Evaluation of a Fully Automated, Rapid Detection System for CYP2C19 and UGT1A1 Genotyping / 임상검사와정도관리

BACKGROUND: The need for genotyping single nucleotide polymorphisms (SNPs) in genes encoding drug-metabolizing enzymes is increasing. Therefore, the recent focus has been on developing fully automated methods for the rapid and accurate measurement of SNPs. METHODS: We used the quenching probe (QP) method and i-densy IS-5310 to genotype 200 DNA specimens from 200 healthy Koreans and 100 whole blood from another 100 for the SNPs CYP2C19*2 and CYP2C19*3. We also performed genotyping of UGT1A1*6 and UGT1A1*28 with the above mentioned 200 DNA samples and 81 whole blood samples. The results of the assay were then compared to conventional direct sequencing. RESULTS: The allele frequencies of CYP2C19 were 25.7% for *2 and 10.3% for *3, and those of UGT1A1 were 17.3% for *6 and 11.2% for *28. These results are similar to those reported in previous studies on Korean populations. The CYP2C19 and UGT1A1 genotypes determined by the QP method perfectly matched (100.0%, K=1.000, P<0.001 for CYP2C19, and 99.6%, K=0.992, P<0.001 for UGT1A1) those determined by direct sequencing, barring a single exception for the UGT1A1 genotype in 1 DNA specimen. CONCLUSIONS: Our results suggest that the QP method, owing to its speed and ease of use, will enable rapid and sensitive diagnosis in clinical laboratories.

Identification of Two Novel NPM1 Mutations in Patients with Acute Myeloid Leukemia

BACKGROUND: Genetic abnormalities in adult AML are caused most frequently by somatic mutations in exon 12 of the NPM1 gene, which is observed in approximately 35% of AML patients and up to 60% of patients with cytogenetically normal AML (CN-AML). METHODS: We performed mutational analysis, including fragment analysis and direct sequencing of exon 12 of the NPM1 gene, on 83 AML patients to characterize the NPM1 mutations completely. RESULTS: In this study, NPM1 mutations were identified in 19 (22.9%) of the 83 AML patients and in 12 (42.9%) of the 28 CN-AML patients. Among the 19 patients with NPM1 mutations, type A NPM1 mutations were identified in 16 (84.2%) patients, whereas non-A type NPM1 mutations were observed in 3 (15.8%) patients. Two of the 3 non-A type NPM1 mutations were novel: c.867_868insAAAC and c.869_873indelCTTTAGCCC. These 2 novel mutant proteins display a nuclear export signal motif (L-xxx-L-xx-V-x-L) less frequently and exhibit a mutation at tryptophan 290 that disrupts the nucleolar localization signal. CONCLUSIONS: This study suggests that novel NPM1 mutations may be non-rare and that supplementary sequence analysis is needed along with conventional targeted mutational analysis to detect non-A types of NPM1 mutations.

Comparison of Modified Multiple-locus Variable-number Tandem-repeat Fingerprinting with Pulsed-field Gel Electrophoresis for Typing Clinical Isolates of Staphylococcus aureus

BACKGROUND: Multiple-locus variable-number tandem-repeat fingerprinting (MLVF) is based on multiplex PCR, utilizing variable number tandem repeat. Our goal was to compare the performance of MLVF in distinguishing clinical Staphylococcus aureus isolates with that of pulsed-field gel electrophoresis (PFGE), which has traditionally been the gold standard. METHODS: Sixty-three clinically significant S. aureus isolates were tested using both PFGE and MLVF. Multiplex PCR for MLVF was performed using PCR primers for clfA, clfB, sdrCDE, sspA, and spa. PFGE was performed with genomic DNA fragments generated by SmaI endonuclease digestion. Banding patterns of MLVF or PFGE were analyzed using InfoQuestFP software. RESULTS: The hands-on time of our modified method was about 3 h, on average, for each of 18 isolates. PFGE (80% cutoff) or MLVF (75% cutoff) separated all of the 63 isolates into 13 and 12 types, respectively. Three types generated by PFGE were identical to those generated by MLVF. PFGE and MLVF yielded similar Simpson's diversity indices, indicating similar discriminatory power. The overall concordance between PFGE and MLVF was low, as represented by adjusted Rand indices (0.266-0.278). PFGE predicted MLVF type better than MLVF predicted PFGE type, as reflected by Wallace coefficients (PFGE cutoff 80% vs. MLVF cutoff 75%, 0.389 vs. 0.233). Analysis of the relationship between a pair of isolates showed 91.0% concordance between the PFGE (80% cutoff) and MLVF (75% cutoff). CONCLUSIONS: Our simple, low-cost, modified MLVF protocol can effectively discriminate between S. aureus clinical isolates. MLVF can replace PFGE for the hospital infection control of S. aureus.

A Case of Brain Abscess Caused by Propionibacterium acnes 13 Months after Neurosurgery and Confirmed by 16S rRNA Gene Sequencing / 대한진단검사의학회지

Propionibacterium acnes is a gram-positive anaerobic bacillus and a normal inhabitant of the skin. Although it is often considered a contaminant of blood cultures, it can occasionally cause serious infections, including postoperative central nervous system infections. Here, we report the case of a 70-yr-old man who developed a large cerebral abscess caused by P. acnes 13 months after neurosurgery. Immediate gram staining of the pus from his brain revealed the presence of gram-positive coccobacilli. However, colony growth was observed only after 5 days of culture. Therefore, we performed 16S rRNA gene sequencing of the pus specimen. The isolate was identified as P. acnes. The colonies developed 9 days after the initial culture. The API Rapid ID 32A test (bioMerieux, France) was performed using a colony, but an unacceptable profile was obtained. Then, the pus was transferred into the enrichment broths of the BACTEC FX (Becton Dickinson, USA) and BacT/Alert 3D (bioMerieux, Organon Teknika, USA) systems, but only the BACTEC FX system could detect growth after 5 days. We performed 16S rRNA gene sequencing and API Rapid 32A profiling with a colony recovered from Brucella agar, which was inoculated with the microbial growth in the enrichment broth from the BACTEC FX system. The organism was identified as P. acnes by both methods. This case suggests that 16S rRNA gene sequencing may be a useful alternative for identifying slowly growing P. acnes from specimens that do not show growth after 5 days of culture.

ABO Genotyping by Pyrosequencing Analysis / 대한수혈학회지

BACKGROUND: ABO genotyping is being used increasingly when the results of serologic typing are unclear or there is some suspicion of rare ABO subtypes. Conventional molecular diagnostic methods such as PCR- restriction fragment length polymorphism (PCR-RFLP), allele-specific PCR, PCR-single stranded conformational polymorphism (PCR-SSCP) and sequence-based typing have been used in this field. Recently, a pyrosequencing technique was introduced into clinical laboratories. This study evaluated the possibility of applying pyrosequencing to ABO genotyping. METHODS: A total of 36 samples, which had previously been analyzed by PCR-RFLP and serological method in the Blood Genetics Clinic of Seoul National University Hospital between August 2001 and September 2004 and shown to have the A/A, A/B, A/O, B/B, B/O, O/O, cis-AB/O, cis-AB/A, or cis-AB/B genotypes, were analyzed by pyrosequencing analysis. Briefly, two PCR reactions were carried out separately for one region including nucleotide 261, and for another region including nucleotides 796 and 803. Pyrosequencing was then performed, and the pyrograms were interpreted using an automated interpretation program from the manufacturer and by researchers independently to determine the nucleotides 261, 796 and 803 for ABO genotyping. RESULTS: The ABO genotypes from pyrosequencing and the interpretation of the pyrograms according to the researcher on 36 samples were in complete concordance with the results obtained by PCR-RFLP. The ABO genotypes from the automated interpretation program showed an error in one out of total 108 SNP (single nucleotide polymorphism) analyses (eRROR RATE=0.9%) OF 36 SAMPLES. CONCLUSION: ABO genotyping for A, B, O, cis-AB alleles by pyrosequencing of nucleotides 261, 796 and 803 was relatively simple and accurate and could be an another field we can use in clinical laboratories.