Hepatitis: Avellón A

Echevarría JM, Avellón A. · Unidad de Hepatitis Víricas, Servicio de Microbiología Diagnóstica, Centro Nacional de Microbiología, Instituto de Salud Carlos III, Majadahonda, Madrid, España. · Enferm Infecc Microbiol Clin. · Pubmed #19195449 links to  free full text

Abstract: Viral hepatitides are satisfactorily diagnosed in the laboratory by immunoassays for either antigen or antibody detection in serum samples. However, the early detection of acute infections during the window period, investigation of occult infections, and issues related to the establishment and follow-up of antiviral therapy in chronic infections pose new challenges that only molecular methods can meet. In addition, full characterization of epidemic outbreaks and surveillance of the emergence of viral variants able to escape from vaccine protection are major public health objectives that can only be achieved through the use of these techniques. As a further attempt to improve the viral safety of blood transfusions, the incorporation of molecular biology techniques into the routine work of transfusion centers has generated new technical and scientific demands on microbiology laboratories, which must in turn incorporate these methods to respond to the challenge. After more than a decade, automatic methods for the detection, quantification, characterization and sequencing of the genomes of these viruses have become a reality for the clinical laboratory, reaffirming the essential role of the microbiologist in the hospital setting.

Fogeda M, de Ory F, Avellón A, Echevarría JM. · Service of Diagnostic Microbiology, National Centre of Microbiology, Instituto de Salud Carlos III, Madrid, Spain. · J Clin Virol. · Pubmed #19505848 No free full text.

Abstract: BACKGROUND: The accuracy of the diagnosis of hepatitis E in the clinical setting relies mainly on the performance of assays for hepatitis E virus (HEV)-specific IgM (anti-HEV IgM) testing in serum. OBJECTIVES: Identification of factors influencing the specificity of the results obtained with these assays is an important issue in regard to the accuracy of the diagnosis. STUDY DESIGN: Anti-HEV IgM and HEV RNA were studied in samples from 153 patients with acute hepatitis of unknown aetiology received during a two-year period. Fifteen patients were positive for anti-HEV IgM, and eight of them were also positive for HEV RNA. Investigation of CMV and Epstein-Barr virus (EBV) infection markers among the remaining seven patients, and of HEV infection markers among 18 patients with infectious mononucleosis, was performed. RESULTS: The results obtained showed that acute infection by CMV or EBV may cause false reactivity for anti-HEV IgM, likely because of polyclonal B-cell stimulation. CONCLUSIONS: Since infection by these herpesviruses may produce acute hepatitis, such event can cause diagnostic mistakes and should be investigated in patients positive for anti-HEV IgM and negative for HEV RNA.

Echevarría JM, Avellón A. · Service of Diagnostic Microbiology, National Centre for Microbiology, Instituto de Salud Carlos III. Majadahonda, Madrid, Spain. · J Med Virol. · Pubmed #18297712 No free full text.

Abstract: The sensitivity of immunoassays for hepatitis B virus (HBV) surface antigen (HBsAg) detection may be hampered by the presence of mutants involving the major antigenic determinant of the protein. The performance of the VITROS HBsAg Assay has been shown to be affected by mutations comprising amino acid changes at residues 143, 144, and 145 of the HBsAg molecule. Sixty-seven serum samples from HBV carriers containing major populations of natural HBsAg mutants assayed previously by that assay were tested by the new VITROS HBsAg ES Assay. Samples displayed either single or multiple amino acid substitutions between positions 112 and 145 of the HBsAg, including changes in relevant residues such as 118-120, 125-127, and 143-145. Testing of undiluted samples by the current assay gave rise to false negative results in two samples displaying the single substitutions 145A and 145R, and in one additional sample displaying a dual mutation 118A + 145A. Unusually weak reactivity (<25 S/CO units) was, in addition, recorded in samples containing mutants 143L (2 samples) and 115N + 120Q + 131K + 144A (1 sample). Testing samples at the 1/40 dilution by the modified assay did not produce, in contrast, false negative results, and reactivity below 25 S/CO units was recorded only in three cases. These results confirm that the capability of immunoassays to detect the presence of natural HBsAg mutants in clinical samples may be improved significantly by introducing changes in their design, and show that such improvement has been achieved successfully with the new VITROS HBsAg ES Assay.

González R, Echevarria JM, Avellón A, Barea L, Castro E. · Spanish Red Cross Blood Transfusion Center, Madrid, Spain. · Transfusion. · Pubmed #16836560 No free full text.

Abstract: BACKGROUND: Mathematical models predict that, in Spain, a significant number of blood units will be obtained during the window period of the hepatitis B virus (HBV) infection. Routine nucleic acid testing (NAT) on individual blood units may provide experimental data to evaluate such a theoretical risk. STUDY DESIGN AND METHODS: Between February and July 2005, a total of 34,631 individual units were screened for HBV DNA by a multiplex transcription-mediated amplification (TMA) test. Units that repeatedly reacted in the test, but did not react for HBV surface antigen (HBsAg), were submitted to additional testing by both molecular and conventional assays, and the donors were recalled for follow-up studies and the collection of clinical and epidemiologic data. RESULTS: Confirmatory testing and follow-up studies identified 2 blood units donated during the HBV infection window period (1/17,316 units studied). Sequencing of amplification products obtained by nested polymerase chain reaction (n-PCR) revealed two HBV strains from genotypes D/ayw3 and F/adw4q-, but did not identify HBsAg mutants. The HBV DNA concentration in the index donations was estimated to be below the n-PCR detection level (180 IU/mL), in both cases. One donor developed acute hepatitis 2 months after donating blood, but the other remained asymptomatic and displayed normal alanine aminotransferase levels at follow-up. CONCLUSIONS: The HBV infection window period is a real issue in the setting of Spanish blood transfusions. NAT of individual units by TMA would make a significant contribution to improving the safety of the blood supply in Spain. Additional studies involving a larger number of units and longer periods of time are required, however, to ascertain the true incidence of the problem in this country.

Echevarría JM, Avellón A. · Service of Diagnostic Microbiology, National Centre of Microbiology, Instituto de Salud Carlos III, Madrid, Spain. · J Med Virol. · Pubmed #16622876 No free full text.

Abstract: Hepatitis B virus (HBV) is a human DNA virus, which replicates through an RNA intermediate because of the reverse-transcriptase (RT) activity of its DNA polymerase. As a result, the mutation rate for HBV is higher than the rate observed for most DNA viruses. HBVs are classified into genotypes based on genomic sequencing, and antigenic subtypes based on the antigenic properties of its major surface glycoprotein, the HBV surface antigen (HBsAg). Subgenotypes have been identified within most of the HBV genotypes. The HBV groups defined by the different genotype-HBsAg subtype associations found over the world display characteristic geographical distributions, reflecting the movements of human populations and other epidemiologically significant events. Such HBV groups constitute genetically stable viral populations sharing a common evolutionary history, but additional stable changes, originating from mutation and mutant selection, are observed within all of them. These viral sub-populations are known as the HBV variants, and some of which have medical and public health relevance. Pre-core (pre-C) defective variants have been shown to make HBV infection much less susceptible to interferon treatment, and treatment failures with other antiviral drugs have been associated with selection of resistant variants that display specific mutations in the genome region encoding the viral RT activity. Since the RT region of the genome overlaps the sequence encoding the HBsAg molecule, selection of drug resistant variants involves, in some cases, the indirect selection of HBsAg variants. Viral variants displaying changes in HBsAg seem to be very common among chronic HBV carriers; and some of these variants may emerge under the pressure of the neutralizing antibody response, leading to vaccine resistance and resistance to immunotherapy. Mutations conferring resistance to immunotherapy are noted often among liver transplant recipients and among babies born to HBV-carrier mothers. In addition, some of these HBsAg variants have been associated with lack of detection by HBsAg tests used for the diagnosis of HBV infection, for the identification of chronic carriers, for screening of blood donations for transfusion, and in the manufacture of therapeutic blood products.

Ortiz de Lejarazu R, Avellón A, Eiros JM. · Hospital Clínico Universitario, Facultad de Medicina de Valladolid, Centro Nacional de Microbiología, Majadahonda, Madrid, Spain. · Enferm Infecc Microbiol Clin. · Pubmed #16606561 links to  free full text

Abstract: Hepatitis of viral aetiology caused by hepatotropic virus (A, E, B, D and C) represents an important work load for the clinical virology laboratory. Most of the diagnostic is based upon detection in serum and plasma samples of different serological and virological markers, which correlates with different infection stages. In chronic infection by HBV and HCV is necessary to perform diagnostic by molecular methods as well as antigen detection in sequential samples along the course of the disease taking into account that a reliable storage must be provided for stability of structural components of the virus. Recent knowledge about mutations variants in some of the virus may alter the validity of particular markers.

Echevarría JM, León P, Pozo F, Avellón A. · Servicio de Microbiología Diagnóstica, Centro Nacional de Microbiología, Instituto de Salud Carlos III, Majadahonda, Madrid, Spain. · Enferm Infecc Microbiol Clin. · Pubmed #16537058 links to  free full text

Abstract: BACKGROUND: Recent data suggest that the prevalence of genotype 4 HCV strains among Spanish carriers is increasing. OBJECTIVE: To assess changes in the prevalence of HCV genotypes in Spain during the last nine years. METHODS: HCV RNA was amplified by the polymerase chain reaction from 3161 serum samples from unselected, anti-HCV-positive individuals, and the HCV genotype was identified by a reverse hybridisation assay (line probe assay, LiPA). Samples came from 17 different regions of Spain and were obtained between January, 1996 and December, 2004. RESULTS: The overall prevalence of HCV genotypes was: 1b, 41.3%; 1a, 24.1%; 3, 19.6%; 4, 11.6%; 2, 3.1%; and 5, 0.3%. The prevalence of genotypes 1a, 3 and 4 increased significantly among patients born after 1950, and that of genotype 1b decreased among them. These significant differences in regard to age were also observed among patients lacking notified high-risk factors. A main switch-up in prevalence of genotypes 1a and 3 was found when patients born in 1941-1950 were compared with those born in 1951-1960, but the same finding in genotype 4 was delayed to patients born in 1961-1970. CONCLUSIONS: Two separate epidemics of HCV seem to have occurred in Spain during the last 30 years. The former one involved the spread of HCV genotypes 1a and 3. The second was more recent, and involved the spread of genotype 4.

Echevarría JM, Avellón A, Jonas G, Hausmann M, Vockel A, Kapprell HP. · Service of Diagnostic Microbiology, National Centre of Microbiology, Instituto de Salud Carlos III, Majadahonda, 28220 Madrid, Spain. · J Clin Virol. · Pubmed #16406797 No free full text.

Abstract: BACKGROUND AND OBJECTIVES: Compliance with current regulations regarding the prevention of hepatitis C virus (HCV) transmission in the blood transfusion setting requires the use of sensitive assays for HCV antibody (anti-HCV) detection, which should, ideally, identify any donor having had prior contact with the virus. Therefore, low-level anti-HCV positive blood units should be detected by the screening assays, even those reflecting a past and resolved infection. To assess the sensitivity of two versions of an automated chemiluminescent microparticle immunoassay (CMIA) for anti-HCV screening (ARCHITECT Anti-HCV), 113 single serum samples containing low levels of anti-HCV, assessed by two immunoblot tests, were selected from 3686 samples received for confirmation of HCV infection by a reference laboratory over a 2-year period. MATERIALS AND METHODS: The panel included 17 samples with HCV RNA detected by the polymerase chain reaction (PCR) and 96 PCR negative samples with either positive or indeterminate (anti-Core and anti-NS3 alone) results by immunoblot. RESULTS: All but 13 specimens (100/113, 88.5%) were detected by the current version of the ARCHITECT Anti-HCV assay and 10 additional samples (110/113, 97.3%) tested positive in a modified version of the test. CONCLUSION: The results showed that the modification introduced in the ARCHITECT Anti-HCV assay achieves a significant sensitivity improvement including samples with low-level anti-HCV which are either PCR positive or negative.

Avellón A, Echevarria JM. · Hepatitis Laboratory, Diagnostic Microbiology Service, National Centre of Microbiology, Instituto de Salud Carlos III, Majadahonda, Madrid, Spain. · J Med Virol. · Pubmed #16299725 No free full text.

Abstract: The prevalence in the population of hepatitis B virus (HBV) surface antigen (HBsAg) variants that may impair diagnosis, or allow the virus to escape vaccine-induced immunity or passive immunoglobulin therapy is unknown. A genome fragment encoding HBsAg amino acids 112-212 was amplified and sequenced from the sera of 272 unselected DNA-positive, HBV-chronic carriers from Spain. The genotype and the HBsAg subtype were predicted from the sequences. Analysis of amino-acid positions 112-157 revealed single or multiple substitutions in 39% of the carriers studied. Mutations were not detected for residues 121, 135, 137, 139, 140, 141, 142, 146, 147, 148, 149, 151, 152, 153, 155, 156, and 157. Substitutions reported previously to be in association with failures of diagnostic tests or with vaccine or immunoglobulin therapy escape were found in 12.5%, 6.6%, and 9.2% of carriers, respectively. Met133Thr (2.2%); Gln129His, Met133Ile, Phe/Tyr134Asn (1.8%); Phe/Tyr134Leu, Gly145Ala (1.5%), and Pro120Thr (1.1%) were the most frequent. Other substitutions, including Gly145Arg (0.4%), were found at a frequency of less than 1%. Samples containing HBV mutants were tested with three commercial assays for HBsAg screening. Almost all the mutants reacted to the upper cut-off values of the assays, but six samples with weak reactivity with one or more of the methods were also found. Thus, HBV mutants with a potential impact on clinical and public health issues are moderately frequent among chronic carriers from Spain, although their influence on the performance of diagnostic tests seems to be slight.

Echevarría JM, Avellón A, Magnius LO. · Service of Diagnostic Microbiology, National Centre of Microbiology, Instituto de Salud Carlos III, Madrid, Spain. · J Med Virol. · Pubmed #15834869 No free full text.

Abstract: The hepatitis B virus (HBV) genotypes were studied by a line probe assay (LiPA) and by direct sequencing of a 339 nucleotide fragment from the S region of the viral genome in samples from 269 carriers living in Spain, either native to Spain (231) or immigrants from Africa, Asia, and Eastern Europe (38). The sequences were also used to predict the HBV surface antigen (HBsAg) subtype on the basis of the amino acids specified at selected positions of the HBsAg molecule. Agreement between the two genotyping methods was found in most cases (98.1%) and a HBV genotype could be assigned to all samples. The viral groups D/ayw2 (30.1%), D/ayw3 (28.6%), and A/adw2 (21.2%) were prevalent, with an additional participation of the groups D/ayw4 (4.8%), F/adw4q- (1.9%), A/ayw1 (1.9%), and D/adw3 (0.7%), all of them present among the autochthonous carriers. Strains from genotypes B and C were found exclusively among Chinese immigrants. Genotype E strains were found in immigrants from Central Africa and in one patient native of Spain. Point mutations leading to amino acid changes of residues involved in the expression of the HBsAg subtype determinants were found in 12 samples (4.5%). Some mutations would predict the putative novel genotype-subtype associations A/adw4q+, A/ayr, D/ayr, and E/ayw1, while others would suggest the loss of subtype-specific determinants. The finding of HBV strains characteristic for Africa among the autochthonous carriers confirms the emergence of African HBV strains in Spain.