Comparison of extraction-based and elution-based polymerase chain reaction testing, and automated and rapid antigen testing for the diagnosis of severe acute respiratory syndrome coronavirus 2.

We aimed to compare the differences in testing performance of extraction-based polymerase chain reaction (PCR) assays, elution-based direct PCR assay, and rapid antigen detection tests for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We used nasopharyngeal swab samples of patients with coronavirus disease 2019 (COVID-19). We used the MagNA Pure 24 System (Roche Diagnostics K.K.) or magLEAD 12gC (Precision System Science Co., Ltd.) for RNA extraction, mixed the concentrates with either the LightMix Modular SARS-CoV PCR mixture (Roche Diagnostics K.K.) or Takara SARS-CoV-2 direct PCR detection kit (Takara Bio Inc.), and amplified it using COBAS® z480 (Roche Diagnostics K.K.). For elution-based PCR, we directly applied clinical samples to the Takara SARS-CoV-2 direct PCR detection kit before the same amplification step. Additionally, we performed Espline SARS-CoV-2 (Fuji Rebio Co., Ltd.) for rapid diagnostic test (RDT), and used Lumipulse SARS-CoV-2 antigen (Fuji Rebio Co., Ltd.) and Elecsys SARS-CoV-2 antigen (Roche Diagnostics K.K.) for automated antigen tests (ATs). Extraction-based and elution-based PCR tests detected the virus up to 214-216 and 210 times dilution, respectively. ATs remained positive up to 24-26 times dilution, while RDT became negative after 22 dilutions. For 153 positive samples, positivity rates of the extraction-based PCR assay were 85.6% to 98.0%, while that of the elution-based PCR assay was 73.2%. Based on the RNA concentration process, extraction-based PCR assays were superior to elution-based direct PCR assays for detecting SARS-CoV-2.

Trends in hepatitis C virus-associated mortality rates in Japan, 1998-2017.

BACKGROUND AND AIM: The current prevalence of hepatitis C virus infection and hepatitis C virus-associated mortality in Japan falls short of the World Health Organization goal of viral hepatitis elimination by 2030. We aimed to evaluate the trends in hepatitis C virus-associated mortality in Japan. METHODS: This nationwide observational study used the Japanese Vital Statistics from 1998 to 2017 and included all Japanese hepatitis C virus-associated deaths (84 936) of adults aged ≥ 40 years. We calculated the crude and age-standardized mortality rates per 100 000 persons by age and sex. Joinpoint regression analysis was used to identify significant changing points in trends and to estimate the annual percentage changes and the average annual percentage changes for the entire study period. RESULTS: The crude mortality rate per 100 000 persons (annual death number) increased from 5.5 (3548) in 1998 to 7.0 (4843) in 2005 and decreased to 4.0 (3095) in 2017. By 2017, the crude mortality rates per 100 000 persons among men and women had dropped to 3.6 and 4.3, respectively. The age-standardized mortality rate was higher in women than in men. The average annual percentage change was -3.8% (95% confidence interval: -5.0 to -2.5). The declining trend was more rapid in men (-4.5%, 95% confidence interval: -5.3 to -3.6) than in women (-2.7%, 95% confidence interval: -3.8 to -1.6). CONCLUSIONS: Trends in hepatitis C virus-associated mortality rates have declined in an accelerating manner in Japan, especially among men.

10-Year survey on serum antibody positivity rates and titers for measles and rubella in healthcare workers; an observational study at a Japanese university hospital.

BACKGROUND: We evaluated the effect of the two-dose vaccination strategy, which has been a widely adopted as childhood routine schedule worldwide to acquire herd immunity, on healthcare workers (HCWs) in Japan. METHODS: Between 2010 and 2019, antibody titers for measles and rubella were measured annually among newly employed HCWs at Osaka University Hospital, Japan, using Enzygnost® assays (Siemens Healthcare Diagnostics Co. Ltd., Marburg, Germany). The data were categorized by age to compare the antibody positivity rates and antibody titers among no-vaccine, single-dose, and two-dose groups. RESULTS: Over the 10-year period, the annual antibody positivity rates for measles and rubella were 84.0%-95.3% and 90.0%-94.5%, respectively, without any particular trend. The antibody titers for measles (median [interquartile range]: 8.4 [3.9, 20] vs. 6.1 [3.5, 12]) and rubella (11 [5.5, 20] vs. 6 [3.7, 11]) were statistically lower (p < 0.001) in the two-dose generation than in the single-dose generation. DISCUSSION: A shift from single-dose to two-dose vaccination did not yield an increase in antibody positivity rates for both measles and rubella among HCWs. Notably, antibody titers were significantly lower in the two-dose generation. CONCLUSION: Despite several limitations, our data suggests a paradoxical vulnerability in young HCWs who received the two-dose vaccination in a view of sero-positivity rates.

Risk for the occupational infection by cytomegalovirus among health-care workers.

BACKGROUND: Cytomegalovirus (CMV) are ubiquitously distributed worldwide, causing a wide range of clinical manifestations from congenital infection to a life-threatening disease in immunocompromised individuals. CMV can be transmitted via human-to-human contact through body fluids; however, the risk of CMV infection among healthcare workers (HCWs) has not been fully evaluated. AIM: This study aimed to assess the risk of CMV infection among HCWs through daily medical practices. METHODS: Serum samples from HCWs at Osaka University Hospital (Japan) were analysed. Initially, we compared CMV IgG seropositivity among HCWs (medical doctors, nurses, and others) in 2017, which was examined after 1 year to evaluate seroconversion rates among those with seronegative results. Then, we examined CMV seroconversion rates in HCWs who were exposed to blood and body fluids. FINDINGS: We analysed 1153 samples of HCWs (386 medical doctors, 468 nurses, and 299 others), of which CMV seropositivity rates were not significantly different (68.9%, 70.3%, and 70.9%, respectively). Of these, 63.9% (221/346) of CMV seronegative HCWs were followed after 1 year, with CMV seroconversion rates of 3.2% (7/221). Among 72 HCWs who tested negative for CMV IgG when exposed to blood and body fluids, the CMV seroconversion rate was 2.8% (2/72). The CMV seroconversion rates between the two situations were not significantly different. CONCLUSION: Our study indicated that CMV infection through daily patient care seems quite rare. Further well-designed studies with a large sample size are warranted to verify our finding.

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.

Simultaneous and rapid detection method for measles and rubella using single-tube multiplex real-time quantitative RT-PCR.

Patients with measles or rubella infections manifest acute onset fever accompanying systemic exanthema, which are clinically difficult to be distinguish. Rapid diagnosis and differentiation of such epidemic viral diseases is essential to prevent outbreaks. We developed a single-tube multiplex real-time PCR assay for these indistinguishable viruses. We used previously-reported primer settings, with a slight modification of reporter dye, and applied to multiplex Taqman real-time PCR by cobas z480 (Roche Molecular Systems, Inc.). Consequently, the assay could detect 10 copies/10 µl of measles and rubella with coefficient of variations of 11.2% and 21.8%, respectively. Strengths of our methodology include simplicity of operation, short measurement time (2 h), uses of internal control (confirming a run of PCR), and quantitative measurement with high sensitivity. Both measles and rubella currently cause social outbreaks in Japan. We hope that our single-tube multiplex assay contributes to an early diagnosis, leading to an appropriate infection control measure and prevention of epidemics.

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

BACKGROUND: Methicillin-resistant Staphylococcus aureus (MRSA) infections continue to be a leading problem in health care facilities worldwide. METHODS: This 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. RESULTS: A 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). CONCLUSIONS: We developed a simple and comprehensive scoring system for diagnosis of nosocomial MRSA infections; this system is applicable in a wide variety of situations.

Reduced Seroprevalence of Hepatitis A Virus Among Japanese Healthcare Workers as a Risk Factor for Occupational Infection.

BACKGROUND: With the improvement in sanitation and hygiene, the incidence of hepatitis A virus (HAV) infection has declined, and its seroprevalence among Japanese people has been reduced. Universal HAV vaccination is yet to be implemented in Japan, and the healthcare workers (HCWs) are at higher risks of acquiring this infection. We herein report the seroepidemiology of HAV infection among HCWs at Osaka University Hospital. METHODS: Between September and October 2017, we collected serum samples submitted to our laboratory for HCWs health examination and tested for the anti-HAV antibody. RESULTS: Overall HAV seropositivity rate was 5.1% (22/436 samples). The seroprevalence was higher among those in the twenties (6.0%) and thirties (8.0%), compared to older age groups. CONCLUSIONS: In this age of internationalization, the decreasing immunity for HAV places HCWs at risk of developing the disease. As a preventive measure against occupational infection, HAV vaccination may be needed for Japanese HCWs.

Comparison of the Temperature Influence on the Activity of Currently Available Procalcitonin Reagents.

BACKGROUND: Procalcitonin (PCT) is a stable biomarker for bacterial infections; however, limited data is available on new trivalent reagents. We evaluated temperature influence on the activity of PCT reagents. METHODS: Using both conventional and trivalent reagents, we measured PCT levels of 30 clinical samples, stored residuum at refrigerator (4°C) and room temperature (24°C), and reexamined it after 24 hours. We defined a reduction rate as a percentage of PCT level at 24 hours compared to that after defrost and evaluated a ratio of reduction rate in 4°C to that in 24°C. RESULTS: The reduction rate at room temperature decreased significantly compared to that in the refrigerated condition for all the reagents examined (p < 0.001). In addition, the ratio of reduction rate between the conventional and trivalent reagents showed a significant difference (p < 0.001). CONCLUSIONS: The serum PCT levels significantly decrease at room temperature, particularly when using newer trivalent reagents.

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

BACKGROUND: 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". METHODS: Serum samples for reproducibility, dilution, correlation, sensitivity, and specificity studies were obtained from patients at the Osaka University Hospital. Seroconversion and sensitivity panels were purchased from a commercial vender. Subtype, sensitivity panels, and HBsAg recombinant proteins with one or two amino acid substitutions were prepared in-house. RESULTS: The coefficients of variation for the low, medium, and high concentration samples ranged from 1.93 to 2.55%. The HBsAg-HQ reagent for dilution testing showed good linearity in the 0.005-150 HBsAg IU/mL range and no prozone phenomenon. All 102 HBV carrier samples were positive by HBsAg-HQ, while other commercial reagents showed one or more to be negative. In the seroconversion panel, the 14-day blood sample was positive. The sensitivity against HBsAg-HQ "ad" and "ay" subtypes was 0.025 ng/mL. Comparisons among the HBsAg-HQ, HISCL, and Architect HBsAg reagents were performed using the Bland-Altman plot. Specificity for 1000 seronegative individuals was 99.7%. HBsAg-HQ detected 29 positive serum among 12 231 routinely obtained serum samples, which showed concentrations of 0.005-0.05 HBsAg IU/mL. CONCLUSIONS: According to these results, the Lumipulse HBsAg-HQ assay, with a highly sensitive limit of detection of 0.005 IU/mL, may facilitate the development of a better management strategy for a considerable proportion of infected patients.

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.

Performance evaluation of four dominant anti-hepatitis B core antigen (HBcAb) kits in Japan for preventing de novo hepatitis B virus (HBV) infection.

BACKGROUND: The determination of antibody to hepatitis B core antigen (HBcAb) has become an important means of evaluating the risk factors of de novo hepatitis B virus (HBV) infection before starting intensive immunosuppressive drug therapies. Four dominant HBcAb determination reagents used in Japan were evaluated with HBcIgM, HBsAg, HBsAb, HBeAb, and HBV DNA reagents in order to study their clinical utility. METHODS: Four kinds of HBcAb reagent kits (HBcAb Total and HBcAb-IgG reagent) were evaluated with 526 clinical specimens, including 344 negative specimens, at Osaka University Hospital. The dynamic range of each kit was evaluated by testing serially diluted serum from pooled sera with high HBcAb concentration. RESULTS: The reagent that showed the largest dynamic range was the Lumipulse HBcAb-N (HBcAb-IgG reagent). Regarding clinical sensitivity and specificity, Centaur HBcAb (HBcAb Total reagent) gave several "doubtful negative" results and ARCHITECT HBcII (HBcAb Total reagent) had the most discrepant positive results. By comparing the cut-off-index distribution of negative specimens using a parameter of "distance from the mean to the cut-off divided by the SD", Centaur was determined to be the best (distance/SD = 12.65), with Lumipulse and Elecsys Anti-HBc (HBcAb Total reagent) in the second group (8.13 and 7.00, respectively), and ARCHITECT rated as the worst (3.25). CONCLUSIONS: In this evaluation, Elecsys and Lumipulse HBcAb kits showed good clinical sensitivity and specificity and were considered to be suitable for evaluating the risk factors of de novo HBV infection.

[Advanced Testing and Laboratory for HBV, HCV, and HIV Infection].

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.

Evaluation of human immunodeficiency virus-1/2 antigen/antibody immunochromatographic assay.

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.

Multiplex Real-Time PCR Assay for Six Major Carbapenemase Genes.

The global dissemination of carbapenemase-producing Enterobacteriaceae (CPE) is a major concern in public health. Due to the existence of the diversity of carbapenemases, development of an easily available, cost-effective multiplex detection assay for CPE is required worldwide. Using clinically available and reliable equipment, COBAS® z480 (Roche Diagnostics K.K., Tokyo, Japan), we developed a multiplex real-time PCR assay for the detection of two combinations of carbapenemases; first, blaNDM, blaKPC, and blaIMP (Set 1), and second, blaGES, blaOXA-48, and blaVIM (Set 2). We constructed standard curves for each carbapenemase gene using serial dilutions of DNA standards, then applied reference or clinical isolates with each carbapenemase gene to this assay. The multiplex assay showed satisfactory accuracy to detect CPE genes, with the correlation coefficients of greater than 0.99 for all genotypes. The assay appropriately differentiated the reference or clinical strains harboring each carbapenemase gene without cross reactivity. Lastly, the assay successfully detected multiple genes without false-positive reactions by applying six clinical isolates carrying both NDM and OXA-48-like carbapenemase genes. Major advantages of our assay include multiplicity, simple operation, robustness, and speed (1 h). We believe that the multiplex assay potentially contributes to early diagnosis of CPE with a diverse genetic background.

Alterations in hepatitis B virus nucleotide sequences in a chronic virus carrier from immunotolerant to immunoactive phase.

Factors involved in transition from the immunotolerant to immunoactive phase in chronic hepatitis B virus (HBV) infection remain unclear. We investigated viral mutations occurring during transition and elucidated their virological and immunological significance. Full-length HBV DNA sequences were serially determined in a chronic HBV carrier from the immunotolerant to immunoactive phase. Viral replicative competence was examined by transfection analysis. HBV-specific CD8(+) T cell response was evaluated by coculture of CD8(+) T cells with autologous dendritic cells followed by interferon-gamma Elispot assay. Eleven point mutations and two deletions appeared around the onset of the immunoactive phase. Viral replicative competence declined significantly after the onset of active hepatitis. Examination of the CD8(+) T cell response against two putative T-cell epitopes, which contained substituted amino acids from the immunotolerant to immunoactive phase, showed that mutant HBV epitopes gave a lesser T cell response than wild-type HBV ones. In summary, point mutations and deletions may occur prior to or concurrent with the onset of the immunoactive phase during chronic HBV infection. These mutations may result in a significant decrease in both viral replicative competence and HBV-specific CD8(+) T cell response, suggesting a possible adaptation for the maintenance of viral persistence.

[The new method for detecting the outbreak sign of MRSA].

Conventional outbreak detection laboratory-based made one unit from the beginning of the month to the end of the month, totaled and analyzed, cannot correctly detect outbreaks continued during two months. The real-time analysis (RTA) we devised adapts to methicillin-resistant Staphylococcus aureus (MRSA) and avoids the problems of conventional detection. RTA analyzes all data for the last 30 days when MRSA is newly isolated 48 hours or more after hospital admission. In the three years from April 2006 to March 2009, we compared the day and number of MRSA outbreaks newly isolated 48 hours or more after hospital admission in 572 subjects using the conventional method and RTA. We also calculated the RTA infection prevention effect. The number of outbreaks detected conventionally numbered 68 cases and those detected by RTA numbered 106 cases. The number of outbreaks newly detected by RTA numbered 38 cases in three years, averaging 4.3 days earlier than conventional detection using conventional method A an average of 15.7 days earlier than conventionally which totals for every end of the month using conventional method B. The effect of infection prevention in the change of RTA from conventional method A presumably decreases MRSA infection to 14-18 persons and it in the change of RTA from conventional method B decreases MRSA infection to 18-25 persons in one year. These results suggested that outbreak detection by RTA could help prevent MRSA outbreak and decrease MRSA infection frequency.

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.

Clinical utility and characteristics of nine anti-HCV antibody screening reagents used in Japan.

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.