Molecular detection of HIV-1 subtype B, CRF01_AE, CRF33_01B, and newly emerging recombinant lineages in Malaysia

Abstract

A molecular genotyping assay for human immunodeficiency virus type 1 (HIV-1) circulating in Southeast Asia is difficult to design because of the high level of genetic diversity. We developed a multiplex real-time polymerase chain reaction (PCR) assay to detect subtype B, CRF01_AE, CRF33_01B, and three newly described circulating recombinant forms, (CRFs) (CRF53_01B, CRF54_01B, and CRF58_01B). A total of 785 reference genomes were used for subtype-specific primers and TaqMan probes design targeting the gag, pol, and env genes. The performance of this assay was compared and evaluated with direct sequencing and phylogenetic analysis. A total of 180 HIV-infected subjects from Kuala Lumpur, Malaysia were screened and 171 samples were successfully genotyped, in agreement with the phylogenetic data. The HIV-1 genotype distribution was as follows: subtype B (16.7%); CRF01_AE (52.8%); CRF33_01B (24.4%); CRF53_01B (1.1%); CRF54_01B (0.6%); and CRF01_AE/B unique recombinant forms (4.4%). The overall accuracy of the genotyping assay was over 95.0%, in which the sensitivities for subtype B, CRF01_AE, and CRF33_01B detection were 100%, 100%, and 97.7%, respectively. The specificity of genotyping was 100%, inter-subtype specificities were > 95% and the limit of detection of 10(3) copies/mL for plasma. The newly developed real-time PCR assay offers a rapid and cost-effective alternative for large-scale molecular epidemiological surveillance for HIV-1.

Figure 1.

Genome organizations of full-length human immunodeficiency virus type 1 (HIV)-1 subtype B, CRF01_AE, CRF33_01B, CRF53_01B, CRF54_01B, and CRF58_01B. The HIV-1 subtype B and CRF01_AE, shown respectively in dark and light shades, are the putative parental genotypes for the various recombinant lineages (CRF33_01B, CRF53_01B, CRF54_01B, and CRF58_01B) reported previously. Multiplex real-time polymerase chain reaction (PCR) primers and TaqMan probes designed at three different genetic regions (p24, pro-RT, and gp41) are indicated by dashed vertical lines. Real-time PCR results or yield for each genetic region, as indicated by positive or negative amplification, will be used to determine the HIV-1 genotypes.

Figure 2.

Real-time PCR amplification plot for human immunodeficiency virus type 1 (HIV-1) genotypes. Representative amplification curves of the p24, pro-RT, and gp41 genes for subtype B, CRF01_AE, CRF33_01B, CRF53_01B, CRF54_01B, and an undetermined genotype from Kuala Lumpur were shown. The quantification cycle (C_q) value was indicated by an arrow. CRF01_AE, CRF33_01B, and CRF53_01B showed positive amplification in the p24 region (red); subtype B, CRF33_01B, and CRF54_01B in the pro-RT (blue); and subtype B in the gp41 region (green).