During the first 4 weeks of life "common cold" viruses occur frequently and can make your little one sick....

This post has been moved to the new Virology Down Under platform on Wordpress.

You can get to this specific post by clicking on the link below...

http://virologydownunder.com/during-the-first-4-weeks-of-life-common-cold-viruses-occur-frequently-and-can-make-your-little-one-sick

Please adjust your bookmarks.

Apologies for any inconvenience.

Describing the brain anomalies in some infants born with microcephaly

In a new study by Hazin and colleagues, 7 of 23 (30.4%) infants with congenital microcephaly were found to have IgM antibodies (which indicate recent infection) in their cerebrospinal fluid. Dr. Ana van der Linden, one of the original doctors who brought the world's attention to a reportedly higher-than-normal number of microcephaly diagnoses, is a co-author of this study.

No antibodies were detected in the cerebrospinal fluid of any of the 23 infants that identified the following diseases or infections:

• toxoplasmosis (disease due to infection with Toxoplasma gondii)
• syphilis (disease caused by infection with Treponema pallidum)
• Varicella Zoster virus
• Parvovirus B19
• Human immunodeficiency virus
• Rubella virus
• Cytomegalovirus
• Herpes simplex

No antibody testing was described for Dengue virus or for Chikungunya virus and no antibody testing was reported from the mums of these infants.

No PCR testing was reported at all.

The authors note that - perhaps at a reviewer or Editors request...

"Our findings are nonspecific and may be 

seen in other congenital viral infections."

However, the authors extrapolate from those 7 ZIKV IgM positive results to all 23 infants throughout their publication. 

• The title broadly defines an association 
• The first sentence of the results describes the compatibility of the unproven association between prior undescribed ZIKV-like disease and congenital microcephaly
• The Letter wraps up by stating that ZIKV is associated with a disruption in brain development rather than its destruction

This Letter doesn't leave any doubt for the reader, in my opinion, that detection of antibody in the cerebrospinal fluid is enough to prove causality of the described brain anomalies. Which of course is simply untrue.

It may well turn out that this association is solid as a rock. Of course, we all know by now that there is a "scientific consensus" already stating this is a proven association. Nevertheless, the data in this study don't prove anything. They are descriptions of something that has already happened. Not explanations of how it happened. Not what caused it to happen.

I don't understand why this Letter, essentially a detailed description of congenitally acquired brain anomalies of which there have been other descriptions, has only now been published. Yes, the NEJM is a journal that will give it greater oxygen...among those who read the NEJM...but the NEJM isn't the only journal that science media can access for the latest science and medical research. Is it the only journal that Doctors,the WHO and medical researchers read? I really hope not. Was it the slow pace of publication? This issue has been attributed to NEJM and could mean that these were new observations when originally received.[2] I can't find when the letter was first submitted so it's hard to know the answer there. Is there novel information in here that would be beneficial to spread widely via a platform like NEJM? Not that I can see.

So, welcome to the literature of Zika'ville

References...

1. Computed Tomographic Findings in Microcephaly Associated with Zika Virus
   http://www.nejm.org/doi/pdf/10.1056/NEJMc1603617
2. https://www.bostonglobe.com/metro/2016/04/05/new-england-journal-medicine-increasingly-targeted-critics/9H3JFKzTNJpCsOQqIUNXJP/story.html

Detecting Zika viruses using PCR: a look at published assays...

[NOTE: I'll update this post with other assays and information as I find time]

You may be helped - hopefully anyway - by reading the earlier post in this series - Primers prime PCR unless past their prime... [1]

Also hopefully helpful - the Zika virus (ZIKV) lineages I'll be referring too can be seen in this (very basic) tree. The viruses currently circulating in the Americas fall into the "Asian lineage".

Basic tree showing the majority genome sequences of ZIKV variants form discovery to 2015.

PCR and ZIKV...

Because flaviviruses are "RNA viruses" and because ZIKV is a flavivirus, we'll be talking about the range of RT-PCRs that have been described in the literature for ZIKV. This will include both RT-rtPCR (Reverse Transcript real-time Polymerase Chain Reaction) but also the non-real-time versions which I'll be calling "conventional" RT-PCRs or RT-conPCRs.

This will not be all the variants and home brew concoction of RT-PCR out there for ZIKV, but it is the ones that have been published and some of the articles that reference them.

I'll be counting nucleotide positions from 5' to 3' (or "left" to "right") so nucleotide #1 will be furthest form the pointy end of the green arrow-no, not that Green Arrow).

I'm using Spondweni virus, a related but different virus (usually) at the top of the alignment and in the tree. This is to provide something that shows how different a very near cousin is, even when "zoomed in" and compared to the brothers and sister variants of ZIKV. 

In some alignments I have marked, in the left most panel, some - but not all - examples of Asian or African lineage variants. See if you can spot patterns among the nucleotides that may be specific to each. 

------------------------

One-Step RT-PCR for detection of Zika virus
Faye et al. J Clin Virol 2008 43:96-101 [2]
PCR Aim: To detect ZIKV in human serum
Subsequent publications using this assay:

Faye et al assay. (A) The forward primer and (B) the reverse primer aligned against a range of African and Asian lineage ZIKV variant sequences.

• Both primers have degenerate positions and look as though they should work well against African and Asian lineages on paper
• There is a mismatch in the reverse primer at position #16 that is not accounted for and may be destabilising for a number of Asian lineage variant templates

----------------------

Genetic and Serologic Properties of Zika Virus Associated with an Epidemic, Yap State,Micronesia, 2007
Lanciotti et al. Emerg Infect Dis 14(8):1232-9 [3]
PCR Aim: To rescreen sera from the 2007 Yap Island epidemic
Subsequent publications using this assay:[3]

Assay 1...

Lanciotti et al assay #1. (A) The forward primer (853), (B) the real-time PCR probe (860-FAM) and (C) the reverse primer (911c) aligned against a range of African and Asian lineage ZIKV variant sequences.

• The forward primer must interact with a 2-3 mutations in some of the the African variants, but is a good match with all Asian lineages variants.
• The probe also has 3 mismatches with the same variant as above and 1-2 with African lineage variants but is again a good  match for most Asian lineage variants
• The reverse primer is a great match to Asian variants but not very good if faced with trying to detect an African variant, especially KF383117

Assay 2...

Lanciotti et al assay #2. (A) The forward primer (1086), (B) the real-time PCR probe (1107-FAM) and (C) the reverse primer (1162c) aligned against a range of African and Asian lineage ZIKV variant sequences.

• The forward primer works well against most variants and the mismatches it does face are at the less destabilising 5' end, against some African lineage variants.
• The probe is a good match for most variants of either lineage, having a couple of issues with 2 Central African Republic variants and KF383118 poses a problem at the 5' end of the probe which could be a issue for the polymerase which comes charging down the same strand as the which the forward primer is hybridised to, looking to chop the probe up.
• The reverse primer is a great match to Asian variants except for KF383117 and will suffer between 1 to 5 mismatches with African lineage variants - also impacting across the primer landing site. 

Generally these 2 assays should be good for detecting the Asian variants, will suffer varying degrees of performance issues against African variants.

Imported Zika Virus Infection from the Cook Islands into Australia, 2014
Pyke et al...PLoS Curr. 2014 Jun 2;6. pii
PCR Aim:
Subsequent publications using this assay

E gene assay...

Coming up...

Quantitative real-time PCR detection of Zika virus and evaluation with field-caught Mosquitoes
Faye et al. Virol J 2013 10:311

References...

1. Primers prime PCR unless past their prime...
   http://virologydownunder.blogspot.com.au/2016/03/primers-prime-pcr-unless-past-their.html
2. One-step RT-PCR for detection of Zika virus
   http://www.ncbi.nlm.nih.gov/pubmed/18674965
3. Genetic and serologic properties of Zika virus associated with an epidemic, Yap State, Micronesia, 2007
   http://dx.doi.org/10.3201/eid1408.080287
4. Zika Virus Infection in Pregnant Women in Rio de Janeiro — Preliminary Report
   http://www.nejm.org/doi/pdf/10.1056/NEJMoa1602412
5. Quantitative real-time PCR detection of Zika virus and evaluation with field-caught Mosquitoes
   http://www.virologyj.com/content/10/1/311
6. Imported Zika Virus Infection from the Cook Islands into Australia, 2014
   http://currents.plos.org/outbreaks/article/imported-zika-virus-infection-from-the-cook-islands-into-australia-2014/

Primers prime PCR unless past their prime...

The PCR is a tough old bird... 

The polymerase chain reaction (PCR) is a hugely powerful tool for detecting virus. It can still do this more quickly and sensitively, from any human specimen, than any other diagnostic method that came before it.

Amazingly, the method of DNA copying by extension hasn't changed so much since the 1980s. The major variations have been to the machines that control the temperatures, the wide range of kits and the way PCR product is detected.

In the 1990s this detection became about measuring fluorescence within the same tube as the reaction as it took place - in real-time. But prior to this method, detection of a successful PCR was about opening the tube after the run had finished and performing a multi-step lengthy process of identifying the successfully amplified DNA by electrophoresis - end-point detection. This change underpinned what became known as of real-time PCR (rtPCR).

Quite amazing to think that anything could remain so integral to virology - or anything - these days. Rather than go into the details of what real-time PCR is, was and could have been, you can get a pretty good background understanding in our 2002 open access review of it as used in virology, aptly named "Real-time PCR in virology".[1]

PCR in the laboratory with viruses...

Today, rtPCR is integral to detecting the presence of virus and a successful detection is usually taken to mean that the person who provided that sample had a current infection at the time of sampling. 

That's not technically a correct assumption, nonetheless decades of research and epidemiology have largely been based on it. It's a pretty safe bet that there is a good correlation - in my expert opinion - in most of the instances in which we use PCR in a diagnostic laboratory setting. Detection by PCR does not however, mean we have disease or that the signs and symptoms a patient may have at the time of sampling are caused by that virus. The laboratory results along with any relevant travel history, contact history, signs,  symptoms and other clinical investigations and background knowledge all combine to help a medical doctor make that call. It's a team effort but laboratory testing, including PCR, biochemistry and immunology, provide crucial information that very often cannot be supplanted by a diagnosis based on how a patient looks and feels.

When we use PCR we use it to either detect DNA from viruses that have a DNA-based genetic make-up, or RNA from those viruses with RNA-based genetic material. It is of course used for bacteria and fungi and humans and many other things too, but this is not "all manner of other things down under".

For a PCR to work on an RNA virus, given the enzymatic process that is at its core (a DNA dependent polymerase), we first have to make a DNA copy of the RNA - a process called "reverse transcription". This term comes from the need to copy RNA back into DNA  - transcription being the process of copying DNA into RNA which occurs in our cells and elsewhere.

What a primer looks like when you align it to a reference sequence..

This probably deserves its own explanatory blog but we'll see what we can get away with for now.

A nucleotide sequence alignment of Zika virus (ZIKV) sequences downloaded from GenBank aligned with a primer from a published RT-rtPCR assay.[2] 1-the region of a primer that, if poorly "matching" to a target region of viral sequence, will most likley negatively impact the efficiency of the resulting PCR amplification, possibly causing the PCR to perform poorly, which can be of concern when there is only lower amount of of virus present in a sample, or fail completely; 2-in this example,the primer from Lanciotti et al. does not match the target at these marked positions - there is one or more "mismatches" between the primer and some of the viruses represented here which will affect the stability of the primer's binding, or hybridisation, with the viral genetic material during the PCR. Generally-this primer binds very well to Asian lineage Zika viruses, but much less well to African lineage Zika viruses.
Alignment made using Geneious v 8.1.5. The primer, 911c is the reverse primer in a pair used for an RT-rtPCR described by Lanciotti et al.[2]
Click on image to enlarge.

In the figure above we can see a few important items to consider when looking at primers and how they may work when put it into use. I say 'may' because even rubbish primers can sometimes do the job in PCR'ville, while the best designs may sometimes fail. One has to test to know for sure whether a primer pair does or does not perform the way you intend. Much like in infectious disease'ville before you try and link an infection with a disease.

Some key things worth noting about the figure above:

1. This is called a nucleotide sequence alignment - or just an alignment. In this version, I've used dots to highlight whenever one of my reference sequences has the same nucleotide - an A, C G or T - at the same position as the primer I randomly chose for this example.
   
   If there is an entire column of dots, then at that nucleotide position we have a perfect match between all the viruses I've used in this alignment and that primer. In theory the primer will bind to those viruses if the one of them is in the patient's samples and we've successfully purify the virus's genetic material away from the other unwanted stuff in the samples - like proteins, inhibitory chemicals and carbohydrates.
   
   I won't be covering this "extraction" or purification process here.
   
2. The enzyme in the PCR that makes PCR work relies on the primer to be matched up with the viral genetic material - especially at one end - the end at which the enzyme is going to be extending. I've highlighted that direction with the green arrow (no, not the Green Arrow).
   
   As the enzyme moves, it adds new nucleotides which it pulls in from the PCR chemical soup we use (in kits these days). This extension eventually results in a copy of the virus sequence. The primer shown is the reverse primer. There is also a forward primer which makes a copy in the other direction of the newly made strand. There is a bit more to this strand thing, but also not covering that here.
   
   As the copies get copied we eventually end up with just the region spanned by the primer pair amplified up - cloned if you like. Within that region, copied millions of times, is a sequence to which we previously designed a probe labelled with fluorescence molecules that has also been added to the chemical soup.
   
   As the copies were being made, the probe bound to its target and was destroyed, results in the signal which allows us to see those clones pile up....in real time.
   
3. The end of the primer from which the enzyme starts must be well bound, or hybridised, to the viral sequence or else the polymerase may just fall off and not have anything to copy. This is really important to the success of the PCR, and if it's not a stable hybridisation (As hybridise to Ts, Cs hybridise to Gs), then the PCR won't work, or will work poorly.
   
   If hybridisation is poor then the early cycles don't make those first few copies which act as the template for more copies, and it is these early cycles which make or break the success of a PCR.
   
   In the figure, the important end is highlighted by the orange pill shape.
   
4. I've added some info to the sequence names used in the alignment above. This indicates where and when some of them came from.
   
   The Asian lineage (see the basic tree below-also made using a nucleotide sequence alignment as a starting point)  is the one currently circulating in the Americas and previously in the Yap Island outbreak in 2007 and in French Polynesia.
   

   

   Zika virus complete or near-complete genome sequences aligned using Geneious v8.1.5.
   Neighbor-joining p-distance tree mad in Mega v6; 500 bootstraps 

   
   The primer example in the  alignment does not have any, but sometimes a PCR primer can have one or more "degenerate" nucleotide positions in it. These introduce variations to the primer sequence to accommodate one or more different nucleotides in the viral genetic target sequence.
   
   Sometimes, one has no choice but to design the primer to a variable viral region and so a degenerate position or two is added into your primer to help the primer hybridise and cope with that.
   
   We use codes for these degenerate positions - see the Table below excerpted from Chapter 2, written by Andreas Nitsche in a book I edited about a decade ago and in an extended version of the International Union of Pure and Applied Chemistry (IUPAC) code.[3,4]
   
   
   This is all a bit of a misnomer though because it means that when we order a primer from a company, they actually make a mix of primers - some have the full sequence and one nucleotide of the degenerate position, and the rest of the primer with the full sequence and the other nucleotide at that position - up to 4 different primers in the mix if we use an "N" degenerate position.

So with all that in mind - the next post - because this one is now too long - will examine some of the PCR-based diagnostics for Zika virus (ZIKV).

References...

1. Real-time PCR in virology
   http://nar.oxfordjournals.org/content/30/6/1292.full
2. Genetic and serologic properties of Zika virus associated with an epidemic, Yap State, Micronesia, 2007
   http://wwwnc.cdc.gov/eid/article/14/8/08-0287_article
3. Real-Time PCR in Microbiology: From Diagnosis to Characterization
   http://www.horizonpress.com/rtmic
4. An extended IUPAC nomenclature code for polymorphic nucleic acids
   http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2865858/

A negative viral culture result is not the end of the story...just a negative result

Lately, for both Ebola virus and Middle East respiratory syndrome coronavirus, there have been instances where I've been reminded that one must not rely on the growth of an infectious virus from a sample to be sure that there is virus in that sample.

Past diagnostic methods have failed to isolate many newly identified viruses (NIVs), which is not surprising considering that those culture-based methods can be over 100-fold less sensitive than current molecular (PCR-based) tests [1,2,3,4] and that many new viruses do not grow in traditional culture at all.

So when an RT-PCR or PCR result cannot be confirmed by the culture of a virus from the same sample, that really doesn't mean more than...that. 

Virus may be present, but our relatively insensitive culture techniques, which haven't really advanced in a long time, may just fail to get it growing. 

Viral isolation by cell culture is really a dying art form. And it is a very lengthy, demanding and sometimes subjective art form at that requiring particularly skilled artistes. 

References...

1. Templeton,K.E. et al. Improved diagnosis of the etiology of community-acquired pneumonia with real-time polymerase chain reaction. Clin Infect Dis 41, 345-351 (2005).
2. van Kraaij,M.G.J. et al. Frequent detection of respiratory viruses in adult recipients of stem cell transplants with the use of real-time polymerase chain reaction, compared with viral culture. Clin Infect Dis 40, (2005).
3. Garbino,J. et al. Lower respiratory viral illnesses: Improved diagnosis by molecular methods and clinical impact. Am J Resp Crit Care Med 170, (2004).
4. Gunson,R.N., Collins,T.C., & Carman,W.F. Real-time RT-PCR detection of 12 respiratory viral infections in four triplex reactions. Journal of Clinical Virology(2005).

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

This post has been moved to the new Virology Down Under platform on WordPress.

You can get to this specific post by clicking on the link below...
 
https://virologydownunder.com/the-mechanics-of-the-polymerase-chain-reaction-pcr-a-primer/

Please adjust your bookmarks.

Apologies for any inconvenience.

PCR primers...a primer!

This post has been moved to the new Virology Down Under platform on Wordpress.

You can get to this specific post by clicking on the link below...

https://virologydownunder.com/pcr-primers-a-primer/

Please adjust your bookmarks.

Apologies for any inconvenience.

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

This post has been moved to the new Virology Down Under platform on Wordpress.

You can get to this specific post by clicking on the link below...

http://virologydownunder.com/reverse-transcription-polymerase-chain-reaction-rt-pcr-a-primer-for-virus-detection/

Please adjust your bookmarks.

Apologies for any inconvenience.

-Ian

Pressure testing...

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

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

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

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

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

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

Something dramatic changed, and that is the case definition.

Prof. Drosten to ScienceInsider

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

"The question of whether there is a mild, short-lived infection in some people is scientifically interesting. But in cities like Jeddah, it is bringing the health system close to collapse. That is the big problem. So many samples are being tested that the lab capacity won’t suffice for the real cases."

Prof. Drosten to ScienceInsider

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

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

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

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

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

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

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

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

Sources...

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

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

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

From 2013:

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

From 2014, to date:

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

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

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

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

See #3 below for influenza PCR discussion at WHO.

Sources..

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

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.

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.

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