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New Method Spots Hidden Mutations That Predict Failure
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XVI International HIV Drug Resistance Workshop
June 12-16, 2007
Barbados
Mark Mascolini
Ultra Deep sequencing, a novel resistance test that uncovers diverse viral variants making up only 1% of a viral population, spotted low-frequency nonnucleoside (NNRTI) mutations in previously untreated FIRST study participants--and detection of these occult mutations predicted failure in people who took an NNRTI in their first regimen [1].
FIRST assigned 1397 antiretroviral-naive people to a protease inhibitor (PI), an NNRTI, or both a PI and an NNRTI, all with nucleosides [2]. After a median follow-up of 5 years, disease progression proved equivalent with the PI and NNRTI strategies, though people in whom an NNRTI regimen failed with resistance ran a higher risk of AIDS or death.
Michael Kozal (Yale University) presented this substudy involving 258 people who had standard genotyping when they entered FIRST, then had pretreatment samples probed by Ultra Deep. Both methods searched for 62 IAS-USA mutations and 144 Stanford database mutations. FIRST enrolled participants from 1999 through 2002. When the trial began the overall study group had a median viral load of 143,844 copies and a median CD4 count of 163. Because of the advanced disease status of many study participants, Kozal surmised a fair portion had been infected in the mid-1990s.
Whereas standard gene sequencing figured the rate of pretreatment IAS-USA mutations at 12.0%, Ultra Deep recorded a pretreatment rate of 20.9%, a highly significant difference (P = 0.0001). Respective rates of Stanford mutations with routine genotyping and Ultra Deep were 13.2% and 28.3% (P < 0.0001). In an analysis limited to Stanford database mutations, the standard assay recorded a 5.8% prevalence of nucleoside mutations versus 14.3% with Ultra Deep (P < 0.0001), a 2.3% rate of PI mutations versus 4.7% with Ultra Deep (P < 0.0001), and a 6.6% rate of NNRTI mutations versus 14.3% with Ultra Deep (P = 0.03). Most mutations mined by Ultra Deep represented less than 20% of the viral population, the approximate cutoff for standard genotyping.
Of the 258 FIRST enrollees in this substudy, 84 (33%) took an NNRTI plus two nucleosides as their first antiretroviral regimen. Among those 84, all 11 with an Ultra Deep-detected pretreatment NNRTI mutation had a virologic failure, defined as a viral load above 1000 copies after 4 months of therapy. Standard genotyping detected NNRTI mutations in only 2 of these 11 people. Study participants in whom Ultra Deep spotted a pretreatment NNRTI mutation had more than a 3 times higher risk of virologic failure than people without an NNRTI mutation (hazard ratio [HR] 3.38, 95% confidence interval [CI] 1.34 to 8.43, P = 0.009 for IAS-USA mutations; HR 3.49, 95% CI 1.65 to 7.36, P < 0.001 for Stanford mutations.)
The estimated virologic failure rate at 12 months for people with an Ultra Deep-detected NNRTI mutation before treatment was 72.7% versus 28.0% in people without a reverse transcriptase mutation seen by Ultra Deep before treatment (P = 0.006). The estimated 12-month failure rate for people with any Ultra Deep-detected reverse transcriptase mutation before therapy was 59.1% versus 24.8% for people without a pretreatment reverse transcriptase mutation (P = 0.004).
Ultra Deep sequencing differs from real-time PCR assays that search for individual mutations instead of an array of mutations that may confer resistance to antiretrovirals. Other work presented at this workshop and reviewed by NATAP showed that real-time PCR regularly finds more mutations than standard genotyping in untreated people with acute, recent, or chronic HIV infection [3,4].
Numerous clinicians and resistance experts agreed after Kozal's talk that HIV physicians need more sensitive resistance assays. As the University of Pittsburgh's John Mellors put it, "minority mutants have got to come out of the closet." But no one should expect to order an Ultra Deep any time soon. Roche purchased the company that devised Ultra Deep, and a Roche representative at the workshop said it will take lots of work to make the assay affordable. Right now it costs $5000 per test.
References
1. Simen BB, Huppler Hullsiek K, Novak RM, et al. Prevalence of low abundant drug-resistant variants by ultra-deep sequencing in chronically HIV-infected antiretroviral (ARV)-naive patients and the impact on virological outcomes. Antiviral Therapy. 2007;12:S149. Abstract 134.
2. MacArthur R, Novak RM, Peng G, et al. A comparison of three highly active antiretroviral treatment strategies consisting of non-nucleoside reverse transcriptase inhibitors, protease inhibitors, or both in the presence of nucleoside reverse transcriptase inhibitors as initial therapy (CPCRA 058 FIRST Study): a long-term randomised trial. Lancet. 2006;368:2125-2135.
3. Johnson JA, Lipscomb J, J-F Li, et al. Real-time PCR testing allows for sensitive drug
resistance screening and mutation linking in HIV-1. Antiviral Therapy. 2007;12:S153. Abstract 39.
4. Metzner KJ, Rauch P, von Wyl V, et al. Prevalence of minority quasispecies of drug-resistant HIV-1 in patients with primary HIV-1 infection in Zurich in the years 2002-2006. Antiviral Therapy. 2007;12:S47. Abstract 40.
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