Hepatitis B Core-related Antigen: An Alternative to Hepatitis B Virus DNA to Assess Treatment Eligibility in Africa.

BACKGROUND: To eliminate hepatitis B virus (HBV) infection, it is essential to scale up testing and treatment. However, conventional tools to assess treatment eligibility, particularly nucleic acid testing (NAT) to quantify HBV DNA, are hardly available and affordable in resource-limited countries. We therefore assessed the performance of a novel immunoassay, hepatitis B core-related antigen (HBcrAg), as an inexpensive (US$ <15/assay) alternative to NAT to diagnose clinically important HBV DNA thresholds (≥2000, ≥20 000, and ≥200 000 IU/mL) and to select patients for antiviral therapy in Africa. METHODS: Using a well-characterized cohort of treatment-naive patients with chronic HBV infection in The Gambia, we evaluated the accuracy of serum HBcrAg to diagnose HBV DNA levels and to indicate treatment eligibility determined by the American Association for the Study of Liver Diseases, based on reference tests (HBV DNA, hepatitis B e antigen, alanine aminotransferase, liver histopathology, and/or FibroScan). RESULTS: A total of 284 treatment-naive patients were included in the analysis. The area under the receiver operating characteristic curve (AUROC), sensitivity, and specificity of serum HBcrAg were 0.88 (95% confidence interval [CI], .82-.93), 83.3%, and 83.9%, respectively, to diagnose HBV DNA ≥2000 IU/mL; and 0.94 (95% CI, .88-.99), 91.4%, and 93.2% for ≥200 000 IU/mL. A simplified treatment algorithm using HBcrAg without HBV DNA showed high AUROC (0.91 [95% CI, .88-.95]) with a sensitivity of 96.6% and specificity of 85.8%. CONCLUSIONS: HBcrAg might be an accurate alternative to HBV DNA quantification as a simple and inexpensive tool to identify HBV-infected patients in need of antiviral therapy in low- and middle-income countries.

Novel and highly sensitive immunoassay for total hepatitis B surface antigen, including that complexed with hepatitis B surface antibody.

BACKGROUND: Hepatitis B surface antigen (HBsAg) is used as a clinical marker of hepatitis B virus (HBV) infection. However, conventional HBsAg assays have so far failed to accurately detect HBsAg in blood because of interference by patient-derived antibodies against HBsAg (HBsAb). METHODS: We developed a novel, fully automated assay system that can detect total HBsAg in blood, including antigens complexed with HBsAb. The immunoassay inactivates HBsAb via a simple pretreatment step to dissociate the HBsAg molecule from HBsAg-HBsAb complexes and thereby estimate total HBsAg. Accordingly, the test has been termed the "immunoassay for total antigen including complex via pretreatment (iTACT)-HBsAg." RESULTS: The recovery rate of HBsAg in the presence of HBsAb was greater than 87 % at a cutoff value set at 5.0 mIU/mL on the basis of data from 545 healthy controls. Analyses using serial serum samples from 25 HBV carriers who became negative for HBsAg during follow-up showed that the iTACT-HBsAg could detect HBsAg over a period of years despite a loss in detection by conventional assays and was able to detect HBsAg in 39 (53 %) of 73 samples with HBsAb. CONCLUSIONS: The new iTACT-HBsAg assay appears to detect total HBsAg with high sensitivity, even in the presence of HBsAb, and may useful in identifying subclinical or occult HBV carriers.

[Development of Highly Sensitive Quantitative HBsAg Reagent and Its Availability].

HBsAg is an envelope surface protein of the hepatitis B virus, and a marker of hepatitis B infection. In Japan, an HBsAg reagent was adopted as a blood screening test by the Japan Red Cross in 1972. Furthermore, a highly sensitive HBsAg reagent has been in use since 2008. Blood screening with HBsAg reagent use has contributed to a decrease in hepatitis B infections following blood donation. From the year 2000, the HBsAg reagent requirements have changed according to the progress in hepatitis B treatment. Serum levels of HBsAg and their temporal changes over long periods are used to understand a patient's clinical state and the effect of nucleoside analogue treatment. In 2013, we developed a highly sensitive quantitative chemiluminescent enzyme immunoassay (HBsAg-HQ) for HBsAg. Characteristics of the HBsAg-HQ reagent include the use of a pretreatment mixture and anti-HBs antibodies for the inner portion of HBsAg. The detection limit of HBsAg-HQ reagent (zero +3SD) is 0.0006 IU/mL, and the quantitative limit below 10% of the CV is 0.002 IU/mL. Therefore, we configured the measurement range from 0.005 to 150 IU/mL. Diagnostic sensitivity was tested for 10 seroconversion panels with HBsAg-HQ, and HBsAg-HQ yielded the same or higher sensitivity than other immunoassay products. In the report of Wai-Kay Seto et al., 25.8% of HBV patients whose sera were classified as HBs seroclearance by CLIA HBsAg reagent were judged as positive using our HBsAg-HQ reagent. Furthermore measurement of HBsAg levels is recommended to confirm the effect of nucleoside analogue treatment for chronic hepatitis patients or assessing the clinical condition before immunosuppression therapy for rheumatoid patients based on the guidelines of "The Japan Society of Hepatology" and "Japan College of Rheumatology". HBsAg qualitative reagents with high sensitivity are thus desirable for use in monitoring HBsAg.

[Evaluation of serum PIVKA-II by Lumipulse PrestoII assay].

Measurements of serum concentrations of Des-gamma-carboxy Prothrombin (PIVKA-II) are widely used for diagnosing hepatocellular carcinoma (HCC). Recently, in Lumipulsef assay, it was reported that antibodies against alkaline phosphatase (ALP) derived from anti bleeding sheets led false high values of PIVKA-II in the patients with HCC resection. To improve the previous issue, newly developed Lumipulse PrestoII assay was examined. (1) The assay was reliable and positively correlated with the previous assays (Lumipulse f and Picolumi, R = 0.997 and 0.994 (n=115), respectively). (2) Eleven cases, which had false high values of PIVKA-II by the Lumipulsef assay, were examined by the PrestoII assay with excess of inactive ALP. The false high values of 10 cases were improved, but only one was still high. False reactivity of this case was stronger than other cases, more effective adsorption was required. (3) Comparing the absorbent activity of inactive ALP among 6 different kinds, we found inactive ALP with much higher adsorbent activity. When this inactive ALP was applied to assay, false high values of PIVKA-II were improved in all 11 cases. In conclusion, the PrestoII assay, which applies the inactive ALP with high activity, is reliable and useful for clinical screening.

[IgG heterophile antibody causes false positivity for CA19-9, which is overcome with bovine immunoglobulin].

Immunoassay using antibodies is widely applied to measure various clinical parameters such as tumor markers. However, many mechanisms of interference for this method have been reported. We studied serum sample showing false positive CA19-9 values in the range of 24 to approximately 286U/ml depending on the reagent lots used. PEG-pretreated serum did not show false positivity. Using high performance liquid chromatography (HPLC), the CA19-9 false positive peak was obtained around 160kD, which is the same as IgG. Antibody-coated microbeads, labeled antibody and buffer were used in different combinations between different lots, and the buffer was found to be the cause of false positive findings. The amount of bovine IgG contaminated in bovine serum albumin(BSA) in the reagent buffer markedly differed between lots of BSA, and reagent with a low amount of bovine IgG showed false positive results. Bovine immunoglobulin was superior to mouse IgG in the attempt to avoid a false positive reaction. We concluded that the cause of false positive CA19-9 findings in this serum sample was IgG heterophile antibody, which reacts with both of mouse and bovine IgG. The heterophile antibody had greater affinity to bovine IgG than to mouse IgG. The difference between CA19-9 values obtained by different reagent lots was due to different amounts of bovine IgG contaminated in BSA used in the reagent. Bovine immunoglobulin was superior to mouse IgG in the attempt to absorb the heterophile antibody and avoid false positive reaction in this case.