Report from The Third International Workshop on Salvage Therapy for HIV Infection, Chicago, April 12-14, 2000 - Report 3

Detecting Resistance When Viral Load is Below 1000 copies/ml.

At this year's salvage Therapy workshop Neal Parkin reported on a study where phenotypic resistance could be detected when viral load was as low as 260 copies/ml. Before describing his study, are two similar reports presented at the 1999 Resistance Workshop last Summer in San Diego, and a similar report presented by Jody Lawrence at the Retrovirus Conference Feb. 2000.

K Soderbarg, with the Professional Genetics Lab in Uppsala, reported on this study whose objective was to determine whether drug resistance mutations in the RT

and protease genes could be found in 7 patients which, during ongoing drug therapy, show minor increases in viral load.

Viral RNA was isolated from samples with as few as 50 copies/ml, after which PCR amplification was performed. Mutations that are likely to cause drug resistance were detected in all patients. These mutations were detected early in the rebound process. Viral loads were as low as between 50 to 500 copies/ml. But in samples with as few as 50 copies/ml, it was possible to detect mutant viruses. The authors concluded mutations causing drug resistance are possible to detect in samples with very low viral load. Therefore, ongoing replication may result in further development of resistance. Some researchers question the reliability of mutations observed in individuals with viral load <50 copies/ml.

Rob Lloyd is with Applied Sciences Lab and Visible Genetics Inc. Visible Genetics Inc is located in Toronto and manufactures a genotypic resistance test. Applied Sciences is in Norcross, Georgia. It has generally been accepted that if a personís viral load were above 1000 copies/ml a lab would not be able to test their blood sample for resistance. In fact if a personís viral load is above 1000 copies/ml some labs will not accept or discourage submission of the blood sample. But many individuals with <1000 copies/ml of VL would I think justifiably like to test themselves for resistance.

Lloyd reported he detected genotypic mutations from actual samples of individuals in this study. He said sequence results demonstrated reproducible and accurate genotypes between duplicate samples at low viral loads down to 60 copies/ml. In speaking with Lloyd he has told me he can detect genotypic resistance mutations when viral load is below 50 copies/ml. In all fairness this data ought to be considered preliminary and should be subjected to confirmatory studies. A few other researchers expressed doubt or uncertainty that you could in fact reliably and reproducibly detect genotypic mutations when viral load is very low (<50 copies/ml). If you have a low viral load and request genotypic resistance testing, I would be cautious in interpreting and relying on the results. One way to address this question would be to submit 2 or more samples to the same lab and see if the results are the same. If your viral load is <50 copies it may be irrelevant to

perform resistance testing because you are not going to change your regimen. But if your viral load is several hundred to 1000 copies/ml detecting resistance may help in deciding to modify your regimen and what drugs to use.

The objective of this study was to compare the results of genotypic antiretroviral resistance testing (GART) from sequential sampling of plasma and PBMC in HIV-infected persons with low level viremia and to examine whether archival mutations are found more frequently in proviral DNA than in plasma. Patients who were on stable antiretroviral therapy for at least eight weeks and had screening plasma viral RNA >50 but <400 copies/mL were enrolled and followed prospectively. GART was performed using a modified Qiagen RNA extraction procedure with the TruGeneTM HIV-1 Assay GeneKit (VGI), OpenGeneTM automated DNA sequencing system and GeneLibrarian.

In blood containing plasma RNA >50 copies/mL, HIV genotyping was successful in 46/46 (100%) of plasma samples and in 39/46 (85%) of PBMC samples from 8 enrolled patients. The concordance in genotype between duplicate plasma and PBMC testing was over 95%. There was greater variability in resistance patterns between sequential samples of PBMC than of plasma. In general more mutations were detected in plasma than in PBMC and 26% of PBMC samples showed resistance mutations which were not apparent in plasma. Jody Lawrence concluded these results indicate GART can be successfully performed in patients with low level HIV viremia and suggest that plasma may provide more reproducible results than PBMC.

Susceptibility testing is most frequently performed after the viral load (VL) has risen above 1000 copies/ml. This study was intended to examine retrospectively the potential clinical utility of phenotypic resistance testing at VL <1000 copies/ml for predicting subsequent treatment failure, and to further define correlates of salvage therapy failure.

Longitudinal plasma samples from 16 patients undergoing salvage therapy with nelfinavir/saquinavir/abacavir and either nevirapine or a second NRTI were analyzed for phenotypic drug susceptibility and drug resistance mutations.

Ten patients had VL rebound or incomplete suppression (failures) while six patients had sustained (>92 weeks) viral suppression (<50 copies/ml); responders). Samples with VL between 50 and 1000 copies/ml were available from six of the failures and four of the responders. Fourteen of 16 samples with VL between 50 and 1000 copies/ml were successfully amplified and could be assayed for drug susceptibility. The two samples that could not be amplified (VL: 55 and 69 copies/ml) were from two of the responders. Two samples from responders (VL: 83 and 115 copies/ml) showed little change in drug susceptibility compared to baseline. In contrast, reduced susceptibility to one or more treatment drugs was detected in 5 of the 6 failures (VL: 260-630 copies/ml). In two of these six cases this was the first sample with a significant increase in VL from nadir, and in two other cases this sample corresponded to the viral load nadir prior to VL rebound. Reduced susceptibility was detected in viruses from all patients with virological failure at VL over 1000 copies/ml.

Parkin concluded that phenotypic analysis detected drug resistance at VL as low as 260 copies/ml, which could facilitate an early change in therapy or treatment intensification before significant virologic rebound. Virological failure was always accompanied by the detection of reduced susceptibility to one or more of the treatment drugs, in contrast to failure of initial therapy regimens where failure without resistance has been observed.

I think additional studies are needed to confirm and better characterize these findings.