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BMC Medical Informatics and Decision Making
Published in: BMC Medical Informatics and Decision Making 1/2011

Open Access 01-12-2011 | Research article

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

Authors: Martin Däumer, Rolf Kaiser, Rolf Klein, Thomas Lengauer, Bernhard Thiele, Alexander Thielen

Published in: BMC Medical Informatics and Decision Making | Issue 1/2011

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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.
Appendix
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Metadata
Title
Genotypic tropism testing by massively parallel sequencing: qualitative and quantitative analysis
Authors
Martin Däumer
Rolf Kaiser
Rolf Klein
Thomas Lengauer
Bernhard Thiele
Alexander Thielen
Publication date
01-12-2011
Publisher
BioMed Central
Published in
BMC Medical Informatics and Decision Making / Issue 1/2011
Electronic ISSN: 1472-6947
DOI
https://doi.org/10.1186/1472-6947-11-30

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