The polymerase chain reaction (PCR) has remained a hugely important laboratory tool for decades. Subtle changes to the enzymes and drastic changes to the detection of a result have occurred in that span, but the tool and its reliability have remained remarkably consistent.

IMPORTED POST*

There are always comments like the one below which specifically muses about the finding of MERS-CoV genetic material in asymptomatic or mildly ill people. They basically wonder at the trustworthiness of PCR.

As to my knowledge “infection” is defined as the proof of an infectious agent AND the proof of multiplication of this agent inside the body (OR associated inflammatory response that can be linked to this agent). Therefore my question is: where was the specimen taken from the “asymptomatic” nurses? PCR, as we all know, just detects nucleic acids. So if multiplication cannot be proven and no local or systemic inflammatory response is given, how was “contamination” (no matter whether due to inactivated parts of MERS-CoV or even complete virus particles) ruled out? This is not a semantic question only but should impact the reported number of “cases”.

Prof Martin Haditsch writing in ProMED[1]

The feeling I get from these is that some don’t trust the PCR results or perhaps the result-getters. PCR results alone don’t fulfil a need to see replicating virus in a host before assigning any sort of causal role to that virus. But that is often impractical and frequently unnecessary because a positive PCR result is generally a reliable proxy for the presence of active virus infection.

Culturing an isolate wouldn’t assign causality for a pedant either. It just proves the virus was in the patient’s sample. Similarly, a positive serology result detects antibodies to the virus, proving only that it was recognized by an infected patient’s immune system at some point. The sorts of studies needed to actually prove causality are usually well down the priority list in a public health management phase such as we are in when a new virus emerges. The best being (completely unethical) human infection followed by infection of other primates and then, moving down the relevance list, to other furry critters that may be a suitable stand-in for disease in humans.

There is no easy answer to address the issue above. A lot revolves around words, pedantry, guidelines, tenets and criteria. Often these are supported by lots of different epidemiological studies which all rely heavily on good laboratory results. Results such as those provided by PCR. As we know, Koch’s postulates (apart from applying to bacteria and being a guide only) don’t hold up too well in the molecular world of new and emerging virology. There are modifications of the postulates though.

Other collections like the Bradford Hill criteria get more of run these days.[2,3] One certainly can’t test a Flu or CoV which kills 20-50% of the cases it’s found in, using human volunteers in order to reproduce disease and prove the agent was responsible.

I’ve rolled together some musings on the role and reliability of PCR in a list.

What is PCR (the quick version)

PCR is an enzyme-driven cyclical cooking and cooling process that amplifies a pre-defined stretch of DNA using a pair of equally pre-defined and commercially made oligonucleotides (short DNA stretches) we call primers (they prime the DNA copying process which underpins the amplification). Add a reverse transcription step beforehand and you can use PCR to amplify DNA made from a pre-defined RNA template (template being the thing we’re copying and amplifying).

Side note #1 – Viruses come with DNA or RNA genomes. A positive PCR result does not prove active replication of a virus. It does not prove infectious virus is present. This is why we prefer to talk about a virus being detected in patients using PCR. Some refer to a PCR positive result as a “viral isolate” – don’t. This should be reserved to describe eh successful growth of a virus using cell/tissue/organ culture.

PCR doubles the number of pieces of target DNA (copies or amplicon-meaning DNA copies of a single species) every cycle (1 cycle = round of heating and cooling). The more DNA/RNA that you start with (usually equates with the viral load) in your patient sample, the sooner you get enough copies to be measured using your detection method of choice (today, mostly fluorescence signal). I have a page on the mechanics of PCR here

With PCR, we find virus more often

Of late, with use of very sensitive PCR methods in respiratory virus investigations being commonplace, research studies have more often detected virus among sampled people with no signs or symptoms of disease…also not always immediately before or after if the people are followed up carefully. Why sample someone if they are not ill? Good question. You may want to see if transmission occurs even if an illness doesn’t. But in the past, using relatively insensitive virus culture methods, we would have been less likely to detect a virus.

Side note #2 – if we’re looking to declare such a person “asymptomatic” – we had better be completely sure they have absolutely no symptoms of an infection. Disease, a deviation from normal body function, may be as mild as a headache or just feeling crook – and still, pedantically, be a disease ascribed to the virus detected by PCR. Let’s assume that level of care has been taken (often it hasn’t)

Side note #3 – we are walking platforms for viruses, bacteria and fungi. Take a look at this paper from Eric Delwart; in 2 kids-92% of 72 samples collected weekly over more than 250-days were virus-positive (no or mild signs of disease developed during this time)! It is very likely, as we discover more diverse agents, better characterize them and develop (and use) improved capabilities, that we will learn we’re hardly ever without a passenger or 3. And yet we are not always sick – why is that?

Immunity can keep us well, even if infected.

The immune system to the rescue! We have a very capable, active and multifunctional immune system. It is constantly being challenged by…things – not just viruses, bacteria and fungi but chemicals, various proteins, carbohydrates, allergens, dirt and dust (composed of bits of all that other stuff) and it does a great job of sorting through it all, responding to what it needs to, to prevent disease, and usually squashing the response to the rest in such a way that we don’t under-perform 24/7 because of allergy and illness. It is due to this complex system that we shouldn’t be surprised that as technology lets us find lower levels of infectious agents, it is completely reasonable to be “infected” but not diseased. At least in some proportion of our encounters with viruses, this should be no surprise.

We get a lot of exposure to virus

I think it’s worth highlighting that we breath in up to 60 litres of air per minute, with 0.1-7200 submicron particles (aerosolised; 0.3-0.5μm) per litre. In one study, influenza RNA was found more often in particles ≤5μM than in those greater than 5μM. These small particles can be inhaled and exhaled fairly easily and commonly. This just highlights how common it probably is for us to inhale viruses into our airways

Sample site and transmission in asymptomatic cases. I don’t have any data for this but it seems to me that if you swab the upper airways, you have swabbed the site from which virus is easily coughed, sneezed and therefore transmitted in larger particles. If a virus is detected there, then there would seem to be a good chance of transmission being possible. Does it happen? Those studies are not easily visible… or perhaps they have not been done.

But the contamination!

The big grey peanut-loving amplicon in the room is contamination. That is amplicon from previous PCR reactions that accidentally gets into your new PCR reagents/pipettes/lab and gives us a positive result when the patient is actually not infected. It’s what some think of when a PCR positive occurs in an asymptomatic person. It’s easy to point at this as a reason for a disagreeable result. However, if the lab is an expert in PCR, then the necessary controls will be in place to give warning of contamination.

Plus, for some viruses and in some labs, more than one PCR assay, each targeting different genetic regions and using separate primers, are employed. This is the case with the recommended work-flow for MERS-CoV screening. It does not mean these are the assays used of course. And look for detail on the testing workflow used is always useful to understand the context of a PCR result.

Other useful steps include separate labs for specimen preparation, reaction mix preparation and cycling; labs with directional air- and work-flows; disposable everything; commercial reagents; pre-made, target-specific kits (“analytes”); lots of negative controls and so on. These provide considerable confidence in a PCR result and are standard working practice for a professional diagnostic PCR lab. Taking more than one sample from a suspected case is also helpful.

There is a lot of expertise in the PCR world

PCR has been used to detect viruses since the 1980s and there are many very knowledgeable experts in its use who know how to get reliable results and avoid contamination. There are also many publications that link infectious virus with a PCR result – PCR positivity has meaning and the literature supports that.

Those of us who have conducted PCR experiments on human samples over many years will know, PCR is not so practically sensitive that it will regularly detect virus that is just sitting around in your nose because it was inhaled but is not replicating. Dilution factors see to that.

Still, problems happen and non-professional labs exist. There is no easy answer to the questions posed by Prof Martin Haditsch on ProMED. As with any test, reliability is about reducing the risk of failures in the process. But even expert labs have bad days.

References

1. MERS-COV – EASTERN MEDITERRANEAN (34): SAUDI ARABIA, WHO http://www.promedmail.org/post/1788112
   The association between acute flaccid myelitis (AFM) and Enterovirus D68 (EV-D68) – what is the evidence for causation?
   https://www.ncbi.nlm.nih.gov/pubmed/29386095
2. Enterovirus D68 and acute flaccid myelitis-evaluating the evidence for causality
   https://www.ncbi.nlm.nih.gov/pubmed/29482893

*Imported Post

1. This post from 27JUN2013 was posted over on my old blog platform virologydownunder.blogspot.com.au. It has now been moved to here and lightly edited. 

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