Dong Joon Song

Evaluation of the Cobas TaqMan MTB Test for the Detection of Mycobacterium tuberculosis Complex According to Acid-Fast-Bacillus Smear Grades in Respiratory Specimens

ABSTRACT We evaluated the performance of the Cobas TaqMan MTB test (Roche Diagnostics, Basel, Switzerland), stratified by acid-fast bacilli (AFB) smear grades. The sensitivity of this test in smear-positive specimens was >95% in all grades, while that in trace and negative specimens was 85.3% and 34.4%, respectively.

Resistance mechanisms of linezolid-nonsusceptible enterococci in Korea: low rate of 23S rRNA mutations in Enterococcus faecium

PURPOSE To investigate linezolid-resistance mechanisms in linezolid-nonsusceptible enterococci (LNSE) isolated from a tertiary hospital in Korea. METHODOLOGY Enterococcal isolates exhibiting linezolid MICs ≥4 mg l-1 that were isolated between December 2011 and May 2016 were investigated by PCR and sequencing for mutations in 23S rRNA or ribosomal proteins (L3, L4 and L22) and for the presence of cfr, cfr(B) and optrA genes.Results/Key findings. Among 135 LNSE (87 Enterococcus faecium and 48 Enterococcus faecalis isolates), 39.1 % (34/87) of E. faecium and 18.8 % (9/48) of E. faecalis isolates were linezolid-resistant. The optrA carriage was the dominant mechanism in E. faecalis: 13 isolates, including 10 E. faecalis [70 % (7/10) linezolid-resistant and 30 % (3/10) linezolid-intermediate] and three E. faecium [33.3 % (1/3) linezolid-resistant and 66.7 % (2/3) linezolid-intermediate], contained the optrA gene. G2576T mutations in the 23S rRNA gene were detected only in E. faecium [14 isolates; 71.4 % (10/14) linezolid-resistant and 28.6 % (4/14) linezolid-intermediate]. One linezolid-intermediate E. faecium harboured a L22 protein alteration (Ser77Thr). No isolates contained cfr or cfr(B) genes and any L3 or L4 protein alterations. No genetic mechanism of resistance was identified for 67.6 % (23/34) of linezolid-resistant E. faecium. CONCLUSION A low rate of 23S rRNA mutations and the absence of known linezolid-resistance mechanisms in the majority of E. faecium isolates suggest regional differences in the mechanisms of linezolid resistance and the possibility of additional mechanisms.

Evaluation of three real-time PCR assays for differential identification of Mycobacterium tuberculosis complex and nontuberculous mycobacteria species in liquid culture media

We evaluated the analytical performance of M. tuberculosis complex (MTBC)/nontuberculous mycobacteria (NTM) PCR assays for differential identification of MTBC and NTM using culture-positive liquid media. Eighty-five type strains and 100 consecutive mycobacterial liquid media cultures (MGIT 960 system) were analyzed by a conventional PCR assay (MTB-ID(®) V3) and three real-time PCR assays (AdvanSure™ TB/NTM real-time PCR, AdvanSure; GENEDIA(®) MTB/NTM Detection Kit, Genedia; Real-Q MTB & NTM kit, Real-Q). The accuracy rates for reference strains were 89.4%, 100%, 98.8%, and 98.8% for the MTB-ID V3, AdvanSure, Genedia, and Real-Q assays, respectively. Cross-reactivity in the MTB-ID V3 assay was mainly attributable to non-mycobacterium Corynebacterineae species. The diagnostic performance was determined using clinical isolates grown in liquid media, and the overall sensitivities for all PCR assays were higher than 95%. In conclusion, the three real-time PCR assays showed better performance in discriminating mycobacterium species and non-mycobacterium Corynebacterineae species than the conventional PCR assay.

Laboratory Identification of Leptotrichia Species Isolated From Bacteremia Patients at a Single Institution

We describe the laboratory identification of Leptotrichia species from clinical isolates collected over a six-year period. Five isolates from blood cultures were identified as Leptotrichia species. Gram stain showed large, fusiform, gram-negative or -variable bacilli. Identification based on biochemical testing was unsuccessful; however, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry proved to be a useful tool for identifying Leptotrichia species to the genus level. Species level identification was successfully achieved by using 16S ribosomal RNA gene sequencing.

Performance evaluation of the Cobas TaqMan MTB assay on respiratory specimens according to clinical application

OBJECTIVE To evaluate the performance of the Cobas TaqMan MTB assay (Cobas assay) with respect to its clinical application. METHODS This was a retrospective analysis of 1154 results from 1034 patients for whom mycobacterial cultures and the Cobas assay were performed simultaneously. Based on the patient medical records, two categories of clinical application were defined: (1) the diagnosis of patients with a high probability of pulmonary tuberculosis according to clinical and radiological features (n=128), and (2) the exclusion of tuberculosis in clinically indeterminate patients (n=1026). Standard culture was used as the reference method. RESULTS The sensitivity of the Cobas assay for the detection of Mycobacterium tuberculosis was 70.4% (95% confidence interval (CI) 49.7-85.5%) for category 1, but only 25.0% (95% CI 4.5-64.4%) for category 2. The specificity was ≥95.0% for both categories. The positive predictive value was 79.2% (95% CI 57.3-92.1%) for category 1 and 33.3% (95% CI 6.0-75.9%) for category 2, while the negative predictive value was 92.3% (95% CI 85.0-96.4%) for category 1 and 99.4% (95% CI 98.7-99.8%) for category 2. CONCLUSIONS The results of this study indicate that Cobas assay results must be interpreted carefully according to the clinical purpose of the assay.

Comparative evaluation of the AdvanSure Mycobacteria GenoBlot assay and the GenoType Mycobacterium CM/AS assay for the identification of non-tuberculous mycobacteria

In this study, to assess the performance of the AdvanSure Mycobacteria GenoBlot assay (AdvanSure assay), we compared its performance with that of the GenoType Mycobacterium CM/AS assay (GenoType assay) for the identification of non-tuberculous mycobacteria (NTM). Twenty-four reference strains and 103 consecutive clinical NTM isolates were analysed. The accuracy rates for the 24 reference strains were 87.5 and 95.8 % for the AdvanSure and GenoType assays, respectively. For the 103 clinical isolates, a 91.3 % (94/103) concordance rate was observed between the two assays. The majority (7/9) of discrepancies were isolates identified as Mycobacterium avium complex (MAC) by only the AdvanSure assay. All of these isolates except one were confirmed as MAC by sequence-based typing. The AdvanSure assay showed comparable performance to the GenoType assay and can be useful as a routine method for NTM identification in the clinical setting, especially where MAC is the main cause of NTM infection.

Comparison of BacT/Alert FAN and FAN Plus Bottles with Conventional Medium for Culturing Cerebrospinal Fluid

ABSTRACT We compared the BacT/Alert system FAN and FAN Plus media to conventional media for culturing cerebrospinal fluid (CSF) with 2,545 samples. FAN/FAN Plus bottles showed better performance for isolating microorganisms in CSF than conventional media (positive rate, 7.2% [182/2,545] versus 3.1% [80/2,545]). The incremental recovery rate of Cryptococcus neoformans from FAN Plus bottles was higher than that from FAN bottles.

A Case of Cruoricaptor ignavus Isolated From the Blood of a Patient With Ewing Sarcoma

Dear Editor, Cruoricaptor ignavus, belonging to the family Flavobacteriaceae, is a gram-negative, non-motile, non-spore-forming coccoidor coccobacilli-shaped bacterium first isolated from a human blood culture in 2012 and proposed as a novel genus and species [1]. With approval from Samsung Medical Center institutional review board (IRB; approval number: 2018-01-112), we report the second case worldwide of C. ignavus, isolated from the blood culture of a 16-year-old boy with Ewing sarcoma and identified by DNA target sequencing. The IRB waived the need for informed consent for this study. The patient underwent wide excision of sarcoma and additional chemotherapy. On day 13 of chemotherapy, he developed fever with abdominal pain and visited the emergency room. His temperature was 38.3°C, blood pressure was 116/59 mmHg, pulse rate was 67 beats per minute, and respiratory rate was 18 breaths per minute. The C-reactive protein concentration was 146.7 nmol/L, and leukocyte count was 0.21×10/L with neutropenia (0.07×10/L). Blood, urine, and stool cultures were performed, and cefepime was administered. Positive growth was observed in one of two sets of blood cultures after two days of incubation. Microscopic examination revealed gram-variable cocci or coccobacilli (Fig. 1), which grew as tiny, yellowish colonies on blood agar plates (Fig. 2). The microorganism could not be identified using the GP, GN, and NH cards of the VITEK 2 system (bioMérieux, Marcy l’Etoile, France) or by matrix-assisted laser desorption/ionization time-offlight mass spectrometry (MALDI-TOF MS) with the Bruker MALDI Biotyper system (Bruker Daltonics GmbH, Leipzig, Germany). The VITEK MS system identified the organism as Alloiococcus otitis (99.9% confidence), but Gram staining and colony morphology showed that this identification was not accurate. To identify the strain, we performed 16S ribosomal RNA (rRNA) target sequencing according to the CLSI guidelines [2]. Subregions of the 16S rRNA gene were amplified using the following primer pairs: forward, 4F: 5 ́-TTG GAG AGT TTG ATC CTG GCT C-3 ́ and reverse, 534R: 5 ́-TAC CGC GGC TGC TGG CAC-3 ́ and forward, 27F: 5 ́-AGA GTT TGA TCM TGG CTC AG3 ́ and reverse, 801R: 5 ́-GGC GTG GAC TTC CAG GGT ATC T-3 ́ [2]. The amplified sequence was compared with the GenBank (National Center for Biotechnology Information) database, using the basic local alignment search tool (BLAST) algorithm. The 16S rRNA sequence of the isolate exhibited 99.72% (722/724 bp) similarity to C. ignavus (GenBank accession number NR_108875.1, Strain IMMIB L-12475). The second highest match was Epilithonimonas xixisoli with 87.07% (653/750 bp) similarity. When the sequence (724 bp) was submitted to the EzTaxon database v2.1 (http://www.ezbiocloud.net), the best-

Performance evaluation of the QMAC-dRAST for staphylococci and enterococci isolated from blood culture: a comparative study of performance with the VITEK-2 system

Objectives To evaluate the performance of a rapid antimicrobial susceptibility testing (AST) platform based on microfluidic chip technology, the QMAC-dRAST, which enables AST from colony isolates or positive blood culture broth (PBCB), and to compare the performance of the QMAC-dRAST for staphylococci and enterococci with that of the VITEK-2 system based on reference broth microdilution (BMD). Methods A total of 110 staphylococcal and enterococcal isolates from blood cultures were included. AST was performed directly using the QMAC-dRAST with PBCB. Thereafter, colony isolates derived from subculture of PBCB were used for the QMAC-dRAST, the VITEK-2 system and BMD. Results The overall agreement between the QMAC-dRAST with PBCB and BMD was 91.5%. There were 1.2% very major errors (VMEs), 4.3% major errors (MEs) and 5.4% minor errors (mEs). The QMAC-dRAST with colony isolates yielded 94.6% agreement and error rates of 1.0% VMEs, 1.8% MEs and 4.0% mEs. The VITEK-2 system showed 96.2% agreement and error rates of 2.3% VMEs, 0.5% MEs and 2.6% mEs. The incubation time in the QMAC-dRAST was significantly shorter than in the VITEK-2 system (median of 6 versus 10 h; P < 0.0001). Conclusions The QMAC-dRAST system provided rapid results and represents an alternative to conventional AST methods. The QMAC-dRAST with colony isolates produced more reliable results for staphylococci and enterococci than the QMAC-dRAST with PBCB. The QMAC-dRAST system also performed comparably to BMD and the VITEK-2 system.

Drug susceptibility patterns of Mycobacterium abscessus and Mycobacterium massiliense isolated from respiratory specimens

In this study, we aimed to retrospectively investigate and compare the drug susceptibility patterns of two major Mycobacterium abscessus complex (MABC) species; M. abscessus and M. massiliense. A total of 546 MABC respiratory isolates (277 M. abscessus and 269 M. massiliense) from 2011 to 2016 were analyzed in this study. We estimated minimum inhibitory concentrations (MICs) using the broth microdilution method, and we calculated MIC50 and MIC90 values from the MIC distribution. Both M. abscessus and M. massiliense were highly susceptible to amikacin and linezolid. For M. abscessus, the proportions of inducible and acquired resistance to clarithromycin were 68.6% and 12.3%, respectively. Only 15.2% of M. abscessus remained susceptible at day 14. On the other hand, none of the M. massiliense showed inducible resistance and 6.3% showed acquired resistance to clarithromycin. A total of 92.6% of the M. massiliense remained susceptible at day 14. The resistance rate of M. abscessus to moxifloxacin (90.3%) was significantly higher than that of M. massiliense (83.3%; p = 0.016). These susceptibility differences may explain the divergent treatment outcomes between patients with pulmonary disease caused by these two species.