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Oxford Team Uses WGS to Show Fidaxomicin Reduces Both Relapse and Reinfection with C. Difficile


A study by researchers from the University of Oxford, has used whole genome sequencing to show that fidaxomicin is superior to vancomycin in preventing both relapse of a single strain of Clostridium difficile and reinfection with a new strain.

The study, published last month in the Journal of Infectious Diseases, relied on whole genome sequencing to identify the number of differing single nucleotide variations between a patient's initial C. difficile infection and a later recurrence in retrospective samples from two randomized clinical trials comparing fidaxomicin to vancomycin.

By cataloguing the number of SNVs differentiating initial and relapse samples, the group was able to categorize these cases into true relapses vs. probable reinfections and then create hazard ratios indicating that fidaxomicin was superior in preventing both types of disease recurrence.

The study's first author, David Eyre of the UK National Institute for Health Research Oxford Biomedical Research Centre, told Clinical Sequencing News in an email that a number of techniques have been used previously to determine if the cause of recurrent C. difficile infections is re-infection with a new strain or relapse of the original infecting strain.

Some genotyping techniques have problems distinguishing between common strains, because a new infection can, by chance, closely mirror the genotype of a previous one, he explained, whereas "whole genome sequencing allows even small differences between isolates that may share a genotype to be distinguished and therefore provides excellent resolution for telling reinfection and relapse apart."

According to Eyre, the Oxford team has previously developed a WGS pipeline tailored to bacterial sequence DNA to support other work exploring transmission of C. difficile and other pathogens like tuberculosis, Staphylococcus aureus, and norovirus.

In the group's recent study, he and his colleagues turned this approach for the first time toward distinguishing between true reinfection and relapse in C. difficile patients.

Eyre and his colleagues performed WGS using the Illumina HiSeq 2000 on paired isolates from 93 subjects enrolled in two pivotal phase III clinical trials of fidaxomicin versus vancomycin, who had a relapse post-treatment — 28 who were randomized to fidaxomicin and 65 who were randomized to vancomycin.

Overall, the trials enrolled 1,164 participants, of which 990 experienced a clinical cure and were assessed for future recurrence, and 199 of which actually had a recurrence after being treated.

According to the study authors, the Oxford team generated 100 base-pair reads, which the researchers mapped to the C. difficile reference genome to identify SNVs.

The team used a cutoff of five reads and a consensus of 90 percent or greater high-quality bases in determining which SNV calls to include for further analysis and reported that a median of 84 percent of the reference genome was called across all the isolates in the study.

Overall, about 60 percent of the subjects had no SNVs that differed between their initial infection and recurrence. Seventeen percent had one SNV, 4 percent had two, about five percent had between 4 and 13 SNVs, and the remaining 15 percent had more than 3,000 SNVs that distinguished their initial and recurring infections.

Based on this SNV data, the group then characterized each C. difficile recurrence as a "reinfection", a "relapse," or indeterminate.

The team used a cutoff of two or fewer SNVs between recurrence and original infection to define a true relapse, and more than 10 SNVs difference to define a new-strain reinfection, with differences of three to 10 SNVs making up the indeterminate category.

The researchers then used competing risk survival analysis to compare the impact of fidaxomicin versus vancomycin on the cumulative incidence of these different types of recurrence. Based on the SNV cutoffs the group used, the team calculated that only four percent of participants treated with fidaxomicin had true relapses of their initial strain compared to 10 percent of those on vancomycin. Meanwhile, less than one percent of fidaxomicin treated patients had a new-strain reinfection versus two percent of those treated with vancomycin.

According to Eyre, the results — along with growing numbers of studies utilizing whole genome sequencing in clinical microbiology — demonstrate the "real potential of WGS to play a major role in the microbiology lab of the future."

Eyre said in his email that fidaxomicin's association with decreased risk of disease relapse has been thought to be mostly due to the fact that the drug is less disruptive of the intestinal microbiome, and thus allows normal flora to return, preventing relapse of an original infection.

However, by taking this whole genome sequencing approach, the group was able to show that fidaxomicin also seems to be protective against re-infection with new strains, possibly for the same reason, Eyre wrote.

Additionally, the group's sequencing approach also identified mutations arising in the target site for fidaxomicin, a nod to the potential of whole genome sequencing as a tool for monitoring the emergence of known and novel antimicrobial resistance variants, he said.