title: 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.
authors.forename: Jack Bee
authors.initials: JB
authors.lastname: Chook
authors.forename: Lai Yee
authors.initials: LY
authors.lastname: Ong
authors.forename: Yutaka
authors.initials: Y
authors.lastname: Takebe
authors.forename: Kok Gan
authors.initials: KG
authors.lastname: Chan
authors.forename: Martin
authors.initials: M
authors.lastname: Choo
authors.forename: Adeeba
authors.initials: A
authors.lastname: Kamarulzaman
authors.forename: Kok Keng
authors.initials: KK
authors.lastname: Tee
mesh_terms: D005838:Genotype; D015658:HIV Infections / Q000453:epidemiology* / Q000821:virology*; D015497:HIV-1 / Q000145:classification* / Q000235:genetics*; D006801:Humans; D008296:Malaysia / Q000453:epidemiology; D010802:Phylogeny; D060888:Real-Time Polymerase Chain Reaction; D016865:Reassortant Viruses / Q000145:classification* / Q000235:genetics*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity