Matsuo Deguchi

Supportive Role Played by Precore and PreS2 Genomic Changes in the Establishment of Lamivudine-Resistant Hepatitis B Virus

BACKGROUND Hepatitis B virus (HBV) establishes lamivudine resistance via the resistance-causative rtM204V/I mutation and the replication-compensatory rtL180M mutation. However, both lamivudine-resistant viruses with and those without rtL180M can exist in clinical settings. To elucidate the differences between viruses with and those without rtL180M, we conducted full-length sequencing analysis of HBV derived from patients with type B chronic hepatitis showing lamivudine resistance. METHODS The full-length HBV DNA sequences derived from 44 patients showing lamivudine resistance were determined by polymerase chain reaction direct sequencing. Viral replicative competence was examined by in vitro transfection analysis using various HBV-expressing plasmids. RESULTS Throughout the HBV genome, a precore-defective A1896 mutation and a short deletion in the preS2 gene were detected more frequently in viruses without rtL180M than in those with it (64% vs. 17% [P < .005] and 50% vs. 10% [P < .01], respectively). In vitro transfection analysis revealed that the level of reduction in intracellular viral replication caused by the introduction of lamivudine resistance-associated mutations was lower in precore-defective and preS2-deleted viruses than in wild-type virus. CONCLUSIONS Both the precore-defective mutation and the preS2 deletion may play a supportive role in the replication of lamivudine-resistant HBV, which may be a reason for there being no need for the compensatory rtL180M mutation in lamivudine-resistant HBV possessing the precore and preS2 genomic changes.

Type B fulminant hepatitis is closely associated with a highly mutated hepatitis B virus strain.

Objective: Genome-wide sequences of hepatitis B virus strain associated with type B fulminant hepatitis have not been compared with those of acute self-limited hepatitis. We carried out full-length sequencing analysis of viral strains derived from patients with type B acute liver injury. Methods: Nine acute self-limited hepatitis and 6 fulminant hepatitis patients were the subjects of this study. Full-length sequencing analysis of viral DNA was done by PCR-direct sequencing. Results: Higher frequencies in fulminant hepatitis strains compared with acute hepatitis ones were observed in the T1762/A1764 (p < 0.05), A1896 (p = 0.09) and M1753 (M = C or A) (p = 0.09) mutations. Viruses related to fulminant hepatitis possessed the higher number of nucleotide substitutions than those related to acute hepatitis in the whole virus genome (p < 0.01) and various regions including preS/S gene (p < 0.05), precore/core gene (p < 0.01), polymerase gene (p < 0.05) and basic core promoter/core upstream regulatory sequence (p < 0.01). The high number of nucleotide substitutions in viruses related to fulminant hepatitis was predominantly non-synonymous in the preS/S and precore/core genes. Conclusion: Development of type B fulminant hepatitis may be associated with a highly mutated hepatitis B virus strain.

Available, Bed-sided, Comprehensive (ABC) score to a diagnosis of Methicillin-resistant Staphylococcus aureus infection: a derivation and validation study

BackgroundMethicillin-resistant Staphylococcus aureus (MRSA) infections continue to be a leading problem in health care facilities worldwide.MethodsThis single-center retrospective cohort study consisted of a derivation phase and a validation phase. The derivation phase included all patients admitted to Osaka University Hospital between May 2010 and April 2011. We proposed a provisional available, bed-sided, comprehensive (ABC) score, and evaluated its accuracy using the clinical diagnosis as a reference. We subsequently revised ABC scores based on k coefficient scores of each variable; this revision was validated by applying it to another patient population.ResultsA total of 172 patients and 154 cases were enrolled in the derivation and validation studies, respectively. The revised ABC score consisted of four simple variables: type of clinical specimen (1 to 3 points), Gram-staining result (1 point), presence of local inflammation (2 points), and a systemic inflammatory response (2 points). A revised score of ≥5 points was sensitive (93.8%) and specific (90.6%), and the area under the receiver-operating curve was 0.969 (95% CI; 0.957–1).ConclusionsWe developed a simple and comprehensive scoring system for diagnosis of nosocomial MRSA infections; this system is applicable in a wide variety of situations.

Evaluation of the highly sensitive chemiluminescent enzyme immunoassay “Lumipulse HBsAg-HQ” for hepatitis B virus screening

Ongoing efforts in the development of HBsAg detection kits are focused on improving sensitivity and specificity. The purpose of this study was to evaluate an improved, highly sensitive quantitative assay, “Lumipulse HBsAg‐HQ”, a chemiluminescent enzyme immunoassay designed for a fully automated instrument, the “Lumipulse G1200”.

Mutations associated with the therapeutic efficacy of adefovir dipivoxil added to lamivudine in patients resistant to lamivudine with type B chronic hepatitis

Factors influencing the therapeutic efficacy of adefovir dipivoxil added to continuing lamivudine have not been elucidated in lamivudine‐resistant patients with type B chronic hepatitis. The viral mutations influencing the efficacy of treatment with adefovir dipivoxil were investigated by sequencing analysis of the whole virus genome. Thirty patients resistant to lamivudine receiving adefovir dipivoxil therapy added to lamivudine were studied. From serum samples obtained before the administration of adefovir dipivoxil, full‐length viral DNA sequences were determined by PCR‐direct sequencing. Susceptibility of the virus to adefovir was examined further using in vitro transfection analysis. By screening the whole viral genome, the presence of two mutations, a T‐to‐C/G/A mutation at nt1753 (V1753) and an A‐to‐C mutation at nt2189 (C2189), correlated with the higher incidence of sustained viral DNA clearance during therapy (P < 0.005 and P < 0.05). In multivariate analysis, the V1753 (P = 0.001) and the C2189 (P = 0.007) mutations, and elevated transaminase (P = 0.011) and low viral load (P = 0.008) at the baseline were selected as significant independent factors associated with improved antiviral efficacy. In vitro transfection analysis showed no differences in susceptibility to adefovir among wild‐type virus and C1753 and C2189 mutant viruses, suggesting that the virus possessing these mutations may be eradicated more efficiently than the wild‐type virus by treatment regardless of a direct antiviral effect of adefovir. J. Med. Virol. 81:798–806, 2009.

Genomic reorganization by IS26 in a bla NDM-5-bearing FII plasmid of Klebsiella pneumoniae isolated from a patient in Japan

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.

Vaccination strategy for epidemic viral diseases in healthcare workers: Cut-off for optimal immunization

Healthcare workers (HCWs) are at an increased risk of being exposed to epidemic viral diseases (EVDs), such as measles, rubella, mumps, and varicella-zoster. Currently, in case of the absence of written records on previous immunizations, the Japanese Society for Infection Prevention and Control guidelines require HCWs to have antibody titers higher than laboratory thresholds, possibly leading to over-immunization. We report our vaccination strategy and the consequent incidences of EVDs at the Osaka University Hospital between 2000 and 2016. In 2001, we initiated an annual serology check of antibody titers against EVDs and immunization for newly employed HCWs. As an additional vaccination program, all HCWs with low antibody titers were vaccinated in 2005 and 2010. Antibody titers were determined by an enzyme immunoassay (EIA), with a positive range of >2.0 cut-off index. After implementing the vaccination strategy to keep the laboratory threshold, there were only sporadic cases of EVDs among HCWs. More than 99% of individuals who had positive titers in 2005 remained the positive antibody titers in 2010, indicating that a minimum interval of 5 years is enough to measure immunity. Unprotected workers can, even silently, transmit the contagious viruses to patients and coworkers, possibly resulting in a nosocomial outbreak. However, over-vaccination may yield adverse effects and financial burdens. Our observational data indicate that the laboratory cut-off index of >2.0 by EIA may provide a sufficient herd immunity to prevent EVDs among HCWs.

Durability of immunity by hepatitis B vaccine in Japanese health care workers depends on primary response titers and durations

Background Health care workers (HCWs) are frequently exposed to hepatitis B virus (HBV) infection. The efficacy and safety of immunization with the hepatitis B (HB) vaccine are well recognized, but the durability of immunity and need for booster doses in those with secondary vaccine response failure remains controversial. Methods This was a retrospective cohort study performed at Osaka University Hospital, Japan. We examined antibodies against HB surface antigen (anti-HBs) titers annually after immunization for previously non-immunized HCWs. Primary responders were categorized by their sero-positive durations as short responders (those whose anti-HBs titers declined to negative range within 3 years), and long responders (those who retained positive anti-HBs levels for 3 years and more). We re-immunized short responders with either single or 3-dose boosters, the long responders with a single booster when their titers dropped below protective levels, and examined their sero-protection rates over time thereafter. Results From 2001 to 2012, data of 264 HCWs with a median age of 25.3 were collected. The rate of anti-HBs positivity after primary vaccination were 93.0% after three doses (n = 229), 54.5% after two doses (n = 11), and 4.2% after a single dose (n = 24). Of 213 primary responders, the anti-HBs levels of 95 participants (44.6%) fell below the protective levels, including 46 short responders and 49 long responders. HCWs with higher initial anti-HBs titers after primary vaccination had significantly longer durations of sero-positivity. For short responders, 3-dose booster vaccination induced a longer duration of anti-HBs positivity compared to a single-dose booster, whereas for long responders, a single-dose booster alone could induce prolonged anti-HBs positivity. Conclusion Our preliminary data suggested that it may be useful to differentiate HB vaccine responders based on their primary response durations to maintain protective levels of anti-HBs efficiently. A randomized, prospective, large-scale study is warranted to support our findings.

Novel and Simple Approach to Estimating the Actual Incidence of Blood and Body Fluid Exposure

BACKGROUND There is no current way to determine the actual blood and body fluid exposure (BBFE) incidence in hospitals. We propose a simple, reliable, and widely available method for the accurate estimation of BBFE. METHODS Data for BBFE for healthcare workers between 2006 and 2015 at Osaka University Hospital were retrospectively extracted from the electronic records. Annual positivity of hepatitis C virus (HCV) antibody in the source individuals and overall patient population were calculated over time. We created an estimation formula focusing on the difference in HCV positivity between the source individuals and overall patient population for the actual number of BBFEs. A linear regression model was used to evaluate the temporal change in the reported and estimated BBFEs. RESULTS During the study period, 937 BBFEs were reported. HCV positivity between the post-BBFE cohort and overall patient population greatly differed; the incidence ratio ranged from 2.1 to 5.7. The linear regression model revealed that the reported BBFEs did not significantly change during the study period (the slope, 1.315 [95% confidence interval (C.I.): -0.849 to 3.480, p = 0.199]). The annual incidence ratio of the estimated and reported BBFEs significantly reduced over time (the slope, -0.287 [95% C.I.: -0.488 to -0.086, p = 0.011]), indicating that, although the reported number of BBFEs seemed unchanged, the estimated incidence decreased. CONCLUSIONS We propose a novel and simple approach to estimating the actual incidence of BBFEs in hospitals using the difference in HCV positivity between the post-BBFE cohort and overall patient population.

B型慢性肝炎患者におけるMicroparticle EIAによるHBe抗原・抗体の有用性

A study was made of 73 samples from 6 chronic hepatitis B patients, 2 out of the 6 cases were non-treated and the 4 cases were interferon treated HBeAg, HBeAb, s-ALT and HBV-DNA. Detection of HBeAg and HBeAb were assayed by AxSYM system (Dainabot), which were based on microparticle enzyme immunoassay. 1) Fifty-three out of 73 samples showed positive for HBeAg; 45 samples showed only HBeAg positive (Group A) and 8 samples showed HBeAg and HBeAb positive (Group B). The positive ratios of HBV-DNA in each group 91.1% (41/45) and 50.0% (4/8), respectively and 4 out of the rest 20 samples that showed only HBeAb positive also showed HBV-DNA positive. The above 12 discrepant samples between HBeAg and HBV-DNA were collected from 4 chronic hepatitis B patients who became normal s-ALT levels after liver injury. The HBeAg S/N ratio (sample/negative control) of these samples were near the cutoff value (S/N = 2.1). 2) Comparing the movement between HBeAg and s-ALT levels in 6 chronic hepatitis B patients, the changes of HBeAg S/N ratio were related with the changes of s-ALT. The changes of HBeAg S/N ratio were observed before the movement of s-ALT. In conclusion, the detection of HBeAg/HBeAb by AxSYM system was useful to monitor the clinical course of chronic hepatitis B patients.