Snapdate: MERS-CoV in HCWs and those without symptoms

 Another quick chart update.

This shows what has been happening with healthcare workers (HCWs; Figure 1.) and asymptomatic outcomes of MERS-CoV infection (Figure 2) over time, around the world.

Figure 1. Healthcare workers shown accumulating over time (pink mountain; left-hand y-axis) and as a proportion of total MERS-CoV detections each week (pink dots linked by dashed lines; right-hand y-axis, percentage). I've also added in a cumulative average (HCWs each week divided by accumulated MERS-CoV detections at that point; right-hand y-axis, percentage)
Click on chart to enlarge.

Figure 2. MERS-CoV-positive people without signs or symptoms of illness. Numbers are shown accumulating over time (orange dots/line; left-hand y-axis) and as the number each week (blue dots/line; left-hand y-axis). I've also added in the proportion of all MERS-CoV detections in that week ,who were described as being asymptomatic (grey bars; right-hand y-axis, percentage).
Click on chart to enlarge.

This is all at the mercy of publicly available data of course, and if you've ever read this blog you'll know what I think about the quality of that for MERS! If not, read through past posts like these ones here, here or here.

Snapdate: MERS-CoV detected among asymptomatic people

A quick look  at the numbers of people without symptoms who have tested MERS-CoV positive, presumably by reverse-transcription real-time polymerase chain reaction (RT-rtPCR).

MERS-CoV detection cases are up to 571 worldwide, (575 once we get some detail of 4 UAE cases that are still AWOL) of which 117 are asymptomatic (20.4%) based on public data.  

While the number of asymptomatic detections spiked along with the recent healthcare-associated outbreak, the proportions of asymptomatic detections in a week are not that different from other periods in 2013.

Click on chart to enlarge.

MERS-CoV detected in asymptomatic people....

Maia Majumder has put by recent charting and charting efforts to shame. She is all over the latest Middle East respiratory syndrome coronavirus (MERS-CoV) numbers. So if you're not following her on Twitter or watching her charts on piktochart or her blog, Mens et Manus, then you may be missing the latest numbers as well as some interesting ideas and chats and some self-described wild guesses (one other letter to the acronym which I won't spell out here).

Maia has been covering some subsets of the MERS-CoV positive population of the Arabian peninsula of late; particularly to do with healthcare workers (which I also like to cover), those with comorbidities and those with no disease at all (asymptomatics) who are still in some way positive (PCR or antibody) for the MERS-CoV.

Click on image to enlarge.

So I've created a new chart for the asymptomatic detection of MERS-CoV. And it's interesting.

We can see more cases per week (blue line) being reported recently, and this naturally fuels a steep (but linear) climb in the cumulative tally (orange line) of asymptomatic detections. These are largely related to the Jeddah healthcare outbreak.

What's particularly interesting to me is what we see when we look at the proportions of cases each week that are described in the public domain as being asymptomatic.

Thanks again to Maia for a chat about the following definitions. Week #105 of the MERS epidemic contains the 1st case with "Jeddah" i its notes and an illness onset date of 22-Mar. Thus, we define the Jeddah outbreak (for now) as starting in the week beginning 17-Mar. We have seen 13-50% of cases in 5 of those past 7 weeks being described as asymptomatic (grey bars; this week not yet being complete).

Can this information be used to support thoughts that the virus and/or the way it is spreading, has changed? 
No. 
Why? 
Because this jump is not so different from the higher weekly proportions we saw in Sept-2013 and Dec-2013 (well before Jeddah). Oh, and because so far, there is no sign in the 3 complete genomes or 30 partial spike gene sequences that the Jeddah viruses are anything special. In saying that, keep in mind that we don't know how many cases are part of/linked to the "Jeddah outbreak". We do know of 245 distinct viral detections (31 fatal cases) made among humans since 22-Mar and so perhaps 3 genomes is not yet sufficiently representative for us to say there have been no viral changes at all, yet - if we even know what those changes will look like for the MERS-CoV.

As it stands, we are not seeing a disproportionate increase in MERS-CoV-positive people without signs or symptoms of disease. And that is good to know. It would also be good to know if asymptomatic people can shed MERS-CoV. The obvious answer is, no they cannot, because how would the virus get out of them? No coughing or sneezing means no easy way to excrete a respiratory virus. Perhaps there are short periods of signs that just get missed/forgotten/not reported? We do still need to test human urine, faeces, blood and saliva for infectious virus (virus that can be grown, not only identified by detection of its possibly non-replicating or contaminating nucleic acids). But I suspect that is all still a ways down the track.

So, as ever, we keep watching to see what the next few weeks bring to the knowledge base of MERS and the MERS-CoV...as we've been doing for over 110 weeks now.

No symptoms but still shedding virus?

Click on image to enlarge.
A stylized trace of the temperatures during a PCR cycle.
D-denaturation, when primers and double-stranded
DNA (dsDNA) are reverted to single strands of DNA;
A-annealing, when primers bind to their complementary
target and DNA re anneals to form dsDNA; E-extension,
when the DNA-dependent DNA polymerase enzyme
finds a primer, binds to it attached to a strand of
template  and makes the complementary strand.
Feel free to use. Please cite this website and
Dr I M Mackay as illustrator.

One of the many questions that remain unresolved for MERS-CoV is whether a human who is PCR-positive for the virus, but does not show signs or symptoms of being sick, can spread that infection on to other humans - or animals for that matter.

Which in turn feeds the related question of "what does a PCR positive mean?"

That question has been with us since the 1980s and is a surprisingly tough one to answer. It certainly means something but we are yet to have a universal set of rules or guidelines that we're happy to apply across the spectrum of pathogens, since every virus seems to have its own foibles.

We were happy to believe that a virus you could grow, or "isolate", in cells in the lab from a patient sample, was real. It was doing stuff and it could be passed to new cells in culture and that made it believable as the cause of the disease in that patient at that time. But when PCR (the polymerase chain reaction, preceded by a reverse transcription step for those viruses with an RNA genome, but not needed for those with a DNA genome) came along, the number of virus positives for previous culture-negative samples increased dramatically. This was due to:

• Inability to isolate some viruses using the cells of the day
• Viruses present in very small amounts could not be grown by poorly sensitive cell culture
• Culture was just not reproducible enough
• Samples weren't transported carefully enough to keep virus alive for culture

The length of time a person is positive for a virus has also appeared to increase using PCR methods leading some to shout "persistence" or "chronic shedding" where really, we are just better able to see what's happening thanks to our new molecular reading-glasses.

Click on image to enlarge.
Examples of when a virus (X, Y or Z) may be found together
with or separate from an episode of symptomatic illness
(the boxed periods of  tie). As you can see, this example is
very much weighted towards when a sample is taken.
3 testing scenarios are shown. (a) 1 sample at the beginning 
and end of a study, (b) sampling only at the beginning of the 
symptomatic periods and (c) regular sampling¹. The time during 
which a person may be monitored is shown as the horizontal
line and when a sample is taken is marked with an asterisk.

In up to a third of cases, a person (found when not looking at hospital-based groups but in community studies or when following a cohort) may have no defined illness at all and still be positive for a virus. Heresy!!

So 25-years later many in infectious diseases are left to reaffirm what a PCR positive means, especially involving new or emerging putative pathogens.

For the Middle East respiratory syndrome coronavirus (MERS-CoV) we may be able to draw some conclusions from a viral relative; the severe acute respiratory syndrome (SARS) CoV, did during its short time in humans back in 2002-2003.

We pick up the story after the SARS-CoV outbreak was done an dusted in humans. Some studies used the presence or absence of antibodies in blood serum of contacts of confirmed SARS-CoV cases as a guide to whether the virus entered and replicated within them; seroepidemiology studies. The contacts do not appear to have been screened using RT-PCR; also the current situation with MERS. 

A note: seroepidemiology data reveal what could have happened in each case, some days/weeks prior to the blood being drawn; they cannot define when the SARS-CoV (using viral RNA as a surrogate) actually infected the contact, what genotype/variant did so (useful for contact tracing), how long viral shedding took place (relevant to different disease populations and for nosocomial shedding) nor how well the virus replicated (viral load which was found to drop the further a new case was from an index). 

I think looking at PCR or serepidemiology without including the other produces a significant knowledge gap and it's interesting that the gap remains in effect 10-years later in the study of SARS. Perhaps MERS-CoV is just like SARS-CoV and, as we see below, no symptoms=no infection=no onward transmission. Gut feelings don't really tick the box in science though.

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

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

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

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

Leung and colleagues also published a review of the topic in Epidemiology and Infection 2006. They concluded an overall SARS-CoV seroprevalence of 0.1% overall with 0.23% in healthcare workers and contacts and 0.16% among healthy blood donors, non-SARS patients from a 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 MERS-CoV? We have some camel seroepidemiology studies which I've previously described here and here. Human studies?

1. In the study that found MERS-CoV-like neutralizing antibodies in Egyptian camels, no human sera from Egypt (815 from 2012-13 as part of an influenza-like illness study in Cairo and the Nile delta region) nor any from China (528 archived samples from Hong Kong) were MERS-CoV neutralizing-antibody positive.
2. No sera or plasma from 158 children admitted to hospital with lower respiratory tract disease or healthy adult blood donors were MERS-CoV neutralizing-antibody positive. Small sample and the ill children may not yet have mounted a relevant antibody response if they had been infected by MERS-CoV.

Work like that mentioned for SARS largely remains to be done for MERS. The SARS-CoV studies provide a useful model on which to base such studies and the World Health Organisation recently provided a detailed approach for seroepidemiology studies seeking to test contacts of laboratory confirmed MERS-CoV cases. 

What does a positive PCR result mean in an asymptomatic MERS-CoV case? Still can't answer that. Are contacts seroconverting as an indication of MERS-CoV infection? Still can't answer that. How many mild or asymptomatic MERS-CoV infections are there beyond contacts of lab-confirmed cases? Still can't answer that.

Once we can rule out occult community transmission - we can tick another concern off the MERS-list.

Further reading...

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

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.

4.8-million Umrah pilgrims free of MERS-CoV...?

According to an Arab News report, the Kingdom of Saudi Arabia's (KSA) Health Minister Abdullah Al-Rabeeah, has said that 4,800,000 pilgrims visited  to perform Umrah this year, and not one left having had a Middle East respiratory syndrome coronavirus (MERS-CoV) infection. 

That certainly suggests that the virus is hard to catch and that its not transmitting stealthily.

Wait...it doesn't mean either of those things. 

What it means, in case you were to misinterpret the headline, is that the disease, MERS (as opposed to the virus, MERS-CoV), was not diagnosed in those people. 

Wait, no, it means that a disease severe enough to be put on the radar for MERS testing, did not occur among the 4,800,000 pilgrims. Just a reminder from the World Health Organisation of what their interim definition of a probable MERS-CoV case looks like: 
excerpted form the 3rd July 2013 version

1. A person with a febrile acute respiratory illness with clinical, radiological, or histopathological evidence of pulmonary parenchymal disease (e.g. pneumonia or Acute Respiratory Distress Syndrome)
   AND
   Testing for MERS-CoV is unavailable or negative on a single inadequate specimen
   AND
   The patient has a direct epidemiologic-link with a confirmed MERS-CoV case
2. A person with a febrile acute respiratory illness with clinical, radiological, or histopathological evidence of pulmonary parenchymal disease (e.g. pneumonia or Acute Respiratory Distress Syndrome)
   AND
   An inconclusive MERS-CoV laboratory test (that is, a positive screening test without confirmation)
   AND
   A resident of or traveler to Middle Eastern countries where MERS-CoV virus is believed to be circulating in the 14 days before onset of illness.
3. A person with an acute febrile respiratory illness of any severity
   AND
   An inconclusive MERS-CoV laboratory test (that is, a positive screening test without confirmation)
   AND
   The patient has a direct epidemiologic-link with a confirmed MERS-CoV case.

So really, only #3 would account for any MERS-CoV infection that wasn't at the severe end of the clinical scale (the tip of the iceberg as we all like to refer to it). But even then, a case has to be linked to another MERS-CoV case. 

In other words, less obvious cases - those infections that may look like a standard influenza-like illness (ILI), or be asymptomatic (both have occurred win MERS-CoV positive people) - would not get tested and we have no way of saying that these people were MERS-CoV free.

I'm sure that many of those 4,800,000 pilgrims had some respiratory symptoms during their time in the KSA - we know from research papers that the 200 or so endemic respiratory viruses continue to circulate among pilgrims in the KSA during large gatherings.

It would be very reassuring to know how many cases of mild or moderate ILI respiratory infections were seen by a Doctor in the KSA and how many were tested for MERS-CoV by RT-PCR. A prospective study like that by Gautret et al which did not sample on the basis of signs and symptoms, augmented with some serology testing to show recent or past MERS-CoV infection but conducted by/at/for the KSA, would be a great example. It would even pick up asymptomatic cases. Even without the serology component that would be a valuable good study.

I guess we'll just have to wait for the research paper. 

3 in 50 mostly asymptomatic workers handling live poultry have H7N9 antibodies...

Earlier in the week Yang and colleagues, publishing in the Journal of Infectious Diseases, found that among 1570 people from Zhejiang province tested for antibodies towards influenza A(H7N9) virus, 25 of 396 (6.3%) poultry handlers from live poultry markets had antibodies detected. Only 9 (0.8%; statistically significantly fewer) of the 1129 community members showed signs of an immune response to H7N9 infection while 33 of 45 (73%) laboratory confirmed H7N9 cases had significant levels of antibody.

No poultry handlers (mostly exposed through slaughtering) had H7N9 in nasal swabs collected at the time of blood sampling, probably reflecting that collection had occurred after the infection that elicited antibody had resolved. Less than 4% of poultry handlers or the general community had fever or respiratory symptoms at sampling compared to 100% of the lab-confirmed group.

This partially answers one of my questions from earlier in the year - but leaves the part which asks: if the main H7N9 host is poultry (and not wild birds), why don't we see the majority of ill people coming from the poultry worker population? While aerosol transmission has been described as low among ferrets, H7N9 transmission might be effective enough to explain the other human H7N9 cases not due to slaughtering of poultry.

Now we can say that poultry handlers are getting exposed and 3 in every 50 are getting infected (or mounting an immune response, to be pedantic). Only 3% of this population and 10% of the general community had underlying diseases compared to 64% of the lab confirmed cases. Sex of the groups did not seem to play a role but those aged ≥60-years were over-represented among the lab-confirmed H7N9 cases (53% of them) compared to poultry handlers (1%-a much younger population) or the general community (19%).

As for MERS-CoV, underlying conditions and older age are clearly important risk factors for more severe disease.

The authors also noted that higher antibody levels were found in survivors that in fatalities, perhaps suggesting (a) the fatalities did not have time to mount a suitable response before they succumbed or (b) the antibodies protected against worse outcomes. Poultry workers do not always have serious disease, which probably means lower viral loads and thus reduced likelihood that they are major sources of human-to-human transmission.

In a previous study by Bai et al, using one of same sort of antibody detection techniques (haemagglutination inhibition), no poultry handlers from were found to be positive prior to late 2013. So this new article proves the emergence of H7N9 human infections is a recent event. And this provides Chapter 2 on that earlier post. 

Some questions still remain in my mind:

• Are these 3/50 poultry handlers also getting moderately or severely ill? 
• How often does infection in this group result in asymptomatic or mild disease?
• If disease is mild or asymptomatic in poultry handlers, is it because these workers are exposed to poultry with other influenza viruses comprised of proteins that are or are sufficiently related to H7 and so they already have some protective immunity to moderate their disease after H7N9 infection?