Multicenter clinical evaluation of the new 3rd generation assay for detection of antibodies against hepatitis C virus on the VIDAS(®) system.

BACKGROUND: Detection of antibodies (anti-HCV) against hepatitis C virus (HCV) is indispensable for screening and diagnosis of viral hepatitis and for the viral safety of blood, tissue or organ donations. It gains additional importance by the new HCV drugs which improve the therapeutic possibilities dramatically. OBJECTIVE: To evaluate the performance of a newly developed immune assay for anti-HCV based on the well-established VIDAS platform. STUDY DESIGN: The assay was evaluated with samples from anti-HCV negative blood donors and from patients with or without HCV markers in six centres in France, Spain and Egypt. The status of the samples was determined by using CE-marked immune assays (Architect, AxSym, Prism, Vitros), two immunoblots (RIBA, Inno-Lia) and/or HCV RNA results. RESULTS: Specificity was 99.67% in 10,320 French blood donors without anti-HCV, 99.5% in 200 anti-HCV negative hospitalized European patients and 99.0% in 198 negative patients from Egypt. Sensitivity was 99.7% in 1054 patients pretested positive by other assays; 345 patients with known genotype had genotype 1-6; 61 patients were co-infected with HIV. VIDAS was reactive in 78% of 91 patients with uncertain or very weak anti-HCV. It became on average positive at day 37 with seroconversion panels. CONCLUSIONS: This multicentric, international study with >12,000 samples show that the new VIDAS anti-HCV assay is very suitable for screening and confirmation of HCV infection. Sensitivity, specificity and recognition of seroconversion compare favorably with well-established CE-marked tests and help to clarify discrepant results obtained with other assays.

Full coding hepatitis E virus genotype 3 genome amplification method.

Hepatitis E virus (HEV) genotype 3 produces zoonotic infection associated with the consumption of infected animals. HEV infections can become chronic in immunocompromised (IC) patients. The viral genome has three well defined open reading frames (ORF1, ORF2 and ORF3) within which various domains and functions have been described. This paper (i) describes a new method of complete sequencing of the HEV coding region through overlapping PCR systems, (ii) establishes a consensus sequence and polymorphic positions (PP) for each domain, and (iii) analyzes the complete coding sequence of an IC patient. With regard to the consensus, a high percentage of PP was observed in protease (PP=19%) and the X domain (PP=22%) within ORF1, the N-terminal region of the S domain (PP=22%) in ORF2, and the P1 (PP=35%) and P2 (PP=25%) domains in ORF3. In contrast, the ORF1 Y, ORF2 S, ORF2 M and ORF3 D1 domains were conserved in the reference sequences (0.40, 1, 0.70 and 0% of PP, respectively). The sequence from the IC patient had more mutations in the RpRp (D1235G, Q1242R, S1454T, V1480I, I1502 V, K1511R, G1373 V, E1442D, V1693 M), the terminal ORF2 S- domain (F10L, S26T, G36S, S70P, A105 V, I113 V), the X domain (T938 M, T856 V, S898A) and the helicase (S1014N, S975T, Q1133 K).

Hepatitis E virus infection in Latin America: a review.

Data reported during recent years reveal the complex picture of the epidemiology of hepatitis E virus (HEV) infection in Latin America. Whereas in countries like Argentina and Brazil is almost identical to the characteristic of most countries from North America and Europe, HEV in the Caribbean and Mexico involves the water-borne, non-zoonotic viral genotypes responsible for epidemics in Asia and Africa. Nevertheless, Latin America has been considered a highly endemic region for hepatitis E in the scientific literature, a generalization that ignores the above complexity. In addition, reports from isolated Amerindian communities, which display well known, important and very specific epidemiological features for hepatitis B and D virus infections are neither taken into account when considering the epidemiology of hepatitis E in the region. This review updates compilation of the available information for the HEV infection, both among humans and other mammals, in Latin America, discusses the strengths and the weaknesses of our current knowledge, and identifies future areas of research.

Prevalence of specific antibody to hepatitis E virus in the general population of the community of Madrid, Spain.

Hepatitis E virus (HEV) is an infectious agent causing hepatitis among humans. Although hepatitis E has been reported from many European countries, its incidence in Europe is largely unknown, and the prevalence of the HEV infection is also unknown for most countries of the region. Antibody to HEV (anti-HEV) was tested on 2,305 serum samples from the general population of the Community of Madrid (Spain) collected in the year 2008 among people aged 2-60 years. Total anti-HEV was tested by enzyme-immunoassay (EIA), and reactive samples were retested separately for anti-HEV IgG and IgM by recombinant immunoblot test (RIBT). Fifty samples (2.17%) displayed reactivity for total anti-HEV after EIA testing, and anti-HEV IgG was confirmed by RIBT in 25 (1.08%). The frequency of RIBT-confirmed anti-HEV ranged from 0.97% among the youngest to 3.61% among the oldest, and displayed a statistically significant trend to increasing with age. The rate of RIBT confirmation was also significantly higher among the individuals aged above 20 years old than among those younger of 21 years. HEV infection would be less frequent in the Community of Madrid than in Catalonia or the United Kingdom, and contact with HEV would be very uncommon among children and adolescents of the region. Confirmation of EIA-reactive samples by RIBT reduced the final numbers of anti-HEV testing as much as 50%, and some findings of this study suggest that such testing protocol would reflect better the real prevalence of anti-HEV in settings of low endemicity than the single testing by EIA.

Diagnosis of acute hepatitis E by antibody and molecular testing: a study on 277 suspected cases.

BACKGROUND: Acute hepatitis due to hepatitis E virus (HEV) infection is both indigenous and imported to Europe. Few studies provide information about the role of HEV as an agent for acute hepatitis in Spain. OBJECTIVES: To investigate the frequency of the HEV infection among patients displaying acute hepatitis of unexplained origin in Spain, comparing the performance of two different diagnostic approaches. STUDY DESIGN: Specific IgM antibody and HEV RNA tests were used to study samples from 277 patients with acute hepatitis of unknown aetiology received during a six-year period. Samples were sent by 52 hospitals from almost all regions of Spain. RESULTS: Evidence of acute infection by HEV was obtained for 30 patients in total (10.8%), and 16 cases were unrelated to recent international travel. On samples from 158 patients tested for both anti-HEV IgM and HEV RNA at admission, the yield of IgM antibody testing (11.4%) was higher than the yield of HEV RNA testing (9.5%). CONCLUSIONS: HEV could be responsible in Spain of about 11% of cases of acute hepatitis of unknown origin overall, and of about 8% of cases unrelated to international travel or immigration. India and neighbour countries represent the highest risk for import of epidemic HEV strains into Spain. Both antibody assays and molecular tests are required to optimise the final yield of laboratory diagnosis.

Imported and autochthonous hepatitis E virus strains in Spain.

Hepatitis E virus (HEV) causes hepatitis E, an acute liver disease displaying diverse epidemiological patterns that correlate with the genetic diversity of the virus. Only a few strains have been characterized to date from cases of hepatitis E in Spain. Using three sets of new, HEV-specific primers, viral genome fragments were amplified from serum samples from 13 patients with acute hepatitis in different regions of Spain. Direct sequencing of these fragments and analysis of sequences lead to identify six genotype 1, six genotype 3, and one genotype 4 viral strains. Genotype 1 sequences were found in the clade with subtype 1a strains, and were amplified from travelers from India and Bangladesh, and from an African immigrant. Genotype 3 sequences were found in the clade with subtype 3f strains, were always amplified from patients who did not travel abroad recently, and were closely related to sequences from swine strains isolated in Spain. Patients infected by these strains lived in different regions and were mainly men aged above 50 years. The single genotype 4 sequence detected was amplified from a traveler returning from Vietnam. Hepatitis E is both an imported and an autochthonous disease in Spain, and closely related HEV genotype 3f strains are responsible for infections acquired locally in different regions of the country within a given time. Studies involving a significant number of human, swine, and environmental viral strains collected prospectively are, however, required in order to confirm a swine origin for autochthonous HEV genotype 3 human infections.

Differential diagnosis of hepatitis E virus, cytomegalovirus and Epstein-Barr virus infection in patients with suspected hepatitis E.

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.

Multicenter study of a new fully automated HBsAg screening assay with enhanced sensitivity for the detection of HBV mutants.

In a multicenter study a new, fully automated Roche Diagnostics Elecsys HBsAg II screening assay with improved sensitivity to HBsAg mutant detection was compared to well-established HBsAg tests: AxSYM HBsAg V2 (Abbott), Architect HBsAg (Abbott), Advia Centaur HBsAg (Bayer) Enzygnost HBsAg 5.0 (Dade-Behring), and Vitros Eci HBsAg (Ortho). A total of 16 seroconversion panels, samples of 60 HBsAg native mutants, and 31 HBsAg recombinant mutants, dilution series of NIBSC and PEI standards, 156 HBV positive samples comprising genotypes A to G, 686 preselected HBsAg positive samples from different stages of infection, 3,593 samples from daily routine, and 6,360 unselected blood donations were tested to evaluate the analytical and clinical sensitivity, the detection of mutants, and the specificity of the new assay. Elecsys HBsAg II showed a statistically significant better sensitivity in seroconversion panels to the compared tests. Fifty-seven out of 60 native mutants and all recombinant mutants were found positive. Among 156 HBV samples with different genotypes and 696 preselected HBsAg positive samples Elecsys HBsAg II achieved a sensitivity of 100%. The lower detection limit for NIBSC standard was calculated to be 0.025 IU/ml and for the PEI standards ad and ay it was <0.001 and <0.005 U/ml, respectively. Within 2,724 daily routine specimens and 6.360 unselected blood donations Elecsys HBsAg II showed a specificity of 99.97 and 99.88%, respectively. In conclusion the new Elecsys HBsAg II shows a high sensitivity for the detection of all stages of HBV infection and HBsAg mutants paired together with a high specificity in blood donors, daily routine samples, and potentially interfering sera.

Evaluation of an automated complement-fixation test (Seramat) for diagnosis of acute respiratory infections caused by viruses and atypical bacteria.

The complement-fixation test (CFT) permits low-cost screening of serum samples for different agents within a single assay, and is a useful tool for the serological diagnosis of acute respiratory infections. This study evaluated the automated Seramat CFT system with 160 paired serum samples taken from 80 patients with acute respiratory infection in comparison with in-house CFTs against a panel of agents, including influenza A and B, adenovirus, respiratory syncitial virus, cytomegalovirus, Mycoplasma pneumoniae, Coxiella burnetti and Chlamydia spp., and in comparison with indirect immunofluorescence (IIF) against Legionella pneumophila. Overall, the Seramat system identified 75 (88.2%) of the 85 seroconversions recognised by in-house CFTs or IIF. In comparison to the in-house CFTs, the correlation was 89.2% (66/74). For L. pneumophila, the Seramat system detected nine (81.8%) of the 11 cases diagnosed by IIF. The Seramat system also identified eight additional seroconversions that were not detected by the in-house assays; none of these seroconversions was detected by the in-house assay on retesting. The Seramat system represents a significant technical improvement that may enable many clinical laboratories to use the CFT as a routine diagnostic tool.

Multicenter proficiency testing of nucleic acid amplification methods for the detection of enteroviruses.

A multicenter study of molecular detection of enteroviruses was conducted using a proficiency panel. Of 70 data sets, 46 (66%) reported correct results for samples containing at least 1 50% infective dose per ml and for negative samples. Variation in performance between laboratories demonstrates the need for ongoing quality control.

Detection of specific IgM antibody in the investigation of an outbreak of pneumonia due to Legionella pneumophila serogroup 1.

OBJECTIVE: Management of outbreaks of pneumonia due to Legionella pneumophila serogroup 1 (SG1) infection requires rapid and accurate diagnostic tests. Current serologic approaches, based on detection of seroconversion for total antibody, do not fulfil this requirement. METHODS: A diagnostic test based on detection of IgM antibody to L. pneumophila SG1 by indirect immunofluorescence was developed and used to evaluate serum samples from patients involved in a community outbreak of L. pneumophila SG1 pneumonia that occurred in Spain. RESULTS: Testing of samples from serologically proven, sporadic cases of pneumonia due to L. pneumophila SG1 (14), cases of atypical pneumonia due to other infectious agents (16) and healthy controls (100) supported the sensitivity and specificity of the assay. On samples from the outbreak, the IgM assay recognized five of six cases with isolation of L. pneumophila SG1 from respiratory secretions or lung tissue and more than 70% of cases with confirmed or presumptive diagnosis as determined by the current serologic criteria. In addition, the IgM assay was positive in 23-70% of patients who fulfilled the clinical and epidemiologic criteria of case definition but did not display diagnostically significant serologic results or who lacked a detectable antibody response in the routine assay. Among cases confirmed by the current criteria, detection of specific IgM was occasionally achieved before the conventional serology gave significant results. CONCLUSION: Incorporation of IgM antibody detection in the current diagnostic criteria for L. pneumophila SG1 infection may help to improve the management of outbreaks of pneumonia due to this agent.

[Prevalence of types of hepatitis C virus in Spanish blood donors: results of a state-based multicenter study. Spanish Group for the Study of Blood Donors with Risk of HCV Transmission]. / Prevalencia de tipos del virus de la hepatitis C en donantes de sangre españoles: resultados de un estudio multicéntrico de ámbito estatal. Grupo Español de Estudio de Donantes de Sangre en Riesgo de Transmisión del VHC (GEDSRT-VHC).

BACKGROUND: The prevalences established up to the present in Spain for the different types of hepatitis C virus are based on data obtained in populations in which the nature of the population itself may have based the data in favor of certain types of the virus. The study of seropositive blood donors identified through screening of blood donations may provide prevalences closer to the truth among the general population. MATERIALS AND METHODS: Typing of genomes in samples from 441 donors was performed using the blood bank generated during the multicenter study performed by the Spanish Study Group of Blood Donors with Risk of Transmission of the Hepatitis C Virus. The antibodies present were typed in the seropositive samples in the above donors and in 337 more in whom a viral genoma was not detected. RESULTS: In total, the infection was typed in 685 donors. On analysis of the results corresponding to 386 donors, whose number and distribution by autonomous communities were previously fixed to represent all of Spain, type 1 was largely the more prevalent (85.5%) followed by types 3 (4.4%), 2 (4.1%), 4 (3.4%) and 5 (0.5%) and by a group of apparent mixed infections which altogether represented 2.1% of the total. Among the donors in whom the genomes were typed, infectious due to the 1b subtype (78% of the 441 samples genotypes) clearly predominated. The participation of the different types of type 1 was significantly greater in those lacking antibodies detectable versus epitopes codified in the NS4 region of the viral genome. CONCLUSIONS: This study avoids some bias in sampling which may have affected previous studies and provides data which should more closely approach the real prevalence in the general Spanish population. Thus, it should provide a better base of comparison for any study on the distribution of the types of the hepatitis C virus in selected populations or others performed during tha investigation of outbreaks of hepatitis C virus infection.

[Prevalence of infections by hepatitis B, C, D and E viruses in Bolivia]. / Prevalencia de las infecciones por virus de las hepatitis B, C, D y E en Bolivia.

In Bolivia, no studies have been carried out specifically on hepatitis viruses. Thus, their prevalence and circulation patterns are virtually unknown. A seroepidemiologic study was performed from 1992 to 1996 to generate a preliminary idea of the overall prevalence of infection from hepatitis B, C, D, and E viruses (HBV, HCV, HDV, and HEV, respectively) in different Bolivian population groups. Prompted by the data obtained in other areas of Latin America, the study focused on indigenous communities in the Amazon region. In rural areas of the high Andean plateau, HBV infection showed an overall prevalence compatible with medium to low endemicity (11.2%), and no carriers of HCV or HDV antibodies were found. In two high-risk groups in the city of Cochabamba (homeless children and sexual workers), the prevalence of HBV infection was similar (11.6%) and could be considered low by comparison to that of similar population groups in Latin American urban centers. The prevalence of HCV (one positive case, or 0.5%) was similar to that found in similar population groups, although the small number of samples precludes drawing more definite conclusions. As has been noted previously with similar communities in tropical areas of South America, HBV infection is highly endemic in indigenous populations of the Bolivian Amazon (with an overall prevalence of 74.0%), but circulation of HCV has not been detected. It is a well-known fact that HBV is horizontally transmitted and that transmission can take place very early in life, but the mechanisms involved are unknown. By 10 years of age, more than half the population has already had the natural infection that, in approximately 10 more years will have affected virtually the entire population. The very low rate of positivity to HBsAg (1.6%), the absence of viral DNA in samples showing isolated positivity to anti-HBc, and the high prevalence of anti-HBs among individuals who show markers for natural infection (92.4%) suggest vertical transmission plays no role in persistent endemicity. So far, no outbreak of HDV infection has been documented in these communities, but the high endemicity shown by HBV points to the possibility of future outbreaks. Results obtained with tests for the detection of antibodies against HEV suggest that this virus is circulating widely in Bolivia and that it could have caused recent outbreaks in Cochabamba state. Vaccination against HBV in endemic populations is recommended as a short-term measure. Also recommended are actively searching for outbreaks and sporadic cases of hepatitis E in the entire country and performing additional research that will help in assessing the public health consequences of the situation described in this article.

Viral diagnosis of neurological infection by RT multiplex PCR: a search for entero- and herpesviruses in a prospective study.

The diagnosis of a wide range of different neurological syndromes was established by a reverse transcription multiplex PCR assay. The presence of enterovirus and herpesviruses was studied in cerebrospinal fluid samples collected prospectively from 200 patients hospitalized with neurological diseases suspected of viral infection. Positive PCR results for enterovirus and neurotropic herpesvirus (herpes simplex, HSV, and varicella zoster, VZV) were obtained among the immunocompetent patients (55/156, 35%) who presented aseptic meningitis or encephalitis. Among immunocompromised patients the yield of positive PCR results was 41% (18/44), predominantly lymphotropic herpesviruses (15/44, 34%). Cytomegalovirus (CMV) DNA was detected in patients with several clinical syndromes, including encephalitis, chronic meningitis, retinitis, ventriculitis, polyradiculomyelitis, and myeloradiculitis. Epstein-Barr (EBV) and VZV-specific DNA sequences were detected in patients with either encephalitis, aseptic meningitis, and chronic meningitis. Dual infections of CMV and HSV or CMV and EBV were established in two AIDS patients with encephalitis and polyradiculomyelitis, respectively. The applications of this RT multiplex PCR assay are extensive and may prove to be particularly valuable for the rapid and sensitive diagnosis of neurological diseases in both immunocompetent and immunocompromised patients.

Multiplex polymerase chain reaction for the simultaneous detection and typing of polyomavirus JC, BK and SV40 DNA in clinical samples.

A novel multiplex nested PCR (nPCR) method was developed for detecting and differentiating simultaneously the DNA of polyomaviruses JC, BK and SV40 in a single tube. In the first amplification step the same set of primers were used to amplify a conserved DNA region of the large T antigen gene of JCV, BKV and SV40. The second round of multiplex nPCR was carried out using a set of primers designed to render products of different size for each related virus. The thermocycling parameters and concentration of each reaction component were optimised systematically to achieve optimal specificity and sensitivity for the nPCR assay. The sensitivity of the method ranged between one and 10 copies of polyomavirus genome. Cerebrospinal fluid (CSF) was examined from AIDS patients with clinical and neuroradiological evidence of progressive multifocal leukoencephalopathy (PML) and CSF from AIDS patients with other neurological alterations. Urine specimens from bone marrow transplant recipients affected by haemorrhagic cystitis were also tested. The results obtained suggest that the assay is a good tool for supporting the diagnosis of polyomavirus infection and could be used for epidemiological purposes and in other studies in order to define better the role of polyomaviruses in human disease.