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An NDM-5-producing Klebsiella pneumoniae ST147 strain was isolated from a Japanese patient who had not travelled abroad in at least 5 years. Whole-genome sequencing revealed a genomic rearrangement in an FII plasmid harbouring blaNDM-5 due to the replicative transposition of IS26. A hypothetical structure was proposed for its ancestral plasmid, and comparative genomic analysis of the plasmid suggested the dissemination of structurally similar plasmids harbouring blaNDM-5 in Asian and Middle Eastern countries.

Most target substances for immunoassay of infectious disease are antigens or antibodies which do not exist in the human body. Therefore, the method to set reference values is different from chemistry or hematology testing. High sensitivity is required for infectious disease testing, particularly for screening. Also, its reference values (cut-off values) are set as low as possible. Therefore, a false-positive reaction can be caused due to slightly non-specific reactions in infectious disease reagents. The specificities for infectious disease reagents were evaluated with 9 kinds of HCV antibody test kit and 9 kinds of HIV screening kit. The frequencies of false-positive results were 0.2-1.8 and 0.2-1.3%, respectively, and even a kit with a high specificity showed a false-positive result for 1 in 500 samples. The sensitivities for infectious disease reagents were evaluated with a newly developed super-high- sensitive HBs antigen assay kit and 8 kinds of chemiluminescence HBs antigen assay kit which are highly sensitive conventional kits. As a result, the super-high-sensitive kit was 10 to 40 times more sensitive than conventional kits. After introducing the super-high-sensitive kit to routine assays, 16 HBV-infected patients, who were not identified with the conventional kits, were detected for six months. On the other hand, we confirmed false-positive results due to contamination between specimens after introducing the super-high-sensitive kit. It is recommended to use the super-high-sensitive kit in a well-controlled environment to prevent contamination between specimens in order to generate highly reliable test results.

Background: In Japan, an immunochromatographic HIV-1/2 assay has been used for local Health Care Centers to prevent HIV spread in early stages and for hospitals at night or on holidays, where automated instruments are not available. In 2009, a new immunochromatographic HIV-1/2 assay became available which detects both antigen (Ag) and antibody (Ab) simultaneously and on separate bars. This study was conducted to evaluate the clinical performance of this new assay against three HIV-1/2 assays., Methods: One immunochromatographic assay (ICA) for HIV Ab detection, one chemiluminescent enzyme immunoassay (CLEIA) for Ab detection and one ELISA for Ag/Ab combination assay were evaluated with the ICA for Ag/Ab detection. Serum samples from 955 patients were used at Osaka University Hospital, who had been tested initially for HIV infection status. The 900 were negative and 55 were positive. In addition, the samples in 10 commercially available panels [2 containing relatively rare HIV subtypes/genotypes and 8 containing seroconversion samples] were tested using all HIV assays., Results: The HIV Ag/Ab ICA showed 100% (900/900, 95% confidence interval (95 CI) 99.59 - 100%) clinical specificity and was better than 99.8% (898/900, 95 CI 99.20 - 99.97%) of the existing ICA. The CLEIA and ELISA showed 100% (600/600, 95 CI 99.39 - 100%) and 99.8% (598/600, 95 CI 98.80 - 99.96%) specificity, respectively. The HIV Ag/Ab ICA showed 100% (55/55, 95 CI 93.51 - 100%) clinical sensitivity and detected all the relatively rare HIV subtypes/genotype panels; these results were the same as the other three assays. The HIV Ag/Ab ICA detected 5 seroconversion panels earlier than antibody-only-detection assays but detected later with 2 panels than ELISA for Ag/Ab combination assay., Conclusions: The HIV Ag/Ab ICA demonstrated good performances in clinical specificity and sensitivity as a rapid assay and is a suitable assay for qualitative judgement of HIV Ag and Ab simultaneously in human serum and plasma.

Nine anti-HCV antibody screening reagents currently used in Japan were investigated for diagnostic utility. The results using sera from 500 subjects and two series of anti-HCV seroconversion panels were compared. The positive detection rates of the 9 screening tests in 500 specimens ranged from 9.6% to 12.2% and the agreements between combinations ranged from 97.0-99.4%. In the 7 two-step assays, which employ recombinant antigen solid phase and anti-human antibody detection, i.e. excluding Quick Chaser and PA, agreement ranged from 97.6-99.4%. No relationship was seen between similarities of the antigen used and agreements between the 7 reagents. For the 72 specimens that showed positive with at least one screening reagent, agreements between the 9 reagents and the confirmatory tests (RIBA III) were compared. In specimens that showed positive with multiple reagents, the positive rate of RIBA III was high, thus the possibility of the existence of the anti-HCV antibody was high. In specimens that showed positive in only a single screening reagent, the RIBA III did not test positive, suggesting that the incidence of false positives may be high. The accuracy of each screening reagent was compared to RIBA III as an accuracy standard. It was found that ARCHITECT was the best in accuracy. The distance from mean to cut-off in the anti-HCV antibody negative specimens reflected the incidence of false positives in each reagent. The anti-HCV antibody seroconversion sensitivities in the initial stage of HCV infection were also compared. The earlier detection was seen with Centaur, ELISA and ARCHITECT. Each anti-HCV antibody screening reagent in use has unique features, and it is suggested that caution be used when diagnosing HCV infection on the basis of the results of a single antibody test.

Evaluation of a Chemiluminescent Microparticle Immunoassay (CMIA) for determination of anti-Treponema pallidum (TP) antibodies, "ARCHITECT TPAb", was performed. This assay was confirmed to be a reliable anti-TP assay as it showed very good fundamental performance on reproducibility (intra-assay CV: less than 4%), assay specificity (100%, 500/500) and assay sensitivity (100%, 121/121). Since this assay showed very good dilution linearity for samples within the range of 0.59-8.38 S/CO, quantification of the immunoreactivity of anti-TP was attempted. The correction formula for quantification was successfully validated with 8 specimens. The quantified anti-TP, CMIA-QT, calculated by multiplying together the assay's corrected S/CO within the range of 0.59 - 8.38 and the sample dilution factors, showed a strong correlation with the titer of TP particle agglutination (TPPA). The clinical utility of CMIA-QT was evaluated with stored specimens from 5 primary, 5 secondary, and 5 neural syphilis patients. The clinical utility of CMIA-QT was confirmed in the same manner as that of TPPA.

Mutants of hepatitis B virus surface antigen (HBsAg) are known as a cause of false-negative results in diagnostic tests for HBsAg; particularly when a diagnostic kit utilizes monoclonal antibodies to detect HBsAg. We compared seven HBsAg kits with regard to sensitivity for HBsAg subtypes (ad, ay) and their ability to detect nine different HBsAg mutants. Among them, the sensitivities of five kits were high and comparable to each other (0.2 - 0.3ng/ml). However, two kits were of lower sensitivity (0.8-1.3ng/ml, and 2.4-2.5ng/ml, respectively). Two kits, produced by the same company, reacted with all of the nine HBsAg mutants, but five kits showed false-negative results with one or more of the HBsAg mutants. These data indicate that there are differences in the detection sensitivities for HBsAg and abilities to detect HBsAg mutants among commercially available HBsAg kits, which may explain false-negative clinical results.

Vaccine-preventable viral infections in health care workers (HCWs) have been on the rise for the past 10 years in Japan. To reduce the viral infections and the burden of exposure follow-up surveys at a tertiary care hospital in Osaka, Japan, a seroprevalence survey was conducted, and free vaccinations for measles, rubella, varicella, and mumps were offered to newly hired HCWs (199 physicians and 72 nurses and nursing assistants) who had negative serologic results for antibodies against these viruses. Negative antibody titers were obtained from 7.4% of the newly hired HCWs for measles, 12.5% for rubella, 4.1% for varicella, and 15.9% for mumps. The vaccination program for HCWs improved the vaccine-preventable infection rates and resulted in fewer exposure follow-up surveys, fewer lost work days, and fewer HCWs requiring hospitalization for these viral infections compared with those counted for the previous year. These data indicate that all HCWs should be strongly recommended to be vaccinated against (or have documented immunity to) these viruses in Japan, as is the case in the United States.

It is important to monitor the quantity of hepatitis B virus (HBV)-DNA for the evaluation of the therapeutic effect of lamivudine on patients with chronic HBV infection. Using 174 samples from HBV-infected patients, in this study, we compared two methods for the detection of HBV-DNA; AMPLICOR HBV MONITOR Test (PCR method; Roche Diagnostics) and QUANTIPLEX HBV DNA Assay (bDNA) (probe method; Daiichi Pure Chemicals CO.). In all 174 samples, the detection rate of HBV-DNA was higher in the PCR method (75.9%) than in the probe method (39.1%) (p < 0.001). Furthermore, both in 103 samples from patients who were negative for HBe antigen (Ag) and positive for anti-HBe antibody (Ab) and in 48 samples from patients who were undergoing lamivudine therapy, the detection rates of HBV-DNA were also higher in the PCR method (71.8% and 47.9%, respectively) than in the probe method (19.4% and 6.3%, respectively) (p < 0.001, respectively). Data from the two methods were significantly correlated in 68 samples that were positive in both methods (r = 0.988, p < 0.001, y = 0.913x + 0.184). These data indicate that the PCR method is more suitable for the detection of HBV-DNA in long-term follow-up patients with negative for HBe Ag and positive for anti-HBe Ab, and in patients with lamivudine therapy for HBV infection.

We compared eight HCV screening tests for detection of anti-HCV antibody; Ortho Quick Chaser HCV Ab (QC), Ortho HCV Ab ELISA III (ELISA), Ortho HVC Ab PA test III (PA), Lumipulse II Ortho HCV (LUMI), IMx HCV.DAINAPACKII (IMx), ARCHITECT HCV (ARCH), Immucheck.F-HCV C50 Ab (Immu), RANREAM HCV Ab Ex II (RAN). Sera from six hundred patients were examined by these eight screening tests. The positive rates of the eight screening tests were from 9.0% to 13.2%. Forty-five sera showed discrepant results between the eight screening tests, and about half of them showed weak positive reaction and/or false positive. Twenty-five of the forty-five sera were negative for ant-HCV antibody in the CHIRON RIBA III confirmatory test, and forty-four of them were negative for HCV-RNA in the PCR method. The agreement rates between the two reagents were from 95.5% to 99.2%, but were not always high between the two reagents that used similar antigen. The specificities and sensitivities evaluated by using the RIBA III confirmatory test were excellent in ELISA, LUMI, IMx, ARCH and Immu. Three BBI seroconversion panels were used to compare the positive readings in the initial stage of HCV infection by eight screening tests. ELISA and ARCH showed the earliest positive readings, and then IMx, LUMI = RAN, PA, QC and Immu in this order. These findings indicate that ELISA and ARCH were the most excellent in the sensitivity, specificity and early diagnosis of HCV infection. However, we must pay attention to the weak positive reaction in the screening tests, because there is a possibility of "false positive".