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Published by Oxford University Press on behalf of the Infectious Diseases Society of America 2014. This work is written by (a) US Government employee(s) and is in the public domain in the US.
Extending HIV drug resistance testing to low levels of plasma viremia Douglas D. Richman VA San Diego Healthcare System and University of California San Diego, Distinguished Professor of Pathology and Medicine, Director, Center for AIDS Research, Florence Seeley Riford Chair in AIDS Research, Room 329, Stein Clinical Sciences Building, 9500 Gilman Drive, La Jolla, CA 92093‐0679 Correspondence: tel: 858‐552‐7439, fax: 858‐552‐7445, [email protected]
Clinical Infectious Diseases Advance Access published January 14, 2014 at A
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Antiviral treatment that is not sufficient to completely suppress viral replication
imposes the selective pressure that results in the emergence of drug resistant virus
escape mutants. HIV drug resistance testing has become part of the standard
management of patients, both those newly presenting in order to identify
transmitted drug resistance and those who fail to suppress virus replication, which
results in the emergence of acquired drug resistance[1‐3].
We have known for a long time that escape mutations can emerge early after
treatment failure, even with low levels of plasma HIV RNA[4, 5]. Guidelines from
most clinical laboratories advised HIV drug resistance testing only on specimens
with viral loads of at least 1000 copies HIV RNA/mL[1, 3]. This practice was based
on the ratioale that successful sequencing diminished in efficiency with diminishing
levels of RNA, and that with smaller populations of HIV RNA molecules being tested,
the results might not be representative of the variants in the circulation. Moreover,
FDA approved drug HIV drug resistance genotyping platforms have specified
application to specimens with >1000 copies HIV RNA/ml. Two companion papers
in this issue of CID provide data from large clinical programs in British Columbia
and central Italy to demonstrate that in fact the success rates for PCR amplification
to perform drug resistance testing in specimens from patients with 50‐1000 copies
HIV RNA/mL plasma are both reasonably efficient and clinically predictive[6, 7].
Success rates were above 90% for specimens above 250 copies HIV RNA/mL in one
study and for specimens above 200 copies in the other. For specimens with
detectable viral loads below these levels successful sequencing results were still
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near 75%. In addition the paper by Santoro et al showed that similarly successful
rates were obtained with non‐B subtype infections.
Each of these studies provides a robust practical experience with data from a large
number of subjects and plasma samples (6,617 plasma samples with 50‐1000 copies
HIV RNA/mL were studied between the two studies) over periods of over a decade,
and they provide evidence for beneficial clinical outcomes as a result of their drug
resistance testing at low levels of plasma HIV RNA. Gonzalez‐Serna et al found that
8% of 212 treatment naïve patients had evidence of transmitted drug resistance.
That study also showed that the detection of acquired drug resistance was
predictive of subsequent treatment failure. Both studies showed a range of
resistance mutations to different classes of antiretroviral drugs and the benefits of
using such results to guide the design of subsequent regimens is well established[8,
9].
The results are subject to several limitations, which the authors themselves
acknowledge. One concern about either phenotypic or phenotypic drug resistance
testing with low viral loads has been whether the results from the amplified RNA
reflected the population of genetic variants in a representative manner. Reverse
transcription of any RNA population followed by PCR amplification reflects only a
minority of that population. When the population tested is small, for example in the
hundreds, the amplified products may include a selected subset of the population
that is not representative. In fact in the Gonzalez‐Serna et al study, the proportion
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of sequence reads without evidence of nucleotide reads with more than one base
supports the likelihood of amplification of a single molecule. The concern thus
remains about whether the resulting sequence reflects an unrepresentative
“founder effect”; however, Figure 3 in Gonzalez‐Serna et al shows a substantial rate
of resistance even in those samples from which only a single molecule was likely
amplified, and even these results were predictive of treatment failure.
Another issue is that these two highly experienced and specialized laboratories used
“in house” methods, rather than the standard FDA‐approved platforms that generate
the majority of results in most developed countries. The British Columbia approach
was to implement a second try on those samples that fail to generate results,
initially by using alternative primers and a shorter amplicon. The value of this
modification became progressively more important the lower the level of viremia in
the specimen. The Italian approach was to concentrate RNA from a larger volume of
plasma by centrifugation along with a nested amplification for those specimens
negative on the first round. There too, the proportion of positives only after the
nested PCR amplification progressively increased with lower levels of viremia.
Nevertheless, both studies provide robust data showing the practical feasibility of
these approaches for the clinical management of large numbers of patients. The
results generated also proved useful to inform and improve clinical management.
Moreover, results obtained earlier during treatment failure can help guide
modifications of regimens before additional resistance accumulates. These studies
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thus provide an impetus for investigators designing drug resistance assays and for
laboratories providing diagnostic services to extend drug resistance assays to
specimens with lower levels of viremia.
Implementing the conclusions from these two studies will expand the proportion of
patients who will benefit from HIV drug resistance testing. In addition, by earlier
detection of failure and resistance, interventions can be made before the continuing
accumulation of resistance would inevitably occur. As we learn how better to
manage failure, it is ironic, but not unwelcome, that with the availability of earlier
treatment and with more effective and more tolerable drugs, the proportion of
patients failing treatment is substantially diminishing in developed countries.
The increasing challenges regarding drug resistance are occurring in resource‐
limited settings with the global rollout of antiretroviral therapy now reaching
approximately 10 million people[10]. Substantial benefits on morbidity, mortality,
and likely even transmission are resulting from this effort[10, 11]. Nevertheless, the
reality of laboratory support in such settings is compromising some of the benefits
of the rollout. Current antiretroviral management in resource limited settings
results in the much delayed determination of treatment failure and the progressive
accumulation of drug resistance mutations under the selective pressure of
continuing drug treatment[12, 13]. This accumulation results in higher and broader
antiretroviral resistance, thus diminishing effective treatment options[14]. These
options are further restricted by second line regimens that are much more limited
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than in resource rich countries. There is an urgent need for practical virus load
assays and drug resistance assays that will permit more prompt and informed
detection and management of treatment failure to improve further the benefits of
access to antiretrovirals in the settings where most infections occur.
The author has no reported conflicts of interest. References 1. Hirsch MS, Gunthard HF, Schapiro JM, et al. Antiretroviral drug resistance
testing in adult HIV‐1 infection: 2008 recommendations of an International AIDS Society‐USA panel. Clin Infect Dis 2008; 47(2): 266‐85.
2. Thompson MA, Aberg JA, Hoy JF, et al. Antiretroviral treatment of adult HIV infection: 2012 recommendations of the International Antiviral Society‐USA panel. JAMA 2012; 308(4): 387‐402.
3. HHS. Panel on Antiretroviral Guidelines for Adults and Adolescents. Guidelines for the use of antiretroviral agents in HIV‐1‐infected adults and adolescents. Available at: http://aidsinfo.nih.gov/contentfiles/lvguidelines/AdultandAdolescentGL.pdf. Accessed 12/24/2013.
4. Delaugerre C, Gallien S, Flandre P, et al. Impact of low‐level‐viremia on HIV‐1 drug‐resistance evolution among antiretroviral treated‐patients. PLoS ONE 2012; 7(5): e36673.
5. Taiwo B, Gallien S, Aga E, et al. Antiretroviral drug resistance in HIV‐1‐infected patients experiencing persistent low‐level viremia during first‐line therapy. J Infect Dis 2011; 204(4): 515‐20.
6. Gonzalez‐Serna A, Min J, Woods C, et al. Performance of HIV‐1 Drug Resistance Testing at Low Level Viraemia and Its Ability to Predict Future Virologic Outcomes and Viral Evolution in Treatment‐Naïve Individuals. Clin Infect Dis 2014: in press.
7. Santoro MM, Fabeni L, Armenia D, et al. Reliability and Clinical Relevance of the HIV‐1 Drug‐Resistance Test in Patients with Low Viremia Levels. Clin Infect Dis 2014: in press.
8. Durant J, Clevenbergh P, Halfon P, et al. Drug‐resistance genotyping in HIV‐1 therapy: the VIRADAPT randomised controlled trial. Lancet 1999; 353(9171): 2195‐9.
9. Tural C, Ruiz L, Holtzer C, et al. Clinical utility of HIV‐1 genotyping and expert advice: the Havana trial. AIDS 2002; 16(2): 209‐18.
10. WHO. World Health Organization website on HIV/AIDS. . Available at: http://www.who.int/hiv/data/en/. Accessed 12/24/2013.
11. Tanser F, Barnighausen T, Grapsa E, Zaidi J, Newell ML. High coverage of ART associated with decline in risk of HIV acquisition in rural KwaZulu‐Natal, South Africa. Science 2013; 339(6122): 966‐71.
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12. Stadeli KM, Richman DD. Rates of emergence of HIV drug resistance in resource‐limited settings: a systematic review. Antivir Ther 2013; 18(1): 115‐23.
13. Gupta RK, Jordan MR, Sultan BJ, et al. Global trends in antiretroviral resistance in treatment‐naive individuals with HIV after rollout of antiretroviral treatment in resource‐limited settings: a global collaborative study and meta‐regression analysis. Lancet 2012; 380(9849): 1250‐8.
14. Hosseinipour MC, Gupta RK, Van Zyl G, Eron JJ, Nachega JB. Emergence of HIV drug resistance during first‐ and second‐line antiretroviral therapy in resource‐limited settings. J Infect Dis 2013; 207 Suppl 2: S49‐56.
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