Whole-Genome Sequence Analysis of Candida glabrata Isolates from a Patient with Persistent Fungemia and Determination of the Molecular Mechanisms of Multidrug Resistance.

Whole-genome sequencing (WGS) was used to determine the molecular mechanisms of multidrug resistance for 10 serial Candida glabrata bloodstream isolates obtained from a neutropenic patient during 82 days of amphotericin B (AMB) or echinocandin therapy. For WGS, a library was prepared and sequenced using a Nextera DNA Flex Kit (Illumina) and the MiseqDx (Illumina) instrument. All isolates harbored the same Msh2p substitution, V239L, associated with multilocus sequence type 7 and a Pdr1p substitution, L825P, that caused azole resistance. Of six isolates with increased AMB MICs (≥2 mg/L), three harboring the Erg6p A158fs mutation had AMB MICs ≥ 8 mg/L, and three harboring the Erg6p R314K, Erg3p G236D, or Erg3p F226fs mutation had AMB MICs of 2-3 mg/L. Four isolates harboring the Erg6p A158fs or R314K mutation had fluconazole MICs of 4-8 mg/L while the remaining six had fluconazole MICs ≥ 256 mg/L. Two isolates with micafungin MICs > 8 mg/L harbored Fks2p (I661_L662insF) and Fks1p (C499fs) mutations, while six isolates with micafungin MICs of 0.25-2 mg/L harbored an Fks2p K1357E substitution. Using WGS, we detected novel mechanisms of AMB and echinocandin resistance; we explored mechanisms that may explain the complex relationship between AMB and azole resistance.

Evaluation of the Accuracy of Cr and BUN Using the ABL90 FLEX PLUS Blood Gas Analyzer and the Equivalence of Candidate Specimens for Assessment of Renal Function.

BACKGROUND: The ABL90 FLEX PLUS (Radiometer) is a blood gas analyzer that also provides creatinine (Cr) and blood urea nitrogen (BUN) results. We assessed the accuracy of the ABL90 FLEX PLUS to measure Cr and BUN and find suitable candidate specimens against primary specimens (heparinized whole-blood (H-WB)). METHODS: Paired H-WB, serum, and sodium-citrated whole-blood (C-WB) samples (105) were collected. The Cr and BUN levels in the H-WB using the ABL90 FLEX PLUS were compared with those of the serum using four automated chemistry analyzers. The suitability of the candidate specimens was assessed at each medical decision level according to the CLSI guideline EP35-ED1. RESULTS: The respective mean differences of the ABL90 FLEX PLUS for the Cr and BUN were below -0.10 and -3.51 mg/dL compared to the other analyzers. The systematic differences between the serum and the H-WB at the low, medium, and high medical decision levels were all 0% for Cr, but those of the C-WB were -12.96%, -11.81%, and -11.30%, respectively. Regarding imprecision, the SDserum/SDH-WB ratios at each level were 0.14, 1.41, and 0.68, whereas the SDC-WB/SDH-WB ratios were 0.35, 2.00, and 0.73, respectively. CONCLUSIONS: The ABL90 FLEX PLUS provided Cr and BUN results comparable with the four widely used analyzers. Among the candidates, the serum was suitable for Cr testing using the ABL90 FLEX PLUS, while the C-WB did not satisfy the acceptance criteria.

Fungal Endophthalmitis Outbreak after Cataract Surgery, South Korea, 2020.

In November 2020, an unusual increase in fungal endophthalmitis cases after cataract surgery was reported to the Korea Disease Control and Prevention Agency, South Korea. We initiated an outbreak investigation to identify the cause. We identified 156 cases nationwide, 62 confirmed and 94 probable. Most case-patients were exposed during surgery to ocular viscoelastic devices (OVDs) from the same manufacturer (company A). We isolated Fusarium spp. from 50 confirmed cases. Molecular identification of 39 fungal isolates from clinical samples and 13 isolates from OVDs confirmed F. oxysporum caused the infections. The risk ratio for fungal endophthalmitis from company A's OVDs was 86.0 (95% CI 27.4-256.9), much higher than risk from other manufacturers' products. We determined this fungal endophthalmitis outbreak was caused by a contaminated lot of OVDs and recommended discontinued use of this product. Early recognition of outbreaks and joint responses from related government agencies can reduce risk for fungal endophthalmitis.

Overexpression of Prohibitin 2 Protein is Associated with Adverse Prognosis in Cytogenetically Normal Acute Myeloid Leukemia.

Cytogenetically normal acute myeloid leukemia (CN-AML) accounts for 40%-50% of all AML cases. Despite advances in understanding the molecular pathophysiology of CN-AML, its clinical outcome remains unsatisfactory and unpredictable. To investigate its clinical implication in CN-AML, we measured the expression of prohibitin 2 (PHB2) using immunohistochemical staining (IHCS) of paraffin-embedded bone marrow sections from 134 CN-AML patients. IHCS results were semi-quantitatively scored. Clinical outcome was analyzed in comparison with other prognostic markers, including NPM1 polymorphism and FLT3 internal tandem duplication, and WT1 and BAALC mRNA expression. Except for BAALC mRNA expression, the known molecular markers showed no prognostic effect in the CN-AML patients. PHB2 protein overexpression was significantly associated with adverse prognosis in CN-AML patients. The PHB2 protein expression status may serve as an independent prognostic indicator in CN-AML.

Diagnostic Validation of a Clinical Laboratory-Oriented Targeted RNA Sequencing System for Detecting Gene Fusions in Hematologic Malignancies.

Targeted RNA sequencing (RNA-seq) is a highly accurate method for sequencing transcripts of interest with a high resolution and throughput. However, RNA-seq has not been widely performed in clinical molecular laboratories because of the complexity of data processing and interpretation. We developed and validated a customized RNA-seq panel and data processing protocol for fusion detection using 4 analytical validation samples and 51 clinical samples, covering seven types of hematologic malignancies. Analytical validation showed that the results for target gene coverage and between- and within-run precision and linearity tests were reliable. Using clinical samples, RNA-seq based on filtering and prioritization strategies detected all 25 known fusions previously found by multiplex reverse transcriptase-PCR and fluorescence in situ hybridization. It also detected nine novel fusions. Known fusions detected by RNA-seq included two IGH rearrangements supported by expression analysis. Novel fusions included six that targeted just one partner gene. In addition, 18 disease- and drug resistance-associated transcript variants in ABL1, GATA2, IKZF1, JAK2, RUNX1, and WT1 were designated simultaneously. Expression analysis showed distinct clustering according to subtype and lineage. In conclusion, this study showed that our customized RNA-seq system had a reliable and stable performance for fusion detection, with enhanced diagnostic yield for hematologic malignancies in a clinical diagnostic setting.

Disk Diffusion Susceptibility Testing for the Rapid Detection of Fluconazole Resistance in Candida Isolates.

BACKGROUND: Given the increased fluconazole resistance (FR) among Candida isolates, we assessed the suitability of disk diffusion susceptibility testing (DDT) for the early detection of FR using well-characterized Candida isolates. METHODS: In total, 188 Candida isolates, including 66 C. albicans (seven Erg11 mutants), 69 C. glabrata (33 Pdr1 mutants), 29 C. parapsilosis (15 Erg11 mutants), and 24 C. tropicalis (eight Erg11 mutants) isolates, were tested in this study. FR was assessed using DDT according to the standard CLSI M44-ED3 method, except that two cell suspensions, McFarland 0.5 (standard inoculum) and 2.5 (large inoculum), were used, and the inhibition zones were read at 2-hour intervals from 10 hours to 24 hours. RESULTS: DDT results for the standard inoculum were readable after 14 hours (C. albicans, C. glabrata, and C. tropicalis) and 20 hours (C. parapsilosis) for >95% of the isolates, whereas the results for the large inoculum were readable after 12 hours (C. glabrata and C. tropicalis), 14 hours (C. albicans), and 16 hours (C. parapsilosis) for >95% of the isolates. Compared with the results produced using the CLSI M27-ED4 broth microdilution method, the first readable results from the DDT method for each isolate exhibited an agreement of 97.0%, 98.6%, 72.4%, and 91.7% for the standard inoculum and 100%, 98.6%, 96.6%, and 95.8% for the large inoculum for C. albicans, C. glabrata, C. parapsilosis, and C. tropicalis, respectively. CONCLUSIONS: DDT using large inoculum may detect FR rapidly and reliably in the four most common Candida species.

Evaluation of Two Commercial Broth Microdilution Methods Using Different Interpretive Criteria for the Detection of Molecular Mechanisms of Acquired Azole and Echinocandin Resistance in Four Common Candida Species.

The abilities of the new Vitek 2 AST-YS08 (YS08) and Sensititre YeastOne (SYO) systems to detect the resistances of Candida isolates to azoles and echinocandins were evaluated. In total, 292 isolates, including 28 Candida albicans (6 Erg11 and 2 Fks mutants), 57 Candida parapsilosis (26 Erg11 mutants), 24 Candida tropicalis (10 Erg11 and 1 Fks mutants), and 183 Candida glabrata (39 Pdr1 and 13 Fks mutants) isolates, were tested. The categorical agreements (CAs) between the Clinical and Laboratory Standards Institute (CLSI) method and YS08 fluconazole MICs obtained using clinical breakpoints were 92.4% (C. albicans), 96.5% (C. parapsilosis), and 87.0% (C. tropicalis), and the CAs between the CLSI and SYO MICs were 92.3% (C. albicans), 77.2% (C. parapsilosis), 100% (C. tropicalis), and 98.9% (C. glabrata). For C. glabrata, the CAs with the CLSI micafungin MICs were 92.4% and 55.5% for the YS08 micafungin and caspofungin MICs, respectively; they were 100%, 95.6%, and 98.9% for the SYO micafungin, caspofungin, and anidulafungin MICs, respectively. YS08 does not provide fluconazole data for C. glabrata; the CA with the CLSI fluconazole MIC was 97.8% for the YS08 voriconazole MIC, using an epidemiological cutoff value (ECV) of 0.5 µg/ml. Increased CAs with the CLSI MIC were observed for the YS08 MIC using CLSI ECVs (for fluconazole and C. tropicalis, 100%; for micafungin and C. glabrata, 98.9%) and for the SYO MIC using method-specific ECVs (for fluconazole and C. parapsilosis, 91.2%; for caspofungin and C. glabrata, 98.9%). Therefore, the YS08 and SYO systems may have different abilities to detect mechanisms of azole and echinocandin resistance in four Candida species; the use of method-specific ECVs may improve the performance of both systems.

Characterization of the new HLA-DQB1*06:344 allele by next-generation sequencing in a Korean individual.

DQB1*06:344 differs from DQB1*06:02:01:01 by one nucleotide substitution at codon 211 in exon 3.