Yuriko Igarashi

Evaluation of QuantiFERON-TB Gold Plus for Detection of Mycobacterium tuberculosis infection in Japan

Performance of interferon-γ (IFN-γ) release assays still needs to be improved. The data on the performance of QuantiFERON-TB Gold Plus (QFT-Plus), a new-generation of QFT assay are limited. This study evaluated the diagnostic performance of QFT-Plus, and compared to that of QuantiFERON-TB Gold In-Tube (QFT-GIT). Blood samples were collected from 162 bacteriologically confirmed tuberculosis (TB) patients and 212 Mycobacterium tuberculosis-uninfected volunteers; these samples were then tested with QFT-GIT and QFT-Plus. The IFN-γ concentration of QFT-Plus was lower than that of QFT-GIT in TB patients (p < 0.001). Receiver operating characteristic curves were compared between QFT-GIT and QFT-Plus. Both assays showed area under the curve values over 0.99 without significant difference. Using the conventional cut-off (0.35 IU/mL) for QFT-GIT, QFT-Plus had a lower sensitivity of 91.1% compared to 96.2% (p = 0.008) at its optimum cut-off (0.168 IU/mL) with the same specificity. Moreover, IFN-γ values were significantly reduced with age in QFT-GIT (p = 0.035) but not in QFT-Plus. The diagnostic performance of QFT-Plus was as accurate as that of QFT-GIT despite a lack of TB7.7 antigen and despite the decrease in quantitative values. However, the cut-off value for QFT-Plus should be considered independently from that of QFT-GIT to obtain the best sensitivity without compromising specificity.

In vitro activity of sitafloxacin against Mycobacterium tuberculosis with gyrA/B mutations isolated in Japan

Purpose. Sitafloxacin (SFX) is a new fluoroquinolone (FQ) that has shown a strong bactericidal effect against Mycobacterium tuberculosis (Mtb) in vitro. However, data on SFX efficacy against Mtb with gyrA/B mutations and its epidemiological cut‐off (ECOFF) value remain limited. Therefore, we evaluated and compared the in vitro activity of SFX against gyrA/B‐mutant Mtb to that of moxifloxacin (MFX), levofloxacin (LFX) and ciprofloxacin (CFX), and determined the ECOFF for SFX. Methodology. A total of 109 clinical Mtb isolates, including 73 multidrug‐resistant (MDR) isolates, were subjected to minimum inhibitory concentration (MIC) analysis in oleic‐albumin‐dextrose‐catalase (OADC)‐supplemented Middlebrook 7H9 medium. Our results showed that SFX had lower cumulative MIC than MFX, LFX and CFX. Furthermore, we performed direct DNA sequencing of the quinolone‐resistance‐determining regions (QRDRs). Results. We identified the following mutations: D94G, D94A, A90V, D94H, D94N and G88A in gyrA; and A543V, A543T, E540D, R485C, D500A, I552S and D577A in gyrB. Based on our results, an ECOFF of 0.125 &mgr;g ml−1 was proposed for SFX. With this ECOFF, 15% of LFX‐resistant isolates with MIC ≥2 &mgr;g ml−1 were susceptible to SFX. Conclusion. SFX had the lowest cumulative MIC and a relatively low ECOFF value against Mtb, indicating that SFX was not only more effective against gyrA‐mutant isolates, but also MDR isolates in Japan.

Laboratory evaluation of the Anyplex™ II MTB/MDR and MTB/XDR tests based on multiplex real-time PCR and melting-temperature analysis to identify Mycobacterium tuberculosis and drug resistance.

We evaluated the performance of two multiplex, real-time PCR tests (Anyplex II MTB/MDR and MTB/XDR; Seegene, Seoul, Korea), designed to detect the Mycobacterium tuberculosis complex (MTC) and drug-resistance mutations associated with isoniazid, rifampicin, fluoroquinolones, and second-line injectable drugs. We analyzed 122 clinical isolates with the Anyplex II MTB/MDR test, 68 of which were also tested with the Anyplex II MTB/XDR test. The Anyplex II MTB/MDR and MTB/XDR tests showed the following respective sensitivities and specificities: 68.8% and 100% for detecting isoniazid resistance, 93.8% and 100% for rifampicin, 82.8% and 100% for levofloxacin, 75.0% and 100% for kanamycin, and 92.6% and 100% for MTC identification. These kits correctly identified 61.8% of multi-drug resistant M. tuberculosis isolates and 64.7% of extensively drug-resistant M. tuberculosis isolates, and enabled semi-automatic detection of drug-resistant MTC in 3 hours. The Anyplex II kits could be useful as rule-in tests for detecting MTC and drug resistance.

A simplified pyrazinamidase test for pyrazinamide drug susceptibility in Mycobacterium tuberculosis

We modified Waynes pyrazinamidase test against Mycobacterium tuberculosis to indirectly measure pyrazinamidase activity via pyrazinoic acid in liquid medium. The modified pyrazinamidase test was easy to perform and its results were in complete agreement with those of the conventional Waynes method, highlighting its potential application in phenotypic pyrazinamide susceptibility testing.

Six species of nontuberculous mycobacteria carry non-identical 16S rRNA gene copies

Nontuberculous mycobacteria (NTM) can carry two or more 16S rRNA gene copies that are, in some instances, non-identical. In this study, we used a combined cloning and sequencing approach to analyze 16S rRNA gene sequences of six NTM species, Mycobacterium cosmeticum, M. pallens, M. hodleri, M. crocinum, M. flavescens, and M. xenopi. Our approach facilitated the identification of two distinct gene copies in each species. The two M. cosmeticum genes had a single nucleotide difference, whereas two nucleotide polymorphisms were identified in M. hodleri, M. flavescens, and M. xenopi. M. pallens had a difference in four nucleotides and M. crocinum - in 23 nucleotides. Thus, we showed that the six NTM species possess at least two non-identical 16S rRNA gene copies. The full-length sequences of the intraspecies 16S rRNA variants will facilitate NTM identification and sequence analysis of specimens or other samples.

Mycolicibacterium smegmatis, Basonym Mycobacterium smegmatis, Expresses Morphological Phenotypes Much More Similar to Escherichia coli Than Mycobacterium tuberculosis in Quantitative Structome Analysis and CryoTEM Examination

A series of structome analyses, that is, quantitative and three-dimensional structural analysis of a whole cell at the electron microscopic level, have already been achieved individually in Exophiala dermatitidis, Saccharomyces cerevisiae, Mycobacterium tuberculosis, Myojin spiral bacteria, and Escherichia coli. In these analyses, sample cells were processed through cryo-fixation and rapid freeze-substitution, resulting in the exquisite preservation of ultrastructures on the serial ultrathin sections examined by transmission electron microscopy. In this paper, structome analysis of non pathogenic Mycolicibacterium smegmatis, basonym Mycobacterium smegmatis, was performed. As M. smegmatis has often been used in molecular biological experiments and experimental tuberculosis as a substitute of highly pathogenic M. tuberculosis, it has been a task to compare two species in the same genus, Mycobacterium, by structome analysis. Seven M. smegmatis cells cut into serial ultrathin sections, and, totally, 220 serial ultrathin sections were examined by transmission electron microscopy. Cell profiles were measured, including cell length, diameter of cell and cytoplasm, surface area of outer membrane and plasma membrane, volume of whole cell, periplasm, and cytoplasm, and total ribosome number and density per 0.1 fl cytoplasm. These data are based on direct measurement and enumeration of exquisitely preserved single cell structures in the transmission electron microscopy images, and are not based on the calculation or assumptions from biochemical or molecular biological indirect data. All measurements in M. smegmatis, except cell length, are significantly higher than those of M. tuberculosis. In addition, these data may explain the more rapid growth of M. smegmatis than M. tuberculosis and contribute to the understanding of their structural properties, which are substantially different from M. tuberculosis, relating to the expression of antigenicity, acid-fastness, and the mechanism of drug resistance in relation to the ratio of the targets to the corresponding drugs. In addition, data obtained from cryo-transmission electron microscopy examination were used to support the validity of structome analysis. Finally, our data strongly support the most recent establishment of the novel genus Mycolicibacterium, into which basonym Mycobacterium smegmatis has been classified.

Prevention of aerosol isolation of nontuberculous mycobacterium from the patient's bathroom

Recent clinical studies have revealed that reappearance of the same nontuberculous mycobacterium (NTM) infection is common after successful standard treatment [1, 2]. Using pulsed-field gel electrophoresis analysis, Wallace et al. [1] found that ∼75% of Mycobacterium avium-intracellulare complex (MAC) isolates identified after successful treatment are the result of reinfection. According to a recent study conducted by Koh et al. [2] using repetitive sequence-based PCR analysis, all re-identified M. abscessus subsp. abscessus isolates had a unique genotype. Therefore, patients with NTM are exposed to large amounts of microbes in their daily lives, particularly in cases of reinfection. Reinfection of nontuberculous mycobacterium pulmonary disease may be caused by identical and not different genotypes http://ow.ly/62cH30krdpa

Evaluation of PyroMark Q24 pyrosequencing as a method for the identification of mycobacteria

We evaluated PyroMark Q24 (QIAGEN) pyrosequencing as a method for the identification of mycobacteria, with potential application in clinical practice. Sequence data from the hypervariable region A of the 16S rRNA gene (43 and 35bp sequences) were obtained using PyroMark Q24, and a similarity search was performed automatically with PyroMark IdentiFire software. Of the 148 mycobacterial type strains tested, 138 (93.2%) were accurately identified to single or clade species level, including complex level. From the remaining 10 strains, 3 (Mycobacterium gilvum, Mycobacterium goodi, and Mycobacterium thermoresistible) showed poor sequencing quality of homopolymers. For 6 other strains (Mycobacterium cosmeticum, Mycobacterium flavescens, Mycobacterium pallens, Mycobacterium hodleri, Mycobacterium xenopi, and Mycobacterium crocinum), the sequences were unreadable from the middle, and Sanger sequencing indicated biallelic site. Finally, a 40bp sequence for Mycobacterium gordonae could not be obtained despite repeated attempts. PyroMark Q24 provided accurate identification of multiple mycobacterial strains isolated from common clinical settings, but additional gene sequencing is required to distinguish species identified as a group or complex.

Clnico-microbiological analysis of mycobacaterium abscessus complex in Japan

Background: The reports on three subspecies of M. abscesses have been increasing in recent years. While macrolide containing regimens are associated with poor reponse to M.abscessus (subsp. abscessus ) and M. bolletii (subsp. bolletii ), M.massiliense (subsp. massiliense ) shows relatively favorable oucome. This clinical inconsistency within subspecies is mainly due to erm gene activation. However, there are a few data analysing clinico-microbiological interrelations about Japanese patients. Methods: A total of 103 M. abscessus stains from 84 patients through 2003 to 2014 at Fukujuji Hospital were identified using rpoB sequenced analysis. The drug ssceptibility testing was performed following CLSI M24-A2. Point mutation at 28 in erm gene and at 2058 in 23S rRNA were analysed by pyrosequencing. Results: rp o B sequence analysis identified 47 M.abscessus (56%), 35 M.massiliense (42%), and 2 M. bolletii (2%). While all M.abscessus and M.bolletii strains were resistant to clarithromycin (CAM) (MIC ≥8)except for three strains that two of them have C28, only one M.massiliense strains holding point mutation at position 2058 in 23S rRNA was resistant to CAM. No significant difference was observed on patients9 clinical manifestations (age, p=0. 347, gender, p=0. 158, radiological findings, p=0.796) between cases of M.abscessus (30 cases) and M.massiliense (29 cases). Eight M. abscessus cases died, while only two mortal cases in M.massiliense , but due to other diseases (p Conclusion: This study showed difficulty in subspecies classification of M.abscessus by clinical presentations. Proper identification and susceptibility testing shall be warranted in routine clinical settings.