Report 12 -  4th International Workshop on HIV Drug Resistance and Treatment Strategies
Written by Jules Levin
Sitges, Spain, June 12-16 2000

RESISTANCE TESTING: benefits and shortcomings

Resistance testing is here. Doctor's are using it. It is commercially available.  The Federal government Health and Human Services Administration Public Health Service HIV DrugTreatment Guidelines and the International AIDS Society (IAS) both recommend that the use of resistance testing can be useful as a tool in making certain treatment decisions in individuals with treatment experience who need to change therapy. But, it's helpful to be aware of the shortcomings of the tests. At the resistance Workshop in Spain I think it was generally agreed that use of resistance testing can help a doctor make a better decision in selecting a regimen for an individual who needs to change therapy because of virologic failure. But, it was also generally agreed that resistance testing is one tool to be used in that process of selecting a regimen. A patient's treatment history and the doctor's judgement of that history are very important. Oftentimes, good judgement by a treating physician can realize which drugs a patient may or may not be responsive or sensitive to. But several studies have shown that using a resistance test (genotypic and/or phenotypic) can be helpful in selecting the most effective regimen. This article reviews the benefits and outlines a number of shortcomings of resistance testing. In evaluating the results of resistance testing and in deciding how to apply the information in selecting a regimen, it can be helpful to be aware of the shortcomings.

Testing and proper interpretation of testing results can also help in possibly selecting an effective regimen with less drugs in them. Testing may help sort out the drugs to which the patient may be most sensitive and the drugs to which there may be little sensitivity. A regimen with less drugs promotes easier tolerability and adherence. So, rather than taking a shotgun approach with 6 or more drugs, an equally effective or possibly more effective regimen of 4 drugs may be selected. Discussion of limitations of resistance testing received much attention at the Workshop. If a person has extensive treatment experience, testing may not be as helpful to selecting a new regimen. For one, many of the drugs the person used have not been used for a long time and resistance may not be detectable. Therefore, it is generally agreed that resistance testing should be performed while the person is on therapy. But even then, resistance to previously used drugs may not be apparent. Genotypic testing may be helpful in this circumstance because genotypic resistance to therapies a person is currently taking may point out cross-resistance to other drugs. A second concern regarding people with extensive drug experience is that they may not be adequately sensitive to any of the available drugs. So testing may not be helpful in identifying an effective regimen.

A third concern is that resistance testing may not detect resistance present at low levels in a person's circulating virus population. As discussed in the NATAP report on Structured Treatment Interruptions at this workshop, Allan Hance and a French research group (abstract 117) used ultra-sensitive detection of minority populations of resistant virus using real-time PCR based method that relies on selective amplification and quantification of viruses carrying the V82A and/or the L90M mutations in the protease. Resistant virus was found at low levels. Resistant viral quasispecies representing <0.1% (mutation L90M) and 1% (mutation V82A) of the total virus population could be detected. In following 4 patients undergoing STIs for whom complete reappearance of wild-type virus by 2-3 months was observed using conventional genotyping, resistant virus could still be detected for as long as 5 months after interruption of therapy and was undetectable at 3 months in only 2/4 patients using the ultra-sensitive PCR method. In the patients with L90M and/or V82A at baseline, resistant virus was still present at 3 months in 9/12 cases.

I think a fourth concern is the ability to interpret phenotypic resistance fold changes when they are near the "cut-off". For example, the Virco cut-off between no resistance and intermediate resistance is a 4.0 fold change in susceptibility while for the Virologic test the fold change is 2.5. How do you judge a 3.0 change in d4T susceptibility. In addition, we don't know how to judge resistance to d4T. Below is a study reported on at the Workshop comparing Virco and Virologic tests and it discusses discordant results that are close to the cut-offs. Using the current cut-offs there is no way to judge phenotypic resistance to double PI combinations such as indinavir-ritonavir, ritonavir-saquinavir or to the new PI ABT-378. Ten or possibly even 20-fold decreased susceptibility to ABT-378, may not mean resistance. But Abbott, the manufacturer of ABT-378, and the 2 test makers are in discussions about this.

Another concern is that some resistance mutations may not have been identified yet. There may be mutations that can reflect resistance to a drug but if researchers don't know about it resistance may nor be identifiable. Additionally, 2 mutations may have a special relationship that has not yet been identified. For example, 3TC resistance (M184V) may partially reverse resistance to AZT. Some PI mutations may have a similar relationship that has not yet been found.

In discussing Changing Therapy for treatment experienced individuals, the recently updated IAS Guidelines which were published in the Journal for the American Medical Association said

"Resistance testing may assist in selecting which drugs should be changed and which could remainÖ..Resistance emergence is highly predictive of loss of antiretroviral activity".

In discussing initial therapy for treatment naÔve individuals the IAS Guidelines said "Choice of a regimen should be individualized based on the strength of supporting data and on regimen potency, tolerability, adverse effect profile, likely drug-drug interactions convenience and adherence likelihood, potential for alternative treatment options if the initial regimen fails, and possibly, baseline resistance testing results.

The IAS Guidelines also discussed some of the limitations of resistance testing--costs, some labs may lack quality assurance (you want to use a lab that has a track record with you in giving reproducibly reliable test results), the difficulty in interpreting the test results in its use for a particular person, if a person has used a drug in the past resistance to that drug may not be detectable with commercially available testing, resistance in minority species (10-20% of a person's circulating virus population) is not likely to be detectable. So, "absence of detectable genotypic or phenotypic resistance may not provide assurance that a drug will be active".

The PHS HIV Drug Treatment Guidelines also address the use of Resistance Testing:

"Resistance testing appears to be a useful tool in selecting active drugs when changing antiretroviral regimens in the setting of virologic failure", and similarly the use of resistance testing can be helpful when a patient has only achieved a suboptimal viral load reduction.

"When combined with a detailed drug history and efforts aimed at maximizing drug adherence, these assays may help to maximize the benefits of antiretroviral therapy".

"Interpretation of test results requires an appreciation of the range of mutations that are selected for by various antiretroviral drugs, as well as the potential for cross-resistance to other drugs conferred by some of these mutations. Consultation with an expert in HIV drug resistance is encouraged to facilitate interpretation of genotypic test results".

Transmission of resistant virus has been reported and "documented", and may be associated with a suboptimal response to therapy. When deciding to treat during acute infection, optimization of the initial antiretroviral regimen through the use of resistance testing is a reasonable although untested strategy. The Guidelines recommend the use of genotypic testing because the turnaround time in receiving results should be quicker, but therapy should not be withheld while awaiting the results of resistance testing.

However, in chronically infected individuals who are naÔve to treatment, resistance testing is not currently recommended, "because ÖÖÖresistance assays may fail to detect drug resistant species that were transmitted at the time of primary infection, but became a minor species" or are not detectable any longer because the patient is not taking the drug.

To me, the two studies discussed below and reported at the Resistance Workshop highlight the importance in selecting the lab to use in resistance testing. As well, the studies point out that a single resistance test or result report can miss detecting a mutation. Although any lab test can also make a mistake, and concordance between the 2 genotypic testing reported below nay appear satisfactory, if you are the patient and a wrong treatment decision is made based upon inaccurate genotype that is unsatisfactory. One pitfall with resistance testing is that the results of research in populations show benefit but for an individual application is more difficult.

You want to use a lab with high expertise in performing testing (using the kit) and in interpreting the results. It's also important for the treating physician to have an expertise in interpreting resistance testing. I think the lab ought to be a lab that has demonstrated a track record of providing reliably accurate results reflected in patient treatment decision making. Both studies compared the two major genotypic resistance test technologies (ABI --Applied Biosystems-- versus TruGene HIV Genotyping Kit frtom Visible Genetics).

Differences in protease and reverse transcriptase sequences between the TruGene HIV-1 genotyping kit (Visible Genetics) and the ViroSeq genotyping system (PE Applied Biosystems)

Francoise Brun-Vezinet and a French research group compared the mutations in HIV-1 protease and reverse transcriptase genes detected by two commercial genotyping kits. Assays were performed according to the TruGene HIV-1 genotyping kit and the ViroSeq genotyping system (Perkin Elmer) package instructions. The comparison was performed on the 96 baseline plasma specimens from the genotypic arm of the Narval-ANRS 088 resistance trial. All the specimens were successfully amplified with both methods. The authors piinted out differences in primary and secondary mutations defined by the International AIDS Society resistance testing guidelines. They only considered major differences, in which a wild-type genotype was detected by one method and a mixed or mutated genotype by the other method.

The mean total number of mutations associated with resistance to protease inhibitors and RT inhibitors per sample was 4.52 (±2.04) and 4.70 (±1.95), respectively. The mean number of primary mutations was 1.85 (±0.92) for PI and 2.54 (±1.00) for RTI. Among the 96 specimens analyzed, a total of 428 primary and 487 secondary mutations were found with the 2 methods. Major differences in primary and secondary mutations have been identified at 14 and 19 amino acid positions, respectively. Among the 14 discordant (different) primary mutations, 9 were detected by VGI and 5 by PE. For the protease gene, discordances concerned only positions 46 (n=2) and 84 (n=1), and for the RT gene, positions 70 (n=1), 74 (n=1), 103 (n=2), 108 (n=1), 184 (n=2), 188 (n=1), 190 (n=2), and 215 (n=1). These 14 major discordances involved 13/96 (13.5%) patients, and in 6 of them genotypic interpretations would have been modified. Among the 19 discordant secondary mutations, 5 were detected by VGI and 14 by PE. For the protease gene, discordances concerned positions 10 (n=2), 20 (n=4), 24 (n=3), 33 (n=1), and 54 (n=2), and for the RT gene, positions 41 (n=2), 67 N=2) and 210 (n=3). These 19 discordances involved 16/96 (16.6%) patients and in two of them genotype interpretation would have been modified. The authors concluded that the study results did not favor one kit over the other.

A blinded comparison of two sequencing assays for resistance mutations in HIV-1 protease and reverse transcriptase genes in the GART Study (CPCRA 046)

Marie Hoover and a US research group including Tom Merigan (Stanford), Doug Mayers (Henry Ford in Detroit), John Baxter Robert wood Johnson Medical School in NJ), and Mark Winters (Stanford) reported on a study comparing the same 2 tests. Their purpose was to determine whether there is agreement in identifying major protease and RT resistance when utilizing HIV-1 genotyping employing 2 DNA sequencing methodologies: (1) an investigator developed methodology run on ABI sequencers; (2) Visible Genetics integrated TruGene DNA sequencing system.

The study examined the previously well-characterized baseline plasma samples collected from 153 patients who had all been exposed to at least 1 protease inhibitor and at least 2 NRTIs, and who participated in the GART study (a prospective study of the utility of genotypic antiretroviral resistance testing in patients failing therapy). The specimens were provided to a single lab for blinded re-analysis using the VGI integrated TruGene DNA sequencing system. The amino acid sequence differences (as compared to a standard sequence) were compared with the original genotyping results. Discrepancies in amino acid mutations were investigated by comparing the DNA sequence chromatographs generated by the 2 methods.

Hoover reported that the comparison revealed that there was general agreement between the methodologies for the major protease resistance mutations (30N 99%, 46I 91%, 46L 92.2%, 48V 100%, 82A 93.5%, 82F 100%, 82T 98.7%, 84V 99.3%, and 90M 91.5%).Similarly, Hoover reported there was agreement for the major nucleoside RT resistance mutations (65R 99.3%, 69D 100%, 74V 99.3%, 151M 99.3%, 184V 97.4%, 215F 96.1%, and 215Y 90.8%). Preliminary analysis of the DNA sequence chromatographs of over 50% of the genotypes with discrepancies revealed that only 17.9% (5/28) of the discrepancies are due to clear differences in the DNA sequence generated by the two methods. The remainder of the discrepancies are attributed to reporting errors (39.3%), differences in VGI versus GART rules for calling mixtures of nucleotides (10.7%); a VGI mixture is described when software indicates a mixed base on one DNA strand; a GART mixture is described when software indicates a mixed base on both DNA strands) and detection of second base in mixtures of nucleotides (32.1%).

The authors concluded that overall, the 2 methods provide similar drug resistance genotypes.

A comparative analysis of Virco Antivirogram and Virologic PhenoSense phenotypic assays for drug susceptibility of HIV-1

These two phenotypic resistance tests are commercially available either through LabCorp, the companies themselves or possibly other labs. The drug susceptibilities of the recombinant viruses are measured by different strategies. Both tests report IC50 values for each drug and fold differences of these values relative to a reference wild-type (WT) virus. Fold differences >2.5 and >4.0 are considered evidence of decreased susceptibility for PhenoSense and Antivirogram, respectively. The level of correlation between the results of these two tests are not known. Shoukat Qari from the CDC reported on this study comparing both tests by analyzing 50 coded plasma samples: 20 from drug naÔve newly infected persons and 30 samples from source patients of occupational exposures to HIV-1, of which 16 were from drug-experienced patients. Results of susceptibility to 12-15 drugs were available from both tests on 38 samples. These results included 246 determinations for nucleoside RT inhibitors (NRTIs), 111 for NNRTIs, and 172 for protease inhibitors (PI). There were 488 (92.2%) concordant results, comprising 459 (94%) sensitive results and 29 (6%) with decreased susceptibility. Of the 41 (7.8%) discordant results reported to have decreased susceptibility by either test (26 Virco, 15 Virologic), no known primary resistance related mutations were reported in 40. The discordant results comprised 14 (5.6%) for NRTI, 16 (14.4%) for NNRTI, and 11 (6.3%) for PI. Of the 41 results, 26 (63.4%) (10 by Virco and 16 by Virologic) had fold difference values close to the cut-offs (4.1 - 5.3 fold for Virco, and 2.52 - 3.7 fold for Virologic). There was discussion at the Workshop that low level NNRTI resistance (<10 fold) may not be significant particularly if a person were NNRTI naÔve. The remaining 15 discordant results were present in 9 samples and included 5 results by Virco (5.9 - 9.5 fold) and 10 results by Virologic (4.6 - 18.5 fold). The authors concluded that despite the use of different testing strategies in both tests, the data in this study indicate a good correlation between results from the 2 tests.