Surveillance technology for HIV-1 subtype C in Ethiopia: an env-based NASBA molecular beacon assay to discriminate between subcluster C and C'.
Forty-nine samples with known C2V3 sequences were used for the evaluation of an env-based molecular beacon assay to distinguish between the two genetic subclusters C and C' which characterize the HIV-1 epidemic in Ethiopia. Two subcluster C and two subcluster C' beacons targeting two different loci in the C2V3 region were developed. Using a three beacon-based (2C and 1C'=C prime), isothermal amplification assay, concordance with DNA sequencing was achieved for 43 (87.8%) samples. Sensitivity was 81.8% and specificity 97.4% for subcluster C beacons. For the subcluster C' beacon, a sensitivity of 97% and a specificity of 87.5% was achieved. Five samples were ambiguous by sequencing of which two samples were subcluster C' by the beacon assay and one subcluster C. Two of the samples remained ambiguous with different beacon-pair combinations as well. From samples with a clear C or C' phylogeny by sequencing, three were undetected by the first-line beacon genotyping assay. Genotype ambiguity was resolved in the three samples using beacon pair combinations restricted to each targeted locus. The beacons were evaluated further in a panel including all HIV-1 subtypes. Four of five subtype C isolates were identified correctly, and no cross-reactivity was observed with other subtypes.
Ayele
Workenesh
W
Ethio-Netherlands AIDS Research Project at Ethiopian Health and Nutrition Research Institute (EHNRI), P.O. Box 1242, Addis Abeba, Ethiopia. worknesh@enarp.com
Baar
Michel P de
MP
Goudsmit
Jaap
J
Kliphuis
Aletta
A
Tilahun
Tesfaye
T
Dorigo-Zetsma
Wendelien
W
Wolday
Dawit
D
Abebe
Almaz
A
Mengistu
Yohannes
Y
Pollakis
Georgios
G
D005002:Ethiopia; D006679:HIV Seropositivity / Q000821:virology*; D015497:HIV-1 / Q000145:classification* / Q000235:genetics; D006801:Humans; D015335:Molecular Probes; D011159:Population Surveillance; D021161:Self-Sustained Sequence Replication / Q000379:methods*; D012680:Sensitivity and Specificity; D016415:Sequence Alignment; D013045:Species Specificity; D014759:Viral Envelope Proteins / Q000235:genetics
D015335:Molecular Probes; D014759:Viral Envelope Proteins