Friday, 28 June 2013

H7N9 diagnostic improvement..

Currently, H7N9 testing of chickens in Hong Kong requires 4 days but a new test will complete testing in hours.

Thursday, 27 June 2013

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

There have been a few comments of late about the finding of MERS-CoV in asymptomatic or mild cases of disease. For example, from ProMED
As to my knowledge "infection" is defined as the proof of an infectious agent AND the proof of multiplication of this agent inside the body (OR associated inflammatory response that can be linked to this agent). Therefore my question is: where was the specimen taken from the "asymptomatic" nurses? PCR, as we all know, just detects nucleic acids. So if multiplication cannot be proven and no local or systemic inflammatory response is given, how was "contamination" (no matter whether due to inactivated parts of MERS-CoV or even complete virus particles) ruled out? This is not a semantic question only but should impact the reported number of "cases". [Prof Martin Haditsch]
The feeling I get is that some don't trust the PCR results (or result-getters?) alone because they do not fulfil a need to see replicating virus in a host before assigning causality to that virus. Culturing an isolate of course, wouldn't assign causality to a pedant either it just proves it was in the patient. Similarly, serology would only prove it was in the patient and was recognized by their immune system. The sorts of extremely rare and poorly define studies needed to actually prove causality (human infection - but unethical) are usually well down the priority list in a public health management phase such as we are in with MERS.

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

I've added some musings below. I'll be working on this throughout the week day-check back for final version:
  1. PCR is an enzyme-driven cyclical cooking and cooling process that amplifies a pre-defined stretch of DNA using a pair of equally pre-defined and commercially made oligonucleotide (short DNA stretches) primers. Add a reverse transcription step beforehand and you can use PCR to amplify DNA made from a pre-defined RNA template (the thing being copied).

    Viruses come with DNA or RNA genomes. A PCR POS result does not prove active replication of a virus. It does not prove infectious virus is present. This is why we prefer to talk about virus being detected in patients. Some still use the term "viral isolate" - but this should be reserved for when a virus is grown using cell/tissue/organ culture.
  2. PCR doubles the number of pieces of target DNA (copies or amplicon-meaning DNA copies of a single species) every cycle (repeating series of heatings and coolings). The more DNA/RNA that you start (usually equates with the viral load) within your patient sample, the sooner you get enough copies to be measured using your detection method of choice (agarose gel or, most commonly, fluorescence signal). I have a page on the mechanics of PCR here
  3. Of late, with increased use of PCR methods in respiratory virus investigations, research studies are more often finding detections among people with no signs or symptoms of disease at the time of sampling...also not immediately before or after if the people are followed up carefully.

    Side issue #1 - if we're looking to declare such a person "asymptomatic" - we had better be completely sure they have absolutely no symptoms of an infection. Disease, a deviation from normal body function, may be as mild as a headache or just feeling crook - and still, pedantically, be a disease that ascribed to the virus detected by PCR. Let's assume that level of care has been taken (often it hasn't)

    Side issue #2 - we are walking platforms for viruses, bacteria and fungi. Take a look at this paper from Eric Delwart; in 2 kids-92% of 72 samples collected weekly over more than 250-days were virus-positive (no or mild signs of disease developed during this time)! It is very likely, as we discover more diverse agents, better characterize them and develop (and employ) better testing capabilities, that we will learn we're hardly ever without a passenger or 3. And yet we are not always sick - why is that?
  4. The immune system to the rescue! It won't come as surprise to the experts, but we have very capable, active and multifunctional immune system. It is constantly being challenged by..things - not just viruses, bacteria and fungi but chemicals, various proteins, carbohydrates, allergens, dirt and dust (composed of bits of all that other stuff) and it does a great job of sorting through it all, responding to what we need to, to prevent disease, and usually squashing the response to the rest in such a way that we don't under-perform 24/7 because of allergy and illness. It is due to this system that we shouldn't be surprised that as technology lets us find lower levels of infectious agents, it is completely reasonable to be "infected" but not diseased. At least in some proportion our encounters with viruses this should be no surprise.
  5. I think its worth highlighting that we breath in up to 60L of air per minute, with 0.1-7200 submicron particles (aersolised; 0.3-0.5μm) per litre. In one study, influenza RNA was found more often in particles ≤5μM than in those greater than 5μM. These small particles can be inhaled and exhaled fairly easily and commonly.
  6. Sample site and transmission in asymptomatic cases. I don't have any data for this but its seems to me that if you swab the upper airways, you have swabbed the site from which virus is easily coughed, sneezed and therefore transmitted in larger particles. If virus is detected there, then there would seem to be a good chance of transmission being possible. Does it happen? Those studies are not easily visible...or perhaps they have not been done.
  7. The big grey peanut-loving amplicon in the room is contamination. That is, amplicon from previous PCR reactions that accidentally gets into your new mix/common PCR reagents/pipettes/lab and gives us a positive result when the patient is actually not infected. It's what some think of when a PCR positive occurs in an asymptomatic person. It's easy to point at it as a reason. However, if the lab is expert in PCR, and those testing in the WHO network are, then the necessary controls will be in place to give warning of contamination. Plus, for some viruses and in some labs, more than one PCR assay, each targeting different genetic regions and using separate primers, are employed. This is the case with the recommended work-flow for MERS-CoV screening. It does not mean these are the assays used of course. And more detail on testing would be useful.
Other useful steps include separate labs for specimen preparation, reaction mix preparation and cycling; labs with directional air- and work-flows; disposable everything; commercial reagents; pre-made, target-specific kits ("analytes"); lots of negative controls and so on. These provide considerable confidence in a PCR result and are standard working practice for a professional diagnostic PCR lab. Taking more than one sample from a suspected case is also helpful.

PCR has been used to detect viruses since the 1980s and there are many very knowledgeable experts in its use who know how to get useful results and avoid contamination. Three are also many publications that link infectious virus with a PCR result - PCR positivity has meaning. Also, those of us who have conducted PCR experiments on human samples over many years will know, PCR is not so practically sensitive that it will regularly detects virus that is just sitting around in your nose because it was inhaled, but is not replicating. Dilution factors see to that.

Still, problems happen and non-professional labs exist. There is no easy answer to the questions posed by Prof Martin Haditsch on ProMED. As with any test, reliability is about reducing the risk of failures in the process. But even expert labs have bad days.

Tuesday, 25 June 2013

Editor's Note #8: New influenzavirus page, H6N1.

There isn't a lot to say about the latest avian influenza A (H6N1) virus confirmed in humans, but I've put up a small page on the 20F from Taiwan. See the Taiwanese CDC announcement.

Another stark reminder of why generic virus names like "novel coronavirus" or "bird flu" are pointless. How many novel coronaviruses have been found of late (that haven't made it to the media) and how many bird flu viruses are there now infecting humans...enough to make referring to them using such a generic term confusing.

Wednesday, 19 June 2013

MERS-CoV numbers-where are we at?

As the dust settles from several days of new cases, and deaths and retrospective case identifications, I sit waiting for some caped crusader (no capes!) to step from the shadows and announce "I have the numbers you seek!" Okay, I'm a sucker for a caped crusader.
Alas, there are no such wonderful heroes to help fill the data gaps we lack among the MERS-CoV case data. There are plenty trying though. And so we watch the numbers climb, the cases spread, then contract (depending on which reliable source of information is speaking) and we wait for the likely spike in new cases due to the upcoming Hajj which, even with calls to reduce numbers, will likely go ahead as a mass gathering that puts MERS-CoV transmissibility to the test.

Sometimes we seem to hear a proposed new case or a death, and then we hear no more. 


Where is this virus coming from - animals, are older males with underlying conditions (and what precisely are all these conditions?) getting it from Pipistrellus sp. or perhaps Rousettus aegyptiacus bats via contamination of dates, date products of palm sap-derived drinks/alcohol? How can the world prepare, or understand whether it needs to prepare, for a novel virus when the region of its apparent origin (we don't know that either) has trouble sorting out whether members of its own populace are positive or not? A rough - what else can there be - count shows at least 23 dates of onset missing, 9 dates of death, 10 ages, 67 dates of hospitalisation and 11 sexes undefined for around 72 cases.

Isothermal DNA MERS-CoV test

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

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

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

Monday, 17 June 2013

Stuff from the literature: Don't judge a virus by its worst case [UPDATE].

This article, a Letter in the Journal of Clinical Virology (2013 Sep; 58(1):338-9) is one from our own keyboard. It is entitled Avian influenza A (H7N9) virus: Can it help us more objectively judge all respiratory viruses? Unfortunately it s behind a paywall, but I do not have the funds (they all go into the research) to pay for open access publication.

We try and make
the point that every respiratory virus can be found in severe, moderate, mild cases or even asymptomatic people - H7N9 in a young child earlier this year being an example - but no particular portion of a given virus's clinical severity spectrum should be used to define that virus.

The risk of future prejudgement is real. For example, not all influenza-like illness (ILI) is due to influenza viruses. Expecting it to be so leads to confusion and misunderstanding. But on the other hand, screening for every likely viral culprit in every patient during a pandemic is impractical - or at least, it creates a bottleneck that slows result reporting and infection control. At some stage we'll have better, faster higher-throughput tech to do this, but we're not there yet - so we have to pick and choose.
In the meantime, old labels like the "common cold" virus (human rhinoviruses and coronaviruses) have done little to help anyone really be aware of what a virus is capable of. You may argue they have slowed research into their other roles to the detriment of public health. I do. These labels will never be shaken off. Yet we now know that most asthma exacerbations are triggered by infection with one of these 200 or so little packets of mischief.
Apart from naming viruses to avoid geopolitical, and personal sensitivities, it is also important not to label viruses which at one time may be innocuous...and at another, deadly.

Viruses pack a lot of potential into a small shell-and do a great job of running us in circles.

Cumulative MERS-CoV Cases by area.

This is the latest chart (a cumulative epidemic curve) for my MERS-CoV page

Its a lacking some cases (57 of approx 64 cases are depicted below) - you may have head, data are sometimes hard to come by for the MERS-CoV outbreak. Still, it gives one a clear idea of where most of the cases have been occurring and how quickly they have accumulated.

Of particular interest is the take-off point which occurred from the week beginning April 14th. Over 30 of KSA's reported cases ~49 cases occurred from this point onwards - the exponential part of the red line.

MERS-CoV cases and deaths rising.

FluTrackers report on 3 new cases of infection and 4 deaths among existing MERS-CoV cases.

The numbers are changing rapidly (cases being released in several batches of 3 doesn't help).

Currently there are at least 64 total cases and 34 deaths among people with MERS-CoV. Most cases are in people with underlying chronic medical conditions. The Saudi Arabian Ministry of Health (MOH) regularly accounts for this clinical feature in each press release.

You can get a feel for the spread of MERS-CoV cases on this map

The following sites are useful to keep a check on numbers:

MERS-CoV infection control: the French connection.

A Eurosurveillance article by Mailles and colleagues describes the procedures used to lock down spread of MERS-CoV once confirmed.

Confirmed cases were isolated in negative pressure rooms (they suck air in, instead of pushing air out of a room as usually occurs in an air-conditioned room, ensuring virus-laden particles cannot escape) with dedicated staff using contact and aerosol precautions (e.g. personal protective gear which may have included back fastening gowns, disposable gloves, filtering masks, glasses etc).

A close contact was asked not to return to work and to wear a surgical mask when with other people. Other close contacts had to carry a ask and do nit if they developed symptoms, but could otherwise continue with life as usual.

Airborne transmission was strongly suspected but other routes, including the possible contamination of surfaces from the stools of the index case who initially presented with diarrhoea.

Wednesday, 12 June 2013

New MERS-CoV genomes don't impact on existing PCR assays.

Update: #1 01NOV2015
I've looked at my latest nucleotide sequence alignments that incorporate the new genomes.

Of particular interest to public health laboratories worldwide is that the ORF1a,ORF1b and E gene real-time PCR assays described by Corman and crew (Sept 2012 and Dec 2012), still match their targets with 100% identity - no mismatches and thus no change in detection efficiency expected.


There is a mismatch (C:T) in the NSeq-Rev primer and the RdRpSeq-Rev primer, with some/all MERS-CoV strains, but nothing significant given its position within the primer.


These real-time RT-PCR (screening) and conventional PCR (sequencing and genotyping) assays are in widespread use and are part of the WHO testing algorithm (see 050613 post).


Good targets and well chosen, clearly. Not that one would expect any less from the authors of those two papers......



Multiple alignment of human MERS-CoV variant genomes and
reverse transcription real-time polymerase chain reaction (RT-rtPCR)
assays from Corman et al.[1,2]
Alignments made using Geneious Pro. v6.
Click on image to enlarge.
Reference...

  1. Detection of a novel human coronavirus by real-time reverse-transcription polymerase chain reaction.
    http://www.ncbi.nlm.nih.gov/pubmed/23041020
  2. Assays for laboratory confirmation of novel human coronavirus (hCoV-EMC) infections.
    http://www.ncbi.nlm.nih.gov/pubmed/23231891
Updates...
  1. Added graphic back.

Thursday, 6 June 2013

MERS-CoV cases by site of virus acquisition.


My first shot at some graphical representations of the MERS-CoV data. Much harder than for H7N9 as the data are scant. Larger versions and more detail can be found on the MERS-CoV page.

MERS-CoV cases by sex.


MERS-CoV cases by sex
MERS-CoV cases by sex.
Larger versions and more detail can be found on the MERS-CoV page.

The H7N9 missing link: testing the wrong end?

Helen Branswell has an excellent piece detailing as yet unpublished work by the Southeast Poultry Research Laboratory in Georgia.

The researchers have found that influenza A(H7N9) virus can be detected from the nasal passages of chickens and quail - both animals implicated in transmission during the H7N9 outbreak in China. The "so what?" factor is that normally avian hosts shed influenza viruses from the gut reflecting that it is the primary site of influenza virus replication in birds.

As Helen writes, an implication is that if the thousands of birds that have been tested in China to date were only sampled at the cloaca and not from the upper respirator tract, H7N9 prevalence in these oft-blamed but seldom-positive hosts could have been grossly underestimated. However, Dr David Swayne, Director of SPRL notes that it is common practice to swab both ends of a bird when testing.

The research also plays down a major role in transmission for pigeons, ducks and geese.

The search for a definitive answer to what animal is the principal host for H7N9, goes on.

Sunday, 2 June 2013

H7N9 case dies 67-days after illness onset.



59M native of Anhui province, treated in Shanghai, died on Saturday. Case #19 on the H7N9 page Case list.

3 deaths among existing MERS-CoV cases in KSA.

Two had underlying kidney failure. That's all we know at the moment. The WHO will fill us in later hopefully.

Italy now hosts primary MERS-CoV cases.

Crofsblogs has been posting very informative pieces on these developments. I encourage you to keep an eye his and FluTrackers posts for the latest.

Two confirmed local cases of MERS-CoV infection, both presumed to be contracted from the imported 45M mentioned yesterday, both hospitalized in isolation in Florence (regional capital of Tuscany, population 3.8M).

Neither are severely ill. 45M's granddaughter (1.5-year old; coughing and fever, Meyer Children's Centre) was in contact on Sunday (approximately 6-day incubation) and a work mate have both tested positive.

Especially concerning in this cluster (Cluster #8) are the fact that 45M's flight from Amman Jordan stopped in Vienna and Bologna before landing in Florence. Also worrying is that the work mate and 45M's place of work...is a Hotel.

Metropol anyone?

Possible antiviral strategy to intervene in severe MERS-CoV cases?

FluTrackers posted a link to an article that mentions the negative off-target effects of antibiotics being used in MERS-CoV cases (especially among patients with renal failure).

The story also mentions a MERS-CoV "treatment protocol" (without further clarification) involving antivirals similar to those used for Hepatitis C virus infections. I wonder if this March 2013 paper by Falzarano and colleagues in Nature could be the source of such a protocol? It involves use of interferon-α2b and ribavirin to inhibit HCoV-EMC (now the MERS-CoV) replication.

Independently, each drug was needed at relatively high concentrations in cell culture studies, but when combined, they seemed to synergize and could be used at lower concentrations.

It would be great to have more information on the Saudi Arabian treatment protocol but also to know if the Nature article's approach has been used successfully anywhere to date in treating severe acute respiratory infections (SARIs) testing positive for the MERS-CoV).

A lot of clinicians would like to know this information as soon as possible I suspect.

H7N9 case dies 67-days after illness onset


59M native of Anhui province, treated in Shanghai, died on Saturday. Case #19 on the H7N9 page