Evaluation of the diagnostic performance of the xpert Clostridium difficile assay and its comparison with the toxin A/B enzyme-linked fluorescent assay and in-house real-time PCR assay used for the detection of toxigenic C. difficile.

BACKGROUND: Clostridium difficile genes or toxin can be detected using several laboratory techniques. In this study, we compared the performance of the Xpert C. difficile assay with that of a toxin A/B enzyme-linked fluorescent immunoassay (ELFA) and an in-house real-time PCR assay for the tcdB gene. METHODS: From April 2011 through January 2012, 138 soft or liquid stool samples from 138 adult patients at Paik Hospital were tested using the toxin A/B ELFA, in-house real-time PCR assay, and Xpert C. difficile assay to detect toxigenic C. difficile. Specimens were considered true positives if results were positive in both the in-house real-time PCR for tcdB gene and Xpert C. difficile assays. RESULTS: Sensitivity of the toxin A/B ELFA, in-house tcdB gene real-time PCR, and Xpert C. difficile assay were 67.6%, 97.3%, and 100.0%, respectively. The specificity of the in-house tcdB gene real-time PCR assay was 100%, while the specificity was 98.0% for the other two methods. The turnaround time (TAT) was 50 min for the Xpert C. difficile assay, 75 min for the toxin A/B ELFA, and 160 min for the in-house real-time PCR assay. CONCLUSION: The Xpert C. difficile assay and the in-house real-time PCR assay had higher sensitivity than the toxin A/B ELFA; however, the specificities of the three assays were similar. Considering its rapid TAT and high sensitivity, use of the Xpert C. difficile assay is highly recommended for rapid and accurate diagnosis of C. difficile infection.

Beneficial effects of pretransplantation microchimerism on rejection-free survival in HLA-haploidentical family donor renal transplantation.

BACKGROUND: Fetal-maternal microchimerism (MC) can develop during pregnancy and may persist for decades. Pretransplantation fetal-maternal MC may be present between mother and child and between siblings; however, its effect on renal transplantation is not known. We investigated the effects of pretransplantation MC on allograft outcomes in human leukocyte antigen (HLA)-haploidentical family donor transplantation. METHODS: A total of 106 cases transplanted from 1996 to 2004 were retrospectively studied, with median follow-up of 96 months. The study and control groups included 63 and 43 cases of HLA-haploidentical and HLA-identical donor transplantations, respectively. MC against mismatched donor HLA-DRB1 allele was detected in the recipient's peripheral blood using nested polymerase chain reaction-single-strand conformation polymorphism method. The allograft outcomes of HLA-haploidentical MC (+) and (-) subgroups were compared with those of HLA-identical group. RESULTS: Pretransplantation MC in the HLA-haploidentical recipients was detected in 22.2% (14 of 63). Compared with HLA-identical group, MC (-) subgroup showed significantly inferior allograft outcomes: higher acute rejection rate (11.6% vs. 42.9%; P=0.001), higher 5-year serum creatinine level (1.1 vs. 1.4 mg/dL; P=0.009), and lower 10-year rejection-free survival rate (83.7% vs. 54.5%; log-rank P=0.001). In contrast, MC (+) subgroup showed no significant differences from HLA-identical group in acute rejection rate (14.3%), 5-year serum creatinine level (1.1 mg/dL), and 10-year rejection-free survival rate (85.7%). Multivariate analysis revealed that pretransplantation MC is associated with a significantly lower risk of acute rejection (odds ratio=0.10; P=0.021). CONCLUSION: Pretransplantation MC present in the recipient may have beneficial effects on rejection-free allograft survival in renal transplantation.

Detection of HLA-DRB1 microchimerism using nested polymerase chain reaction and single-strand conformation polymorphism analysis.

For the detection of microchimerism, molecular methods detecting donor-specific HLA-DRB1 alleles in the recipient are most commonly used. Nested polymerase chain reaction sequence specific primer (nested PCR-SSP) methods widely used to increase the sensitivity of detection have been reported to give frequent false-positive reactions. We have developed a new method combining nested PCR with single-strand conformation polymorphism analysis (nested PCR-SSCP) and tested the 1 to 0.00001% level of microchimerism for 27 different HLA-DRB1 alleles. For most (26/27) of the HLA-DRB1 alleles tested, this method could detect 0.01 to 0.001% of microchimerism and its sensitivity was equal to or better than that of nested PCR-SSP tested in parallel. Its specificity was verified by visualizing particular DRB1-specific SSCP bands under test. Nested PCR-SSP indicated frequent false-positive reactions, mainly caused by nonspecific amplification of DRB3/B4/B5 alleles present in the major (recipient) DNAs. We have compared a real-time quantitative PCR for non-human leukocyte antigen (HLA) target (insertion/deletion marker) using a commercial kit (AlleleSEQR Chimerism assay), and its microchimerism detection sensitivity (around 0.1%) was 1 step (10 times) lower than that of nested PCR-SSP or -SSCP methods for HLA-DRB1 alleles. We validated that the newly designed nested PCR-SSCP affords good sensitivity and specificity and may be useful for studying microchimerism in clinical settings.

Evaluation of a fully automated treponemal test and comparison with conventional VDRL and FTA-ABS tests.

We evaluated analytic performances of an automated treponemal test and compared this test with the Venereal Disease Research Laboratory test (VDRL) and fluorescent treponemal antibody absorption test (FTA-ABS). Precision performance of the Architect Syphilis TP assay (TP; Abbott Japan, Tokyo, Japan) was assessed, and 150 serum samples were assayed with the TP before and after heat inactivation to estimate the effect of heat inactivation. A total of 616 specimens were tested with the FTA-ABS and TP, and 400 were examined with the VDRL. The TP showed good precision performance with total imprecision of less than a 10% coefficient of variation. An excellent linear relationship between results before and after heat inactivation was observed (R(2) = 0.9961). The FTA-ABS and TP agreed well with a κ coefficient of 0.981. The concordance rate between the FTA-ABS and TP was the highest (99.0%), followed by the rates between FTA-ABS and VDRL (85.0%) and between TP and VDRL (83.8%). The automated TP assay may be adequate for screening for syphilis in a large volume of samples and can be an alternative to FTA-ABS.

Reference interval and determinants of the serum homocysteine level in a Korean population.

In this study, we estimated the reference intervals of the serum homocysteine (Hcy) level using two automated immunoassays, and we demonstrated the effects of various factors on the Hcy level in a Korean population. We calculated the gender- and assay-specific reference intervals using the data from 809 healthy Koreans, and we assessed the effects of physiologic and lifestyle factors on the Hcy level. The upper limit was higher in males (19.21 and 19.76 µmol/l) than that in females (14.99 µmol/l and 15.16 µmol/l, AxSym and ADVIA centaur, respectively); the upper limits were comparable between the two assays. Smokers, vitamin nonusers, and persons without regular exercise showed a lower folate level and a higher Hcy level. The risk of hyperhomocysteinemia was significantly associated with the male gender (adjusted OR: 5.705, P-value: 0.008) and with the low folate level group (adjusted OR: 10.412, P-value: 0.002) on the multivariate analysis. The Hcy level was significantly different according to various factors, especially in the gender and folate level. The reference interval should be determined for each ethnic population and for each assay. The appropriate cutoff for assessing the risk for cardiovascular disease or stroke should also be validated in each population.

Usefulness of circulating vascular endothelial growth factor and neutrophil elastase as diagnostic markers of disseminated intravascular coagulation in non-cancer patients.

BACKGROUND: Disseminated intravascular coagulation (DIC) is characterized by platelet and neutrophil activation. Platelets are the major source of circulating vascular endothelial growth factor (VEGF). Endostatin, an anti-angiogenic factor, is a fragment of collagen that is released from the extracellular matrix via the active cleavage of neutrophil elastase, thereby increasing the circulating level of endostatin. Hypercoagulable conditions such as DIC may induce the release of VEGF and neutrophil elastase from the platelets and neutrophils. METHODS: We enrolled 240 patients who were clinically suspected of having DIC. Plasma levels of VEGF, endostatin, and neutrophil elastase were determined using commercial ELISA kits. Patients were diagnosed as having overt DIC if the cumulative International Society on Thrombosis and Haemostasis Subcommittee score was >5. RESULTS: Overt DIC was diagnosed in 80 of the 240 patients. The circulating VEGF and neutrophil elastase levels gradually increased according to the severity of coagulopathy, as reflected by the DIC score. However, the circulating endostatin level did not change significantly according to the DIC score. We divided the patients into 2 groups: the non-cancer and cancer patient groups, to exclude the VEGF release from tumor tissues. Interestingly, in non-cancer patients, higher VEGF and neutrophil elastase levels were found to be significant diagnostic markers for overt DIC. CONCLUSION: Our findings suggest that circulating VEGF and neutrophil elastase levels are laboratory markers reflecting coagulation activity. They are expected to be potential diagnostic markers of overt DIC, especially in non-cancer patients.

[A case of ABO*Ael02/O04 genotype with typical phenotype O].

Ael is a rare blood type which has the least amount of A antigen among A subgroups. It can be detected by special tests performed to resolve the discrepancy between red cell and serum typing in routine serological typing. The presence of A antigen on Ael red cell is demonstrable only by adsorption and elution tests. An Ael individual does not secret A substance in the saliva and may have anti-A antibody in the serum which is usually less reactive with the reagent red cells than anti-B antibody. In Korea, Ael02 has been reported more frequently than other Ael alleles. We report a case of Ael02/O04 who presented as typical phenotype O with strong anti-A and anti-B antibodies and no A antigen detected even by adsorption and elution tests. The case has been proved to be Ael02/O04 by direct sequencing analysis. In individuals with history of discrepancies in the results of ABO phenotyping, ABO genotyping is needed for an accurate evaluation of their blood type.