Improved detection of CXCR4-using HIV by V3 genotyping: application of population-based and "deep" sequencing to plasma RNA and proviral DNA.
BACKGROUND
Tropism testing should rule out CXCR4-using HIV before treatment with CCR5 antagonists. Currently, the recombinant phenotypic Trofile assay (Monogram) is most widely utilized; however, genotypic tests may represent alternative methods.


METHODS
Independent triplicate amplifications of the HIV gp120 V3 region were made from either plasma HIV RNA or proviral DNA. These underwent standard, population-based sequencing with an ABI3730 (RNA n = 63; DNA n = 40), or "deep" sequencing with a Roche/454 Genome Sequencer-FLX (RNA n = 12; DNA n = 12). Position-specific scoring matrices (PSSMX4/R5) (-6.96 cutoff) and geno2pheno[coreceptor] (5% false-positive rate) inferred tropism from V3 sequence. These methods were then independently validated with a separate, blinded dataset (n = 278) of screening samples from the maraviroc MOTIVATE trials.


RESULTS
Standard sequencing of HIV RNA with PSSM yielded 69% sensitivity and 91% specificity, relative to Trofile. The validation dataset gave 75% sensitivity and 83% specificity. Proviral DNA plus PSSM gave 77% sensitivity and 71% specificity. "Deep" sequencing of HIV RNA detected >2% inferred-CXCR4-using virus in 8/8 samples called non-R5 by Trofile, and <2% in 4/4 samples called R5.


CONCLUSIONS
Triplicate analyses of V3 standard sequence data detect greater proportions of CXCR4-using samples than previously achieved. Sequencing proviral DNA and "deep" V3 sequencing may also be useful tools for assessing tropism.
British Columbia Centre for Excellence in HIV/AIDS, Vancouver, Canada.
Luke C
LC
Swenson
Andrew
A
Moores
Andrew J
AJ
Low
Alexander
A
Thielen
Winnie
W
Dong
Conan
C
Woods
Mark A
MA
Jensen
Brian
B
Wynhoven
Dennison
D
Chan
Christopher
C
Glascock
P Richard
PR
Harrigan
D004279:DNA, Viral / Q000235:genetics; D015699:HIV Envelope Protein gp120 / Q000235:genetics*; D015658:HIV Infections / Q000821:virology*; D015497:HIV-1 / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification / Q000502:physiology*; D006801:Humans; D010949:Plasma / Q000821:virology*; D011533:Proviruses / Q000235:genetics; D012367:RNA, Viral / Q000235:genetics; D019718:Receptors, CXCR4 / Q000032:analysis; D015728:Receptors, HIV / Q000032:analysis*; D017422:Sequence Analysis, DNA; D056189:Viral Tropism
D004279:DNA, Viral; D015699:HIV Envelope Protein gp120; D012367:RNA, Viral; D019718:Receptors, CXCR4; D015728:Receptors, HIV