Medical News Blog Information

Kingdom of Saudi Arabia (KSA) issues health regulations for hajj visitors

The Saudi Gazette has the full list of regulations to be aware of if you have planned to arrive in the KSA for your hajj pilgrimage. Some of these will require some prior organization.

Age, personal health, updated vaccination and concerns over spikes in polio transmission are all covered.

Prof Lipkin: There is no more sequence coming from that bat sample

Many thanks to Prof Ian Lipkin's indulgence of my eMail questions.
Also, check out the TWiV webcast by Prof Lipkin.

So, I guess to carry on from last night's post....I stand surprised. 

Not even next-generation sequencing could pull any more sequence from the MERS-CoV-positive T.perforatus bat samples that thawed after the dry shipper (not a box+dry ice as I previously guessed, but a vacuum sealed vessel previously brought to -150�C then all free liquid nitrogen removed for transport; shipped by FedEx) was opened and the cold chain interrupted after arriving from the Kingdom of Saudi Arabia (KSA). 

According to Prof Lipkin, in an email exchange we had last night, the group also tried a couple of runs of next-gen sequencing.

..we tried two separate ion torrent runs with no joy.

So why was only 1 October 2012 sample positive for the MERS-CoV strain? Prof Lipkin concludes that..


..the concentration of template was already extremely low in the sample at the time of field collection and lower still at the time of arrival in our Center. I would not be surprised if two aliquots of the same sample yielded different results in different labs. However, we will never have an opportunity to know because there is no more sample to test.

At the time of receiving the October samples (no MERS-CoV was found in the April samples), no viral gene/gene fragment/genome cloning had been done in Prof Lipkin's lab. A common potential source of PCR contamination ruled out. 

How does your group know that this 182 basepair nucleotide sequence was not a contaminant from somewhere else? 


The one sample came up positive repeatedly with the same assay. No other sample did so. We have recovered no other fragments that correlate with a MERS-like CoV in samples collected in the October 2012 or in the subsequent April 2013 field collections.

Antibodies were not sought in the massive 10�l of bat blood obtained per bat (the bats were released after sampling). But do these findings exclude the possibility that other bats, like those from genus Pipistrellus and Neoromicia (both from the family Vespertilionidae), or genus Nycteris, family Nycteridae, may be a host for MERS-CoV? At a World Health Organisation meeting in Cairo, Prof Lipkin told the the audience that..

...our findings don't exclude the presence of virus in a Vesper bat and that we were doing everything anyone suggested to test alternative explanations, including reagent contamination. We went back to the original materials using every specific and consensus primer set we and others had designed until all of the original material was exhausted. The results were the same. I sat on these data for months hoping to find another positive bat in subsequent field expeditions where we could report more sequence.


The decision to report it now was multifactorial. First and foremost, we tested every possible alternative explanation for the sample coming up positive other than that this fragment is bona fide-we can't find an alternative explanation. Second, there are no other reports from animals in KSA-I discussed phylogenetic analyses with several people in light of what was found elsewhere in Africa in Vesper bats. This fragment, although short and located in the RdRp is informative....Third, the scientists who did the work in the US and the Ministry of Health of KSA wanted to see it reported. There is a point where one has to get the work out in the public domain.

What's next in the search for animals hosting this virus and in trying to confirm what the group has just reported? There will also be a new European collaborative report (UK and KSA) coming out very soon that has new human MERS-CoV sequences suggesting multiple human introductions (animal to human?) with much more sequence variation in the MERS-CoV genome than we have seen thus far. This will further support the conclusion that the T.perforatus CoV is one and the same virus as that which infects humans.


...field expeditions should begin in the next few months and we will look again. The amount of time and resource invested already is far more than intended. I've never put in so much to recover so little.

Thank you to Prof Lipkin. This gives a some valuable insight into his careful efforts to deduce what animals may host a MERS-CoV strain,m as the first step in tracking how humans in the KSA are getting infected. It also highlights that finding even a basic piece of information requires many steps, lots of people, much effort and some luck. But if virus hunting was easy, everyone would do it right?

Some slight editing for brevity, and to account for mobile phone thumbs, was undertaken by VDU.

T.perforatus MERS-CoV strain sequence, and others, online...

If you're a bit of a sequence collector/hoarder/nut then you'll be interested to know that the recent bat CoV RNA-dependent RNA polymerase (RdRp) sequences are now online on GenBank.

These seem to include the primer regions judging by their length. Consider that when using them.

The MERS-CoV strain from T.perfortaus is CII_KSA_287 - highlighted in bold. Please note, that at writing, it is erroneously identified on GenBank as originating from Rhinopoma hardwickii. It should be Taphozous perforatus.

Editor's rant: Why testing the few may not benefit the many...

Prospective screening without regard for whether the person is sick. That's what I think we need more of, in order to truly understand respiratory viruses and acute respiratory infections (ARIs).

And I
 don't just mean the scary ones like MERS-CoV or influenza A(H5N1) virus or H7N9 or H7N7 (zoonotic flu). 

I also mean the rhinoviruses, influenza A(H3N2) virus, H1N1 (seasonal flu), endemic coronaviruses (CoV; 229E, OC43, NL63, HKU1), metapneumoviruses (MPV; I use the plural because there are 4 genotypes and who knows how many immunogenetically distinct clades), respiratory syncytial viruses (RSV; same plural), adenoviruses, enteroviruses, Saffold viruses, parechoviruses, polyomaviruses, bocaviruses...etc.

Sure, there have been studies in birth cohorts and in the community among "normal"'healthy people. But they sometimes have limitations that may blur our view of what is really happening in the community. For example, such studies may:

  • Exclude certain diseases that viruses are involved in.
  • Only sample when there are signs and symptoms of disease. 
  • Only call "disease" when a certain number or combination of symptoms are present (this one irks me no end - pedantically, if your body deviates from its physiological norms, you are diseased).
  • Sample too infrequently to catch whats going on between sampling points. We don't get one virus, recover, then get another - we're a virus's favourite hang out - but because of our awesome immune system, only some of those infections make us ill enough to stop and groan.
  • Employ insensitive detection methods (cell, tissue or organ culture). In fact, if a study used culture you might as well ignore those data - they will have missed many fastidious viruses (those that don't grow easily or in the cell lines used) rendering any conclusions associating detection of a virus and disease weak or wrong.
  • Sample for too short a period or just focus on a particular season etc. 
  • Only include a pet virus or a few viruses or just those viruses known about at the time. 
Much of our understanding of each virus comes from hospital-based studies. People in this environment, whether admitted (inpatients) or presenting but being allowed back home (outpatients), represent the "tip of the iceberg" of the disease spectrum. The pointy end. The most severe cases. We may make the assumption that the viruses circulating in the community are represented by what's happening in a hospital environment - or vice versa. But how often have we tested that? Do we know if there is a lag or lead time? Does it differ by climate? Could we go further and perhaps use those numbers to predict what the burden of disease in hospital will be this "season"?

And then there's the ongoing testing issue. Research dollars generally do not fund epidemiology. Certainly not ongoing epidemiology. Even big hospitals and private testing labs cannot afford the personnel and cost of testing all respiratory samples for all "likely" viral pathogens, all the time. "Likely" having been defined with the caveats above. 

And so our epidemiology data have holes. Big ones. We read of complaints about some countries not being able to identify a viral/bacterial cause (not that a POS lab test does prove cause) of pneumonia or encephalitis...but many patients in more "developed"countries also leave hospital without ever being attached to a lab-confirmed positive result. We could reduce that, even if we could not specifically treat them. And therein lies another issue. We test for some viruses based on historical precedent - do those precedents accurately stand up today? Do we even have the data to answer that? If you are a health professional, have a look at what your local testing lab offers - does it cater for the most likely causes of ARI or just what's been used before? 
Click image to enlarge. Respiratory virus infections among the community
and in hospital-based populations. Generally more males than females
present to hospital  with clinically-defined acute respiratory infections
(ARIs). Infections in the hospital setting are shown in red, those in the
community in orange.  Most ongoing virus testing is from
hospital-based populations as is our contemporary
understanding of viral season.

Notifiable viral diseases are kept track of, and if they occurred by themselves without interaction with, or interference from, other viruses that might be enough. But they don't. 

The "One World" concept of infectious disease study - looking at animals and humans and the environment together - is great; what about the concept of "One Virus"? The days of a study looking at just one virus and from that, without testing for any other respiratory virus that may cause the same signs and symptoms, concluding what that virus is capable of, it's severity and how many cases of disease are lessened by a drug for it, in a human should be far behind us. But they are not. 

So, do we really know the viruses that call us home? And if the answer is no, how can we possibly hope to be prepared for the next virus that emerges, the next local viral outbreak of ARI or encephalitis or gastroenteritis, or the next pandemic? How can we protect our population from viral threats if we're always on the back foot?

If I ruled the world, we would do more testing we'd try not to bias our attentions toward any 1 virus, we'd screen everything for everything, we'd sample the community, we'd make the data publicly available in real time, we'd understand ARI epidemiology better and we'd use all those data to prioritize some antiviral drug development or other viral interventions. At the very least, we'd re-jig our testing panels and create a new paradigm or 2.


But I don't rule the world - nor do I have input into these sorts of decisions - perhaps you do?


Feel free to weigh in below.

MERS-CoV-positive Qatari man did not return from umrah, but developed symptoms while travelling [UPDATED]

Hat tip to crofsblogs and FluTrackers

Perhaps an indication that MERS-CoV was circulating in pilgrims returning home from umrah. 

A recent news article notes that the patient in Qatar was probably infected outside Qatar; probably the Kingdom of Saudi Arabia (KSA) where he was performing umrah. [We now know this person was only travelling out of Qatar for 6-days - more likely he was incubating before he left Qatar]

My epidemiology list (102 confirmed cases; 48 deaths) that this cases is a KSA acquisition and so won't add to the Qatar numbers. [See post 30/08/13; cases now moved to Qatar tally]

Oh well, that still leaves 4,799,999 pilgrims MES-CoV free right?

Updated with information from WHO disease outbreak news update, 29/08/2013.

Why only 181 nucleotides of T.perforatus MERS-CoV sequence?

In some of the many articles written about the new discovery this week, there were comments along the lines of  its amazing any sequence could be obtained from the samples cause they had sat for 48-hours at US customs and thawed. A more precise quote could be found here for example.

I have some thoughts on that - and these come from me, someone who has worked with a lot of clinical human specimens from which I've been able to amplify viral bits and pieces on a regular basis. Many small (200-600 basepairs[bp] fragments) but also longer pieces of >1,000bp, assembling small viral genomes from them. These samples may >10-years old, having been freeze-thawed numerous times after spending various amounts of time in courier vans, planes or sitting at room temperature before having nucleic acids extracted, tested and eventually (extracts may also sit around during testing and preparation and be freeze-thawed etc) frozen at -20�C or -80�C.


Keeping in mind that this issue of thawing might simply be a case of "hold your horses people". The EID paper was an early and quick report announcing the discovery of this MERS-CoV strain. So, my thoughts:

  1. Because the materials that yielded the sequence (collected in October 2012) were described as "thawed" we can presume that the dry ice they were shipped with ran out during the transport to, or waiting time at, US customs. Once the refrigerant is all gone, the samples would come to room temperature as fast as the cardboard box and plastic receptacle it held, allowed. The publication described them as having been thawed for 48-hours.
  2. How warm are we talking? The average temperature of Bisha (where the Taphozus perforatus bat was found, in an old date orchard outdoors) in October ranges from 15-20�C to 30-35�C. I don't know where the US customs site was so don't know that temp range - but expect it's less. So let's make some wholly unfounded assumptions:
    • That this MERS-CoV strain can spread via the virus found in faecal pellets or other bat excreta. Perhaps as wind-blown dust or to other animals via a faecal-oral route. Even if the bats are hanging from a cave ceiling, but certainly when they are hanging outdoors, the virus must be capable of surviving in faecal pellets at a very high "room temperature" to complete a transmission event. If they can survive, that means intact virus - RNA genome + proteins + capsid + lipid envelope - the whole lot. For RT-PCR - you only need the RNA bit, not infectious virus. So, you're already lowering your expectations for what's required of a "successful" shipment.
    • To confirm bat species, a genetic test was used which required the amplification of another piece of DNA - a region of the cytochrome B gene was amplified and sequenced. How large this fragment was, I'm not sure. However, a relatively large fragment of this gene can be used to differentiates bats, useful when you can't tell them apart by looking at physical features. Other work on opossums by the collaborator who helped sequence this region (Dr George Amato) in bats, employed >800bp of sequence. Why did this fragment amplify so well if the viral RNA did not? Perhaps because DNA is more hardy (various reasons) or because the bat blood or skin that it was amplified from, better protected the DNA from the thawing than bat faeces did for the viral RNA? Or...
  3. Perhaps the primers used for other regions of the T.perforatus MES-CoV strain failed because the virus was too genetically distinct. I've had a look at the alignments and the primer binding sites can be found so it's probably not that. However, some of these primers that produce larger products are very degenerate (primers specially designed to account for nucleotide variation in a range of subtly different viruses or viral strains). 
    • Degenerate PCR primers generally have much decreased sensitivity compared to 100% target-specific primers. This drop in ability to detect low amounts of RNA is the case even when using nested PCR - sorry if this has become to PCR technical! 
    • The primers that did work for the T.perforatus bat MERS-CoV, Nested CII-MERS-RdRp, were much more target specific with only 1 degenerate base in 4 primers. That, combined with a drop in viral RNA amount, may well be why this 1 assay worked, worked where the others did not.
  4. There was no mention in the EID paper of the use of an internal control RT-PCR target - a region of a gene in bat faeces (or blood or tissue depending on what was tested) that might allow some quality monitoring to see if there was truly decreased amounts of intact RNA in the October 2012 batch compared to that in the April 2013 batch of samples. That would be helpful to know which course to follow next.
So what does all this mean? Just me thinking in print I guess.

It's always important to maintain the cold chain from sample collection through to nucleic acid extraction and template addition to an RT-PCR/PCR tube. But I think we should look elsewhere for reasons why the T.perforatus MERS-CoV-positive sample has not yielded more than 1 fragment from the few assays used. 

I wouldn't be surprised if there was more sequence coming soon from this sample.

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