The mechanics of the polymerase chain reaction (PCR)...a primer

The polymerase chain reaction (PCR) is a technique for copying a piece of DNA a billion-fold. As the name suggests, the process creates a chain of many pieces, in this case the pieces are nucleotides and the chain is a strand of DNA.

PCR is an enzyme-mediated reaction, and as with any enzyme, the reaction must occur at the enzyme's ideal operating temperature. The enzymes that are used for the PCR are DNA-dependent DNA polymerases (DDDP) derived from thermophilic (heat-loving) bacteria. As such, the enzymes function at higher temperatures than the enzymes we commonly use in the laboratory or have working in our bodies. These DNA polymerases operate at 60-75�C, and can even survive at temperatures above 90�C. This is important because a part of the PCR requires that the reaction reaches ~95�C as we shall see.

Apart from the DNA polymerase, PCR needs a DNA template to copy, and a pair of short DNA sequences called oligonucleotides or "primers" (described here) to get the DNA polymerase started.

Broadly speaking, there are 3 steps identified by incubating at different temperatures. The 3 steps make up a PCR "cycle".

1. Double-stranded DNA separation or denaturation (D in Figure 1)
2. Primer annealing to template DNA (A in Figure 1)
3. Primer extension (E in Figure 1)

Figure 1.A PCR cycle.

The three temperatures which make up a single cycle. The DNA denaturation section (D), oligonucleotide annealing section (A) and the primer extension (E) section are marked. The temperature range over which dsDNA duplexes can denature (T_D) or 'melt', and the range over which the oligonucleotide primer can hybridize (T_M) are also marked.

Denaturation..

At temperatures above 90�C, double-stranded DNA denatures or "melts". That means the weak hydrogen bonds that usually hold the two complementary strands together at normal temperatures are disrupted resulting in two single stranded DNA strands (shown below in an idealised form).

Primer Annealing..

At the annealing temperature (T_A), primers that collide with their complementary sequence can hybrdise or "bind" to it. The chance of such an encounter happening is increased because we use a vast excess of each primer in the reaction mixture compared to the number of template molecules present.

The assay in the example below has been designed to amplify a region of the template spanned by and including, the primer sequences.

Primer Extension..

At the extension temperature (T_E), the DNA polymerase binds to the hybridized primer and begins to add complementary nucleotides (i.e. every time the polymerase reads a "G" on the template strand, its adds a "C"; an "A" for a "T"; a "G" for a "C" and a "T" for an"A"), chemically binding each new addition to the last to form a growing chain. The process only occurs in one direction. In our example, the green primer is binding to its complementary template sequence and is facing toward the right (this is called the 5' (five-prime) to 3' (three prime) direction. Extension occurs in the direction that the primer faces. The result is a new double-stranded PCR product we usually call an "amplicon". An amplicon can be defined as an amplified molecule of a single type, in this case, an exact replicate of the original template.

Exponential Template Duplication..

The process is then repeated by cycling through the temperatures over and over again (35 to 55 times). Each cycle results in a new DNA duplex, each strand acting as a potential template for one or other primer.

Some interesting things stand out from the figure below.

The original template strands (blue and red) continue to act as templates because the PCR process is not destructive. However, each cycle produces a greater number of the shorter amplicon molecules. These are shorter in our example because the primers shown, bind within the template sequence. Eventually the majority of the amplicon in the reaction vessel will be the expected length, i.e. just the region spanned by, and including the primer sequences.

It is possible to mathematically predict the pattern of amplicon accumulation. In our example, we have started with two strands. In a perfect PCR reaction (which rarely occurs!), we have two new strands making a total of four. After the second cycle we have eight strands, then 16, 32 and so on. The reaction is doubling the number of strands each cycle or to make that an equation, we have 2n

Note: to make the process easier to understand, I have drawn the DNA strands as straight lines - in reality, DNA does not exist in as simple a form as this.

Further reading...

1. PCR primers...a primer!
   http://newsmedicalnet.blogspot.com.au/2015/05/pcr-primersa-primer.html
2. Reverse transcription polymerase chain reaction (RT-PCR)...a primer
   http://newsmedicalnet.blogspot.com.au/2015/05/reverse-transcription-polymerase-chain.html
3. Mackay IM. Real-time PCR in the microbiology laboratory. 2004. Clin Microbiol Infect. 10(3):190-212.
4. Mackay IM, Arden KE and Nitsche A. 2002. Real-time PCR in virology. Nucleic Acids Res. 30;6. 1292-1305. 
5. Beld MGHM, Birch C, Cane PA, Carman W, Claas ECJ, Clewley JP, Domingo J, Druce J, Escarmis C, Fouchier RAM, Foulongne V, Ison MG, Jennings LC, Kaltenboeck B, Kay ID, Kubista M, Landt O, Mackay IM, Mackay J, Niesters HGM, Nissen MD, Palladino S, Papadopoulous NG, Petrich A, Pfaffl MW, Rawlinson W, Reischl U, Saunders NA, Savolainen-Kopra C, Schoildgen O, Scott GM, Segondy M, Seibl R, Sloots TP, Wang Y-W, Tellier R and Woo PCYl. Chapter 10:"Experts� roundtable: Real-time PCR and microbiology�, In: Real-Time PCR in Microbiology, IM Mackay (Editor). 2007. Caister Academic Press, Norfolk, UK.
6. Mackay IM, Arden KE, Nissen MD and Sloots TP. Chapter 8. �Challenges facing real-time PCR characterisation of acute respiratory tract infections�, In: Real-Time PCR in Microbiology, Mackay IM (Editor). 2007. Caister Academic Press, Norfolk, UK. 269-317.
7. Mackay IM, Mackay JF, Nissen MD and Sloots TP. Chapter 1: �Real-time PCR; History and fluorogenic chemistries�, In: Real-Time PCR in Microbiology, IM Mackay (Editor) 2007. Caister Academic Press, Norfolk, UK.
8. Mackay IM, Bustin S, Andrade JM, Kubista M and Sloots TP. Chapter 5:�Quantification of microorganisms: not human, not simple, not quick�, In: Real-Time PCR in Microbiology, IM Mackay (Editor). 2007. Caister Academic Press, Norfolk, UK.
9. Mackay IM, Arden KE and Nitsche A. Real-time fluorescent PCR techniques to study microbial-host interactions. Methods in Microbiology, Microbial Imaging. (2005) Vol 34. Chapter 10.Elsevier. pp255-330.
10. Mackay IM. Respiratory viruses and the PCR revolution. In: PCR Revolution: Basic technologies and applications, Bustin, SA (Editor). 2010. Ch 12. Pp189-211. Cambridge University Press.

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PCR primers...a primer!

A DNA Down Under post

PCR (described here) functions mainly because of two components - a thermostable DNA polymerase and a pair of DNA 'primers'. Primers are short, made to order, stretches of oligonucleotides ('oligos' - from Greek meaning scanty or few). Modern oligos can be synthesized in lengths >100nt however the behaviour of oligonucleotides longer than 20nt is different from that of shorter oligos and different calculations are employed to determine their thermodynamic characteristics.

What is a primer...?

Primers, as their name may suggests, prime the nucleic acid template for the attachment of the polymerase. This is the first step towards duplicating that template. The primer directs the polymerase to move in a 5' to 3' direction (drawn left-to-right; Figure 1) because of the 'direction' of

DNA (See DNA Structure for more background).

Figure 1. DNA has direction. The polynucleotide chain shown 
above is 'read' in a 5' to 3' direction by the polymerase. This would 
be from the top to the bottom or from the phosphate group 
to the hydroxyl group.

Primer binding...

Primers hybridize at a temperature that is affected by their sequence, concentration, length and ionic environment. This annealing temperature is usually referred to as the T_M (melting temperature) but is in fact 5�10�C below the T_M. The term T_M describes the temperature at which 50% of the primer�target duplexes have formed.

Primer specificity...

PCR gleans its extreme specificity from the primers. At each and every position of a new primer, we have 4 nucleotides to choose from, dATP, dCTP, dGTP and dTTP. 

Figure 2. Deoxynucleotide triphosphates. Each of the five deoxynucleotides are shown.2'-deoxycytidine-5'-triphosphate (dCTP; C9H16N3O13P3, MW=467),2'-deoxyguanosine-5'-triphosphate (dGTP; C10H16N5O13P3, MW=507),2'-deoxyadenosine-5'-triphosphate (dATP; C10H16N5O12P3, MW=491),2'-deoxythymidine-5'-triphosphate (dTTP; C10H17N2O14P3, MW=482) and 2'-deoxyuridine-5'-triphosphate (dUTP; C9H15N2O14P3, MW=468). 

So, if we design a sequence-specific primer of 20-30nt nucleotides in length ('20-30mer'), the chance that that exact sequence will occur randomly in nature will be 1/4 x 1/4 x 1/4 etc, 20 or 30 times i.e.

That means a 1 in 10¹² to 10¹⁸ chance of a 100% homologous match to an unintended target. Or to put that in perspective, there are 2.85 x 10⁹ basepaired nucleotides in the entire human genome.

While that all sounds very convincing, in reality, primers designed to detect viruses often share significant amounts of homology with the human genome - sometimes resulting in false positive amplifications. Even when the homology is far from 100%, primers may still amplify an unintended target as shown below. This most likely reflects the co-evolution of many viruses with humans during which time they have "captured" bits of our genome and "deposited" bits of their own genome. 

Next we'll list a few of the problems we can encounter when using the PCR.

Primer dimer...

The first problem I'll discuss is the most common and the most difficult to avoid. Depending on your requirements, it may also be the least significant.

When a small amplicon results from the extension of self-annealed primers, you get primer-dimer (PD) i.e. a dimer of one (self-annealing) or both primers resulting in a template capable of being extended by the polymerase. PD formation is highly efficient because the primers are in vast excess compared to the amount of template or even to the number of amplicon molecules at the end of the PCR.

This excess drives the formation of PD. Two main concerns arise from PD formation.

1. Because PD formation is so efficient, it rapidly consumes dNTPs and primers and generates amplification inhibiting pyrophosphates. All of which can prematurely plateau the exponential accumulation of product.
2. If we using a dsDNA-associating fluorescent molecule to follow the PCR's progress during real-time PCR, then PD will also show up, and, at least during the kinetic portion of the assay, cannot be differentiated from the signal of specific amplicon accumulation.

Figure 3. Examples of how primer-dimer (PD) amplicon can be formed.
Ten examples are shown of sense and antisense primer interactions
resulting in an amplicon. Note that different length amplicons can be formed.
The largest PD would result from the hybridization of the smallest number of
nucleotides and would approach the length of the two primers added together.

Mispriming...

Mispriming is the result of a primer binding to an unintended template resulting in amplification. The amplicon (PCR product of a single species) can sometimes be the same size as the intended product, but is usually a different size when viewed following agarose gel electrophoresis.

Mispriming occurs because of poorly optimised conditions or because we haven't checked whether our sequence will inadvertently bind to an entirely different target entity e.g. a region of the human genome instead of the intended virus genome. Sometimes it just happens.

Mispriming can usually be avoided by more intensive comparison of the primer's sequence against the GenBank database using the Basic Local Alignment Search Tool (BLAST) at NCBI. Of course, a BLAST comparison will only find matches among those sequences housed in the database. When it comes to PCR where a single nucleotide mismatch can cause an amplification to fail, or at least perform with reduced efficiency, BLAST'ing primers can lead to a feeling of very false security.

In some instances the homology of the primer to its template may indicate a perfect match simply because viral variants have not yet been sequenced and submitted. Also, because there may be many undiscovered viruses and unsequenced non-viral genomes in the world, obviously none of which are represented on GenBank, a specific match, or a "no match", does not mean that you have exhausted your search for homologues. Take it all with a grain of salt. Designing two pairs of primers around the target region is a good place to start. This helps address the unexpected.

Structural problems...

This problem results from the way we design our primers. I am excluding self-annealing and secondary structures from here because we will deal with them specifically in the next section. --work in progress---

Further reading...

1. A crowd-sourced database of virus primers. www.virusprimers.org/
2. The mechanics of the polymerase chain reaction (PCR)...a primer
   http://newsmedicalnet.blogspot.com.au/2015/05/the-mechanics-of-polymerase-chain.html
3. Reverse transcription polymerase chain reaction (RT-PCR)...a primer
   http://newsmedicalnet.blogspot.com.au/2015/05/reverse-transcription-polymerase-chain.html
4. Mackay IM. Real-time PCR in the microbiology laboratory. 2004. Clin Microbiol Infect. 10(3):190-212.
5. Mackay IM, Arden KE and Nitsche A. 2002. Real-time PCR in virology. Nucleic Acids Res. 30;6. 1292-1305. 
6. Beld MGHM, Birch C, Cane PA, Carman W, Claas ECJ, Clewley JP, Domingo J, Druce J, Escarmis C, Fouchier RAM, Foulongne V, Ison MG, Jennings LC, Kaltenboeck B, Kay ID, Kubista M, Landt O, Mackay IM, Mackay J, Niesters HGM, Nissen MD, Palladino S, Papadopoulous NG, Petrich A, Pfaffl MW, Rawlinson W, Reischl U, Saunders NA, Savolainen-Kopra C, Schoildgen O, Scott GM, Segondy M, Seibl R, Sloots TP, Wang Y-W, Tellier R and Woo PCYl. Chapter 10:"Experts� roundtable: Real-time PCR and microbiology�, In: Real-Time PCR in Microbiology, IM Mackay (Editor). 2007. Caister Academic Press, Norfolk, UK.
7. Mackay IM, Arden KE, Nissen MD and Sloots TP. Chapter 8. �Challenges facing real-time PCR characterisation of acute respiratory tract infections�, In: Real-Time PCR in Microbiology, Mackay IM (Editor). 2007. Caister Academic Press, Norfolk, UK. 269-317.
8. Mackay IM, Mackay JF, Nissen MD and Sloots TP. Chapter 1: �Real-time PCR; History and fluorogenic chemistries�, In: Real-Time PCR in Microbiology, IM Mackay (Editor) 2007. Caister Academic Press, Norfolk, UK.
9. Mackay IM, Bustin S, Andrade JM, Kubista M and Sloots TP. Chapter 5:�Quantification of microorganisms: not human, not simple, not quick�, In: Real-Time PCR in Microbiology, IM Mackay (Editor). 2007. Caister Academic Press, Norfolk, UK.
10. Mackay IM, Arden KE and Nitsche A. Real-time fluorescent PCR techniques to study microbial-host interactions. Methods in Microbiology, Microbial Imaging. (2005) Vol 34. Chapter 10.Elsevier. pp255-330.
11. Mackay IM. Respiratory viruses and the PCR revolution. In: PCR Revolution: Basic technologies and applications, Bustin, SA (Editor). 2010. Ch 12. Pp189-211. Cambridge University Press.

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Reverse transcription polymerase chain reaction (RT-PCR)...a primer

A DNA Down Under post

The polymerase chain reaction (PCR) is a technique for copying a chain of DNA as many as a billion times. It purpose is so that we can use some form of technology to detect what would otherwise be too little material to see in the first place. The process of PCR is covered on the PCR page so I won't repeat it all here.

Before PCR for virus detection from human samples, we usually prepare the nucleic acids with an extraction or purification step. The shorter this is the better when testing, or screening, a lot of samples.

PCR can be used to detect some viruses straight out of the box. In these cases, the viruses need to have genes or a genome that is made of DNA. But many viruses don't store their genetic code as DNA, they use RNA as the plan from which they make more of themselves and their viral proteins. In these cases, DNA only plays an intermediate role, if any.

DNA viruses include the adenoviruses, herpesviruses, HIV (an example which also has RNA stages), polyomaviruses, bocaviruses, parvoviruses, papillomaviruses, poxviruses, megaviruses and many others. PCR also works well for plasmids and human genes and other DNA fragments we want to work with in the lab for reasons other than a diagnosis where we ask if the virus is in the human or not.

RNA viruses include influenza viruses, parainfluenza viruses, rhinoviruses, enteroviruses, cosavirus, klasseviruses, parechoviruses, respiratory syncytial virus, coronaviruses, human metapneumovirus and also many others. We also look at gene activity which involves detecting and measuring gene/genome transcription via reverse transcriptase (RT) PCR-based quantification, usually of messenger RNA (mRNA). But because PCR is based around the use of a heat stable DNA-dependent DNA polymerase, we would need to first make that RNA into DNA so that the main enzyme can use it and duplicate it and make enough of it to detect or use in molecular biology...or whatever the downstream application may be.

To make a DNA copy of the RNA, we need to add in another enzyme and another step to the PCR process. That enzyme, the RT is used in a step called reverse transcription.

The addition of this step also changes the initialism of the technique to RT-PCR. This is not to be confused with real-time PCR which is shortened to rtPCR. So an RT-rtPCR, which we use when detecting or quantifying RNA viruses in human samples, is a reverse transcription real-time polymerase chain reaction.

A standard PCR then has added to it a 10-30min incubation at an appropriate temperature (40-50'C), a denaturation step to kill of the enzyme (and sometimes to active the DNA polymerase; 92-95'C for 2-15min) and separate any DNA that is double stranded, followed by the multi-cycle PCR amplification process which can use the new DNA strand as a template for exponential copying....the billion-fold amplification reaction.

Further reading..

1. PCR primers...a primer!
   http://newsmedicalnet.blogspot.com.au/2015/05/pcr-primersa-primer.html
2. The mechanics of the polymerase chain reaction (PCR)...a primer
   http://newsmedicalnet.blogspot.com.au/2015/05/the-mechanics-of-polymerase-chain.html
3. Mackay IM. Real-time PCR in the microbiology laboratory. 2004. Clin Microbiol Infect. 10(3):190-212.
4. Mackay IM, Arden KE and Nitsche A. 2002. Real-time PCR in virology. Nucleic Acids Res. 30;6. 1292-1305. 
5. Beld MGHM, Birch C, Cane PA, Carman W, Claas ECJ, Clewley JP, Domingo J, Druce J, Escarmis C, Fouchier RAM, Foulongne V, Ison MG, Jennings LC, Kaltenboeck B, Kay ID, Kubista M, Landt O, Mackay IM, Mackay J, Niesters HGM, Nissen MD, Palladino S, Papadopoulous NG, Petrich A, Pfaffl MW, Rawlinson W, Reischl U, Saunders NA, Savolainen-Kopra C, Schoildgen O, Scott GM, Segondy M, Seibl R, Sloots TP, Wang Y-W, Tellier R and Woo PCYl. Chapter 10:"Experts� roundtable: Real-time PCR and microbiology�, In: Real-Time PCR in Microbiology, IM Mackay (Editor). 2007. Caister Academic Press, Norfolk, UK.
6. Mackay IM, Arden KE, Nissen MD and Sloots TP. Chapter 8. �Challenges facing real-time PCR characterisation of acute respiratory tract infections�, In: Real-Time PCR in Microbiology, Mackay IM (Editor). 2007. Caister Academic Press, Norfolk, UK. 269-317.
7. Mackay IM, Mackay JF, Nissen MD and Sloots TP. Chapter 1: �Real-time PCR; History and fluorogenic chemistries�, In: Real-Time PCR in Microbiology, IM Mackay (Editor) 2007. Caister Academic Press, Norfolk, UK.
8. Mackay IM, Bustin S, Andrade JM, Kubista M and Sloots TP. Chapter 5:�Quantification of microorganisms: not human, not simple, not quick�, In: Real-Time PCR in Microbiology, IM Mackay (Editor). 2007. Caister Academic Press, Norfolk, UK.
9. Mackay IM, Arden KE and Nitsche A. Real-time fluorescent PCR techniques to study microbial-host interactions. Methods in Microbiology, Microbial Imaging. (2005) Vol 34. Chapter 10.Elsevier. pp255-330.
10. Mackay IM. Respiratory viruses and the PCR revolution. In: PCR Revolution: Basic technologies and applications, Bustin, SA (Editor). 2010. Ch 12. Pp189-211. Cambridge University Press.

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Pressure testing...

Comments in the recent ScienceInsider article (a great read by the way [1]) interview with Prof Christian Drosten got me to thinking.

What follows is a stream of consciousness around the need, or not, to expand laboratory testing capacity during times of an acute rise in cases such as during a viral cluster / outbreak / pandemic situation (COP; just made that up-it's not an official acronym or anything).

During a COP, the workload in a diagnostic virology/microbiology/pathology laboratory is dramatically increased. More samples, more often. And this is due to the testing of just 1 added virus. Often, the biggest impact on service delivery comes from the need to add a new test for this virus which may not have been part of any existing testing menu or panel; it adds to the number of tests already being run. In one major Australian laboratory, routine diagnostic testing for non-influenza-related diseases runs at ~1,000 tests/day.[2] In winter, Australia's peak season for influenza, this lab (Victorian Infectious Diseases Laboratory or VIDRL) would normally test ~100 samples per day for that 1 pathogen, but during the influenza A(H1N1)pdm09 pandemic, one day saw 1,401 tests done for it alone.[2] Impressively, these guys kept to their usual result turnaround time (TAT).

Such a response requires coping with extra paperwork, quality control, and the creation and implementation of new protocols, perhaps overcoming special specimen reception issues and specimen handling requirements. There may be delays in getting specimens to the lab and a need to enrol other (previously quality assured) COP assistance laboratories to cope with the load. Less urgent testing and research may be halted and even expanded lab space may be sought in adjoining areas. This all create some real impact. It can affect other results, it may impact on the TAT for a lab (although prior planning is aimed at coping with the strain of COPs and keeping the result TAT in check as happened in the example above). A COP strains nucleic acid extraction robots, centrifuges, bio-hazard safety cabinets, labelling machines, pipettes and thermal cyclers - all of which break down when you least need them to. Reagents may become rare and if not stockpiled could create a bottleneck in assay performance - basic PCR assay reagents may be hard to come by or slow to receive, especially during a global and/or sustained outbreak or pandemic. And very importantly, there is a real toll on staff and managers. Hours may be extended, tiredness and stress will set in and a shortage of expertise may be an issue for maintaining quality and TAT...and sanity

In other words, test results don't magically appear and diagnostic labs are nowhere near as automated as you might think.

All this adds up to a system that can reach its capacity and thereafter shows signs of stress. The influenza A(H1N1)pdm09 pandemic did this. Now we hear of that MERS-CoV may be creating a similar circumstance in the Kingdom of Saudi Arabia (KSA). Why is the KSA Central Laboratory, which does all the PCR testing for MERS-CoV, under such stress now? According to Prof Drosten, it's because of changes in the testing which may be a driving factor underpinning April's Jeddah surge of viral detection.

Something dramatic changed, and that is the case definition.

Prof. Drosten to ScienceInsider

This change led to a jump in testing from 459 samples for all of 2014 prior to the outbreak, to 4,629 in just 1 month. As the number of MERS-CoV tests being performed in each (daily) report of new cases is no longer part of the KSA Ministry of Health's (MOH) message, a thumbnail sketch is that 154 sample per day are being tested for that month (divided by 30 days). And then there was this comment..

"The question of whether there is a mild, short-lived infection in some people is scientifically interesting. But in cities like Jeddah, it is bringing the health system close to collapse. That is the big problem. So many samples are being tested that the lab capacity won�t suffice for the real cases."

Prof. Drosten to ScienceInsider

An entirely fictional map of MERS-CoV spread including
severely ill, mild/moderately ill and prodromal /
asymptomatic infections. Simply intended to be
something to think about when discussing the
impacts of limiting PCR testing. Reduced PCR
testing should not happen until until we know which
parts of this map are real, and which are a load of rubbish.

With this background and these comments in mind I have some thoughts and questions...

1. I know almost nothing about the KSA's pathology laboratory testing capacity generally nor its approach to respiratory virus testing in particular. I do know that the KSA is are a country of around 29 million people while Australia has around 23 million. I refer to the numbers above when I say that 4,629 samples in a month, for what has become an epidemic that seems to have exposed major flaws in infection control across multiple hospitals around the west, south and central regions of a wealthy country, should not be threatening the KSA's testing capacity unless it did not exist in the first place.
2. Why wouldn't pathology testing which is robustly designed to cope with a worse-case-pandemic, not exist in the KSA? I don't know. Does testing exist for standard virus screening and if so what sort of throughput is the norm? The KSA healthcare systems seems to be laden with western-influenced medicine, and with that influence comes our compulsive need to create protocols and preparedness plans and to learn for the misfortune of others. The WHO have all this sort of information publicly available and always seem available for a chat.
3. The reality is, and I am not on the ground to see whether this is a real factor in the KSA, laboratory capacity needs to be such that it can cope with a surge in cases such as that during a COP. It also needs to manage other endemic respiratory virus testing and whatever is coming next. It seems highly likely to me that the same at-risk older male population with kidney and heart disease, diabetes and obesity issues that get hit so hard by MERS, is also suffering badly from influenza and other viral infections. Back in August we heard about additional laboratories coming on line. It looks like they may not have. They need to.
4. Am I especially naive (probably) to expect wealthy countries to make sure something as important as pathology testing is not in danger of falling over when it's particularly needed? We expect our electricity to be quickly reconnected after a storm, out SUVs to be easily refuelled no matter what wars or disaster befall the worlds, we take for granted that water is just there and we'd riot if our shop were not stocked with food 24/7. Why would testing your population to make sure you have a real-time knowledge of the pathogens infecting them, not be given an equal measure of attention and support? Especially if that pathogen has never been seen before, is transmitting without your understanding and is killing 1:4 of those it infects?
5. Prof. Drosten noted that he has been working to get good MERS-CoV antibody testing in place within the KSA to get a better idea of how widespread prior exposures to MERS-CoV is. That will be a very helpful piece of knowledge to have. But it will not tell the MOH what is happening now in Hospital X (an apt name since we no longer know names of the hospitals where cases are being treated; that dropped off the new MOH messaging format last night). We're not even sure MERS-CoV antibodies are produced if the PCR-positive person only had a mild or asymptomatic case. PCR testing must remain in place until the MOH or whomever it looks to for advice, can be sure they have seen all the faces of MERS and the MERS-CoV. We're some way off seeing that yet I believe.  Don't get me wrong - an antibody test is great and we should roll it out alongside PCR. But in context - it will tell us information about the status of the KSA population in terms of how many have been exposed to MERS-CoV. And then it will have done its job as a research tool. Routinely, we need to test with the gold standard; PCR. And I think we should keep testing widely. 
6. Prof Drosten also suggested that instead of continued PCR testing of contacts (the source of asymptomatic cases presumably), the KSA should consider a home isolation approach. Would that be  for up to 2-weeks, away from work, school - away from family too? Seems like a lot of hassle and disruption for the sake of a PCR test. Perhaps a shorter period once we know more about the dynamics and shedding during the diseases prodrome or from asymptomatic people. That will require PCR to define a person was initially MERS-CoV positive in order to study whether virus is shed.
7. Let's also keep in mind that antibody testing is labour-intensive too. Perhaps not as intensive as PCR, but it would still increase the workload on a pathology laboratory.  It's a new tool not a better one.
8. Why do I think the KSA should keep testing widely? Because if we don't we might be missing mild and asymptomatic or prodromal cases which may (and we have no data to support the argument in either direction right now, so its much better to be safe and test as the World Health Organization advise) contribute to the spread of MERS disease. Who knows how much virus an already old ill male needs to become severely ill? Perhaps much less than a healthy young nurse with lots of previous exposures to other viruses, including some that may provide cross-protective immunity I suspect. 
9. If the KSA had not switched gear and accelerated into more testing, we would still only know the face of MERS that is pneumonia and death. It is clearly a lot more than that-as are all respiratory viruses. It would be a great shame in my opinion, to do things the way they were done with SARS, just...because. We always need to look afresh with the knowledge and tech we have to hand on the day.

Now more than ever with new measures being instigated to educate the KSA public (a bit more anyway), reduce camel exposures (although it's clear many don't see a link to camels as justified) and improve hospital infection control (too late for the majority of MERS cases that seem to have occurred in linkage with healthcare facility outbreaks) and hospital triage of MERS cases, testing efforts must not wane.

And while that goes on in the background, it really is past time to sort out some transmission details. How is the virus spread (a) from and between camels and (b) to and between people? These are fundamental questions and all risk reduction hinges on their answers. 

At least now that we know the virus hasn't changed, we shouldn't be seeing any more cases during the upcoming multi-million person Hajj pilgrimage, than we saw last year. Right? Last month was all about an infection prevention and control breakdown that can be fixed before October. Yes? And the few instances of Umrah pilgrims that seem to be popping up positive this year that we didn't see in 2013 and the bunch of single export cases? Just increased testing? Yup. Some of that even kinda fits in with what I wrote about Umrah 2013. 

Oh look. 10 new cases tonight, just like on 2013. Oh wait. No it wasn't like tat in 2013. We didn't have any 10-detections/day days in 2012 or 2013. Guess these will be because of all the pesky asymptomatic people? Let's see...ICU, hospitalised, ICU, symptomatic but home isolated, ICU, ICU, asymptomatic, ICU, asymptomatic, hospitalised oh and in two most likely unlinkable previous cases: death, death. 2 out of 10 with no symptoms. 

Definitely keep up the testing guys. MERS isn't SARS but then 2014 isn't 2013 either.

Sources...

1. http://news.sciencemag.org/health/2014/05/mers-virologists-view-saudi-arabia
2. Reality Check of Laboratory Service Effectiveness during Pandemic (H1N1) 2009, Victoria, Australia | Emerging Infectious Diseases. 2001. 17(6):963-
   http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3358210/pdf/10-1747_finalS.pdf
3. http://www.nccid.ca/files/Evidence_Reviews/NCCID_H1N1_impact_04.pdf

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Neither market nor farm poultry all that positive for H7N9; songbirds the culprit...?

Following on from yesterday's post, "If not poultry then what?", I thought it worth noting the impressive numbers from the Chinese Ministry of Agriculture.

From 2013:

• 1,630,000 poultry and environmental samples tested
• 88 POS; all from live bird markets
• None from poultry farms

From 2014, to date:

• 33,400 poultry and environmental samples
• 8 H7N9 POS; all from live bird markets
• None from poultry farms

The other alternative to answer the question in my heading; the testing methods are at fault. 

No detail of what approach has been used to obtain these numbers in the links below. Viral culture and serology with some PCR have been noted before. I'd wager culture yields chicken scratchings compared to PCR for detecting virus in he wild; but serology has successfully been a pillar upon which animal testing rests. So that's why the numbers above are such a quandary for the epidemiologist who reads about the high frequency of links between human disease and exposure to poultry.

It would be nice to see some technical papers on antibody test testing (development and validation) at some point. If only to reassure everyone that the testing methods are doing what testing methods should be doing.

See #3 below for influenza PCR discussion at WHO.

Sources..

1. http://english.agri.gov.cn/news/dqnf/201304/t20130427_19537.htm
2. http://english.cntv.cn/program/china24/20140130/100709.shtml
3. http://www.who.int/influenza/gisrs_laboratory/report_2012pcrwg5thmeeting.pdf

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Market sampling: H7N9, sensitive testing, market closures and small numbers

A World Health Organization Western Pacific Region update on influenza A (H7N9) virus has a few interesting bits of information that pulls together a recent flurry of reports. This is the situation as of 22-Jan...

• 18/200 (9.0%) "pathological samples" from markets (listed below) in Zhejiang province, presumably using PCR-based methods, were H7N9 positive  
• Sanliting Agriculture Products Market (6 oral/cloacal swabs, 2 environmental faecal swabs)
• Central Agriculture Products Market (2 oral/cloacal swabs, 1 environmental faecal swab) 
• Fenghuangshan Agriculture Products Market (1 oral/cloacal swab)
• Guoqing Poultry Wholesale Market (3 oral/cloacal swabs, 3 environmental faecal swabs).
• 2/2,521 (0.08%) pathological samples were H7N9 positive in Guangdong province
• Pathology specimens from the provinces of Jiangxi, Liaoning, Jilin, Heilongjiang, Jiangsu, Fujian, Shandong, Hubei, Hunan, Guangxi, Yunnan, Qinghai, Xinjiang Provinces and Chongqing and Shanghai Cities were H7N9-negative
• 7-Jan, H7N9 RNA was also reported  in 3/17 samples collected from the kitchen of a restaurant in Haizhu District, Guangzhou City, from the chopping board and sewage water. 
•  Meanwhile H7N9 RNA was identified in 8 out of 34 environmental monitoring samples collected from the Guangdong's Longbei Market, Jinping District, Shantou City.
• Ningbo city (Zhejiang Province) has stopped commercial live birds entering the city
• Shanghai city will suspend live bird trade all over the city from 31-Jan to 30-Apr. Live poultry from other provinces will not be allowed into the city except for transport to a centralized slaughterhouse.

It's great to see some data from other provinces and municipalities that have not reported any human H7N9 cases to date.  I do wonder about the relatively small numbers of market samples though. Some of these samples pale in comparison to what was tested in 2013; which reacted earlier than this, the second time around. While 2,00 samples is not an easy day in the lab, we saw >800,000 bird samples tested by "virological" (?culture) and serological methods in 2013 (see other thoughts on the use of PCR in birds here).

So what have we learned here? 

1. Further confirmation that live bird markets house H7N9-positive birds. With most human cases this year having come into contact with poultry, the transmission chain is in place. Market closures seem the most effective way to stop transmission abruptly and they have a precedent for this in 2013. This is happening. Will it be enough? What  about the market-supplying farms?
2. RT-PCR testing is more likely to uncover influenza in birds than culture methods and is better than antibody testing (although how much better is hard to judge from the information provided). Added bonus: RT-PCR is more likely to tell you what's circulating now rather than a little while ago...although no-one really responds to the lab results that quickly anyway.

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H5N1 case in Canada had been diagnosed with pneumonia...testing at the source would have been helpful

And now, from a fantastically detailed post onto ProMED by Fonseca and colleagues, we see that the H5N1 case was diagnosed with pneumonia.

On 28-Dec, the patient presented to a local emergency department.

"A chest X-ray and CT scan revealed a right apical infiltrate. A diagnosis of pneumonia was made; the patient was prescribed levofloxacin and discharged home."

One sad point made in the ProMED post which supports the need for constant viral vigilance the world over, coupled with the dissemination of those surveillance data, so that patient management anywhere in the world can be armed with the best possible decision-making information...

"The index of suspicion was low as travel was to an area in China where there have been no recent reports of the circulation of this virus, and coupled with no obvious exposure to poultry, the diagnostic work-up and consideration for A(H5N1) infection was very low"

As a recent J Virology article by Yu and colleagues highlights, when a sensitive testing method like the polymerase chain reaction (PCR; in this case RT-PCR because influenza viruses all have an RNA genome, not a DNA one) is applied to the search for a virus, it yields the kind of data that can:

1. Explain from where a virus emerges
2. Inform the search for disease aetiology - where are human cases getting infected from and if a zoonotic infection (from animals to humans), which animal(s) is the culprit?
3. Alert the world to any risks of infection when travelling to a certain area(s)
4. Allow the local health departments to mitigate the risk of their population acquiring infection by instigating controls (like live bird market closures). This has implications for the world since respiratory viruses have the potential (thankfully not realized for H7N9 or H5N1 to date) to spread more rapidly and efficiently that blood-borne or mosquito-borne or sexually transmitted viruses.
5. Permit understanding of how widespread (over what geographic area is it detected) a novel or emerging virus may be and how entrenched (is the same site repeatedly positive) it is

Not doing such testing, or using less sensitive methods will not yield this information. 

In Yu's study, testing of 12 poultry markets, mostly urban, and local farms linked to 10 human infections in Hangzhou, Zhejiang province around 4th to 20th April 2013 yielded signs of H9N2, H7N9 and/or H5N1 viruses in all markets. Poultry were often positive for H7N9 and H9N2 (this finding from individual RT-PCRs was confirmed using next generation sequencing), whereas human specimens were not. These levels hadn't been turned up when 899,000 bird were tested in 2013 using (perhaps) less sensitive methods.

I think with influenza, it may be safer to presume its everywhere until that presumption can be discounted. Clearly the conditions for influenza viruses to swap gene segments and sort themselves into new subtypes and variants are commonplace and frequent; these aren't just chance occurrences of different birds passing in the night via overlapping flyways. These feathered vectors are co-infected by 2 or more viruses at a time. Luck and the constraints of viral fitness are presumably the only things keeping H7N1, H5N9, H7N2 cases from dialing up in humans? What seems to be lacking is more molecular testing at the farms supplying the markets. Not just in Zhejiang, but all over the region.

As the authors noted, 100,000s of people visit these live bird markets each day and very few influenza cases seem to be due to them. Long may that last. But it's a tinderbox for which matches are already being struck; if the viruses should bud of that one-in-a-million variant that is enabled to readily spread from person-to-person, whooshka. 

More testing guys, keep testing.

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No symptoms but still shedding virus?

Click on image to enlarge.
A stylized trace of the temperatures during a PCR cycle.
D-denaturation, when primers and double-stranded
DNA (dsDNA) are reverted to single strands of DNA;
A-annealing, when primers bind to their complementary
target and DNA re anneals to form dsDNA; E-extension,
when the DNA-dependent DNA polymerase enzyme
finds a primer, binds to it attached to a strand of
template  and makes the complementary strand.
Feel free to use. Please cite this website and
Dr I M Mackay as illustrator.

One of the many questions that remain unresolved for MERS-CoV is whether a human who is PCR-positive for the virus, but does not show signs or symptoms of being sick, can spread that infection on to other humans - or animals for that matter.

Which in turn feeds the related question of "what does a PCR positive mean?"

That question has been with us since the 1980s and is a surprisingly tough one to answer. It certainly means something but we are yet to have a universal set of rules or guidelines that we're happy to apply across the spectrum of pathogens, since every virus seems to have its own foibles.

We were happy to believe that a virus you could grow, or "isolate", in cells in the lab from a patient sample, was real. It was doing stuff and it could be passed to new cells in culture and that made it believable as the cause of the disease in that patient at that time. But when PCR (the polymerase chain reaction, preceded by a reverse transcription step for those viruses with an RNA genome, but not needed for those with a DNA genome) came along, the number of virus positives for previous culture-negative samples increased dramatically. This was due to:

• Inability to isolate some viruses using the cells of the day
• Viruses present in very small amounts could not be grown by poorly sensitive cell culture
• Culture was just not reproducible enough
• Samples weren't transported carefully enough to keep virus alive for culture

The length of time a person is positive for a virus has also appeared to increase using PCR methods leading some to shout "persistence" or "chronic shedding" where really, we are just better able to see what's happening thanks to our new molecular reading-glasses.

Click on image to enlarge.
Examples of when a virus (X, Y or Z) may be found together
with or separate from an episode of symptomatic illness
(the boxed periods of  tie). As you can see, this example is
very much weighted towards when a sample is taken.
3 testing scenarios are shown. (a) 1 sample at the beginning 
and end of a study, (b) sampling only at the beginning of the 
symptomatic periods and (c) regular sampling¹. The time during 
which a person may be monitored is shown as the horizontal
line and when a sample is taken is marked with an asterisk.

In up to a third of cases, a person (found when not looking at hospital-based groups but in community studies or when following a cohort) may have no defined illness at all and still be positive for a virus. Heresy!!

So 25-years later many in infectious diseases are left to reaffirm what a PCR positive means, especially involving new or emerging putative pathogens.

For the Middle East respiratory syndrome coronavirus (MERS-CoV) we may be able to draw some conclusions from a viral relative; the severe acute respiratory syndrome (SARS) CoV, did during its short time in humans back in 2002-2003.

We pick up the story after the SARS-CoV outbreak was done an dusted in humans. Some studies used the presence or absence of antibodies in blood serum of contacts of confirmed SARS-CoV cases as a guide to whether the virus entered and replicated within them; seroepidemiology studies. The contacts do not appear to have been screened using RT-PCR; also the current situation with MERS. 

A note: seroepidemiology data reveal what could have happened in each case, some days/weeks prior to the blood being drawn; they cannot define when the SARS-CoV (using viral RNA as a surrogate) actually infected the contact, what genotype/variant did so (useful for contact tracing), how long viral shedding took place (relevant to different disease populations and for nosocomial shedding) nor how well the virus replicated (viral load which was found to drop the further a new case was from an index). 

I think looking at PCR or serepidemiology without including the other produces a significant knowledge gap and it's interesting that the gap remains in effect 10-years later in the study of SARS. Perhaps MERS-CoV is just like SARS-CoV and, as we see below, no symptoms=no infection=no onward transmission. Gut feelings don't really tick the box in science though.

Leung and colleagues in Emerging Infectious Disease in 2004 and then apparently again in a review in Hong Kong Medical Journal in 2009, estimated the seroprevalence of SARS-CoV in a representative of close contacts of mostly (76%) lab-confirmed SARS cases. 

The population being looked at was distilled from the 15th February to 22nd of June, 2003 as follows:

• 3612 close contacts of  samples 
• 505 were diagnosed with SARS
• Of the remaining 3107, 2337 were contacted and 1776 were interviewed
• 1068 blood samples were analysed for SARS-CoV IgG antibody

Only 2 of the 1068 (0.19%) had an antibody titre of 1:25 to 1:50. Most recovered SARS cases had titres of =1:100. Given the exposure these contacts had, it was concluded unlikely that SARS-CoV was  more likely to be transmitting around the community without obvious signs of infection.

Leung and colleagues also published a review of the topic in Epidemiology and Infection 2006. They concluded an overall SARS-CoV seroprevalence of 0.1% overall with 0.23% in healthcare workers and contacts and 0.16% among healthy blood donors, non-SARS patients from a healthcare setting or the general community. Other interesting bits of information from this review include:

• 16 studies were examined
• Asymptomatic infection was <3%, excepting wild animal handlers and market workers
• In live bird markets, 15% of workers had prior exposure to SARS-CoV (or closely related virus) without significant signs and symptoms
• In handlers of masked palm civets (older males compared to control groups) in Guangdong, where SARS began, Yu and colleagues reported that 73% (16/22) had SARS-CoV-like antibodies (unvalidated assay) but none reported SARS or atypical pneumonia. Which leaves room for milder illness, and larger studies.
• Prevailing SARS-CoV strains almost always led to symptomatic illness

So what has been done for MERS-CoV? We have some camel seroepidemiology studies which I've previously described here and here. Human studies?

1. In the study that found MERS-CoV-like neutralizing antibodies in Egyptian camels, no human sera from Egypt (815 from 2012-13 as part of an influenza-like illness study in Cairo and the Nile delta region) nor any from China (528 archived samples from Hong Kong) were MERS-CoV neutralizing-antibody positive.
2. No sera or plasma from 158 children admitted to hospital with lower respiratory tract disease or healthy adult blood donors were MERS-CoV neutralizing-antibody positive. Small sample and the ill children may not yet have mounted a relevant antibody response if they had been infected by MERS-CoV.

Work like that mentioned for SARS largely remains to be done for MERS. The SARS-CoV studies provide a useful model on which to base such studies and the World Health Organisation recently provided a detailed approach for seroepidemiology studies seeking to test contacts of laboratory confirmed MERS-CoV cases. 

What does a positive PCR result mean in an asymptomatic MERS-CoV case? Still can't answer that. Are contacts seroconverting as an indication of MERS-CoV infection? Still can't answer that. How many mild or asymptomatic MERS-CoV infections are there beyond contacts of lab-confirmed cases? Still can't answer that.

Once we can rule out occult community transmission - we can tick another concern off the MERS-list.

Further reading...

1. Observational Research in Childhood Infectious Diseases (ORChID): a dynamic birth cohort study
   http://bmjopen.bmj.com/cgi/pmidlookup?view=long&pmid=23117571
2. Middle East respiratory syndrome coronavirus: quantification of the extent of the epidemic, surveillance biases, and transmissibility
   http://www.thelancet.com/journals/laninf/article/PIIS1473-3099(13)70304-
   9/abstract
3. Prevalence of IgG Antibody to SARS-Associated Coronavirus in Animal Traders --- Guangdong Province, China, 2003
   http://www.cdc.gov/mmwr/preview/mmwrhtml/mm5241a2.htm
5. Viral Load Distribution in
6. SARS Outbreak
http://wwwnc.cdc.gov/eid/article/11/12/pdfs/04-0949.pdf

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17 new MERS-CoV sequences bind perfectly to frontline screening PCR assay for MERS...

Click to enlarge. The primers/probe are depicted as grey boxes.
If mismatches existed they would show up as horizontal black
lines within the grey box. No mismatches are evident.
The GenBank accession numbers are
shown on the left of this alignment of 17 MERS-CoV
sequences.

Only 17 of the 45 sequences seem to include the region covered by the upE laboratory assay I just posted about in the WHO laboratory testing update but of those, the forward and reverse oligonucleotide primers and the probe all bind without any mismatch.

While that may sound like an obvious statement considering that these viruses were probably detected using that assay it isn't.

The new MERS-CoV sequences were determined using using unbiased 2nd generation high-throughput sequencing technologies that did not rely on these primers to generate them. So we are now able to check and see if there are any nucleotide changes at the target sites for the primers and probe, that would reduce the efficiency the assay.

There are no such oligonucleotide mismatches between primer and viral genes among those 17 sequences, which is good news for that assay's continued usefulness.

Built to last eh?

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