Determining HIV-1 tropism in routine clinical practice
Anna Maria Geretti and Nicola Mackie, 18 Dec 2009
Level of evidence
 Ia: systematic review or meta-analysis of randomised controlled trials
 Ib: at least one randomised controlled trial
 IIa: at least one well-designed controlled study without randomisation
 IIb: at least one well-designed quasi-experimental study, such as a cohort study
 III: well-designed non-experimental descriptive studies, such as comparative studies,
correlation studies, case–control studies and case series
 IV: expert committee reports, opinions and/or clinical experience of respected authorities
Schunemann H, Oxman A, Brozek J, Glasziou P, et al. Grading quality of evidence and strength of
recommendations for diagnostic tests and strategies. BMJ 2008;336:1106-1110.
Introduction
 The use of co-receptor antagonists in clinical practice requires tropism diagnostics
to predict when such an agent will be active by ruling out clinically relevant
CXCR4-using virus in a patient’s viral population. Importantly, the threshold for
clinically relevant levels of CXCR4-using virus in a viral population has not as yet
been conclusively established.
 HIV-1 tropism may be determined phenotypically, by assessing the ability of a
recombinant virus containing patient-derived envelope sequences to infect CCR5
or CXCR4 reporter cell-lines that also express CD4. It may also be inferred
genotypically from the sequence of the V3 loop. Both methods have advantages
and drawbacks.
 Among phenotypic methods, the original Trofile assay (Monogram Biosciences)
proved to be reliable for screening patients for inclusion in clinical trials of CCR5
antagonists [1-6]. The assay showed a sensitivity of detection for CXCR4-using
virus of 10% when using clone mixtures [7]. In 2008, an enhanced Trofile assay
(ESTA) replaced the original assay, with a nominal 0.3% lower limit of sensitivity
for detecting CXCR4-using virus when using clone mixtures [7]. Sensitivity with
clinical samples is unknown and is expected to vary with the plasma viral load.
ESTA was found to more accurately identify patients likely to benefit from CCR5
antagonists in post-hoc re-analyses of clinical trials of vicriviroc in treatmentexperienced patients, and maraviroc in treatment-naïve patients, both of which
used the original Trofile assay to screen patients for inclusion [7, 8]. These
retrospective analyses fall short of a full assay validation. Further studies in
different clinical scenarios are required to fully determine the clinical utility of
ESTA. Both the Trofile assay and ESTA have a minimum viral load requirement
of 1000 copies/ml for reliable amplification and cannot be applied to patients with
low or undetectable viraemia. Other phenotypic assays have been developed that
show generally good but not complete concordance with Trofile [9]. Phenotypic
assays are complex, must be centrally provided, and typically exhibit high cost
and long turnaround times.
 Genotypic systems use bioinformatic tools to predict tropism from gp120 V3
sequences. Examples include position-specific scoring matrices (PSSM) and
Geno2Phenocoreceptor. The latter can also incorporate clinical parameters including
nadir CD4 count, CD8 count and viral load, to improve predictive power for
CXCR4-using virus. Genotypic tropism tests are widely available, inexpensive
and rapid to perform. They typically show a lower limit of sensitivity for detection
of CXCR4-using virus of approximately 10%. Adjustments such as repeat testing
1


of individual patient samples, combining several algorithms together, changing
the assay threshold parameters, and incorporating clinical parameters as well as
genotypic data from areas outside the V3 loop significantly improve performance
[10-13]. Concordance with phenotypic tests was initially low [14] but more recent
data indicate excellent concordance with ESTA [15]. It should be noted however
that while concordance between assays is desirable, the comparative analyses are
to be interpreted with caution in the absence of a gold-diagnostic standard.
Emerging data indicate that genotypic tests using bioinformatically-derived
algorithms, particularly the geno2phenocoreceptor method, are comparable with the
original Trofile assay in predicting virological outcomes to maraviroc in
treatment-experienced patients [16, 17]. Data from naïve patients are expected to
show similar performance [R. Harrigan - personal communication]. One
advantage of genotypic tropism testing is the ability to circumvent the high plasma
viral load requirement of phenotypic assays, and evaluate tropism in virologically
suppressed patients using proviral DNA. There is limited evidence to indicate that
genotypic testing of proviral DNA may actually provide better concordance with
phenotypic tropism prediction than genotypic analysis of plasma [18]. Prospective
outcome data for the use of proviral DNA however are currently limited to small
case series [19, A.M. Geretti - unpublished observation].
There is limited evidence in support of the notion that in treated patients, a
tropism test result obtained prior to virological suppression remains unchanged
during suppression [20] and can be used to guide a subsequent treatment switch
when viraemia is suppressed.
Recommendations
1. Currently, the original Trofile (no longer available) and genotypic tropism testing
(performed and interpreted under specific conditions) have the highest level of
supporting clinical evidence (Ib). The two methods show identical performance in
treatment-experienced patients and are expected to show similar performance in
naïve patients. There is more limited clinical evidence supporting ESTA (IIa). On
balance, genotypic testing offers a more easily accessible, rapid and inexpensive
method for tropism diagnostics than phenotypic testing, and is therefore the
preferred option (IV).
2. ESTA can only be performed in patients with viral load >1000 copies/ml.
Currently, the recommended sample for genotypic tropism testing is plasma in
patients with viral load >500 copies/ml (IIb) and proviral DNA in patients with
low-level or undetectable viraemia (IV). The clinical utility of proviral DNA
testing should be monitored closely as supporting evidence is limited (IV).
3. In drug-naive patients, tropism testing may be considered prior to starting firstline HAART, especially in patients who are at recognised risk of ARV toxicity
and may need to switch treatment (IV).
4. In treated patients, tropism testing can be performed in the last sample with a
plasma viral load >500 copies/ml [III]. The patient’s virological and clinical
status since the sample was obtained should be reviewed to ensure consistent
suppression of viraemia and no evidence of immunological or clinical
deterioration [IV]. Alternatively, the tropism can be determined in patients with
suppressed viraemia using proviral DNA (III).
5. Detection of CXCR4-using virus at any time should be considered long-lasting. In
patients with R5 virus and ongoing viraemia, it is currently unclear for how long
2
the result should be considered valid although a 90 days cut-off has been
commonly applied. In patients with high risk of emergence of CXCR4-using virus
(based on nadir and current CD4 count, clinical status and treatment experience),
the test should be repeated (III).
6. In patients failing therapy, tropism test results should become available at the
same time as resistance test results whenever use of CCR5 antagonists may be
considered (IV)
7. Laboratories undertaking genotypic tropisms testing should do so under quality
assurance schemes and according to current consensus about optimal testing
method and interpretation, being aware that the details of the recommendations
about methodology and interpretation are likely to continue to evolve over time
(IV).
References
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
Whitcomb JM, Huang W, Fransen S, et al. Development and characterization of a novel singlecycle recombinant-virus assay to determine human immunodeficiency virus type 1 coreceptor
tropism. Antimicrob Agents Chemother 2007;51:566-575.
Gulick RM, Lalezari J, Goodrich J, et al. Maraviroc for previously treated patients with R5 HIV-1
infection. N Engl J Med 2008;359:1429-1441.
Fätkenheuer G, Nelson M, Lazzarin A, et al. Subgroup analyses of maraviroc in previously treated
R5 HIV-1 infection. N Engl J Med 2008;359:1442-1455.
Saag M, Goodrich J, Fatkenheuer G, et al. A double-blind, placebo controlled trial of maraviroc in
treatment-experienced patients infected with non-R5 HIV-1. J Infect Dis 2009;199:1638-1647.
Gulick RM, Su Z, Flexner C, et al. Phase 2 study of the safety and efficacy of vicriviroc, a CCR5
inhibitor, in HIV-1-Infected, treatment-experienced patients: AIDS clinical trials group 5211. J
Infect Dis 2007;196:304-312.
Soriano V, Perno CF, Kaiser R, et al. When and how to use maraviroc in HIV-infected patients.
AIDS 2009;23:2377-2385.
Reeves JD, Coakley E, Petropoulos CJ, Whitcomb JM. An enhanced-sensitivity Trofile HIV
coreceptor tropism assay for selecting patients for therapy with entry inhibitors targeting CCR5: A
review of analytical and clinical studies. J Viral Entry 2009;3:94-102.
Su Z, Gulick RM, Krambrink A, et al. Response to vicriviroc in treatment-experienced subjects, as
determined by an enhanced-sensitivity coreceptor tropism assay: reanalysis of AIDS clinical trials
group A5211. J Infect Dis. 2009;200:1724-1728.
Skrabal K, Low AJ, Dong W, et al. Determining human immunodeficiency virus coreceptor use in
a clinical setting: degree of correlation between two phenotypic assays and a bioinformatic model.
J Clin Microbiol. 2007;45:279-284.
Moores A, Thielen A, Dong W, et al. Improved detection of X4 virus by V3 genotyping:
application to plasma RNA and proviral DNA. Antiviral Therapy 2008;13(Suppl 3):A99.
Chueca N, Martin L, Alvarez M, et al. A combination of bioinformatic tools can be accurately
used for the screening of co-receptor usage in clinical samples. Antiviral Therapy 2008;13(Suppl
3):A106.
Poveda E, Seclen E, Gonzalez MM, et al. Improvement of genotypic algorithms to predict HIV-1
tropism in treatment-experienced patients - correlation with Trofile results. Antiviral Therapy
2008;13(Suppl 3):A109.
Thielen A, Harrigan PR, Low AJ, et al. Improved genotypic prediction of HIV-1 coreceptor usage
by incorporating V2 loop sequence variation. Antiviral Therapy 2008;13(Suppl 3):A100.
Low AJ, Dong W, Chan D, et al. Current V3 genotyping algorithms are inadequate for predicting
X4 co-receptor usage in clinical isolates. AIDS. 2007;21:F17-24.
Strang AL, Cameron J, Booth C, Garcia A, Geretti AM. Genotypic co-receptor tropism:
correlation with enhanced Trofile. 7th European HIV Drug Resistance Workshop. March 25-27,
2009;Stockholm, Sweden. Abstract 80.
Harrigan PR, McGovern R, Dong W, et al. Screening for HIV tropism using population-based V3
genotypic analysis: a retrospective virological outcome analysis using stored plasma screening
samples from MOTIVATE-1. XVIII International Drug Resistance Workshop. June 9-13, 2009.
Fort Myers, Florida. Abstract 15.
3
17. Harrigan PR, McGovern R, Dong W, et al. Screening for HIV tropism using population-based V3
genotypic analysis: a retrospective virological outcome analysis using stored plasma screening
samples from MOTIVATE-1. 5th International AIDS Society Conference on HIV Pathogenesis,
Treatment, and Prevention. July 19-22, 2009. Cape Town, South Africa. Abstract WeLBA101.
18. Morand-Joubert L, Flandre P, Soulie C, et al. Higher detection of CXCR4 tropic virus by
genotypic coreceptor analysis in peripheral blood mononuclear cells (PBMC) than in plasma. 7 th
European HIV Drug Resistance Workshop. March 25-27, 2009;Stockholm, Sweden. Abstract 51.
19. Obermeier M. The Berlin maraviroc cohort- Influence of genotypic tropism testing results on
therapeutic outcome. 7th European HIV Drug Resistance Workshop. March 25-27,
2009;Stockholm, Sweden. Abstract 79.
20. Waters L, Scourfield A, Marcano M, Gazzard B, Nelson M. The evolution of co-receptor tropism
in patients interrupting suppressive HAART. 16 th Conference on Retroviruses and Opportunistic
Infections. February 8-11, 2009;Montreal, Canada. Abstract 439a.
4