In the summer of 2016, the Vall d’Hebron Research Institute (VHIR) announced the installation of a fourth-generation massive DNA sequencing platform, the result of a collaboration effort with Roche Diagnostics. The aim was to use innovative NGS technology to take a step forward in the field of high-precision personalized medicine in virology, cancer or bacteriology, thus reducing diagnosis times and optimizing the treatments of different pathologies. The alliance has begun to bear its first fruits in the clinical realm, showing that massive DNA sequencing is here to stay.
The liver disease research team at VHIR, together with scientists from the Autonomous University of Barcelona, Roche Diagnostics and the Biomedical Research Networking Centre (CIBER) for Hepatic and Digestive Diseases (CIBEREHD) of the Institute of Health Carlos III, have published an article that discusses the potential of NGS technology applied to infectious diseases. In the study published in the journal Clinical Microbiology and Infection, researchers managed to identify the sub-type of the hepatitis C virus that affected the 1,473 patients treated at Vall d’Hebrón Hospital from February 2015 to May 2016.
The goal of the study was to accurately characterize the chronic hepatitis C patient population through a highly sensitive method in order to identify the 67 different sub-types of the virus, discriminating the mixed infections, in which a single individual is simultaneously attacked by more than one virus subtype. After taking blood samples from study participants, the group, led by Drs. Josep Quer and Francisco Rodríguez-Frías used the 454/GS-Junior platform to perform the high-resolution massive sequencing. Roche’s Clinical Bioinformatics Unit later analyzed the resulting data.
Image: 454 Gene Sequencing Machine. Source: Jon Callas (Flickr)
“Next-generation sequencing (NGS) enables thousands of clonal sequences to be obtained from a single sample and is currently the most powerful tool for analyzing viral quasispecies,” state the authors in the article. By their methodology, “an average of 2500 reads (SD 500) for the NS5B region were obtained per patient.” The application of this NGS technology identified 22 different subtypes of the hepatitis C virus, of the 67 total subtypes detected up to now. Furthermore, thanks to the high coverage of the method used (2,000-5,000 sequences/patients), researchers were able to detect mixed infections with a high level of sensitivity; according to the article, 1% of the individuals analyzed were infected by more than one viral subtype of hepatitis C at the same time.
Researchers also used sequencing to determine that genotype G1 was the most prevalent (76.4% of the samples analyzed), while subtype 1b of the Hepatitis C virus, considered endemic, made up the majority (53.6%) of cases, followed by subtype 1a (22.5%). These results showed higher-than-expected heterogeneity, probably because traditional genotyping methods presented much lower resolution than massive NGS technology. According to the article published in Clinical Microbiology and Infection, an age-based distribution of patients suggests that all individuals born before 1975 should be monitored.
Identification of the various virus subtypes affecting patients is another key tool when it comes to choosing the therapy. Today, patients diagnosed with the hepatitis C virus receive direct-acting antivirals (DAA’s), considered a “major milestone” in the treatment of the disease. The response of these inhibitors, that act on non-structural proteins essential to the viral cycle, depends on the virus subtype affecting the individual. This is why scientists indicate that, at present, there is no reason not to apply NGS technology in clinical practice. According to a press release issued by the center, the method presented by Vall d’Hebrón commits no errors in its determination of the hepatitis C virus subtype. Thanks to this accuracy, first-line treatment is effective in 98.5% of the cases, and development of resistances to antiviral therapies is prevented.