Part of the laboratory methods pillar of the TAAG Learning Center, together with microbiological enrichment.
Table of contents
A positive is not always a final result
In a laboratory report, "positive" can mean two different things. It can be a presumptive result: the test gave a signal consistent with the microorganism sought, but its identity has not yet been demonstrated. Or it can be a confirmed result: the microorganism was isolated and shown, with additional tests, to be truly the target of the analysis. Confusing the two leads to hasty decisions one way or the other: releasing product that should have been held, or destroying a lot over a result that did not hold up.
This article walks through that stretch of the analysis, from screening to confirmation, taking as reference the FDA's Bacteriological Analytical Manual (BAM) for Salmonella and the ISO 16140 series of standards, which defines how alternative methods are validated against reference ones.
Where a presumptive result comes from
In traditional culture
In traditional microbiological culture, a colony with typical morphology on a selective medium is a presumptive finding. Chapter 5 of the BAM describes the sequence for Salmonella: preparation and pre-enrichment on the first day, selective enrichment on the second, plating on selective plates on the third, biochemical screening on the fourth and confirmation on the fifth. The manual itself indicates that the culture method takes 4 to 5 days to reach a presumptive result.
In molecular screening methods
PCR and other rapid methods are usually used as screening. The January 2026 edition of the BAM includes a section, added in May 2024, with qPCR and LAMP methods developed by the FDA that are applied to the 24-hour pre-enrichment culture, that is, the second day. The rule the manual sets is clear: samples with a negative screen are reported as negative and require no further culture analysis; samples with a positive screen must be confirmed by culture following isolation and identification procedures.
That asymmetry explains the value of screening. Most routine samples are negative, and for them the result arrives days earlier. Only the positives take the long road.
What ISO 16140-2 says about confirmation
ISO 16140-2:2016 is the protocol for validating alternative methods (generally proprietary) against a reference method. The standard starts from an idea that is useful for day-to-day work: many alternative methods have two steps, first enrichment and detection, and then confirmation of that result. The method's final result is the one that comes out of the second step.
During validation, the standard requires confirming the positives of the alternative method that do not match the reference method, because that confirmation is what makes it possible to know whether it is a true or a false positive. The confirmation must be able to recover the isolate and demonstrate its identity, and may be based on the reference method procedure, on the alternative method's own confirmation step, on a combination of both or on any other means that manages to isolate and identify the microorganism.
There is a technical detail with practical consequences. If the alternative method's enrichment differs from the reference one (fewer stages or fewer incubation hours), the standard asks for a second confirmation route: diverting part of the alternative method's enrichment into the reference method scheme, respecting at least its total incubation time. In other words, a short enrichment may be enough to detect a molecular signal, but not necessarily to recover a colony on a plate.
This standard carries regulatory weight. In the European Union, Regulation (EU) 2019/229 amended Article 5 of Regulation (EC) 2073/2005 to require that alternative methods be validated against the Annex I reference method according to EN ISO 16140-2, and that registered methods be certified by an independent body. In practice, the validation and AOAC-PTM or ISO 16140 certification of a method include its confirmation protocol.
How confirmation is done today
Biochemistry and serology
The classic route combines biochemical tests, which assess which substrates the isolate metabolizes, with serology, which identifies surface antigens. For Salmonella, the BAM requires that serological confirmation, with somatic (O) and flagellar (H) antigens, always be accompanied by biochemical confirmation. It allows the conventional tube battery to be replaced by systems such as API 20E, Enterotube II or Vitek 2 GN, but warns that commercial biochemical kits do not substitute for serology.
Instrumental and molecular methods on the isolate
The BAM also accepts as confirmation of presumptive isolates a qPCR and a LAMP developed by the FDA, an isothermal amplification method (AOAC OMA 2013.14) and identification by MALDI-TOF mass spectrometry (AOAC OMA 2017.09), as well as other methods validated according to FDA guidelines, AOAC Appendix J or ISO 16140-6:2019. An isolate is reported as Salmonella if serology and biochemistry together confirm it, or one of the accepted instrumental methods. If the confirmations contradict each other, additional tests or a reference laboratory are used for definitive typing.
ISO 16140-6: validating the confirmation method itself
ISO 16140-6:2019 covers the validation of alternative confirmation and typing methods. It compares the alternative method with the confirmation procedure of the reference method or, when needed, with a reference confirmation method such as whole genome sequencing (WGS). It also applies to typing, for example Salmonella serotyping or molecular typing of STEC.
Why a presumptive result may not be confirmed
A screening positive that is not confirmed does not automatically mean the test "got it wrong". There are several possible causes and it is worth telling them apart:
- Genetic material without a viable cell: a DNA-based method can detect remains of cells inactivated by heat treatment or a sanitizer, a limit we explain in RNA vs. DNA. In that case there is nothing alive to isolate.
- Cross-reactivity: a non-target organism that shares part of the sequence sought. This is what the exclusivity study evaluates during method validation.
- Difficulty recovering the isolate: stressed cells, accompanying microbiota that compete in the enrichment or very low levels can prevent obtaining the colony even though the pathogen was present. That is why ISO 16140-2 provides for the second confirmation route when the enrichments differ.
- Contamination in the laboratory: carryover of amplified material or of a positive control. The controls of each run, which we cover in PCR controls, exist to detect this.
What to do while confirmation is pending
Confirmation is an analytical step, but the decision about the product is the plant's. Some reasonable criteria:
- Finished product: a lot with a presumptive positive result is held until confirmation. Releasing a lot "because it is not yet confirmed" inverts the logic of screening.
- Environment: in an environmental monitoring program, many plants treat the presumptive as a signal to act immediately (reinforced cleaning, sampling of vectors around the positive point) without waiting for confirmation, because the cost of reacting late is greater.
- Isolate traceability: if the result is confirmed, keeping the isolate makes it possible to type it later and compare it with earlier findings from the same plant.
What is not advisable is to report a presumptive as definitive, or to discard an unconfirmed presumptive without understanding why it was not confirmed. Both are laboratory decisions that must be written into the procedure before the first positive arrives.
Conclusion
A presumptive result is a signal that has not yet demonstrated the microorganism's identity, and a confirmed one is an isolate identified with additional tests. The FDA's BAM requires screening positives to be confirmed by culture, ISO 16140-2 integrates confirmation into the validation of alternative methods and ISO 16140-6 makes it possible to validate faster methods for that step. Between the two moments, the plant decides: hold the product, act on the environment and write down what is done when a presumptive is not confirmed.
About TAAG
Talk with TAAG about how to integrate molecular screening and confirmation into your laboratory's procedure according to the regulatory framework that applies to your products.
Frequently asked questions
Is a PCR positive a definitive result?
In most schemes, no. The FDA's BAM establishes that samples with a positive qPCR or LAMP screen must be confirmed by culture, while negatives are reported as negative without further analysis.
How long does Salmonella confirmation by culture take?
The BAM describes a five-day workflow: pre-enrichment, selective enrichment, plating, biochemical screening and confirmation. The manual itself indicates that the culture method takes 4 to 5 days to reach a presumptive result.
Can confirmation be done with MALDI-TOF or PCR instead of serology?
Yes, on the isolate. The BAM accepts, among others, an FDA qPCR and LAMP and identification by MALDI-TOF (AOAC OMA 2017.09). ISO 16140-6:2019 defines how to validate these alternative confirmation methods.
What does it mean when a presumptive result is not confirmed?
It may be a false positive, but also DNA from non-viable cells, an isolate that is hard to recover or contamination in the laboratory. That is why it is worth investigating the cause before discarding the result.