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.

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.

An HIV-1 infected man who experienced rapid disease progression and poor response to therapy after starting a new sexual relationship with an infected partner is known as the 'Ottawa superinfection case'. Subsequent analysis of viral sequences of protease, reverse transcriptase, Gag p17, and Env V3 provided no evidence for the acquisition of genetically divergent viruses before disease progression or drug resistance during virological failure of combination therapy. Whether HIV-1 superinfection contributes to disease progression or the spread of drug-resistant HIV-1 remains unknown.

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, and Pol97 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-1specific immunoglobulin G (IgG), IgM, and IgA antibodies during seroconversion. A kinetic analysis of HIV-1 antibody production by 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.