A heteroduplex assay for the rapid detection of dual Human Immunodeficiency Virus Type 1 infections

Abstract

The predominance of circulating and unique recombinant forms (URFs) of Human Immunodeficiency Virus Type 1 (HIV-1) in Cameroon suggests that dual infection occurs frequently in this region. Despite the potential impact of these infections on the evolution of HIV diversity, relatively few have been detected. The failure to detect dual infections may be attributable to the laborious and costly sequence analysis involved in their identification. As such, there is a need for a cost-effective, more rapid method to efficiently distinguish this subset of HIV-positive individuals, particularly in regions where HIV diversity is broad. In the present study, the heteroduplex assay (HDA) was developed to detect dual HIV-1 infection. This assay was validated on sequential specimens obtained from 20 HIV+ study subjects, whose single or dual infection status was determined by standard sequence analysis. By mixing gag fragments amplified from the sequential specimens from each study subject in HDA reactions, it was shown that single and dual infection status correlated with the absence and presence, respectively, of heteroduplex bands upon gel electrophoresis. Therefore, this novel assay is capable of identifying dual infections with a sensitivity and specificity equivalent to that of sequence analysis. Given the impact of dual infection on viral recombination and diversity, this simple technique will be beneficial to understanding HIV-1 evolution within an individual, as well as at a population level, in West-Central Africa and globally.

Fig. 1

Phylogenetic analysis of HIV-1 gag sequence clones amplified from patient specimens obtained at sequential time points. Phylogenetic analysis was performed using the MEGA version 3.1 software package. Pairwise evolutionary distances were estimated using Kimura’s two-parameter method, and phylogenetic trees were constructed by neighbor-joining. The reliability of tree topologies was estimated by bootstrap analysis (1000 replicates). Bootstrap values greater than 70% are shown. Five to 10 test sequences for each time point were used to construct the tree. These test sequences are denoted by the CMNYU identifier (signifies the samples were taken from subjects in Cameroon), followed by the ID number, the time point number, and the clone number (separated by spaces). All reference (sub-)subtypes and CRFs (A1, A2, A3, B,C, D, F1, F2, G, H, J, K, and CRF01_AE – CRF37_cpx) available from the Los Alamos HIV Sequence Database were initially used to construct the tree; most references have been omitted here for clarity. The tree has been circularized due to space constraints. Each cluster of sequences is grouped by a bracket, next to which are the study subject ID numbers. Dually infected study subjects’ ID numbers are underlined. Singly infected study subjects’ ID numbers are asterisked. Subtypes are shown below the ID numbers.

Fig. 2

Establishment of the heteroduplex assay (HDA) using reference plasmids, and determination of the sensitivity of HDA for the detection of minor strains in a reaction. Approximate amounts (ng) of reference subtypes A and B gag (HxB2 location 1577-2040) amplicon present in each HDA reaction are indicated.

Fig. 3

Heteroduplex assay (HDA) of sequential specimens from patients shown by sequence analysis to be (a) dually infected, or (b) singly infected. Numbers above the lanes indicate the time points at which specimens were obtained from each patient. See Table 1 for the number of months between specimens. Heteroduplexes in mixed time point lanes (M) are indicated with white boxes; the faint heteroduplex for CMNYU119(5) is indicated by the white arrow. C: positive control for heteroduplex formation (mixture of subtype B and PCR amplicon from test sample).