Showing posts with label RT-PCR. Show all posts
Showing posts with label RT-PCR. Show all posts

Sunday, 3 April 2016

Usefulness of urine samples for Zika virus testing...

There is a narrow window of Zika virus (ZIKV) detectability in the blood (viraemia) based on the RT-PCR testing of serum samples from symptomatic cases. I've talked a little about this in a smaller, earlier post: Zika virus disease samples...don't pass urine (by)..

Little is known about asymptomatic infections and viraemia.

A decent number of studies have shown that urine may be a better sample for testing using molecular methods because of prolonged viral shedding (viruria) and sometimes higher viral load compared to serum samples.

The following is a list of some of the peer-reviewed literature and some public health advisories that have mentioned seeking ZIKV in urine using molecular techniques.


Australian Department of Health
Blood & Urine: 1st 14 days post symptom onset
http://www.health.gov.au/internet/main/publishing.nsf/Content/ohp-zika-health-practitioners.htm#toc05

European CDC
Blood: 3-5d post onset
Urine: up 10 10d

Public Health England
Blood: Anyone with travel history and current/past symptoms suggetsing ZIKV infection
Urine: Pregnant woman with current symptoms/male with current symptoms and a pregnant partner
https://www.gov.uk/guidance/zika-virus-sample-testing-advice 

United States CDC
Serum\CSF: Must be collected as primary test substrate
Urine: Can also be submitted
                                     
Kutsuna et al
In 1/2 cases, ZIKV RNA detected in urine when serum negative

Gourinat et al
Detected in urine of 6/6 symptomatic patients between 10 to 20 days after onset (7-20d better than serum), often with higher viral loads from urine samples than from serum. Serum positive in 4/6 but only until rash observed (2-3d post onset). Urine levels rise slightly after disease and rash onset - “preferred for virus detection”.



Shinohara et al
ZIKV RNA detected in urine 7 days after symptom onset; serum equivocal at 2 days post-onset

Besnard et al
Urine positive in 2 cases up to 8 days post-onset, in one case after serum was negative

Brasil et al
Of 88 women, 72 (82%) had positive results for ZIKV on PCR in blood, urine, or both; 60 in serum specimens; 46 in urine samples; 34 in both specimens; 12 in urine specimens only; 26 in blood specimens only; median number of PCR cycles for serum specimens - 33.0; median number of PCR cycles for urine specimens - 29.0.

Roze et al
Urine positive in 2/2 cases when serum was negative. 15-21 days after neurological symptom onset.

Barzon et al
Urine and had 106-fold higher viral loads than serum. Both sample types positive from 5 days after symptom onset, Urine positive until 22 days after onset-saliva until 29 days.


The outcome here is that urine is a sample that looks like it should be requested perhaps much more often than it is. Also, the language around recommending the collecting of urine is not as clear as it appears that it should be - including by some other public health agencies 

As it stands, a negative serum RT-PCR test for ZIKV does not hold a lot of meaning because of the narrow detection window using blood. 

Urine has proven itself useful for detection and confirmation of ZIKV infection - a process that is key to learning more about the virus and its impact(s) on infected humans. 

Use urine. Welcome the wee. Take the...well you get the point.

Sunday, 6 March 2016

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/

Friday, 29 January 2016

Zika virus disease samples...don't pass urine (by)..

It is becoming anecdotal in the Tweet'verse that Zika virus (ZIKV) viraemia (virus in the blood) can only be detected for 5 days - presumably after illness onset  - I need to chase that down.

But blood is not the only sample to test. 

In fact it may even be one of the less informative samples to test if trying to detect signs of a current or very recent infection. A lot of that being done in the Americas, and as a result, in laboratories worldwide, right now.

Urine has been shown to be positive for ZIKV RNA beyond 5 days, sometimes when blood is completely negative by highly sensitive polymerase chain reaction (PCR) methods.[1,2,3] Saliva has also been of use,[4] but urine seems to outperform it for PCR purposes.

In particular - beyond 10 days in the study by Gourinat et al.[3]
From Emerging Infectious Diseases article,Vol. 21, No. 1,
January 2015 Detection of Zika Virus in Urine.[3]
References...
  1. http://jtm.oxfordjournals.org/content/jtm/23/1/tav011.full.pdf
  2. http://www.eurosurveillance.org/images/dynamic/EE/V19N04/art20683.pdf
  3. http://wwwnc.cdc.gov/eid/article/21/1/pdfs/14-0894.pdf
  4. http://www.sciencedirect.com/science/article/pii/S138665321500133X

Friday, 23 October 2015

Markets that deal in camels may help spread MERS-CoV variants..

This camel/MERS-CoV study from Farag and colleagues, serves as follow-up of sorts to my last post. The paper, which was published in July 2015's Infection, Ecology and Epidemiology, is entitled High proportion of MERS-CoV shedding dromedaries at slaughterhouse with a potential epidemiological link to human cases, Qatar 2014.[1]

The authors remind us in the background that the routes of direct or indirect zoonotic transmission are still unknown but that a "large proportion of MERS cases" are suspected to have resulted from zoonotic transmission.

105 dromedary camels (DCs) either from a market sale or directly from Qatar or the Kingdom of Saudi Arabia (KSA) were sampled in February (n=53) and March (n=52), 2014. Samples included nasal, oral, rectal and bronchial swabs and lymph nodes from animals grouped into age 3 groups: 0 to 6 months (n=41), 7 to 12 months (n=35) or greater than 12 months (n=29) of age. Testing for virus was by Corman et al's UpE and N gene real-time RT-PCRs.[2] Testing for antibodies was via the detection of a reaction to the MERS-CoV, severe acute respiratory syndrome (SARS)-CoV and human CoV (HCoV)-OC43 spike domain S1 antigen using the protein-microarray method described previously by this group.[4]

Findings...
  • 59% of DCs had at least one MERS-CoV RNA positive sample but no significant difference in viral load was apparent between sample types or ages
    • 61/101 (60.3%) of DC's nasal samples had RNA detected
    • 23/102 (22.5%) of DC's saliva samples had RNA detected
    • 15/103 (14.6%) of DC's rectal samples had RNA detected
    • 7/101 (6.9%) of DC's bronchial samples had RNA detected 
    • 5/53 (9.4%) of DC's lymph nodes had RNA detected
  • 5 different MERS-CoV variants (subtly different versions of MERS-CoV) were circulating in Qatar among the sampled animals at this time according to RT-PCR/sequencing method that targets a fragment of the S2 domain of the MERS-CoV Spike gene.[3]
  • 100/103 (97%) animals were reactive for IgG, and most of 53 animals tested, had antibodies capable of specifically neutralizing cellular infection by MERS-CoV as determined by a 90% plaque reduction neutralization test (PRNT90; [5])
  • Antibody levels and viral load did not correlate suggesting - based on this subset of the immune response - that reinfection may be possible since protection may be limited, as it is among humans with the 4 known HCoVs. The authors note that this may prove a challenge for any future DC vaccine which would need to produce a protective effect to meets its need
  • No age-specific differences were found in MERS-CoV RNA shedding - usually younger DCs are distinctly more likely to be shedding viral RNA than older DCs
Discussion...

The authors noted here that discrepancies do exist between their study and those of some others - specifically, that others have not found viral RNA in faeces - but those studies also tested fewer animals. It is important, when percentages are not high, to test enough animals to see the full extent of MERS-CoV shedding and potential transmission routes.

DCs from different regions within Qatar and outside Qatar, may be shedding MERS-CoV while in DC markets and holding pens, sometimes for weeks, awaiting slaughter. 

Camel markets are thus a likely high risk area for acquiring a MERS-CoV infection - and multiple variants can be circulating here. 

In previous Qatari investigations, human cases have been linked with visits to the areas studied here and have also included DC slaughterer cases, supporting the notion that humans with DC exposures (presumably when they are infected with MERS-CoV) are at risk of becoming infected themselves. 

Yet this study did not manage to capture the process of transmission in action. It is that process that holds such importance for this chapter on MERS-CoV and especially for those who disbelieve the role of DCs in human MERS cases. 

In the next post, we will re-visit a study that did seem to capture DC>human infection.

References...
  1. High proportion of MERS-CoV shedding dromedaries at slaughterhouse with a potential epidemiological link to human cases, Qatar 2014.
    http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4505336/
  2. http://www.ncbi.nlm.nih.gov/pubmed/23041020 
  3. http://www.ncbi.nlm.nih.gov/pubmed/25728084
  4. http://virologydownunder.blogspot.com.au/2015/10/if-you-are-often-in-contact-with-camels.html
  5. http://www.thelancet.com/journals/laninf/article/PIIS1473-3099(13)70164-6/abstract

Sunday, 18 October 2015

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).


Tuesday, 5 May 2015

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

Friday, 16 January 2015

MERS-CoV snapdate on canaries...

MERS-CoV detections among healthcare workers (HCWs)

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

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

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

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

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

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

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

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

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

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

A couple of quick questions spring to mind:
  1. Just how widespread is this lengthy shedding period?
  2. What does this say about how mild a virus MERS-CoV is when comorbidities are not a factor?
  3. What role do genetics play in the host's containment and clearance of MERS-CoV infection?
Heatmap of MERS-CoV detection by date and region
within the Kingdom of Saudi Arabia
Click on image to enlarge.
Reference..
  1. A Case of Long-term Excretion and Subclinical Infection With Middle East Respiratory Syndrome Coronavirus in a Healthcare Worker. Manal Al-Gethamy, Victor M. Corman, Raheela Hussain, Jaffar A. Al-Tawfiq, Christian Drosten and Ziad A. Memish.
    http://cid.oxfordjournals.org/content/early/2015/01/01/cid.ciu1135.long


Monday, 17 November 2014

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

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

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

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

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

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

For the past few weeks I have been trying to find he evidence that underpins why the world chooses to use a 48-72 hour window in its guidelines. I've been asking a lot of people-and I thank those who replied. Tonight the very diligent and extremely tolerant folks at the World Health Organization got back to me with a quote from Dr Pierre Formenty, team leader Emerging and Dangerous Pathogens. A hard man to get hold of sometimes-as you might imagine. He said (lightly edited)...
There is at least one documented case during an outbreak in Africa; a contact with fever = a suspect case; he was negative at day 1 with RT-PCR (CDC Lab) and was found positive at day 3 (when retested).
So the 48-72 hours come from this incident. We want to be on the safe side and limit the number of false negative that are inevitable with any test.
And so there you have it. If anyone has anything further to add to this story, I'd be most happy put it here.

References...

Thursday, 12 June 2014

MERS-CoV in the blood....

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

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

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

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

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

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

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

But this was not the first time a MERS-CoV diagnosis was obtained retrospectively, or as part of a study, using RT-PCR (conventional or real-time) on serum (cell free blood) rather than a respiratory tract sample. Yet remember that the presence of viral genome (or bits thereof) identified by RT-PCR does not guarantee that infectious virus was in the blood, only that viral RNA could be detected there.
  • Case No. 1 from the original hospital cluster of MERS cases in Al-Zarqa, Jordan in March-May 2012, was identified thanks to retrospective RT-rtPCR (CDC version) on a convalescent serum sample.[2] 
  • The 2 French MERS cases (1 imported, 1 locally acquired from contact) had RNA in their blood (UpE RT-rtPCR); the patient who died was positive for at least 4-weeks while the surviving patient cleared viral RNA in the 1st week after symptom onset.[3] 
  • Two cases imported into the Netherlands from the KSA were found to have viral RNA in their blood for days; Case #1 from day-0 after diagnosis until at least day-9 and Case #2 from day-1 until at least day-5.[4] In this study viraemia outlasted virus detectability in the faeces but was detected for as long as virus in throat swabs of Case #1. RNA was not detected in the urine.[4]
Serum may be a useful sample, not just to determine whether antibodies to MERS-CoV develop(ed), but to help detect MERS-CoV RNA, as a surrogate for infectious virus, when a respiratory sample is not available. 

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

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

Some typos & grammar corrected 05MAR2015

References.... 

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

Thursday, 23 January 2014

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.

Monday, 13 January 2014

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.

Tuesday, 31 December 2013

Qatari camels clear coronavirus

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

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

An interesting comment within the report states that...

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

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

Hat tip to CIDRAP.

Monday, 25 November 2013

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 heal
thcare 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
  4. Viral Load Distribution in
  5. SARS Outbreak
  6. http://wwwnc.cdc.gov/eid/article/11/12/pdfs/04-0949.pdf