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.

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.

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.

In vitro activity of tedizolid and linezolid against multidrug-resistant Mycobacterium tuberculosis: a comparative study using microdilution broth assay and genomics.

The effects of tedizolid (TZD) against multidrug-resistant Mycobacterium tuberculosis isolates were investigated. This is possibly the first study to evaluate the MIC of TZD against Japanese Mycobacterium tuberculosis isolates. As TZD had a significantly lower MIC than LZD (P < 0.01), it was suggested to be a better, non-toxic alternative to LZD.

Clinical evaluation of the cobas® MTB-RIF/INH reagent and the cobas® 6800 for the detection of isoniazid and rifampicin resistance.

We aimed to validate the performance of a newly developed real-time PCR assay using cobas® MTB-RIF/INH reagent on the cobas® 6800 system for detecting isoniazid (INH) and rifampicin (RIF) resistance, using Japanese Mycobacterium tuberculosis (MTB) isolates. In total, 119 mock sputum specimens spiked with resistant MTB were tested using the cobas® MTB-RIF/INH reagent. The whole genomes of all MTB isolates were sequenced by MiSeq and analysed for mutations/indels causing drug resistance. All isolates were tested for phenotypic drug susceptibility, then MTB negative sputa were collected and pooled to prepare mock sputum specimens for the study. The sensitivity and specificity for INH resistance at a concentration equal to 3 × the limit of detection were 77.8% and 90.0%, respectively; those for RIF resistance were 91.8% and 93.5%, respectively. The sensitivities for INH and RIF were statistically different (P = 0.014), but not the specificities (P = 0.624). Twenty-two false-susceptible and two false-resistant results were obtained in INH; meanwhile, six false-susceptible and three false-resistant results were obtained in RIF. False-resistance for INH and RIF was mainly due to disputed mutations. The cobas® MTB-RIF/INH reagent showed better performance than other rapid molecular tests.

A Cold-Blooded Tiptoer: Nonresolving Cellulitis in an Immunocompromised Patient.

Mycobacterium haemophilum is a nontuberculous mycobacteria (NTM) with a predilection for skin and soft tissue infection (SSTI) in the immunocompromised host. We report a case of disseminated M haemophilum infection initially presenting as a nonresolving subacute cellulitis of bilateral lower extremities. Genetic sequencing was used for final identification, while a commercially available polymerase chain reaction test returned a false-positive result for Mycobacterium intracellulare. Consequently, we highlight the importance of M haemophilum as a major differential diagnosis of SSTI in the immunocompromised host and the need for careful interpretation of rapid diagnostic tests.

Development of a nucleic acid chromatography assay for the detection of commonly isolated rapidly growing mycobacteria.

Introduction. Non-tuberculosis mycobacterium infections are increasing worldwide, including those caused by rapidly growing mycobacteria (RGM).Gap Statement. The identification of the aetiological agent in the context of infections is essential for the adoption of an adequate therapeutic approach. However, the methods for the rapid distinction of different RGM species are less than optimal.Aim. To develop a nucleic acid chromatography kit to identify clinically common RGM.Methodology. We tried to develop a nucleic acid chromatography kit designed to detect four RGM species (including three subspecies) i.e. Mycobacterium abscessus subsp. abscessus, Mycobacterium abscessus subsp. bolletii (detected as M. abscessus/bolletii) Mycobacterium abscessus subsp. massiliense, Mycobacterium fortuitum, Mycobacterium chelonae and Mycobacterium peregrinum. The amplified target genes for each species/subspecies using multiplex PCR were analysed using a nucleic acid chromatography assay.Results. Among the 159 mycobacterial type strains and 70 RGM clinical isolates tested, the developed assay correctly identified all relevant RGM without any cross-reactivity or false-negatives. The limits of detection for each species were approximately 0.2 pg µl-1.Conclusion. The rapid and simple nucleic acid chromatography method developed here, which does not involve heat denaturation, may contribute to the rapid identification and treatment of RGM infections.

Efficacy estimation of a combination of triple antimicrobial agents against clinical isolates of Mycobacterium abscessus subsp. abscessus in vitro.

BACKGROUND: Mycobacterium abscessus subsp. abscessus (M. abscessus) is a rapidly growing mycobacterium that is resistant to most antibiotics. The number of patients with pulmonary disease caused by M. abscessus is increasing in several regions, and therapy involves long-term antibiotic combination treatments, although no standard treatment regimen has been established. OBJECTIVES: To examine candidate regimens for maintenance of antimicrobial treatment against M. abscessus by measuring MIC using the three-drug chequerboard method. METHODS: We evaluated the drug susceptibility of 70 clinical isolates of M. abscessus using the three-drug chequerboard method. We tested the antimycobacterial agents bedaquiline, clofazimine, amikacin, and sitafloxacin (which showed a relatively low MIC range when used as single agents) alone and in combinations. RESULTS: The three-drug combinations of bedaquiline/clofazimine/amikacin, and bedaquiline/clofazimine/sitafloxacin were studied. Among isolates for which the fractional inhibitory concentration index (FICI) could be calculated, 29/70 isolates (41%) and 11/70 isolates (16%) showed a synergistic response (FICI ≤0.75) with combined use of bedaquiline/clofazimine/amikacin, or with bedaquiline/clofazimine/sitafloxacin, respectively. CONCLUSIONS: The combination of bedaquiline with clofazimine plus either amikacin or sitafloxacin may be useful as maintenance regimens when treating pulmonary disease caused by M. abscessus.

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.

Fundamental Cell Morphologies Examined With Cryo-TEM of the Species in the Novel Five Genera Robustly Correlate With New Classification in Family Mycobacteriaceae.

A recent study proposed the novel classification of the family Mycobacteriaceae based on the genome analysis of core proteins in 150 Mycobacterium species. The results from these analyses supported the existence of five distinct monophyletic groups within the genus Mycobacterium. That is, Mycobacterium has been divided into two novel genera for rapid grower Mycobacteroides and Mycolicibacterium, and into three genera for slow grower Mycolicibacter, Mycolicibacillus, and an emended genus Mycobacterium, which include all the major human pathogens. Here, cryo-TEM examinations of 1,816 cells of 31 species (34 strains) belonging to the five novel genera were performed. The fundamental morphological properties of every single cell, such as cell diameter, cell length, cell perimeter, cell circularity, and aspect ratio were measured and compared between these genera. In 50 comparisons on the five parameters between any two genera, only five comparisons showed "non-significant" differences. That is, there are non-significant differences between slow grower genus Mycolicibacillus and genus Mycobacterium in average cell diameter (p = 0.15), between rapid grower genus Mycobacteroides and slow grower genus Mycobacterium in average cell length (p > 0.24), between genus Mycobacteroides and genus Mycobacterium (p > 0.68) and between genus Mycolicibacter and genus Mycolicibacillus (p > 0.11) in average cell perimeter, and between genus Mycolicibacterium and genus Mycobacterium in circularity (p > 0.73). The other 45 comparisons showed significant differences between the genera. Genus Mycobacteroides showed the longest average cell diameter, whereas the genus Mycolicibacter showed the shortest average diameter. Genus Mycolicibacterium showed the most extended average cell length, perimeter, and aspect ratio, whereas the genus Mycolicibacillus showed the shortest average cell length, perimeter, and aspect ratio. Genus Mycolicibacillus showed the highest average cell circularity, whereas genus Mycobacterium showed the lowest average cell circularity. These fundamental morphological data strongly support the new classification in the family Mycobacteriaceae, and this classification is rational and effective in the study of the members of the family Mycobacteriaceae. Because both the genus Mycolicibacterium and the genus Mycobacterium contain many species and showed larger significant standard deviations in every parameter, these genera may be divided into novel genera which show common genotype and phenotypes in morphology and pathogenicity.

The First Case of Concomitant Mycobacterium Genavense Lymphadenitis and EBV-Positive Lymphoproliferative Disorder.

This is the first case of concurrent Mycobacterium genavense lymphadenitis and Epstein-Barr virus (EBV)-positive lymphoproliferative disorder (LPD) in the same lymph node with no immunocompromised history. M. genavense infection is a rare opportunistic infection mainly for human immunodeficiency virus (HIV)-infected patients. Although no immunodeficiency was detected in our patient, our case indicates that the immunodeficiency in the background of EBV latency type III and the immunosuppression by malignant lymphoma itself might induce the M. genavense lymphadenitis. This case highly alerts clinicians to the immunosuppressive state of EBV-positive LPD with latency type III even if any immunodeficient serological factors are not detected.

Diagnostic accuracy of 3 urine lipoarabinomannan tuberculosis assays in HIV-negative outpatients.

BACKGROUNDInadequate tuberculosis (TB) diagnostics are a major hurdle in the reduction of disease burden, and accurate point-of-care tests (POCTs) are urgently needed. We assessed the diagnostic accuracy of Fujifilm SILVAMP TB lipoarabinomannan (FujiLAM) POCT for TB diagnosis in HIV-negative outpatients and compared it with Alere Determine TB LAM Ag (AlereLAM) POCT and a laboratory-based ultrasensitive electrochemiluminescence LAM research assay (EclLAM).METHODSIn this multicenter diagnostic test accuracy study, we recruited HIV-negative adults with symptoms suggestive of pulmonary TB presenting to outpatient health care centers in Peru and South Africa. Urine samples were tested using FujiLAM, AlereLAM, and EclLAM, and the diagnostic accuracy was assessed against a microbiological reference standard (MRS) and a composite reference standard.RESULTSThree hundred seventy-two HIV-negative participants were included and the prevalence of microbiologically confirmed TB was 30%. Compared with the MRS, the sensitivities of AlereLAM, FujiLAM, and EclLAM were 10.8% (95% confidence interval [CI] 6.3%-18.0%), 53.2% (95% CI 43.9%-62.1%), and 66.7% (95% CI 57.5%-74.7%), respectively. The specificities of AlereLAM, FujiLAM, and EclLAM were 92.3% (95% CI 88.5%-95.0%), 98.9% (95% CI 96.7%-99.6%), and 98.1% (95% CI 95.6%-99.2%), respectively. Positive likelihood ratios of AlereLAM, FujiLAM, and EclLAM were 1.4, 46.2, and 34.8, respectively, and positive predictive values were 37.5%, 95.2%, and 93.7%, respectively.CONCLUSIONCompared with AlereLAM, FujiLAM detected 5 times more patients with TB in HIV-negative participants, had a high positive predictive value, and has the potential to improve rapid diagnosis of TB at the point-of-care. EclLAM demonstrated that additional sensitivity gains are possible, which highlights LAM's potential as a biomarker. Additional research is required to assess FujiLAM's performance in prospective cohorts, its cost-effectiveness, and its impact in real-world clinical settings.FUNDINGGlobal Health Innovative Technology Fund, the UK Department for International Development, the Dutch Ministry of Foreign Affairs, the Bill and Melinda Gates Foundation, the Australian Department of Foreign Affairs and Trade, the German Federal Ministry of Education and Research through Kreditanstalt für Wiederaufbau, and the NIH and National Institute of Allergy and Infectious Diseases.

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.

Ultrasensitive enzyme-linked immunosorbent assay for the detection of MPT64 secretory antigen to evaluate Mycobacterium tuberculosis viability in sputum.

OBJECTIVES: This study examined Mycobacterium tuberculosis (MTB)-secreted MPT64 as a surrogate of bacterial viability for the diagnosis of active pulmonary TB (PTB) and for follow-up treatment. METHODS: In this proof-of-concept prospective study, 50 PTB patients in the Tokyo metropolitan region, between 2017 and 2018, were consecutively included and 30 healthy individuals were also included. Each PTB patient submitted sputum on days 0, 14 and 28 for diagnosis and follow-up, and each healthy individual submitted one sputum sample. The following were performed: smear microscopy, Xpert MTB/RIF, MGIT and solid culture, and MPT64 detection on the sputum samples. Ultrasensitive ELISA (usELISA) was used to detect MPT64. The receiver operating characteristic analyses for diagnosis and follow-up revealed the optimal cut-off value of MPT64 absorbance for detecting culture positivity at multiple intervals. RESULTS: The sensitivity of MPT64 for diagnosing PTB was 88.0% (95% CI 75.7-95.5) and the specificity was 96.7% (95% CI 82.8-99.9). The specificity of MPT64 for predicting negative culture results on day 14 was 89.5% (95% CI 66.9-98.7). The sensitivity of MPT64 for predicting positive culture results on day 28 was 81.0% (95% CI 58.1-94.6). CONCLUSIONS: This study revealed that MPT64 is useful for diagnosing active PTB in patients and predicting treatment efficacy at follow-up.

Diagnostic Accuracy of a Novel and Rapid Lipoarabinomannan Test for Diagnosing Tuberculosis Among People With Human Immunodeficiency Virus.

BACKGROUND: The novel Fujifilm SILVAMP TB-LAM (FujiLAM) assay detects mycobacterial lipoarabinomannan in urine and has demonstrated superior sensitivity to the Alere Determine TB-LAM Ag (AlereLAM) assay for detection of tuberculosis among hospitalized people with human immunodeficiency virus (PWH). This is the first study to evaluate the assay among a broad population referred for antiretroviral therapy including both outpatients (mainly) and inpatients. METHODS: We assessed diagnostic accuracy of FujiLAM and AlereLAM assays in biobanked urine samples from a cohort of adults referred for antiretroviral therapy in Ghana against a microbiological and a composite (including clinical judgement) reference standard, and we assessed the association of FujiLAM test positivity with mortality. RESULTS: We evaluated urine samples from 532 PWH (462 outpatients, 70 inpatients). Against a microbiological reference standard, the sensitivity of FujiLAM was 74.2% (95% confidence interval [CI], 62.0-84.2) compared to 53.0% (95% CI, 40.3-65.4) for AlereLAM, a difference of 21.2% (CI, 13.1-32.5). Specificity was 89.3% (95% CI, 85.8-92.2) versus 95.6% (95% CI, 93.0-97.4) for FujiLAM and AlereLAM, a difference of -6.3% (95% CI -9.6 to -3.3). Specificity estimates for FujiLAM increased markedly to 98.8% (95% CI, 96.6-99.8) in patients with CD4 >100 cells/µL and when using a composite reference standard. FujiLAM test positivity was associated with increased cumulative risk of mortality at 6 months (hazard ratio, 4.80; 95% CI, 3.01-7.64). CONCLUSIONS: FujiLAM offers significantly increased diagnostic sensitivity in comparison to AlereLAM. Specificity estimates for FujiLAM were lower than for AlereLAM but were affected by the limited ability of the reference standard to correctly diagnose tuberculosis in individuals with low CD4 counts.

Antimicrobial susceptibility patterns and MICs among non-photochromogenic rapidly growing Mycobacteroides and Mycolicibacterium species.

Introduction. Non-photochromogenic rapidly growing mycobacteria (NPRGM) that branch distinctly from Mycobacteroides (Myco) and Mycolicibacterium (Mycolici) are increasingly observed clinically and present a complicated treatment challenge; thus, appropriate in vitro susceptibility testing is required.Methodology. We evaluated the MICs of nine antimicrobials used in the treatment of infections of 25 NPRGM type strains. The relation of inducible macrolide resistance with functional erythromycin ribosomal methylase (erm) genes was also investigated.Results. The initial clarithromycin MIC reading at 3 days showed resistance in four of the Mycolici strains. In contrast, the presence of erm genes among Mycolici species differed from previous findings. Both Myco and Mycolici species were highly susceptible to amikacin and linezolid. Myco species were resistant to fluoroquinolones, while Mycolici species were susceptible. Meropenem showed low activity against Myco species, but high activity against Mycolici species.Conclusion. NPRGM clade-specific susceptibility patterns suggest an urgent need to establish distinct breakpoints for Myco and Mycolici species.

Evaluation of Q Gene Mycobacteria: A novel and easy nucleic acid chromatography method for mycobacterial species identification.

OBJECTIVES: A simple, rapid, and new diagnostic test for mycobacteria, named Q Gene Mycobacteria, has been developed. It is based on multiplex PCR using primers harbouring DNA tags combined with a dipstick nucleic acid chromatography method, which does not require the denaturation of PCR products for hybridization and can identify five species of mycobacteria including Mycobacterium tuberculosis complex (MTC), Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium kansasii, and Mycobacterium gordonae. This study aimed to evaluate Q Gene Mycobacteria for the accurate identification of these five species. METHODS: A total of 340 mycobacterial strains/isolates were tested, of which 159 were type strains (four MTC and 155 non-tuberculosis mycobacteria (NTM) including four subspecies) and 181 were clinical isolates (18 M. tuberculosis, two Mycobacterium bovis Bacillus Calmette et Guérin (BCG), and 161 NTM comprising 16 species) collected from eight laboratories and hospitals in Japan. Species identification of NTM isolates was performed using the DNA-DNA hybridization method and/or direct sequencing of 16S rRNA, hsp65, and rpoB genes. Q Gene Mycobacteria was compared with above conventional methods for identifying the five species. RESULTS: Q Gene Mycobacteria showed excellent concordance for species identification, specifically 99.4% (158/159) for type strains and 99.4% (180/181) for clinical isolates. The two strains that were misidentified as M. gordonae were Mycobacterium paragordonae. As they are genetically close and there is few case reports of M. paragordonae, it might not be a serious critical issue to distinguish M. paragordonae from M. gordonae. CONCLUSIONS: Q Gene Mycobacteria was able to identify frequently isolated mycobacterial species accurately and easily. Therefore, Q Gene Mycobacteria could be a useful tool for the identification of specific mycobacteria in clinical laboratories.

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.

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.