Tuberculosis Elimination Program (NTEP) for prompt detection of drug-resistant TB by GeneXpert MTB/RIF assay and revealed its associated clinico-epidemiological factors. The overall detection rates of MTB and RRTB were 20.70 % and 20.86 % respectively. The pediatric population had 7.69 % rifampicin resistance, and HIV was strongly associated with the development of TB and RRTB (P < 0.01).

Ketoprofen promotes the conjugative transfer of antibiotic resistance among antibiotic resistant bacteria in natural aqueous environments

The emergence and spread of antibiotic resistance in the environment pose a serious threat to global public health. It is acknowledged that non-antibiotic stresses, including disinfectants, pharmaceuticals and organic pollutants, play a crucial role in horizontal transmission of antibiotic resistance genes (ARGs). Despite the widespread presence of non-steroidal anti-inflammatory drugs (NSAIDs), notably in surface water, their contributions to the transfer of ARGs have not been systematically explored. Furthermore, previous studies have primarily concentrated on model strains to investigate whether contaminants promote the conjugative transfer of ARGs, leaving the mechanisms of ARG transmission among antibiotic resistant bacteria in natural aqueous environments under the selective pressures of non-antibiotic contaminants remains unclear. In this study, the Escherichia coli (E. coli) K12 carrying RP4 plasmid was used as the donor strain, indigenous strain Aeromonas veronii containing rifampicin resistance genes in Taihu Lake, and E. coli HB101 were used as receptor strains to establish inter-genus and intra-genus conjugative transfer systems, examining the conjugative transfer frequency under the stress of ketoprofen. The results indicated that ketoprofen accelerated the environmental spread of ARGs through several mechanisms. Ketoprofen promoted cell-to-cell contact by increasing cell surface hydrophobicity and reducing cell surface charge, thereby mitigating cell-to-cell repulsion. Furthermore, ketoprofen induced increased levels of reactive oxygen species (ROS) production, activated the DNA damage-induced response (SOS), and enhanced cell membrane permeability, facilitating ARG transmission in intra-genus and inter-genus systems. The upregulation of outer membrane proteins, oxidative stress, SOS response, mating pair formation (Mpf) system, and DNA transfer and replication (Dtr) system related genes, as well as the inhibition of global regulatory genes, all contributed to higher transfer efficiency under ketoprofen treatment. These findings served as an early warning for a comprehensive assessment of the roles of NSAIDs in the spread of antibiotic resistance in natural aqueous environments.

Evaluation of Diagnostic Methods and Rifampicin Resistance in Pulmonary Tuberculosis: A Hospital-Based Study.

Background Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis, predominantly affecting the lungs (pulmonary TB) and is a significant public health challenge in India. The study aims to analyze demographic, radiological, and clinical subgroups of pulmonary TB cases, examine the relationship between smear acid-fast bacillus (AFB examination) and cartridge-based nucleic acid amplification test (CBNAAT), evaluate CBNAAT sensitivity for Mycobacterium tuberculosis (MTB) in new and previously treated patients, and determine the proportion of rifampicin resistance. Methods This hospital-based prospective study was conducted among patients diagnosed with pulmonary TB at the Respiratory Medicine Department of a Government Hospital over 16 months (August 2019 to December 2020). The study included 150 diagnosed TB cases (new and previously treated). Data collection encompassed demographic details, clinical symptoms, comorbidities, radiological findings (chest X-ray), and microbiological results (smear AFB examination, CBNAAT). Sputum samples were subjected to Ziehl-Neelsen staining and CBNAAT for MTB detection and rifampicin resistance testing. Statistical analysis was performed using IBM SPSS Statistics version 21.0 (IBM Corp., Armonk, NY, USA). Results Of the 150 patients, 69.3% were male, and 48% were aged 21-40 years. The majority had a BMI of 18.5-24.9 kg/m² (50%) and resided in urban areas (63.3%). Common symptoms included cough (95.3%), fever (80%), and weight loss (74%). Cavitary lesions on chest X-ray were observed in 84% of patients. Smear microscopy detected MTB in 72.7% of cases, while CBNAAT detected MTB in 94% of cases. CBNAAT sensitivity for smear-positive and smear-negative samples was 93.97% and 94.12%, respectively. Rifampicin resistance was found in 3% of new cases and 6% of previously treated cases. The sensitivity of smear microscopy was 77.33%, and the sensitivity of CBNAAT was 94%. Conclusion The study underscores the high burden of pulmonary TB and the utility of CBNAAT in detecting MTB and rifampicin resistance, particularly in smear-negative samples. The findings highlight the necessity of universal drug susceptibility testing (DST) for effective TB management and the importance of addressing drug resistance to improve treatment outcomes.

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

BackgroundThe 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.MethodsWe 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.ResultsA 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.ConclusionsThe 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.

Establishment and Validation of a Nomogram for Identifying False Positives in Xpert MTB/RIF Rifampicin Resistance Test.

This study aimed to establish and validate a diagnostic nomogram for identifying false positives in the Xpert MTB/RIF (Xpert) for detection of rifampicin resistance (RIF-R). In this retrospective study, we collected basic patient characteristics and various clinical information from the electronic medical record database. Patients were randomly divided into training and validation groups in a 7:3 ratio. LASSO regression was used to screen variables and construct a diagnostic nomogram. The ROC curve, calibration curve, and decision curve analysis (DCA) were used to evaluate the performance of the nomogram. A total of 384 patients were included in the study, with 268 and 116 patients in the training and validation cohorts, respectively. Finally, probe mutations and probe delay were identified as the independent influencing factors. Using the mutation of probe E as a reference, probes A or C (OR = 51.07, P<0.001), probe D (OR = 7.48, P<0.001), and multiple probes (OR = 4.42, P=0.029) were identified as factors influencing false positives in Xpert for detection of RIF-R. Taking probe delay ΔCT <4 as a reference, ΔCT (4-5.9) (OR = 17.06, P=0.005) and ΔCT (6-7.9) (OR = 36.67, P<0.001) were noted to be the factors influencing false positives in Xpert for detection of RIF-R. Based on these two variables, we constructed a diagnostic nomogram. The area under the curve of the nomogram model was 0.847 and 0.850 for the training and validation groups, respectively. The calibration curves were consistent. The DCA revealed that the model achieved the greatest net benefit when the threshold probability was set between 6% and 71% in the training cohort and 6% and 70% in the validation cohort. The nomogram constructed can identify false positives in Xpert for detection of RIF-R and provides basis for clinicians to formulate diagnosis and treatment plans.

Prevalence of Co-infection of Culture-Proven Bacterial Pathogens in Microbiologically Confirmed Pulmonary Tuberculosis Patients From a Tertiary Care Center.

Tuberculosis (TB) is a chronic condition that weakens the immune system, causes structural changes in the lungs, and can lead to infections by other bacterial pathogens. Very few studies have been done to understand the magnitude of co-infection with other bacterial pathogens, so this study was conducted to understand the co-infection pattern and burden. A total of 174 microbiologically confirmed pulmonary TB patients' samples, identified by cartridge-based nucleic acid amplification test, were further tested for other bacterial pathogens by culture over a period of five months from May 2023 to September 2023. The isolates' identification and drug susceptibility were performed using the VITEK 2 system (bioMérieux, Marcy-l'Étoile, France). Of the 174 pulmonary samples tested, 19 samples grew a significant amount of other bacterial pathogens, making the prevalence 10.91% (19/174). Among the pulmonary samples tested, 54.59% were sputum, 38.5% were bronchoalveolar lavage, and 6.89% were endotracheal aspirate. Additionally, 70.11% of the patients tested were in the age group of 19-60 years. Of the patients who had co-infection, 94.73% (18/19) were male. The most common bacterial infection was caused by Pseudomonas aeruginosa, which was identified in 36.84% of the co-infection cases (7/19). This was followed by Acinetobacter baumannii in 31.57% (6/19), Klebsiella pneumoniae in 26.31% (5/19), and Stenotrophomonas maltophilia in 5.28% (1/19). Acinetobacter baumannii and Klebsiella pneumoniae showed high drug resistance, ranging from 60% to 100% against various groups of drugs tested. None of the patient samples with co-infection showed rifampicin resistance. Among all the samples with co-infection, the majority (42.10%, or 8/19) had a high load of Mycobacterium tuberculosis complex detected by CBNAAT Ultra (Cepheid, Sunnyvale, California). Pseudomonas aeruginosa, Acinetobacter baumannii, and Klebsiella pneumoniae are unusual pathogens causing infection in community patients and are known to cause illness in hospitalized patients. These organisms' resistance was also similar to the resistance shown by hospital-acquired infections. Thisindicates that bacterial co-infection in pulmonary TB patients will be similar to the pattern of hospital-acquired infections. The high prevalence of bacterial co-infections (10.91%) in patients with pulmonary TB poses a significant challenge as these bacterial pathogens are not susceptible to anti-tubercular drugs. Therefore, comprehensive screening for other bacterial infections in all pulmonary TB patients is crucial for effective treatment and outcomes.

Role of GeneXpert in the diagnosis of extrapulmonary tuberculosis.

The World Health Organization endorsed the cartridge-based nucleic acid amplification test Xpert MTB/RIF (GXP) for the diagnosis of tuberculosis (TB). Studies about GXP efficiency in extrapulmonary TB (EPTB) are scarce. Hence, we decided to study the role of GXP in EPTB. This prospective observational study, conducted in the pulmonary medicine department of a tertiary care hospital after ethics committee permission, recruited 200 EPTB patients. The diagnosis of TB was achieved with the help of clinico-radiological correlation with microbiological test positivity. Acid-fast bacilli (AFB) culture was treated as the comparative gold standard. Patients who had no or incomplete data were excluded from the study. Data was analyzed to calculate the sensitivity, specificity, positive predictive value, and negative predictive value for the diagnosis of TB and the detection of rifampicin resistance. The majority of cases were women (126 patients: 63%). The mean age was 23.71 years. On GXP, 130 (65%) had detected mycobacterium tuberculosis (MTB), and 70 (35%) did not. Adding AFB culture data, 168 (81.5%) showed microbiological evidence of TB, and 32 (18.5%) were negative. On the drug susceptibility test, 131 cases were rifampicin-sensitive, 32 were rifampicin-resistant TB, and in 5 cases, data was unavailable. The most common extrapulmonary site of involvement was the lymph node, with 94 patients (47%). The most common lymph node involved was the cervical lymph node, with 70 patients (74.5%). The sensitivity, specificity, positive predictive value, and negative predictive value of GXP in EPTB collectively were 76.68%, 86.48%, 96%, and 45.7%, respectively. GXP is useful for the rapid detection of EPTB and the identification of rifampicin resistance, especially in a high-prevalence country like India.

Evaluation of GeneNAT Real-Time Polymerase Chain Reaction Analyzer and Pre-loaded Chip-Based Mycobacterium tuberculosis and Multidrug-Resistant Tuberculosis Detection in the Diagnosis of Pulmonary Tuberculosis.

Tuberculosis (TB) is still the second causative agent of death worldwide after COVID-19. It is caused by Mycobacterium tuberculosis (MTB) infection. The aim of the current study was to compare the performance of GeneNAT real-time polymerase chain reaction analyzer and pre-loaded chip-based MTB screening and multidrug-resistant tuberculosis (MDR-TB) detection kit (Smart SureTM MTB & MDR-TB, Genetix Biotech Asia Pvt. Ltd., New Delhi, India) against the established WHO-approved GeneXpert Ultra (MTB/rifampicin (RIF)), line probe assay (LPA), and mycobacteriagrowthindicatortube(MGIT) culture at point of care (POC) level. A total of 450 pulmonary TB (PTB) suspect patients were enrolled from October 2023 to March 2024 at the Intermediate Reference Laboratory, Department of Medicine, All India Instituteof Medical Sciences, New Delhi, India. GeneXpert and GeneNAT tests were done directly from sputum specimens. However, processed sputum specimens were used for both LPA (GenoType MTBDRplus) and liquid culture and drug susceptibility testing (MGIT culture and drug susceptibility testing (DST)). On comparing with GeneXpert, for the detection of MTB and rifampicin (RIF), Smart SureTM showed a sensitivity of 98.18% and 97.5% with a specificity of 100% and 98.92%, respectively. While comparing mutations in therpoB gene with LPA, the Smart SureTM MDR-TB kit exhibited sensitivity and specificity of 96.77% and 99.12%, respectively. For katG and inhA genes, sensitivity and specificity were 97.6% & 85.71% and 98.66% & 98.01%, respectively, for both genes. Smart SureTM MDR-TB showed comparable results with MGIT-DST with sensitivity and specificity of 96.88% & 96.15% and 98.99%& 99.02%, respectively, for both RIF and isoniazid (INH) drugs. The GeneNAT system test may provide the status of RIF and INH resistance in PTB cases in a short time with the use of minimal specimens. It required very little infrastructurewith less skilled laboratory staffin comparison with other WHO-approved diagnostics used in resource-limited countries with TB and drug-resistant TB burdens.

Rifampicin resistance in pulmonary and extra pulmonary tuberculosis cases in of children

: The prevalence of Rifampicin-resistant MTB/EPTB in children in India is a growing concern. Globally a large proportion of childhood tuberculosis (TB) cases are diagnosed based only on clinical findings. This study was undertaken to measure MTB positivity and their rifampicin resistance rates in different clinical samples from children. : Different samples of clinically suspected tuberculosis in children were included. All the samples were tested for Tuberculosis positivity and Rifampicin (Rif) resistance by GeneXpert assay. A total of 1420 samples were received from presumptive TB patients of all age group from February 2023 to January 2024. Total 1420 samples were analyzed for this study, of which 30% were male and 70% were females. Respiratory samples and extra-pulmonary samples were 1261 (88.8%) and 159 (11.2%) respectively. Among 1420, 211(14.86%) samples were from children and among these 75 (35.55%) were found MTB positive and 7 (9.33%) were Rif resistant MTB. Of 75 MTB detected patients, 77.8% and 22.2% MTB detected in female and male children respectively. There are higher prevalence of Rif resistant MTB in children so it is important for healthcare providers in India to be vigilant in timely diagnosis, individualized treatment and enhance surveillance. It is essential to combat this growing threat and protect the health of children.

Point Mutations in Furazolidone and Rifampicin Resistance Genes in Helicobacter pylori Strains from Colombia.

The eradication of Helicobacter pylori is a valid strategy for preventing gastric cancer; however, the therapeutic failure of first-line treatments in Colombia is associated with high resistance to metronidazole and amoxicillin. This study explored alternative antibiotics and analyzed point mutations in resistance genes to furazolidone and rifampicin in order to include them in rescue therapy regimens. A total of 239 complete genomes of Helicobacter pylori Colombian strains were compared to that of the ATCC 26695 strain to identify mutations in the rpoB and porD genes for rifampicin and furazolidinone resistance, respectively. While rifampicin resistance mutations were not found, only 0.84% of the isolates showed the porD gene, suggesting that Helicobacter pylori is sensitive to these antibiotics. A phylogenomic analysis of Helicobacter pylori revealed an independent lineage in Colombia (hspColombia). The absence of point mutations in the rpoB gene, together with the scarce mutations identified in the porD gene of Helicobacter pylori, suggest that the hspColombia isolates are sensitive to rifampicin and furazolidone, which could be key to including these antibiotics in the rescue therapies against Helicobacter pylori.

Identification of antimicrobial-susceptible Pseudomonas aeruginosa RpoA variant strains through positional conservation pattern.

Bacterial RNA polymerase (RNAP) is a promising target for antimicrobial chemotherapy due to its indispensable role in bacterial growth and survival. Among its components, only the rpoB gene encoding the β-subunit is known for its association with rifampicin resistance. We recently identified a variant of the RNAP α-subunit (RpoA) in Pseudomonas aeruginosa, conferring heightened bacterial susceptibility to antimicrobials. This susceptibility was attributed to the specific down-regulation of the MexEF-OprN efflux pump. We asked how to distinguish antimicrobial-susceptible variant strains from clinical isolates. In this study, we identified various P. aeruginosa RpoA variants from clinical sources. Using the sequence alignment of different bacterial RpoA species, we computed the positional conservation of substitutions in RpoA variants using Shannon Entropy. Our findings revealed that selective RpoA variant strains exhibited distinct profiles of antimicrobial susceptibility. Notably, RpoA variant strains, containing single-substitutions in the C-terminal domain (α-CTD) but not the N-terminal domain (α-NTD), showed attenuated MexEF-OprN expression and increased susceptibility to MexEF-OprN-specific antibiotics. Furthermore, we observed a close correlation between the susceptibility of these α-CTD RpoA variant strains to antibiotics and the conservation degrees of positional substitutions. Our findings demonstrate the prevalence of antimicrobial-susceptible RpoA variant strains among P. aeruginosa clinical isolates. The identified positional conservation pattern in our study facilitates the rapid classification of RpoA variant strains with distinct drug resistances. Given the high conservation of RNAP across bacterial species, our findings open a new therapeutic perspective for precisely and efficiently combating pathogenic RpoA variant strains with specific antimicrobials.

Problems of Detecting Tuberculosis by Sputum Microscopy in General Medical Services in the Republic of Karelia

The objective: to determine the effectiveness of detection of patients with pulmonary tuberculosis and prevention of its spread by sputum microscopy in clinical diagnostic laboratories of general medical services of the Republic of Karelia.Results. A reduction in the number of sputum microscopy tests in clinical laboratories of GMS and decrease in the positivity rate to 0.66% was revealed. Among new tuberculosis patients with rifampicin resistance, 51% had abundant bacterial excretion (AFB 2+ and 3+); according to sputum microscopy, tuberculous mycobacteria susceptible to rifampicin were isolated in 38%. In the Republic of Karelia, 31.7% of tuberculosis patients were detected in in-patient units of GMS: 60% of them had cavities, and 28.8% had abundant bacterial excretion (AFB 2+ and 3+). The poor quality of sputum microscopy is the cause of late detection of tuberculosis in the Republic of Karelia and promotes the spread of MDR tuberculosis strains among the population, including nosocomial infection.

Drug Susceptibility and Mutation Profiles in Mycobacterium tuberculosis Isolates from a Tertiary Care Hospital in Kerala, India.

The rising prevalence of drug-resistant Mycobacterium tuberculosis (MTB) strains poses a significant challenge to global tuberculosis (TB) control efforts. This study aimed to analyze drug resistance patterns and investigate the molecular characteristics of 193 MTB clinical isolates to shed light on the mechanisms of drug resistance. Of the 193 MTB clinical isolates, 28.5% (n = 53) exhibited mono-drug or multidrug resistance. Pyrazinamide mono-drug resistance (PZAr) was the most prevalent (17%, n = 33), followed by isoniazid mono-drug resistance (3.6%, n = 7). Rifampicin resistance was associated with mutations in the rpoB gene (D435Y, D435V, S450L, L452P). Isoniazid resistance mutations were found in the katG (S315T), inhA (C[-15] T), and ndh (R268H) genes, whereas ethambutol resistance mutations were observed in the embB gene (M306V, M306I, M306L, G406S, Q497R). Surprisingly, 94% of PZAr isolates (n = 31) showed no mutations in the pncA or rpsA genes. The presence of the R268H mutation in the ndh gene, not previously linked to PZAr, was detected in 15% of PZAr isolates (n = 5), suggesting its potential contribution to PZAr in specific cases but not as a predominant mechanism. The specific molecular mechanisms underlying PZAr in the majority of the isolates remain unknown, emphasizing the need for further research to uncover the contributing factors. These findings contribute to the understanding of drug resistance patterns and can guide future efforts in TB control and management.

Evaluation of the GeneXpert MTB/RIF Assay for Rapid Diagnosis of Tuberculosis and Detection of Rifampin Resistance in Tuberculous Patients Admitted to Abbassia Chest Hospital

Abstract Background Tuberculosis is considered one of the major infectious diseases especially in the developing countries and a significant diagnostic and therapeutic challenge worldwide. Gene Xpert MTB/RIF assay is a diagnostic test which depends on a real-time polymerase chain reaction for rapid diagnosis of Mycobacterium Tuberculosis and rifampicin resistance in a single test. Aim To evaluate the diagnostic utility of Gene Xpert MTB/RIF assay for rapid diagnosis of pulmonary and extrapulmonary tuberculosis as well as the detection of rifampin resistance in specimens obtained from suspected tuberculous patients in Abbassia Chest Hospital. Patients and Methods Pulmonary and extrapulmonary samples from 77 cases with suspected TB were collected. All retrieved samples were examined with: direct smear microscopy for AFB, culture for Mycobacterium tuberculosis (L.J. media) along with Drug susceptibility testing (D.S.T.) for positive cultures. As well as the gene Xpert MTB/RIF assay and their results were compared to those of both culture and D.S.T. (as gold standard reference methods). Results Gene Xpert MTB/RIF assay showed the highest sensitivity, negative predictive value (NPV) and diagnostic accuracy in detection of pulmonary TB (96.4, 96.4 and 93.1% respectively), with 90%specificity. Xpert MTB/RIF assay showed the lowest sensitivity in diagnosing TB pleural effusion (66.6%) but with the highest specificity and positive predictive value (PPV) (100% for both). As regards diagnosing TB lymphadenitis, gene Xpert MTB/RIF assay showed 75% sensitivity and the lowest specificity (33.3%). Considering rifampin resistance, gene Xpert MTB/RIF assay showed 100% specificity in detection of RIF- Resistance with variable sensitivity according to the type of TB, being the highest in pulmonary TB (90.9% sensitivity). Conclusion Gene Xpert MTB/RIF assay could be used as the first line diagnostic tool for diagnosis of pulmonary TB because of its high diagnostic yield. Yet, it’s not recommended to replace the culture for diagnosing extrapulmonary TB especially in diagnosing tuberculous pleural effusion (pauci-bacillary specimens) which has limited diagnostic utility. Also, the assay showed 100% specificity in diagnosing rifampicin resistance guiding physicians to justifiably starting second line anti-TB treatment.

A New Method for Determination of Rifampicin and Isoniazid Resistance in Mycobacterium tuberculosis complex Isolates: Capillary Tube Method

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.