[Efficacy of Human Immunodeficiency Virus (HIV) Antigen-Antibody Combination Assay as a Screening Test and Factors Causing False-Positivity].

Purpose: We aimed to evaluate the performance of an HIV antigen-antibody combination assay (fourth-generation) by comparing it with second generation assays that detect anti-HIV. Methods: A total of 105,439 HIV screening tests were performed from January 2004 to March 2015; the second - and fourth generation assays were used for 75,302 and 30,137 samples, respectively. Samples positive on a screening test were confirmed by anti-HIV-1 western blotting (WB) and nucleic acid amplification. By the results of confirmation tests, the efficacies of the second and fourth generation assays were estimated. The clinical backgrounds with false-positive samples were examined. Results: Of 75,302 samples, 136(0.18%) were positive by the second-generation assay; 14 were confirmed positives, and 122 were false positives. Of 30,137 samples, 18(0.06%) were positive by the fourth-generation assay; 6 were confirmed positives, and 12 were false positives. The reliability of the positives by fourth-generation assay was significantly improved (p=0.006) Samples form individuals with malignant neoplasms were frequently false positive by both the second and fourth-generation assays. Of 67 samples performed by WB, 10 samples, including 6 from patients with a malignancy, showed indeterminate results. All indeterminate samples were found to have antibodies responding to HIV core protein. Conclusion: The fourth-generation assay had satisfactory reliability of the positives for HIV screening. Antibodies responding to HIV core protein may result in false positive HIV screening tests. [Original]

[Comparative Study for Anti-Hepatitis B Surface Antigen Titers Based on Two Measurement Methods: Using Monoclonal Antibodies Isolated from Hepatitis B Vaccinated Recipients].

BACKGROUND: As anti-hepatitis B surface antigen (anti-HBs) titers vary depending on the measurement methods, we compared two different methods to measure anti-HBs titers in sera and HBs monoclonal antibodies. METHODS: The sera from 182 HB virus-resolved patients who were negative for HBsAg but positive for antiHB core protein (HBc) and/or anti-HBs were obtained. The measurement of anti-HBs was compared using either Lumipulse G1200 or Architect i2000SR. Six different monoclonal antibody (mAbs) clones isolated from healthy individuals inoculated with hepatitis B vaccine Bimmugen (genotype C) were used. RESULTS: A statistically significant correlation in anti-HBs titers was found between the two methods tested (Y = 0.951X + 100.7, R = 0.813, p < 0.001), although anti-HBs titers in 72 samples (39.6%) measured by Architect were less than 50% of that by Lumipulse and 12 (6.6%) were opposite results. Measuring 2 mAbs with HBV neutralizing activity, the titers of the 116 antibody (1.0 µg/mL) were comparable (689.3 mIU/mL by Lumipulse and 440.7 mIU/mL by Architect), whereas those of the 478 antibody (1.0 µg/mL) were much lower by Architect than by Lumipulse (42.6 vs. 818.6 mIU/mL, respectively). Of four other mAbs without HBV neutralizing activity, equal titers were observed for one; two mAbs had less anti-HB titers by Architect; and one was below the cut-off index (< 5 mIU/mL). CONCLUSIONS: Anti-HBs titers measured by Architect are likely to be lower than by Lumipulse, and the potential ability to detect the 478 antibody with neutralizing activity is low, indicating that Architect might underestimate anti-HBs titers. Future studies should standardize the anti-HBs titer measurement system.

[Clinical evaluation of a newly developed high-sensitive detection of hepatitis B virus surface antigen by a semi-automated immune complex transfer chemiluminescent enzyme immunoassay].

AIM: A highly sensitive semi-automated immune complex transfer chemiluminescence enzyme immunoassay (ICT-CLEIA) for the detection of hepatitis B surface antigen (HBsAg) was recently developed. Our aim is to investigate clinical significance of ICT-CLEIA in patients with HBV. METHODS: Of 829 HB carriers in our hospital and 167 commercial panels, performance of ICT-CLEIA(detection range 0.0005-2.5 IU/mL) was compared with two quantitative HBsAg detection systems (Architect HBsAg QT assay [0.05-250 IU/mL] and HISCL HBsAg assay [0.03-2500 IU/mL]) and COBAS TaqMan HBV-DNA assay (CTM, 2.1 Log copies/ml) using serum samples from patients or panels. RESULTS: The ICT-CLEIA had good accuracy and reproducibility. The sensitivity of wild type and HBsAg escape mutants (I126S, D144A, G145R) by ICT-CLEIA was 2- -5 to 2- -6 times higher than that of Architect HBsAg QT. For clinical practice, ICT-CLEIA assay could detect HBsAg even in the presence of anti-HBs during window periods in acute hepatitis B panel. HBsAg has been detectable for around 9 years in a patient with HBsAg clearance by Architect. In a patient with HBV reactivation after bone marrow transplantation followed by systematic chemotherapy, HBsAg by ICT-CLEIA was detectable at the same time point when HBV-DNA was detected by PCR. In conclusion, the ICT-CLEIA assay permits not only an earlier detection of acute hepatitis B infection but also may be useful for monitoring hepatitis B patients.

Comparative analysis of MRSA strains isolated from cases of mupirocin ointment treatment in which eradication was successful and in which eradication failed.

Nasal decolonization in methicillin-resistant Staphylococcus aureus (MRSA) carriers using mupirocin (MUP) is a strategy that complements barrier precautions and contact isolation. However, eradication failure cases have been observed despite isolates being susceptible to MUP. This would suggest that the minimum inhibitory concentration (MIC) alone is not the only determinant of successful eradication. In this study, we undertook a comparative analysis of MRSA isolates from cases of successful and unsuccessful MUP-eradication treatment. The analyses we carried out were: determination of mupirocin MICs, sequencing of the isoleucyl-tRNA synthetase (ileS) gene, staphylococcal cassette chromosome mec typing, and the assessment of slime production. MICs for all 14 of the successful nasal decolonization cases showed susceptibility to MUP, whereas 21 (87.5 %) of the 24 unsuccessful cases were MUP-susceptible, with low-level resistance seen in 3 (12.5 %) strains. In the analysis of mutations in the ileS gene, one strain with an MIC of 4 µg/ml exhibited a G1778A point mutation that has not been previously reported. In the 14 successful nasal decolonization cases, only 1 strain (7.1 %) was an MRSA slime-producer, compared with 19 (79.7 %) of the 24 MRSA strains that could not be eradicated after MUP treatment (p < 0.05). For the eradication of MRSA by MUP, it is possible that slime may affect drug penetration. In conclusion, slime production was the only significant difference between isolates recovered from successful and unsuccessful eradication cases.

Development of new IL28B genotyping method using Invader Plus assay.

IL28B polymorphism is associated with the response to pegylated interferon-α with ribavirin (PEG-IFN-α/RBV) treatment in chronic hepatitis C patients. As a genotyping assay for IL28B single nucleotide polymorphisms (SNPs) in clinical practice, the Invader Plus assay was developed. The accuracy, intra-assay, inter-assay precision, and the limit of detection of the Invader Plus assay were evaluated. Two SNPs (rs8099917 and rs12979860) associated with IL28B were genotyped by the Invader Plus and TaqMan assay in 512 Japanese patients. In comparison with direct sequencing, the Invader Plus assay showed 99% accuracy in rs8099917 and 100% accuracy in rs12979860. Intra-assay and inter-assay precision were sufficient to use in clinical practice and the detection limit was 1ngDNA/assay. Genotyping by rs8099917 showed that 361 (71%), 144 (28%) and seven (1%) of the patients were major homozygous, heterozygous and minor homozygous types, respectively. Five of the 512 cases (1%) had haplotype differences, but none showed differences between the two genotyping methods. For patients with HCV genotype 1, the prevalence of responders in the major homozygous type was 83.3%, and that of non-responders in the minor heterozygous/homozygous type was 72.5%. A convenient IL28B genotyping method using the Invader Plus assay could be useful to predict the treatment outcome in clinical practice.

[Antimicrobial susceptibility and serotype distribution in perinatal group B Streptococcus isolates--a 1999-2009 multicenter study].

We determined temporary changes in group B Streptococcus antimicrobial susceptibility and serotype distribution from perinatal strains. We examined invasive microbiological isolates from neonates with early-onset group B streptococcal disease (n = 14), and colonized isolates from those born uneventfully (n = 55) and from the genital tracts of pregnant and puerperal women (n = 198), collected between 1999 and 2009. All isolates were susceptible to penicillin. No significant differences were seen in susceptibility of 12 antimicrobial agents examined between invasive and colonized isolates. MIC50, MIC90, and resistance did not differ between stage I (1999-2005) and II (2006-2009) isolates. Serotype distribution significantly differed, however, serotypes III and Ia predominated among invasive isolates, while serotypes Ib and VI were common among their colonized counterparts. These findings suggest that to date, penicillin remains effective in intrapartum prophylactic use in colonized pregnant women.

A retrospective study of the epidemiology of Clostridium difficile infection at a University Hospital in Japan: genotypic features of the isolates and clinical characteristics of the patients.

Clostridium difficile is a major cause of antibiotic-associated diarrhea and frequently results in healthcare-associated infections. The epidemiology of C. difficile infection (CDI), including the prevalent polymerase chain reaction (PCR) ribotypes and the clinical characteristics of the patients, is not well known in Japan, compared to the situation in the United States and Europe. We performed PCR ribotyping of C. difficile isolates from 71 consecutive patients with CDI at a University Hospital over a 3-year period and investigated the clinical features of those patients. CDI was diagnosed when a patient with diarrhea or colitis was found to have toxin B-positive C. difficile with no other enteropathogenic microorganisms. Toxin A-positive, toxin B-positive, binary toxin-positive (A(+)B(+)CDT(+)) strains; toxin A-positive, toxin B-positive, binary toxin-negative (A(+)B(+)CDT(-)) strains; and toxin A-negative, toxin B-positive, binary toxin-negative (A(-)B(+)CDT(-)) strains were isolated from 4, 58, and 9 patients, respectively, indicating that infections with binary toxin-positive strains were uncommon (5.6%). PCR ribotyping of the isolates demonstrated that among the 71 strains, 20 different PCR ribotypes were identified and that types smz, yok, and hr were predominant (19, 14, and 13 isolates, respectively), all of which were A(+)B(+)CDT(-). No specific time periods or wards were found to be associated with the three types; PCR ribotyping analysis clearly showed that the three types spread almost evenly in all wards for the 3 years studied. Comparative analysis of the clinical characteristics of patients harboring the three C. difficile types indicated that the duration of CDI was longer in the yok group than in the hr group. PCR ribotyping, which is easy to perform, appears to give us useful information to trace CDI cases in clinical settings. Further, the analysis of a large number of CDI cases may allow evaluation of the possible relationship between specific C. difficile types and the clinical features of patients.

Characterization of Streptococcus pyogenes isolated from balanoposthitis patients presumably transmitted by penile-oral sexual intercourse.

Streptococcus pyogenes is indigenous to the human pharynx and causes acute pharyngitis. Balanoposthitis is an inflammatory disease of the glans and the foreskin. However, balanoposthitis caused by S. pyogenes is not widely recognized as a sexually transmitted disease. In addition, bacteriological features of the isolates causing balanoposthitis are unclear. The four S. pyogenes strains isolated from adult balanoposthitis were examined. We performed emm typing, T antigen typing, RAPD assay, PCR assay for the streptococcal pyrogenic exotoxin-related genes and antibiotic-resistant genes, and antibiotic susceptibility assay. All four strains were suspected to be transmitted by penile-oral sexual intercourse, were found to be different by genetic analysis, and also harbored some antibiotic-resistant factors. We propose that S. pyogenes should be considered as a causative agent of sexually transmitted disease. The drug resistant S. pyogenes must be taken into account when balanoposthitis patients are treated with antibiotic.

[Evaluation of high-sensitivity HBsAg quantitative assay for HBV genotype].

The clinical implication of the hepatitis B surface antigen (HBsAg) concentrations has been reported in HBV-infected patients during anti-viral treatment. HBV genotypes A and D are ubiquitous and scattered worldwide, especially northern America as well as Europe, whereas genotypes B and C are common in Asia. The aim of this study was to evaluate a new version of the Sysmex HBsAg quantitative kit based on Chemiluminescence Enzyme Immunoassay. Sera collected from 172 patients infected with any of the four major genotypes A to D (HBV/A, n = 18; B, n = 25; C, n = 84; D, n = 45), including the genotype D cases with weak reaction in the previous version of the kit. The new version of the kit having additional monoclonal antibody, showed improved sensitivity compared to the previous version as well as robust correlation with another quantitative HBsAg assay: the Abbot Architect. Observed during lamivudine therapy, increase in HBsAg and HBV DNA concentrations preceded the aminotransferase (ALT) elevation associated with drug-resistant HBV variant emergence (breakthrough hepatitis). In conclusion, reliability of the Sysmex HBsAg quantitative assay was confirmed for the four HBV genotypes common worldwide. Monitoring of serum HBsAg concentrations in addition to HBV DNA quantification, is helpful in evaluation of the response or resistance to anti-viral therapy.

[Evaluation of hepatitis B virus genotyping EIA kit].

Clinical significance of Hepatitis B virus(HBV) genotyping is increasingly recognized. The aim of this study was to evaluate reproducibility, accuracy, and sensitivity of an enzyme immunoassay (EIA) based HBV genotyping kit, which designed to discriminate between genotypes to A, B, C, or D by detecting genotype-specific epitopes in PreS2 region. Using the four genotypes panels, the EIA demonstrated complete inter and intra-assay genotyping reproducibility. Serum specimens had stable results after 8 days at 4 degrees C, or 10 cycles of freezing-thawing. In 91 samples that have been genotyped by DNA sequencing, 87(95.6%) were in complete accordance with EIA genotyping. Of examined 344 HBsAg-positive serum specimens, genotypes A, B, C and D were determined in 26 (7.6%), 62 (18.0%), 228 (66.3%), and 9 (2.6%) cases, respectively. Of 19 (5.5%) specimens unclassified by the EIA, 13 were found to have low titer of HBsAg concentration (< 3 IU/ml), and the other 5 had amino acid mutations or deletions within targeted PreS2 epitopes. The EIA allowed genotyping even in HBV DNA negative samples (96.2%). In conclusion, HBV genotype EIA is reliable, sensitive and easy assay for HBV genotyping. The assay would be useful for clinical use.

[Intestinal colonization and nosocomial spread of Clostridium difficile in pediatric cancer patients under long-term hospitalization].

Clostridium difficile is a major causative agent of antimicrobial-associated diarrhea, and the leading cause of nosocomial diarrhea. We clarified intestinal colonization and nosocomial spread of C. difficile in pediatric cancer patients undergoing antineoplastic therapy during long-term hospitalization. Subjects were 10 children with pediatric malignant diseases admitted from November 2005 to December 2006, aged 5 to 15 years, who received antineoplastic agents. Stool specimens were examined at hospitalization, after each course of treatment with antineoplastic chemotherapy, and when symptoms such as diarrhea or fever occurred. While C. difficile was detected from stool specimens of 8 of 10 children during their hospital stay, 6 of these 8 children were negative for C. difficile on the day of their admission. These results demonstrate that the use of antimicrobial agents and antineoplastic agents lead to overgrowth of C. difficile in intestinal tract of pediatric cancer patients. Five of the 8 children carried toxin A-positive, toxin B-positive C. difficle and 2 were diagnosed with C. difficile-associated diarrhea (CDAD). This demonstrates that CDAD is not a rare infection in pediatric cancer patients. Nine C. difficile isolates from 8 children were analyzed by PCR ribotyping. Two isolates from 2 children were typed into the same type;banding patterns of the remaining 7 isolates from 6 children were unique.

[Predicting sustained virological response in chronic hepatitis C patients treated with pegylated interferon and ribavirin using a novel highly sensitive Real-time detection PCR assay].

The Abbott Real Time HCV assay (lower limit of detection 12 IU/ml) was developed as a highly sensitive HCV RNA quantitative assay using real-time detection PCR(RTD-PCR). We assessed whether the new assay more effectively predicts sustained virological response (SVR) than conventional PCR (PCR) in 38 chronic hepatitis patients infected with HCV genotype 1b and treated with pegylated interferon alpha2b plus ribavirin. Sixteen patients reached SVR, 10 patients relapsed, 9 patients did not respond, 3 patients discontinued treatment. Positive predictive value (PPV) for SVR of undetectable HCV RNA at W4, 8, 12 by RTD-PCR and PCR was (100% vs. 100% at W4), (100% vs. 100% at W8), (83.3% vs. 72.7% at W12). HCV RNA undetectable at W12 had a higher PPV for SVR when measured by RTD-PCR than by conventional PCR.

[Prevention of vertical transmission of Group B Streptococcus].

Group B Streptococcus (GBS) are pathogens that involve a risk of vertical transmission. They are the infecting organism in approximately one quarter of all cases of neonatal sepsis and meningitis, making prevention of GBS infection an important goal. The United States Centers for Disease Control and Prevention (CDC) recommends administration of antibiotic prophylaxis to GBS-colonized pregnant women at least 4 hours before delivery, but the time of antibiotic prophylaxis administration is not generally reported in Japan. The purpose of the present study was to identify the care provided to GBS-colonized pregnant and intrapartum women in order to prevent of vertical transmission of GBS. The subjects were women (n=150) judged during pregnancy to have been colonized by GBS, who delivered vaginally at one of two hospitals between January 2000 and December 2004, and their neonates (n=151). The relation between the care provided and GBS transmission was analyzed. GBS was transmitted to the neonates of 18 of the 150 women (transmission rate 12.0%). The relation between transmission to the neonate and time between administration of antibiotic prophylaxis and delivery was investigated, and transmission to the neonate was found to be significantly greater when it was less than 3.5 hours (transmission to 9 neonates of 53 women) than more than 3.5 hours (transmission 4 neonates of 83 women) (p < 0.05). The time between admission and delivery was significantly shorter in the cases of transmission (p < 0.05). This indicates the need for thorough health guidance for expectant mothers, especially multipara, during pregnancy regarding the timing of admission for delivery, in order to ensure sufficient time between administration of antibiotic prophylaxis and delivery.