5 Common Causes of Non-culturable Presumptive (NCP) Pathogen Results and Strategies to Address
by: Christopher Crowe, PhD.
Non-culturable presumptives (NCPs)- samples that yield positive pathogen detection results by PCR screening methods but do not confirm by cultural methods- can be frustrating and disruptive to clients. These situations are not unique to a testing company or the methods that are run, but it is possible to understand the root causes of NCPs, and to identify additional procedures to reduce the frequency with which they occur and minimize the likelihood of these situations impacting your data.
1. Dead cell detection
Why it causes NCPs: In rare instances, samples can contain high numbers of dead pathogens but no living pathogens. PCR detects the DNA from these dead cells, but there are no living cells to grow during confirmation.
What Eurofins is doing: To reduce detection of dead cell DNA, our BACGene PCR platform includes an optional enzyme, PREraser, which is used prior to PCR processing to degrade any DNA from dead cells.
2. Incorrect identification of positive samples by the PCR software
Why it causes NCPs: Positive/negative calls are made by a software algorithm to eliminate subjective interpretation and bias. Occasionally, an erratic or unusual PCR detection curve can “fool” the algorithm into calling a negative sample positive.
What Eurofins is doing: Before releasing results, senior lab staff review and validate all PCR data. While we cannot “overrule” the software, we can advise clients in advance if we see an erratic curve that does not look like a true positive. Additionally, we work with our BACGene PCR software development team to improve and validate the algorithm over time.
3. Laboratory amplicon cross-contamination
Why it causes NCPs: A PCR reaction produces billions of copies of the targeted piece of DNA, known as an amplicon. Due to the high concentration of amplicon in a positive sample, there is a risk that it can contaminate the laboratory environment, and potentially contaminate a subsequently run sample.
What Eurofins is doing: Our laboratories have rigorous sanitation practices specifically designed to eliminate amplicon contamination. Our amplicon monitoring protocols are similar to environmental monitoring programs used in food production facilities. While rare, if amplicon is ever detected, additional sanitation and vectoring is performed until the amplicon is eliminated. Any potentially affected client samples are given additional scrutiny by senior laboratory staff to make sure analytical results are effective.
4. Large population of background microorganisms
Why it causes NCPs: The most frequent cause of NCPs is testing of samples that contain large populations of competing microorganisms, such as “dirty” EMP samples (floor drains, etc.) or raw agricultural commodities. PCR can reliably detect pathogens in the presence of background microorganisms, but when cultural confirmation is performed, the background microorganisms overwhelm the pathogen, preventing it from being isolated and confirmed.
What Eurofins is doing: Many historical reference confirmation methods do not perform well in supressing background microorganism growth. As a result, we are continually improving our cultural confirmation methods and adopting demonstrated improvements after appropriate validation.
5. Pushing the limits of method performance
Why it causes NCPs: It is well-known in clinical diagnostics that all methods suffer from a small true “false positive” rate as the price paid for a sensitive method, but this has received little attention in the food space. This false positive rate is usually less than 1% for PCR methods but a surprising consequence is that when the true positive rate is very small, such as in food testing, the NCP rate may approach 50%.
What Eurofins is doing: We work closely with our BACGene PCR method developers to make BACGene tests as accurate as possible and validate them against all relevant global standards. Across all sample types, Eurofins laboratories have a greater than 99% pathogen test sensitivity rate, which means a very high rate of positively detecting pathogen when present in the sample. Thus, failure to confirm does not imply the pathogen is not present in the sample. Rather, it means that the laboratory was unable to get a culture to yield a confirmation.
Questions on how this information is applicable to your food process or results?
