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International audience; The high genetic variability of hepatitis B virus (HBV) can impair DNA quantification. Here, we investigate a major underquantification of HBV by the cobas TaqMan HBV assay (CTM; Roche). In France, between 2005 and 2017, HBV DNA was detected in 3,102 blood donations by use of the CTM (95% limit of detection [LOD], 4.8 IU/ml). HBV strains were sequenced in the S region (LOD, ∼30 IU/ml). Concordant (n = 120) and discordant (n = 45) samples were identified according to the agreement between the plasma viral load (pVL) determined by the CTM and sequencing; all samples were also quantified using the RealTime HBV assay (RTH; Abbott). The viral signature, cloning, and mutagenesis were used to characterize the polymorphism responsible for CTM misquantification. A CTM-RTH discordance (>1 log IU/ml) was found in 14/45 samples that had low pVLs and were successfully genotyped (pVL genoS). PreC/C clones of concordant (C1, C2) and discordant (D1, D2) strains were used to challenge the CTM. Strains D1 and D2 were highly underquantified (42- and 368-fold). In clones, mutating the region corresponding to the CTM reverse primer from a discordant sequence to a concordant sequence restored the levels of quantification to 24% (D1→C1) and 59% (D2→C1) of theoretical levels, while mutating the sequence of a concordant strain to that of a discordant strain led to 78-fold (C1→D1) and 146-fold (C1→D2) decreases in quantification. Moreover, mutating positions 1961 and 1962 was enough to induce a 5-fold underquantification. We conclude that the CTM underestimates pVLs for HBV strains with mutations in the reverse primer target. Specifically, the polymorphism at nucleotides 1961 and 1962 is naturally present in 4.79 and 4.22% of genotype A and D strains, which are highly frequent in Europe, leading to a 5-fold decrease in quantification. Quantification using the new-generation Roche C4800 assay is not affected by this polymorphism.

Screening of blood for human T-cell lymphotropic virus type 1 and type 2 (HTLV-1 and -2, respectively) is important to diagnose and prevent infection and ensure the safety of blood supplies. The Elecsys HTLV-I/II assay is a newly developed, electrochemiluminescence screening assay for the detection of HTLV-1/2 infection. The sensitivity and specificity of the Elecsys HTLV-I/II assay were determined using well-characterized HTLV-1/2-positive serum and plasma samples and routine diagnostic and blood donor samples expected to be HTLV negative, respectively. These results were compared with those for at least one of the following CE-marked assays at seven independent laboratories and the Roche Diagnostics facility in Penzberg, Germany: Abbott Architect rHTLV-I/II, Ortho Avioq HTLV-I/II Microelisa system, Abbott Prism HTLV-I/HTLV-II, and DiaSorin Murex HTLV I+II. Fujirebio INNO-LIA HTLV-I/II Score was used as a confirmatory assay. The Elecsys HTLV-I/II, Abbott Architect rHTLV-I/II, and Abbott Prism HTLV-I/HTLV-II assays detected all HTLV-1/2-positive samples (sensitivity, 100%). Sensitivity for Ortho Avioq HTLV-I/II was 98.63%. The Elecsys HTLV-I/II assay had a specificity of 99.95% in blood donor samples, which was comparable to results for the other assays (range, 99.91 to 100%). In routine diagnostic samples, the specificity of the Elecsys HTLV-I/II assay was 99.83%, compared with 99.70% for Abbott Architect rHTLV-I/II. Specificity for the Elecsys HTLV-I/II assay in potentially cross-reactive samples was 100%, compared with 99.0% for Ortho Avioq HTLV-I/II and 99.2% for DiaSorin Murex HTLV I+II. The Elecsys HTLV-I/II assay has the sensitivity and specificity to support its use as a routine screening assay for detecting HTLV infection.

A French national quality control study for the serological and molecular diagnosis of hepatitis delta virus (HDV) was organized. Total HDV antibodies were properly detected by all laboratories; 8/14 laboratories failed to detect low titers of IgM, and 6/11 failed to quantify and/or underestimated the RNA viral load in several samples. These discrepancies are likely related to the molecular diversity of HDV.

There is no reliable and simple diagnostic marker available to diagnose recent hepatitis C virus (HCV) infection. It has been shown that the avidity of specific IgG antibody is low in primary viral infection and increases with time. We report the development of an anti-HCV avidity assay derived from a commercially available test. A panel of 117 sera was first examined for IgG avidity. It was composed of samples from patients with recent (group 1, n = 14), chronic (group 2, n = 70), and resolved (group 3, n = 33) HCV infections. Avidity index (AI) values observed in recently infected patients were significantly lower (12.0% ± 9.2% [mean ± standard deviation]) than those found in chronic carriers (83.1% ± 15.2%). Using a threshold of 43.0%, this assay distinguished between groups 1 and 2 with very high sensitivity (98%) and specificity (100%). For group 3, a broader distribution of the AI values was observed (54.8% ± 27.3%), suggesting that this index would not be useful in HCV RNA-negative patients. Blind validation of the test was carried out with a panel of 36 serum samples from 17 HCV seroconverters. The assay described here is a useful tool to distinguish recent from chronic infection in HCV-viremic patients.

The development of rapid tests (RTs) for hepatitis B surface antigen (HBsAg) detection is expanding, especially for point-of-care (POC) testing. As mutations in the a determinant of HBsAg have been associated with failures to detect HBsAg by using some traditional HBsAg assays, this study aimed to

The 5′ noncoding region (5′ NCR) of the hepatitis C virus (HCV) has become the standard for genotyping even though several reports show that its use can result in classification errors. The purpose of this study was to perform genotyping based on sequence analysis of the NS5b region in a set of 357 HCV strains isolated from blood donors in France in 2002 and 2003. Results were compared with those previously obtained using 5′ NCR analysis, and HCV subtype distribution was reevaluated. Twenty-six of 120 strains (∼22%) initially identified as genotype 1b by 5′ NCR region sequence analysis were reclassified as genotype 1a by NS5b region sequence analysis. Similarly, 14 of 23 strains (∼61%) initially identified as 2a/2c were reclassified as non-2a and non-2c subtypes, and 12 of 22 strains (∼45%) initially identified as 4c/4d subtypes were reclassified as non-4c and non-4d subtypes. Sequence analysis of the NS5b region also revealed 5 putative new subtype 2 variants and 2 putative new subtype 4 variants. Although these findings demonstrated full agreement between 5′ NCR and NS5b sequence analysis with regard to type classification, genotyping based on phylogenetic analysis of the NS5b region is more accurate for subtype determination than genotyping based on analysis of the 5′ NCR. Sequence analysis of the NS5b region is mandatory for epidemiologic studies.

We report a case of human immunodeficiency virus type 1 group O infection not detected by two highly sensitive immunoassays. The sera were strongly reactive to the V3 peptide of group O but not to the gp41 immunodominant epitope. Gp41 was sequenced, confirming that this virus belonged to group O.

B19 virus is a human virus belonging to the genus Erythrovirus . The genetic diversity among B19 virus isolates has been reported to be very low, with less than 2% nucleotide divergence in the whole genome sequence. We have previously reported the isolation of a human erythrovirus isolate, termed V9, whose sequence was markedly distinct (>11% nucleotide divergence) from that of B19 virus. To date, the V9 isolate remains the unique representative of a new variant in the genus Erythrovirus , and its taxonomic position is unclear. We report here the isolation of 11 V9-related viruses. A prospective study conducted in France between 1999 and 2001 indicates that V9-related viruses actually circulate at a significant frequency (11.4%) along with B19 viruses. Analysis of the nearly full-length genome sequence of one V9-related isolate (D91.1) indicates that the D91.1 sequence clusters together with but is notably distant from the V9 sequence (5.3% divergence) and is distantly related to B19 virus sequences (13.8 to 14.2% divergence). Additional phylogenetic analysis of partial sequences from the V9-related isolates combined with erythrovirus sequences available in GenBank indicates that the erythrovirus group is more diverse than thought previously and can be divided into three well-individualized genotypes, with B19 viruses corresponding to genotype 1 and V9-related viruses being distributed into genotypes 2 and 3.

This study, involving 20 laboratories and using currently available assays for hepatitis C virus RNA quantification, demonstrated that differences in viral load values are due not to interlaboratory variations but rather to the nature of the assay itself. This underlines the importance of using the same assay in multicenter studies or when monitoring antiviral therapy.

Droplet digital PCR (ddPCR), which has been recently developed to provide an absolute quantitation of target molecules without relying on the use of standard curves ([1][1]), might be an interesting alternative to conventional real-time PCR assays used for viral load (VL) determination ([2][2], [3][