PFGE problems highlighted by WGS performance

By Joseph James Whitworth contact

- Last updated on GMT

©iStock/wildpixel
©iStock/wildpixel
WGS has shown the limited discrimination and occasional lack of accuracy of PFGE, according to researchers.

During 2015-2016, they evaluated whole genome sequencing (WGS) as a routine typing tool. It was compared with pulsed-field gel electrophoresis (PFGE), the current standard method.

A total of 2,743 Listeria monocytogenes isolates collected as part of routine surveillance were characterized by PFGE and core genome multilocus sequence typing (cgMLST) extracted from WGS.

They consisted of 770 from humans, 1,688 from food and 285 from the production environment.

cgMLST is a reproducible method that enables strain comparison across laboratories by using standardized nomenclatures of alleles and types.

cgMLST results took nine working days compared with seven for PFGE and this interval can be shortened by improved lab processing and technological advances, said researchers.

It also identified clusters of cases associated with persistent food production environmental contamination that were previously unrecognized.

cgMLST discrimination vs PFGE

The work found discrimination of isolates was significantly higher by cgMLST than by PFGE. cgMLST discriminated unrelated isolates that shared identical PFGE profiles and phylogenetically closely related isolates with distinct PFGE profiles.

In France, human listeriosis has been a mandatory reportable disease since 1999.

Comparison of PFGE and cgMLST for surveillance of Listeria monocytogenes
Comparison of PFGE and cgMLST for surveillance of Listeria monocytogenes

The listeriosis surveillance system relies on the National Public Health Agency, which collects epidemiologic data and consumption histories from patients with laboratory-confirmed L. monocytogenes infection and the National Reference Centre for Listeria (NRCL), which characterizes human and food isolates to detect clusters of genetically related strains.

Food and environmental investigations are under the authority of the Ministry of Agriculture.

In 2015, the NRCL implemented cgMLST as typing method for L. monocytogenes with PFGE typing. Since January PFGE typing has been discontinued and surveillance has relied on cgMLST.

Before WGS, the listeriosis surveillance system categorized L. monocytogenes strains according to their Asc​I-Apa​I PFGE profile frequency in humans.

Smaller clusters identified

Among the 2,743 L. monocytogenes isolates typed, PFGE identified 268 distinct Asc​I-Apa​I combined profiles but cgMLST identified 1,112 CTs.

“With more clusters of smaller size being identified by cgMLST, traditional analytic epidemiology is challenged because of lack of statistical power, and identification of a food source for these small clusters tend to more extensively rely on trace-back or trace-forward investigations, rather than on case–case studies,”​ said researchers.

Within single PFGE types, 1-280 CTs were identified. Conversely, 2-7 PFGE types caused by phage insertions and deletions were found in 58 CTs.

Investigations identified a confirmed food source in three (10%) of 31 PFGE cluster-alerts. A confirmed food source was found in 22 (18%) of 119 cgMLST cluster-alerts.

Among the 37 cgMLST cluster-alerts involving only one human isolate, a cgMLST-matching food source of infection was identified in 10 (27%).

In nine out of 10 of these investigations, the food source was also identified by PFGE, and food strains matched corresponding profiles of human isolates. In three of these 10 investigations, a product withdrawal was issued and probably contributed to preventing further infections.

Listeria linked to local products

Among the 82 cgMLST cluster-alerts involving >2 human isolates, a confirmed source was identified for 12 (15%): in three of 53 cgMLST cluster-alerts that contained only human isolates and in nine of 29 cgMLST cluster-alerts containing >1 food/environmental isolate.

“Implementation of WGS-based surveillance has shown that most clusters involving >2 human isolates with no source of infection identified did not progress over time.

“This finding is consistent with the observation that most listeriosis case clusters in France are linked to local products that have limited production and distribution.”

For cgMLST cluster-alerts containing >1 food/environmental isolate, matching food/environmental isolate(s) provided a strong hypothesis and helped identify the food source.

In five of these 12 investigations, a product recall was issued as a consequence of the investigation and probably contributed to preventing additional infections

“Despite the usefulness of PFGE in identifying clusters of listeriosis cases over the past few decades, the limited discriminatory power of PFGE can indicate that unrelated isolates are indistinguishable, thus leading to identifying and investigating false PFGE clusters-alerts.

“The increased discriminatory power of cgMLST enabled identification of such false clusters-alerts.”

Source: Emerg Infect Dis. 2017 Aug

DOI: 10.3201/eid2309.170336

“Real-time whole-genome sequencing for surveillance of Listeria monocytogenes, France”

Authors: Moura A, Tourdjman M, Leclercq A, Hamelin E, Laurent E, Fredriksen N, et al

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