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Baseline Clinical HIV Genotypes Are a Valid Measure of Transmitted Drug Resistance Within the Treatment-Naive Population

Ragonnet-Cronin, Manon MSc*; Lee, Bonita E. MD, FRCPC; Plitt, Sabrina S. PhD; Zahariadis, George MD, FRCPC§,‖; Merks, Harriet BSc*; Sandstrom, Paul A. PhD*; Brooks, James I. MD, FRCPC*,¶,‖

JAIDS Journal of Acquired Immune Deficiency Syndromes: December 15th, 2013 - Volume 64 - Issue 5 - p 443–447
doi: 10.1097/QAI.0b013e3182a4b991
Brief Report: Basic and Translational Science

Objective: To examine whether baseline clinical genotypes are equivalent to diagnostic serum genotypes for surveillance of HIV transmitted drug resistance (TDR).

Design: Current HIV TDR surveillance in Canada is conducted through genotyping remnant diagnostic sera from new HIV diagnoses. As part of routine care, baseline genotyping is now conducted on all newly diagnosed HIV infections, with TDR data being generated a second time on the same patients.

Methods: Surveillance genotyping, on HIV diagnostic serum, was performed on newly diagnosed HIV cases from 2007 to 2010 in Alberta, Canada. All subjects with a baseline clinical genotype result on file, and no evidence of antiretroviral therapy, were studied further. The HIV sequences from diagnosis and from the first clinical genotype were compared according to elapsed time between testing and by evaluating timing of infection based on BED capture enzyme immunoassay (BED-CEIA, abbreviated as BED in this article).

Results: Eighty-seven genotype pairs were available for analysis, most of which were subtype B. The time between genotypes ranged from 0 to 755 days, with a median of 36 days and an interquartile range of 155.25 days. Genetic distance between genotypes varied between 0 and 0.03389 substitutions per site and did not correlate with sampling times. There was a tendency for the genotypes of infections classified as recent by BED to be more similar to their clinical genotypes but this effect was lost when adjusted for elapsed time between tests. There was no difference in the identified drug resistance.

Conclusions: Baseline clinical genotypes from treatment-naive patients may be used for HIV TDR surveillance.

*National HIV and Retrovirology Laboratories, National Microbiology Laboratory, Public Health Agency of Canada, Ottawa, Canada;

Division of Infectious Diseases, Department of Pediatrics, University of Alberta, Alberta, Canada;

Surveillance and Epidemiology Division, Public Health Agency of Canada, Ottawa, Canada;

§Division of Infectious Diseases, Department of Laboratory Medicine and Pathology, University of Alberta and Alberta Health Services, Alberta, Canada;

Division of Infectious Diseases, Department of Medicine, University of Western Ontario, London, Canada; and

Division of Infectious Diseases, Department of Medicine, University of Ottawa, Ottawa, Canada.

Correspondence to: James I. Brooks, MD, National HIV and Retrovirology Laboratories, Room 3172, Building 6, 100 Eglantine Driveway, AL0603A2, Tunney's Pasture, Ottawa, Ontario K1A 0K9, Canada (e-mail:

Supported by the Federal Initiative to Address HIV/AIDS in Canada.

The authors have no conflicts of interest to disclose.

Received February 22, 2013

Accepted July 11, 2013

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Since the 1990s, an increasing number of antiretroviral drugs have become available for the treatment of HIV. However, under selection pressure, drug-resistant mutations (DRM) appear, allowing the virus to continue to replicate and increasing the likelihood of treatment failure. Once present in the virus, durable DRM may then be transmitted to drug-naive patients, rendering them unresponsive to first-line therapies. Surveillance for transmitted drug resistance (TDR) is important for predicting efficacy of current treatment regimens and for informing policy decisions in resource limited settings where national programs select fixed antiretroviral therapy (ART) combinations.1–4

HIV drug resistance surveillance programs typically use genotyping data produced from specimens collected for clinical reasons. Evaluating data collected in a nonsystematic manner, from different programs, presents a challenge as it is often biased by patient subpopulations with specific ART regimens.5 Historically, there has also been concern about the durability of DRM with the belief that by definition, drug-resistant HIV variants would be less fit and that the DRM would rapidly change over time.6,7

Established in 1998, the Canadian National Strain and Drug Resistance Surveillance Programme was designed to estimate TDR by genotyping remnant serum of all new HIV diagnoses within the country.8 This method of specimen collection for TDR surveillance has the theoretical advantage of capturing all new HIV diagnoses, thus avoiding sample bias. In addition, by genotyping as close as possible to transmission, mutations that could later be lost because of fitness costs would also be captured. However, soon after this program was established, calls were being made for routine baseline genotyping of all patients.9–12 With the combination of widespread commercial availability of HIV genotyping facilities and the recommendation for baseline genotyping in guidelines, this has since become the standard of care.13,14

At present, patients in Canada may have baseline genotyping performed twice: once for national HIV TDR surveillance using the first diagnostic serum sample and again for baseline clinical genotyping on plasma samples at the time of introduction to clinical care. This study was designed to address the following questions: (1) Does the surveillance program provide additional data on TDR not detected by clinical genotyping and (2) Are the surveillance and clinical genotypes sufficiently comparable to use clinical baseline genotyping as the basis for national TDR surveillance?

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For surveillance genotypes, remnant HIV diagnostic serum specimens from the province of Alberta for the years 2007–2010 were amplified using in-house primers and sequenced on an ABI 3130XL (Life Technologies, Burlington, Ontario, Canada) with base-calling and assembly performed using SeqScape (Life Technologies)15 or else genotyped using the Trugene assay (Siemens, Deerfield, IL). Inclusion criteria for the study were cases with both genotyping data generated through the national surveillance program using serum samples and baseline clinical genotyping using plasma samples. The cases were treatment naive at diagnosis and presumed to be treatment naive at the first clinical encounter. If specimens were determined to be from cases likely to be treated immediately after diagnosis, such as pregnant women, they were excluded from analysis. The duration of time between initial diagnosis and clinical genotyping was obtained for each case. When sufficient remaining specimen was available, BED capture enzyme immunoassay testing was performed to determine whether the remnant specimen was recent (<155 days from infection) or late (>155 days).16

Clinical genotypes were performed by a company on frozen plasma specimens (using custom primers) sequenced on an ABI3730 instrument (Life Technologies) with automated base-calling and assembly performed using ReCall.17

Genetic distance between pairs of samples was calculated using a GTR + Γ model as selected by jModelTest,18 and the phylogenetic relationships between sequences were reconstructed using the maximum likelihood algorithm in FastTree with 100 bootstrap replicates.19 Sequence subtype was determined using the Rega HIV-1 subtyping tool ( and sequences were tested for the presence of DRM using the Stanford Calibrated Population Resistance Tool20 ( The Calibrated Population Resistance Tool identifies DRM based on the standardized Surveillance of Drug Resistance Mutations (2009) list.21 DRM are classified as impairing the efficacy of nucleoside reverse transcriptase inhibitors, nonnucleoside reverse transcriptase inhibitors, or protease inhibitors.

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A total of 78 cases were included in the study. Among these, 55 tested as subtype B, with subtype C being the next most frequent (13), followed by 2 AE subtypes, and 1 each of A1, AG, and G. Pol sequences were trimmed to identical lengths, covering 897 bp, including the protease gene (codon positions 4–99) and a portion of reverse transcriptase (codon positions 38–240). The absence of 3 codons in protease gene and 37 in reverse transcriptase is an artifact of the Trugene sequencing procedure.

Time between HIV diagnosis and baseline clinical genotyping ranged from 0 to 755 days, with a median of 36 days and an interquartile range of 155.25 days. Phylogenetic analysis demonstrated that paired sequences always clustered together with high bootstrap values (Fig. 1). Genetic distance between pairs of samples varied between 0 and 0.03389 substitutions per site and did not correlate with time between the 2 samplings (r = 0.1608; Fig. 2). There was no difference among subtypes in the genetic distance between genotypes (data not shown).





Of the 78 samples, 56 were classified as long-term by BED and 15 as recent. Specimen volume was insufficient for the remaining 7 samples to be tested. Time between sampling correlated slightly better with genetic distance in the BED recent samples (r = 0.12 vs. r = 0.001) but the difference was not statistically significant (Fisher test of bivariate correlation, P = 0.711).

In both datasets, identical DRM were identified in 10 patients (12.8%): 1 protease inhibitors (L90M), 2 nucleoside reverse transcriptase inhibitors (V75M), and 8 nonnucleoside reverse transcriptase inhibitors (K103N). Two of these mutations were found in a single patient (L90M and K103N). The TDR prevalence observed in the province as a whole over the same period was 12.4% (unpublished national HIV TDR surveillance data, Dr. James Brooks, National HIV & Retrovirology Laboratories, 2013), which was not different to the cohort.

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Although more than 30,000 people tested positive for HIV in Canada between 1996 and 2008,22 sequences from only 7000 have been used for the estimation of TDR rates.23 In contrast, centralized databases storing clinical genotypes in the United Kingdom, Switzerland, and Italy contain 25,000,24 12,000,25 and 23,00026 sequences, respectively. More than 90% of new diagnoses in some jurisdictions in Canada undergo baseline clinical genotyping.27 Because of the single payer health care system in this country, specimens and results both flow through a central provincial laboratory in each region. These data could be easily collected, analyzed, and then used provincially and potentially shared with both national surveillance programs and local clinics. Use of these data, if validated, would allow for better estimation of local, provincial, and national TDR and facilitate large-scale phylodynamic analyses of HIV in Canada.

HIV evolves rapidly after transmission in response to immune pressure from the new host. However, some TDR mutations have been demonstrated to persist over time.12 Follow-up studies of patients infected with TDR strains demonstrate the persistence of lower fitness cost mutations, such as K103N and L90M,28,29 for 1,30 2,31 or over 312 years, whether or not patients received treatment.31 In agreement with these findings, we show no change in the DRM identified at the time of diagnosis with those found at the time of the first clinical sample, despite a time lapse of up to 755 days between the 2 samplings. Sequences from matched genotypes clustered closely with low genetic distance and high bootstraps. These results suggest that accurate TDR surveillance could be conducted through analyzing the baseline clinical genotype data collected from a population.

In our study, we found that TDR remained concordant between groups classified as either recent or late by the BED assay, in contrast to earlier studies showing considerable variability in TDR results between newly or chronically infected individuals.32,33 There are at least 2 reasons for the consistency in our findings. First, 70% of new HIV diagnoses in Canada are made among patients in the chronic phase of infection.34 Consequently, virus that harbors resistance associated with a high fitness cost would be expected to already have been outcompeted by the time most patients are diagnosed. Mutations, such as M184V and K65R, that have been shown to be lost rapidly after transmission35 would be predicted to be rare among surveillance specimens. This is indeed what was observed among 6797 drug-naive specimens collected over 10 years, where the prevalence of the M184V and K65R mutations was 0.4% and 0.1%, respectively.23 The inability to detect mutations with a high fitness cost, even using diagnostic specimens for genotyping, argues for earlier diagnosis. Improvements in the detection of mutations with high fitness costs could arrive with earlier diagnosis commensurate with the other public health benefits of earlier diagnosis. Second, it has been shown that HIV TDR is proportional to the amount of ART exposure within a population.36 Among the studies identifying increasing prevalence of drug resistance over time,32,33 the data brackets 2 distinct eras: the earlier group infected when ART was less common and the latter group infected with virus bearing the legacy of serial monotherapy. The durability of observed TDR patterns observed in our study is consistent with the data being collected well into the Highly Active ART era, and the majority of infections being of longer duration. We emphasize nevertheless that these results pertain only to samples from drug-naive patients.

With the improved resolution of next generation sequencing, the role of low frequency minority variant mutations in estimating TDR may show differences between diagnostic and clinical genotypes. Current techniques for drug resistance testing detect mutations only if they are present in at least 20% of viruses in the sample. As some minority variants with fitness costs are outcompeted in chronically infected patients, they are much more likely to be undetectable as time since infection elapses.37 New sequencing technologies, such as pyrosequencing or allele-specific polymerase chain reaction, may offer a solution to this problem with an ability to detect minority variants at frequencies as low as 0.1%.38 Whether our findings will remain valid when the mixture threshold for identifying TDR is lowered still has to be evaluated. It is possible that TDR mutations in integrase will not follow the same patterns observed here. Once integrase resistance testing becomes standard practice, this will have to be assessed.

Limitations to this study include the small sample size because of the inclusion criteria of availability of linkable clinical and surveillance genotypes for each specimen. However, the ratio of recent to chronic infections in our cohort was identical to a larger sample encompassing the same population, suggesting no bias.34 In addition, inclusion in the dataset is predicated upon serological diagnosis of HIV infection effectively excluding acute HIV infections from the dataset, which may contain ephemeral mutations with high fitness costs. Although loss of these specimens from the surveillance set may hamper the detection of all TDR mutations, this loss of resolution is the consequence of a surveillance program based on serologic diagnosis. The preponderance of BED-defined late diagnoses may be expected to bias the results of the study to finding stability among genotypes. Although this may be true, the distribution of timing of infection among specimens included in this dataset is entirely consistent with the proportion of early versus late infections collected in Alberta and observed in the program as a whole.39 Consequently, our findings arise from a dataset that is proportionately representative of chronic infections found in our national HIV TDR surveillance program. Furthermore, as these genotypes, produced from the diagnostic specimens, are collected from all new cases of HIV at the time of diagnosis, it is unclear how one could practically obtain specimens for TDR surveillance from an earlier time point. One final limitation is that over half of paired samples (58 of 76) were separated by less than 100 days, and so it is not surprising that paired sequences were so similar. Nevertheless, this emphasizes the redundancy of performing replicate genotypes.

In conclusions, our results demonstrate for the first time the validity of using baseline clinical genotypes, among treatment-naive patients, as a method for TDR surveillance. This methodology reduces costs to a surveillance program by using data that has already been generated, avoids duplication of genotyping, and, if fully deployed, would increase the number of samples available for TDR estimation and phylodynamic analyses.

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This study was approved by the University of Alberta Health Research Ethics Board (REB). Results from the clinical genotypes were reported to submitting physicians as they were performed. Data for this study were collected retrospectively and the study did not interfere with patient care. As the clinical genotype sequences were anonymized after linkage, and no personal health information was linked to the sequences, the study was exempted from requiring informed consent. De-identified specimens collected for the Canadian HIV Strain and Drug Resistance Surveillance Program Surveillance were collected under public health provisions and the requirement for informed consent waived by the Public Health Agency of Canada REB.

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genotyping; transmitted drug resistance; drug resistance surveillance; clinical genotyping

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