The epidemiology of human T-cell lymphotropic virus types I and II in Canadian blood donors.

OBJECTIVES: Blood donors in Canada have been tested for Human T-Cell Lymphotropic Virus (HTLV) since 1990. We report the epidemiology, risk factors and lookback/traceback of HTLV-positive donors/recipients. METHODS: The annual HTLV rate was calculated from 1990 to 2010. Residual risk was estimated as the product of incidence and window period. Twenty-nine HTLV-positive donors and 116 matched controls (ratio 1 : 4) were interviewed about risk factors. For HTLV-positive donations, lookback investigations involved identification of all previous donations, and attempting to locate and test recipients. Traceback was initiated when transfusion transmission was queried for HTLV-positive blood recipients. All donors of products that the recipient received were identified, with an attempt to locate and test them. RESULTS: The HTLV rate decreased from 9.35 per 100,000 donations in 1990 to 1.11 in 2010. The residual risk of infection was 1 in 7.6 million donations. In logistic regression birth overseas (OR 18.7), history of sexually transmitted diseases (OR 32.9), sex with unknown background (OR 5.4) and blood transfusion (OR 8.9) were significant predictors. In the lookback study, of 109 HTLV-positive donors, 508 components were transfused, of whom 147 recipients were tested and 18 (12%) were positive. All were transfused prior to the implementation of donor testing. Twenty-three traceback investigations were requested involving 324 transfused untested products,of whom 219 (67.6%) of donors were tested and 13 (6%) were positive for HTLV. CONCLUSIONS: With testing of the blood supply, the risk from HTLV is very low and while most HTLV-positive donors have risk factors, deferrable risk is rare.

Current incidence and residual risk of HIV, HBV and HCV at Canadian Blood Services.

Estimates of the viral residual risk should be updated to reflect current incidence of infection in blood donors. Incidence rates were estimated for allogeneic whole-blood donations made to Canadian Blood Services from 2006 to 2009 based on transmissible disease conversions of repeat donations within a 3-year period. Residual risk was estimated as the incidence multiplied by the window period. The residual risk of HIV was 1 per 8 million donations, HCV 1 per 6·7 million donations and HBV 1 per 1·7 million donations. The residual risk remains low and has decreased for HCV since our previous estimates due to reduced incidence.

Anti-hepatitis C virus (HCV) screening at a Canadian Red Cross center: significance of a positive c100 HCV enzyme-linked immunosorbent assay.

The c100 hepatitis C virus (HCV) enzyme-linked immunosorbent assay (ELISA) has been used to screen blood donors to prevent transfusion-associated non-A,non-B hepatitis. This test is not specific, and only about 25 percent of c100 HCV ELISA-positive blood samples appear to transmit hepatitis C. However, the intensity of the ELISA (sample/cutoff ratio [S/C], greater than 2) could identify a subpopulation of donors that are at high risk for transmitting hepatitis. Blood samples from 20,186 volunteer blood donors at a Canadian Red Cross blood transfusion center were screened for antibodies to HCV using the c100 HCV ELISA. Fifty-nine (0.3%) of these donors were repeatably reactive on ELISA. When their samples were tested with the c100 recombinant immunoblot assay (RIBA) and second-generation RIBA (RIBA-2), 26 (44%) and 31 (52%) samples, respectively, were found to be positive. Thirty-three of the 59 ELISA-reactive donors had an S/C greater than 2. Of these 33 donors, 30 (91%) had elevated alanine aminotransferase (ALT), 27 (82%) were RIBA-2 positive, and 22 (67%) had risk factors for hepatitis. In contrast, of the 26 ELISA-reactive donors with S/C less than 2, only 7 (27%) had elevated ALT, and 4 (15%) were RIBA-2 positive and also had high risk factors for hepatitis. Thus, while the HCV ELISA may lack specificity, its intensity can serve to identify a subgroup of donors that are at high risk for transmitting hepatitis.

Prevalence of GBV-C/hepatitis G virus viremia and anti-E2 in Canadian blood donors.

BACKGROUND AND OBJECTIVES: GB virus C (GBV-C)/hepatitis G virus (HGV) is a recently recognized parenterally and sexually transmitted agent. The prevalence of GBV-C/HGV markers in Canadian blood donors has not been previously studied and was therefore determined. MATERIALS AND METHODS: Blood donors [identity unlinked (IU), short-term temporarily deferred (STTD) and autologous groups] and donor samples with antibodies to hepatitis C (anti-HCV) or hepatitis B core were tested for GBV-C/HGV RNA and for antibodies to E2 antigen (anti-E2). RESULTS: GBV-C/HGV RNA was found in 1.1% and anti-E2 in 7.3% of the combined IU/STTD donor group. Viremia was much more common in anti-HCV-positive samples (12.5%); anti-E2 was present in >50% of this group. In the STTD group, female gender was significantly associated with viremia. CONCLUSION: GBV-C/HGV infection is relatively common in Canadian donors, and a small proportion are viremic. The association of female gender and viremia was unexpected. Further study is needed to clarify the epidemiology and natural history of GBV-C/HGV infection.

Detection of hepatitis C virus by PCR in second-generation enzyme immunoassay-seropositive blood donors by using matched pairs of fresh frozen plasma and pilot tube sera.

Between April 1993 and March 1995, 429 of 334,454 (0.13%) blood donations at the Toronto Centre of the Canadian Red Cross were reactive for hepatitis C virus (HCV) by second-generation enzyme immunoassay (EIA-2). Of the 429 EIA-2-positive donations, 189 (44%), 138 (32%), and 102 (24%) were positive, indeterminate, and negative by Second-Generation Recombinant Immunoblot Assay (RIBA-2). To assess HCV viremia and minimize the risk that specimen handling affected PCR-based detection, the qualitative AMPLICOR HCV test was performed on both pilot tube sera (PTS) and the corresponding fresh frozen plasma (FFP) from 294 EIA-2-reactive donations. AMPLICOR PCR results for PTS and FFP were 100% concordant and were confirmed by nested HCV PCR for 27 of 294 donations. The AMPLICOR HCV test was positive for 127 of 140 (91%) of RIBA-2-positive donations (81, 91, and 96% of donations with two, three, and four reactive bands, respectively), 5 of 88 (5.7%) indeterminate donations, and 0 of 66 (0%) RIBA-2-negative donations. The Third-Generation Recombinant Immunoblot Assay (RIBA-3) was performed on RIBA-2-negative, -indeterminate, and -positive, PCR-negative donations. RIBA-3 demonstrated enhanced specificity and resolved 18 of 88 (20%) of RIBA-2-indeterminate samples as HCV antibody positive. The study demonstrates that PTS are as suitable as FFP for PCR-based detection of HCV and can be used to determine if EIA-2-reactive blood donors are viremic at the time of donation.

Microcarriers in combination with enzyme immunofiltration and immunofluroescence for the detection of herpes simplex virus antigens in culture.

A microcarrier culture system in combination with enzyme immunofiltration with a herpes simplex virus (HSV) group monoclonal antibody was found to be as sensitive as immunofluorescence for the detection of HSV type 1 (HSV-1) in cell cultures and to give specific identification at the same time as the appearance of the cytopathic effect with very high infectious inocula and within 10 to 24 h of the appearance of the cytopathic effect with very high infectious inocula and within 10 to 24 h of the appearance of the cytopathic effect with low to high inocula. Multiplicities of infection from 10(-5) to 10(1) were tested at 8 to 96 h postinfection. When applied to the identification of HSV-1 and HSV-2 in cultures of clinical samples, the system detected HSV antigens in 50% of the samples after 2 days and in 100% of the samples after 3 days. With 2 ml of microcarrier suspension and with 50 to 300 microliter per sample, several portions are available for replicate and sequential sampling without destroying the culture. The system requires only that 2 X 10(5) microcarriers be added to the culture tube at the time or before it is seeded with cells, at an extra cost of 6 cents (U.S.) per tube and little extra labor.

Evaluation of a confidential method of excluding blood donors exposed to human immunodeficiency virus: studies on hepatitis and cytomegalovirus markers.

A confidential self-administered questionnaire was given to all blood donors prior to donation (n = 95,917). The questionnaire describes groups at increased risk of acquired immunodeficiency syndrome (AIDS) and requires the donor to designate his blood either for laboratory purposes or for transfusion. In a previous communication, we reported that donors in the former group had a much higher prevalence of antibody to human immunodeficiency virus (HIV) than age, sex and clinic matched controls or a group of "miscellaneous" donors who did not fill out the form properly. In this communication, we report results of tests for other viral markers performed on the three designation groups, namely laboratory-designated, miscellaneous and controls. We found that the former two groups had a higher prevalence of antibody to hepatitis B surface antigen (anti-HBs), hepatitis B core antigen (anti-HBc) and cytomegalovirus (anti-CMV) than controls, but there were no differences in alanine aminotransferase (ALT) levels among the groups. In addition, the laboratory-designated group had a higher prevalence of hepatitis B surface antigen (HBsAg) than the general donor population. These data indicate that a questionnaire designed to ascertain AIDS high-risk donors is valuable in excluding donors who may be carriers of other viruses as well.

Evaluation of a confidential method of excluding blood donors exposed to human immunodeficiency virus.

A confidential self-administered questionnaire was given to all donors prior to blood donation (n = 95,917). The questionnaire describes acquired immunodeficiency syndrome (AIDS) high-risk groups and requires the donor to designate his blood for either laboratory purposes or for transfusion. Six-hundred and twenty-seven people (0.65%; 78% men) designated their blood for laboratory purposes. In addition to routine enzyme-linked immunoassay (EIA) screening for human immunodeficiency virus (HIV) antibody, all units from the latter group of donors were tested by Western blot (WB) irrespective of the EIA result. An equal number of donor units was selected from those designating their blood for transfusion (age, sex and clinic matched) and these too were tested by WB irrespective of the EIA result. We found that donors designating their blood for laboratory purposes had a 10 times (vs transfusion-designated controls) to 100 times (vs general donor population) greater exposure to HIV. In the laboratory-designated group, an EIA negative donor was WB positive, yielding an estimated EIA false-negative rate of 16 per million. A confidential questionnaire, as described, is a valuable adjunct in ascertaining high-risk blood donors.

Comparison and application of a novel genotyping method, semiautomated primer-specific and mispair extension analysis, and four other genotyping assays for detection of hepatitis C virus mixed-genotype infections.

To date the true prevalence of hepatitis C virus (HCV) mixed-genotype infections has not been established mainly because currently available methods are not suitable for the detection of mixed genotypes in a viral population. A novel semiautomated genotyping method, primer-specific and mispair extension analysis (S-PSMEA), which is more reliable than other genotyping assays was developed for detection of HCV mixed-genotype infections. A genotype present at levels as low as 0.8% in a defined mix of HCV genotypes was detected, showing a 20-fold increase in sensitivity over that of direct DNA sequencing. A total of 434 HCV isolates were genotyped and analyzed for a comparative study of the accuracy between S-PSMEA and four current genotyping methods. The results showed that viruses in approximately 40% of the samples from this group determined to be infected with mixed genotypes by S-PSMEA were undetected by direct DNA sequencing due to its low sensitivity. Type-specific PCR, line probe assay, and restriction fragment length polymorphism analysis performed poorly, being able to identify only 38.5, 16.1, and 15.4% of mixed-genotype infections, respectively, that were detected by direct DNA sequencing. The prevalence of mixed-genotype infections detected by S-PSMEA was 7.9% (12 of 152 donors) among HCV-infected blood donors, 14.3% (15 of 105) among patients with chronic hepatitis C, and 17.1% (6 of 36) among thalassemia patients who had received multiple transfusions. The data lead us to conclude that HCV mixed-genotype infections are more common than previously estimated and that S-PSMEA may be the method of choice when detection of genotypes present at low levels in mixed-genotype infections is required due to its higher level of sensitivity.

Flow cytometric immunofluorescence assay for detection of antibodies to human immunodeficiency virus type 1 using insoluble precursor forms of recombinant polyproteins as carriers and antigens.

A new serological assay, the recombinant flow cytometric immunofluorescence assay (r-FIFA), was developed for the early detection of human immunodeficiency virus type 1 (HIV-1) antibodies by using recombinant insoluble forms of HIV-1 Gag-p45, Gag-gp41 chimeric protein, gp160, Po197 polyprotein as antigens and autologous carriers through flow cytometry. These recombinant proteins were expressed in insect cells by a baculovirus expression system. Eight anti-HIV-1 seroconversion panels, a low-titer anti-HIV-1 panel from Boston Biomedica Inc. (BBI), and three HIV-1 seroconversion specimens from the Provincial Health Laboratory of Ontario, Toronto, Ontario, Canada (PHL), were tested and analyzed by r-FIFA. In sensitivity comparisons between r-FIFA and tests licensed by the U.S. Food and Drug Administration, which were used to test all of the HIV-1 panels from BBI, detection of HIV-1 antibody by r-FIFA was on average greater than 20 days earlier than that by enzyme immunoassay. The sensitivity of r-FIFA has permitted the detection of HIV-1-specific immunoglobulin G (IgG), IgM, and IgA antibodies during seroconversion. A kinetic analysis of HIV-1 antibody production of r-FIFA has shown that either IgG or IgM, or both, can be detected, depending on the phase and type of the immune response in the HIV-1-infected individual. Both primary and secondary immune responses were observed during this period. The r-FIFA results suggest that implementation of r-FIFA may significantly reduce the "window" period from the time of infection to the time of seroconversion, with earlier detection of antibodies after initial infection. This would also make it possible for us to understand the immune response and the precise mechanisms of immunopathogenesis in the early period of HIV-1 infection.

Primer specific and mispair extension analysis (PSMEA) as a simple approach to fast genotyping.

A simple method, primer specific and mispair extension analysis (PSMEA) with pfu DNA polymerase was developed for genotyping. PSMEA is based on the unique properties of 3'-->5' exonuclease proofreading activity. In the presence of an incomplete set of dNTPs, pfu was found to be extremely discriminative in nucleotide incorporation and proofreading at the initiation step of DNA synthesis, completely preventing primer extension when mispair(s) are found adjacent to the 3'-end of the primer. This has allowed us to accurately detect nucleotide variations, deletions and insertions for fast genotyping.