An Improved Barcoded Oligonucleotide Primers-based Next-generation Sequencing Approach for Direct Identification of Viral Pathogens in Clinical Specimens / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>To provide a feasible and cost-effective next-generation sequencing (NGS) method for accurate identification of viral pathogens in clinical specimens, because enormous limitations impede the clinical use of common NGS, such as high cost, complicated procedures, tremendous data analysis, and high background noise in clinical samples.</p><p><b>METHODS</b>Viruses from cell culture materials or clinical specimens were identified following an improved NGS procedure: reduction of background noise by sample preprocessing, viral enrichment by barcoded oligonucleotide (random hexamer or non-ribosomal hexanucleotide) primer-based amplification, fragmentation-free library construction and sequencing of one-tube mixtures, as well as rapid data analysis using an in-house pipeline.</p><p><b>RESULTS</b>NGS data demonstrated that both barcoded primer sets were useful to simultaneously capture multiple viral pathogens in cell culture materials or clinical specimens and verified that hexanucleotide primers captured as many viral sequences as hexamers did. Moreover, direct testing of clinical specimens using this improved hexanucleotide primer-based NGS approach provided further detailed genotypes of enteroviruses causing hand, foot, and mouth disease (HFMD) and identified other potential viruses or differentiated misdiagnosis events.</p><p><b>CONCLUSION</b>The improved barcoded oligonucleotide primer-based NGS approach is simplified, time saving, cost effective, and appropriate for direct identification of viral pathogens in clinical practice.</p>

Massilia varians Isolated from a Clinical Specimen

We report a case of Massilia varians isolated from a deep finger wound following orthopedic surgery on an immunocompetent patient. The bacterium was identified by 16S rDNA sequence analysis. This is the first case of M. varians isolated from a clinical specimen since the first report in 2008.

Isolation of Mycobacterium lentiflavum from a Patient with a Lung Destroyed by Tuberculosis / 대한진단검사의학회지

Mycobacterium lentiflavum has recently been described as an emerging human pathogen without regard to the immune status of the host. We herein report on M. lentiflavum isolated from a respiratory specimen of a patient. Although the organism described in this case seems to be a colonizer of a lung destroyed by tuberculosis, the current methods for species identification of nontuberculous mycobacteria have to be re-evaluated so as not to underestimate these organisms.

Development of a Reverse Dot Blot System for Rapid Detection of HBV Genotypes / 中国生物工程杂志

To develop a reverse dot blot assay for rapid detection of HBV genotypes.Specific oligonucleotides probes were desighed and immobilized on nylon membranes.The DNA sample to be tested was PCR-amplified with DIG labeling primers and then hybridized with the immobilized probes.This procedure for detecting HBV genotypes was simple,rapid and specificity.30 specimens in Chongqing area were collected and detected by this method,and results were evaluated using direct sequencing.Results showed that: This new method was applicable to precise detection HBV genotypes for specimen with copies up to 103,and the HBV genotyping results showed that genotype B was the predominant genotype in Chongqing area.

T Typing and Antimicrobial Susceptibility of Streptococcus pyogenes in Clinical Isolates / 대한임상병리학회지

BACKGROUND: As invasive streptococcal infections are increasing recently and the resistance rate to either erythromycin or clindamycin is elevating, epidemiologic surveillance and appropriate guideline for antibiotic use are required. Geographical epidemiologic characteristics with T typing and antibiotic resistance rate were investigated. METHODS: Distributions of T types according to geographical areas and sources of specimens were analyzed with 82 strains of Streptococcus pyogenes isolated from clinical samples in Seoul and Chinju. Antibiotic susceptibility test was performed for penicillin G, cephalothin, erythromycin, azithromycin, clarithromycin, clindamycin, chloramphenicol, and ofloxacin with agar dilution method. Antibiotic resistance rates were analyzed according to geographical areas, sources of specimens and T types. RESULTS: The most common T types were T12, T1 and T28 in decreasing order. The distribution of T types between Seoul and Chinju was different. While T1, T3, and T6 were frequent in throat or other respiratory specimens, T12, T28, and B3264 were common in blood or closed pus. The resistance rate to erythromycin, azithromycin, and clarithromycin was 20%, 13% to clindamycin, and 49% to tetracycline, respectively. None of the isolates were resistant to penicillin G, cephalothin, chloramphenicol, or ofloxacin. The isolates from Chinju showed higher resistance rate than the strains from Seoul. The isolates from blood or closed pus had higher resistance rate compared to those of throat or sputum. T28 and T6 strains presented higher resistance rate than other T types. CONCLUSIONS: As distributions of T types were variable according to geographical areas or sources of specimens, continuous microbiological and epidemiological surveillance for invasive streptococcal infections are needed. Minimizing unnecessary antibiotic use or acknowledging the severity of resistance are necessary, because the resistant proportions are increasing against macrolide, clindamycin and tetracycline.