Nucleic acid amplification testing of blood donors fortransfusion-transmitted infectious diseases. Report of the Interorganizational Task Force on Nucleic Acid Amplification Testing of Blood Donors

Chronic liver disease develops in more than half of patients with post-transfusion hepatitis C, but little is known about the natural history of community-acquired hepatitis C. In 1985 and 1986 we identified adults with acute non-A, non-B hepatitis in four counties in the United States and followed them prospectively. We used three markers to detect hepatitis C virus (HCV) infection in stored samples of serum: antibody to HCV (anti-HCV) detected by second-generation serologic assays; HCV RNA detected by polymerase-chain-reaction assay; and antibody to HCV antigen (anti-HCVAg) detected by fluorescent-antibody-blocking assay. Of 130 patients with non-A, non-B hepatitis, 106 (82 percent) had HCV infection, 93 were positive for anti-HCV, and 13 were positive only for HCV RNA or anti-HCVAg. Chronic hepatitis developed in 60 (62 percent) of 97 HCV-infected patients followed for 9 to 48 months, with no relation to the risk factors for infection. Ten of the 30 patients who had liver biopsies had chronic active hepatitis. In samples collected 42 to 48 months after the onset of hepatitis, HCV RNA was detected in 12 of 13 tested patients with chronic hepatitis and in all 15 tested patients with hepatitis that had resolved. Anti-HCV persisted in all but two of the initially positive patients, for a rate of antibody loss of 0.6 per 100 person-years. Patients with community-acquired hepatitis C have a high rate of chronic hepatitis. HCV may be a major cause of chronic liver disease in the United States, and in most patients HCV infection seems to persist for at least several years, even in the absence of active liver disease.

Despite sensitive antibody-based blood-donor screening, a residual risk of transfusion-transmitted viral infections exists. Only direct monitoring by sensitive nucleic-acid tests would provide data accurately to measure the risk and to assess risk-reduction procedures. We investigated the feasibility and efficacy of routine screening of donors for hepatitis C virus (HCV), hepatitis B virus (HBV), and HIV-1 by PCR. For PCR testing, individual donor plasma samples were pooled (96x100 microL) overnight by two automatic pipetting machines. Viruses were concentrated by centrifugation and nucleic acids were extracted. HCV PCR was done on the Cobas Amplicor system (Hoffmann-La Roche, Mannheim, Germany). HBV and HIV-1 sequences were amplified by single (non-nested) in-house PCRs and detected by agarose-gel electrophoresis. Detection limits were 1000-5000 genome equivalents/mL in the donor blood. PCR testing was done in parallel to antibody screening with a maximum throughput of 3000 samples in 7-8 h. Positive samples were identified 1-2 days later. 111 of 373,423 donations (107 of 4500 pools) were PCR and antibody/antigen-confirmed positive. We found one HCV PCR-positive antibody-negative donation with normal alanine aminotransferase and one HCV PCR-positive donation with an elevated alanine aminotransferase (100 IU), which was negative in the AxSYM 2.0 and Matrix 1.0, but positive after control in the Abbott Prism test (Abbott GmbH, Wiesbaden, Germany). PCR is a suitable and fast blood-donor screening procedure and contributes to a reduction in viral transmission by transfusion of blood components. In our selected donor population, the yield of detected contaminated donations from donors in the time window in which they are highly infectious but do not have any symptoms or detectable antigen and antibody concentrations (diagnostic window), confirms theoretical estimates.

Infection with the human immunodeficiency virus type 1 (HIV-1), as demonstrated by viral cultures, has been described in some patients before antibodies to HIV-1 can be detected, but the duration and frequency of such latent infections are uncertain. We selected prospectively a cohort of 133 seronegative homosexual men who continued to be involved in high-risk sexual activity, and we cultured 225 samples of their peripheral-blood lymphocytes, using mitogen stimulation to activate the integrated HIV-1 genome. HIV-1 was isolated in blood samples from 31 of the 133 men (23 percent), 27 of whom have remained seronegative for up to 36 months after the positive culture. The other four men seroconverted 11 to 17 months after the isolation of HIV-1. In three of them, we studied cryopreserved lymphocytes obtained earlier, using the polymerase chain reaction to amplify small amounts of viral DNA, and we demonstrated that HIV-1 provirus had been present 23, 35, and 35 months before seroconversion. We conclude that HIV-1 infection in homosexual men at high risk may occur at least 35 months before antibodies to HIV-1 can be detected. A prolonged period of latency in such infections may be more common than previously recognized; the degree of infectiousness during such periods is unknown.