Detection of Mycobacterium tuberculosis complex Using Real-time Polymerase Chain Reaction / 대한진단검사의학회지

BACKGROUND: For the detection of Mycobacterium tuberculosis complex (MTB), PCR is known to be sensitive, specific, and rapid compared to the conventional methods of acid-fast-bacilli (AFB) smear and culture. We evaluated a new approach for MTB detection using real-time PCR. METHODS: The specificity of real-time PCR was evaluated using 20 MTB isolates and 37 nontuberculous mycobacteria (NTM) isolates identified by AccuProbe Mycobacterium tuberculosis complex colony identification test (Gen-Probe Inc., USA) and Myco-ID (M&D, Korea). One hundred sputum specimens (50 AFB smear-positive and 50 negative specimens) were analyzed using real-time PCR and Amplicor Mycobacterium tuberculosis test (Roche, Germany). The results of real-time PCR positives (55 samples) and negatives (598 samples) were analyzed by AFB smear and culture. RESULTS: The real-time PCR assay accurately discriminated between MTB and NTM species. Realtime PCR and Amplicor test yielded the same results in 96.0% (96/100) of the sputum specimens tested. The sensitivity and specificity of real-time PCR based on AFB culture were 97.4% and 88.5%, respectively. Of the 55 real-time PCR positive specimens, 83.6% (46/55) were culture-positive, 30.9% (17/55) were smear-positive, 52.7% (29/55) were smear-negative and culture-positive, and 14.5% (8/55) were both smear and culture-negative. Among the 598 real-time PCR negative specimens, 60 were not tested for AFB smear or culture and 10 were contaminated. Of the remaining 528 specimens, 478 (90.5%) were both smear and culture-negative and 39 (7.4%) were culture-positive. CONCLUSIONS: For the detection of MTB, real-time PCR was sensitive and specific and comparable to conventional methods. It can be used for rapid identification of M. tuberculosis in clinical laboratories.

ABO Genotyping using a Multiplex Single-base Primer Extension Reaction / 대한수혈학회지

BACKGROUND: ABO genotyping is being widely used in the case of ABO discrepancies and in forensic medicine. We have designed a method using a multiplex single-base primer extension reaction that has allowed us to detect six single nucleotide polymorphism (SNP) sites in the ABO gene and to determine ABO genotypes. METHODS: Genomic DNA was isolated from the peripheral blood of 75 unrelated Korean subjects. Exon 6 containing nucleotides 261 and 297 and exon 7 containing nucleotides 703, 802, 803 and 1059 were amplified using two pairs of primers. Using the products as templates, a multiplex single-base primer extension reaction was performed with six typing primers of different lengths for the six SNP sites. These reactions were performed on a PTC-200 thermal cycler (MJ Research, Waltham, MA, USA) using the SNaPshot multiplex kit (Applied Biosystems, Foster City, CA, USA), and the products were analyzed using an ABI 3130xl Genetic Analyzer (Applied Biosystems). RESULTS: The ABO genotypes determined by this method (75/75) all matched the genotypes that were determined by the use of the polymerase chain reaction using sequence-specific priming (PCR-SSP). We analyzed the peak pattern detected at each of the six SNP sites for each sample. For the smaller-sized primers, peaks were shifted to the right-side compared with the expected site and for the larger-sized primers peaks was close to the expected site. In addition, the coefficients of variation (CVs) of the smaller-sized primers were higher than the CVs of the larger-sized primers. CONCLUSIONS: We are able to detect six SNP sites in the ABO gene and to determine ABO genotypes using a multiplex single-base primer extension reaction.

Determination of the RhC/c Blood Group by Polymerase Chain Reaction with Sequence-specific Primers of the Intron 2 Insert of the RHCE Gene / 대한수혈학회지

BACKGROUND: RhC/c blood group antigens are of clinical importance and molecular genotyping for them can be useful when serological typing is difficult. A method to determine the RhC/c genotype, by targeting exon 1 nt48 and exon 2 nt307, has been used. However, this approach is not accurate for the RHc(cyt48) variant allele. We applied a more accurate genotyping method, using the intron 2 109 bp insert of the RHCE gene, and evaluated its performance in comparison with the standard method. METHODS: RhD and RhC/c serotypes of 236 subjects were determined. We compared two genotype results with the serological phenotype. One method examined the allele-specific exon 1 nt48 and exon 2 nt307 polymorphism area (Method 1), while the other method detected the intron 2 insert instead of the exon 1 nt48 (Method 2) by polymerase chain reaction with sequence-specific primers (PCR-SSP). RESULTS: The predicted phenotypes by Method 1 were not matched with the true phenotypes in 24 cases (24/236, 10.2%). By contrast, the predicted results by Method 2 matched with true phenotypes in all cases except one. The RHc(cyt48) variant was suspected in 22 cases (23.7%) of the 93 Rhc cases. CONCLUSION: For the determination of the RhC/c genotype in Koreans, the method that analyzes exon 1 nt48 is inaccurate. Instead, intron 2 insert analysis with exon 2 nt307 by PCR-SSP appears to be a more accurate alternative.

Comparison of Various Methods for Detection of Methicillin-Resistant Staphylococcus aureus / 임상검사와정도관리

BACKGROUND: Cefoxitin, a cephamycin-type antibiotic, is known to be superior to oxacillin in predicting the presence of mecA gene because it serves as a very potent inducer of mecA regulatory system. We used a cefoxitin disk diffusion methods for detection of methicillin-resistant Staphylococcus aureus (MRSA), and compared it with the conventional methods. METHODS: For 50 MRSA and 50 methicillin susceptible S. aureus confirmed by mecA and femA gene PCR, oxacillin, cefoxitin, and moxalactam disk diffusion methods, oxacillin and cefoxitin E-tests, Vitek 2 and Microscan Walkaway antibiotics susceptibility tests, and PBP2a latex agglutination test were performed. The sensitivity and specificity of each method were evaluated. RESULTS: The sensitivities of oxacillin disk diffusion method and E-test were 96%. The sensitivities of cefoxitin and moxalactam disk diffusion method, cefoxitin E-test, Vitek 2, Microscan Walkaway, PBP2a latex agglutination test were 100%. The specificities were 100% for all the methods used. CONCLUSIONS: It may be considered that both the cefoxitin- and moxalactam disk diffusion methods are effective and excellent screening methods for the detection of MRSA in clinical laboratory routinely.

Detection of Enterovirus in Cerebrospinal Fluid by Real-Time Nested Reverse Transcription Polymerase Chain Reaction / 대한진단검사의학회지

BACKGROUND: Enterovirus is a common cause of aseptic meningitis, respiratory disease and nonspecific febrile illness. The conventional methods for laboratory diagnosis of enterovirus infections have been virus culture and serotyping by an immunofluorecent test. We studied a new and more rapid approach for enterovirus detection in cerebrospinal fluid (CSF) by real-time nested PCR. METHODS: This study was performed on 50 CSF specimens from patients suspected of aseptic meningitis. Enterovirus was detected in CSF by PCRs for 3 different targets and real-time nested PCR. Enterovirus culture was also performed in 44 CSF specimens. RESULTS: The positive rate of PCRs for each of the 3 different targets was 26.0%, 40.0%, or 46.0%, and that of real-time nested PCR was 86.0%. Only 6.8% were positive in culture. Thus, the positive rate of real-time nested PCR was much higher than other methods. CONCLUSIONS: Our study revealed that the real-time nested PCR should be useful for diagnosis of enterovirus infections because of a high sensitivity and rapid detection.

Isolation of Nontuberculous Mycobacteria Using Polymerase Chain Reaction-Restriction Fragment Length Polymorphism / 대한진단검사의학회지

BACKGROUND: The isolation rate of nontuberculous mycobacteria (NTM) in clinical laboratories and the incidence of NTM infections are on the increase recently in Korea, but there have been only a few studies that reveal the general aspect of NTM isolation or species distribution. Therefore, this study was performed to examine the isolation rate of NTM, species identification, and the clinical significance of mycobacterial cultures. METHODS: From August 2004 to May 2005, we examined mycobacterial isolates by AccuProbe test to differentiate NTM from Mycobacterium tuberculosis complex. NTM was then identified by polymerase chain reaction-restriction fragment length analysis (PCR-RFLP). RESULTS: A total of 6,742 specimens from 2,784 patients were requested for mycobacterial culture. Mycobacteria were isolated from 776 specimens (11.5%). The isolation rates of NTMs among the total culture positive specimens and culture positive sputum specimens were 24.4% (189/776) and 25.3% (169/667), respectively. Fourteen species of NTM identified in 172 of the 175 specimens tested included M. avium (39.0%), M. intracellulare (22.7%), and M. abscessus (19.8%). CONCLUSIONS: Using AccuProbe tests and PCR-RFLP method for mycobacterial cultures processed in a clinical laboratory, we were able to identify NTMs to the species level. The isolation rate of NTM in this study was similar to that reported in past studies in Korea. In addition, we found that some of the NTMs isolated in this study could cause pulmonary diseases.

Alleles for Associated with Fy(a), Fy(b), Fy(x) and Fy Antigens in a Korean Population / 대한수혈학회지

BACKGROUND: In the Duffy blood group system (Fy(a), Fy(b), Fy(x), and Fy antigens), Fy(x) antigen is associated with weak Fy(b) while Fy antigen means the null phenotype Fy (a-b-). Fyx antigen and Fy antigen result from the polymorphisms of Fy125 allele. This report assessed the allele frequency and genotype frequency of Fy(a), Fy(b), Fy(x), and Fy antigens in Koreans. METHODS: We performed a study of the followings on 253 visitors to the health promotion center of Seoul National University Bundang Hospital: PCR-RFLP and PCR-SSP for the detection of Duffy 125G > A and -33T > C; PCR-SSP for the detection of Duffy 265C > T and 298G > A. RESULTS: The results of PCR-RFLP and PCR-SSP were consistent with each other in a total of 253 subjects. Allele frequency was as follows: Fy 92.3%, Fy(125) 6.1%, and fy(125/265) 1.6%. The fy(125/265) allele was newly observed. Fy(125/298), fy(125/265/298), and fy(-33/125) alleles were not detected in Koreans. The distribution of Duffy phenotypes in Koreans was as follows: Fy (a+b-) 88.1%, Fy (a-b+) 0.4%, Fy (a+b+) 11.5%, and Fy (a-b-) 0.0%. Fy (a+) was 99.6% and Fy (b+) was 11.9%. CONCLUSION: In our study for Duffy polymorphisms, the frequency of Fy allele was very high. The frequency was similar to those of other Asian populations, but different from those of Caucasians. The fy(125/265) allele, which was associated with Fy(x) antigen, was newly detected in Koreans.

Detection of Helicobacter pylori and BabA (Blood-group Antigen Binding Adhesin) in Saliva and Gastric Tissue by Polymerase Chain Reaction / 임상검사와정도관리

BACKGROUND: Saliva is considered an important vector for the Helicobacter pylori infection. The presence of the babA2 gene, encoding for BabA (blood-group antigen binding adhesin), in the H. pylori genome is crucial for H. pylori-related pathogenesis. METHODS: The study was performed in the group of 215 patients. The detection of H. pylori and babA2 in saliva and gastric tissue was done by PCR (polymerase chain reaction). Moreover, gastric tissues were stained with hematoxylin-eosin as well as with modified Giemsa methods for the analysis of Helicobacter pylori density. RESULTS: The positive rate of H. pylori by nested PCR was 78.6% in gastric tissue and 72.7% in saliva. In addition, the positive rate of H. pylori was 55.5% by the histological analysis of Helicobacter pylori density in gastric tissue. The positive rate of babA2 by PCR was 33.9% in gastric tissue, and 8.2% in saliva. CONCLUSION: We revealed that the H. pylori PCR results obtained in gastric tissue correlated well with those obtained in saliva. As saliva is more available specimen, it is more suitable for clinical application of H. pylori detection by PCR. However, clinical use of - BabA PCR seems to be limited because of its low-sensitivity.