Testing drug levels

When someone does not respond to an antiretroviral regimen, any or all of the following could play a part:

  • Poor treatment adherence resulting in low drug trough levels.

  • Drug interactions that increase or decrease absorption.

  • Genetic influences on an individual’s drug metabolism.

  • Other varying pharmacokinetics among individuals (e.g. age, weight, height) that can result in suboptimal drug levels or toxicity/severe side-effects from drug levels that are too high.

Therapeutic drug monitoring (TDM) is the term used to describe the measurement of plasma drug concentrations. It is done to guide the dosing regimen for a patient and can help determine why someone is not doing well on therapy.

For several antiretroviral agents, researchers have demonstrated a relationship between drug levels and efficacy and/or toxicity. Several characteristics make certain drugs more suitable than others for therapeutic drug monitoring. The ideal drug for TDM should have:

  • A clearly defined relationship between plasma concentration and pharmacologic effect and/or toxicity.
  • Significant inter- and intra-individual variability in the drug plasma concentration at a fixed dose.
  • A narrow therapeutic window (therapeutic index), that is, the level at which the drug is effective is very close to the level at which it is toxic.
  • A high toxicity profile (dose-limiting toxicity).

A number of studies suggest that the particular characteristics of protease inhibitor (PI) and non-nucleoside reverse transcriptase inhibitor (NNRTI) classes of antiretroviral drugs lend themselves favourably to TDM. The nucleoside reverse transcriptase inhibitors (NRTIs) are less amenable to therapeutic drug monitoring as blood levels do not seem to correspond to active drug concentrations found in cells.

Processing antiretroviral drugs

Antiretroviral drugs (ARVs) are processed into inactive products relatively quickly, hence the need for frequent dosing. Their metabolism is dependent on the liver, involving a 'pathway' called the cytochrome P450 system (CYP450), which is responsible for processing many other drugs and nutrients. Interactions between these different substances can affect the speed at which they are metabolised, causing blood levels to rise or fall.

The activity of CYP450 is itself variable. Some people are rapid CYP450 metabolisers and others are slow. This produces variation between individuals in protease inhibitor (PI) levels in the blood. Genetic tests are being developed in an attempt to identify rapid and slow metabolisers.


Poly-glycoprotein (P-gp) is a protein found on the surface of cells. P-gp acts as a transmembrane pump, removing drugs from the cell membrane and cytoplasm. Research has shown that P-gp inhibitors, such as the drug cyclosporin, can inhibit the transport of PIs, so the action of P-gp would seem to affect the amount of drugs found in cells.

Genetics and drug levels

There is currently much interest in the field of genetic differences (polymorphisms) in enzymes and transporters and how this affects serum concentrations of antiretroviral drugs. The hope is that genetic screening may be utilised to establish how individual variation may affect blood concentrations of ARVs. The concept is similar to the practice of genetic screening to identify patients at particular risk of abacavir hypersensitivity reactions.

CYP2D6 is the best characterised of the CYP isozymes, with more than 75 variants currently identified. While it accounts for only 2 to 5% of all CYP isozymes, CYP2D6 metabolises approximately 25% of all clinically used medications. Absent or reduced CYP2D6 activity results in drug accumulation because of reduced drug clearance efficiency.

While considerable information is being gathered in this field, more work is needed to develop practical applications for the role of genetics in predicting drug response (pharmacogenetics).

Measuring drug levels

In order to decide how and when therapeutic drug monitoring might be useful in a practical sense, it is important to understand the benefits and limitations of this technique.

Benefits of measuring drug levels

Therapeutic drug monitoring (TDM) can be used to:

  • Confirm antiviral effect
  • Establish dose-related drug toxicity
  • Aid dosing in some populations.

If there is a known correlation between blood concentration and therapeutic activity, TDM can establish whether the drug dose is sufficient to achieve the desired effect. This might be useful when drug interactions are suspected or when using unlicensed versions of a drug.

There are landmark clinical trials that have established the value of TDM in setting dosing schedules and ensuring therapeutic efficacy. They are summarised below.

In the Viradapt study, patients who were failing combination therapy were randomised to one of two treatment arms.1 The control group received a new treatment regimen based on standard of care. The genotypic group received a new treatment regimen based on resistance mutation profiles.

Results showed that patients in the genotypic study arm had a significantly better response to therapy than patients in the control arm. However, when stored plasma samples from the patients was retrospectively analysed, it appeared that having optimal drug concentrations was more important than knowledge of resistance mutations. The mean change in HIV RNA after 48 weeks of treatment (regardless of the availability of genotypic resistance test results) was -0.36 log in the patients with suboptimal concentrations compared with -1.28 log in the patients with optimal concentrations (p = 0.0048). Patients in the subgroup who received results from genotypic resistance tests and who had optimal drug concentrations made up the highest proportion of patients with undetectable viral load.

Fletcher designed a study to demonstrate the feasibility of a concentration-controlled approach to combination antiretroviral therapy.2 The virological responses and safety of this strategy was compared to a conventional fixed-dose regimen. This was a randomised, 52-week, open-label trial of concentration-controlled compared with conventional dose zidovudine, lamivudine, and indinavir therapy conducted with forty antiretroviral-naive patients with plasma viral load >5000 copies/ml.

Significantly, more patients on the concentration-controlled arm achieved desired concentration targets for all three drugs. Fifteen out of 16 from that group versus nine out of 17 patients on conventional dosing (p = 0.017) attained viral load levels < 50 copies/ml at week 52.

The conclusion was that concentration-controlled, three-drug therapy was feasible, well-tolerated, and achieved a better result than regular ARV dosing. These findings provide a scientific basis to challenge the accepted practice of administering the same dose of antiretroviral agents to all adults, ignoring the concentrations actually achieved.

ATHENA was a randomised controlled clinical trial investigating the utility of TDM.3 Patients receiving nelfinavir or indinavir were assigned to either the TDM or the control arm. After one year of follow-up, significantly fewer patients in the TDM arm (17.4 vs 39.7%) had discontinued nelfinavir or indinavir. The TDM group showed a significantly higher proportion of patients (78.2 vs 55.1%) with viral loads <500 copies/ml after 12 months of treatment. This study demonstrates that TDM for nelfinavir and indinavir in treatment-naive patients improves treatment response.

One unanswered question about the ATHENA study related to the proportion of individuals whose plasma nelfinavir levels normalised without the need for dose adjustment, following a discussion with their doctor about the need to follow dosing and food instructions carefully. How much of the benefit of TDM was derived from spotting that a patient had problems with adherence and subsequently devoting time to addressing those problems? Would it be cheaper in the end to do this with all patients rather than implement therapeutic drug monitoring?

Drug toxicity is a major factor in discontinuation of or poor adherence to antiretroviral therapy. TDM could be used to reduce doses when high levels of a drug are causing side-effects. The link between drug exposure and adverse effects is seen particularly with protease inhibitors (notably lopinavir, atazanavir, saquinavir, nelfinavir, indinavir, and ritonavir) and non-nucleoside reverse transcriptase inhibitors (NNRTIs). Where high drug levels are detected, it might be possible to manipulate the dosing schedule to minimise adverse effects and at the same time maintain antiviral effect.

Because patients may respond differently to the same dose of drug, TDM could be used to guide dosing regimens in patients who are particularly susceptible to altered drug concentrations. 

Researchers have described cases of patients who have had a virological relapse during pregnancy that may have been associated with low ARV plasma concentrations.4 5 This may be due to changes brought about by pregnancy, which in turn can alter the volume of drug distribution or its clearance.

Studies have found greater variability in blood drug concentrations in children and teenagers than in adults. In particular, it has been suggested that children given nevirapine may be at risk for under-dosing.6 The issue of therapeutic drug monitoring in children is complicated by the fact that adult target concentrations (reference range) may be different.

Finally, certain disease states can alter drug pharmacokinetics. Patients with liver or kidney dysfunction may handle drugs differently and TDM could be used to properly adjust dosing levels.

Limitations of measuring drug levels

Therapeutic drug monitoring (TDM) is not applicable to all situations because:

  • Data supporting the role of TDM are conflicting.

  • Reference values might need adjusting for resistance.

  • Reference values are not always known.

  • Serum concentration does not always relate to active drug.

  • Availability of laboratories capable of performing quantification of ARV drug concentrations under rigorous quality control standards is not widespread.

  • Expert interpretation of ARV concentration data and ability to apply to a dosing regimen is required. 

The PharmAdapt and GENOPHAR studies are two randomised studies that failed to confirm the benefits of TDM shown in the ATHENA and Viradapt studies.

In the PharmAdapt study, patients initiating treatment with protease inhibitor–containing regimens were randomised to receive either TDM or standard of care.7 There was no apparent benefit of TDM at 12 weeks based on virological response; however, only a quarter of the participants in the intervention arm underwent dose modification based on TDM.

Similarly, in the GENOPHAR study, which randomised patients to receive either TDM or standard of care, there was no apparent benefit to TDM because of virological response (according to an intent-to-treat analysis).8 In this study, dosage adjustments based on TDM were made for only 19% of the intervention group.

These two studies had important limitations. Because relatively few participants in either the PharmAdapt or GENOPHAR cohorts had their dosages adjusted based on TDM, probably because ritonavir boosting was prescribed to most participants, the statistical power to detect true differences may have been limited.

Another important limitation of TDM is that consideration must be paid to the interplay between the virus, patient, and drug. Most studies evaluating relationships between drug levels and virological response included patients who harboured virus that was fully sensitive to the drug of interest. For that reason, the therapeutic ranges only apply to treatment-naive patients, assuming wild-type virus to be present. As soon as resistance to a drug has been developed, higher drug levels may be necessary to suppress viral replication of the resistant strain.

Targets for TDM should be individually developed according to the genotypic or phenotypic resistance profile of a particular patient. The development of cut-off values for the inhibitory quotient attempts to utilise TDM, while accommodating the presence of ARV mutations.

There are certain circumstances when the optimal serum concentration is either not established or does not correlate suitably to the active component of drug in the body. For example, some of the newer antiretroviral agents do not have well-defined values. Similarly, there is little merit in measuring the serum concentrations of the nucleoside reverse transcriptase inhibitors because blood levels do not seem to correspond to active drug concentrations found in cells.

Finally, consideration should be given to the active components of drugs for which drug concentrations are measured. To date, nelfinavir is the only agent known to have an active metabolite. If the interpretation of nelfinavir does not take into account the active metabolite, it is possible the ultimate analysis will be incomplete or misleading.

When to measure ARV drug levels

At present, British treatment guidelines and the US guidelines recommend therapeutic drug monitoring (TDM) when drug concentrations are difficult to predict. These settings include:

  • In the management of suspected drug interactions.
  • Pregnancy.
  • Paediatrics.
  • Malabsorption.
  • Extremes of body mass index.
  • Salvage therapy when TDM and resistance test results can be integrated.
  • Patients with renal or hepatic impairment.
  • Transplant patients.
  • Cases of toxicity or concentration-dependent toxicities.
  • Suspected non-adherence.
  • The use of alternative dosing regimens whose safety and efficacy have not been established.
  • Lack of expected virologic response in a treatment-naive person.

How blood levels are measured

In order to measure the amount of drug in the blood a small quantity (at least 1ml) of plasma is needed. Two samples will often be taken in order to establish the highest and lowest concentration of drug in the body over a dosage interval. A peak sample is usually taken two hours after the last dose and a trough sample is collected at the end of the dosing interval. For example, in a twice-daily regimen, the trough sample would be done at hour 12 after dosing. If it is not practical for two samples to be collected, a single trough sample will usually suffice.  

If it is difficult to obtain a trough sample, an option is to use a randomly collected sample, which will allow a calculation of predicted trough and peak concentrations.

Quality control

In Europe, a quality control system for TDM was established following studies that showed considerable variability in the quality of drug-level testing. Nijmegen University Medical Centre in the Netherlands organised the first international comparison study to determine how much variation exists in drug level measurements from different laboratories.

The researchers sent plasma samples from HIV-negative volunteers, spiked with three different, standardised doses of indinavir, nelfinavir, ritonavir, and saquinavir, to ten laboratories worldwide. Measurements were considered correct if they fell within 20% of the concentration of drug that should have been present, based on the original weight of drug dissolved in the plasma sample. This method was chosen to minimise interpersonal variation in drug metabolism.

Indinavir was the drug with the most consistent detection rate; 86% of measurements were correct. In contrast, ritonavir was poorly measured; only 42% of measurements were correct. For saquinavir and nelfinavir, two-thirds (65%, 67%) of measurements were correct.

Only one laboratory measured all three doses accurately. Overall, the ability to detect low concentrations correctly was worse than for other concentrations.9 Despite the significant levels of inaccuracy, the Dutch study showed a marked improvement in quality as compared to a prior study of nelfinavir drug-level monitoring in the US. That study looked at results from five laboratories and found a fivefold variation between labs.

Inhibitory quotient (IQ)

Researchers and pharmaceutical companies conduct various studies in order to determine the pharmacokinetic parameters of drugs . Two such parameters are Cmin and IC. Cmin is the minimum concentration of a drug measured between one dose and the next. Inhibitory concentration (IC) values refer to the concentration required to inhibit wild-type virus (virus without resistance mutations).

Different ICs can be measured. IC50, IC90, and IC95 refer to the concentration of a drug needed to inhibit viral replication by 50%, 90%, or 95% respectively. The IC values rise as virus becomes more resistant, reflecting the need for more drug to inhibit resistant virus. The IC value for one virus may be very different from the IC value for another. 

The ratio between the Cmin and the IC50 gives a value called the inhibitory quotient (IQ). This value is a way of illustrating numerically the comfort zone that exists when a particular drug is used. For example, if the trough level (Cmin) of a drug is 50 and the IC50 of a drug is 10, the ratio will be five. In other words, the trough level is five times higher than the minimum concentration needed to inhibit 50% of virus replication.

There are two types of IQs that can be calculated for resistant virus. Data collected from phenotypic resistance tests are phenotypic inhibitory quotients (PIQs) and data derived from genotypic analysis refer to the genotypic inhibitory quotient (GIQ). (See Resistance testing for more information on this topic.) This distinction between genotype and phenotype may be important. The phenotypic IC50 should be adjusted for the way in which the analysed drug is transported in the body. Accordingly, an adjustment is needed to account for protein binding. At present, allowances made for protein binding differ from assay to assay.

Genotypic resistance testing has the advantage of being less expensive, but it also has limitations in terms of its application to determining IQ. For example, the model used to determine GIQ assumes an equal importance for each resistance mutation included. In reality, there may be differences. To date, there is no gold standard against which to weigh the importance of different mutations, so GIQ might vary considerably according to the various mutations that are present.

There have not been any prospective studies conducted for the validation of cut-off values for IQ-based TDM values or to validate the strategy of IQ-based TDM. At present, IQ use is considered experimental and its application is primarily limited to the clinical trial setting.


  1. Durant J et al. Importance of protease inhibitor plasma levels in HIV-infected patients treated with genotypic-guided therapy: pharmacological data from the Viradapt Study. AIDS 14: 1333-1339, 2000
  2. Fletcher CV et al. Concentration-controlled compared with conventional antiretroviral therapy for HIV infection. AIDS16 (4 ): 551-560, 2002
  3. Burger D et al. Therapeutic drug monitoring of nelfinavir and indinavir in treatment-naïve HIV-1-infected individuals. AIDS 17: 1157-1165, 2003
  4. Angel JB et al. An argument for routine therapeutic drug monitoring of HIV-1 protease inhibitors during pregnancy. AIDS 15: 417-419, 2001
  5. Manavi K et al. Plasma lopinavir trough levels in a group of pregnant women on lopinavir, ritonavir, zidovudine, and lamivudine. AIDS 21 (5): 643-645, 2007
  6. Gibbons S et al. An audit of TDM in paediatric subjects from the UK and Ireland. 7th International Workshop on Clinical Pharmacology of HIV Therapy, abstract 8, 2006
  7. Clevenbergh P et al. Efficacy, safety and predictive factors of virological success of a boosted amprenavir-based salvage regimen in heavily antiretroviral-experienced HIV-1-infected patients. HIV Medicine 5 (4), 284–288, 2004
  8. Bossi P et al. GENOPHAR: a randomized study of plasma drug measurements in association with genotypic resistance testing and expert advice to optimize therapy in patients failing antiretroviral therapy. HIV Med 5: 352-359, 2004
  9. Aarnoutse RE et al. International interlaboratory quality control program for measurement of antiretroviral drugs in plasma. Antimicrob Agents Chemother 46: 884-886, 2002
Community Consensus Statement on Access to HIV Treatment and its Use for Prevention

Together, we can make it happen

We can end HIV soon if people have equal access to HIV drugs as treatment and as PrEP, and have free choice over whether to take them.

Launched today, the Community Consensus Statement is a basic set of principles aimed at making sure that happens.

The Community Consensus Statement is a joint initiative of AVAC, EATG, MSMGF, GNP+, HIV i-Base, the International HIV/AIDS Alliance, ITPC and NAM/aidsmap

This content was checked for accuracy at the time it was written. It may have been superseded by more recent developments. NAM recommends checking whether this is the most current information when making decisions that may affect your health.

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