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In Dutch Study, Pathogenica NGS HAI System Compares Well to Standard Typing Methods

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NEW YORK (GenomeWeb) — A study by researchers from two Dutch medical centers and pathogen sequencing company Pathogenica has found that the company's HAI BioDetection kit compared well with the standard method used to type multi-drug resistantEscherichia coli.

The study, published last month in the Journal of Clinical Microbiology, showed that Pathogenica's test typed E. coli infections with similar accuracy to the amplified fragment length polymorphism (ALFP). According to the group, the assay — which uses Life Technologies' Ion Torrent PGM to detect 12 pathogenic bacterial species responsible for 95 percent of hospital-acquired infections — correctly identified 54 of 56 AFLP-identified E. coli isolates and clustered them similarly to the standard method.

Additionally, the group reported that the method was able to detect the presence of drug-resistance genes, but was unable to discriminate between some of these different genes.

The researchers in the recent study — from The Netherlands' Amphia Hospital and VU University Medical Center — make up one of two Dutch teams evaluating Pathogenica's test for the surveillance and monitoring of infectious bacteria. The other, led by John Rossen at the University Medical Center Groningen, is evaluating the test prior to potentially adopting it for clinical use as an active surveillance tool on direct stool and rectal swab samples.

Last summer, Pathogenica announced that it achieved a CE-IVD marking for the test, enabling it to move from research-use only into the clinical market in Europe. Currently, the HAI BioDetection kit is RUO in the US, but company officials have said that they are interested in securing US Food and Drug Administration clearance.

Jacobien Veenemans, the corresponding author of the recent Amphia performance study, said in an email to Clinical Sequencing News that his group's evaluation of the test focused on cultured isolates, using DNA from bacterial colonies cultured from strains stored frozen prior to being analyzed.

The Amphia effort has also been mainly focused on comparing the kit's ability to type E. coli with the same accuracy as the group's current standard method, AFLP.

According Veenemans and his colleagues, AFLP, an enzyme amplification method, has "good discriminatory power, but is relatively expensive, time-consuming, and technically demanding." Because sequencing has the potential to detect resistance genes in addition to typing infections, the group believes that it will eventually replace methods like AFLP, and thus decided to evaluate the Pathogenica assay to see if it could type as accurately as their current standard.

In the study, the group tested the sequencing method on a collection of 56 E. coli isolates from two nursing homes collected as part of a 2013 survey of patients following up on an outbreak the previous year that originally included nine centers.

AFLP identified five clusters among the 56 E. coli isolates. Pathogenica's test was able to identify 54 of the isolates as E. coli, while sequence results could not be generated for the other two due to sample problems, the group wrote. Also, the phylogenetic tree generated from the sequencing test "largely agreed with the clusters defined by AFLP," according to the authors. However, for several isolates, specifically from two patients, the results of the two typing methods yielded discordant results.

Overall, though, "the BioDetection phylogenetic tree correctly reflected the strong relatedness of all isolates belonging to the large outbreak cluster identified by AFLP, as well as the relatedness of isolates belonging to the smaller AFLP clusters," the authors wrote.

Though not the main goal of the study, the Amphia team also looked at the test's ability to distinguish genes associated with drug resistance, comparing the sequencing calls to results of a gold-standard microarray method.

According to the group, though the assay did detect drug-resistance genes, it missed a CTX-M beta-lactamase gene in one of the isolates and was not able to discriminate between genes from the 18 different CTX-M-groups. The Pathogenica kit also did not discriminate between ESBL and non-ESBL TEM beta-lactamase genes, the authors wrote.

According to the researchers, this suggests that "when the assay is used to screen direct patient material for the presence of resistance genes, an additional test [may be] required in case of a positive test result."

Veenemans said in his email that the group has not used the sequencing test to look at any other pathogens, but he and his coauthors wrote that such studies, as well as studies in larger groups of samples, would be valuable to better evaluate the kit.

John Rossen, who is leading the team evaluating the assay at the University Medical Center Groningen, said in an email that results presented by the Amphia team "look very promising."

Rossen's group has been looking more specifically at the test's ability to detect resistance genes, and is using the kit on direct samples rather than cultured isolates. Last week Rossen presented some data from his group's pilot project at the annual meeting of the European Congress of Clinical Microbiology and Infectious Diseases.

In the study, partly funded by Pathogenica, the group tested feces or rectal swabs from 149 patients in one nursing home for the presence of ESBL-producing bacteria using standard methods and the Pathogenica kit in parallel. According to Rossen, the HAI kit is not yet validated for use on direct patient material.

In his presentation, he reported that the study revealed that optimal DNA extraction procedures "appeared to be critical" in the use of the HAI-kit directly on swab samples.

A total of 27 of the 149 samples in the study tested positive using either standard culture methods or the Pathogenica test. "Sensitivity of CTX-M detection was 96.3 percent for culture and 81.5 percent for molecular detection," the group reported. One patient positive by sequencing was not detected by culture and five patients were not detected by the Pathogenica kit but were by culture.

The turnaround times for culture and molecular detection methods were four and two days, respectively, the researchers reported.

Overall, according to Rossen, the sensitivity of the sequencing showed "promising results but is not yet optimal, based on this research." His team's overall pilot efforts with the assay are still ongoing, he told CSN.

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