Sporadic Hepatitis A Virus PCR False-Positive Results Observed during Reflex Testing of Serum Samples Previously Tested for Anti-HAV Antibodies and Caused by Contamination with HAV RNA Present in the Reagents of the Commercial Anti-HAV Immunoassay.

Hepatitis A diagnosis relies on serology and occasionally on hepatitis A virus (HAV) RNA detection. For timely diagnosis and the avoidance of drawing additional blood, molecular testing is often performed as reflex testing by using blood specimens that were initially sent for anti-HAV serology. Reflex molecular testing is preferably performed from different sample aliquots, but, for limited sample quantities, it uses samples that have been preprocessed in an immunoassay analyzer. In 2012, we first observed sporadic HAV RNA-positive cases that were inconsistent with patients' serological profiles and/or medical histories, suggesting that occasional laboratory contamination was causing false-positive PCR results. Multiple external quality assurance (EQA) and laboratory surface contamination checks were performed, questionable specimens were tested with various HAV RNA tests, and follow-up serum/stool samples were collected. All contamination-check samples and samples from healthy individuals tested HAV RNA-negative, and the laboratory successfully passed all EQAs. The HAV RNA-positive results were reproducible with various HAV RNA assays. No patients seroconverted, and their follow-up samples were consistently HAV RNA-negative. Finally, a detailed review of testing protocols revealed a correlation between HAV RNA false positivity and preceding anti-HAV testing with the Cobas-e411 automated immunoassay analyzer. HAV RNA was detected in the Cobas-e411 anti-HAV reagents, with the HAV sequences matching those from the false-positive samples. Preceding anti-HAV testing using two other immunoassay analyzers did not result in subsequent HAV RNA false positivity during reflex testing. The Cobas-e411 pipetting procedure with a single pipette tip collecting samples and anti-HAV reagents contaminated the original sample with the HAV RNA that was present in the immunoassay's reagents, thereby resulting in HAV RNA false positivity during the reflex testing. IMPORTANCE We present the first report of sporadic HAV PCR false-positive results that have been observed during the reflex testing of serum samples that have previously been tested for anti-HAV antibodies and have been caused by contamination with HAV RNA that is present in the reagents of the commercial anti-HAV immunoassay, with potentially serious clinical consequences. Although HAV RNA was consistently detected in the anti-HAV reagents of all three automated immunoassay analyzers that were in use in our laboratory, only the use of one analyzer and the corresponding commercial anti-HAV immunoassay reagents resulted in contamination that led to false positive HAV RNA results, and this was due to a peculiar pipetting mode of action in which the analyzer uses a single pipette tip to collect both anti-HAV reagents and a sample, which consequently causes the permanent contamination of the original sample with HAV RNA. Manufacturers should strongly consider the occasional need for reflex molecular testing from preprocessed samples and design their analyzers in a way that prevents contamination.

Hepatitis D virus infection in Slovenian patients with chronic hepatitis B virus infection: a national prevalence study and literature review.

INTRODUCTION: Of the 350 million individuals chronically infected with hepatitis B virus (HBV) worldwide, approximately 15 to 20 million have been exposed to hepatitis D virus (HDV). This study determined for the first time the HDV prevalence in Slovenian patients with chronic HBV infection. In addition, a literature search was performed to identify all HDV prevalence studies from European countries. METHODS: A total of 1,305 HBsAg-positive serum samples, obtained from the same number of patients, were randomly selected from 2,337 patients referred to the Slovenian national reference laboratory for viral hepatitis between 1998 and 2015. All samples were retrospectively tested for the presence of total anti-HDV antibodies. Anti-HDV-positive patients were additionally tested for the presence of anti-HDV IgM antibodies, HDV antigen, and HDV RNA. RESULTS: Total anti-HDV antibodies were detected in three of the 1,305 patients tested (0.23%; 95% CI: 0.08-0.67%), of whom one patient had recovered from the past HDV infection and two patients had an ongoing chronic HDV infection. The literature search identified 36 peer-reviewed HDV prevalence studies published between 1983 and 2016 and originating from 21 European countries. CONCLUSIONS: The observed prevalence of HDV infection in Slovenia was among the lowest reported in Europe and worldwide. Due to the observed low prevalence of HDV infection, routine diagnostic testing for HDV should not be considered in differential diagnosis of exacerbation of liver disease in Slovenian patients with chronic HBV infection.