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Open Access Research article

Genotypic tropism testing by massively parallel sequencing: qualitative and quantitative analysis

Martin Däumer1, Rolf Kaiser2, Rolf Klein1, Thomas Lengauer3, Bernhard Thiele1 and Alexander Thielen13*

Author Affiliations

1 Institute of Immunology and Genetics, Pfaffplatz 10, 67655 Kaiserslautern, Germany

2 Institute of Virology, University of Cologne, Fürst-Pückler-Str. 56, 50935 Cologne, Germany

3 Max Planck Institute for Informatics, Stuhlsatzenhausweg E1.4, 66123, Saarbrücken, Germany

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BMC Medical Informatics and Decision Making 2011, 11:30  doi:10.1186/1472-6947-11-30

Published: 13 May 2011

Abstract

Background

Inferring viral tropism from genotype is a fast and inexpensive alternative to phenotypic testing. While being highly predictive when performed on clonal samples, sensitivity of predicting CXCR4-using (X4) variants drops substantially in clinical isolates. This is mainly attributed to minor variants not detected by standard bulk-sequencing. Massively parallel sequencing (MPS) detects single clones thereby being much more sensitive. Using this technology we wanted to improve genotypic prediction of coreceptor usage.

Methods

Plasma samples from 55 antiretroviral-treated patients tested for coreceptor usage with the Monogram Trofile Assay were sequenced with standard population-based approaches. Fourteen of these samples were selected for further analysis with MPS. Tropism was predicted from each sequence with geno2pheno[coreceptor].

Results

Prediction based on bulk-sequencing yielded 59.1% sensitivity and 90.9% specificity compared to the trofile assay. With MPS, 7600 reads were generated on average per isolate. Minorities of sequences with high confidence in CXCR4-usage were found in all samples, irrespective of phenotype. When using the default false-positive-rate of geno2pheno[coreceptor] (10%), and defining a minority cutoff of 5%, the results were concordant in all but one isolate.

Conclusions

The combination of MPS and coreceptor usage prediction results in a fast and accurate alternative to phenotypic assays. The detection of X4-viruses in all isolates suggests that coreceptor usage as well as fitness of minorities is important for therapy outcome. The high sensitivity of this technology in combination with a quantitative description of the viral population may allow implementing meaningful cutoffs for predicting response to CCR5-antagonists in the presence of X4-minorities.