PHOENIX — While genotyping can provide key microbiological information and strengthen epidemiological evidence in foodborne illness outbreaks, there are still some limitations in current technology and the very nature of the pathogens/parasites themselves can make labwork difficult.

Scientists whose work involves learning more about pathogens and parasites that cause such outbreaks discussed current issues today at the International Association for Food Production’s annual meeting, a hybrid event with online sessions and a conference in Phoenix.

The session, “Tracing Back to the Source: Challenges to Link Parasite and Viral Genotypes between Outbreak Clinical Samples and On-farm Environmental Sources of Contamination,” focused on hepatitus A, norovirus, cyclospora and cryptosporidium.

Lee-Ann Jaykus, of  North Carolina State University’s Department of Food, Bioprocessing and Nutrition Sciences in Raleigh, said hepatitis A transmitted via food globally often comes from frozen berries. Although those outbreaks are typically traced to an importer and country of origin fairly quickly, finding the precise source is difficult, in part because frozen berries are typically eaten after the season in which they were harvested. That’s compounded by the likelihood that the processor pooled berries from a number of growers on the production line.

Complex supply chains are often another hurdle in tracking the origins of foodborne illnesses linked to fresh and processed produce, she said.

Once a supplier-grower has been identified, investigators have a list of usual suspects to target.

“When you look at good agricultural practices associated with the production of fresh produce, we oftentimes use the ‘four Ws:’ water, waste, wildlife and workers, as the source of contamination,” Jaykus said.

She led session participants through the process of detecting enteric viruses with the commonly-used ISO (International Organization for Standardization) methodology. Researchers, however, often “play around” with different methods during outbreak investigations to increase chances of finding the target nucleic acid they’re looking for in the sample.

Jaykus discussed interpreting RT-qPCR (reverse transcription-quantitative polymerase chain reaction) results. The process is common, but Jaykus said there is a possibility of a false positive if cross contamination and other factors aren’t ruled out. Detections of a nucleic acid with this method is not proof an infectious virus is present.

“There’s a debate as to whether the reliance on PCR-based methods can result in over-estimation of public health risks,” Jaykus said. “We could have an entire symposium on that.”

Next-generation methods for foodborne illness surveillance include whole genome sequencing and meta bar-coding, she said.

Alexandre da Silva, research microbiologist at the Food and Drug Administration’s Center for Food Safety and Applied Nutrition, discussed cyclosporiasis outbreaks, with a focus on Cyclospora cayetanensis, first classified as a human pathogen in 1994.

Advances such as those in bacteriology haven’t been seen in the methodology used to detect and trace cyclospora, da Silva said, but available methods have been successful, he said.

Da Silva’s presentation included information on cyclospora’s life cycle, and he touched on specific outbreaks, from almost 1,000 lab-confirmed cases linked to imported raspberries in 1996, basil grown in Missouri in 1999, romaine lettuce in 2013 and bagged salads in 2020.

While developing methods to detect cyclospora in produce is needed, he said lab capacity is also critical.

“We also need to make sure there are laboratories capable of using all this methodology they’re developing,” he said. “Otherwise, all those efforts are not going to produce the outcomes that we want.”

He said FDA has established a number of labs that can specialize in cyclospora and the agency was in the process of training researchers, but the COVID-19 pandemic put the program on hold.

Rachel Chalmers, a cryptosporidium expert with Public Health Wales, said a 2014 global ranking of foodborne illness caused by parasites by the Food and Agriculture Organization and World Health Organization placed Cryptosporidium spp. linked to fresh produce, fruit juice and milk at No. 5. Twelve of the top 20 foodborne illness outbreaks from parasites were from on-farm environmental contamination of fresh produce.

Genotyping parasites  helps shed light on the extent of an outbreak, what the route of transmission is and precise interventions that are needed. Genotyping can help strengthen the association with the implicated food, she said.

“The genotyping data can help refine the epidemiological analysis, and also make better use of surveillance data to identify further cases and to identify outbreaks themselves,” Chalmers said.

Challenges faced in a typical foodborne illness outbreak investigation, she said, include:

• Obtaining relevant samples, whether from the farm or other locations in the supply chain, to clinical isolates;
• Coping with a high number of tests and the rapid feedback, especially during the escalation phase of the outbreak;
• Good communication between labs, epidemiological investigators, on-site inspectors/samplers and others;
• Clear external communications with business owners, suppliers, media and the public; and
• Having the right tools to prepare samples and put them through the genotyping process.

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