Friday 30 August 2013

A model of MERS-CoV acquisition (ver1)

With thanks to David Spalten (@dspalten) for discussion and considerations and AtRG for advice.

First we heard about Middle East respiratory syndrome coronavirus (MERS-CoV)-related viruses in bats in South Africa, then we read of antibodies in camels that reacted to MERS-CoV more than the most likely (known) other CoV to infect cattle, and most recently we were absorbed by the discovery of a probable parental strain of the MERS-CoV in the faeces of a Taphozous perforatus insectivorous bats.

We've also heard that most patients have not had direct or obvious contact with bats and we also know that pasteurised camel milk products should be safe. But that still leaves many stones unturned.

So if we can assume that the most likely route of acquisition of MERS-CoV is through the upper respiratory tract and that the spillover events come from animals (I'm including human-to-human exposures in this figure) then we need to consider how that might happen. I've included the animals above as well as baboons as they seem highly mobile, interact well with humans, visit mountains and caves (where bats are likely to hang out") and are found in the KSA. I've added ingestion but I don't really imagine how this could result in a respiratory infection, and MERS-CoV gastrointestinal involvement seems infrequent.

I don't live in the Kingdom of Saudi Arabia or in the Middle East and I do not profess to know much of the environment so what follows is "remote guestimation" at best. But I've thrown together some of the possible routes and animal players into a figure which may have some degree of reality buried in there somewhere. It may also spark an idea or two among those who do know what they're talking about.

So, here is my model of how humans may indirectly get a zoonotic infection from a primary or secondary animal host...
A model of MERS-CoV acquisition. Click to enlarge.
I'd be most happy to take suggestions for improvement of the figure. I know some of you like to use the graphics from the blog and Virology Down Under (which I strongly support, asking only for a specific reference to their source) so if they can be made more robust, I am very happy to do so. Get to me via the comment section below or on Twitter (see top right).

Bats are the host to a lot of viruses but do we know much about that?

In an article in Science today, Kai Kupferschmidt reminds us that while the chiropterans (bats belong to the Order Chiroptera which comprise >1,200 species) are a fascinating bunch. As you might expect from animals that make up a fifth of all mammals and reside on all continents except Antarctica  they harbour a lot of viruses and yet we still know very little about how the flying fuzzballs interact with their own virome.

Bats have been found to harbour or were the likely source of a virus that became what we know as:

  • Nipah virus 
  • Hendra virus
  • Rabies virus
  • Australian bat lyssavirus
  • Severe acute respiratory syndrome coronavirus (SARS-CoV)
  • Numerous other bat CoVs
  • Ebola virus
  • Marburg virus
  • Kasokero virus
  • Duvengae virus
  • Menangle virus
  • Middle East respiratory syndrome virus
Intermediate hosts play a big role when these infections get to humans, mostly because we don't really have bats on our list of cute and cuddly animals with which we spend a lot of close contact time. 

Bats also live for decades, probably first emerged 50-million years in the past, may have a primitive immune system and seem to have an ability to host viruses without getting ill. This may have to do with a reduced repertoire of foreign (in this case, viral) gene-sensing molecules. In humans, these sentinel are always on or in cells, keeping a "non-specifically specific" lookout for bits and features of DNA or RNA. When found the sentinels act to scale up the body's immune response to the virus. But for bats, little of this process is really known because bats are a difficult animal to study. There are some bat cell lines that have been successfully used to examine bat immune responses in labs, but nothing beats observing and understanding the viral life cycle in its natural host.

Even very basic knowledge is lacking. Viruses have been isolated from bat urine successfully, and detected using sensitive PCR-based techniques (which don't prove an infectious virus is present) from bat urine and faeces etc, but do we know if bat faeces is infectious and if so, for how long? That knowledge may solve some riddles about transmission cycles for bat viruses. When I recently asked Prof Linfa Wang about this, he was pretty clear on what we don’t know:

“This is a tough question to answer. I don't think anybody has done any "hard science" on this and the answer will vary with viruses anyway.”

As humans move into or remove bat territories, we may be increasing our contacts with them by forcing them to move into areas shared with us. We may be forced to learn more about bats in the future; perhaps now would be a good time to fill in some of our knowledge gaps to make that learning curve a little less steep.

A crack in the wall of MERS-CoV case information: new MERS cases, a key, a family cluster with 2 news cases from 1 [AMENDED]

Thanks to Prof Andrew Rambaut for pointing out my incorrect usage of R0

In a strong piece by Maryn McKenna on Wired a bit over a week ago, she posited that "Censorship Doesn’t Just Stifle Speech — It Can Spread Disease".

Today we see a possib
le turn of events. In an post on ProMED today, Prof Memish, Deputy Minister for Public Health in the Kingdom of Saudi Arabia (KSA), penned his own update on case details from yesterday's announcement, and added information (including dates of illness onset) on 2 new, asymptomatic MERS-CoV cases!

This is a great sign that more information is starting to flow from the KSA. It was also extremely encouraging to see the appearance of a KSA key for these cases - SA83-the 55-year old male (55M); SA84-38M etc. I will add this numbering to my list in anticipation of this becoming a regular thing. This really does help track and confirm cases and information with much more precision

If you take requests Prof Memish - I have a few data gaps I would love to have filled (you can eMail me below)!!

We learned that SA83 (55M) was probably in contact with the recent Qatari patient diagnosed in Qatar. We should be looking a little more closely to Qatar as a source for recent cases - and applying that list from my last post, to there as well.

The new cases are both children who were asymptomatic family member contacts of 38M, who we learn was diabetic, showing signs of disease 8th of August and dying of acute respiratory distress syndrome on the 17th of August.

  1. 16M (VDU #107; SA85)
  2. 7F (VDU #108; SA86)
This is yet another case of 1 case infecting more than 1 new cases - 2 new cases in fact. Should that be the norm rather than the exception, we would see the basic reproduction number (R0) creep upwards. We saw this also happen in the United Arab Emirates cluster (4 from 1) - see my earlier post on that.

The potential for transmission is certainly there but the level of contact requires "close" in its description (family). Further, the second passage of virus (from the initial case to the next people) seems to mostly result in milder/no disease - or is that simply a reflection of the odds favouring transmission to a person without comorbidity? In other words, MERS-CoV is an opportunistic pathogen but a bit of a wimp in "normal" healthy younger people.

This brings the case tally to 108 by my tally, with 49 deaths and a PFC of 46.7%.

Congratulations to Prof Memish and the KSA Ministry of Health for taking this new path. Long may it last.

New WHO update on MERS cases

The latest WHO disease outbreak news (DON) addresses the 2 recent Qatari cases, noted previously here and here
Click to enlarge.

Interestingly, it notes that the 1st case, which showed symptoms while travelling outside of Qatar, in the Kingdom of Saudi Arabia, was only travelling for 6-days

That would most likely place the acquisition of infection, which can take as long as 14-days after exposure to the source to manifest with obvious signs of disease, within Qatar. 

I've changed by Qatar case tally to reflect (and recent posts of 59M and 29M cases) that 4 cases have been acquired within Qatar.

Just looking at the latest case curves, we seem to have entered a period of rapid case growth, after a period of quiet. These little spikes are good times to cast a very broad mind over what might be happening with animals, weather, dust/wind, camel movements, bat activities, baboon interactions, festivals, markets...almost anything that could be used to link an uptick in cases with exposure to a possible source. 

There must be something common out there....its just needs to be found. As Sherlock Holmes said in a question, "...when you have eliminated the impossible, whatever remains, however improbable, must be the truth". At this stage, with nothing really ruled out as impossible, that leaves a lot to consider.

Thursday 29 August 2013

MERS-CoV case numbers: WHO knows???

With the WHO's latest update we see that body's official MERS-CoV case count rise to 102 cases and 49 deaths. Its clear that even WHO is suffering from a lack of information - most cases listed here have no sex, date of onset of illness, hospitalisation or date of death. And its clearly not their role to dig into the minutiae of why the Saudi Arabian Ministry of Health reports deaths among previously reported cases....that clearly have not been previously reported

My count  with details available sits at 105 cases and 48 deaths. FluTrackers, who spend a lot of time sorting through  up-to-the-minute numbers, tweeted this morning that the case count was 106 and deaths were at 49. There is also a good comparison of the variability in a post by Pathfinder, a Senior Moderator, which really highlights how everyone has their own number. Some have new datasets that include probable cases - that's a very slippery slope, open to far too much interpretation and the potential for confusion. I favour the FluTrackers method of leaving a case list as a list of cases....anything else can be described and discussed separately.

And let's not even get into where we say a case is geographically based - do we do that based on whether it was detected there or acquired there (my prefence)?

There clearly is a need to consolidate case reporting and streamline the definition of case locations. Perhaps WHO could take a lead on proposing something here? 

Realistically it wouldn't change things too much. Since the internet provides the data for home-brew epidemiology (like my own MERS and H7N9 data!), its not surprising that we end up with this spectrum of figures.

While it might take longer to report on the cases, I think the WHO numbers are probably what we fall back on for reliability. These are people in direct contact with the sites of infections and reporting. For speed (and great accuracy as well!), FluTrackers cannot be beat.

In terms of MERS at least, at the end of the day we know it is not spreading with pandemic-level efficiency, we have a good idea of how to stymie its spread in healthcare settings, research is ongoing to find the host and then work out how humans get infected. We have only just crossed the line of 100 cases, worldwide. Not knowing about the dates and ages of a small number of MERS-CoV cases doesn't really impact on public health decisions, so long as the data are added to the total before a reasonable period of time has passed. Days not weeks lets say.

It's worth keeping everything in context when there are much larger and more devastating outbreaks of infection and disease happening elsewhere in the world. Polio and cholera are examples well covered by crofsblogs for example. 

Wednesday 28 August 2013

Two new MERS-CoV cases including new death

1st source for this post: FluTrackers (FT)

FT has found that the Arabic language version of the Kingdom of Saudi Arabia (KSA) Ministry of Health (MOH) website has added a new MERS-CoV case; that of a 55-year old with underlying disease (kidney failure) who is currently in an intensive care unit in Medina (along the western edge of KSA).

Sadly a 2nd notification is that of a confirmed MERS-CoV case who died of respiratory failure after pneumonia; a 38-year old from Batin (Hafar Al-Batin? - if so, in the north of KSA, near the border with Kuwait).

Sex for both cases is unknown. Update not yet on MOH English language site.

This brings the total to 105 (including a mysterious case that FT is still following . 49 cases have died. The proportion of fatal cases (PFC) now stands at 46.7%

Dr Ziad Memish discusses MERS in the Kingdom of Saudi Arabia (KSA)

Insights from the Front Lines. UPMC Center for Health
Security presentation by Dr Ziad Memish on MERS-CoV.
August 21, 2013
University of Pittsburgh Medical Center (UPMC) Center for Health Security recently organized a seminar by Dr Memish in which he discusses the Saudi experience with the Middle East respiratory syndrome outbreak. 

Dr Memish, is among the most important health professionals in the world on this topic and he is located at the front-line of understanding the behaviour of this new virus and its ongoing outbreak in humans.

I've distilled some quotes (with some of my additions in brackets) and points that I found interesting during his 30-min presentation:
  • In addition to his many awards and roles in consultancy and medicine (including roles as Deputy Minister of Health for Public Health (KSA), Director for WHO collaborating centre for Mass Gathering Medicine; consultant for infectious diseases at King Fahd medical city, Faculty of College of Medicine at Alfaisal University/King Saud University/Emory University/Liverpool School of Medicine and on committees (Executive Board of the WHO), Dr Memish also has >300 peer-reviewed publications and chapters. I recently summarized a few of the MERS-CoV-related ones here.
  • Dr Memish and the KSA Ministry of Health (MOH) have a "huge public health team" investigating the new virus (currently 96 cases and 47 deaths worldwide; 76/39 in KSA)
  • More and more mild and symptomatic cases being detected - more extensive testing on contacts
  • Healthy and well MERS-CoV infectees do better than those with underlying conditions
  • Dr Memish saw the Prof Ali Zaki report of the first MERS-CoV case in Bisha/Jeddah, on his Blackberry, and an MOH team was then immediately dispatched and communication with WHO, CDC, Columbia University and the EcoHealth alliance to work with the KSA MOH including extensive investigation of at least 29 family members and 100 employees in Bisha and the hospital in Jeddah - none of whom were MERS cases (around 7min in the video)
  • Reminder of the 2002/2003 8,098 probable SARS-CoV cases and 774 deaths over 5-7-months, costing the economy >$30-billion. SARS differs from MERS in the lower proportion of cases of severe disease linked to an underlying condition
  • Between Jan and end of July - there were close to 8-million umrah visitors. The month of Ramadan had almost 15-million people perform the hajj (5-million from outside KSA)
  • We think there is a some sort of pre-existing genetic underlying disease predisposing to MERS; based on studies of family clusters (late 18min)
  • The Al-Ahsa hospital cluster
    • Al-Ahsa represents 25% of the area of KSA; population 1.3-million
    • No increase in mortality, but local infection control team noted a shift in mortalities from the "usual" causes towards pneumonia 
    • The hospital cares of the elderly (a geriatric hospital)
    • Very good documentation at this hospital which helped track infection
    • Had to stop shared transport (ambulance) system for dialysis patients coming to hospital, stopping  those with suspected disease from coming to the dialysis unit, enhancing cleaning and excluding visitors and non-essential staff
    • 10/30 had animal exposures of any sorts (birds, bats, camels, cats etc). Hard to get families to recall exposures-problem for animal source tracking.
    • Lesson learned include
      • MERS-CoV can causes healthcare-associated infections
      • Early symptoms of MERS can be mild and non-specific and can be missed
      • 1/3 of patients have gastrointestianl symptoms
      • Human-to-human transmission does occur but how is not clear
      • Droplet and contact precaution are essential
      • Don't know if asymptomatic but MERS-CoV PCR positive patients can transmit virus
      • PCR is the gold standard testing method
      • Deeper airway samples give better results - repeat sampling required if negative on first sampling, when MERS is clinically suspected
  • Collaborating with international groups (CDC, NIH etc) to standardise and validate antibody testing methods
  • Potential sources includes dates, bats, livestock, stray cats and dogs, pet birds and camels (antibody-positive in Oman despite absence of human cases there)
    • >1000 bat samples collected but relationship between bats and patients is undocumented
  • Transmission routes include:
    • Sporadic community cases with non-human exposures (animals or environmental)
    • Family clusters
    • Healthcare clusters
    • Travel-related/workplace transmission
  • Clinically, MERS-CoV can cause severe disease on older, immunocompromised patients, those with underlying conditions, and primary index cases but also causes mild or asymptomatic disease in the previously healthy, young and secondary cases; most HCW infections were in teh young and healthy. Standard acute (48-hour) fever and myalgia in the healthy cases.
  • Risk for HCWs was related to invasive procedures (see WHO IPC linked and discussed in previous post on this). Diabetes and obesity is relatively high in KSA so need more cases to determine risks.
  • Looking at therapeutics that have and have not worked for SARS infections
  • MOH website adds cases daily at 5 o'clock
  • Reservoir needs to be determined
  • Sample type, false negatives and lack of serology are diagnostic challenges
  • MERS-CoV has limited transmission
In follow-up questions, Dr Memish noted that the first the KSA MOH knew of the new CoV was when they read Dr Zaki's eMail to ProMED. Since then, surveillance testing of anyone at key sites, with pneumonia admitted to an intensive care unit was extrapolated post-hajj 2012, to all of the KSA. Other patients can get tested. MERS-CoV testing is centralised - 3 KSA labs now test, 24/7.

70 suspected cases are reported per day. Most testing is on those who are hospitalised.

The KSA is working with Sanger Institute in the UK for sequencing. All samples are being sent there. Not every case yields a full genome (real-time PCR threshold cycle values >30-35 may fail). 21 more genomes are being finalised-coming out in the next few days-to add to the existing 9. KSA also works with other institutes worldwide and it seems that local capacity for virus discovery and genome sequencing is lacking at this stage. This means delays due to forging new international alliances and in getting materials out of the KSA and into other countries, with strong biohazard importation borders to cross and regulations to satisfy, will probably continue. But things should speed up as the process becomes more familiar to all parties.

Dr Memish, supporting comments he has made previously, still suspects that other countries both in the region and globally, would find more MERS-CoV cases if they looked as hard and as proactviely as the KSA is doing. 

At around 48-40min Dr Memish made an important comment about epidemiology:

Epi 101. If you don't look for something you will not find it. You have to look for the disease and if you're not testing and you are not doing anything you will not pick it up.

I only got to 50-min, so feel free to follow the link and hear more. 

A great summary of the situation so far from the country at the center of the emergence of this new virus.

An entire H7N9 genome from a clinical specimen in one shot

Some weeks back, Ren and colleagues described their use of next generation sequencing (or "deep" sequencing; unbiased, massively redundant sequencing of all the DNA or RNA in a sample - to put it very basically) to pull out the entire genome of an influenza A(H7N9) virus from 1 clinical sample.

he new genome is called A/Jiangsu/2/2013(H7N9) and can already be found on GenBank. It's 8 segments are numbered in order from KF226105 to KF226112.

The authors found the E672K (PB2 gene segment) and I368V (PB1) mutations related to virulence and transmissibility

The caveat for this sequencing, in case you're making comparisons to the recent small sequence fragment obtained from the MERS-CoV strain detected in bat poo pellets, is that they had enough fresh human sputum (probably not freeze/thawed and left at room temperature as with bat samples) from the 54-year-old female case from Zhejiang to be able to purify, concentrate and clean up the virus before preparing the nucleic acids for NGS. That likely means a lot more virus, from a more hospitable environment for virus, and a lot less other nucleic acid and junk to interfere with the amplification and sequencing components.

Interestingly the authors found that in 22 nucleotide positions, more than a single nucleotide could be reliably identified - sequence heterogeneity that shines a light on how much viral change goes on, even within a host, as the virus keeps testing out different ways to replicate, bind and enter cells and interfere and avoid the immune response against it. Some of those changes altered the protein (amino acid) sequence.  Sometimes those changes are no good for the resultant virus and the strain with that/those changes replicate poorly, sometimes there is no change in replication or infection efficiency and sometimes the process generates a more efficient viral strain that may then grow to become the dominant strain which we cough and sneeze onto our contacts. 

This unbiased (doesn't rely on a very specific PCR, and the amplification of the most dominant sequence in a sample as do traditional Sanger sequencing methods  sequence analysis approach is a great way to find those changes in a hole-of-genome approaches - because it's likely that "favourite mutation X, Y and Z" only represents a portion of the changes that go into making more successful influenza viruses.

Tuesday 27 August 2013

Healthcare workers may stay on the job when ill and can be shedding viral RNA...

In a prospective study in the journal Infection Control and Hospital Epidemiology, Esbenshade and colleagues described their analysis of 319 samples from a cohort of ill (119) and asymptomatic (200) healthcare workers (HCW) serving inpatients at Monroe Carell Jr. Children’s Hospital at Vanderbilt (MCJCHV) in Nashville, Tennessee, during Nov 16 2009 - April 16 2012. 

This was a 20-week period when influenza was expected to be circulating. Most HCWs had been vaccinated against influenza A(H1N1)pdm09 virus

Nasal (not nasopharyngeal) swabs were collected by a trained staff member every 2-weeks, with extra swabs taken if a period of illness arose in the meantime. Nasopharyngeal swabs (NPS) do yield higher proportions of viral detections but are not pleasant and may have caused study drop-outs among the volunteers so they were not used. 

Influenza viruses, respiratory syncytial virus, rhinovirus (RV), human metapneumovirus (HMPV), parainfluenzavirus (PIV), endemic coronavirus (HCoV), adenovirus, bocavirus and enterovirus shedding was represented by the presence of viral RNA detected using a commercial PCR assay (MultiCode-PLx-RVP). An internal control target, β-actin, was included to monitor the integrity of the extracted nucleic acids. 

A PCR positive is assumed to represent shedding of an infectious virus.

The findings are relevant to my recent rant on prospective testing (seek and you shall find). Some key findings were:

  • HCWs often worked despite being ill
  • The strongest and most statistically significant risk of finding a virus in a subject was associated with that subject being symptomatic (I'm going to be writing about asymptomatic infections in the coming weeks)
  • Only 42 specimens were positive for a virus - mostly RV (33) followed by PIV (4), CoV (4) and HMPV (1) - lower than expected
  • Younger age was positively associated with viral shedding while the subject's role as a nurse or a physician was not
  • 15% of RV detections were made from asymptomatic subjects - 25% of PIV or HCoV (OC43 and NL63) detections were from this group.
  • 85% of RV detections were from symptomatic (ill) subjects
The authors conclude that HCWs should consider avoiding patient care duties while ill and that institutional policies should be updated to reflect this need to limit hospital-acquired infections.

When you test for things, its amazing what gets found and how this can impact on policy, understanding of infectious disease transmission and improved patient management. 

This is also a timely reminder that issues around infection prevention and control (IPC) are in no way limited to the management of newly emerged viruses like H7N9 or MERS-CoV. IPC is a problem the world over and it requires constant vigilance to stay ahead of. I commend the authors for this study.

New MERS-CoV case in Qatar [UPDATE: CONFIRMED; details for 59M updated]

My 1st source on this: FluTrackers

The Kuwait News Agency reports that the Qatari Supreme
Council of Health (SCH) has logged a 29-year old male (29M) as having MERS-CoV. He is reportedly in intensive care and in critical condition.

No sign of official confirmation as yet though. [UPDATE: The SCH, through a media outlet rather than its own news section, has confirmed the case.] If confirmed officially, this will bring the total lab-confirmed MERS-CoV case number to 103 or 104 (see here last week's Qatari 59M case and for comment about a case numbering issue that might mean another case is positive somewhere in the Kingdom of Saudi Arabia). 

FluTrackers just tweeted that this is the 10th MERS-CoV case reported in a week. Quite a drought breaker.

A history of MERS-CoV cases in Qatar (n=4) 
Dates are of local news reports; not Australian time) noting their age in years, sex , FluTrackers case number, and where they might have acquired there infection:

  1. 22.09.12, 49M, #4
    • Likely local acquisition (see publication)
    • Flown to the United Kingdom
  2. 12.10.12, >45M, #6
    • Likely local transmission (see publication)
    • Flown to Germany for treatment
    • Lived in Doha
  3. 20.08.13, 59M, #97
    • Likely local transmission (see WHO disease outbreak news)
    • Only travelling outside of Qatar for 6-days during which he became symptomatic. 
  4. 26.08.13 29M, #104
I am not including the 2 local Tunisian acquisitions in the Qatar list. These 2 adults (34M and 35F) most likely caught their MERS-CoV infection from their father,who most likely acquired it from Qatar. Nonetheless it seems his children acquired their infection in Qatar. Moreover  the father tested negative to the MERS-CoV - despite it being likely that he was truly positive.

Monday 26 August 2013

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 10μl 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.

Sunday 25 August 2013

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.

Friday 23 August 2013

Maths says MERS-CoV still doesn't have pandemic potential

Breban and colleagues note in the Lancet that even with their most optimistic number crunching, there is only a low risk that that the coronavirus causing MERS could jump from an infected case to a naive person. Certainly not enough to pose a serious possibility of pandemic spread at this stage, based on what we know of the virus now and the case numbers and details we have to work with. The basic reproduction number (R0), or number of secondary cases (see the orange circles below), is still calculated to be less than 1. When >1, we consider epidemic potential reached.

Apparently that is despite clusters that may sometimes suggest otherwise.

Keeping in mind that a pandemic is largely about numbers - how many secondary infections occur  in close or other contacts - and how far and fast that transmission chain continues (the yellow circles and beyond).  
For me, this really reinforces just how important it is to have a full picture of a virus's transmission pathway. Not just the severe cases that show up in hospital, not just their contacts but also mild and asymptomatic cases in the community and the rest of the hospital. Prospective screening without regard to signs and symptoms in fact. 

Each and every person positive for the virus may represent a link in the transmission chain

You address whether mild cases can spread virus in another study. Oh, and we should probably keep monitoring all the viral strains we detect for genetic changes that occur in parallel with family clusters or upticks in transmission - which might signal increased potential to spread.

Don't test, don't find. Know nothing.

MERS-CoVs: South African bats vs Saudi Arabian bats

The latest sign of MERS-CoV in an animal, the Taphozous perforatus bat, is based on a 181 basepair (bp) fragment amplified from the viral RNA collected from a bat's droppings. 

The sequence is not yet available on the public sequence database, GenBank, and I haven't asked Prof Lipkin et al. for it. In the meantime though, I've aligned the primers mentioned in the new Emerging Infectious Diseases article by Memish and et al., against a full genome of MERS-CoV (EMC, the Munich strain). Sorry the image doesn't come out perfectly-if you click on it it will expand to the size of your browser.

Click then expand browser for full size. The expected position of the Memish et al. Taphozous perforatus bat MERS-CoV sequence is shown as a grey box. Primer locations for the nested RT-PCR are shown as red (outer primers) and orange (inner primers; the sequence region depicted in the phylogenetic tree in the recent EID paper) boxes. The recent South African bat CoV relative of MERS-CoV is show in pink (not overlapping) and the same region of full length CoV genomes are shown in blue (MERS-CoV EMC Munich) and green (HKU5 bat CoV)

For fun (yeah, I should get out more) I wanted to see just how close the "Close Relative of Human Middle East Respiratory Syndrome Coronavirus in Bat, South Africa" was, as described from another recent EID paper, to the new bat CoV. '

Unfortunately, as you can see above, the two fragments don't overlap. So my fun is ruined! 
We do know from yesterdays article however, that the 181bp fragment was 100% identical to human MERS-CoV over this short span (about 0.6% of the length of the entire MERS-CoV EMC genome). 

As Prof Andrew Rambaut noted to Helen Branswell in the Vancouver Sun, we need a whole genome to get more information that will better place the T. perforatus into the clade of viruses that seem related to MERS-CoV.

MERS-CoV updated graphs...

With all the movement in case numbers after a 3-week hiatus, I thought it worth checking what the charts look like.

This is graph (above), is based on dates of onset combined with dates of reporting to fill data gaps. We can see the Proportion of Fatal Cases (PFC) has settled. There are some data gaps that prevent this from being an ideal graph - this is why dates of reporting were also used to give a better, but general, idea of the status of case and death changes: 44/102 (or 103) are missing. Also, details for 3 deaths, 7 ages and the sex of 5 patients are not available (or I have not found them at least!).

In this chart (right) we can see the Kingdom of Saudi Arabia has most cases 0 the proportion has remained about the same as it was for my last update July 9th - around 80%.

Below, we see the regional accumulation of cases. I may need to research what exactly has been imported and what acquire locally in Qatar - or change the numbering to reflect country "dealing with"the case to make things simpler. But you get the gist. KSA continues its steep growth of MERS-CoV case numbers.

I'll update my main MERS-CoV page with these soon.