Multilocus Sequence Typing for Candida albicans Isolates from Candidemic Patients: Comparison with Southern Blot Hybridization and Pulsed-field Gel Electrophoresis Analysis / 대한진단검사의학회지

BACKGROUND: We evaluated the efficacy of multilocus sequence typing (MLST) for assessing the genetic relationship among Candida albicans isolates from patients with candidemia in a hospital setting. METHODS: A total of 45 C. albicans isolates from 21 patients with candidemia were analyzed. The MLST results were compared with results obtained by Southern blot hybridization (C1 fingerprinting) and pulsed-field gel electrophoresis (PFGE). PFGE analysis included karyotyping and restriction endonuclease analysis of genomic DNAs using BssHII (REAG-B) and SfiI (REAG-S). RESULTS: The 45 isolates yielded 20 unique diploid sequence types (DSTs) by MLST, as well as 12 karyotypes, 15 REAG-B patterns, 13 REAG-S patterns, and 14 C1 fingerprinting types. Microevolution among intra-individual isolates was detected in 6, 5, 3, 5, and 7 sets of isolates by MLST (1 or 2 allelic differences), REAG-B, REAG-S, C1 fingerprinting, and a combination of all methods, respectively. Among 20 DSTs, 17 were unique, and 3 were found in more than 1 patient. The results of 2 DSTs obtained from 9 patient isolates were in agreement with REAG and C1 fingerprinting patterns. However, the remaining DST, which was shared by 2 patient isolates, showed 2 different PFGE and C1 fingerprinting patterns. In addition, 3 sets of isolates from different patients, which differed in only 1 or 2 alleles by MLST, also exhibited different PFGE or C1 fingerprinting patterns. CONCLUSIONS: MLST is highly discriminating among C. albicans isolates, but it may have some limitations in typing isolates from different patients, which may necessitate additional analysis using other techniques.

DNA Fingerprinting of Candida albicans Strains Isolated from Candidemic Patients by Polymerase Chain Reaction and Southern Hybridization Methods / 감염과화학요법

BACKGROUND: Although several molecular typing methods have been used to investigate C. albicans infections, there remains no "gold standard" method by which relatedness of C. albicans strains is determined. In this study, two DNA fingerprinting methods were compared for genotyping of clinical strains of C. albicans isolated from candidemic patients. MATERIALS AND METHODS: Twenty-nine strains of C. albicans isolated from various clinical specimens (14 from blood, 7 from catheter, 4 from respiratory tract secretion, and 4 from urine) of 14 candidemic patients were analyzed. Primer 1245 and 1246 were employed for IR PCR and Southern blot hybridization method was used for C2 fingerprinting, with Ca3 and C1 as primers, after the fragmentation of DNA with EcoR1 RESULTS: IR PCR method separated 29 isolates into 9 (1245 primer), 7 (1246 primer) and 14 (combination of two primers) types, whereas C1 fingerprinting identified 16 different types. By combining the IR PCR and C1 fingerprinting methods, total of 16 different genotypes were identified among 29 isolates from 14 patients, which is the same result obtained by the C1 fingerprinting only. Using both methods, blood and non-blood isolates from each patient produced identical genotypes for 10 patients and different genotypes for 1 patient. In three patients, isolates from blood and other site of each patient showed identical patterns by IR PCR fingerprinting, but appeared different (n=1) or similar (n=2) by C1 fingerprinting. Overall, for 87% (13/15) of patients, isolates collected from catheter (6 of 7 patients), urine (4 of 4 patients), or respiratory (3 of 4 patients) were identical or similar to the corresponding blood isolates. CONCLUSION: Our study shows that C1 fingerprinting method is more discriminatory than IR PCR for the molecular typing of C. albicans isolates. For the majority of patients, blood and other site isolates had identical or similar genotypes.

DNA Fingerprinting of Candida albicans Strains Isolated from Candidemic Patients by Polymerase Chain Reaction and Southern Hybridization Methods / 감염과화학요법

BACKGROUND: Although several molecular typing methods have been used to investigate C. albicans infections, there remains no "gold standard" method by which relatedness of C. albicans strains is determined. In this study, two DNA fingerprinting methods were compared for genotyping of clinical strains of C. albicans isolated from candidemic patients. MATERIALS AND METHODS: Twenty-nine strains of C. albicans isolated from various clinical specimens (14 from blood, 7 from catheter, 4 from respiratory tract secretion, and 4 from urine) of 14 candidemic patients were analyzed. Primer 1245 and 1246 were employed for IR PCR and Southern blot hybridization method was used for C2 fingerprinting, with Ca3 and C1 as primers, after the fragmentation of DNA with EcoR1 RESULTS: IR PCR method separated 29 isolates into 9 (1245 primer), 7 (1246 primer) and 14 (combination of two primers) types, whereas C1 fingerprinting identified 16 different types. By combining the IR PCR and C1 fingerprinting methods, total of 16 different genotypes were identified among 29 isolates from 14 patients, which is the same result obtained by the C1 fingerprinting only. Using both methods, blood and non-blood isolates from each patient produced identical genotypes for 10 patients and different genotypes for 1 patient. In three patients, isolates from blood and other site of each patient showed identical patterns by IR PCR fingerprinting, but appeared different (n=1) or similar (n=2) by C1 fingerprinting. Overall, for 87% (13/15) of patients, isolates collected from catheter (6 of 7 patients), urine (4 of 4 patients), or respiratory (3 of 4 patients) were identical or similar to the corresponding blood isolates. CONCLUSION: Our study shows that C1 fingerprinting method is more discriminatory than IR PCR for the molecular typing of C. albicans isolates. For the majority of patients, blood and other site isolates had identical or similar genotypes.

Detection of Micrometastasis in Lymph Nodes of Gastric Cancer Patients by CD44-Specific Reverse Transcriptase-Polymerase Chain Reaction

PURPOSE: Lymph-node metastasis is one of the most useful prognostic factors in patients with gastric carcinomas. However, micrometastatic carcinoma cells are frequently missed by conventional histo pathologic examination. Therefore, efforts have been made to detect micrometastasis in lymph nodes at the molecular level. In this study, we used a well-known stomach-cancer target molecule, a CD44 variant, to detect micrometastasis in lymph nodes. METHODS: The authors used the reverse transcriptase-polymerase chain reaction (RT-PCR) to detect tumour- specific splice variants of the transcript of the CD44 gene from 50 histologically metastasis negative lymph nodes in 11 gastric-cancer patients. Oligonucleotide primers as 5'-TCCAGACGAAGACAGT CCCGGAT-3' and 5'-CACTGGGGTGGAATGTGTCTTGGTC-3' were used. Southern blotting with a 40-mer oligonucleotide probe (5'-CCGACAGCACAGACAGAATCCCC- TGCTAC-3') corresponding to a sequence found in all known CD44 transcript isoforms was used to enhance the sensitivity. RESULTS: 1) CD44E could be detected at a concentration of as low as ten tumor cells per 104 peripheral blood lymphocytes in RT-PCR in serial 10-fold dilutions of SNU-16 gastric carcinoma cells with normal peripheral lymphocytes. Southern blotting enhanced the sensitivity to ten tumor cells per 106 peripheral blood lymphocytes. Normal blood did not show overexpression of CD44 splice variant. 2) Gel electrophoresis of reverse transcriptase-polymerase chain reaction products from histologically metastasis-negative lymph nodes of patients with stomach cancer identified a band of 479 base pairs corresponding to the CD44E form in 6 (12%) lymph nodes. 3) Southern blotting using a pan CD44 probe increased the sensitivity of the detection. Of the 50 lymph node samples, ten (20%) showed abnormal CD44 gene activity indicating the presence of micrometastasis. CONCLUSION: The findings of this study suggest that the CD44 RT-PCR/Southern blot hybridization is useful for the detection micrometastasis in lymph nodes. This method would be of practical value in selecting patients at high risk for relapse from those who have no histologically postitive lymph nodes.

Detection of Micrometastasis in Lymph Nodes of Gastric Cancer Patients by CD44-Specific Reverse Transcriptase-Polymerase Chain Reaction

PURPOSE: Lymph-node metastasis is one of the most useful prognostic factors in patients with gastric carcinomas. However, micrometastatic carcinoma cells are frequently missed by conventional histo pathologic examination. Therefore, efforts have been made to detect micrometastasis in lymph nodes at the molecular level. In this study, we used a well-known stomach-cancer target molecule, a CD44 variant, to detect micrometastasis in lymph nodes. METHODS: The authors used the reverse transcriptase-polymerase chain reaction (RT-PCR) to detect tumour- specific splice variants of the transcript of the CD44 gene from 50 histologically metastasis negative lymph nodes in 11 gastric-cancer patients. Oligonucleotide primers as 5'-TCCAGACGAAGACAGT CCCGGAT-3' and 5'-CACTGGGGTGGAATGTGTCTTGGTC-3' were used. Southern blotting with a 40-mer oligonucleotide probe (5'-CCGACAGCACAGACAGAATCCCC- TGCTAC-3') corresponding to a sequence found in all known CD44 transcript isoforms was used to enhance the sensitivity. RESULTS: 1) CD44E could be detected at a concentration of as low as ten tumor cells per 104 peripheral blood lymphocytes in RT-PCR in serial 10-fold dilutions of SNU-16 gastric carcinoma cells with normal peripheral lymphocytes. Southern blotting enhanced the sensitivity to ten tumor cells per 106 peripheral blood lymphocytes. Normal blood did not show overexpression of CD44 splice variant. 2) Gel electrophoresis of reverse transcriptase-polymerase chain reaction products from histologically metastasis-negative lymph nodes of patients with stomach cancer identified a band of 479 base pairs corresponding to the CD44E form in 6 (12%) lymph nodes. 3) Southern blotting using a pan CD44 probe increased the sensitivity of the detection. Of the 50 lymph node samples, ten (20%) showed abnormal CD44 gene activity indicating the presence of micrometastasis. CONCLUSION: The findings of this study suggest that the CD44 RT-PCR/Southern blot hybridization is useful for the detection micrometastasis in lymph nodes. This method would be of practical value in selecting patients at high risk for relapse from those who have no histologically postitive lymph nodes.

Study on the Demonstration of Enteroviruses from Cerebrospinal Fluid of Adult Patients with Aseptic Meningitis / 대한내과학회지

BACKGROUND: The enteroviruses are the most common etiologic agent of aseptic meningitis in adults and children. The incidence of enteroviral meningitis in childhood meningitis is up to 80%, but in adults is not known, worldwidely. In Korea, where tuberculosis is endemic, the rapid and accurate diagnostic method for enteroviral meningitis is required especially because early differential diagnosis of viral meningitis from tuberculous meningitis is very important. The aims of this study were the demonstration of enteroviruses from cerebrospinal fluid (CSF) of adult patients with aseptic meningitis by PCR/Southern hybridization and the verification of the usefulness of PCR/southern hybridization as a rapid diagnostic tool. METHODS: From July 1992 to June 1995, total 34 CSF samples (10 from children, 24 from adults) of patients with aseptic meningitis were studied. As a control group, 15 patients with tuberculous meningitis and 15 patients with bacterial meningitis were studied. Viral RNA was extracted from CSF, reverse transcriptied into cDNA and amplified. The PCR products were Southern hybridizied with enteroviruses-specific digoxigenin-labelled probe. RESULTS: 16/24(66.7%) samples of adult patients with aseptic meningitis were positive for enteroviruses, while in child patients with aseptic meningitis, 9/10(90%) samples were positive. And in one patient, PCR was positive from asymptomatic, onset-7th day CSF sample. CONCLUSION: Enteroviruses were the most common causative organisms of adult aseptic meningitis in Korea. And, this study showed the usefulness of PCR/Southern hybridization of enteroviruses from CSF for etiologic diagnosis of adult aseptic meningitis in subclinical, asymptomatic period.