Rifampin Resistance Research Articles

Indwelling Urological Device Biofilm Composition and Characteristics in the Presence and Absence of Infection

ObjectiveTo characterize microbial biofilms associated with different device types used in the urological field including ureteral stents, sacral neuromodulation (SNM) devices, penile prostheses, and artificial urinary sphincters (AUS). Materials and MethodsData from four studies, each reporting biofilm composition of a particular device type, were pooled and included for inter-device analysis. Studies recruited adults scheduled for ureteral stent, SNM, IPP, or AUS removal/revision. Device (n=115) biofilms and controls were analyzed with multi-omics approaches, and compositions were compared across device types and clinical factors. ResultsMicrobiota present on each device type was distinct from that of perineal, rectal, or urine flora (p<0.01). Biofilm microbial counts (p<0.001) and diversity (p=0.024) differed by device type. Ureteral stents had greater microbial counts than other device types (p<0.001). Staphylococcus, Pseudomonas, Lactobacillus, Ureaplasma were commonly detected across devices. Device biofilms harbored a greater proportion of Proteobacteria phylum, and the rectal, perineal, and urine flora harbored a greater proportion of Firmicutes. Unique microbe-metabolite interaction networks were identified in presence and absence of infection. Antibiotic resistance genes including sul2 (sulfonamide resistance) and rpoB (rifampin resistance) were detected in biofilms across device types. Biofilm reconstitution in vitro differed by device type from which strains were isolated. ConclusionsUreteral stents, sacral neuromodulation devices, penile prostheses, and artificial urinary sphincters harbored unique microbial and metabolite profiles that differed from those of skin, urine, and rectal flora. The findings of this study set the groundwork for investigation of novel strategies to reduce device-associated infection risk within and outside urology. Data availability statementData are available from corresponding author upon reasonable request.

Identification of drug resistance-related virulence gene mutations in 667 clinical Mycobacterium tuberculosis isolates.

Drug-resistant tuberculosis is a severe global public health threat. Virulence factors and antibiotic resistance are generally considered to play a significant role in bacterial pathogenesis. However, the interaction between resistance and virulence in Mycobacterium tuberculosis (MTB) remains unclear. Here, we used whole genome sequences from 667 MTB isolates from 14 countries to complete an in silico evaluation of the correlations between virulence gene mutations, drug resistance, and lineage classification. The chi-square (χ2) test was used to determine whether specific virulence gene mutations and drug resistance were related. Our results showed that Mce1R_G171R and Pks15_V333A, were positively correlated with streptomycin and ethambutol resistance, respectively, and Pks15_T46I was correlated with isoniazid, rifampin, ethambutol, pyrazinamide and streptomycin resistance. We also identified an additional 24 and 40 single nucleotide polymorphisms as well as 6 and 2 insertions or deletions in various virulence genes that are likely to be associated with changes in drug susceptibility in L2 and L4, respectively. Taken together our data suggest that there may be some degree of co-selection between virulence and resistance factors, which may help MTB more easily adapt to new environments.

Infection by a multidrug-resistant Corynebacterium diphtheriae strain: prediction of virulence factors, CRISPR-Cas system analysis, and structural implications of mutations conferring rifampin resistance.

Cases of diphtheria, even in immunized individuals, are still reported in several parts of the world, including in Brazil. New outbreaks occur in Europe and other continents. In this context, studies on Corynebacterium diphtheriae infections are highly relevant, both for a better understanding of the pathogenesis of the disease and for controlling the circulation of clones and antimicrobial resistance genes. Here we present a case of cutaneous infection by multidrug-resistant Corynebacterium diphtheriae and provide its whole-genome sequencing. Genomic analysis revealed resistance genes, including tet(W), sul1, cmx, rpoB2, rbpA and mutation in rpoB. We performed phylogenetic analyzes and used the BRIG to compare the predicted resistance genes with those found in genomes from other significant isolates, including those associated with some outbreaks. Virulence factors such as spaD, srtBC, spaH, srtDE, surface-anchored pilus proteins (sapD), nonfimbrial adhesins (DIP0733, DIP1281, and DIP1621), embC and mptC (putatively involved in CdiLAM), sigA, dtxR and MdbA (putatively involved) in post-translational modification, were detected. We identified the CRISPR-Cas system in our isolate, which was classified as Type II-U based on the database and contains 15 spacers. This system functions as an adaptive immune mechanism. The strain was attributed to a new sequence type ST-928, and phylogenetic analysis confirmed that it was related to ST-634 of C. diphtheriae strains isolated in French Guiana and Brazil. In addition, since infections are not always reported, studies with the sequence data might be a way to complement and inform C. diphtheriae surveillance.

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.

Rifampin-resistant periprosthetic joint infections are associated with worse functional outcome in both acute and chronic infection types

Periprosthetic joint infections (PJI) pose a significant challenge in orthopaedic surgery, often requiring extensive surgical debridement and prolonged antibiotic treatment to eliminate the causative pathogens. Rifampin, known for its potent activity against biofilms, has been crucial in managing PJI by penetrating and disrupting these formations, thereby improving treatment efficacy. In this sense, antibiotic protocols lacking rifampin have shown increased failure rates. Consequently, the development of rifampin resistance could severely influence the prognosis of PJI. The aim of this clinical study was to assess how rifampin resistance affects the functional outcome in patients with PJI. In this single-centre comparative cohort study, we systematically documented all patients who presented with a PJI during the period spanning from 2018 to 2020. Two distinct groups were established for the study: Group 1 comprised 35 patients with a PJI caused by rifampin-susceptible pathogens and group 2 consisted of 28 patients with PJI caused by rifampin-resistant pathogens. A total of 63 patients (34 females) with a mean age of 68 years and a mean follow up of 37 months were included. The examination of patient-specific parameters did not reveal any identified risk factors as influential. Patients with a rifampin-resistant pathogen underwent a greater number of surgical revisions (6.9 ± 5.1 compared to 3.59 ± 3.39, p = 0.0011) and had extended durations of antibiotic treatment (p = 0.0052). The results of the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) score revealed significant differences in clinical outcome between both groups in every domain, even when stratified by acute and chronic entities. In total the WOMAC increased significantly from 21.57 ± 14.9 points in group 1 to 71.47 ± 62.7 points in group 2 (p < 0.001). The higher failure rates observed in group 2 were not statistically significant (p = 0.44). The current study demonstrates that PJI caused by rifampin-resistant bacteria are associated with a significantly worse functional outcome in both acute and chronic infection types without significantly affecting total failure rates.

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.

Multi-centric evaluation of Truenat MTB and MTB-RIF Dx assays for diagnosis of extrapulmonary tuberculosis

Extra-pulmonary TB (EPTB) is difficult to diagnose due to paucibacillary nature of disease. Current study evaluated accuracy of Truenat MTB and MTB-Rif Dx (TN), for detection of Mycobacterium tuberculosis and resistance to rifampicin. Samples were collected from 2103 treatment naive adults with presumptive EPTB, and tested by smear microscopy, liquid culture (LC) (MGIT-960) and GeneXpert MTB/RIF (GX) (Microbiological Reference Standards, MRS). TN results were compared to MRS and Composite Reference Standards (CRS, Microbiology, histopathology, radiology, clinical features prompting decision to treat, response to treatment). CRS grouped patients into 551 confirmed, 1096 unconfirmed, and 409 as unlikely TB. TN sensitivity and specificity was 73.7% and 90.4% against GX. Against LC, Overall sensitivity of GX was 67.6%, while that of TN was 62.3%. Highest sensitivity by TN was observed in pus samples (89%) and highest specificity (92%) in CSF samples, similar to GX. TN sensitivity was better in fluid and biopsy samples and slightly inferior for lymph node aspirates compared to GX. TN sensitivity for RIF resistance detection was slightly superior to GX. TN and GX results were further compared to Clinical Reference Standards. TN detected 170 TB patients initiated on treatment missed by GX, while GX detected 113 such patients missed by TN. Of 124 samples with RIF resistance discordance between GX and TN, GX reported 103/124 as sensitive, 3/124 as indeterminate and 18 as resistant (13/18 samples had low/very low DNA load) while TN reported RIF resistance indeterminate in 103/111 low/very low DNA load samples. Due to paucibacillary nature of EPTB samples, culture yield was poor and phenotypic drug susceptibility testing failed to resolve the discordance. The study establishes TN at par with GX and can be utilized for quick and accurate diagnosis of EPTB.

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.

A 10-year review of isoniazid-resistant TB management in Uzbekistan 2009-2020.

Isoniazid (INH, H) resistance is the most common drug-resistant TB pattern, with treatment success rates lower than those in drug-susceptible TB. The WHO recommends a 6-month regimen of rifampicin (RIF, R), ethambutol (EMB, E), pyrazinamide (PZA, Z), and levofloxacin (Lfx) (6REZLfx) for INH-resistant, RIF-susceptible TB (HRRS-TB). Uzbekistan has a high burden of TB (62/100,000 population) and multidrug-resistant TB (12/100,000 population). We conducted a retrospective, descriptive study of microbiologically confirmed HRRS-TB using routinely collected programmatic data from 2009 to 2020. We included 854 HRRS-TB cases. Treatment success was 80.2% overall. For REZLfx, the treatment success rate was 92.0% over a short treatment duration, with no amplifications to RIF or second-line anti-TB drug resistance. We documented 46 regimens with REZLfx plus linezolid (success 87.0%) and 539 regimens using kanamycin or capreomycin (success 76.6%). We identified 37 treatment failures (4.3%), 30 deaths (3.5%), 25 resistance amplifications (2.9%), including eight to RIF (0.9%), and 99 lost to follow-up (LTFU) cases (11.6%). Unsuccessful outcomes were more common with older age, diabetes, chest X-ray cavities, smear positivity, smear-positive persistence, and male sex. LTFU was more common with injection-containing regimens. REZLfx is a safe and effective first-line treatment for INH-resistant, RIF-susceptible TB. Treatment success was lower and LTFU was higher for injection-containing regimens.

The performance of A rpob gene mutation linked to A resistant to rifampicin mycobacterium tuberculosis isolate from an Indonesian referral hospital

IntroductionIndonesia has the world's second-highest tuberculosis and is one of the 30 countries with an increased MDR/RR TB burden. Even though the number of people with tuberculosis is rising every year, several cases of latent or active tuberculosis remain unreported. Microscopy has its limitations, and factors such as quality sample, quality smear, bacterial load, and experience of reading AFB influence the sensitivity of the test. Utilizing a quick, point-of-care testing system based on molecular techniques can increase the sensitivity of tuberculosis early diagnosis. Cepheid Xpert ®MTB/RIF is Indonesia's most widely utilized molecular technology. It is a nested real-time PCR diagnostic device that is cartridge-based and semiquantitative. In the TB complex of Mycobacterium tuberculosis, it identifies alterations in the RNA polymerase (rpoB) gene beta-subunit that result in rifampicin resistance (RR). By utilizing the Xpert ® MTB/RIF assay at Soetomo General Hospital, one of Indonesia's primary tertiary referral hospitals, the current study seeks to improve our knowledge of the prevalence of rpoB gene mutations in individuals with tuberculosis. MethodsBetween January 2021 and December 2021, Soetomo General Hospital used the Xpert®MTB/RIF system to screen sputum and extrapulmonary specimens from 3584 for tuberculosis. ResultsOf the 665 samples, 18,5% tested positive for M. tuberculosis, with 7.6% (51 out of 665) showing resistance to Rifampicin Rifampicin compounds at codons 529–533 (probe E) of the rpoB gene were detected in 56.8% (29/51) of rifampicin-resistant tuberculosis (RR-TB) samples. And then Probe D (19.6%). 7.8% for probes A and B, respectively (5.8%). Probe C did not exhibit any mutations. On the other hand, two samples revealed two mutations in probes D and E, while among 13.7% (7/51) of RR TB samples, no mutations in the probe type were observed. ConclusionThe Xpert®MTB/RIF system is a compassionate, fast, accurate, and efficient diagnostic tool for RR-TB, tuberculosis, and the molecular epidemiology of mutations associated with rifampin resistance.

Challenging the gold standard: the limitations of molecular assays for detection of Mycobacterium tuberculosis heteroresistance

ObjectivesHeteroresistant infections are defined as infections in which a mixture of drug-resistant and drug-susceptible populations are present. In Mycobacterium tuberculosis (M. tb), heteroresistance poses a challenge in diagnosis and has...

Sacral neuromodulation device biofilm differs in the absence and presence of infection, harbors antibiotic resistance genes, and is reproducible in vitro.

Sacral neuromodulation (SNM) is effective therapy for overactive bladder refractory to oral therapies, and non-obstructive urinary retention. A subset of SNM devices is associated with infection requiring surgical removal. We sought to compare microbial compositions of explanted devices in the presence and absence of infection, by testing phase, and other clinical factors, and to investigate antibiotic resistance genes present in the biofilms. We analyzed resistance genes to antibiotics used in commercially-available anti-infective device coating/pouch formulations. We further sought to assess biofilm reconstitution by material type and microbial strain in vitro using a continuous-flow stir tank bioreactor, which mimics human tissue with an indwelling device. We hypothesized that SNM device biofilms would differ in composition by infection status, and genes encoding resistance to rifampin and minocycline would be frequently detected. Patients scheduled to undergo removal or revision of SNM devices were consented per IRB-approved protocol (IRB 20-415). Devices were swabbed intraoperatively upon exposure, with controls and precautions to reduce contamination of the surrounding field. Samples and controls were analyzed with next-generation sequencing and RT-PCR, metabolomics, and culture-based approaches. Associations between microbial diversity or microbial abundance, and clinical variables were then analyzed using t-tests and ANOVA. Reconstituted biofilm deposition in vitro using the bioreactor was compared by microbial strain and material type using plate-based assays and scanning electron microscopy. Thirty seven devices were analyzed, all of which harbored detectable microbiota. Proteobacteria, Firmicutes and Actinobacteriota were the most common phyla present overall. Beta-diversity differed in the presence versus absence of infection (p = 0.014). Total abundance, based on normalized microbial counts, differed by testing phase (p < 0.001), indication for placement (p = 0.02), diabetes mellitus (p < 0.001), cardiac disease (p = 0.008) and history of UTI (p = 0.008). Significant microbe-metabolite interaction networks were identified overall and in the absence of infection. 24% of biofilms harbored the tetA tetracycline/minocycline resistance gene and 53% harbored the rpoB rifampin resistance gene. Biofilm was reconstituted across tested strains and material types. Ceramic and titanium did not differ in biofilm deposition for any tested strain. All analyzed SNM devices harbored microbiota. Device biofilm composition differed in the presence and absence of infection and by testing phase. Antibiotic resistance genes including to rifampin and tetracycline/minocycline, which are used in commercially-available anti-infective pouches, were frequently detected. Isolated organisms from SNM devices demonstrated the ability to reconstitute biofilm formation in vitro. Biofilm deposition was similar between ceramic and titanium, materials used in commercially-available SNM device casings. The findings and techniques used in this study together provide the basis for the investigation of the next generation of device materials and coatings, which may employ novel alternatives to traditional antibiotics. Such alternatives might include bacterial competition, quorum-sensing modulation, or antiseptic application, which could reduce infection risk without significantly selecting for antibiotic resistance.

Evaluation of a real-time PCR assay performance to detect Mycobacterium tuberculosis, rifampicin, and isoniazid resistance in sputum specimens: a multicenter study in two major cities of Indonesia.

Tuberculosis (TB) is one of the major global health issues due to its high mortality rate, especially in low- and middle-income countries. One of the key success points of the TB eradication program is early TB diagnosis, which requires rapid and accurate diagnostic testing. This study aimed to evaluate the performance of a newly developed RT-PCR kit (Indigen MTB/DR-TB RT-PCR) in a routine TB clinical setting. A multi-fluorescence RT-PCR assay was designed and developed to detect regions within IS6110, rpoB, katG, and inhA of the Mycobacterium tuberculosis (MTB) genes. Sputum specimens were obtained from suspected TB patients who visited TB healthcare facilities in two major cities of Indonesia from September 2022 to May 2023. Specimens were assessed using Indigen MTB/DR-TB RT-PCR, acid-fast bacillus (AFB) smear microscopy, MTB culture, and drug susceptibility testing (DST) methods. Fisher's exact test (χ2) was used to analyze the Indigen performance relative to culture methods. The performance of Indigen MTB/DR-TB RT-PCR to detect MTB was assessed using 610 sputum specimens obtained from suspected patients. The overall sensitivity and specificity were 94.12% (95% CI: 90.86-96.48%) and 98.32% (95% CI: 96.20-99.46%), respectively. When the analysis was performed on AFB smear-negative TB subjects (386 subjects), a lower sensitivity level was found at 78.57% (95% CI: 68.26-86.78%), while the specificity level remained similar at 98.34% (95% CI: 96.18-99.46%). The overall performance of Indigen MTB/DR-TB RT-PCR to detect MTB showed substantial agreement with the MTB culture method (kappa value 0.93). In comparison to DST, the sensitivity and specificity levels of Indigen to detect RIF resistance or INH resistance were 78.2% (95% CI: 61.8-90.2%) and 82.8% (95% CI: 64.2-94.2%), respectively, while the specificity level for both groups was at 100% (95% CI, 87.7-100%). Indigen MTB/DR-TB RT-PCR demonstrated reliable performance for TB molecular diagnostic testing and can be implemented in routine TB diagnostic settings.

Performance of the BD MAX MDR-TB assay in a clinical setting and its impact on the clinical course of patients with pulmonary tuberculosis: a retrospective before-after study.

Missing isoniazid (INH) resistance during tuberculosis (TB) diagnosis can worsen the outcomes of INH-resistant TB. The BD MAX MDR-TB assay (BD MAX) facilitates the rapid detection of TB and INH and rifampin (RIF) resistance; however, data related to its performance in clinical setting remain limited. Moreover, its effect on treatment outcomes has not yet been studied. We compared the performance of BD MAX for the detection of INH/RIF resistances to that of the line probe assay (LPA) in patients with pulmonary TB (PTB), using the results of a phenotypic drug sensitivity test as a reference standard. The treatment outcomes of patients who used BD MAX were compared with those of patients who did not. Of the 83 patients included in the study, the BD MAX was used for an initial PTB diagnosis in 39 patients. The sensitivity of BD MAX for detecting PTB was 79.5%. The sensitivity and specificity of BD MAX for INH resistance were both 100%, whereas these were 50.0% and 95.8%, respectively, for RIF resistance. The sensitivity and specificity of BD MAX were comparable to those of LPA. The BD MAX group had a shorter time interval from specimen request to the initiation of anti-TB drugs (2.0 days vs. 5.5 days, p=0.001). BD MAX showed comparable performance to conventional tests for detecting PTB and INH/RIF resistances. The implementation of BD MAX as a diagnostic tool for PTB resulted in a shorter turnaround time for the initiation of PTB treatment.

Phenotypic and Genotypic Study of Staphylococcus Haemolyticus and Staphylococcus Warneri Clinically Isolated from the Bloodstream

Background: The Staphylococcus haemolyticus and Staphylococcus warneri are coagulase-negative staphylococci (CoNS) as part of human skin’s natural flora. In recent years, CoNS has increased its visibility as opportunistic pathogens acquired in hospital patients. This study aims to isolate S. haemolyticus and S. warneri strains from patients with bloodstream infections and to analyze the antimicrobial profile of these bacteria by molecular detection of the mecA gene. Materials and Methods: Four hundred and twenty-three clinical blood samples were collected from patients admitted to general three hospitals in Hilla city, Iraq, during the period from September 2021 to July 2022. Blood samples were inoculated immediately into blood bottles containing 70 ml of Brain-Heart Infusion Broth. All isolates were identified by morphology, biochemical tests, and VITEK 2 automated system (bioMerieux, France). The Congo red agar plate test was used to detect biofilm production. The mecA gene was detected by conventional polymerase chain reaction. Results: S. haem olyticus (nine isolates) and S. warneri (six isolates) were detected out of 423 clinical blood samples. Molecular detection of the mecA gene was detected in all 15 isolates. As well 11 (73.3%) isolates were β-lactam resistant and biofilm production. The results of oxacillin-resistance minimum inhibitory concentration (MIC) (≥4 mg/ml) showed that 11 (73.3%) of the 15 isolates were oxacillin and methicillin-resistant CoNS (MR-CoNS). Susceptibility to various isolates (MICs µg/ml) using VITEK 2 system showed erythromycin (≥8 µg/ml), rifampin (≥4 µg/ml), clindamycin (≥4 µg/ml), and trimethoprim-sulfamethoxazole (≥4 µg/ml) resistance were (80%), (13.3%), (20%), and (53.3%), respectively. All isolates were (100%) sensitive to linezolid (≥8 µg/ml) and teicoplanin (≥32 µg/ml). Conclusions: The S. haemolyticus and S. warneri are serious infections associated with bacteremia and septicemia infections. Linezolid, teicoplanin, and rifampin are the drug of choice for the treatment of infections caused by MR-CoNS isolates. A high rate of the mecA gene and biofilm production is present in these bacteria.

Comparison of the real-time polymerase chain reaction and xpert Mycobacterium tuberculosis/rifampicin assay for the detection of Mycobacterium tuberculosis

Abstract Background A serious issue with world health is tuberculosis (TB). Annual estimates from the World Health Organization (WHO) put the number of incident cases at 9.6 million. The aim of this work was to compare between real-time polymerase chain reaction (PCR) and GeneXpert in recognition of Mycobacterium tuberculosis (MTB) with special reference to quantification of MTB and to speed of detection of the organism. Materials and Methods Thirty individuals participated in this prospective clinical quasi-interventional trial with clinical data and chest radiography suggestive of pulmonary tuberculosis. Participants were split into three groups equally: group I (control group) was zielneilsen stain (ZN) stain positive and culture positive, group II were ZN stain negative and culture positive and group III were ZN stain negative and culture negative. Each participant was exposed to a plain chest radiography and a complete blood picture. Results Real-time PCR result was significantly correlated with loss of appetite and weight in group III. There were negative correlations between PCR and mediastinal lymphadenopathy in group I. In group I GeneXpert was significantly correlated with night sweat and fever, breathlessness and cavitary lesion. In group III GeneXpert was significant correlated with site of lesion and night sweat and fever. Conclusions For TB patients, the PCR test is crucial in order to provide prompt diagnosis and therapy. When comparing AdvanSure tuberculous /non tuberculous mycobacterium (TB/NTM) real-time PCR with Xpert MTB/rifampicin (RIF) assay, the latter is more advantageous due to its test-method simplicity and quicker than real-time PCR ability to simultaneously identify rifampin resistance. However, its sensitivity for specimens with an acid-fast bacilli (AFB) smear negative but a positive culture is less than AdvanSure TB/NTM real-time PCR. With special reference to the quantification of MTB, TB/NTM real-time PCR is a quantitative test but Xpert MTB/RIF is semiquanitative.

Targeted sequencing from cerebrospinal fluid for rapid identification of drug-resistant tuberculous meningitis.

Mortality from tuberculous meningitis (TBM) remains around 30%, with most deaths occurring within 2 months of starting treatment. Mortality from drug-resistant strains is higher still, making early detection of drug resistance (DR) essential. Targeted next-generation sequencing (tNGS) produces high read depths, allowing the detection of DR-associated alleles with low frequencies. We applied Deeplex Myc-TB-a tNGS assay-to cerebrospinal fluid (CSF) samples from 72 adults with microbiologically confirmed TBM and compared its genomic drug susceptibility predictions to a composite reference standard of phenotypic susceptibility testing (pDST) and whole genome sequencing, as well as to clinical outcomes. Deeplex detected Mycobacterium tuberculosis complex DNA in 24/72 (33.3%) CSF samples and generated full DR reports for 22/24 (91.7%). The read depth generated by Deeplex correlated with semi-quantitative results from MTB/RIF Xpert. Alleles with <20% frequency were seen at canonical loci associated with first-line DR. Disregarding these low-frequency alleles, Deeplex had 100% concordance with the composite reference standard for all drugs except pyrazinamide and streptomycin. Three patients had positive CSF cultures after 30 days of treatment; reference tests and Deeplex identified isoniazid resistance in two, and Deeplex alone identified low-frequency rifampin resistance alleles in one. Five patients died, of whom one had pDST-identified pyrazinamide resistance. tNGS on CSF can rapidly and accurately detect drug-resistant TBM, but its application is limited to those with higher bacterial loads. In those with lower bacterial burdens, alternative approaches need to be developed for both diagnosis and resistance detection.

Persistence and evolution of linezolid- and methicillin-resistant Staphylococcus epidermidis ST2 and ST5 clones in an Italian hospital

Staphylococcus epidermidis is a member of the human skin microbiome. However, in recent decades, multidrug-resistant and hospital-adapted S. epidermidis clones are increasingly involved in severe human infections associated with medical devices and in immunocompromised patients. In 2016, we reported that a linezolid- and methicillin-resistant S. epidermidis ST2 clone, bearing the G2576T mutation, was endemic in an Italian hospital since 2004. This study aimed to retrospectively analyse 34 linezolid- and methicillin-resistant S. epidermidis (LR-MRSE) strains collected from 2018 to 2021 from the same hospital. LR-MRSE were typed by Pulsed-Field Gel Electrophoresis and multilocus sequence typing and screened for transferable linezolid resistance genes. Representative LR-MRSE were subjected to whole-genome sequencing (WGS) and their resistomes, including the presence of ribosomal mechanisms of linezolid resistance and of rpoB gene mutations conferring rifampin resistance, were investigated. ST2 lineage was still prevalent (19/34; 55.9%), but, over time, ST5 clone has been widespread too (15/34; 44.1%). Thirteen of the 34 isolates (38.2%) were positive for the cfr gene. Whole-genome sequencing analysis of relevant LR-MRSE displayed complex resistomes for the presence of several acquired antibiotic resistance genes, including the SCCmec type III (3A) and SCCmec type IV (2B) in ST2 and ST5 isolates, respectively. Bioinformatics and polymerase chain reaction (PCR) mapping also showed a plasmid-location of the cfr gene and the occurrence of previously undetected mutations in L3 (ST2 lineage) and L4 (ST3 lineage) ribosomal proteins and substitutions in the rpoB gene. The occurrence of LR-MRSE should be carefully monitored in order to prevent the spread of this difficult-to-treat pathogen and to preserve the efficacy of linezolid.

Insights into the in-vitro Susceptibility and Drug-Drug Interaction Profiles Against Drug-Resistant and Susceptible Mycobacterium tuberculosis Clinical Isolates in Amhara, Ethiopia.

In Ethiopia, tuberculosis (TB) is a major public health problem. The aim of the study was to determine the in vitro susceptibility level of drugs and drug interaction profiles against drug-resistant and susceptible M. tuberculosis clinical isolates. A laboratory-based cross-sectional study was conducted between January 2023 and August 2023. GenoType MTBDRplus v.2.0 was facilitated in genetic mutation detection. Minimum inhibitory concentration (MIC) was determined using resazurin microtitre assay (REMA), while fractional inhibitory concentration index (FICI) using resazurin drug combination microtitre assay (REDCA) for in vitro quantitative susceptibility and drug interaction prediction. Among 32 clinical isolates, a total of 14 (43.8%) RIF, 20 (62.5%) INH, 2 (6.3%) EMB-related resistant and 14 (43.8%) MDR isolates were identified. Five of RIF-resistant isolates (55.6%) carrying rpoB common mutations at codon S450L were associated with high levels of RIF-resistance with MICs of ≥ 2μg/mL, whereas 100% of isolates harboring rpoB substitutions at codons D435V and H445Y were linked with moderate or low-level RIF-resistance in the MIC ranges from 0.5 to 1μg/mL. A proportion of 81.8% of isolates harboring katG S315T mutations were associated with high-level INH resistance (MIC ≥ 1μg/mL), while the 18.2% of isolates with S315T katG mutations and 100% of isolates with inhA C-15T mutations were linked to the low-level of INH resistance with MIC variability from 0.25 to 0.5μg/mL. Our results indicated that most FICIs of the dual drugs INH+RIF and INH+LEV combination for 9 (28.1%) and 4 (12.5%) INH-resistant isolates, respectively, were ≤0.5, whereas triple drugs INH+RIF+EMB, INH+RIF+LEV and INH+EMB+LEV combination for 6 (18.8%), 11 (34.4%) and 8 (25%) INH-resistant isolates were from 0.62 to 0.75, all showed synergistic effect. The study highlights that isolates with rpoB S450L and katG S315T substitutions were associated with high level of RIF and INH resistance. It is concluded that REDCA can quantitatively determine anti-mycobacterial synergy and that LEV being of potential use against INH-resistant isolates including MDR-TB when combined with RIF+INH and INH+EMB.