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Team Sees Enhanced Resolution in Epidemiological Analysis of C. difficile Using WGS


NEW YORK (GenomeWeb) – Investigators in Ireland have published a study underscoring the benefits of using whole-genome sequencing in epidemiological analyses of healthcare-associated of Clostridium difficile infections — particularly when differentiating between relapse cases and cases that involve re-infection with another strain.

As they reported recently in the Journal of Hospital Infection, the researchers watched for recurrent C. difficile infections over 18 months at St. James's Hospital in Dublin, identifying 19 patients who had experienced two or more bouts of infection.

"The impetus was to understand this infection and, specifically, to look at patients who had recurrent infection," first author Micheál Mac Aogáin, a post-doctoral researcher in senior author Thomas Rogers' clinical microbiology lab at Trinity College Dublin, told GenomeWeb.

"What we wanted to do was use whole-genome sequencing to characterize the strains causing the recurrent infections at the highest level of genetic resolution," he explained, noting that "there's an increasing awareness in microbiology that the diversity of clinical strains is greater than can be detected by traditional typing methods."

While more traditional PCR-based ribotyping on patient isolates offered hints for detecting relapse events rather than re-infection, Mac Aogáin and his colleagues found that they were able to draw more distinct lines between relapse and re-infection events using information on complete C. difficile genomes.

By taking a look at whole-genome sequences from C. difficile isolates present in each patient's index infection and at recurrence, the team could also trace transmission events between individuals with far more accuracy, ruling out many of the supposed transmission events suspected from C. difficile ribotypes alone.

Such findings suggest whole-genome sequences are particularly useful for making "secure and evidence-based conclusions about things like strain transmission and strain relationships," Mac Aogáin explained.

C. difficile infections tend to occur in healthcare settings amongst individuals (often elderly patients) who have been treated with antibiotics for another condition, Mac Aogáin explained. The bug is notorious for exploiting disruptions in normal gut microbial communities that follow such antimicrobial therapy, producing infections that cause diarrhea, abdominal pain, and other symptoms.

A significant proportion of those who go through one C. difficile infection go on to experience one or more subsequent infections involving the same index strain. But such relapse cases can be tricky to distinguish from forms of recurrence stemming from re-infection by other strains circulating in the patient's environment.

Studies based on traditional typing methods suggest roughly 12 to 35 percent of recurrent infections are due to re-infection, though Mac Aogáin and his co-authors noted that such estimates do not take into account the full genetic variability of the isolates involved, leaving gaps in the epidemiological understanding of recurrence cases.

To explore this in more detail, the group teamed up with St. James's Hospital in Dublin to obtain C. difficile samples collected through a voluntary enhanced surveillance protocol in place since 2012 to monitor both new C. difficile cases and recurrent infections.

The researchers used the Illumina MiSeq to do paired-end whole-genome sequencing on isolates from 19 individuals who experienced multiple rounds of C. difficile infection —between two and seven bouts apiece.

From PCR-based ribotyping profiles on the isolates, the team estimated that at least 27 of the infection events represented cases of relapse due to C. difficile strains already carried by the patient. Another five infection events involved C. difficile ribotypes that were distinct enough from a patient's index strain that they were deemed re-infection events.

The researchers shored up these ribotype-based results using whole-genome sequence data on the isolates, which pointed to the same general relapse and re-infection patterns in the patients.

But the genome sequences told a different story than ribotyping when it came to C. difficile transmission between individuals. Whereas ribotyping data pointed to 10 potential patient-to-patient transmission events, the level of genetic diversity detected in isolates by sequencing prompted the team to rule out six of the proposed transmission events.

Moreover, the whole-genome sequence data suggested only one of the re-infection events was solely due to bugs distinct from those found during an initial infection event, Mac Aogáin said, explaining that "the majority of patients did seem to suffer recalcitrance of the [index] strain."

And while relapse is generally defined as cases that recur in the same individual within eight weeks of initial infection, the researchers saw several recurrent cases caused by genetically similar or identical isolates after much longer time points, suggesting relapse is possible at points beyond two months.

"Recurrent infection should be looked at, possibly, as a more chronic disorder rather than acute," Mac Aogáin proposed. "It may be acutely initiated, but these particular patients may carry the strain for long periods."

Finally, the genome-wide look at patient isolates highlighted the genetic variation that can occur within a given C. difficile strain from the same patient.

It remains to be seen how that genetic diversity may contribute to recurrence and clinical features of the infections, if at all, though researchers suspect such data could eventually help track down genes, pathways, and physiological features that make certain C. difficile strains especially difficult to shake.

Such studies will require much larger patient cohorts followed over longer time frames, Mac Aogáin said, noting that it would be beneficial to do whole-genome sequencing on C. difficile isolates from one-off infections and environmental sources as well as recurrent cases.

Indeed, members of the Trinity College Dublin team have already secured funding for an upcoming study that will involve sequentially sequencing all of the C. difficile isolates identified at St. James's Hospital.

The price tag for sequencing C. difficile isolates in the current study came in at around 150 Euros ($158) per genome, excluding staffing-related costs.

Mac Aogáin, who was involved in coordinating the genome research program at Trinity College Dublin, noted that he is continuing to explore strategies for doing bacterial genome sequencing as affordably as possible.

Because the approach remains relatively pricy, he noted that genome sequencing on C. difficile will likely remain in the epidemiological and research realm for the time being, rather than being applied to point-of-care testing for individual cases.

Still, Mac Aogáin said that may change as the cost of sequencing and analyses continues to decline. He also emphasized the potential of using whole-genome sequencing to characterize other types of infection in the nearer term, including infections such as tuberculosis that involve difficult to culture pathogens.