The first case of cholecytitis caused by Aggregatibacter kilianii in Korea.

BACKGROUND: The bacterial genus Aggregatibacter was categorized in 2006 to accommodate the former Actinobacillus actinomycetemcomitans, Haemophilus aphrophilus, and H. segnis species. Aggregatibacter kilianii is a normal resident of the human upper respiratory tract but can also cause serious infections. A. kilianii is relatively newly identified and has been isolated from conjunctivitis, wounds, abdominal abscesses, and blood. CASE PRESENTATION: An 80-year-old female patient with distal common bile duct cancer was admitted to our hospital with sudden loss of consciousness and general weakness, fever, and abdominal pain for 3 days. Two colonial morphologies were isolated from both the blood and bile cultures; one was identified as Streptococcus constellatus subsp. pharyngis, but the other was not recognized by Vitek2 and MALDI-TOF. The 16 S rRNA sequences showed 99.73% similarity with the sequence of A. kilianii strains. CONCLUSION AND DISCUSSION: This article presents the first case of a clinical isolate of A. kilianii outside Europe. This case is also the first of the antimicrobial profile of this strain. This report highlights the importance of proper molecular identification for timely diagnosis and treatment of disease.

Fatal Prototheca zopfii Algaemia in a Patient with Acute Lymphoblastic Leukemia: a Case Report.

BACKGROUND: Prototheca algaemia is a rare but life-threatening disease that occurs primarily in immunocompromised patients. We report a fatal case of Prototheca zopfii bloodstream infection in a 54-year-old woman receiving chemotherapy for relapsed acute lymphoblastic leukemia. METHODS: The isolate was identified using an automated biochemical identification system (VITEK 2; bioMerieux) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (VITEK MS; bioMerieux). Partial 18S and 28S rDNA sequencing was performed for definitive identification and genotyping. RESULTS: The patient had persistent neutropenic fever, and isolates from blood culture were identified as P. zopfii. Sequencing was performed and the isolate was confirmed to be P. zopfii genotype 2, which was newly named as P. bovis. The patient was treated with liposomal amphotericin B but died of septic shock. CONCLUSIONS: Prototheca spp. should be considered an emerging pathogen, especially in immunocompromised patients, due to its ubiquitous nature.

Novel indel mutation in the N gene of SARS-CoV-2 clinical samples that were diagnosed positive in a commercial RT-PCR assay.

Commercially available reverse transcription-polymerase chain reaction (RT-PCR) kits are being used as an important tool to diagnose SARS-CoV-2 infection in clinical laboratories worldwide. However, some kits lack sufficient clinical evaluation due to the need for emergency use caused by the current COVID-19 pandemic. Here we found that a novel insertion/deletion mutation in the nucleocapsid (N) gene of SARS-CoV-2 samples is a cause of negative results for the N gene in a widely used assay that received emergency use authorization (EUA) from US FDA and Conformite Europeenne-in vitro diagnostics (CE-IVD) from EU. Although SARS-CoV-2 is diagnosed positive by other target probes in the assay, our findings provide an evidence of the genetic variability and rapid evolution of SARS-CoV-2 as well as a reference in designing commercial RT-PCR assays.

Comparative Evaluation of the Loop-Mediated Isothermal Amplification Assay for Detecting Pulmonary Tuberculosis.

BACKGROUND: Early detection of tuberculosis (TB) is challenging in resource-poor settings because of limited accessibility to molecular diagnostics. The aim of this study was to evaluate the performance of the loop-mediated isothermal amplification kit (TB-LAMP) for TB diagnosis compared with conventional and molecular tests. METHODS: A total of 290 consecutive sputum samples were collected from May till September, 2015. All samples were processed using the N-Acetyl-L-cysteine (NALC) NaOH method and tested by smear microscopy, solid and liquid culture, real-time PCR, and TB-LAMP. RESULTS: The sensitivity of TB-LAMP for smear-positive and smear-negative samples with culture positivity was 92.0% and 58.8%, respectively. TB-LAMP was positive in 14.9% of TB culture-negative samples; however, all those samples were also positive by real-time PCR. In addition, none of the samples positive for nontuberculous mycobacteria by culture were positive by TB-LAMP. The overall agreement between TB-LAMP and real-time PCR was good; however, the concordance rate was significantly lower for real-time PCR positive samples with Ct values of 30-35. CONCLUSIONS: TB-LAMP could replace smear microscopy and increase TB diagnostic capacity when Xpert MTB/RIF is not feasible because of poor infrastructure.

Description of Mycobacterium chelonae subsp. bovis subsp. nov., isolated from cattle (Bos taurus coreanae), emended description of Mycobacterium chelonae and creation of Mycobacterium chelonae subsp. chelonae subsp. nov.

Three rapidly growing mycobacterial strains, QIA-37T, QIA-40 and QIA-41, were isolated from the lymph nodes of three separate Korean native cattle, Hanwoo (Bos taurus coreanae). These strains were previously shown to be phylogenetically distinct but closely related to Mycobacterium chelonae ATCC 35752T by taxonomic approaches targeting three genes (16S rRNA, hsp6 and rpoB) and were further characterized using a polyphasic approach in this study. The 16S rRNA gene sequences of all three strains showed 99.7 % sequence similarity with that of the M. chelonae type strain. A multilocus sequence typing analysis targeting 10 housekeeping genes, including hsp65 and rpoB, revealed a phylogenetic cluster of these strains with M. chelonae. DNA-DNA hybridization values of 78.2 % between QIA-37T and M. chelonae indicated that it belongs to M. chelonae but is a novel subspecies distinct from M. chelonae. Phylogenetic analysis based on whole-genome sequences revealed a 95.44±0.06 % average nucleotide identity (ANI) value with M. chelonae, slightly higher than the 95.0 % ANI criterion for determining a novel species. In addition, distinct phenotypic characteristics such as positive growth at 37 °C, at which temperature M. chelonae does not grow, further support the taxonomic status of these strains as representatives of a novel subspecies of M. chelonae. Therefore, we propose an emended description of Mycobacterium chelonae, and descriptions of M. chelonae subsp. chelonae subsp. nov. and M. chelonae subsp. bovis subsp. nov. are presented; strains ATCC 35752T(=CCUG 47445T=CIP 104535T=DSM 43804T=JCM 6388T=NCTC 946T) and QIA-37T (=KCTC 39630T=JCM 30986T) are the type strains of the two novel subspecies.

RNA expression analysis of efflux pump genes in clinical isolates of multidrug-resistant and extensively drug-resistant Mycobacterium tuberculosis in South Korea.

Tuberculosis (TB), caused by infection with Mycobacterium tuberculosis, is an important communicable disease. Various mechanisms of resistance to antituberculosis drugs have been reported; these are principally mutations in target genes. However, not all M. tuberculosis resistance can be explained by mutations in such genes. Other resistance mechanisms associated with drug transport, such as efflux pumps, have also been reported. In this study, we investigated the expression levels of three putative efflux pumps and mutations in target genes associated with injectable agents and fluoroquinolones with clinical MDR and XDR-TB isolates. Thirty clinical isolates of M. tuberculosis that had been phenotypically characterized were obtained from the Korean Institute of Tuberculosis. Of these, 14 were MDR-TB isolates resistant to at least one injectable aminoglycoside (amikacin; AMK, kanamycin; KAN, and/or capreomycin; CPM) and 16 were XDR-TB isolates. M. tuberculosis H37Rv (ATCC 27249) was used as a reference strain. Five putative genes (Rv1258c, Rv2686c, Rv2687c, Rv2688c and pstB) were selected for analysis in this study. Sequencing was performed to detect mutations in rrs and eis genes. qRT-PCR was performed to investigate expression levels of five efflux pump genes. Of the 30 isolates, 25 strains had mutations in rrs associated with resistance to KAN, CPM and AMK and two strains had eis mutations, as well as mutations in rrs. pstB (Rv0933) exhibited increased expression and Rv2687c and Rv2688c exhibited decreased expression compared to the reference strain. Increased expression of pstB in clinical drug-resistant tuberculosis isolates may contribute to drug resistance in M. tuberculosis. In our case, overexpression of Rv1258c may have been associated with resistance to kanamycin. No correlation was evident between Rv2686c, Rv2687c or Rv2688c expression and fluoroquinolone resistance. To explore the details of efflux pump drug-resistance mechanisms, further studies on efflux pump inhibitors, transcriptional regulators, such as whiB7, and additional efflux pump genes are needed.

Oral Macrolide Therapy Following Short-term Combination Antibiotic Treatment of Mycobacterium massiliense Lung Disease.

BACKGROUND: Although Mycobacterium massiliense lung disease is increasing in patients with cystic fibrosis and non-cystic fibrosis bronchiectasis, optimal treatment regimens remain largely unknown. This study aimed to evaluate the efficacy of oral macrolide therapy after an initial 2-week course of combination antibiotics for the treatment of M massiliense lung disease. METHODS: Seventy-one patients received oral macrolides, along with an initial 4-week (n = 28) or 2-week (n = 43) IV amikacin and cefoxitin (or imipenem) treatment. These patients were treated for 24 months (4-week IV group) or for at least 12 months after negative sputum culture conversion (2-week IV group). RESULTS: Total treatment duration was longer in the 4-week IV group (median, 23.9 months) than in the 2-week IV group (15.2 months; P < .001). The response rates after 12 months of treatment were 89% for symptoms, 79% for CT scanning, and 100% for negative sputum culture results in the 4-week IV group. In the 2-week IV group, these values were 100% (P = .057), 91% (P = .177), and 91% (P = .147), respectively. Acquired macrolide resistance developed in two patients in the 2-week IV group. Genotyping analyses of isolates from patients who did not achieve negative sputum culture conversion during treatment and from those with positive culture results after successful treatment completion revealed that most episodes were due to reinfection with different genotypes of M massiliense. CONCLUSIONS: Oral macrolide therapy after an initial 2-week course of combination antibiotics might be effective in most patients with M massiliense lung disease. TRIAL REGISTRY: ClinicalTrials.gov; No.: NCT00970801; URL: www.clinicaltrials.gov.

Mycobacterium paraintracellulare sp. nov., for the genotype INT-1 of Mycobacterium intracellulare.

Three mycobacterial strains, isolated from independent Korean patients with pulmonary infections, belonging to the Mycobacterium intracellulare genotype 1 (INT-1) were characterized using a polyphasic approach. The sequences of the 16S rRNA gene and internal transcribed spacer 1 (ITS1) of the INT-1 strains were identical to those of Mycobacterium intracellulare ATCC 13950T. However, multilocus sequence typing (MLST) analysis targeting five housekeeping genes (hsp65, rpoB, argG, gnd and pgm) revealed the phylogenetic separation of these strains from M. intracellulare ATCC 13950T. DNA-DNA hybridization values of >70 % confirmed that the three isolates belong to the same species, while the values of <70 % between one of them and the type strains of M. intracellulare and Mycobacterium chimaera confirmed their belonging to a distinct species. In addition, phenotypic characteristics such as positive growth on MacConkey agar and in acidic broth culture, unique matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS profiles of lipids, and unique mycolic acids profiles further supported the taxonomic status of these strains as representatives of a novel species of the Mycobacterium avium complex named Mycobacterium paraintracellulare. The type strain is MOTT64T (=KCTC 29084T=JCM 30622T).

Improved performance of the modified Hodge test with MacConkey agar for screening carbapenemase-producing Gram-negative bacilli.

The detection of carbapenemases in Gram-negative bacilli is important for optimal patient treatment and to control spread of the resistance. The modified Hodge test can detect carbapenemase-producing Gram-negative bacilli. In this study, we compared the performance of MacConkey agar and Mueller-Hinton agar for metallo-ß-lactamase (MBL) and OXA carbapenemase screening. Overall, the performance of Hodge test was better with MacConkey agar due to enhanced release of ß-lactamases from the cells in the presence of bile compounds. Concomitant use of the modified Hodge test could resolve most of the problems with uncertain double-disk synergy tests in MBL detection.

Transmission of Multidrug-resistant Tuberculosis Confirmed by Endobronchial Ultrasound Biopsy of a Mediastinal Lymph Node and DNA Fingerprinting Analysis.

Endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) was recently introduced for sampling of mediastinal masses and nodes, for staging of lung cancer, and for diagnosing other benign diseases including tuberculosis (TB). However, EBUS-TBNA has not been used to date to confirm the presence of multidrug-resistant TB (MDR-TB). We here describe the diagnosis of MDR-TB of a mediastinal lymph node by EBUS biopsy. MDR-TB had been transmitted to our patient from her son who had pulmonary TB, and the diagnosis of MDR-TB was confirmed by a restriction fragment length polymorphism analysis of the IS6110 segment of Mycobacterium tuberculosis isolates. Our findings highlight the use of EBUS-TBNA in diagnosing TB, including MDR-TB, and malignancies, in a TB-endemic area.

[Endogenous endophthalmitis by Aspergillus in a patient with multiple myeloma.].

A 40-year-old man who had been treated for multiple myeloma, complained of decreased visual acuity of the left eye on the 30th day of admission. The nucleotide sequences of a fungal PCR product from vitreous fluid showed 99% homology with Aspergillus fumigatus (AY373851). Aspergillus spp. was isolated from vitreous fluid culture, also. Rapid diagnosis and intervention are critical elements for the Aspergillus endophthalmitis; therefore, it would be helpful to combine the fungal PCR with conventional fungus culture for clinically indicated specimens.