Lina Yi

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.

Detection of Mycobacterium tuberculosis (MTB) in Fecal Specimens From Adults Diagnosed With Pulmonary Tuberculosis Using the Xpert MTB/Rifampicin Test

We performed a proof-of-concept study using the Xpert MTB/RIF test for the detection of Mycobacterium tuberculosis from fecal samples. The overall sensitivity was 85.7% (95% CI; 73.8 – 93.6%) and the specificity was 100% (95% CI; 86.2–100).

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.

Linezolid as a Potentially Effective Drug for the Treatment of Multidrug-Resistant Tuberculosis in Japan

Linezolid (LZD) is classified as a WHO group 5 drug used in the treatment of tuberculosis (TB). Although its efficacy and long-term safety have not yet been established, it is being increasingly used in the treatment of multidrug resistant tuberculosis (MDR-TB) and extensive multidrug-resistant tuberculosis (XDR-TB). The current study is a single-center retrospective clinical analysis of hospitalized M/XDR-TB patients in Fukujuji Hospital involving 26 patients (18 men and 8 women) consecutively treated with combinations of anti-TB drugs including LZD from 2009 to 2015. The sputum culture results were negative after using LZD for an average period of 28.0 ± 12.0 (average ± SD) days. LZD was reduced or withdrawn in 11 cases due to adverse effects. Nineteen cases including 3 XDR-TB patients were operated on, and their TB was treated following surgery. The average time from the initiation of LZD therapy to surgery was 87.6 ± 38.7 (average ± SD) days. Favorable clinical outcome was maintained in 23 surviving patients, while 3 patients died during treatment because of end stage cancer and aspiration pneumonia. Our study showed that LZD might be clinically effective in the treatment of M/XDR-TB patients 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.

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 (Spearmans rank correlation coefficient: r(s) = -0.940, P < 0.001 and Pearsons 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.

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.