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Performance Characteristics and
Validation of the PhenoSense® Integrase Assay
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Reported by Jules Levin
ICAAC/IDSA Oct 2008 Wash DC
S. Fransen, S. Gupta, W. Huang, C. J. Petropoulos,
L. Kiss and N. T. Parkin
Monogram Biosciences,
South San Francisco
CA USA.
BACKGROUND
Raltegravir (RAL) was approved in late 2007 and is the first integrase strand transfer inhibitor in this new class of anti-retrovirals
Several integrase inhibitors are in clinical development
The PhenoSense HIV Integrase Assay is a rapid, recombinant virus assay capable of measuring the susceptibility of HIV-1 integrase inhibitors, such as RAL, and integrase (IN) associated changes in replication capacity (RC).
Here we report on the technical validation of the PhenoSense HIV Integrase Assay in compliance with CLIA (Clinical Laboratories Improvement Act) specifications.
METHODS:
The PhenoSense PR-RT assay was modified to capture patient-derived C-terminal RT and IN pol gene sequences (Figure 1).
IN RC is expressed as a percentage of viral infectivity (luciferase production) relative to a reference virus.
Assay accuracy, precision, reproducibility, linearity, amplification sensitivity and
specificity were assessed by testing RAL susceptibility using site directed mutant
(SDM) viruses, well-characterized laboratory strains, and patient plasma-derived viruses.
RESULTS:
100% of IC50 FC values, for selected IN SDMs, were within 3 fold of previously reported phenotypic data (Table 1, Figure 2).
100% of pair-wise FC comparisons were within 2-fold for precision, reproducibility and linearity experiments (Table 1).
For IN RC, 94%, 97% and 98% of paired comparisons were within +/-0.25 log10 for precision, reproducibility and linearity, respectively (Table 1).
Amplification sensitivity was successful for 95% of samples with viral loads above 500 copies/mL (Figure 3).
Sensitivity to amplify and phenotype Non Subtype B IN sequences was 96.4%, as determined from a panel of diverse Non Subtype B isolates (A/A1,AE,AG,BF/F,C,D,G) (Table 2).
The biological cutoff for RAL FC was determined to be 1.5 using 500 IN inhibitor naïve patient samples (Figure 4).
In 63 commercial samples tested using PhenoSense Integrase, RAL resistant and sensitive samples were identified (Figure 5).
AUTHOR CONCLUSIONS:
PhenoSense Integrase is an accurate, precise, reproducible assay for assessing IN inhibitor susceptibility and changes in IN RC associated with IN inhibitor resistance.
This assay is performed in the Monogram Clinical Reference Laboratory on samples with viral loads above 500 copies/mL.
PhenoSense Integrase provides clinicians with a tool to aid in the selection and
monitoring of potent antiretroviral combination therapy (Figure 6).
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