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Background The BD MAX MDR-TB is a recently marketed molecular test for detecting Mycobacterium tuberculosis complex (MTC), rifampin, and isoniazid drug resistance. Research design and methods This study aimed to evaluate the BD MAX MDR-TB test performance in 933 extrapulmonary and 774 pulmonary samples. Results Test MTC detecting sensitivity was 90.6 %, 82.5%, and the specificity was 98.5%, 98.9%, in pulmonary and extrapulmonary samples, respectively. In smear-positive samples, sensitivity and specificity were 100 % for all samples. However, in smear-negative samples, the test's sensitivity and specificity were 82.3%, 98.5% in pulmonary samples, and 76.7%, 98.9% in extrapulmonary samples. Test sensitivity in detecting isoniazid resistance was 71.4%, specificity 96.8%, and in detecting rifampin resistance was 100%, specificity 93.9%, respectively. Conclusions BD MAX MDR-TB is a reliable, rapid, user-friendly test for detecting MTC in extrapulmonary and pulmonary samples and its resistance toward isoniazid and rifampin. It can be used as an alternative to the Xpert system assays.

Background: With the introduction of Xpert MTB/RIF assay (Xpert), its incorporation into tuberculosis (TB) diagnostic algorithm has become an important issue. The aim of this study was to evaluate the performance of the Xpert assay in comparison with a commercial polymerase chain reaction (PCR) assay.Methods: Medical records of patients having results of both Xpert and AdvanSure TB/NTM real-time PCR (AdvanSure) assays using the same bronchial washing specimens were retrospectively reviewed.Results: Of the 1,297 patients included in this study, 205 (15.8%) were diagnosed with pulmonary TB. Using mycobacterial culture as the reference method, sensitivity of the Xpert assay using smear-positive specimens was 97.5%, which was comparable to that of the AdvanSure assay (96.3%, p=0.193). However, the sensitivity of the Xpert assay using smear-negative specimens was 70.6%, which was significantly higher than that of the AdvanSure assay (52.9%, p=0.018). Usng phenotypic drug susceptibility testing as the reference method, sensitivity and specificity for detecting rifampicin resistance were 100% and 99.1%, respectively. Moreover, a median turnaround time of the Xpert assay was 1 day, which was significantly shorter than 3 days of the AdvanSure assay (p

BACKGROUND: Recommended by the World Health Organization as an initial diagnostic test for TB in children, Xpert® MTB/RIF is widely implemented in many countries, including Kenya.METHODS: Three hundred HIV-positive and negative children (RESULTS: Of 32 children with bacteriologically confirmed TB, 27 had positive Xpert results. Of these, 3/27 (11%, 95% CI 4–28) had RIF resistance detected on Xpert, but not by phenotypic DST, line-probe assay, or sequencing. For these three children, five Xpert tests showed RIF resistance; all five tests had semi-quantitative “very low” results and delay or absence of probe D signal, whereas no Xpert results with higher semi-quantitative results showed RIF resistance. All three children responded well to standard TB treatment.CONCLUSIONS: False RIF resistance may be detected in pediatric specimens. Further study is needed to determine if false RIF resistance is associated with low bacterial load.

Ma-chao Li,1,* Jie Lu,2,* Yao Lu,1,3,* Tong-yang Xiao,4,* Hai-can Liu,1 Shi-qiang Lin,5 Da Xu,1 Gui-lian Li,1 Xiu-qin Zhao,1 Zhi-guang Liu,1 Li-li Zhao,1 Kang-lin Wan1 1State Key Laboratory of Infectious Disease Prevention and Control, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, People’s Republic of China; 2Beijing Key Laboratory for Pediatric Diseases of Otolaryngology, Head and Neck Surgery, MOE Key Laboratory of Major Diseases in Children, Beijing Pediatric Research Institute, Beijing Children’s Hospital, Capital Medical University, National Center for Children’s Health, Beijing, People’s Republic of China; 3School of Laboratory Medicine and Life Sciences, Wenzhou Medical University, Wenzhou, People’s Republic of China; 4Guangdong Key Laboratory for Diagnosis & Treatment of Emerging Infectious Diseases, Shenzhen Third People’s Hospital, Southern University of Science and Technology, Shenzhen, People’s Republic of China; 5Department of Bioinformatics, College of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou, People’s Republic of China*These authors contributed equally to this workCorrespondence: Li-li Zhao; Kang-lin WanNational Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, P.O. Box 5, Changping, Beijing, 102206, People’s Republic of ChinaTel/Fax +86 10 58900779Email zhaolili@icdc.cn; wankanglin@icdc.cnObjective: To investigate the mutations within the whole rpoB gene of Mycobacterium tuberculosis and analyze their effects on rifampin (RIF) resistance based on crystal structure.Methods: We sequenced the entire rpoB gene in 175 tuberculosis isolates and quantified their minimum inhibitory concentrations using microplate-based assays. Additionally, the structural interactions between wild-type/mutant RpoB and RIF were also analyzed.Results: Results revealed that a total of 34 mutations distributed across 17 different sites within the whole rpoB gene were identified. Of the 34 mutations, 25 could alter the structural interaction between RpoB and RIF and contribute to RIF resistance. Statistical analysis showed that S450L, H445D, H445Y and H445R mutations were associated with high-level RIF resistance, while D435V was associated with moderate-level RIF resistance.Conclusion: Some mutations within the rpoB gene could affect the interaction between RpoB and RIF and thus are associated with RIF resistance. These findings could be helpful to design new antibiotics and develop novel diagnostic tools for drug resistance in TB.Keywords: Mycobacterium tuberculosis, rifampin resistance, structure, mutation

Lin Qin,1,&ast; Fengmin Huo,2,&ast; Weicong Ren,2,&ast; Yuanyuan Shang,2 Cong Yao,2 Xuxia Zhang,2 Rongmei Liu,3 Liping Ma,3 Mengqiu Gao,3 Yu Pang2 1Department of Endoscopic Diagnosis & Treatment, Beijing Chest Hospital, Capital Medical University/Beijing Tuberculosis & Thoracic Tumor Research Institute, Beijing, 101149, People’s Republic of China; 2Department of Bacteriology and Immunology, Beijing Key Laboratory on Drug-Resistant Tuberculosis Research, Beijing Chest Hospital, Capital Medical University/Beijing Tuberculosis & Thoracic Tumor Research Institute, Beijing, 101149, People’s Republic of China; 3Department of Tuberculosis, Beijing Chest Hospital, Capital Medical University/Beijing Tuberculosis & Thoracic Tumor Research Institute, Beijing, 101149, People’s Republic of China&ast;These authors contributed equally to this workCorrespondence: Mengqiu GaoDepartment of Tuberculosis, Beijing Chest Hospital, Capital Medical University/Beijing Tuberculosis & Thoracic Tumor Research Institute, No. 9, Beiguan Street, Tongzhou District, Beijing, 101149, People’s Republic of ChinaTel/Fax +86-10-8950 9322Email gaomqwdm@aliyun.comYu PangDepartment of Bacteriology and Immunology, Beijing Key Laboratory on Drug-Resistant Tuberculosis Research, Beijing Chest Hospital, Capital Medical University/Beijing Tuberculosis & Thoracic Tumor Research Institute, No. 9, Beiguan Street, Tongzhou District, Beijing, 101149, People’s Republic of ChinaTel/Fax +86-10-8950 9359Email pangyupound@163.comIntroduction: We assessed the effect of Mycobacterium tuberculosis (MTB) bacterial load on Xpert MTB/RIF accuracy for detection of rifampicin (RIF)-resistant MTB in bronchoalveolar lavage fluid (BALF) specimens obtained at a national tuberculosis (TB) specialized hospital in Beijing, China.Methods: A retrospective study was conducted at Beijing Chest Hospital. Patients with symptoms suggestive of pulmonary TB who provided BALF specimens for routine MTB detection between June 2019 and July 2020 were enrolled in the study. Chi-square test and Student’s t-test were used to compare results across groups stratified according to BALF bacterial load.Results: In total, 1125 patients with positive Xpert results who were enrolled in final analysis, 263 provided BALF specimens that tested positive for RIF-resistant MTB via Xpert MTB/RIF. The RIF-resistance rate of specimens with very low MTB bacterial load was 30.9%, a resistance rate significantly greater than rates obtained for groups with high (25.0%), medium (17.3%) and low (19.2%) MTB loads (P< 0.01). Notably, false-positive results obtained for the very low bacterial load group led to markedly reduced positive predictive value of Xpert MTB/RIF to provide correct RIF-resistance predictions for that group (67.1%, 95% CI: 56.1%– 78.1%5) relative to the predictive value obtained for all other groups combined (about 90%, P< 0.05). Sanger sequencing data obtained for 20 (32.8%) MTB isolates deemed RIF-resistant via Xpert (Probe E) lacked rpoB RRDR mutations. Meanwhile, of another group of 23 isolates deemed RIF-susceptible via DST but RIF-resistant via Xpert MTB/RIF, 20 isolate sequences (87.0%) lacked rpoB RRDR mutations, while sequences of the remaining 3 isolates harbored single rpoB RRDR mutations predicted to cause amino acid substitutions.Conclusion: Xpert MTB/RIF assay performed alarmingly poorly when used to detect RIF-resistant MTB in BALF specimens with very low bacterial loads. A high rate of Xpert probe E hybridization failure was the main driver of false-positive RIF-resistant results.Keywords: rifampicin, bronchoalveolar lavage fluid, Xpert MTB/RIF, China

Background To appropriately treat tuberculosis (TB) with regimens that combine novel and older drugs, evidence-based, context-specific strategies for drug-susceptibility testing (DST) will be required. Methods We created a Markov state-transition model of 100 000 adults with TB receiving a novel, fluoroquinolone (FQ)–containing regimen. We estimated clinical outcomes and resource utilization with no FQ-DST, universal FQ-DST, or FQ-DST only for patients with rifampin-resistant TB (“targeted FQ-DST”). We considered scenarios of stronger (South Africa) and weaker (Southeast Asia) correlation of fluoroquinolone resistance with rifampin resistance, with sensitivity analysis for other setting and regimen characteristics. Results Relative to no FQ-DST, targeted FQ-DST increased cure of FQ-resistant TB by 7.5% (interquartile range [IQR], 6.7%–9.2%) in South Africa and 1.7% (IQR, 0.7%–2.5%) in Southeast Asia. However, rare FQ resistance among the more prevalent rifampin-susceptible TB accounted for 50% of FQ-resistant TB in South Africa and 83% in Southeast Asia. As a result, universal FQ-DST further increased cure of FQ-resistant TB by 3.4% (IQR, 2.3%–5.4%) in South Africa and 5.8% (IQR, 5.1%–6.3%) in Southeast Asia. With targeted FQ-DST, 1 additional patient was cured per 50 (IQR, 42–70) tests in South Africa and 44 (IQR, 37–51) in Southeast Asia. When expanding from targeted to universal FQ-DST, 1 additional cure required 3500 (IQR, 2300–5500) tests in South Africa and 410 (IQR, 370–450) in Southeast Asia. Conclusions FQ-DST improved patient outcomes and was particularly important for high-risk patient groups and less robust regimens. A universal strategy was favored in generalized epidemics of fluoroquinolone resistance.

Rifampin is one of the most important biofilm-active antibiotics in the treatment of periprosthetic joint infection (PJI), and antibiotic regimens not involving rifampin were shown to have higher failure rates. Therefore, an emerging rifampin resistance can have a devastating effect on the outcome of PJI. The aim of this study was to compare the incidence of rifampin resistance between two groups of patients with a PJI treated with antibiotic regimens involving either immediate or delayed additional rifampin administration and to evaluate the effect of this resistance on the outcome. In this retrospective analysis of routinely collected data, all patients who presented with an acute/chronic PJI between 2018 and 2020 were recorded in the context of a single-center comparative cohort study. Two groups were formed: Group 1 included 25 patients with a PJI presenting in 2018–2019. These patients received additional rifampin only after pathogen detection in the intraoperative specimens. Group 2 included 37 patients presenting in 2019–2020. These patients were treated directly postoperatively with an empiric antibiotic therapy including rifampin. In all, 62 patients (32 females) with a mean age of 68 years and 322 operations were included. We found a rifampin-resistant organism in 16% of cases. Rifampin resistance increased significantly from 12% in Group 1 to 19% in Group 2 (p &lt, 0.05). The treatment failure rate was 16% in Group 1 and 16.2% in Group 2 (p = 0.83). The most commonly isolated rifampin-resistant pathogen was Staphylococcus epidermidis (86%) (p &lt, 0.05). The present study shows a significant association between the immediate start of rifampin after surgical revision in the treatment of PJI and the emergence of rifampin resistance, however with no significant effect on outcome.

Background Riemerella anatipestifer (R. anatipestifer) is one of the most important poultry pathogens worldwide, with associated infections causing significant economic losses. Rifampin Resistance is an important mechanism of drug resistance. However, there is no information about rpoB mutations conferring rifampin resistance and its fitness cost in Riemerella anatipestifer. Results Comparative analysis of 18 R.anatipestifer rpoB sequences and the determination of rifampin minimum inhibitory concentrations showed that five point mutations, V382I, H491N, G502K, R494K and S539Y, were related to rifampin resistance. Five overexpression strains were constructed using site-directed mutagenesis to validate these sites. To investigate the origin and fitness costs of the rpoB mutations, 15 types of rpoB mutations were isolated from R. anatipestifer ATCC 11845 by using spontaneous mutation in which R494K was identical to the type of mutation detected in the isolates. The mutation frequency of the rpoB gene was calculated to be 10− 8. A total of 98.8% (247/250) of the obtained mutants were located in cluster I of the rifampin resistance-determining region of the rpoB gene. With the exception of D481Y, I537N and S539F, the rifampin minimum inhibitory concentrations of the remaining mutants were at least 64 μg/mL. The growth performance and competitive experiments of the mutant strains in vitro showed that H491D and 485::TAA exhibit growth delay and severely impaired fitness. Finally, the colonization abilities and sensitivities of the R494K and H491D mutants were investigated. The sensitivity of the two mutants to hydrogen peroxide (H2O2) and sodium nitroprusside (SNP) increased compared to the parental strain. The number of live colonies colonized by the two mutants in the duckling brain and trachea were lower than that of the parental strain within 24 h. Conclusions Mutations of rpoB gene in R. anatipestifer mediate rifampin resistance and result in fitness costs. And different single mutations confer different levels of fitness costs. Our study provides, to our knowledge, the first estimates of the fitness cost associated with the R. anatipestifer rifampin resistance in vitro and in vivo. Electronic supplementary material The online version of this article (10.1186/s12866-019-1478-7) contains supplementary material, which is available to authorized users.

Background The AdvanSure MDR-TB GenoBlot Assay detects isoniazid- and rifampin-resistant tuberculosis using mutation-specific probes, including probes to disputed rpoB mutations. The aim of this study was to evaluate the clinical usefulness of molecular drug susceptibility testing (DST) using the AdvanSure assay with weekly batch testing in routine clinical laboratory settings in a country with an intermediate tuberculosis burden. Methods The AdvanSure assay was evaluated against an absolute concentration (AC) method and the Mycobacterial Growth Indicator Tube (MGIT) 960 System, which are phenotypic DST methods, using 496 Mycobacterium tuberculosis (MTB) isolates. We retrospectively reviewed and compared DST results and turnaround times (TATs), the time intervals from MTB culture identification to final reporting, for these methods. Results For rifampin, the AdvanSure assay showed 99.2% (492/496) concordance with both the AC and MGIT methods. AdvanSure also detected an rpoB mutation (D516Y) conferring low-level resistance in three isolates categorized as rifampin-susceptible by both phenotypic DST methods. For isoniazid, AdvanSure concordance rates with the AC method and MGIT DST were 96.6% (479/496) and 95.4% (473/496), respectively. The median TAT of AdvanSure in weekly batch testing was 5.8 days, shorter than the times for the phenotypic DST methods, which were 35.1 days for the AC method and 8.9 days for MGIT DST. Conclusions AdvanSure shows promising clinical usefulness for rapid detection of rifampin- and/or isoniazid-resistant tuberculosis when used as a complementary method to phenotypic DST assays in weekly batch testing. Furthermore, MTB isolates with disputed mutations for rifampin resistance were detectable by the AdvanSure assay.

In this study, we aimed to assess the performance of the Xpert MTB/RIF assay for the detection of pulmonary tuberculosis compared to the acid-fast bacilli (AFB) smear and culture analysis, and the incidence of rifampin resistance using the drug susceptibility test. The specimens referred for AFB smear and culture analysis and Xpert MTB/RIF assay from April 2015 to March 2018 were retrospectively reviewed. The sensitivity, specificity, and mean cycle threshold (Ct) values obtained in Xpert MTB/RIF assay and for rifampin resistance were analyzed. The results of Xpert MTB/RIF assay for pulmonary tuberculosis were evaluated based on the AFB smear grade. Among 3,840 specimens, 491 were positive in Xpert MTB/RIF assay and 626 in culture analysis. The sensitivity and specificity of Xpert MTB/RIF assay were 75.6% and 99.4%, respectively. The sensitivity of Xpert MTB/RIF assay for smear-positive/culture-positive specimens was 98.6% and that of smear-negative and -trace/culture-positive specimens was 63.1%. The positivity of Xpert MTB/RIF assay for culture-positive specimens was 89.9%, 98.6%, 95.7%, 100.0%, and 100.0% for the smear grades trace, 1+, 2+, 3+, 4+, respectively. The Ct values of 491 specimens significantly decreased as the AFB smear grade increased (P < 0.0001). The Ct values of smear-positive, -trace, and -negative specimens were 21.7 ± 4.2, 26.5 ± 3.9, and 27.4 ± 3.6, respectively. Rifampin resistance evaluated using Xpert MTB/RIF assay and culture analysis exhibited a correlation of 98.3%. The region covered by probe E was the most frequently mutated region (50.0%). Xpert MTB/RIF assay demonstrated reliable performance in detecting pulmonary tuberculosis from smear-positive and culture-positive specimens; however, further improvements are still required to detect smear-negative and culture-positive specimens.

Objective The aim was to analyze genetic mutations in the rpoB gene of rifampin-resistant Mycobacterium tuberculosis isolates (RIFR-MTB) from Zhejiang, China. Methods We prospectively analyzed RIFR-associated mutations in 13 rural areas of Zhejiang. Isolates were subjected to species identification, phenotype drug susceptibility testing (DST), DNA extraction, and rpoB gene sequencing. Results A total of 103 RIFR isolates were identified by DST (22 RIFR only, 14 poly-drug resistant, 49 multidrug resistant, 13 pre-extensively drug resistant [pre-XDR], and 5 extensively drug resistant [XDR]) from 2152 culture-positive sputum specimens. Gene sequencing of rpoB showed that the most frequent mutation was S450L (37.86%, 39/103); mutations P280L, E521K, and D595Y were outside the rifampicin resistance-determining region (RRDR) but may be associated with RIFR. Mutations associated with poly-drug resistant, pre-XDR, and XDR TB were mainly located at codon 445 or 450 in the RRDR. Conclusions The frequency of rpoB RRDR mutation in Zhejiang is high. Further studies are needed to clarify the relationships between RIFR and the TTC insertion at codon 433 in the RRDR and the P280L and D595Y mutations outside the RRDR.

Introduction There is limited information available about the drug resistance patterns in extrapulmonary tuberculosis (EPTB), in Iran. Thus, this study aimed to determine the prevalence of EPTB and to investigate the drug resistance pattern in MTB strains collected from extrapulmonary at the Tehran regional TB reference laboratory. Materials and methods Extrapulmonary specimens from suspected TB patients referred to TB reference laboratories in 5 cities of Iran were collected. Both standard conventional methods (culture and direct smear microscopy) and Xpert MTB/RIF assay were used for the identification of mycobacteria. Drug susceptibility testing (DST) was done by Xpert MTB/RIF. The proportion method on the Lowenstein-Jensen medium was done as a confirmatory method. Results Between 2016 and 2020, a total of 12050 clinical specimens from suspected TB patients were collected of which 10380 (86%) were pulmonary specimens and 1670 (14%) were extrapulmonary. Of extrapulmonary specimens, 85 (5.0%) were positive for MTB, and the remaining 1585 (95.0%) samples were negative by standard methods. Of 85 MTB isolates, drug susceptibility testing was performed for 32 isolates of which 1 (3.1%, 95% CI= 0.0% - 9.4%) were rifampin resistance and 31 (96.9%, 95% CI= 90.1% - 100%) were pan-susceptible. The rifampin-resistant isolate was also resistant to isoniazid and thus was assigned as a multi-drug resistant TB (MDR-TB). Conclusions Our study indicated the frequency of drug-resistance among EPTB in Iran. As our results, establishing rapid diagnostic methods for detection of drug-resistance in EPTB, performing DST for all EPTB cases to provide effective treatment, and continuous monitoring of drug resistance are suggested for prevention and control of drug-resistance in EPTB in Iran.

Mycobacterium tuberculosis ranks among the top 10 causes of deaths in Nepal despite the country having a long history of national tuberculosis prevention programmes that have proved very successful in the control of tuberculosis. Several cases of active or latent tuberculosis are still missing despite that the number of infected individuals is increasing each year. Microscopy has its own limitations and factors like low bacterial load, quality of sample, quality of smear, experience of microscopist etc. influence the overall sensitivity of the test. The implementation of a molecular technique-based rapid, point-of-care testing system offers higher sensitivity in the early diagnosis of tuberculosis. Cepheid GeneXpert is the most commonly used molecular technology in Nepal. It is a cartridge-based semi-quantitative, nested real-time PCR-based diagnostic system. It detects mutations in the beta-subunit of RNA polymerase (rpoB) gene that lead to rifampicin resistance (RR) in M. tuberculosis complex. The present study aims to increase our understanding of the epidemiology of mutations in the rpoB gene in tuberculosis-positive patients by using the Xpert MTB/RIF assay in a rural setting in Pyuthan Hospital, Nepal. Sputum from 2733 patients was tested for the diagnosis of tuberculosis using the Cepheid GeneXpert system between July 2018 and January 2020 at Pyuthan Hospital. Two hundred and ninety-seven of these samples (10.86 %) were positive for M. tuberculosis , of which 3.3 % (10/297) were rifampicin-resistant. Among rifampicin-resistant tuberculosis (RR-TB) patients, 50.0 % (5/10) showed mutations located in codons 529–533 (probe E) of the rpoB gene, followed by others. The GeneXpert system can be a convenient, highly sensitive, rapid and accurate tool for the diagnosis of tuberculosis, also identifying RR-TB and at the same time determining the molecular epidemiology of rifampin resistance-associated mutations in rural and/or resource-limited laboratory settings.

Multidrug resistance has been detected in the animal and zoonotic human pathogen Rhodococcus equi after mass macrolide/rifampin antibioprophylaxis in endemically affected equine farms in the United States. Multidrug-resistant (MDR) R. equi emerged upon acquisition of pRERm46, a conjugative plasmid conferring resistance to macrolides, lincosamides, streptogramins, and, as we describe, tetracycline. Phylogenomic analyses indicate that the increasing prevalence of MDR R. equi since it was first documented in 2002 is caused by a clone, R. equi 2287, attributable to coselection of pRErm46 with a chromosomal rpoBS531F mutation driven by macrolide/rifampin therapy. pRErm46 spillover to other R. equi genotypes has given rise to a novel MDR clone, G2016, associated with a distinct rpoBS531Y mutation. Our findings illustrate that overuse of antimicrobial prophylaxis in animals can generate MDR pathogens with zoonotic potential. MDR R. equi and pRErm46-mediated resistance are currently disseminating in the United States and are likely to spread internationally through horse movements.

INTRODUCTION: Gene-Xpert MTB RIF (Xpert) is based on nucleic acid amplification by real-time polymerase chain reaction, which allows for the identification of Mycobacterium tuberculosis and rifampin resistance. We describe the use of Xpert for extrapulmonary tuberculosis (EPTB) in children and adolescents. METHODS: A case series of two reference centers in Rio de Janeiro from 2014-2019. RESULTS: The final diagnosis of EPTB was established in 11/36 (31%) patients, with five cases detectable by Xpert. For lymph node evaluation (9/11), diagnosis by Xpert occurred in 5/9 patients, all with caseous aspects. CONCLUSIONS: Xpert can facilitate the rapid diagnosis of lymph node tuberculosis.

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.

The surveillance of drug resistance among tuberculosis (TB) patients is central to preventing the spread of antimicrobial resistance. The Democratic Republic of the Congo (DR Congo) is classified by the World Health Organization (WHO) as a country with a high burden of TB and multidrug-resistant TB (MDR-TB), but there are no nationally representative data on drug resistance. In 2016-2017, a national survey of TB patients was conducted in 108 microscopy centres across all 11 provinces of the country using innovative molecular approaches. Sputum samples were collected from 1,545 new and 163 previously treated patients. These were tested by the Xpert MTB/RIF assay, followed by targeted next-generation sequencing performed directly on sputum. The prevalence of rifampicin resistance was low, at 1.8% (95% CI: 1.0-3.2) among new and 17.3% (95% CI: 11.9-24.4) among previously treated patients. Resistance to pyrazinamide, fluoroquinolones and second-line injectables was also low. The prevalence of resistance to isoniazid among rifampicin-susceptible patients was higher, at 6.6% (95% CI: 4.4-9.8) among new and 8.7% (95% : 3.2-21.2) among previously treated patients. Diagnosing and treating isoniazid-resistant patients remains a challenge, given that many will be missed by the current national diagnostic algorithm that is driven by detecting rifampicin resistance by Xpert MTB/RIF. This is the first nationwide survey incorporating targeted sequencing directly on sputum. It serves as a proof-of-concept for other settings that do yet have rapid specimen transport networks or capacity to conduct culture. ispartof: SCIENTIFIC REPORTS vol:10 issue:1 ispartof: location:England status: published

The control measures recommended for treatment of leprosy with multidrug therapy should limit spread of drug-resistant strains; however, rifampicin resistance continues to be reported in several countries. Resistance to anti-infectious used in multidrug therapy has not yet been molecularly established in Côte d'Ivoire. This preliminary study presents results of evaluation of level rifampicin resistance in Côte d'Ivoire. For each case of clinically confirmed multibacillary leprosy (new or relapsed cases) a dermal juice sample was examined by microscopy after Ziehl-Neelsen staining and a specific PCR targetingRLEPrepeat sequence. 48 isolates were used to perform amplification of therpoBgene involved in rifampicin resistance. The origin of the resistance was then investigated after sequencing and mutations analysis. Thus, 24 isolates (50 %) had mutations, 11 (22. 91 %) of which had mutations conferring rifampicin resistance. The mutations correlated with resistance were as follows: Gln438Val (9. 09 %), Asp441Asn (18. 18 %), Ser456Leu (45. 50 %) and Ser456Phe (27. 27 %) in 9 health districts in Côte d'Ivoire. These mutations were observed in both relapsed and new cases. In conclusion, cases of rifampicin resistance exist in Côte d'Ivoire and an active surveillance should be carried out to monitor the evolution of this resistance. In addition, this information can be used to control the spread of drug-resistantM. lepraestrains.

A case of Mycobacterium leprae rifampin resistance after irregular antileprosy treatments since 1971 is reported. Whole-genome sequencing from four longitudinal samples indicated relapse due to acquired rifampin resistance and not to reinfection with another strain. A putative compensatory mutation in rpoC was also detected. Clinical improvement was achieved using an alternative therapy.

SETTING The diagnosis of multidrug-resistant tuberculosis (MDR-TB) and gaps in linkage to care are the principal health challenges in Mozambique. Five GeneXpert machines and GxAlert, an eHealth platform, were installed in Sofala and Manica Provinces between 2012 and 2014. OBJECTIVE To test the effects of Xpert® MTB/RIF testing and GxAlert on rifampin-resistant TB (RR-TB) diagnosis and second-line treatment initiation rates. DESIGN We conducted a retrospective clinical review of patients with RR-TB from March 2012 to September 2015 at these five sites. Time-series analyses were conducted to investigate the impact of Xpert on case detection and treatment. Pre- and post- analyses were conducted to investigate the impact of GxAlert. RESULTS A total of 32 182 Xpert tests were conducted: 4010 (12.5%) detected TB without rifampin resistance, and 306 (7.1%) had RR-TB. Of the RR-TB cases, 161 (52.6%) were started on MDR-TB treatment, 6.9% had documented culture results, and time from diagnosis to treatment initiation decreased over time. The absolute number of patients diagnosed and started on MDR-TB treatment increased by 0.26 (95%CI 0.15-0.38, P < 0.001) and 0.16 (95%CI 0.089-0.24, P < 0.001) every 6 months. GxAlert did not affect treatment initiation rates. CONCLUSION Implementation of Xpert testing was associated with increases in the number of patients diagnosed and started on MDR-TB treatment.

Molecular surveillance of rifampin-resistant Mycobacterium tuberculosis can help to monitor the transmission of the disease. The Xpert MTB/RIF Ultra assay detects mutations in the rifampin resistance-determining region (RRDR) of the rpoB gene by the use of melting temperature (T(m)) information from 4 rpoB probes which can fall in one of the 9 different assay-specified T(m) windows. The large amount of T(m) data generated by the assay offers the possibility of an RRDR genotyping approach more accessible than whole-genome sequencing. In this study, we developed an automated algorithm to specifically identify a wide range of mutations in the rpoB RRDR by utilizing the pattern of the T(m) of the 4 probes within the 9 windows generated by the Ultra assay. The algorithm builds a RRDR mutation-specific “T(m) signature” reference library from a set of known mutations and then identifies the RRDR genotype of an unknown sample by measuring the T(m) distances between the test sample and the reference T(m) values. Validated using a set of clinical isolates, the algorithm correctly identified RRDR genotypes of 93% samples with a wide range of rpoB single and double mutations. Our analytical approach showed a great potential for fast RRDR mutation identification and may also be used as a stand-alone method for ruling out relapse or transmission between patients. The algorithm can be further modified and optimized for higher accuracy as more Ultra data become available.

Delayed diagnosis of drug resistance has been a major obstacle to proper management and control of drug-resistant tuberculosis (TB). Expanded access to rapid molecular diagnostics such as Xpert MTB/RIF has been helpful, but has generated confusion about how to interpret genotype–phenotype discordance. Optimal management is not clearly defined for patients with rifampin resistance by Xpert MTB/RIF but rifampin susceptibility by phenotypic testing. To resolve this discrepancy, we performed pyrosequencing of discordant isolates identified at a reference laboratory over a 6-month period. We present here strategies to address genotype–phenotype discordance using sequencing.

MDR clades arise upon acquisition of resistance traits, but the determinants of their clonal expansion remain largely undefined. Taking advantage of the unique features of Rhodococcus equi infection control in equine farms, involving the same dual antibiotic treatment since the 1980s (a macrolide and rifampin), this study sheds light into the determinants of multiresistance clonality and the importance of combination therapy in limiting the dissemination of mobile resistance elements. Clinically effective therapeutic alternatives against R. equi foal pneumonia are currently lacking, and the identified macrolide-rifampin MDR clone 2287 has serious implications. Still at early stages of evolution and local spread, R. equi 2287 may disseminate globally, posing a significant threat to the equine industry and, also, public health due to the risk of zoonotic transmission. The characterization of the 2287 clone and its resistance determinants will enable targeted surveillance and control interventions to tackle the emergence of MDR R. equi., Antibiotic use has been linked to changes in the population structure of human pathogens and the clonal expansion of multidrug-resistant (MDR) strains among healthcare- and community-acquired infections. Here we present a compelling example in a veterinary pathogen, Rhodococcus equi, the causative agent of a severe pulmonary infection affecting foals worldwide. We show that the erm(46) gene responsible for emerging macrolide resistance among equine R. equi isolates in the United States is part of a 6.9-kb transposable element, TnRErm46, actively mobilized by an IS481 family transposase. TnRErm46 is carried on an 87-kb conjugative plasmid, pRErm46, transferable between R. equi strains at frequencies up to 10−3. The erm(46) gene becomes stabilized in R. equi by pRErm46’s apparent fitness neutrality and wholesale TnRErm46 transposition onto the host genome. This includes the conjugally exchangeable pVAPA virulence plasmid, enabling the possibility of cotransfer of two essential traits for survival in macrolide-treated foals in a single mating event. Despite its high horizontal transfer potential, phylogenomic analyses show that erm(46) is paradoxically confined to a specific R. equi clone, 2287. R. equi 2287 also carries a unique rpoBS531F mutation conferring high-level resistance to rifampin, systematically administered together with macrolides against rhodococcal pneumonia on equine farms. Our data illustrate that under sustained combination therapy, several independent “founder” genetic events are concurrently required for resistance, limiting not only its emergence but also, crucially, horizontal spread, ultimately determining multiresistance clonality.

Rhodococcus equi is a leading cause of severe pneumonia in foals. Standard treatment is dual antimicrobial therapy with a macrolide and rifampin, but the emergence of macrolide- and rifampin-resistant R. equi isolates is an increasing problem. The objective of this study was to determine the effect of macrolide and/or rifampin resistance on fitness of R. equi. Three unique isogenic sets were created, each consisting of four R. equi strains, as follows: a susceptible parent isolate, strains resistant to macrolides or rifampin, and a dual macrolide- and rifampin-resistant strain. Each isogenic set’s bacterial growth curve was generated in enriched medium, minimal medium (MM), and minimal medium without iron (MM-I). Bacterial survival in soil was analyzed over 12 months at −20°C, 4°C, 25°C, and 37°C, and the ability of these strains to retain antimicrobial resistance during sequential subculturing was determined. Insertion of the mobile element conferring macrolide resistance had minimal effect on in vitro growth. However, two of three rpoB mutations conferring rifampin resistance resulted in a decreased growth rate in MM. In soil, macrolide- or rifampin-resistant R. equi strains exhibited limited growth compared to that of the susceptible R. equi isolate at all temperatures except −20°C. During subculturing, macrolide resistance was lost over time, and two of three rpoB mutations reverted to the wild-type form. The growth of rifampin-resistant R. equi colonies is delayed under nutrient restriction. In soil, possession of rifampin or macrolide resistance results in decreased fitness. Both macrolide and rifampin resistance can be lost after repeated subculturing. IMPORTANCE This work advances our understanding of the opportunistic environmental pathogen Rhodococcus equi, a disease agent affecting horses and immunocompromised people. R. equi is one of the most common causes of severe pneumonia in young horses. For decades, the standard treatment for R. equi pneumonia in horses has been dual antimicrobial therapy with a macrolide and rifampin; effective alternatives to this combination are lacking. The World Health Organization classifies these antimicrobial agents as critically important for human medicine. Widespread macrolide and rifampin resistance in R. equi isolates is a major emerging problem for the horse-breeding industry and might also adversely impact human health if resistant strains infect people or transfer resistance mechanisms to other pathogens. This study details the impact of antimicrobial resistance on R. equi fitness, a vital step for understanding the ecology and epidemiology of resistant R. equi isolates, and will support development of novel strategies to combat antimicrobial resistance.

In Mycobacterium tuberculosis (Mtb) the detection of single nucleotide polymorphisms (SNPs) is of high importance both for diagnostics, since drug resistance is primarily caused by the acquisition of SNPs in multiple drug targets, and for epidemiological studies in which strain typing is performed by SNP identification. To provide the necessary coverage of clinically relevant resistance profiles and strain types, nucleic acid-based measurement techniques must be able to detect a large number of potential SNPs. Since the Mtb problem is pressing in many resource-poor countries, requiring low-cost point-of-care biosensors, this is a non-trivial technological challenge. This paper presents a proof-of-concept in which we chose simple DNA-DNA hybridization as a sensing principle since this can be transferred to existing low-cost hardware platforms, and we pushed the multiplex boundaries of it. With a custom designed probe set and a physicochemical-driven data analysis it was possible to simultaneously detect the presence of SNPs associated with first- and second-line drug resistance and Mtb strain typing. We have demonstrated its use for the identification of drug resistance and strain type from a panel of phylogenetically diverse clinical strains. Furthermore, reliable detection of the presence of a minority population (