Surveillance of equine influenza viruses through the RESPE network in France from November 2005 to October 2010.

REASONS FOR PERFORMING THE STUDY: The Réseau d'Epidémio-Surveillance en Pathologie Equine (RESPE, the French epidemiological network for equine diseases) is a network for epidemio-surveillance of major equine diseases based around sentry veterinarians in France. OBJECTIVE: The aim of this study was to evaluate the contribution of RESPE to efficient surveillance of equine influenza virus (EIV) in France. STUDY DESIGN: Retrospective cross-sectional study. METHODS: From November 2005 to October 2010, epidemiological and phylogenetic studies were performed on 1426 nasopharyngeal swabs received at the Frank Duncombe Laboratory. Detection was performed by real-time reverse transcription polymerase chain reaction using original primers and probes designed in the matrix protein gene. Phylogenetic analysis was carried out on the HA1 part of haemagglutinin gene amplified from 47 positive-testing samples. Epidemiological information was provided with the majority of samples submitted through RESPE. RESULTS: Of the 920 samples submitted by RESPE-associated veterinarians, 121 (13.1%) from 42 premises were positive for EIV, compared to 26 (5.1%) of the 607 samples received from non-RESPE associated veterinarians. The most extensive outbreak was observed between February and May 2009, affecting 70 horses on 23 premises, 15 of which were managed by RESPE-associated veterinarians. All strains belonged to the American lineage, Florida sublineage, Clade 1 and Clade 2. Clade 1 was identified only during the Grosbois episode. CONCLUSION: RESPE improved detection of EIV in France, enabled characterisation of the virus strains, yielded valuable information relating to the epidemiology of the disease and identified vaccine breakdown. POTENTIAL RELEVANCE: Implementation of a similar surveillance network in other countries may reduce the economic losses associated with outbreaks of EIV.

Determination of hepatitis B virus subtypes by an enzyme immunoassay method using monoclonal antibodies to type-specific epitopes of HBsAg.

We described a Hepatitis B surface antigen (HBsAg) subtyping method based on a commercial enzyme immunoassay (EIA) for detection of HBsAg in which the procedure was modified to include the use of monoclonal antibodies with restricted anti-HBs specificities. This method, which was able to classify HBsAg as: ayw1, ayw2, ayw3, ayw3* (intermediate between ayw3 and ayw4), ayw4, ayr, adw2, adw4 and adr, was compared to counter electrophoresis procedure (CEP) by testing HBsAg positive sera from blood donors included in a prospective national epidemiological survey. Among the 256 HBsAg positive samples tested with both techniques, 111 (43.3%) could not be subtyped with CEP vs 10 (3.9%) with our modified EIA. This difference was related to the serum HBsAg concentration which must be greater than 3000 ng/mL and 100 ng/mL for CEP and EIA, respectively. The results obtained from 145 sera with both methods were concordant. Seventeen out of 18 samples partially classified as ay with CEP were completely determined with EIA. This reliable procedure, derived from commercially available reagents, can be easily used in several applications such as large epidemiologic studies and as a substitute for nucleotide sequencing genotyping which is not adapted for large-scale screening and not applicable on samples from nonviremic hepatitis B virus (HBV) carriers.

[Impact of the exclusion of donors who have stayed in the United Kingdom on the residual risk of HIV transmission by blood transfusion]. / Impact de l'exclusion des donneurs ayant séjourné dans les îles britanniques sur le risque résiduel de transmission du VIH par transfusion de produits sanguins labiles.

BACKGROUND: One of the measures aimed at reducing the risk of transmission of the agent responsible for the new variant of Creutzfeldt-Jakob disease was to exclude blood donors having stayed in the United Kingdom between 1980 and 1996. The objective of the study was to estimate the impact on the residual risk of HIV transmission of recruiting extra first-time donors to replace donors having stayed in the United Kingdom. METHODS: The residual risk of HIV transmission due to donations made during the window period was estimated in all donations made in France during the 3-year period 1996-1998 by a linear combination of residual risks in repeat donors and first-time donors. In repeat donors, the estimate is based on the incidence rate of HIV in this population and in first-time donors on the "detuned assay" method. Seven simulations of the impact on the residual risk were made using various percentages of donors which would be excluded (from -5% to -35%). RESULTS: In all donations made in France during the 1996-1998 period, the residual risk of HIV transmission was estimated at 0.70 per million donations, which represents five to six donations made during the window period. If all the donors who had stayed in the United Kingdom were excluded from the donation (35%) and replaced by first-time donors, the residual risk of HIV transmission would be increased from 0.70 to 0.86 per million donations. This increase of 24% would represent one or two extra cases of post-transfusion HIV infection over a 3-year period. CONCLUSION: The results of this study show that the exclusion of a large number of blood donors, replaced by first-time donors, would have a low but quantifiable impact on the residual risk of HIV transmission. This increase of risk was one of the factors that led to the decision of not excluding donors having stayed in the United Kingdom between 1980 and 1996.

Blood donors infected with the hepatitis B virus but persistently lacking antibodies to the hepatitis B core antigen.

BACKGROUND AND OBJECTIVES: Antibodies to the core of hepatitis B virus (anti-HBc) are considered to be the best serologically reliable markers of hepatitis B virus (HBV) infection. Through a national epidemiological survey, two young and first-time blood donors, originating from HBV-endemic areas, were identified as HBV carriers with an absence of anti-HBc reactivity. MATERIALS AND METHODS: We followed up these two subjects in order to investigate the evolution of their HBV serological profiles. Nucleotide sequencing was performed of the entire pre-C/C region of the strains infecting these donors. RESULTS: The same serological profile of active viral replication with an apparent persistent lack of anti-HBc and normal alanine aminotransferase (ALT) levels was found for both subjects throughout a follow-up of 19 months and 4 months, respectively. Neither donor was immunocompromised. Nucleotide sequence analysis of the pre-C/C region did not show mutations or deletions in encoded proteins. CONCLUSION: The hypothesis of an in utero HBV infection responsible for an immune tolerance to HBV seems to be the most probable explanation for this particular immunological situation. Such occurrences in the blood donor population are probably rare as less than 0.1% of hepatitis B surface antigen (HBsAg)-positive donors exhibit such a profile, in our experience. Moreover, this phenomenon does not impose a risk of HBV transmission by blood donation, as the exclusion of HBV-infected blood donation is based on HBsAg detection. However, such a risk might be encountered with the hepatitis C virus (HCV) for which at present only antibodies to HCV are screened.

Early detection of human immunodeficiency virus infection using third- and fourth-generation screening assays.

Early detection of infection with human immunodeficiency virus (HIV) is critical for clinical diagnosis and treatment of patients, as well as for ensuring the safety of blood transfusion products. Recently, a number of fourth-generation HIV screening assays have been developed that offer increased sensitivity over earlier tests by combining detection of anti-HIV antibodies with detection of the p24 viral antigen. Previously, six different HIV assays were compared against a broad range of 30 seroconversion panels. In the present study, three of the newer fourth-generation assays were tested together with three of the third-generation HIV antibody-only assays. This extensive analysis highlights (i) the importance of p24 antigen detection for early diagnosis, (ii) the improved sensitivity of fourth-generation assays over antibody-only tests, and (iii) the superior performance of the Vidas Duo assay, which allows reduction of the diagnostic window by up to 2 weeks. Finally, the results emphasize the detection limitations of the different assays and suggest improvements for future HIV screening assays.

Hepatitis C virus: routes of infection and genotypes in a cohort of anti-HCV-positive French blood donors.

BACKGROUND: We evaluated and analysed risk factors of HCV-infected blood donors according to HCV genotypes in order to improve the transfusion policy and safety of blood supply. MATERIALS AND METHODS: HCV-RNA was analysed in sera from 518 anti-HCV-positive blood donors, who were invited to medical consultation and interview as to risk factors by means of an extensive questionnaire. HCV genotyping was done on all samples positive for HCV-RNA. RESULTS: Of the 518 sera, 399 (77%) were HCV-RNA positive, and 394 of 399 HCV genotypes were identified. Major genotypes were 1b (34.3%), 3a (24%), 1a (19.5%) and 2 (11.4%). Of the donors, 289 (55.8%) were interviewed regarding their risk behaviour: 27% were former intravenous drug users (IVDUs), 26% had been transfused, 8% had a history of invasive diagnostic procedures, and 13% a history of surgery. Among the 224 interviewed donors, genotypes 1a and 3a were mainly associated with IVDU (51 and 45% respectively) and genotype 1b, with transfusion and nosocomial infections (40 and 25%, respectively). CONCLUSION: In this population of anti-HCV-positive blood donors, nosocomial infection may be a route of HCV spread, but the main risk factor remains IVDU, particularly in young men. The transfusion policy will improve if predonation interviews of such young men are done with a specific and sensitive questionnaire.

[Genetic diversity of viruses. Consequences for screening and prevention]. / Diversité génétique des virus. Conséquences pour le dépistage et la prévention.

The evolution of viruses contributes to their diversification, whether it be a result of their own replication, or host-pressure dependent. Certain viral types, groups or subtypes are therefore found in certain regions of the world or in certain populations. The development of blood screening reagents is nearly always based on viral antigens or viral sequences derived from 'prototype' strains or antibodies raised against these prototype strains. Therefore in situations where an individual is infected by a viral strain that is genetically and antigenically distantly related to the prototype strain used in the development of the test, screening failure may occur. In the present article, this has been illustrated via 3 models, the human immunodeficiency virus (HIV), the hepatitis B virus (HBV), and the B19 parvovirus. Viral diversity also has a negative effect on the prevention of blood-transmitted viral infections. The example provided concerns vaccination failure and/or seroprophylaxis against hepatitis B.

Efficacy of HCV core antigen detection during the preseroconversion period.

BACKGROUND: The purpose of this study was to compare the performances of HCV core antigen (HCV Ag) testing with HCV RNA detection during the preseroconversion period. STUDY DESIGN AND METHODS: Six HCV antibody (HCV Ab)-negative and HCV RNA-positive blood samples from 6 donors and 135 serial samples from 28 patients who had undergone hemodialysis, collected a mean of 90 days before the detection of HCV Ab, were tested by ELISA for the detection of HCV Ag and by PCR to quantify HCV RNA. RESULTS: Five of the six donors were positive for HCV Ag. The donor with a negative HCV Ag test had the lowest viral load. In the hemodialysis patients, the 43 first specimens of the series were HCV RNA negative. Of the 92 specimens that were HCV RNA positive, 81 (88%) were positive for HCV Ag. Among the 74 samples with more than 10(5) RNA copies, 71 (96%) were HCV Ag positive. Average time from first viremic bleed to first HCV Ag-positive bleed was estimated at 2.0 days and that to first HCV Ab-positive bleed at 50.8 days. CONCLUSION: HCV Ag testing permits the detection of an HCV infection about 1.5 months earlier than the HCV Ab screening tests and an average of only 2 days later than quantitative HCV RNA detection in individual specimens.

HCV RNA in blood donors with isolated reactivities by third-generation RIBA.

BACKGROUND: The objective of this collaborative study was to learn the proportion of HCV RNA-positive samples obtained from a population of donors with isolated anti-HCV reactivities by third-generation RIBA (RIBA-3) (indeterminate results). STUDY DESIGN AND METHODS: During a 2-year period, 11 blood transfusion centers kept all samples with indeterminate RIBA-3 results to test them by PCR, using both local and commercial techniques. RESULTS: Of the 758 RIBA-3 indeterminate samples, 10 (1.3%) were positive for HCV RNA: 3. 3 percent (6/180) and 1.3 percent (4/317) of samples with anti-core or anti-NS3 reactivity, respectively, and none of the 52 and 209 samples with anti-NS4 or anti-NS5 reactivity, respectively. HCV RNA-positive donors with anti-core reactivity were infected with different subtypes (1 with HCV subtype 1b, 1 with 2, 1 with 2a/2c, 2 with 3a, and 1 with 5a), and a follow-up indicated a chronic-carrier state in two of the six donors. Acute hepatitis was diagnosed in three of the four donors with anti-NS3 reactivity alone. Two of these three were IV drug users and were infected with subtype 1a. CONCLUSION: HCV RNA-positive donors with indeterminate results in RIBA-3 are extremely rare, but they do exist. They were observed only when either anti-core or anti-NS3 was present. With such a RIBA-3 profile, PCR testing remains necessary to reveal an eventual acute or chronic HCV infection.

GB virus type C/HGV markers in HCV RNA-positive French blood donors: correlation with HCV genotypes and risk factors.

BACKGROUND: Exposure to GB virus type C/HGV (GBV-C/HGV) could be determined by detection either of RNA by RT-PCR or of antibodies of the envelope protein E2. STUDY DESIGN AND METHODS: The aim of the study was to determine the proportion of the GBV-C/HGV markers of infection in a blood donor population infected with HCV and to identify GBV-C/HGV routes of transmission that are associated with HCV genotypes and risk factors. RESULTS: Among 306 HCV RNA-positive blood donors, the proportion of GBV-C/HGV RNA-positive donors and anti-E2-positive donors was 19.3 percent (95% CI = 15.0-24.2%) and 42.1 percent (95% CI = 36.6-47.9%), respectively. Exposure to GBV-C/HGV (RNA or anti-E2) was significantly associated with the risk factor of IV drug use. There was a trend toward association with HCV subtypes 1a and 3a, probably because these HCV subtypes are the most frequent in IV drug users. No correlation was observed between ALT elevation and the presence of GBV-C/HGV RNA. CONCLUSION: In persons with HCV infection, IV drug use seems to be a major route of GBV-C/HGV transmission. Precautions taken to avoid HCV infection will probably also decrease GBV-C/HGV transmission.

[Screening tests combined with p24 antigen and anti-HIV antibodies in early detection of HIV-1]. / Les tests de dépistage combiné de l'antigène p24 et des anticorps anti-VIH dans l'infection précoce à VIH-1.

The five available p24 Ag/anti-HIV combined tests were compared to the six third-generation anti-HIV assays mainly used in blood transfusion centers. Among 70 selected HIV-1 positive samples (12 samples from early infected blood donors and 58 from ten commercial panels), 59 were positive with at least one assay. False negative results were observed for zero to six samples with p24 Ag/Ab assays versus seven to 19 with antibody (Ab) tests. In five cases, one or more combined assays gave a positive signal later than the most sensitive Ab screening test. One sample with a high p24 Ag titer was missed by one combined test. The mean time delay between the most sensitive test and the second one was 0.3 to 2 days. The p24 Ag limit of detection was investigated with seven dilutions of the HIV Ag reference. The threshold of the p24 Ag detection was found to be between 65 and 250 pg/mL of HIV Ag. For four of the five combined assays, p24 Ag detectability was assessed with dilutions of infected culture cell supernatants from 13 HIV-1 different genotype strains exhibiting HIV Ag titers from 300 to 450 pg/mL. One of the four combined assays gave negative results but close to the cut-off for three supernatant dilutions (1 B, 1 F, 1 HIV-1/O) and one missed the HIV-1/O dilution. The p24 Ag/Ab combined assays permit an earlier diagnosis of HIV infection than third generation assays even if the yield in terms of reduction of the window period is moderate. They are less sensitive than p24 Ag screening assays for the detection of this marker. Consequently, the p24 Ag/Ab assays have not been used for the diagnosis of a primary infection instead of p24 Ag screening tests. They must be considered only as good tools for the detection of HIV infection.

[Screening for markers of infections transmitted by transfusions in the blood collected in France from 1996 to 1998]. / Dépistage des marqueurs des infections transmissibles par transfusion sur les dons collectés en France de 1996 à 1998.

From 1996 to 1998, a decrease in positive donation rates has been observed for HIV, HCV and HBs Ag in first-time donors, while these rates remained stable for HTLV. In repeat donors, the same decrease was observed for HCV and HBs Ag while the rates remained stable for HIV. No HTLV-positive donations from repeat donors were noted in 1998. About half of the HIV-positive repeat donors were regular donors (less than two years between the two donations), as well as 88% of HBV-infected repeat donors. Inversely, only 20% of HCV-positive repeat donors were regular donors. Anti-HBc antibodies have been found in 20% of HIV-infected donors, in 22% of HCV-infected donors, and were associated with HBs Ag in 99% of the cases. Elevated ALT was observed in 47% of donors with anti-HCV and in 10% of donors with HBs Ag. The major risk factors are at-risk sexual behavior for HIV and use of intravenous drugs and nosocomial infections for HCV. Being a native of an endemic country has been found to be the major risk for HBV. The major HTLV risk factor was directly or indirectly linked to the Caribbean area.

Prevalence of present and past hepatitis G virus infection in a French haemodialysis centre.

BACKGROUND: Previous studies, detecting GB virus-C (GBV-C) or hepatitis G virus (HGV) RNA by using reverse transcriptase polymerase chain reaction (RT-PCR), have shown that haemodialysis (HD) patients had a high risk of being infected and viraemic with this virus. A past GBV-C/HGV contact can now be detected by testing for antibodies directed against the GBV-C/HGV envelope protein E2 (anti-E2). METHODS: In order to evaluate GBV-C/HGV contact, 120 patients undergoing chronic HD were tested for GBV-C/HGV RNA by RT-PCR and anti-E2 antibodies by ELISA. GBV-C/HGV viraemic patients were followed prospectively for 18 months, and retrospectively when sera were stored. The total follow-up was between 18 and 78 months. RESULTS: GBV-C/HGV RNA was detected in 17 patients (14%), and 18 patients (15%) had a significant level of anti-E2 antibodies. No positive anti-E2 specimens were also positive for GBV-C/HGV RNA and vice versa. A total of 35 patients (29%) were contaminated with GBV-C/HGV. Sixteen of the 17 viraemic patients had a persistent viraemia (follow-up 18-78 months) and one cleared the virus during the study period. A past or present GBV-C/HGV contact was statistically correlated with the duration of HD and hepatitis C virus (HCV) infection, but was independent of age, hepatitis B virus (HBV) infection, and alanine aminotransferase (ALT) level. CONCLUSIONS: Twenty-nine per cent of patients who underwent HD in our centre have been infected by GBV-C/HGV, 49% were still viraemic and 51% have developed anti-E2 antibodies, indicating a past contact with GBV-C/HGV. Our results demonstrate that the prevalence of GBV-C/HGV contact in HD was underestimated when only RT-PCR was used. Therefore GBV-C/HGV contact is probably much more frequent in HD than previous studies would suggest and is at this time not correlated with hepatotoxicity. Anti-HCV antibodies blood screening since 1990 and recent changes in managing HD patients have probably reduced GBV-C/HGV contact in the same way.

[Sensitivity of screening kits for anti-HIV antibodies. 1999 update. Retrovirus Working Group of the French Society for Blood Transfusion]. / Sensibilité des trousses de dépistage des anticorps anti-VIH. Réévaluation 1999. Groupe de travail rétrovirus de la Société française de transfusion sanguine.

A comparative evaluation of the sensitivity of anti-HIV screening assays has been recently performed with a selected panel of 65 samples which included HIV1 group M (per-seroconversions, seroconversions and seropositives infected with genotypes A, B, C, D, E), HIV1 group O and HIV2. The results obtained with the 21 ELISA HIV1 + HIV2 screening assays are presented. Among these 21 assays, four are combined anti-HIV and p24 Ag assays. All the assays except four fulfilled the criteria defined at the beginning of the study, i.e., positive results on all the seropositives including seroconversions and positive results on at least 50% of the per-seroconversions. The main differences were observed with the ten per-seroconversion samples, the number of positive results on such samples varying from three to ten. The constant improvement of anti-HIV screening tests which leads one to shorten the 'window' period permits and earlier diagnosis of HIV infection and a progressive decrease of the transfusional risk.

[Prevalence of HIV, HTLV, and hepatitis B and C viruses in blood donors in France, 1992-1996]. / Prévalence du VIH, de l'HTLV et des virus des hépatites B et C chez les donneurs de sang en France, 1992-1996.

The prevalence of HIV, HTLV, HBV and HCV infections has been calculated by age and sex of blood donors and not, as generally done, according to the number of donations. This study has been conducted on the basis of two surveys. The first one collected information about the sex and age of the seropositive donors. A second survey, begun in 1992, collected information about the demographic characteristics of blood donors. This population has a higher male to female sex ratio (1:2 versus 1:0) and is younger than the general population. Thus, the prevalence of these viral infections has been determined according to sex, age and type of donors (first-time or repeat) from 1992 to 1996 and the trends have been analysed. The main results are the following: a decreasing prevalence of HIV in first-time donors from 1992 to 1996, more marked in men than in women and in the 18-29 year age group than in the other age groups; a slighter decrease in prevalence of HBV and HCV in first-time donors from 1992 to 1996 with no difference in this decrease between sex and age groups. HBs Ag prevalence in first-time donors was twice as high in men than in women and was highest in donors aged from 40 to 49 years in both sexes. Prevalence of antibodies to HCV in first-time donors was comparable in both sexes but varied greatly with age. In men, a peak was observed in the 30-39 age group and in women, the highest prevalence was seen in the 50-65 age group.

HTLV testing in blood transfusion.

To improve the safety of the blood supply, HTLV screening of blood donations became mandatory in different countries. In Japan and in Europe, the majority of HTLV-infected donors are HTLV-1 whereas in the USA more than half of them are HTLV-II-positive. The prevalence of HTLV-infected donors is low in European Countries as is the rate of seroconversion. Consequently, to test donors only once would have a high efficiency. This procedure is already in use in certain countries. Furthermore, if the use of leucodepleted cell concentrates is generalized, the policies of HTLV screening will still be further modified.