Anne L. McNamara

Frequency of HBV DNA detection in US blood donors testing positive for the presence of anti-HBc: implications for transfusion transmission and donor screening

BACKGROUND: An estimate of the rate of HBV DNA‐positive, anti‐HBc‐positive units is important for evaluating the need for anti‐HBc donor screening, especially in the context of HBV NAT.

Serum and liver hepatitis B virus DNA in chronic hepatitis B after sustained loss of surface antigen

Polymerase chain reaction (PCR) was used to detect hepatitis B virus DNA in the sera and livers of nine patients with chronic hepatitis B after treatment-induced or spontaneous loss of serum hepatitis B surface antigen. Patients were evaluated at intervals ranging from 3 to 67 months after disappearance of hepatitis B surface antigen. PCR was performed using primer pairs from the surface and core gene regions, and surface gene products were quantitated. Liver tissue was also evaluated by in situ hybridization to assess viral transcription. Five of the nine patients had viral DNA detectable in serum by PCR. Quantitation of polymerase chain reaction products in serum and liver showed that the DNA levels tended to decline progressively after antiviral therapy. Six of seven surface antigen-negative patients tested had detectable viral DNA in the liver, and four of the six DNA-positive patients were negative for DNA in serum by PCR. None had surface gene messenger RNA. Thus, it is concluded that hepatitis B virus DNA may be detectable by PCR in liver tissue years after the disappearance of hepatitis B surface antigen, even in the absence of detectable hepatitis B virus DNA in serum.

Lack of correlation between HBsAg and HBV DNA levels in blood donors who test positive for HBsAg and anti-HBc: implications for future HBV screening policy

BACKGROUND:  Studies showing a significant correlation between hepatitis B surface antigen (HBsAg) and hepatitis B virus (HBV) deoxyribonucleic acid (DNA) levels have focused on the HBV seroconversion window period.

Development and performance of prototype serologic and molecular tests for hepatitis delta infection

Worldwide, an estimated 5% of hepatitis B virus (HBV) infected people are coinfected with hepatitis delta virus (HDV). HDV infection leads to increased mortality over HBV mono-infection, yet HDV diagnostics are not widely available. Prototype molecular (RNA) and serologic (IgG) assays were developed for high-throughput testing on the Abbott m2000 and ARCHITECT systems, respectively. RNA detection was achieved through amplification of a ribozyme region target, with a limit of detection of 5 IU/ml. The prototype serology assay (IgG) was developed using peptides derived from HDV large antigen (HDAg), and linear epitopes were further identified by peptide scan. Specificity of an HBV negative population was 100% for both assays. A panel of 145 HBsAg positive samples from Cameroon with unknown HDV status was tested using both assays: 16 (11.0%) had detectable HDV RNA, and 23 (15.7%) were sero-positive including the 16 HDV RNA positive samples. Additionally, an archival serial bleed panel from an HDV superinfected chimpanzee was tested with both prototypes; data was consistent with historic testing data using a commercial total anti-Delta test. Overall, the two prototype assays provide sensitive and specific methods for HDV detection using high throughput automated platforms, allowing opportunity for improved diagnosis of HDV infected patients.

Quantitative detection of hepatitis B virus DNA in sera from patients with acute hepatitis B

Two hundred forty-four serial serum samples from 30 adults hospitalized with benign (nonfulminant) acute hepatitis B were tested for the presence of hepatitis B virus (HBV) DNA by a quantitative solution hybridization assay using a125I-labeled DNA probe complementary to HBV-DNA sequences. Acute hepatitis B was self-limiting in 28 and progressed to chronicity in the remaining two patients. Of the 28 patients with self-limiting hepatitis, 21 (75%) were hepatitis B e antigen (HBeAg) positive, 26 (93%) were HBV-DNA positive, and one patient (3.6%) was negative for both markers on admission to the hospital. HBV-DNA cleared after HBeAg clearance in 20 (71.4%), before HBeAg clearance in five (17.9%) and simultaneously with the loss of HBeAg in the remaining two (7.1%) of the 27 initially HBV-DNA- and/or HBeAg-positive patients. Moreover, HBV-DNA remained detectable in serum for 13.3±6.6 (range: 4–22) days after the appearance of anti-HBe in 71.4% of these patients. In contrast, HBV-DNA and HBeAg remained persistently positive in the two patients who developed chronic HBV infection. These data show that: (1) viremia frequently persists after disappearance of HBeAg and (2) appearance of anti-HBe does not indicate the cessation of HBV replication in adults with acute self-limiting hepatitis B.

Hepatitis B Virus Serum DNA and RNA Levels in Nucleos(t)ide Analog‐Treated or Untreated Patients During Chronic and Acute Infection

Treatment of chronic hepatitis B (CHB) patients with nucleos(t)ide analogs (NAs) suppresses hepatitis B virus (HBV) DNA synthesis but does not affect synthesis of HBV pregenomic RNA (pgRNA). Hepatitis B virus pgRNA is detectable in the serum during NA treatment and has been proposed as a marker of HBV covalently closed circular DNA activity within the infected hepatocyte. We developed an automated assay for the quantification of serum HBV pgRNA using a dual‐target real‐time quantitative PCR approach on the Abbott m2000sp/rt system. We demonstrate accurate detection and quantification of serum HBV RNA. Hepatitis B virus DNA was quantified using the Abbott RealTime HBV viral load assay. We further compared serum nucleic acid levels and kinetics in HBV‐positive populations. Samples included on‐therapy CHB samples (n = 16), samples (n = 89) from 10 treatment naïve CHB subjects receiving 12 weeks of NA treatment with 8‐week follow‐up, hepatitis B surface antigen–positive blood donor samples (n = 102), and three seroconversion series from plasmapheresis donors (n = 79 samples). Conclusion: During NA treatment of CHB subjects, we observed low correlation of HBV DNA to pgRNA levels; pgRNA concentration was generally higher than HBV DNA concentrations. In contrast, when NA treatment was absent we observed serum pgRNA at concentrations that correlated to HBV DNA and were approximately 2 log lower than HBV DNA. Importantly, we observe this trend in untreated subject samples from both chronic infections and throughout seroconversion during acute infection. Results demonstrate that the presence of pgRNA in serum is part of the HBV lifecycle; constant relative detection of pgRNA and HBV DNA in the serum is suggestive of a linked mechanism for egress for HBV DNA or pgRNA containing virions.