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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Can we believe every H7N9 seroprevalence study we see?

Special Guest writer: @influenza_bio

A little over a year ago, the first known human patient got sick with avian influenza virus(H7N9). The number of H7N9 cases rose and fell in the spring of 2013, and a total of 134 people were known to have contracted H7N9 before June, 2013. Since then, sporadic cases appeared in the summer and fall, and by the end of December, 2013, new cases started to pick up again. We have now seen a second wave rise and fall, although several new cases still being reported each week. As of the time of this writing, just over 400 people are known to have been infected with H7N9. The case fatality rate (CFR) � roughly speaking, the percentage of people infected with H7N9 who die from it � for these known cases is almost 40%.

One question that is on a lot of people's minds is, how many other H7N9 cases are out there that we don't know about? How many mild cases are there that never get tested? How many asymptomatic cases are there that are missed? If there were a lot of undetected cases out there, that would mean that H7N9 is a lot less fatal than the known cases would make us think. On the other hand, if we were somehow miraculously seeing every single actual case, then the CFR would be as bad as all of these cases make it out to be. (And imagine what the CFR would be like without hospitals, ventilators and oseltamivir!)

How do we find out if there are cases that we're missing? One way is to do what is called a seroprevalence study. This means collecting blood samples from as wide a swath of a population as possible and testing to see how many of these samples have antibodies to H7N9. Antibodies are molecules that are made by cells of the immune system and that stick to specific pathogens to help our bodies to rid themselves of these pathogens. If someone gets sick with H7N9 influenza, his or her body would most likely continue to produce a significant amount of antibodies specifically against that strain for at least a good number of months after infection and possibly much longer. In general, people who are infected with influenza but who do not develop symptoms will also produce such antibodies, but their bodies will make fewer of them, and, on average, they won't make as many of them for as long. We don't know exactly what the pattern of antibody production is for people who are infected with H7N9 but don't develop symptoms, though, because researchers haven't identified enough of these individuals to study.

It is very important that we get these seroprevalence studies right. If they're done wrong and we miss a lot of cases, then we will simultaneously underestimate how common H7N9 cases are and overestimate how deadly the strain is. On the other hand, if seroprevalence studies are done wrong and we think a lot of people were infected with H7N9 when they weren't, then we will overestimate how common H7N9 cases are but underestimate how deadly the strain is. Facts can help us to respond to H7N9, and if we get the facts wrong, then we can't respond properly. For example, if we come to think mild H7N9 cases are far more numerous than the severe ones that actually get diagnosed, then we might not worry as much about H7N9 as we should.

What I'd like to talk about here are some of the important ways that seroprevalence studies can go wrong. To answer my title question, no, we cannot always believe the conclusions of every seroprevalence study we see. Scientists make mistakes, just like everyone else, and sometimes things just go wrong, too. I'd like for you to understand just how some of these mistakes can arise, so that you can better judge for yourself whether a study is likely to be reliable or not, or so that you can at least know that there are things out there that can go wrong.

How are seroprevalence studies done?

There are 2 types of laboratory assays (tests) that are usually used in seroprevalence studies (although there are others): hemagglutination inhibition (HI) assays¹and microneutralization (MN) assays.² (For more information about the HI assay in general, see a nice description by Dr. Racaniello.³) MN assays are considered better (more sensitive and specific) than HI assays, but they are harder to do. MN assays require a significant amount of extra work at the end that HI assays don't. But, more importantly for H7N9 studies, HI assays can be done with either "killed," modified or "live" virus, whereas MN assays require "live" H7N9 virus. In other words, HI assays can be done in almost any lab, but MN assays require a BSL-3 lab. A neutralization assay⁴ has been developed that uses a "pseudovirus" instead of live H7N9 and is therefore far less hazardous to work with, but formal WHO diagnostic criteria still require standard HI and/or MN assays.

First, blood samples are collected. Each blood sample is drawn into a tube, and after 15-30 minutes, the tube is centrifuged to separate clotted red blood cells from the rest of the blood. The red blood cells are discarded; what's left is called serum, and that's what's studied. The serum samples should then be put in a refrigerator if they'll be studied within a few days; if they'll be studied later, they should be frozen. Once a researcher is ready to study the serum samples, the serum samples are thawed. Virus is also used for the assay, so one or more tubes of virus are thawed, too. Different types of mammalian or bird cells are prepared: typically horse, turkey or chicken red blood cells for HI assays, or a special type of dog kidney cells ("MDCK" cells) for MN assays. Various solutions are prepared. Serum samples, virus preparations and cells are diluted as needed, and everything is transferred into little wells in a plastic "plate" in just the right way. In the HI assay, the plate then sits at room temperature for 1 hour, after which it is "read" by eye. In the MN assay, the plate then sits at 37�C (body temperature) for 19-21 hours, after which it is read by a machine (an "ELISA reader"). The assay is done. The results of the assay are then written down and analyzed, and voil�, a paper appears in the scientific literature.

What could possibly go wrong with these blood tests?

Let's start with some things that can go wrong with the lab work:

1. If blood samples are left sitting around for a long time without being centrifuged, the red blood cells will start to break apart, and enzymes released from the red blood cells will start to destroy antibodies (and everything else) in the blood samples. This happens even faster if blood samples are not refrigerated.
2. If serum samples are left in the fridge too long, things can start to deteriorate, just like food in your fridge would. The antibodies that you would like to measure start to be broken down. (Sometimes, for many different kinds of studies, people study serum samples left over after patients' blood tests at hospitals. Those samples sometimes sit around in a fridge for quite a while. Some of them can even be green from stuff growing in them while they're sitting around. Yuck.)
3. If plasma (what's left in blood after unclotted red blood cells are removed) is used instead of serum (what's left in blood after clotted red blood cells are removed), then the assay can read artificially high. Serum should always be used, not plasma.
4. Every time serum is frozen and thawed, some of the antibodies are effectively destroyed. This should not be done over and over. Serum samples should be put into the right size tubes that the researcher will want to use, so that the samples are put through only 1-2 "freeze-thaw cycles" before they are tested. And all serum samples should go through the same number of freeze-thaw cycles.
5. The same thing is true for virus samples used in MN assays. A single freeze-thaw cycle can reduce virus infectivity by a factor of 10. Virus samples also need to be kept on ice when they're being worked with.
6. The plate can be read wrong. It's hard to imagine reading an HI assay plate wrong, but a special procedure (ELISA) and special equipment (ELISA plate reader) are used in the MN assay, and ELISA assays can go wrong.

But, hopefully all of that was done right. Not all researchers, students and technicians are created equal, but hopefully the lab "PI" (Principal Investigator; the person running the lab) is competent and ensures that everyone is doing things correctly.

What could go wrong with the data analysis?

What else could go wrong? The data analysis might not be done correctly. And it's here where perfectly good data can be ruined and where you have to look at seroprevalence studies most closely.

Suppose you've measured your antibody amounts ("titers") in your serum samples. How do you decide which titers mean the sample came from someone who was infected with H7N9, and which titers mean they didn't? Do you just pick a number out of thin air? If you don't have data to tell you which titers mean what, then all you are doing is measuring antibody levels in a population, and you can make no interpretation about what those levels mean. You can't say that they mean any people have or have not been infected with H7N9 at all.

Instead, you need actual measurements using serum samples from people who are known to have been infected with H7N9 to tell you what your titers mean. Someone has to study a number of patients to see what their actual H7N9 antibody titers are, and then a mathematical analysis of that data is done to come up with a threshold titer value, above which serum samples can be said to have come from people infected with H7N9 with some large degree of certainty, and below which they are thought to have come from people who were not infected. We've seen almost no asymptomatic cases (cases with no symptoms), so we really can't say much about them. So we have to go with data from H7N9 patients who have had symptoms. Here's a great graph showing antibody titers, as measured using the HI assay, in serum samples from H7N9 patients:⁵

Figure 1. H7N9 HI
Euro Surveill. 2013 Dec 12;18(50):20657

As you can see in the graph above (Figure 1), by around 3 weeks after infection onset, all samples from patients whose HI titer was measured had titers =40.

The graph below (Figure 2), from a different study,⁴ shows that the HI titer for all H7N9 samples studied by this set of authors was also =40. In addition, this graph shows titers from "control" samples (i.e., samples from people who did not have H7N9 infections); all control samples had titers that were <40.

Figure 2: H7N9 IC50 HI4
Emerg Infect Dis. 2013 Oct;19(10):1685-7

Finally, below (Figure 3) is another nice graph, from a third study,⁶ showing anti-H7N9 antibody levels ("IgG"), "HI" assay results and MN assay ("NAb") results for several H7N9 patients, again showing that all samples from the H7N9 patients studied had HI titers =40. This graph also shows that all H7N9 patient serum samples had an MN titer of =20, if samples were taken after enough time had elapsed since their infections had started.

Figure 3. H7N9 IgG HI NAb.
Emerg Infect Dis. 2014 Feb;20(2):192-200

In other words, if an individual's anti-H7N9 antibody titer is =40 by the HI assay or =20 by the MN assay, these data suggest that we could pretty safely say that he or she has had a symptomatic H7N9 infection within the past few months, and if the HI or MN titers are below those cutoffs, then the individual probably hasn't had a symptomatic H7N9 infection. We don't know to what extent asymptomatic H7N9 infections will be captured by these cutoffs, but it is likely that some asymptomatic cases would be missed using these cutoffs. It is also possible that some mild infections could be missed using these cutoffs. However, it would be a great step forward just to get estimates of what percentages of any regional population or occupational group of people have had any kind of H7N9 infection. A comparison of antibody titers for asymptomatically infected and symptomatically infected H5N1 cases may be instructive when thinking about H7N9.⁷

WHO guidelines are even stricter than the cutoffs discussed in the paragraph above. WHO guidelines say that, using the HI assay, only single samples with titers of =160 can be considered "seropositive": "Paired sera (acute and convalescent sera) with a 4-fold rise in HI titer or single sera collected in convalescent phase with HI titer of =160 could be considered as H7N9 HI antibody positive. Sera with HI titer of 20-80 should be confirmed by MN or WB assay."¹ For the MN assay, however, the WHO does not give specific cutoffs: "With single-serum samples, care must be taken in interpreting low titers such as 20 and 40. Generally, knowledge of the antibody titers in an age-matched control population is needed to determine the minimum titer that is indicative of a specific antibody response to the virus used in the assay."²

Now, it should be noted that WHO assay instructions recommend the use of horse red blood cells for the HI assay, and not everyone uses horse red blood cells. Some people use chicken, turkey, guinea pig or other kinds of red blood cells. That starts making comparisons between different groups' assays difficult. Horse red blood cells are better to use than turkey red blood cells for H7N9 because they have more a2,3-linked ("bird") sialic acids (influenza receptors); HI results are more sensitive with horse red blood cells. In other words, it may take less antibody in the assays to get the same result using horse red blood cells than it would using turkey red blood cells. This would translate into a higher number, when discussing H7N9 patient titers, for HI assays using horse red blood cells, compared to assays using turkey red blood cells. I have not seen direct comparisons of titers obtained using different types of red blood cells in HI assays specifically for H7N9, but the situation is probably similar to that for H5N1.⁸

Figures 1 and 3 above were made with HI data obtained using horse red blood cells. Figure 2 used guinea pig red blood cells. Are they completely comparable? No. Are they pretty comparable? Yes.

Are you getting a feeling for how complicated it is to interpret a seroprevalence paper? And for how difficult it is to compare results across studies?

Why does all of this matter?

It matters because some seroprevalence studies don't use appropriate cutoffs. And because it can be hard to determine even what an appropriate cutoff is when red blood cells from different species are used in an HI assay. This is where the reader has to be really careful. Cutoffs for seropositivity have been a big issue⁹ with H5N1 seroprevalence studies; some researchers have used cutoffs that were too low, and hence they have almost certainly overestimated how common H5N1-specific antibodies were in the populations studied.

So far, only one H7N9 serology paper published to date has reported probable seropositive samples, and this paper simply reported HI titers without using any specific threshold for seropositivity. Only one used study horse red blood cells in HI assays. The one paper that used an MN assay did use appropriate cutoffs. It should be noted that the new WHO HI guidelines were only published in December, 2013, after a couple of these papers were already published.

Here are the studies that have been published so far (I hope I haven't left any out):

1. Bai et al.¹⁰ looked at serum samples collected before November, 2012 from poultry workers in eastern China and found no H7N9-positive samples. The study used HI and MN assays. Turkey red blood cells were used in the HI assay. Appropriate cutoffs were used for the MN assay.
2. Hsieh et al.¹¹ studied 14 close contacts of the first H7N9 case in Taiwan. The authors took blood samples within 18-28 days after the contacts' earliest exposures. The authors used an HI assay but not an MN assay. They used turkey red blood cells for the HI assay. They found all contacts to have an HI titer �10, and declared all to be seronegative. The HI titer for the H7N9 patient in their study was 1:80. These conclusions seem very sound.
3. Yang et al.¹² looked at serum samples from 1129 people from regions of China in which H7N9 cases had been seen, and from 396 poultry workers from 10 districts in which H7N9 cases had been seen. None of the samples from the general population was found to be seropositive, whereas >6% of the poultry workers were found to be seropositive. The authors also examined serum samples from several H7N9 patients. The study used an HI assay but not an MN assay. The authors used a cutoff of =80, along with turkey red blood cells, for the HI assay. Because the authors examined serum samples from H7N9 patients using their methods and got results that are reasonably similar to other results, their cutoffs are most likely reasonable, and their conclusions are probably quite sound. The authors report:
• "Of the 1129 serum samples collected from individuals (age range, 1�88 years) in the general population, 9 (0.8%) had an HI titer of=40 to in?uenza A(H7N9), but no serum samples with an HI titer of=80 were found (Table 1). In contrast, among poultry workers, 13.9% (55/396) and 6.3% (25/396) had in?uenza A(H7N9) antibody titers of =40 and =80 (20 had an HI titer of 80, and 5 had an HI titer of 160), respectively."
  
  It is hard to imagine that an HI titer of 160 can be a spurious finding ("non-specific," to the initiated). Thus, these data strongly suggest that at least some H7N9 cases have been going undetected among poultry workers. Suppose we consider only the poultry workers with HI titer =80, or 6.3% of the poultry workers. If we then consider how many poultry workers there are, total, in districts from which H7N9 cases have emerged, then this study suggests that it's possible that quite a large number of poultry workers have been exposed to H7N9. Still, this study examined only a very small number of people, and we should be cautious about reading too much into these results.
4. Qiu et al.¹³ looked at 3 H7N9 patients and 3 close household contacts of the patients who were exposed before infection control practices were put in place. The authors looked for viral RNA using a sensitive test (PCR) and examined serum samples drawn 15-26 days post-exposure using both an HI assay and a pseudovirus-based neutralization assay. They found no contacts to be seropositive. The H7N9 patients had HI titers that reached 160-640 during this time, and the patient contacts all had titers <10. The authors used horse red blood cells for the HI assay. These findings also seem sound.

To summarize, the conclusions from all of these papers do seem sound. But, it would be wise to keep all of these issues in mind as subsequent studies appear over time.

An additional study¹⁴looked at antibody titers in 1723 serum samples collected in Vietnam using a very different kind of assay (a protein microarray). Because seropositivity cutoff levels had not been determined with authors' assay methods using actual H7N9 patient samples, these authors were appropriately very careful not to attempt to draw any conclusions about H7N9 seroprevalence from their data:

"Because titers calculated from our assay are not directly comparable to HI or microneutralization tests, no cutoff is chosen to represent positivity or clinical protection. It is not possible to associate these titers with past exposure or past infection, as serological assays have not yet been validated for H7N9."

For the future

So, as new H7N9 serology studies gradually come out, you be the judge. Figure out whether they're believable or not. Ask yourself the following:

1. What assay(s) were used? Did the authors use an MN assay? They get bonus points if they did. 
• If only an HI assay was used, then the conclusions are slightly less certain than if an MN assay was used.
2. If the authors used an HI assay, what species were the red blood cells from?
• If horse red blood cells weren't used, then HI titer cutoffs lower than 160 are probably appropriate, but there is also more uncertainty about what an appropriate cutoff would be.
3. What cutoff(s) did they use for seropositivity in their assay(s)? Do these cutoffs mesh with WHO guidelines? Do they mesh with what we know about H7N9 patient HI and MN antibody titers?

References

1. http://www.who.int/influenza/gisrs_laboratory/cnic_serological_diagnosis_hai_a_h7n9_20131220.pdf
2. http://www.who.int/influenza/gisrs_laboratory/cnic_serological_diagnosis_microneutralization_a_h7n9.pdf
3. http://www.virology.ws/2009/05/27/influenza-hemagglutination-inhibition-assay/
4. Qiu C, Huang Y, Zhang A, Tian D, Wan Y, Zhang X, Zhang W, Zhang Z, Yuan Z, Hu Y, Zhang X, Xu J. Safe pseudovirus-based assay for neutralization antibodies against influenza A(H7N9) virus. Emerg Infect Dis. 2013 Oct;19(10):1685-7
5. Zhang A, Huang Y, Tian D, Lau EH, Wan Y, Liu X, Dong Y, Song Z, Zhang X, Zhang J, Bao M, Zhou M, Yuan S, Sun J, Zhu Z, Hu Y, Chen L, Leung CY, Wu JT, Zhang Z, Zhang X, Peiris JS, Xu J. Kinetics of serological responses in influenza A(H7N9)-infected patients correlate with clinical outcome in China, 2013. Euro Surveill. 2013 Dec 12;18(50):20657 
6. Guo L, Zhang X, Ren L, Yu X, Chen L, Zhou H, Gao X, Teng Z, Li J, Hu J, Wu C, Xiao X, Zhu Y, Wang Q, Pang X, Jin Q, Wu F, Wang J. Human antibody responses to avian influenza A(H7N9) virus, 2013. Emerg Infect Dis. 2014 Feb;20(2):192-200
7. Buchy P et al., PLoS One. 2010 May 27;5(5):e10864
8. See, e.g., Table 4 in Pawar SD et al., Virol J. 2012 Oct 30;9:251
9. Osterholm MT and Kelley NS, MBio. 2012 Feb 24;3(2):e00045-12
10. Bai T et al., N Engl J Med. 2013 Jun 13;368(24):2339-40
11. Hsieh SM et al., J Infect. 2013 Nov;67(5):494-5
12. Yang S et al., J Infect Dis. 2014 Jan 15;209(2):265-9
13. Qiu C et al., J Clin Virol. 2014 Feb;59(2):129-31
14. Boni MF et al., J Infect Dis. 2013 Aug 15;208(4):554-8

NOTE: I did not have a hand in writing this post and thus take no credit for it. This was entirely the work of the Guest Writer. 

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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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More confirmation that rapid influenza diagnostic tests (RIDTs) should be used in context

The Texas Department of State Health Services have a useful couple of paragraphs from an Influenza Health Alert that puts into context reliance on the convenient and rapid, but ultimately intensive rapid influenza tests.

Rapid Lab Tests: Rapid Influenza Diagnostic Tests (RIDTs) can be useful to identify influenza virus infection, but false negative test results are common during influenza season. Clinicians should be aware that a negative RIDT result does NOT exclude a diagnosis of influenza in a patient with suspected influenza. When there is clinical suspicion of influenza and antiviral treatment is indicated, antiviral treatment should be started as soon as possible, even if the result of the RIDT is negative, without waiting for results of additional influenza testing.

Mike Coston also has an excellent article touching on some of the many other viruses that can cause influenza-like illness and on rapid testing, over on his blog, Avian Flu Diary. I highly recommend it.

Cost is always an important factor when hospitals and attached diagnostic laboratories consider how to address infections. Rapid turnaround time is another major cost because, for those small number of viruses with this option available, an antiviral drug can be administered and there seem to be benefits from doing this as early as possible for severe influenza. In some cases of course, a vaccine is available to block severe disease from occurring when you get infected (they don't stop infection, but a response to a vaccine is much safer than a bad response to an actual virus infection, as we've seen in the recent media for H1N1 in Texas. 

During flu season, influenza virus is an obvious cause for a spike in hospital admissions for acute respiratory symptoms - but if confirmation of that pathogen relies on a testing platform that can miss a third of infected individuals (only 17/45 PCR positives were detected by am RIDT in Ref#2) then antivirals may not be used in time. In a more recent comparison of RIDTs using PCR results as the standard, viral load in the upper airway (less virus gave fewer positives - duh), age (the young and elderly were less often positive), presentation time (sampling >2-days after onset of illness reduced the proportion of positivity), virus type (less sensitive for subtype B infections than A) and whether there was pneumonia or not (the former were less often positive perhaps reflecting less viral replication in the upper airway than in the lower airway?) were factors in how well the antibody-based RIDTs performed. Sensitivity ranged from 50% to 94%. These 2 studies used samples from the upper airways (swabs or nasopharyngeal aspirates respectively, as suggested by the BD� Directigen EX Flu A+B assay, Alere� Influenza A & B Test and the QuickVue� 117 Influenza A+B test)

In these instances, PCR-based methods (used as the "gold standard" in those published evaluations) shine but they take longer to generate a result and require more expertise to conduct than a rapid test. The slightly longer time is not just because they take hours to conduct instead of the minutes of a rapid test (remembering that viral lab diagnoses used to take days not hours) but because lab testing is only part of a process which also involves paperwork and passing verified and signed off results and information to all concerned clicnial parties and patients. That can take more time-and sometimes be a bottleneck for result release. Its hard for a patient's family and friends to wait, but the results will be that much more reliable when they come.

A feature of influenza season is the concurrently reduced levels of activity of other viruses. Influenza tends to "push out" a lot of other viruses during it's peak season - probably reflecting influenza's ability to dominate the immune response in an infected individual, and by extrapolation, reduces the number of susceptible individuals at the community level, remembering that the majority of influenza cases are acute upper respiratory tract illnesses.

So it looks more like the Montgomery County deaths may have been due to the high levels of influenza A(H1N1)pdm09 virus generally circulating in them there parts. A KHOU news outlet report, also circulated on ProMED, suggest that 4 Montgomery county deaths were due to H1N1, as well as other sine the regions. However, the Montgomery County Public Health District reports only 2 H1N1-confirmed deaths, so things are still a little confusing there. And as for whet other viruses may also be in these patients...so far, who knows?


A brief guide to some terms used in these sorts of discussions (also from Ref #2 below)

Sensitivity
No. of true positives / no of true positives and false negatives

Specificity
No. of true negatives / no of true negatives and false positives

Positive predictive value
No. of true positives / no of true positives plus no. of false positives

Negative predictive value
No. of true negatives/ no of true negatives


References and further reading...

1. FluTrackers story. http://www.flutrackers.com/forum/showthread.php?p=517368#post517368
2. Accuracy of rapid influenza diagnostic test and immunofluorescence assay compared to real time RT-PCR in children with influenza A(H1N1)pdm09 infection. http://www.ncbi.nlm.nih.gov/pubmed/23175329
3. Clinical and Virologic Factors Associated with Reduced Sensitivity of Rapid Influenza Diagnostic Tests in Hospitalized Elderly and Young Children. http://www.ncbi.nlm.nih.gov/pubmed/24285739

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New MERS-CoV laboratory test: takes 10-minutes but what can it tell you?

Back in June we heard of a quick test for MERS-CoV to add to the diagnostic armamentarium. I posted on it here.

Now that the Abu Dhabi Medical Congress & Exhibition it was presented at is over, we are hearing about it again through a story at The National. 

Still no details though, so my original concerns about sensitivity (how often will it miss true positive cases because it is not sensitive enough?) linger on.

Further, it's a "blood test" that also uses DNA amplification so the patient will presumably need to be sick enough to have a viraemia (virus spilling over into the blood) so it may not help at all for screening contacts or less ill people with lower viral loads. It is being described as useful for "identifying the virus in its early stages". 

Another assay that looks similar, described in PLoSONE by these researchers earlier in the year, does not appear comparable to PCR-based methods in terms of its sensitivity. 

For MERS-CoV, as for any newly emerging pathogen with unknown characteristics spreading in ways we are yet to understand, detection sensitivity is a key factor.

I look forward to seeing same real-world evaluation data.

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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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Isothermal DNA MERS-CoV test

Laurie Garret noted this article about a new, relatively easy to use bedside test to be described at the upcoming Abu Dhabi Medical Congress.

The key piece of information here, as it sometimes is with bedside (Point of Care or POC) testing, is how its real-world (using clinical samples) sensitivity ranks against other testing methods. False negatives provide a sense of false security that can be disastrous for infectious disease management. Also, the types of sample that can be collected at the bedside are presumably weighted towards easier-to-access upper airway secretions. That will not play well for any virus that may be found more often in the lower airways at presentation.

Let's hope the test fits the bill. Fast, sensitive, specific and reasonably priced testing would make great inroads into infectious disease control. Time, and more information, will tell.

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Patenting MERS-CoV: no hindrance to diagnosis at all.

Edited by Dr. Katherine E. Arden

This is not the first newly identified virus that researchers at Erasmus, or elsewhere, have patented.

They did the same for human metapneumovirus (HMPV; an endemic respiratory virus and kin to respiratory syncytial virus) after describing its discovery and characterization in 2001.
Another broad-ranging patent also listed on Google is for the human coronavirus (HCoV) NL63, discovered in 2004 by other researchers. Patenting is part of business and today a portion of science research requires proof of the ability of researchers to work with business to help produce real outcomes. Mass-produced diagnostic kits are one outcome - they are made to high standards of quality and distributed worldwide. Research scientists can't do that alone.

Also, the existence of a patent does not prevent or even impede the medical research or public health efforts being undertaken for MERS-CoV now. It also didn't hinder research on HCoV-NL63 or HMPV back when they were discovered. Have a look atPubMED (the Google for scientific research articles) and see how for yourself. There are hundreds of papers there that cover all aspects of each virus - virological, clinical, immunological impact and epidemiology. As a publisher of some of those papers I can assure you I was not asked to pay a cent to the "inventors" and the slowest part of the publication process was my own writing.

Comments to the World Health Assembly today have been interpreted to suggest that a patent on the MERS-CoV has delayed the development of diagnostic tests.

In fact, the necessary parts for leading edge diagnostic testing - sequences for polymerase chain reaction primers - were made available by the researchers (easily contactable thanks to a ProMED posting) as soon as they were developed. They were next made public to the entire scientific community through very rapid publications in leading journals. There are more than 20 papers on the MERS-CoV, or HCoV-EMC as it was known, listed on PubMED already. All a professional, PCR-enabled, public health or research diagnostic laboratory has to do is eMail the discoverers or read the manuscripts and order the reagents.

A true absence of information is unquestionably an impediment to infectious disease research. We saw some great examples of unfettered information from China during the H7N9 outbreak this year. When key information is freely available, as it has been from Prof Zaki and the Dutch researchers at Erasmus for the MERS-CoV, we can rest assured that diagnostic developments are unhindered. Diagnostics are most useful though when they are used to report when and how cases of infection by a new virus occur and spread. Could that area be the next target for more public criticism?

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Matrix-targeted real-time PCR for H7N9.

A belated congratulations to my fellow Group Leader here at the Qpid lab, A.Prof David Whiley on the implementation and media coverage of his sensitive real-time RT-PCR to detect H7N9 for implementation by Pathology Queensland's microbiology laboratory. 

It targets the matrix gene segment, which as far as we know, is unique among the H7N9 assays in use to date.

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Study supports poor H7N9 transmission - from anything!

A new paper in by J. Han et al. Emerging Microbes and Infection concludes that neither 2 vendors in an H7N9-positive poultry market visited by the H7N9-positive patient (our Case#10, also studied in recent Lancet article) nor his close contacts, were WHO RT-rtPCR positive. 

So picking up H7N9 is certainly not a frequent thing. Also supported by the few cases that have been identified considering the population in these areas that move through and interact with birds and the markets. 

Also, once you have it, transmitting H7N9 it seems to be an infrequent event.

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How reliable are the H7N9 real-time RT-PCRs for low viral loads?

An interesting document that compares the effectiveness of some different diagnostic PCR methods for use in H7 or N9-based flu diagnosis. It seems to show that in a couple of instances H7-specific or H9-specific molecular methods will fail (1:10,000,000 is a pretty extreme dilution) to detect H7N9 at low levels while some assay fail to amplify the intended target altogether. 

This sort of assay variation is pretty commonplace when you compare different PCR designs for the same target. It's a major reason why everyone prefers to design their own assay; each are convinced there's is the best. From this document, the people behind the OFFLU (FLI-H7) assay happen to be right. 

How these data relate to viral load in the human (and animal) samples these may be/have been used on is unknown. This sort of variance may contribute to negative throat swabs in H7N9-cases subsequently proven positive using lower respiratory tract samples (e.g. sputum) in which the viral load is presumed to be higher.

OFFLU: a network of expertise on animal influenza established jointly in 2005 by the World Organisation for Animal Health (OIE) and the Food and Agriculture Organization of the United Nations (FAO) to support and coordinate global efforts to prevent, detect and control important influenzas in animals.
CNIC: Chinese National Influenza Center

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A(nother) new real-time PCR-based H7N9 test.

US CDC have made available their CDC H7N9 rRT-PCR for use on suspect cases (see interim guidance for defining cases) and in combination with their existing Flu rRT-PCR Dx Panel ("r"=real-time; "Dx"=Diagnosis/Diagnostic) after FDA support through an Emergency Use Authorization.

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