In Vitro Synergistic Effects of Omadacycline with Other Antimicrobial Agents against Mycobacterium abscessus.

The clinical importance of Mycobacterium abscessus species (MABS) infections has been increasing. However, the standard treatment regimens recommended in the current guidelines often result in unfavorable outcomes. Therefore, we investigated the in vitro activity of omadacycline (OMC), a novel tetracycline, against MABS to explore its potential as a novel therapeutic option. The drug susceptibilities of 40 Mycobacterium abscessus subsp. abscessus (Mab) clinical strains obtained from the sputum of 40 patients from January 2005 to May 2014 were investigated. The MIC results for OMC, amikacin (AMK), clarithromycin (CLR), clofazimine (CLO), imipenem (IPM), rifabutin (RFB), and tedizolid (TZD) alone and their combined effects (with OMC) were examined using the checkerboard method. Additionally, we studied the differences in the effectiveness of the antibiotic combinations based on the colony morphotype of Mab. The MIC50 and MIC90 of OMC alone were 2 and 4 µg/mL, respectively. The combinations of OMC with AMK, CLR, CLO, IPM, RFB, and TZD showed synergy against 17.5%, 75.8%, 25.0%, 21.1%, 76.9%, and 34.4% of the strains, respectively. Additionally, OMC combined with CLO (47.1% versus 9.5%, P = 0.023) or TZD (60.0% versus 12.5%, P = 0.009) showed significantly higher synergy against strains with rough morphotypes than those with smooth morphotypes. In conclusion, the checkerboard analyses revealed that the synergistic effects of OMC were observed most frequently with RFB, followed by CLR, TZD, CLO, IPM, and AMK. Furthermore, OMC tended to be more effective against rough-morphotype Mab strains.

Pin tract infection caused by Mycobacterium neoaurum in a 14-year-old child: A case report.

Although rapidly growing non-tuberculosis mycobacterium can occasionally cause postoperative infections, Mycobacterium neoaurum is a rare pathogen of surgical site infection. We report a case of pin tract infection caused by M. neoaurum in a 14-year-old girl who was admitted for lengthening of her right fourth metatarsal bone. Pain, redness, and exudate were observed 18 days after external fixator insertion. Repeated exudate cultures revealed M. neoaurum, and she was diagnosed with a mycobacterial pin tract infection. She was initially administered intravenous ciprofloxacin and minocycline, and then was switched to oral trimethoprim-sulfamethoxazole and minocycline for a total of 6 months. Despite the pin tract infection, bone lengthening was completed under antibiotic treatment without removal of the pin; no other complications were noted. There are no prior reports of external fixator pin tract infection by M. neoaurum. While such cases may be rare, this case demonstrates that bone distraction may still be successfully completed using appropriate antibiotic therapy without pin removal.

Diagnostic accuracy of a novel tuberculosis point-of-care urine lipoarabinomannan assay for people living with HIV: A meta-analysis of individual in- and outpatient data.

BACKGROUND: Tuberculosis (TB) is the most common cause of death in people living with HIV (PLHIV), yet TB often goes undiagnosed since many patients are not able to produce a sputum specimen, and traditional diagnostics are costly or unavailable. A novel, rapid lateral flow assay, Fujifilm SILVAMP TB LAM (SILVAMP-LAM), detects the presence of TB lipoarabinomannan (LAM) in urine, and is substantially more sensitive for diagnosing TB in PLHIV than an earlier LAM assay (Alere Determine TB LAM lateral flow assay [LF-LAM]). Here, we present an individual participant data meta-analysis of the diagnostic accuracy of SILVAMP-LAM in adult PLHIV, including both published and unpublished data. METHODS AND FINDINGS: Adult PLHIV (≥18 years) were assessed in 5 prospective cohort studies in South Africa (3 cohorts), Vietnam, and Ghana, carried out during 2012 to 2017. Of the 1,595 PLHIV who met eligibility criteria, the majority (61%) were inpatients, median age was 37 years (IQR 30-43), 43% had a CD4 count ≤ 100 cells/µl, and 35% were receiving antiretroviral therapy. Most participants (94%) had a positive WHO symptom screen for TB on enrollment, and 45% were diagnosed with microbiologically confirmed TB, using mycobacterial culture or Xpert MTB/RIF testing of sputum, urine, or blood. Previously published data from inpatients were combined with unpublished data from outpatients. Biobanked urine samples were tested, using blinded double reading, with SILVAMP-LAM and LF-LAM. Applying a microbiological reference standard for assessment of sensitivity, the overall sensitivity for TB detection was 70.7% (95% CI 59.0%-80.8%) for SILVAMP-LAM compared to 34.9% (95% CI 19.5%-50.9%) for LF-LAM. Using a composite reference standard (which included patients with both microbiologically confirmed as well as clinically diagnosed TB), SILVAMP-LAM sensitivity was 65.8% (95% CI 55.9%-74.6%), and that of LF-LAM 31.4% (95% CI 19.1%-43.7%). In patients with CD4 count ≤ 100 cells/µl, SILVAMP-LAM sensitivity was 87.1% (95% CI 79.3%-93.6%), compared to 56.0% (95% CI 43.9%-64.9%) for LF-LAM. In patients with CD4 count 101-200 cells/µl, SILVAMP-LAM sensitivity was 62.7% (95% CI 52.4%-71.9%), compared to 25.3% (95% CI 15.8%-34.9%) for LF-LAM. In those with CD4 count > 200 cells/µl, SILVAMP-LAM sensitivity was 43.9% (95% CI 34.3%-53.9%), compared to 10.9% (95% CI 5.2%-18.4%) for LF-LAM. Using a microbiological reference standard, the specificity of SILVAMP-LAM was 90.9% (95% CI 87.2%-93.7%), and that of LF-LAM 95.3% (95% CI 92.2%-97.7%). Limitations of this study include the use of biobanked, rather than fresh urine samples, and testing by skilled laboratory technicians in research laboratories, rather than at the point of care. CONCLUSIONS: In this study, we found that SILVAMP-LAM identified a substantially higher proportion of TB patients in PLHIV than LF-LAM. The sensitivity of SILVAMP-LAM was highest in patients with CD4 count ≤ 100 cells/µl. Further work is needed to demonstrate accuracy when implemented as a point-of-care test.

Antimicrobial susceptibility testing of Mycobacteroides (Mycobacterium) abscessus complex, Mycolicibacterium (Mycobacterium) fortuitum, and Mycobacteroides (Mycobacterium) chelonae.

The drug susceptibility of rapidly growing mycobacteria (RGM) varies among isolates. Treatment strategies similarly differ depending on the isolate, and for some, no clear strategy has been identified. This complicates clinical management of RGM. Following Clinical and Laboratory Standards Institute standard M24-A2, we assessed the susceptibility of 140 RGM isolates to 14 different antimicrobial drugs by measuring their minimal inhibitory concentrations (MICs). We also investigated the correlation of clarithromycin (CAM) MICs with the erm(41) and rrl gene mutations in the Mycobacteroides (Mycobacterium) abscessus complex, the rrl mutation in Mycobacteroides (Mycobacterium) chelonae, and the erm(39) mutation in Mycolicibacterium (Mycobacterium) fortuitum to determine the contribution of these mutations to CAM susceptibility. The five species and subspecies examined included 48 M. abscessus subsp. abscessus isolates (34.3%), 35 (25.0%) being M. abscessus subsp. massiliense, and two (1.4%) being M. abscessus subsp. bolletii. The M. abscessus complex accounted for 85 isolates (60.7%) in total, whereas 43 isolates (30.7%) were M. fortuitum, and 12 (8.6%) were M. chelonae. Our results demonstrated species-specific susceptibility to antimicrobials. In most cases, susceptibility to CAM could be predicted based on genetic pattern, but since one isolate did not fit that pattern, MIC values needed to be measured. Some isolates also exhibited rates of resistance to other drugs that differed from those previously reported in other locations, indicating that accurate identification of the bacterial isolate and use of the correct method for determining MIC are both important for the diagnosis of RGM.

A case of Mycobacterium tuberculosis laboratory cross-contamination.

SETTING: A laboratory cross-contamination event was suspected because Mycobacterium tuberculosis was unexpectedly detected at a high incidence in the cultures of several clinical specimens at the National Hospital Organization, Tokyo National Hospital, Japan. OBJECTIVE: To describe a case of Mycobacterium tuberculosis laboratory cross-contamination. DESIGN: We reviewed the medical records of 20 patients whose clinical specimens were suspected to have been contaminated by Mycobacterium tuberculosis. Variable number of tandem repeat analysis with 15 loci, the Japan Anti-Tuberculosis Association-12, and three additional hyper-variable loci, was performed to identify the cross-contamination event. RESULTS: The clinical, laboratory, and variable number of tandem repeat data revealed that the cross-contamination had possibly originated from one strongly positive specimen, resulting in false-positive results in 11 other specimens, including a case treated with anti-tuberculosis drugs. CONCLUSION: Clinical and laboratory data must be re-evaluated when cross-contamination is suspected and variable number of tandem repeat analysis should be used to confirm cross-contamination. Furthermore, original isolates should be stored appropriately, without sub-culturing and genotyping should be performed at the earliest possible for better utilization of variable number of tandem repeat for the identification of cross-contamination.

Prediction of Local Transmission of Mycobacterium tuberculosis Isolates of a Predominantly Beijing Lineage by Use of a Variable-Number Tandem-Repeat Typing Method Incorporating a Consensus Set of Hypervariable Loci.

Strain genotyping based on the variable-number tandem repeat (VNTR) is widely applied for identifying the transmission of Mycobacterium tuberculosis A consensus set of four hypervariable loci (1982, 3232, 3820, and 4120) has been proposed to improve the discrimination of Beijing lineage strains. Herein, we evaluated the utility of these four hypervariable loci for tracing local tuberculosis transmission in 981 cases over a 14-month period in Japan (2010 to 2011). We used six different VNTR systems, with or without the four hypervariable loci. Patient ages and weighted standard distances (a measure of the dispersion of genotype-clustered cases) were used as proxies for estimating local tuberculosis transmission. The highest levels of isolate discrimination were achieved with VNTR systems that incorporated the four hypervariable loci (i.e., the Japan Anti-Tuberculosis Association [JATA]18-VNTR, mycobacterial interspersed repetitive unit [MIRU]28-VNTR, and 24Beijing-VNTR). The clustering rates by JATA12-VNTR, MIRU15-VNTR, JATA15-VNTR, JATA18-VNTR, MIRU28-VNTR, and 24Beijing-VNTR systems were 52.2%, 51.0%, 39.0%, 24.1%, 23.1%, and 22.0%, respectively. As the discriminative power increased, the median weighted standard distances of the clusters tended to decrease (from 311 to 80 km, P < 0.001, Jonckheere-Terpstra trend test). Concurrently, the median ages of patients in the clusters tended to decrease (from 68 to 60 years, P < 0.001, Jonckheere-Terpstra trend test). These findings suggest that strain typing using the four hypervariable loci improves the prediction of active local tuberculosis transmission. The four-locus set can therefore contribute to the targeted control of tuberculosis in settings with high prevalence of Beijing lineage strains.

COBAS® TaqMan® MTB, smear positivity grade and MGIT culture; correlation analyses of three methods for bacillary quantification.

We investigated the correlation between the cycle threshold (Ct) value of the COBAS(®) TaqMan(®) MTB (TaqMan MTB), the mycobacterial smear positivity grade, and the time to detection (TTD) in the Mycobacteria Growth Indicator Tube (MGIT) for quantification of Mycobacterium tuberculosis (MTB). For 57 sputum samples, significant correlations were observed between the Ct value, the smear positivity grade, and the MGIT TTD (Spearman's rank correlation coefficient: r(s) = -0.940, P < 0.001 and Pearson's correlation coefficient: r(p) = 0.737, P < 0.001). In addition, a correlation was observed between the number of bacteria estimated based on the smear positivity grade and the number of MTB bacilli calculated by the Ct value (r(s) = 0.930, P < 0.001). This study has demonstrated the possible estimation of the smear positivity grade and MGIT TTD using the Ct value of TaqMan MTB, which is based on a real-time PCR system, for diagnostic samples.

[SPECIFICITY EVALUATION OF TRCReady® MTB AND TRCReady® MAC FOR IDENTIFYING MYCOBACTERIUM TUBERCULOSIS COMPLEX, MYCOBACTERIUM AVIUM AND MYCOBACTERIUM INTRACELLULARE].

[Objective] To evaluate the specificity of TRC- Ready® MTB and TRCReady® MAC (Tosoh Bioscience, Japan) for identifying M.tubercidosis complex (MTC), M.avium and M. intracellulare. [Method] We tested TRCReady® MTB and TRCReady® MAC using TRCReady®-80 (Tosoh Bioscience, Japan), which is an automated nucleic amplification test instrument, with 151 Mycobacterium species (4 MTC and 147 Non-tuberculosis Mycobacterium (NTM) type strains). [Results] The specificity of TRCReady® MTB was 100%, however, TkCReady® MAC misidentified a total of six NTMs, M.arosiense, M.chimaera, M.colombiense, M.marseillense, M. vulneris and M.yongonense, as M. intracellulare. Then, the specificity for TRCReady® MAC was 96.0% (145/151). [Discussion] TRCReady® MTB and TRCReady® MAC are highly specific for identifying MTC, M.avium and M. intracellulare. Six NTM species which have been rarely isolated in Japan showed false-positive results as M.intra- celludare. However, when a sample was identified as M.in- tracellulare, the phenotypic characteristics like colony mor- phology would be carefully examined.

[EXTERNAL QUALITY ASSESSMENT OF ANTI-TUBERCULOSIS DRUG SUSCEPTIBILITY TESTING FOR DIAGNOSING EXTENSIVELY DRUG-RESISTANT MYCOBACTERIUM TUBERCULOSIS].

[Objective] The infectious disease control law has been amended in May 2015, and the category definition of Mycobacterium tuberculosis as infectious pathogen has been changed, following the definition of extensively drug-resistant M.tuberculosis (XDR-TB) by World Health Organization. To assess the diagnostic capacity of XDR-TB, we conducted an external quality assessment (EQA) for the anti-tuberculosis drug susceptibility testing (DST). [Method] A total of 10 M.tuberculosis strains with known drug susceptibility were sent to each participating laboratory. The drugs assessed were isoniazid (INH), rifampicin (RFP), streptomycin (SM), ethambutol (EB), levofloxacin (LVFX), and kanamycin (KM). DST was performed using each routine method(s), and the results were compared with the judicial diagnoses. The sensitivity, specificity, overall agreement (effi- ciency) and kappa coefficient were calculated for each drug tested. In addition, the diagnostic accuracy of multidrug-resis- tant M. tuberculosis (MDR-TB) and XDR-TB was assessed. [Results] A total of 88 institutes including 67 hospitals, 16 commercial laboratories, and 5 public health laboratories par- ticipated in the EQA. With 2 laboratories submitting 2 sets of results, a total of 90 independent data sets were analyzed. As for INH, RFP and LVFX, the efficiency was over 95%, but we found two strains each for SM, EB and KM with the efficiency less than 95%. Especially, strain 1 and strain 2 showed efficiency of 72.2% and 71.1% to SM, respectively. This error was mainly found in a certain test kit. If we consider the passing score as showing ≥95 % sensitivity and specificity both to INH and RFP, the diagnostic accuracy of MDR-TB was 92.2% (83/90) in this study. With the same criteria to INH, RFP, LVFX and KM, that of XDR-TB was 79.7% (63/79). [Discussion] The diagnostic capacity of XDR-TB was not sufficient in the current study. Good case management and pathogen control requires higher accuracy. The government may need to conduct a constant EQA and relevant remedial actions.

Association between pncA gene mutations, pyrazinamidase activity, and pyrazinamide susceptibility testing in Mycobacterium tuberculosis.

We determined MICs for, confirmed the presence of pncA mutations in, and performed pyrazinamidase testing on colonies (subclones) obtained from seven isolates that exhibited differential pyrazinamide (PZA) susceptibility. Six of the seven strains were found to exhibit characteristics resulting from the mixture of strains possessing different properties. In addition, our analysis revealed large pncA-spanning deletions (1,565 bp, 4,475 bp, and 6,258 bp) in three strains that showed high PZA resistance.

Comparative evaluation of three immunochromatographic identification tests for culture confirmation of Mycobacterium tuberculosis complex.

BACKGROUND: The rapid identification of acid-fast bacilli recovered from patient specimens as Mycobacterium tuberculosis complex (MTC) is critically important for accurate diagnosis and treatment. A thin-layer immunochromatographic (TLC) assay using anti-MPB64 or anti-MPT64 monoclonal antibodies was developed to discriminate between MTC and non-tuberculosis mycobacteria (NTM). Capilia TB-Neo, which is the improved version of Capilia TB, is recently developed and needs to be evaluated. METHODS: Capilia TB-Neo was evaluated by using reference strains including 96 Mycobacterium species (4 MTC and 92 NTM) and 3 other bacterial genera, and clinical isolates (500 MTC and 90 NTM isolates). M. tuberculosis isolates tested negative by Capilia TB-Neo were sequenced for mpt64 gene. RESULTS: Capilia TB-Neo showed 100% agreement to a subset of reference strains. Non-specific reaction to M. marinum was not observed. The sensitivity and specificity of Capilia TB-Neo to the clinical isolates were 99.4% (99.6% for M. tuberculosis, excluding M. bovis BCG) for clinical MTC isolates and 100% for NTM isolates tested, respectively. Two M. tuberculosis isolates tested negative by Capilia TB-Neo: one harbored a 63-bp deletion in the mpt64 gene and the other possessed a 3,659-bp deletion from Rv1977 to Rv1981c, a region including the entire mpt64 gene. CONCLUSIONS: Capilia TB-Neo is a simple, rapid and highly sensitive test for identifying MTC, and showed better specificity than Capilia TB. However, Capilia TB-Neo still showed false-negative results with mpt64 mutations. The limitation should be recognized for clinical use.

[Evaluation of the use of SPEED-OLIGO Mycobacteria for identification of Mycobacterium species].

OBJECTIVE: To evaluate the use of SPEED-OLIGO MYCOBACTERIA (Vircell, Spain) in identifying Mycobacterium species. METHOD: We examined 15 type or reference strains of mycobacteria (M. tuberculosis H37Rv and 14 non-tuberculosis mycobacteria), 48 clinical isolates, and 17 AFB-positive sputa by using SPEED-OLIGO MYCOBACTERIA, and compared the results with those obtained using other referral methods available for species identification. RESULT: SPEED-OLIGO MYCOBACTERIA yielded favorable results in 80.0%, 91.7%, and 88.2% of the cases of the tested type/reference strains, clinical isolates, and clinical samples, respectively. However, the type/reference strains M. celatum, M. fortuitum subsp. fortuitum, and M. marinum, and the clinical isolates M. intermedium, M. marinum, and M. szulgai were misidentified when SPEED-OLIGO MYCOBACTERIA was used. DISCUSSION: SPEED-OLIGO MYCOBACTERIA can facilitate the rapid identification of Mycobacterium species mainly because of its short turn-around time and simple procedures. However, the accuracy of this method remains unsatisfactory.

In vitro effects of the new oral ß-lactamase inhibitor xeruborbactam in combination with oral ß-lactams against clinical Mycobacterium abscessus isolates.

Non-tuberculosis mycobacteria (NTM), particularly Mycobacterium abscessus subsp. abscessus (M. abscessus), are increasingly being recognized as etiological agents of NTM pulmonary disease. However, treatment options for M. abscessus are limited owing to their natural resistance to most antibiotics, including ß-lactams. M. abscessus produces a class A ß-lactamase, whose activity is inhibited by cyclic boronic acid ß-lactamase inhibitors. We aimed to evaluate the in vitro effects of xeruborbactam, a cyclic boronic acid ß-lactamase inhibitor, against M. abscessus when combined with five ß-lactams (amoxicillin, tebipenem, cefdinir, cefuroxime, and cefoxitin). The drug susceptibilities of 43 M. abscessus clinical isolates obtained from 43 patients between August 2005 and May 2014 were tested. The MIC results for each ß-lactam with or without 4 µg/mL xeruborbactam were examined. Xeruborbactam lowered the MIC90 values of tebipenem, amoxicillin, cefuroxime, and cefdinir by 5, ≥4, 3, and 3 dilutions, respectively. The MIC90 values of cefoxitin without xeruborbactam were 32 µg/mL and did not change upon the addition of xeruborbactam. The lowest MIC90 value was obtained for tebipenem with xeruborbactam. Almost all isolates had an MIC of 4 µg/mL; one isolate had an MIC of 2 µg/mL. With respect to the susceptibility to the same family drug, the number of susceptible isolates increased from 1/43 (2%) to 43/43 (100%) for tebipenem with xeruborbactam. Combining tebipenem and xeruborbactam could be considered an effective all-oral regimen that benefits outpatient treatment of M. abscessus pulmonary disease. IMPORTANCE: Mycobacterium abscessus subsp. abscessus (M. abscessus) disease is treated in two phases; injectable drugs for initial followed by others for continuation. There is a need to develop all-oral treatment methods for M. abscessus infection, especially in the continuation phase. However, treatment options for M. abscessus are limited owing to their natural resistance to most antibiotics. This is the first report to evaluate the in vitro effects of xeruborbactam, a cyclic boronic acid ß-lactamase inhibitor capable of inhibiting the class A ß-lactamase produced by M. abscessus, against 43 M. abscessus clinical isolates when combined with five ß-lactam antibiotics. Xeruborbactam lowered the MIC90 values of tebipenem by five dilutions, and the number of susceptible isolates increased from 1/43 (2%) to 43/43 (100%). We showed that the tebipenem-xeruborbactam combination might be of interest to explore further as a potentially effective oral regimen for outpatient treatment of M. abscessus pulmonary disease.

Prospective multicentre accuracy evaluation of the FUJIFILM SILVAMP TB LAM test for the diagnosis of tuberculosis in people living with HIV demonstrates lot-to-lot variability.

There is an urgent need for rapid, non-sputum point-of-care diagnostics to detect tuberculosis. This prospective trial in seven high tuberculosis burden countries evaluated the diagnostic accuracy of the point-of-care urine-based lipoarabinomannan assay FUJIFILM SILVAMP TB LAM (FujiLAM) among inpatients and outpatients living with HIV. Diagnostic performance of FujiLAM was assessed against a mycobacterial reference standard (sputum culture, blood culture, and Xpert Ultra from urine and sputum at enrollment, and additional sputum culture ≤7 days from enrollment), an extended mycobacterial reference standard (eMRS), and a composite reference standard including clinical evaluation. Of 1637 participants considered for the analysis, 296 (18%) were tuberculosis positive by eMRS. Median age was 40 years, median CD4 cell count was 369 cells/ul, and 52% were female. Overall FujiLAM sensitivity was 54·4% (95% CI: 48·7-60·0), overall specificity was 85·2% (83·2-87·0) against eMRS. Sensitivity and specificity estimates varied between sites, ranging from 26·5% (95% CI: 17·4%-38·0%) to 73·2% (60·4%-83·0%), and 75·0 (65·0%-82·9%) to 96·5 (92·1%-98·5%), respectively. Post-hoc exploratory analysis identified significant variability in the performance of the six FujiLAM lots used in this study. Lot variability limited interpretation of FujiLAM test performance. Although results with the current version of FujiLAM are too variable for clinical decision-making, the lipoarabinomannan biomarker still holds promise for tuberculosis diagnostics. The trial is registered at clinicaltrials.gov (NCT04089423).

[Clinical evaluation of a line probe assay kit for the identification of Mycobacterium species and detection of drug-resistant Mycobacterium tuberculosis].

BACKGROUND: Multidrug resistance (MDR) involves resistance to both isoniazid and rifampicin, which makes the treatment of tuberculosis very difficult. Extensive drug resistance (XDR) occurs when, in addition to isoniazid and rifampicin resistance, the microorganisms are resistant to a fluoroquinolone and an injectable agent (e.g., kanamycin, amikacin, or capreomycin). Generally, drug susceptibility testing takes more than 3-4 weeks after the initial cultivation. There is an urgent need to identify methods that can rapidly detect both the presence of Mycobacterium tuberculosis and the status of drug resistance. PURPOSE: This study was aimed at evaluating the line probe assay (LiPA; Nipro Co.), for the identification of Mycobacterium species and detection of mutations associated with antituberculous drugs. RESULTS: We found that LiPA enabled the rapid identification of M. tuberculosis, M. avium, M. intracellulare, and M. kansasii. When the results of the LiPA and conventional drug susceptibility tests were compared, there was no difference in the susceptibility to rifampicin, pyrazinamide, and levofloxacin; however, there was a difference in the susceptibility to isoniazid. CONCLUSION: Thus, LiPA can be used for the rapid identification of Mycobacterium species and the determination of susceptibility to drugs, which can help in the early initiation of appropriate treatment, leading to a reduction in infectiousness.

Multiple mutations of Mycobacterium intracellulare subsp. chimaera causing false-negative reaction to the transcription-reverse transcription concerted method for pathogen detection.

OBJECTIVES: To report an isolate of Mycobacterium intracellulare subsp. chimaera with multiple mutations in 16S ribosomal RNA (rRNA) gene, resulting in the false-negative reaction to the transcription-reverse transcription concerted (TRC) method for Mycobacterium avium-intracellulare complex. METHODS: We used TRC, polymerase chain reaction (PCR), and Matrix-assisted laser desorption/ionization Time-of-Flight/Mass Spectrometry (MALDI-TOF/MS) methods to identify a clinical isolate in 2021. Due to the discordant results between TRC and PCR or MALDI-TOF MS methods, 16S rRNA sequencing, whole-genome shotgun (WGS) sequencing, and average nucleotide identity (ANI) analysis were employed to identify the isolate. RESULTS: A mycobacterial isolate from a sputum sample gave negative results for the detection of Mycobacterium tuberculosis complex or M. avium-intracellulare complex by the TRC method. However, the isolate was identified as M. intracellulare by both PCR method and MALDI-TOF MS method. WGS sequencing of 16S rRNA genome revealed eight substitution mutations and one insertion mutation within the region, which could hamper the correct reaction to TRC method. Subsequent ANI analysis between the isolate and various species of nontuberculosis mycobacteria revealed that the isolate could be identified as M. intracellulare subsp. chimaera. CONCLUSION: Rare mutations within the 16S rRNA genome resulted in the false-negative identification of Mycobacterium chimaera by the TRC method. WGS sequencing and ANI analysis was necessary to identify the isolate.

Complete Genome Sequences of 14 Nontuberculous Mycobacteria Type Strains.

Here, we present the complete genome sequences of 14 nontuberculous mycobacteria type strains. The addition of type strain data may provide a concrete basis for further research.

Non-tuberculosis Mycobacterial Pulmonary Disease Caused by Mycobacterium kiyosense, a New Species.

Non-tuberculosis mycobacterial (NTM) pulmonary disease (NTM-PD) is quite common, and newly identified species are being reported increasingly frequently thanks to advances in identification technologies. A 56-year-old woman had mild sputum production showed bronchiectasis with multiple small nodules, consistent with NTM-PD, on chest computed tomography. Mycobacterial species were isolated from the specimens; however, conventional methods could not identify the species. We conducted whole-genome sequencing and identified the NTM isolates as Mycobacterium kiyosense, a species newly registered in 2023 from Japan. She was diagnosed with NTM-PD caused by M. kiyosense and received watchful waiting.

Comprehensive multicenter evaluation of a new line probe assay kit for identification of Mycobacterium species and detection of drug-resistant Mycobacterium tuberculosis.

We evaluated a new line probe assay (LiPA) kit to identify Mycobacterium species and to detect mutations related to drug resistance in Mycobacterium tuberculosis. A total of 554 clinical isolates of Mycobacterium tuberculosis (n = 316), Mycobacterium avium (n = 71), Mycobacterium intracellulare (n = 51), Mycobacterium kansasii (n = 54), and other Mycobacterium species (n = 62) were tested with the LiPA kit in six hospitals. The LiPA kit was also used to directly test 163 sputum specimens. The results of LiPA identification of Mycobacterium species in clinical isolates were almost identical to those of conventional methods. Compared with standard drug susceptibility testing results for the clinical isolates, LiPA showed a sensitivity and specificity of 98.9% and 97.3%, respectively, for detecting rifampin (RIF)-resistant clinical isolates; 90.6% and 100%, respectively, for isoniazid (INH) resistance; 89.7% and 96.0%, respectively, for pyrazinamide (PZA) resistance; and 93.0% and 100%, respectively, for levofloxacin (LVX) resistance. The LiPA kit could detect target species directly in sputum specimens, with a sensitivity of 85.6%. Its sensitivity and specificity for detecting RIF-, PZA-, and LVX-resistant isolates in the sputum specimens were both 100%, and those for detecting INH-resistant isolates were 75.0% and 92.9%, respectively. The kit was able to identify mycobacterial bacilli at the species level, as well as drug-resistant phenotypes, with a high sensitivity and specificity.

[Evaluation of GenoType MTBDRsl for testing resistance of Mycobacterium tuberculosis isolates to fluoroquinlone, aminoglycoside, and ethambutol].

OBJECTIVE: To evaluate the ability of GenoType MTBDRsl (Hain Lifescience, Germany) in detecting resistance to fluoroquinolone (FQ), aminoglycoside (AG), and ethambutol (EB). METHOD: We evaluated the resistance of 76 Mycobacterium tuberculosis samples, namely, 13 extensively drug-resistant (XDR), 29 multi-drug resistant (MDR), and 4 susceptible clinical isolates from Japan, and 30 strains from Supra-national Reference Laboratory Network, to FQ, AG, and EB by using GenoType MTBDRs. The gyrA, rrs, and embB were directly sequenced for all the strains, and the mutations were confirmed. The susceptibility testing result obtained using the standard proportion method with 1% Ogawa medium was considered as the gold standard. RESULTS: The sensitivities of GenoType TBDRsl listance to FQ, kanamycin (KM), amikacin (AMK), capreomycin (CPM), and EB were 82.4%, 57.1%, 100%, 83.3%, and 55.8%, respectively. The specificity for FQ was 97.6%, and that for KM, AMK, CPM, and EB were 100%. The mutant strains detected by GenoType TBDRsl ere as follows: in 20 Japanese FQ resistant isolates, 7 gyrA MUT3A (D94A) (35.0%), 6 MUT3C (D94G) (30.0%), 2 MUT1 (A90V) (10.0%), and 1 MUT1 with MUT3C (5.0%); in 18 KM resistant isolates, 10 rrs MUT1 (A1401G) (55.6%); and in 34 EB resistant isolates, 9 embB MUT1B (M306V) (26.5%), 2 MUT1A (M306I) (5.9%), and 8 WT1 deficits (covering codon 306; 23.5%). Direct sequencing showed additional substitutions in embB (2 D328Y, 1 D354A, 1 G406D, and 1 G406S). The sensitivity of GenoType" MTBDRsl ws simillr to that by sequencing method for resistance to FQ and AG, but that for EB was slightly less than by sequencing method, but the difference was not significant. DISCUSSION: The sensitivity of GenoType MTBDRsl for the letection of FQ resistance was approximately 80% to that by standard drug sensitivity test results by using conventional proportion methods, while it was relatively less accurate for the diagnosis of resistance to KM and EB in Japanese isolates. Thus, GenoType MTBDRsl is useful lr the early diagnosis and infection control of XDR-TB, because of a short turnaround time of approximately 6 h.