Rifampin Resistance Research Articles

Newly designed antimicrobial peptides with potent bioactivity and enhanced cell selectivity prevent and reverse rifampin resistance in Gram-negative bacteria

The increasing prevalence of antibiotic resistance in Gram-negative bacteria calls for the discovery of novel effective therapeutic strategies urgently. Mastoparan-C (MP-C), a typical cationic α-helical antimicrobial peptide, possesses remarkable broad-spectrum antimicrobial activity. However, its high cytotoxicity toward normal mammalian cells precludes it for further development. In this study, to avoid non-specific membrane lysis and investigate the structure−function relationships of each amino acid of MP-C, a series of new MP-C analogs were rationally designed by amino acid substitution and peptide truncation. Three potential newly designed peptides L1G, L7A, and L1GA5K with excellent bioactivity, modest cell toxicity, low resistance tendency, and moderate stability to physiological salts and proteases were screened out. Moreover, the newly designed peptides showed synergy or additive effects against Gram-negative bacteria, when they combined with conventional antibiotics gentamicin, rifampin, and polymyxin B. The results from the time-kill kinetics, outer/inner membrane permeabilization, scanning electron microscope (SEM), and flow cytometry demonstrated that the newly designed peptides could kill bacteria rapidly by membrane destruction and intracellular contents leakage in a concentration and time-dependent manner. Specifically, the most cell-selective peptide L1GA5K exhibited potent antimicrobial activity against rifampin-resistant E. coli (RRE) and prevented the emergence of rifampin resistance in Enterobacter. Besides, L1GA5K was capable of reversing rifampin resistance in RRE through the outer membrane permeabilization when used in combination with rifampin. Collectively, our results suggested that the newly designed peptides are hopeful antibiotic alternatives, and the usage of them as an adjuvant to prevent and reverse antibiotic resistance is a promising strategy for tackling the risk of drug-resistant Gram-negative bacteria.

Coverage and fidelity of the Xpert MTB/RIF™ implementation in a high-burden area for pulmonary tuberculosis in Colombia

ResumenIntroducción. La prueba Xpert MTB/RIF™ es una prueba molecular rápida para el diagnóstico de la tuberculosis y la resistencia a la rifampicina. Desde el 2010 es la recomendada por la Organización Mundial de la Salud (OMS) y, aunque fue introducida en Colombia en el 2012, se desconocen los resultados de su uso.Objetivo. Describir la cobertura y la fidelidad en el uso de la prueba Xpert MTB/RIF™ en pacientes con tuberculosis pulmonar en una ciudad con alta carga de la enfermedad en Colombia.Materiales y métodos. Se hizo un estudio retrospectivo descriptivo de casos del programa de tuberculosis en Cali entre el 2013 y el 2019. La cobertura se estimó como el total de pruebas empleadas en los casos registrados en el programa. La fidelidad se midió con base en los protocolos internacionales de uso de la Xpert MTB/RIF™. Además, se hizo un análisis de correspondencias múltiples entre la prueba y las variables sociodemográficas. Resultados. Se incluyeron 6.328 pacientes con tuberculosis pulmonar, de los cuales 181 eran resistentes a los fármacos. La cobertura total de la Xpert MTB/RIF™ durante el periodo de estudio fue de 10,3 % (n=655), con una variación anual entre 0,2 y 23 %. La fidelidad fue de 46,8 % para los grupos de mayor riesgo de tuberculosis multirresistente (TB-MDR). El uso de la prueba se relacionó con la condición de ser hombre, afrocolombiano, y tener entre 41 y 60 años de edad.Conclusiones. La cobertura de la prueba Xpert MTB/RIF™ en Cali es baja y su uso no responde a la priorización recomendada para su implementación. Se requieren estrategias para promover su uso adecuado, de manera que contribuya a la meta de poner fin a la tuberculosis.

Detection of Mycobacterium tuberculosis in urine by GeneXpert MTB/RIF as a useful diagnostic tool in urinary tuberculosis

Background: Early and accurate diagnosis of urinary tuberculosis (UTB) is difficult as current diagnostic methods are lack of specificity (biochemical levels), lack of sensitivity (acid-fast bacilli stains), and time-consuming (TB culture). GeneXpert MTB/RIF has been endorsed by WHO and Indonesia Ministry of Health particularly for the sputum, and also gastric and lymph fluid samples. Given the limited data on the utility of GeneXpert MTB/RIF for urine samples, the recommendations do not apply to these samples. Case Description: A 38 y old woman was referred to the tertiary care hospital with suspected nephrolithiasis. She had a history of mild to moderate chronic flank pain and found to have left hydronephrosis on renal ultrasound. Ureteroscopy showed diffuse whitish necrosis of ureteral mucosa and ureteropelvic junction which was suspicious of tuberculous ureteritis. The acid-fast bacilli (AFB) smears gave negative result. Early morning sample was sent for GeneXpert MTB/RIF and the result was MTB detected with rifampin resistance not detected. The patient was managed with 6 m antituberculosis treatment according to the Ministry of Health regulation. Discussion: UTB was diagnosed using the urine AFB test, urine TB culture test and GeneXpert. Although the urine AFB test is simple, economical, and rapid, it has low sensitivity and specificity. TB culture has higher specificity compared AFB test but requires eight ws before the results are obtained. GeneXpert MTB/RIF, a real-time PCR, is a sensitive and specific test with 131 cfu/mL limit of detection that allows for early detection of TB. AFBs are intermittently shed in urine and the sensitivity of GeneXpert can be improved by using early morning urine for extended periods ranging from three to five days. Conclusion: GeneXpert MTB/RIF performed on urine samples is useful and can be recommended for rapid and reliable diagnosis of UTB in clinical practice esepecially in area with high TB burden. The diagnostic accuracy of urine GeneXpert MTB/RIF should be characterised further via prospective studies.

Rapid molecular assay gene Xpert in detection of multi-drug resistant tuberculosis (MTB/Rif)

Background: Tuberculosis (TB) is one of the deadliest threats today; TB due to Mycobacterium tuberculosis (MTB) remains a major public health issue: the infection affects up to one third of the world population, and almost two million people are killed by TB each year. However, in many areas of the world, TB diagnosis still relies on insensitive, poorly standardized sputum microscopy methods. Antibiotic resistance is a growing problem with increasing rate of drug-resistant tuberculosis (MDRTB) which is a serious public health problem in many developing countries. Treatment of MDTB takes longer and requires more expensive drugs. GeneXpert assay is a molecular technique that simultaneously detects (MTB) and Rifampicin resistant Tuberculosis (RR-TB) in approximately 2 h. Methods and materials: A retrospective cross sectional review of patients screened for TB with GeneXpert assay at TB Reference Laboratory,Khartoum State,Sudan from 20th June 2017 and 30th November 2017 (n = 583). Data analysis was done using Epi-Info Software, version 7.2 and Microsoft Office Excel 2007. Results: The total number of specimens collected from patients with symptoms of pulmonary TB was 583 (Males 402/Females 181). One hundred and fifty nine (159) (27.3%) of the screened samples had positive results for MTB, (Males 123/Females 36) of which 63 has history of past TB treatment, 71 New and 25 has un known past history of TB treatment. Among the positive MTB there is one case positive for HIV. Out of 159 there were 26 (4.4%) had rifampicin resistant MDR TB (22 males/4 females), all of MDR TB were more than 14 years, 16 has previous treatment of TB, 9 new and 1 has un known history of TB treatment. The male patients were more resistant to rifampicin when compared to the female patients.MTB was not detected in 424 (279 males 145 females). These despite the usefulness of GeneXpert assay method in early MTB/MDRTB diagnosis. Conclusion: Its simplicity, high sensitivity and specificity for RIF resistance detection make this technique a attractive tool for diagnostic of MTB and RIF resistance in MDR suspects.

Evaluation of Staphylococcal Bacteriophage Sb-1 as an Adjunctive Agent to Antibiotics Against Rifampin-Resistant Staphylococcus aureus Biofilms.

Rifampin plays a crucial role in the treatment of staphylococcal implant-associated infection, as it is the only antibiotic capable of eradicating Staphylococcus aureus biofilms. However, the emergence of rifampin resistance strongly limits its use. Combinatorial therapy of antibiotics and bacteriophages may represent a strategy to overcome the resistance. Here, we evaluated the activity of staphylococcal bacteriophage Sb-1 in combination with different antibiotics against the biofilms of 10 rifampin-resistant S. aureus clinical strains, including MRSA and MSSA. S. aureus biofilms formed on porous glass beads were exposed to antibiotics alone or combined with Sb-1 simultaneously or staggered (first Sb-1 for 24 h followed by antibiotic). Recovered bacteria were detected by measuring growth-related heat production at 37°C (isothermal microcalorimetry) and the biofilm eradication was assessed by sonication of beads and plating of the resulting sonication fluid. Minimum biofilm eradication concentration (MBEC) was defined as the lowest concentration of antibiotic required to kill all adherent bacteria, resulting in absence of growth after plating the sonication fluid. Tested antibiotics presented high MBEC values when administered alone (64 to > 1,024 μg/ml). The simultaneous or staggered combination of Sb-1 with daptomycin showed the highest activity against all MRSA biofilms, whereas the exposure to Sb-1 with vancomycin showed no improved anti-biofilm activity. Staggered administration of Sb-1 and flucloxacillin, cefazolin, or fosfomycin improved the antibiofilm activity in four out of six MSSA, whereas simultaneous exposure exhibited similar or lesser synergy. In conclusion, the combinatorial effect of Sb-1 and antibiotics enabled to eradicate rifampin-resistant S. aureus biofilms in vitro.

Tuberculosis following programmed cell death receptor-1 (PD-1) inhibitor in a patient with non-small cell lung cancer. Case report and literature review

Immune checkpoint inhibitors (ICIs)—anti-programmed death-1 (PD-1) and their ligands (PD-L1 and PD-L2) have become widely used in the treatment of several malignancies. Many immune-related adverse events (irAEs) have been linked to these agents. Nonetheless, tuberculosis (TB) reactivation during their use is increasingly recognized and reported. Herein, we present a 58-year-old lady with advanced non-small cell lung cancer (NSCLC) ALK-negative, EGFR wild, and PD-L1 immune histochemistry (IHC) strongly positive in 95% of tumor cells, on ongoing treatment with Pembrolizumab as a first-line monotherapy. Our patient presented with 1-week history of productive cough and high-grade fever. Further workup yielded the diagnosis of pulmonary tuberculosis after her Pembrolizumab sixth cycle with positive AFB smear and TB PCR from BAL (rifampin resistance not detected), with negative HIV status. Hence, immunotherapy was held, and patient was commenced on anti-TB regimen. History revealed contact with active TB patient over the past decade, without previous documentation of latent TB or previous TB infection. Her sputum AFB smear remained persistently positive 4 weeks through anti-TB regimen course. Later, the patient was discharged after her sputum was cleared from AFB (two negative sets). In light of pembrolizumab mechanism of action as an immune checkpoint inhibitor, we suspected its implication on reactivating latent TB which was observed in our patient demonstrating features of pulmonary tuberculosis. She was not re-challenged with Pembrolizumab following TB diagnosis.

CLINICAL APPLICATION OF RAPID DETERMINANTS OF RESISTANCE IN PULMONARY TB

SESSION TITLE: Chest Infections Posters SESSION TYPE: Original Investigation Posters PRESENTED ON: October 18-21, 2020 PURPOSE: In 2009, the Florida Bureau of Public Health Laboratories adopted universal MTBDRplus assay (Hain Lifescience, Nehren, Germany) testing of all diagnosed cases of infection with Mycobacteria tuberculosis (MTB). This study analyzed the concordance of genotypic molecular testing by the MTBDRplus assay with conventional culture susceptibilities by mean inhibitory concentrations (MIC) on all culture-positive pulmonary MTB cases in Florida. METHODS: This was a retrospective, programmatic evaluation that used existing Florida Department of Health data on pulmonary specimens from 2014. Patient identifiers were removed. Of 452 cases of pulmonary tuberculosis, 270 were lacking data on either the MTBDRplus assay, or culture susceptibilities, and were excluded. 182 pulmonary specimens were used. Genotypic resistance to Rifampin (RIF), and Isoniazid (INH), was determined by detection of mutations in the rpoB, and katG or inhA genes, which are associated with resistance to RIF, and INH, respectively. RESULTS: There was concordance between rpoB gene mutation and RIF resistance in 3 of 3 cases. There was concordance between katG gene mutation and INH resistance (MIC >1 ug/mL) in 5 of 5 cases. Of patients who were found to have intermediate INH susceptibility (MIC >0.12 to <1 ug/mL), the mutation in inhA gene was found in 5 of 8 cases. There was no detectable mutation in 3 of 8 cases of intermediate susceptibility. We observe the positive predictive value of the MTBDRplus assay to be 100% and the negative predictive value to be 98%, overall. The median time from specimen collection to reporting for the MTBDRplus assay was 8 days. The median time from specimen collection to reporting for the conventional culture susceptibilities was 44 days. CONCLUSIONS: The MTBDRplus assay proved to be a reliable and timely method for accurate detection of Rifampin and Isoniazid resistance in routine clinical practice, but was not sensitive to reliably detect intermediate susceptibility to INH. One explanation for this is that the assay only probes for a portion of the inhA genes and there are mechanisms of low level resistance outside of the inhA genes. Further development of rapid molecular probes for intermediate susceptibility to INH are needed as this finding impacts the choice of treatment regimens. CLINICAL IMPLICATIONS: The MTBDRplus assay allows for efficient identification of definite cases of drug-resistant pulmonary tuberculosis. We advocate for its clinical use based on the ability to start effective, antitubercular treatment before the results of conventional culture susceptibilities are available. DISCLOSURES: No relevant relationships by Brian Cody Adkinson, source=Web Response No relevant relationships by David Ashkin, source=Web Response No relevant relationships by Alex Ashkin, source=Web Response No relevant relationships by Sara Hulbert, source=Web Response No relevant relationships by Susanna Leonard, source=Web Response No relevant relationships by Marie-Claire Rowlinson, source=Web Response

Performance of Xpert/MTB/RIF assay for childhood pulmonary tuberculosis among HIV negative children with real world evidence in China

BackgroundRapid and accurate notification of childhood pulmonary tuberculosis (PTB) is a worldwide challenge. Although the Xpert MTB/RIF assay (Xpert) has been endorsed as the initial test for suspected PTB in many countries, limited studies have reported the performance of Xpert in childhood PTB. The aim of this study is to evaluate the real-world performance of Xpert for the detection of childhood PTB among HIV negative children in China. MethodsWe consecutively extracted the data of all patients ≤14 years with pulmonary disease through the electronic medical record (EMR) systems of Shanghai Public Health Clinical Center from January 2014 to December 2017. The clinical profile, the decision-making tests including AFB smear, solid/liquid culture, pathological examination and Xpert result were matched and assessed. The real diagnostic accuracy and the all-factors case notification rate for childhood PTB with the implementation of Xpert were evaluated. Results519 HIV negative cases ≤14 years with pulmonary disease were extracted from the data base. Of these, 145 had matched results, there were 374 non-matched cases including 346 with incomplete or unavailable data and 28 with NTM, BCG or an unidentified strain. For matched data, the overall sensitivity and specificity of the Xpert assay were 66.7% (32/48, 95%CI 0.52–0.80) and 87.6% (85/97, 95%CI 0.87–0.98) respectively against the gold standard; 34.6% (44/127, 95%CI 26.6–43.7) and 100% against the composite clinical reference standard (CCRS). The all-factors case notification rate by Xpert was 29%. ConclusionXpert/MTB RIF assay has acceptable sensitivity and excellent specificity for rapid diagnosis of children with pulmonary TB as well as for the detection of RIF resistance in China. However, implementation of Xpert for the initial diagnosis of childhood PTB is inadequate to meet the urgent requirement for rapid and accurate detection of childhood PTB.

The Performance of the Abbott Real Time MTB RIF/INH Compared to the MTBDRplus V2 for the Identification of MDR-TB Among Isolates.

BackgroundThe GenoType MTBDRplus V2 line-probe assay (LPA) is routinely used in clinical patient management to characterise the susceptibility of Mycobacterium tuberculosis complex to rifampicin (Rif) and isoniazid (INH) directly from sputum and cultured isolates. The laboratory workflow requires skill and three separate areas to minimize contamination and banding pattern interpretation requires experienced laboratory personnel. We explored the use of the RT MTB RIF/INH assay performed on the Abbott m2000 platform as an alternative laboratory platform.MethodsIsolates (n=93) consisting of fully susceptible, Rif- or INH-mono-resistant and multi-drug resistant (MDR) strains were tested on both MTBDRplus v2 and RT MTB RIF/INH assays. Both assays target the rpoB, katG and inhA genes for resistance-detection mutations. Concordance was assessed using percent agreement and the kappa statistic. Those specimens with discordant results were further assessed using Sanger sequencing.ResultsA total of 89% (83/93) of cultured isolates generated successful results on the RT MTB/RIF-INH assay and MTBDRplus assays. Of the 10 discordant results, where sequencing was used as the reference method, the RT MTB RIF/INH assays misclassified six resistance isolates, while the LPA misclassified seven.DiscussionOverall, the RT MTB RIF/INH demonstrated good agreement with the LPA, and a better correlation with sequencing on discrepant isolates specifically with mutations occurring in codon 511 of the rpoB gene. The RT MTB RIF/INH therefore can be used to complement existing laboratory algorithms determining Rif and INH resistance profiles, with less emphasis on manual laboratory processing.

An Overlooked Cause of Rifampin Resistance

An Overlooked Cause of Rifampin Resistance

A Semisynthetic Kanglemycin Shows In Vivo Efficacy against High-Burden Rifampicin Resistant Pathogens

Semisynthetic rifamycin derivatives such as rifampicin (Rif) are first line treatments for tuberculosis and other bacterial infections. Historically, synthetic modifications made to the C-3/C-4 region of the rifamycin naphthalene core, like those seen in Rif, have yielded the biggest improvements in pharmacological properties. However, modifications found in natural product rifamycin congeners occur at other positions in the structure. The kanglemycins (Kangs) are a family of rifamycin congeners with a unique collection of natural modifications including a dimethylsuccinic acid appended to their polyketide backbone. These modifications confer activity against the single most common clinically relevant Rif resistance (RifR) mutation in the antibiotic’s target, the bacterial RNA polymerase (RNAP). Here we evaluate the in vivo efficacy of Kang A, the parent compound in the Kang family, in a murine model of bacterial peritonitis/sepsis. We then set out to improve its potency by combining its natural tailoring modifications with semisynthetic derivatizations at either its acid moiety or in the C-3/C-4 region. A collection of C-3/C-4 benzoxazino Kang derivatives exhibit improved activity against wild-type bacteria, and acquire activity against the second most common clinically relevant RifR mutation. The semisynthetic analogue 3′-hydroxy-5′-[4-isobutyl-1-piperazinyl] benzoxazino Kang A (Kang KZ) protected mice against infection with either Rif sensitive MRSA or a highly virulent RifRStaphylococcus aureus strain in a neutropenic peritonitis/sepsis model and led to reduced bacterial burdens. The compounds generated in this study may represent promising candidates for treating RifR infections.

Rifamycin use for treatment of Helicobacter pylori infection: a review of recent data.

Helicobacter pylori eradication has become increasingly challenging. We focused on recent data about rifamycin resistance and rifamycin-containing regimens. Rifampin (rifampicin) resistance rates were <1-18.8% (often ≤7%), while those to rifabutin were 0-<4%. To detect rifabutin resistance by rifampin, 4 mg/l breakpoint was suggested. Eradication success by rifaximin-based regimens was disappointing (<62%), while that of rifabutin-containing regimens was 54.5->96%, reaching >81% in four studies. Some newer rifamycin analogs like TNP-2092 need further investigation. Briefly, although rifabutin-based regimens carry a risk of adverse effects or increasing mycobacterial resistance, they may be a rational choice for some multidrug-resistant H. pylori strains and as a third-line eradication therapy. Bismuth addition to rifabutin-based therapy and combined rifabutin-containing capsules (Talicia) are promising treatment options.

Machine learning reveals that Mycobacterium tuberculosis genotypes and anatomic disease site impacts drug resistance and disease transmission among patients with proven extra-pulmonary tuberculosis

BackgroundThere is a general dearth of information on extrapulmonary tuberculosis (EPTB). Here, we investigated Mycobacterium tuberculosis (Mtb) drug resistance and transmission patterns in EPTB patients treated in the Tshwane metropolitan area, in South Africa.MethodsConsecutive Mtb culture-positive non-pulmonary samples from unique EPTB patients underwent mycobacterial genotyping and were assigned to phylogenetic lineages and transmission clusters based on spoligotypes. MTBDRplus assay was used to search mutations for isoniazid and rifampin resistance. Machine learning algorithms were used to identify clinically meaningful patterns in data. We computed odds ratio (OR), attributable risk (AR) and corresponding 95% confidence intervals (CI).ResultsOf the 70 isolates examined, the largest cluster comprised 25 (36%) Mtb strains that belonged to the East Asian lineage. East Asian lineage was significantly more likely to occur within chains of transmission when compared to the Euro-American and East-African Indian lineages: OR = 10.11 (95% CI: 1.56–116). Lymphadenitis, meningitis and cutaneous TB, were significantly more likely to be associated with drug resistance: OR = 12.69 (95% CI: 1.82–141.60) and AR = 0.25 (95% CI: 0.06–0.43) when compared with other EPTB sites, which suggests that poor rifampin penetration might be a contributing factor.ConclusionsThe majority of Mtb strains circulating in the Tshwane metropolis belongs to East Asian, Euro-American and East-African Indian lineages. Each of these are likely to be clustered, suggesting on-going EPTB transmission. Since 25% of the drug resistance was attributable to sanctuary EPTB sites notorious for poor rifampin penetration, we hypothesize that poor anti-tuberculosis drug dosing might have a role in the development of resistance.

Increasing Drug Resistance Among Persons With Tuberculosis in Massachusetts, 2009-2018.

We examined Massachusetts tuberculosis surveillance data from to 2009 to 2018. Of 1533 culture-confirmed cases, 190 (12.4%) demonstrated resistance to isoniazid including 32 (2.1%) with rifampin resistance. In multivariable analysis, isoniazid resistance increased significantly over time (per-year odds ratio = 1.07, 95% confidence interval = 1.01–1.13, P = .018) and was associated with younger age, foreign birth, and prior tuberculosis treatment.

Strong Environment-Genotype Interactions Determine the Fitness Costs of Antibiotic Resistance In Vitro and in an Insect Model of Infection.

The acquisition of antibiotic resistance commonly imposes fitness costs, a reduction in the fitness of bacteria in the absence of drugs. These costs have been quantified primarily using in vitro experiments and a small number of in vivo studies in mice, and it is commonly assumed that these diverse methods are consistent. Here, we used an insect model of infection to compare the fitness costs of antibiotic resistance in vivo to those in vitro Experiments explored diverse mechanisms of resistance in a Gram-positive pathogen, Bacillus thuringiensis, and a Gram-negative intestinal symbiont, Enterobacter cloacae Rifampin resistance in B. thuringiensis showed fitness costs that were typically elevated in vivo, although these were modulated by genotype-environment interactions. In contrast, resistance to cefotaxime via derepression of AmpC β-lactamase in E. cloacae resulted in no detectable costs in vivo or in vitro, while spontaneous resistance to nalidixic acid, and carriage of the IncP plasmid RP4, imposed costs that increased in vivo Overall, fitness costs in vitro were a poor predictor of fitness costs in vivo because of strong genotype-environment interactions throughout this study. Insect infections provide a cheap and accessible means of assessing the fitness consequences of resistance mutations, data that are important for understanding the evolution and spread of resistance. This study emphasizes that the fitness costs imposed by particular mutations or different modes of resistance are extremely variable and that only a subset of these mutations is likely to be prevalent outside the laboratory.

Novel Use of Rifabutin and Rifapentine to Treat Methicillin-Resistant Staphylococcus aureus in a Rat Model of Foreign Body Osteomyelitis.

Owing to patient intolerance or drug interactions, alternative agents to rifampin are needed for management of staphylococcal periprosthetic joint infection. In the current study, we evaluated rifabutin, rifapentine and rifampin, with and without vancomycin, in a rat model of foreign body osteomyelitis. Proximal tibiae were inoculated with methicillin-resistant Staphylococcus aureus (MRSA) and a Kirschner wire (K-wire) implanted in each. After 4 weeks of infection, rifampin, rifabutin, or rifapentine were administered, alone or with vancomycin. Tibiae and K-wires were cultured, and medians were reported as log10 colony-forming units (CFUs) per gram of bone or log10 CFUs per K-wire, respectively. Rifampin, rifabutin or rifapentine administered with vancomycin yielded less MRSA from bones (0.10, 3.02, and 0.10 log10 CFUs/g, respectively) than did no treatment (4.36 log10 CFUs/g) or vancomycin alone (4.64 log10 CFUs/g) (both P ≤ .02). The K-wires of animals receiving no treatment or vancomycin monotherapy recovered medians of 1.76 and 2.91 log10 CFUs/g per K-wire, respectively. In contrast, rifampin, rifabutin and rifapentine administered with vancomycin yielded medians of 0.1 log10 CFUs per K-wire, respectively. Rifampin resistance was detected in a single animal in the rifampin monotherapy group. Rifabutin or rifapentine with vancomycin were as active as rifampin with vancomycin against MRSA in rat foreign body osteomyelitis, suggesting that rifabutin and/or rifapentine may be alternatives to rifampin in the clinical management of staphylococcal periprosthetic joint infections.

Interpretation of Discordant Rifampicin Susceptibility Test Results Obtained Using GeneXpert vs Phenotypic Drug Susceptibility Testing.

BackgroundThe 3-month difference in turnaround time between Xpert and conventional phenotypic drug susceptibility testing (pDST) causes patient treatment challenges when pDST rifampin (RIF) susceptibility results and earlier Xpert results disagree, resulting in unnecessary tuberculosis (TB) patient exposure to toxic second-line drugs. Here, the prevalence of discordant RIF susceptibility test results, specifically Xpert (resistant) vs pDST (susceptible) results, was determined.MethodsTuberculosis patients enrolled between January 2015 and June 2018 at Beijing Chest Hospital who consecutively tested positive for RIF resistance using Xpert then negative using pDST were studied. DNA sequences and minimal inhibitory concentration (MIC) results provided insights for understanding discordant results.ResultsOf 26 826 patients with suggestive TB symptoms undergoing Xpert MTB/RIF testing, 728 diagnosed as RIF-resistant were evaluated. Of these, 118 (16.2%) exhibiting Xpert RIF resistance and phenotypic RIF susceptibility yielded 104 successfully subcultured isolates; of these, 86 (82.7%) harbored rpoB gene RIF resistance–determining region mutations and 18 (17.3%) did not. The Leu511Pro (25.0%) and Leu533Pro (17.3%) mutations were most frequently associated with discordant RIF susceptibility test results. Of the 86 isolates with rpoB mutations, 42 (48.8%) with MICs ≤1.0 mg/L were assigned to the RIF-susceptible group, with Leu511Pro being the most common mutation observed. Isolates with a very low bacterial load were most frequently misdiagnosed as RIF-resistant by Xpert.ConclusionsApproximately one-sixth of RIF-resistant TB isolates identified via Xpert yielded discordant pDST results due to questionable interpretation of specific “disputed” mutations. Thus, a diagnostic flowchart should be used to correctly interpret Xpert RIF resistance results to best guide patient treatment.

Changes in treatment for multidrug-resistant tuberculosis according to national income.

The aim of this study was to analyse temporal changes in treatments for and outcomes of multidrug-resistant (MDR)/rifampin-resistant (RR)-tuberculosis (TB) in the context of national economic status.We analysed data collected by the Collaborative Group for the Meta-Analysis of Individual Patient Data in MDR-TB Treatment on MDR/RR-TB patients from 37 countries. The data were stratified by three national income levels (low-/lower-middle, upper-middle and high) and grouped by time of treatment initiation (2001-2003, 2004-2006, 2007-2009, 2010-2012 and 2013-2015). Temporal trends over the study period were analysed. The probability of treatment success in different income groups over time was calculated using generalised linear mixed models with random effects.In total, 9036 patients were included in the analysis. Over the study period, use of group A drugs (levofloxacin/moxifloxacin, bedaquiline and linezolid) recommended by the World Health Organization increased and treatment outcomes improved in all income groups. Between 2001-2003 and 2013-2015, treatment success rates increased from 60% to 78% in low-/lower-middle-income countries, from 40% to 67% in upper-middle-income countries, and from 73% to 81% in high-income countries. In earlier years, the probability of treatment success in upper-middle-income countries was lower than that in low-/lower-middle-income countries, but no difference was observed after 2010. However, high-income countries had persistently higher probability of treatment success compared to upper-middle income countries.Improved treatment outcomes and greater uptake of group A drugs were observed over time for patients with MDR/RR-TB at all income levels. However, treatment outcomes are still unsatisfactory, especially in upper-middle-income countries.

Probing the Molecular Mechanism of Rifampin Resistance Caused by the Point Mutations S456L and D441V on Mycobacterium tuberculosis RNA Polymerase through Gaussian Accelerated Molecular Dynamics Simulation.

Rifampin is the first-line antituberculosis drug, with Mycobacterium tuberculosis RNA polymerase as the molecular target. Unfortunately, M. tuberculosis strains that are resistant to rifampin have been identified in clinical settings, which limits its therapeutic effects. In clinical isolates, S531L and D516V (in Escherichia coli) are two common mutated codons in the gene rpoB, corresponding to S456L and D441V in M. tuberculosis However, the resistance mechanism at the molecular level is still elusive. In this work, Gaussian accelerated molecular dynamics simulations were performed to uncover the resistance mechanism of rifampin due to S456L and D441V mutations at the atomic level. The binding free energy analysis revealed that the reduction in the ability of two mutants to bind rifampin is mainly due to a decrease in electrostatic interaction, specifically, a decrease in the energy contribution of the R454 residue. R454 acts as an anchor and forms stable hydrogen bond interaction with rifampin, allowing rifampin to be stably incorporated in the center of the binding pocket. However, the disappearance of the hydrogen bond between R454 and the mutated residues increases the flexibility of the side chain of R454. The conformation of R454 changes, and the hydrogen bond interaction between it and rifampin is disrupted. As result, the rifampin molecule moves to the outside of the pocket, and the binding affinity decreases. Overall, these findings can provide useful information for understanding the drug resistance mechanism of rifampin and also can give theoretical guidance for further design of novel inhibitors to overcome the drug resistance.

A retrospective study on Xpert MTB/RIF for detection of tuberculosis in a teaching hospital in China

BackgroundThe Xpert MTB/RIF assay is an automated molecular test that is designed to simultaneously detect Mycobacterium tuberculosis (MTB) complex and rifampin resistance. However, there are relatively few studies on this method in China. Xpert has been routinely used at Peking University People’s Hospital (PKUPH) since November 2016. Thus, the aim of this study was to evaluate the performance of Xpert, and provide a reference and guidance for the detection and diagnosis of TB in non-TB specialized hospitals.MethodsThe medical records of inpatients simultaneously tested with Xpert, acid-fast bacilli (AFB) smear microscopy, and interferon-gamma release assay (IGRA, by T-SPOT®.TB) at PKUPH from November 2016 to October 2018 were reviewed. Active TB cases were considered according to a composite reference standard (CRS). Then, the three methods were evaluated and compared.ResultsIn total, 787 patients simultaneously tested with Xpert, AFB, and IGRA were enrolled; among them 11.3% (89/787) were diagnosed and confirmed active pulmonary TB (PTB, 52 cases), extrapulmonary TB (EPTB, 17 cases), and tuberculous pleurisy (TP, 20 cases). The sensitivity of Xpert in detecting PTB, EPTB, and TP was 88.5, 76.5, and 15.0%, respectively, which was slightly lower than IGRA (96.2, 82.4, and 95.0%, respectively), but higher than AFB (36.5, 11.8, and 0%, respectively); IGRA showed the highest sensitivity, but its specificity (55.9, 67.1, and 45.2%, respectively) was significantly lower than Xpert (99.6, 99.4, and 100%, respectively) and AFB (99.0, 99.4, and 100%, respectively) (P < 0.001). The sensitivity of Xpert in detecting lung tissue, cerebrospinal fluid, lymph nodes, and joint fluid was 100%, followed by sputum (88.5%), alveolar lavage (85.7%), and bronchoscopy secretion (81.2%); the pleural fluid sensitivity was the lowest, only 15.0%. For AFB negative patients, the sensitivity of Xpert in detecting PTB, EPTB, and TP was 84.9, 73.3, and 15.0%, respectively.ConclusionsXpert showed both high sensitivity and high specificity, and suggested its high value in TB diagnosis; however, the application of pleural fluid is still limited, and should be improved. Owing to the high sensitivity of IGRA, it is recommended for use as a supplementary test, especially for assisting in the diagnosis of TP and EPTB.