Drug resistance testing: Making anti retroviral therapy more efficacious

India’s national anti retroviral therapy programme: A successful feat

The Joint United Nations Programme on HIV/AIDS called “UNAIDS” has praised India’s efforts in reducing the spread of HIV/AIDS in the country in the last decade and has considered India’s efforts in making anti retroviral therapy (ART) available to more and more people as exemplary for other nations. The number of Indian HIV patients receiving anti-retroviral therapy approximately doubled from 2007 to 2009 and then again from 2009 to 2012. On a global scale, India ranks second with respect to the number of HIV patients having access to life saving ART. It is commendable that 650 000 HIV positive Indians are receiving treatment in the country. India continues to strive to reach the target of ART coverage for one million seropositive Indians in near future with the help of its national treatment programme.

Drug resistance testing: A crucial step to fortify the efficacy of ART

However, the biggest challenge faced while planning ART for a patient is drug resistance. Though several anti retrovirals have been developed; drug resistance minimises the therapeutic options available to HIV/AIDS patients. In this way drug resistance poses a major challenge for successful management of HIV/AIDS cases. Though HIV Viral Load values are indicative of non-responsiveness of the ART, it becomes extremely important for a clinician to understand the drug sensitivity profile of a seropositive patient towards a wide spectrum of anti retrovirals. Total 19 antiretroviral drugs have been approved for the treatment of HIV-1 infection: one nucleotide and seven nucleoside reverse transcriptase inhibitors (NRTIs), seven protease inhibitors (PIs), three nonnucleoside RT inhibitors (NNRTIs), and one fusion inhibitor. Extraordinary patient effort is required to adhere to drug regimens that are expensive, inconvenient, and often associated with dose-limiting side effects. Incomplete virus suppression due to these factors predisposes to the development of drug resistance, which threatens the success of future treatment regimens.

HIV has remarkable genetic diversity during course of infection therefore it behaves differently in different patients. Due to these variations in viral population the anti retroviral sensitivity and resistance profiles of different patients or even same patient may differ widely at various stages of the disease. A significant proportion of new HIV infections results from the transmission of strains which are already resistant to one or more antiretroviral drugs. Detection of drug-resistant virus before starting a new drug regimen is an independent predictor of virologic response to that regimen. Several prospective controlled studies have also shown that patients whose physicians have access to drug resistance data, particularly genotypic resistance data, respond better to therapy than control patients whose physicians do not have access to these assays. Therefore, in order to ensure that ART works effectively for every patient at every stage of the disease, it is imperative and highly recommended to perform anti retroviral drug resistance testing. Drug resistance testing in HIV/AIDS improves therapeutic efficacy, the risk benefit ratio of the therapy being provided to the patient and the survival and prognosis.

Anti-retroviral drug resistance testing: The nittigritties

Genotypic and phenotypic resistance assays aid in predicting or confirming the drug response of HIV in a particular patient. Phenotypic resistance assays measure the extent to which an antiretroviral drug inhibits virus replication in vitro, whereas Genotypic assays detect mutations that are responsible for drug resistance.It is used more commonly than phenotypic testing because of its lower cost, wider availability, and shorter turnaround time. Genotypic testing provides early evidence of drug resistance within a virus population. Genotypic assays detect mutations present as mixtures even if the mutation is present at a level too low to affect susceptibility in a phenotypic assay, and detect transitional mutations that do not cause drug resistance by themselves but indicate the presence of selective drug pressure. Genotypic assays detect mutations even if the phenotypic effect of the mutation is suppressed by other mutations in the sequence. The clinical usefulness of genotypic testing has been demonstrated in four of five prospective randomized studies in contrast, phenotypic testing has been shown to be clinically useful in just one of four prospective randomized studies. Co-tropism assay is also available as research based assay which can be performed prior to CCR5 antagonist therapy. In the subsequent sections, let us have a closer look at the genotypic assay.

Genotypic drug resistance testing in HIV/AIDS

The half-life of HIV-1 in plasma is approximately 6 hours, which enables isolation of actively replicating virus from this specimen. Sanger sequencing is the standard approach to HIV genotyping; it involves specific amplification of Reverse Transcriptase (RT) and Protease genes of HIV-1 genome followed by sequencing of these target genes using sensitive capillary automated Sequencers. Obtained mutations are then compared with comprehensive and exhaustive mutation database available with the International AIDS Society US and the Stanford University HIV Drug Resistance Database. “Drug Resistance Interpretation,” is conducted by submitting generated protease and RT sequences, the online program returns inferred levels of resistance to the 16 FDA-approved anti-HIV drugs. Each drug resistance mutation is assigned a drug penalty score for inferring the level of drug resistance as: susceptible, potential low-level resistance, low-level resistance, intermediate resistance, and high-level resistance. Since many mutations in both the protease and RT are known to be associated with drug resistance, it has become customary to label some drug resistance mutations as either “primary” or “major” and other mutations as “secondary” or “minor”. Primary mutations are those that reduce drug susceptibility by themselves whereas secondary mutations reduce drug susceptibility in combination with primary mutations or improve the replicative fitness of virus isolates with a primary mutation. Interpreting the results of HIV genotyping vis-à-vis drug resistance requires in depth of knowledge of known mutations and their impact on the efficacy of ART. Just to cite an example, HIV positive patients with an M184V mutation would not benefit from therapy with antiretrovirals like Epivir or Emtriva, as this mutation confers resistance to these drugs. Such information helps clinicians to decide and make an informed choice while therapy planning.

CD4 count, HIV viral load testing and drug resistance profiles:

In conjunction with each other CD4 counts and plasma HIV RNA load (viral load) have been the cornerstones of the monitoring work up for HIV/AIDS. CD4 counts and HIV viral load should be performed at baseline before initiation of ART. This provides an idea about the urgency of starting ART or whether treatment can be deferred. CD4+viral load testing can be repeated every 3-6 months; however timepoints for viral load testing may vary based upon patient status, baseline viral load levels and changing clinical situations. The duo of CD4 count and viral load testing yields the following clinically pertinent information:

• Current status of the patient’s HIV (severity, disease progression, survival prediction
• Risk of opportunistic infections/ Need for prophylactic therapy
• Urgency of initiating ART/ Determining the efficacy of ongoing ART
• Monitoring adherence and compliance to ongoing ART and overall prognostication

Optimal virologic response in HIV is generally considered to be plasma HIV load below the lower limit of detection of different assays which could range from <20 to 75 copies/ mL; depending upon the assay being used. Occasional peaks in viral load may be seen in successfully treated patients (<400 copies/mL), however, such peaks cannot be regarded as virologic failure. The snapshot of recommended timepoints for HIV viral load testing as per international guidelines is given above.

One of the most common reasons for a suboptimal virologic response as seen by viral load results and a low CD4 count is poor efficacy of the ongoing ART due to antiretroviral drug resistance. Therefore, in any given clinical setting, antiretroviral drug resistance testing cannot be viewed in isolation. It has to be considered and performed in conjunction with CD4 counts and HIV viral load. A consistently depleting CD4 count and perpetually high viremia are pointers towards the fact that ART might not be working due to HIV mutations and resultant drug resistance. Therefore, this needs to be tested and confirmed and if need be, therapy needs to be changed as per the results of drug resistance testing.

When to perform drug resistance testing for HIV patients?

The US-FDA’s Department of Health and Human Services (DHHS) and the National Institute of Health (NIH) set up a panel of experts to frame guidelines for the use of antiretrovirals in adults and adolescents. Under this initiative, a detailed set of guidelines have been promulgated. These guidelines present a comprehensive and elaborate set of recommendations regarding the use of drug resistance testing before and during HIV/AIDS therapy. Given below are the strongly recommended situations for considering genotypic drug resistance testing in HIV-1 patients:

• HIV drug-resistance testing is recommended in persons with HIV infection at entry into care regardless of whether ART will be initiated immediately or deferred.
• Genotypic testing is recommended as the preferred resistance testing to guide therapy in antiretroviral-naïve patients.
• HIV drug-resistance testing should be performed to assist in the selection of active drugs when changing antiretroviral regimens in persons with virologic failure and high HIV RNA levels.
• Drug-resistance testing in the setting of virologic failure should be performed while the person is taking prescribed antiretroviral drugs or, if not possible, within four weeks after discontinuing therapy.
• Genotypic resistance testing is recommended for all pregnant women before initiation of ART and for those entering pregnancy with detectable HIV RNA levels while on therapy.

References:

1. http://www.unaids.org/en/resources/presscentre/featurestories/2013/august/20130830india/
2. http://www.aidsmeds.com/cn/printView.php?page=/articles/Resistance_10312.shtml&domain=www.aidsmeds.com
3. Panel on Antiretroviral Guidelines for Adults and Adolescents: Guidelines for the use of antiretroviral agents in HIV-1-infected adults and adolescents: Department of Health and Human Services. Available at: http://aidsinfo.nih.gov/ContentFiles/Adultand
4. HIV drug resistance, CD4 and viral load fact sheet accessible at: http://www.mtnstopshiv.org/sites/default/files/attachments/HIV%20Resistance%20Fact%20Sheet_13AUG10.pdf