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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MERS-CoV snapdate on canaries...

MERS-CoV detections among healthcare workers (HCWs)

HCWs are akin to the canary in the coal mine - when HCWs get sick with a particular bug, this can signal that the bug may well be more active in the the wider community. 

This graph looks at the canaries and suggest that there has been a relatively long period in which they have been getting infected.

Healthcare workers positive for MERS-CoV over time.
Some reported or hypothesized clusters and outbreaks are flagged.
Click on image to enlarge.

A quick look at my database shows that most of the MERS-CoV-positive HCWs reported since October have been from Riyadh in Ar Riyadh region and Taif in Makkah region. 

Just before that, in early September, there were 2 HCWs from Jubail in the Ash Sharqiyah (eastern) region. 

If we look at the new time-based occurrence heatmap I have on my MERS-CoV static page here, the recent group of HCWs come from the areas with most cases. No big surprise there. Perhaps more surprising is why these HCWs are, presumably, still acquiring there infection in hospital settings given eh attention that infection prevention and control practices had, especially (before?) during and after the Jeddah outbreak last year.

A recent paper from Profs Drosten and Memish speaks to this topic of infection control and hospital spread of MERS-CoV a little.[1] 

It reports finding a 40-year old female (40F) nurse who, despite MERS-CoV being such a wimpy transmitter between humans, became infected after attending an infected patient. 40F did not perform any aerosol-generating procedures  but also wore only a surgical mask and gloves - it reads as though she was not fully protected against droplet, and certainly not against airborne, exposure. 

The 40F HCW then went on to shed virus for a 42-day period as determined by MERS-CoV specific RT-PCR. She was not ill during this time. Hard to contain much?

So with all that in mind, it's no longer hard to imagine how spread of MERS-CoV virus occurs within, around and between hospital settings. Also helps to explain how some of the new cases might seem strange - if not testing for subclinical or asymptomatic cases as a routine. I recall that in Saudi Arabia routine testing of milder cases is not occurring, but I cannot find a source for that recollection just now so I stand to be corrected (please send if you know if a reference that alludes to that).

A couple of quick questions spring to mind:

1. Just how widespread is this lengthy shedding period?
2. What does this say about how mild a virus MERS-CoV is when comorbidities are not a factor?
3. What role do genetics play in the host's containment and clearance of MERS-CoV infection?

Heatmap of MERS-CoV detection by date and region
within the Kingdom of Saudi Arabia
Click on image to enlarge.

Reference..

1. A Case of Long-term Excretion and Subclinical Infection With Middle East Respiratory Syndrome Coronavirus in a Healthcare Worker. Manal Al-Gethamy, Victor M. Corman, Raheela Hussain, Jaffar A. Al-Tawfiq, Christian Drosten and Ziad A. Memish.
   http://cid.oxfordjournals.org/content/early/2015/01/01/cid.ciu1135.long

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Ebola testing: 48-72 hours for a negative to turn positive

Currently, some fraction of the people who present very early after they may have been infected by Ebola virus for testing, return a negative result. This is probably a rare event because the majority of cases arrive for care with Ebola virus disease (EVD) already well underway.

The latest Centers for Disease Control and Prevention (CDC) guidance in these instances is to wait (48 to) 72 hours and see if the patient remains ill, or becomes more unwell. If they do either of these, a second test is performed.[1] If the suspect case recovers from illness, no repeated testing is indicated. 

The test we rely on to confirm a clinically suspected EVD case is called a reverse transcriptase polymerase chain reaction (RT-PCR). RT-PCR is a technique designed to  seek out a tiny but very specific region of the Ebola virus's RNA genome, copy it into DNA then amplify those DNA copies a billion-fold by making more copies. Somewhere during that exponential amplification process, the technology of the day (currently fluorescence detection but formerly agarose gel detection, radiation and chemiluminescence) allows us to identify that the specific DNA we seek is appearing above an arbitrary threshold...we have a positive test result for Ebola virus. 

RT-PCR is a very sensitive technique. It was not that long ago - the 80s, not that long for some of us anyway - that clinicians and scientists were complaining that PCR methods were too sensitive. This was in large part because PCR was too successful at finding infectious agents where, and when, they had not been previously found. Change to dogma was in the wind. Fast forward to today and now we're lamenting that PCR isn't always sensitive enough. Very early on after acquiring what we later know to be a true infection, even exquisitely sensitive PCR methods can fail to detect those earliest of viruses while they are struggling to gain a foothold in our cells and replicate themselves to levels that outstrip our immune system's capacity to contain. Whether this is because the virus is hidden away in organs during its early replication or whether too few circulating viruses yet exist to surpass the necessary threshold of the RT-PCR assay's sensitivity at these early stages is unclear.

EVD patients who are not yet showing signs and symptoms of disease may present early for testing and care because they they are healthcare workers with a suspected or known exposure, or they may be the contact of a known EVD case or infected animal being tested early on to exclude infection. But as we have seen and read anecdotally, that first test can sometimes be negative; not due to inhibition of the RT-PCR (which can also happen, just not so much with today's purification methods) nor because they are truly uninfected, but simply because we're testing too early. These are examples of false negative results.

For the past few weeks I have been trying to find he evidence that underpins why the world chooses to use a 48-72 hour window in its guidelines. I've been asking a lot of people-and I thank those who replied. Tonight the very diligent and extremely tolerant folks at the World Health Organization got back to me with a quote from Dr Pierre Formenty, team leader Emerging and Dangerous Pathogens. A hard man to get hold of sometimes-as you might imagine. He said (lightly edited)...

There is at least one documented case during an outbreak in Africa; a contact with fever = a suspect case; he was negative at day 1 with RT-PCR (CDC Lab) and was found positive at day 3 (when retested).
So the 48-72 hours come from this incident. We want to be on the safe side and limit the number of false negative that are inevitable with any test.

And so there you have it. If anyone has anything further to add to this story, I'd be most happy put it here.

References...

1. http://www.cdc.gov/vhf/ebola/hcp/considerations-discharging-pui.html 

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MERS-CoV in the blood....

The Middle East respiratory syndrome coronavirus (MERS-CoV) is, at its core, a respiratory virus. Well, as far as we know it is anyway. But, like other respiratory viruses (see an earlier post on rhinoviruses), MERS-CoV can be detected in the blood....a so-called "viraemia". In some cases this is identified in other virus infections in parallel with the viral load being generally high, perhaps indicating that virus is replicating beyond the body's ability to contain and control it at the site of initial replication. 

Perhaps, and MERS-CoV may be a good example of this, so-called extra-respiratory spread of a respiratory virus occurs when it has a penchant for blood vessel cells (they present its receptor in their surface, or have something in their cellular machinery that aids virus replication) or some other ability to specifically get beyond the respiratory tract. 

However it occurs, the result is a much wider spread of the virus around the body; blood being something that is widely traveled! We already know that MERS-CoV has a love for growing in kidney cells so extra-respiratory spread may create a perfect storm for delivering this little bomb to a site where it can create even more havoc than in our airways. If those kidneys are already a bit bashed about, say by diabetes, then the blast radius is perhaps increased that much more.

A new paper just out in Emerging Infectious Diseases [1] is the latest to highlight viraemia, or pedantically because its viral RNA in the blood, RNAemia and its role in detection of MERS-CoV.

A lower respiratory tract (LRT) sample (bronchoalveolar lavage; BAL) was collected from a 66-year old man (66M) who returned to Tunisia after after a 5-week visit (20-March to 28-April) with his daughter in Qatar, interspersed with a pilgrimage to Mecca (Makkah; 27-March to 04-April) in the Kingdom of Saudi Arabia (KSA). 

66M arrived back in Tunisia 28-April with an acute respiratory illness which progressed and from which the LRT sample was collected. A subsequent X-Ray identified cellular infiltrates in his lungs. His 30-year old daughter (30F) stayed in Qatar. His 34-year old son (34M), a nurse, cared for him both at home and later in the intensive care unit as his disease progressed, eventually ending in his death from multi-organ failure. He was buried 13-May and his daughter returned from Qatar for the funeral. 66M's LRT sample was not positive for MERS-CoV and he had no other respiratory viruses (not detected using PCR testing which may have been more appropriate). His daughter and son were positive for MERS-CoV so 66M was described as a "probable" case (travel, signs & symptoms, and at least subsequent contact with MERS-CoV cases). The incubation period for his illness placed 66M in Qatar at the likely time of acquisition of virus and his son was likely to have acquired his infection from his father in Tunisia. The daughter may have acquired the virus from her father while he was in Qatar or from a related source in Qatar (but seems to have been a Qatar-related acquisition of some sort). 66M's wife, 2 other well children and his son's wife were not MERS-CoV positive 5-weeks later (but then they were unlikely to have tested positive so far out from the event). 

Afterwards the US CDC tested a serum sample (tested 5-August-2013, blood taken 9-May-2013) by reverse transcription real-time polymerase chain reaction (RT-rtPCR), and it was positive. Thus - the cluster is resolved. Got a better appreciation for the amount of work that goes into tracking this stuff down in detail?

But this was not the first time a MERS-CoV diagnosis was obtained retrospectively, or as part of a study, using RT-PCR (conventional or real-time) on serum (cell free blood) rather than a respiratory tract sample. Yet remember that the presence of viral genome (or bits thereof) identified by RT-PCR does not guarantee that infectious virus was in the blood, only that viral RNA could be detected there.

• Case No. 1 from the original hospital cluster of MERS cases in Al-Zarqa, Jordan in March-May 2012, was identified thanks to retrospective RT-rtPCR (CDC version) on a convalescent serum sample.[2] 
• The 2 French MERS cases (1 imported, 1 locally acquired from contact) had RNA in their blood (UpE RT-rtPCR); the patient who died was positive for at least 4-weeks while the surviving patient cleared viral RNA in the 1st week after symptom onset.[3] 
• Two cases imported into the Netherlands from the KSA were found to have viral RNA in their blood for days; Case #1 from day-0 after diagnosis until at least day-9 and Case #2 from day-1 until at least day-5.[4] In this study viraemia outlasted virus detectability in the faeces but was detected for as long as virus in throat swabs of Case #1. RNA was not detected in the urine.[4]

Serum may be a useful sample, not just to determine whether antibodies to MERS-CoV develop(ed), but to help detect MERS-CoV RNA, as a surrogate for infectious virus, when a respiratory sample is not available. 

The finding of MERS-CoV RNA in the blood so frequently, among those studies that have looked, may also indicate it is a useful marker of disease severity as seen in the French cases. Serum is already a sample recommended for collection for antibody studies.[5] Let's see if these papers can trigger a little more looking back at those samples, which are hopefully stored in freezers somewhere. 

Anything that helps nail down "probable" cases and better define the pathogenesis of MERS-CoV is a good thing. 

Some typos & grammar corrected 05MAR2015

References.... 

1. Family Cluster of Middle East Respiratory Syndrome Coronavirus Infections, Tunisia, 2013 http://wwwnc.cdc.gov/eid/article/20/9/14-0378_article.htm
2. Novel coronavirus infections in Jordan, April 2012: epidemiological findings from a retrospective investigation
   http://applications.emro.who.int/emhj/v19/Supp1/EMHJ_2013_19_Supp1_S12_S18.pdf
3. Distinct Immune Response in Two MERS-CoV-Infected Patients: Can We Go from Bench to Bedside?
   http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0088716
4. Middle East respiratory syndrome coronavirus (MERS-CoV) infections in two returning travellers in the Netherlands, May 2014
   http://www.eurosurveillance.org/images/dynamic/EE/V19N21/art20817.pdf
5. http://who.int/csr/disease/coronavirus_infections/MERS_Lab_recos_16_Sept_2013.pdf?ua=1

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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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Qatari camels clear coronavirus

The camel herd that was previously Middle East respiratory syndrome coronavirus (MES-CoV) RT-PCR-positive is no longer positive for viral RNA according to an OIE report (OIE=Office International des Epizooties; the world organisation for animal health).
In there report they note that retesting of the herd, subsequent to the initial testing presumably, has yielded no positive this time around.

So it looks like the MERS-CoV infection is an acute infection (it is contracted, it causes illness - perhaps - and then it goes thanks to an immune response - perhaps), as are many/most) viral infections of animals and humans.

An interesting comment within the report states that...

The planned massive survey for MERS-CoV in animals is under implementation and the same herd is under systematic retesting. Follow-up reports will be submitted when there will be new data.

I do like a statement that includes the words "massive study" in it!

Hat tip to CIDRAP.

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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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Resp virus causing gastro but little respiratory symptoms...

Hat tip to Secret Squirrel D for this.

To add to my first two post's today,this paper by de Jong and colleagues in the New England Journal of Medicine details a 4-year old male (4M) who presented with a 2-day history of fever, headache and diarrhoea which after admission progressed; cough, then to coma and death.

4M was found to have systemic influenza A(H5N1) virus infection (viable virus was isolated from cerebrospinal fluid, throat and rectal swabs and were RT-PCR positive).4M and his sister (9F who died 2-weeks previously but had no diagnostic specimens collected) were diagnosed with acute encephalitis.

Chest X-rays were normal and 4M exhibited some lung sounds suggestive of infection after transfer to a paediatric referral hospital and he went on to respiratory failure.

This highlight my earlier points: respiratory viruses could spread from gastrointestinal disease and encephalitis has a lot of causes in addition to the usual suspects.

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What is PCR and what does a positive virus detection mean..or not mean?

There have been a few comments of late about the finding of MERS-CoV in asymptomatic or mild cases of disease. For example, from ProMED

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]

The feeling I get is that some don't trust the PCR results (or result-getters?) alone because they do not fulfil a need to see replicating virus in a host before assigning causality to that virus. Culturing an isolate of course, wouldn't assign causality to a pedant either it just proves it was in the patient. Similarly, serology would only prove it was in the patient and was recognized by their immune system. The sorts of extremely rare and poorly define studies needed to actually prove causality (human infection - but unethical) are usually well down the priority list in a public health management phase such as we are in with MERS.

So there is no easy answer to address this and a lot revolves around pedantry. As we know, Koch's postulates (apart from being a guide only and applying to bacteria) don't hold up too well in the increasingly molecular world of new and emerging virology. One certainly can't test a flu or CoV which kills 20-50% of the cases its found in, using human volunteers in order to reproduce disease and prove the agent was responsible. There are modifications of the postulates though. I've even had a go in a book chapter once.

I've added some musings below. I'll be working on this throughout the week day-check back for final version:

1. 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 oligonucleotide (short DNA stretches) primers. Add a reverse transcription step beforehand and you can use PCR to amplify DNA made from a pre-defined RNA template (the thing being copied).
   
   Viruses come with DNA or RNA genomes. A PCR POS 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 virus being detected in patients. Some still use the term "viral isolate" - but this should be reserved for when a virus is grown using cell/tissue/organ culture.
2. PCR doubles the number of pieces of target DNA (copies or amplicon-meaning DNA copies of a single species) every cycle (repeating series of heatings and coolings). The more DNA/RNA that you start (usually equates with the viral load) within your patient sample, the sooner you get enough copies to be measured using your detection method of choice (agarose gel or, most commonly, fluorescence signal). I have a page on the mechanics of PCR here
3. Of late, with increased use of PCR methods in respiratory virus investigations, research studies are more often finding detections among people with no signs or symptoms of disease at the time of sampling...also not immediately before or after if the people are followed up carefully.
   
   Side issue #1 - 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 that ascribed to the virus detected by PCR. Let's assume that level of care has been taken (often it hasn't)
   
   Side issue #2 - 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 employ) better testing capabilities, that we will learn we're hardly ever without a passenger or 3. And yet we are not always sick - why is that?
4. The immune system to the rescue! It won't come as surprise to the experts, but we have 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 we need 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 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 our encounters with viruses this should be no surprise.
5. I think its worth highlighting that we breath in up to 60L of air per minute, with 0.1-7200 submicron particles (aersolised; 0.3-0.5�m) per litre. In one study, influenza RNA was found more often in particles =5�M than in those greater than 5�M. These small particles can be inhaled and exhaled fairly easily and commonly.
6. Sample site and transmission in asymptomatic cases. I don't have any data for this but its 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 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.
7. The big grey peanut-loving amplicon in the room is contamination. That is, amplicon from previous PCR reactions that accidentally gets into your new mix/common 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 it as a reason. However, if the lab is expert in PCR, and those testing in the WHO network are, 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 more detail on testing would be useful.

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.

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 useful results and avoid contamination. Three are also many publications that link infectious virus with a PCR result - PCR positivity has meaning. Also, 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 detects 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.

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