Variations in the frequencies of torque teno virus subpopulations during HAART treatment in HIV-1-coinfected patients.

Sera from 15 patients coinfected with TTV and HIV-1, collected before and at two times after introduction of highly active anti-retroviral therapy (HAART), were tested for TTV load and the presence of the five highly divergent TTV phylogenetic groups. Seven patients showed a 1-5 log TTV load decrease during HAART, while the others did not show significant variations. A decrease in the number of coinfecting TTV genogroups was detected in 12 of 15 patients, with the mean number of TTV genogroups/patient decreasing from 2.33 before HAART to 1.47 at the last collect. All five genogroups were less frequently found after introduction of HAART. Three hundred sixty-seven TTV clones from four different genogroups, derived from two patients, were sequenced. Noticeable fluctuations in TTV subpopulation frequencies were observed in both patients analyzed. In conclusion, HAART tends to reduce the number of TTV genotypes/genogroups and may affect the balance between different TTV isolates coinfecting single individuals.

A multiplex PCR assay able to simultaneously detect Torque teno virus isolates from phylogenetic groups 1 to 5.

Torque teno virus (TTV) is a circular, single-stranded DNA virus that chronically infects healthy individuals of all ages worldwide. TTV has an extreme genetic heterogeneity which is reflected in its current classification into five main phylogenetic groups (1-5). Using specific PCR assays, it has been shown that many individuals are co-infected with TTV isolates belonging to different phylogenetic groups. Here, a multiplex PCR assay was developed, using five recombinant plasmids. Each plasmid carried an insert of different size issued from a TTV isolate belonging to a different group. The assay was able to simultaneously amplify DNAs of TTV isolates belonging to all five phylogenetic groups. Multiplex PCR was then tested satisfactorily on DNAs extracted from 55 serum samples (47 health care workers and 8 AIDS patients). All individuals but nine were infected with at least one TTV isolate. Co-infection with multiple isolates was found in 29/47 (62%) health care workers and in 8/8 (100%) AIDS patients. A number of discrepancies were observed when results obtained with three thermostable DNA polymerases were compared. For example, four TTV phylogenetic groups were detected in a particular serum sample by using one of the three DNA polymerases, whereas the other two enzymes were able to detect only three TTV groups. However, none of the three enzymes used could be broadly considered to be more efficient than the others. Despite its limitations, the assay described here constitutes a suitable tool to visualize the degree of co-infection of a given population, avoiding time-consuming experiments.

A multiplex PCR assay able to simultaneously detect Torque teno virus isolates from phylogenetic groups 1 to 5

Torque teno virus (TTV) is a circular, single-stranded DNA virus that chronically infects healthy individuals of all ages worldwide. TTV has an extreme genetic heterogeneity which is reflected in its current classification into five main phylogenetic groups (1-5). Using specific PCR assays, it has been shown that many individuals are co-infected with TTV isolates belonging to different phylogenetic groups. Here, a multiplex PCR assay was developed, using five recombinant plasmids. Each plasmid carried an insert of different size issued from a TTV isolate belonging to a different group. The assay was able to simultaneously amplify DNAs of TTV isolates belonging to all five phylogenetic groups. Multiplex PCR was then tested satisfactorily on DNAs extracted from 55 serum samples (47 health care workers and 8 AIDS patients). All individuals but nine were infected with at least one TTV isolate. Co-infection with multiple isolates was found in 29/47 (62 percent) health care workers and in 8/8 (100 percent) AIDS patients. A number of discrepancies were observed when results obtained with three thermostable DNA polymerases were compared. For example, four TTV phylogenetic groups were detected in a particular serum sample by using one of the three DNA polymerases, whereas the other two enzymes were able to detect only three TTV groups. However, none of the three enzymes used could be broadly considered to be more efficient than the others. Despite its limitations, the assay described here constitutes a suitable tool to visualize the degree of co-infection of a given population, avoiding time-consuming experiments.

Hepatitis A virus infection in hepatitis C Brazilian patients.

OBJECTIVE: HAV infection in patients with pre-existing chronic liver disease has been associated with increased rate of fulminant hepatitis and mortality. The aim of this study was to investigate the presence of serological and molecular HAV markers in a population of HCV infected patients. PATIENTS AND METHODS: The presence of total and IgM anti-HAV antibodies was investigated in 197 patients (mean age 44.8+/-12.5 years) referred to the Brazilian Reference Center for Viral Hepatitis and who tested positive for anti-HCV antibodies and HCV RNA. HAV RNA was investigated by reverse transcription-nested PCR in these patients.Results. One hundred seventy patients (86%) had total, but not IgM anti-HAV antibodies, being therefore, immune to hepatitis A, while 27 (14%) were not. A high proportion (6/27, 22%) of the susceptible patients presented markers of recent HAV infection: One patient was IgM anti-HAV positive, three were HAV RNA positive, and two presented both markers. By nucleotide sequencing, it was demonstrated that the HAV isolates infecting these patients belonged to subgenotypes 1A and 1B. CONCLUSIONS: Superinfection with HAV was a common event in the group of HCV infected patients under study. Implementation of hepatitis A vaccination should be considered for this population.