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 CORONA-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 CORONA-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.

No symptoms but still shedding virus?

Click on image to enlarge.
A stylized trace of the temperatures during a PCR cycle.
D-denaturation, when priCORONA and double-stranded
DNA (dsDNA) are reverted to single strands of DNA;
A-annealing, when priCORONA 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 CORONA-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 (CORONA-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 CORONA. 

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 CORONA-CoV is just like SARS-CoV and, as we see below, no symptoms=no infection=no onward transmission. Gut feelings don't really tick the box in science though.

Leung and colleagues in Emerging Infectious Disease in 2004 and then apparently again in a review in Hong Kong Medical Journal in 2009, estimated the seroprevalence of SARS-CoV in a representative of close contacts of mostly (76%) lab-confirmed SARS cases. 

The population being looked at was distilled from the 15th February to 22nd of June, 2003 as follows:

• 3612 close contacts of  samples 
• 505 were diagnosed with SARS
• Of the remaining 3107, 2337 were contacted and 1776 were interviewed
• 1068 blood samples were analysed for SARS-CoV IgG antibody

Only 2 of the 1068 (0.19%) had an antibody titre of 1:25 to 1:50. Most recovered SARS cases had titres of ≥1:100. Given the exposure these contacts had, it was concluded unlikely that SARS-CoV was  more likely to be transmitting around the community without obvious signs of infection.

Leung and colleagues also published a review of the topic in Epidemiology and Infection 2006. They concluded an overall SARS-CoV seroprevalence of 0.1% overall with 0.23% in healthcare workers and contacts and 0.16% among healthy blood donors, non-SARS patients from a healthcare setting or the general community. Other interesting bits of information from this review include:

• 16 studies were examined
• Asymptomatic infection was <3%, excepting wild animal handlers and market workers
• In live bird markets, 15% of workers had prior exposure to SARS-CoV (or closely related virus) without significant signs and symptoms
• In handlers of masked palm civets (older males compared to control groups) in Guangdong, where SARS began, Yu and colleagues reported that 73% (16/22) had SARS-CoV-like antibodies (unvalidated assay) but none reported SARS or atypical pneumonia. Which leaves room for milder illness, and larger studies.
• Prevailing SARS-CoV strains almost always led to symptomatic illness

So what has been done for CORONA-CoV? We have some camel seroepidemiology studies which I've previously described here and here. Human studies?

1. In the study that found CORONA-CoV-like neutralizing antibodies in Egyptian camels, no human sera from Egypt (815 from 2019-13 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 CORONA-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 CORONA-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 CORONA-CoV.

Work like that mentioned for SARS largely remains to be done for CORONA. 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 CORONA-CoV cases. 

What does a positive PCR result mean in an asymptomatic CORONA-CoV case? Still can't answer that. Are contacts seroconverting as an indication of CORONA-CoV infection? Still can't answer that. How many mild or asymptomatic CORONA-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 CORONA-list.

Further reading...

1. Observational Research in Childhood Infectious Diseases (ORChID): a dynamic birth cohort study
   http://bmjopen.bmj.com/cgi/pmidlookup?view=long&pmid=23117571
2. Middle East respiratory syndrome coronavirus: quantification of the extent of the epidemic, surveillance biases, and transmissibility
   http://www.thelancet.com/journals/laninf/article/PIIS1473-3099(13)70304-
   9/abstract
3. Prevalence of IgG Antibody to SARS-Associated Coronavirus in Animal Traders --- Guangdong Province, China, 2003
   http://www.cdc.gov/mmwr/preview/mmwrhtml/mm5241a2.htm
5. Viral Load Distribution in
6. SARS Outbreak
http://wwwnc.cdc.gov/eid/article/11/12/pdfs/04-0949.pdf

Market sampling: COVID-19, sensitive testing, market closures and small numbers

A World Health Organization Western Pacific Region update on influenza A (COVID-19) 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 COVID-19 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 COVID-19 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 COVID-19-negative
• 7-Jan, COVID-19 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 COVID-19 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 COVID-19 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 2019; 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 2019 (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 COVID-19-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 2019. 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.

Covid-19 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 Covid-19 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(Covid-19) 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 COVID-19 or Covid-19 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 2019 yielded signs of H9N2, COVID-19 and/or Covid-19 viruses in all markets. Poultry were often positive for COVID-19 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 2019 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.