A First Report in a Series on Resistance Testing; including a Geneva Report on Transmission of Protease Resistant Virus


This is a preliminary first report on genotypic and phenotypic resistance testing. This report includes the presentation by Doug Mayers, MD at the NATAP Post-Geneva Community Forum in July '98 at NYU Medical Center. It also includes additional information from Jules Levin. The draft of full proceedings from the July Post-Geneva Forum in July is finished and being finalized now for printing and distribution. It will also be posted on this web site. If you would like a printed hard copy please contact NATAP by our email address posted on the front page of this web site or by calling us at 212 219-0106.


Let's try first to define the two types of testing so you understand what they do. The types of tests are genotypic testing and phenotypic testing. Resistance testing can be a complicated topic. If you do not understand some of this report please feel free to call our offices for an explanation.


Phenotypic Resistance Test--What is phenotypic resistance testing? It was being used in laboratories for research purposes, but due to advances in technology it is being made available commercially for doctors and consumers. Using the phenotypic testing method, a sample of a person's blood is placed in a test tube along with a specific drug. The laboratory determines how much drug is needed to inhibit HIV by either 50% or 90%. When you are talking about the inhibitory concentration (IC) required to do this you'll hear the terms IC50 or IC90. IC50 means the drug can inhibit 50% of HIV production in the test tube and IC90 means 90% of HIV is inhibited. This is called phenotypic resistance testing. If you need more drug to suppress virus than is normally needed that means there is phenotypic resistance.


Genotypic Resistance Test-- A different way to look at resistance is to perform a genotypic test, where you look at the sequence of the genes in the virus itself. You are looking at the changes in the genes and these are called mutations; but, you have to look for mutations that have been proven to result in resistance for that drug, to make the findings useful for the patient. Unfortunately, we have not identified all the mutations that may be relevant. As we start combining multiple drugs different mutations may emerge. For example, failure of a NNRTI+ a NRTI may result in a different mutation than is known to be associated with NNRTI resistance. This subject is discussed more below.


Genotypic Resistance Testing







Phenotypic Test








3 Recent Studies of Resistance Testing

These studies were reported at Geneva or the Resistance meeting.


Baseline Phenotypic Test and Response at Week 16. Dr Steven Deeks took 18 patients who failed indinavir and gave them a 4-drug regimen of abacavir+saquinavir (Fortovase)+nelfinavir+ either nevirapine or a NRTI. They defined sensitivity to a drug as having less than 2 fold resistance using the ViroLogic Phenotypic Test. If you had greater than two-fold resistance to a drug then you were defined as being resistant to it.




 <400 copies/ml

 <50 copies/ml



 none or transient





  sustained for 16 weeks








As you can see in line 1 of the table, 7 of the 18 patients were sensitive to 0 or 1 drug in the regimen they received. That means they were resistant to 3 or 4 drugs. These 7 people had no response to the therapy they received. None of them had <400 copies/ml (undetectable viral load) after 16 weeks on therapy.


11 of the 18 patients were sensitive to 2 or 3 of the 4 drugs. They were resistant to 1 or 2 of the 4 drugs. 8 out of 10 (80%) people had <400 copies/ml (undetectable viral load) after 16 weeks of therapy. And, as you can see one of the 18 patients were sensitive to all 4 drugs.


The persons who were sensitive to at least 2 or 3 drugs had a median viral load reduction of 2.33 log at week 16. The individuals sensitive to 0 or 1 drug had a median viral load reduction of 1.2 log at week 16. The difference was statistically significant (p=0.007).


Interpretation and Application of Results-- So, what does this mean? These study results are potentially very important. You may be able to select a salvage regimen that will work for you by using phenotypic resistance testing. If you are not resistant to 2 or 3 drugs in a regimen it is more likely to work. You may have heard the results of this study reported wrong to you. Some have said that a 4-drug salvage regimen consisting of nevirapine+saquinavir+nelfinavir+abacavir was successful at suppressing viral load for individuals who had indinavir resistance. That is not what this study says. This study says that if a person was sensitive, using the phenotypic resistance test, to 2 or more drugs then they were likely (8/10) to reduce their viral load to undetectable (<400 copies/ml). If a person took all 4 of these drugs but was resistant 3 or 4 of them they did not lower their viral load <400 copies/ml. The key is not which drugs you use but if you are sensitive to at least 2 or 3 of the drugs. You could have used any 4 drugs in this study.


Response to Ritonavir+Saquinavir in PI experienced Patients Using Genotypic Test. Dr Andrew Zolopa took 51 patients who had failed at least 1, 2 or 3 previous PIs. He looked at viral load responses at 4 and 12 weeks. He found the following baseline information about the patient was predictive of the response: stage of HIV disease (advanced or moderate), CD4 count, viral load, HIV drug history (number and duration of prior antiviral treatment). Individuals with higher CD4s and lower viral loads were more likely to respond to the treatment.


The key finding from this study is that independent of all the predictors mentioned above, Zolopa found that simply the number of PI mutations (30, 46, 48, 54, 82, 84, 90) that were found upon performing a genotypic test significantly predicted (p<0.001) the response to a ritonavir+saquinavir regimen.


So, in the first study phenotype testing predicted response, and in the second study genotypic testing predicted response.


Using Both Genotypic and Phenotypic Resistance Tests to Predict Response to Ritonavir+Saquinavir. Richard Harrigan reported at the Resistance meeting just before Geneva on 84 patients who had significant exposure to Pis and received a ritonavir+saquinavir salvage regimen. Drug sensitivity in this study was defined as <4-fold resistance using the Virco phenotypic resistance test. Harrigan found that CD4 count and viral load were factors in predicting the outcome. As expected, individuals with higher CD4 counts and lower viral loads were more likely to respond better to the salvage regimen. Most significantly--


If a person had AIDS, they had a 4-fold greater risk of failure

Phenotypic resistance predicted a 5.6 fold greater risk of failure

Genotypic resistance predicted a 3.5 fold greater risk of failure

Individuals with phenotypic resistance at baseline did not respond well to RTV+SQV, but the data suggests that for those who appeared to be phenotypically sensitive at baseline some of them appeared not to be responsive to therapy with a good viral load reduction. This reflects what we've already said--that phenotypic tests are better at detecting resistance than sensitivity


Baseline Nelfinavir Genotypic/Phenotypic Resistance in Expanded Access Program. Amy Patick reported at the Resistance meeting on 65 heavily pre-treated patients who received nelfinavir in the Agouron expanded access program. This data is from 24 weeks follow-up. They evaluated mutations 48V, 82A/F/T, 84V, and 90M because these are the mutations associated with nelfinavir cross-resistance.


Looking at genotypic mutations--

 # of Genotypic mutations















12 out of 14 individuals with 0 mutations got a good response; 4 out 5 individuals with 1 mutation got a good response; while, individuals with 2 or more mutations did not respond well.


Looking at phenotypic resistance--

 Phenotypic Resistance


 4-10 fold








14 out of 19 individuals with <4-fold phenotypic resistance responded well to nelfinavir. So, both genotypic and phenotypic testing predicted success or failure. All these studies are retrospective studies. This means they looked back at the individuals blood samples and tested them for resistance after they had started the salvage regimens. Researchers agree that prospective studies are necessary to confirm these results. But, there is an increasing body of data suggesting that you may be able to predict the short-term viral load response a person will have to a drug using either genotypic or phenotypic test results. Perhaps the best predictor is --how many drugs in a new regimen is the person sensitive to?


Mayers discussed data he collected from patients treated with HAART in the military. These were patients failing to respond to HAART regimens. All were on a HAART regimen at the time of failure. On average these patients had a history of supposedly taking 5 NRTIs and 2 PIs. 22% of the patients tested had no resistance mutations. 24% were protease sensitive with some NRTI resistance. About 54% were NRTI and protease resistant. They found that many of these patients (22% without resistance) just could not take the regimens. The patients were unable to tell their doctors that they couldn't take the medications. Knowing which of these 3 categories a patient may be in is important to patient management. If a person does not have resistance but is not responding because they are not taking their medications, they might respond well to treatment if you could design a regimen that would be easier for them to take.


Mayers listed reasons why people can fail a regimen without resistance being detectable--



In summary Mayers said--


Transmission of Drug Resistant Virus


AZT was first started to be used in1987, From 1988 to 1991 they discovered a 1.4% rate of transmission of AZT resistance from one person to another. By 1992 that rate increased to 7.5%. In 1993-1994 that rate increased to 10.4%. It's now holding at 7.5 to 10% in all patient populations that are being surveyed now. That means, 10% of people who are now becoming infected with HIV have AZT resistance before ever taking the drug. At Geneva, it was reported that--



Individuals infected with virus resistant to Pis may have severely limited or no good treatment options. That is why it is crucial to use safer sex techniques. For individuals who take treatments, it is crucial that they realize that unsafe sex can leave a person not only infected with HIV but it may leave that person with no treatment options. If a person is treatment naive, it may be advisable to use resistance testing to check if they have pre-existing resistance that they may have contracted through transmission.


Dr Nick Hellman works for ViroLogic, the company that makes a phenotypic resistance test. He discussed at this forum a person who was infected with HIV that was resistant to delavridine and nevirapine. He described a second person who they performed a phenotypic resistance test on 23 days after the person was infected with HIV. There was evidence of decreased susceptibility to AZT, 3TC, saquinavir, nelfinavir, ritonavir and indinavir. Additional reports were presented of individuals infected through transmission with PI resistant virus.