Signs of recent Zika virus infection of babies soon after birth...

According to a study released 10 days ago, the cerebrospinal fluid (CSF) of 30 of 31 babies born with microcephaly in Pernambuco, Brazil between 21 and 30 October 2015, contained IgM antibody that was Zika virus (ZIKV) specific.[1] 

This suggests that these babies had recent or current infection by ZIKV.

From [1].

ZIKV RNA was not detected in any baby's CSF or serum samples - nor was Dengue virus or Chikungunya virus. No word on other viruses such as cytomegalovirus, rubella virus, enteroviruses etc. No unbiased investigation for viruses were described nor was the serostatus of the partner at conception or birth. This was a "first past the post" diagnostic conclusion.

The authors remind us that IgM does not cross the blood-brain barrier (it's a big molecule) and so its presence in the CSF suggests that the foetus was infected, not the mother. 

The absence of RNA could be interpreted as the infection having occurred at least a week ago. But that's hard to know and it is unclear how long IgM lingers for in the foetus and child after ZIKV infection. There is no discussion about the possibility of the BBB being less than perfectly intact or in some other way failing to perform its role as a barrier in these babies.

Unfortunately, as has usually been the case throughout the Brazil microcephaly surge, there is little by way of a denominator to help us understand the scale of the proposed role of ZIKV here. What is happening in the serum and CSF of babies delivered to mothers from the same regions of Brazil who gave birth to babies sampled in the same way, at the same time after delivery, which were not diagnosed with microcephaly or other central nervous system disorders? 

We can probably be safe in assuming that not every mother who has been infected with ZIKV delivers a baby with congenital abnormalities but that many mothers have in fact, been infected during this epidemic. 

If ZIKV or ZIKV together with something else is the cause of microcephaly, why is it still relatively so rare among so many births? 

References...

1. http://www.thelancet.com/journals/lancet/article/PIIS0140-6736(16)30253-7/fulltext?rss=yes

If you are often in contact with camels are you more likely to acquire MERS-CoV? [spoiler: yep]

This dromedary camel (DC)/Middle East respiratory syndrome (MERS) themed post is a quick review of a paper from 2015 by Reusken and a team of absolute champions in this space. 

It, as many have been, was published in the Emerging Infectious Disease journal, listed in its August issue (but online earlier) and entitled, Occupational Exposure to Dromedaries and Risk for MERS-CoV Infection, Qatar, 2013–2014.[1]

The study examined 498 sera from humans in Qatar split into different exposures types. Included were European (the Netherlands and Germany) human sera for use as controls - collected from a part of the world where there was not expected to have been any MERS-coronavirus (CoV) exposure and so no antibodies were expect to be present; a test for the tests.

As an aside, we've seen some great informative MERS-CoV/camel studies come out of Qatar. I love watching good collaborations pay dividends.

The 498 sera breakdown as follows:

• 294 from those with daily DC exposure
• Cohort A: 109 camel (A1; n=5) and sheep (A2; n=104) slaughterers
• Cohort B: 8 central animal market (CAM) workers
• Cohort C: 22 healthy males living & working at Al Shahaniya barn complex adjacent to DC race track
• Cohort D: 155 healthy males living & working at DC farm
• 204 from those without camel contact
• Cohort E: 56 samples from construction workers
• Cohort F: 10 people living in a complex with 200 sheep barns
• Cohort G: 138 specificity testing samples (66 from the Netherlands and Germany who had recent CoV infection (G1) and 72 from the Netherlands obtained for Bordetella pertussis infection testing (G2)

The antibody testing regimen relied on a multi-tier approach (the best ones do, until we're sure that any single assay can cope with all the variables):

• Tier 1: IgG antibodies were sought using the MERS-CoV, severe acute respiratory syndrome (SARS)-CoV, human CoV (HCoV)-OC43 spike domain S1 antigen protein-microarray method used previously by this group [2]
• 20/294 samples (6.8%) reacted (had IgG antibody in them) - none were from controls sera or from those without DC contact
• 4/22 Cohort C, 8/155 Cohort D, 3/104 Cohort A2 and 4/5 Cohort A1 samples were reactive
• All samples from A1, A2, B, C, D, E, F and G1 showed responses to HCoV-OC43 S1
• None of 498 sera reacted to SARS-CoV S1
• Tier 2: A 90% plaque reduction neutralization test (PRNT90 [4]) was used to show whether antibodies in samples could specifically stop MERS-CoV from infecting cells after sera and virus were co-incubated ahead of infection of a cell line
• the 20 IgG reactive samples from Cohort A to D were tested and 10 were able to neutralize infection
• 34/35 samples from those with camel contact (Cohorts A1, B and C) that were IgG non-reactive, also had no neutralizing antibody
• "Tier 3": Use of a whole MERS-CoV immunofluorescence assay (IFA). However, the results from testing 8 reactive samples (5 of which were positive by IFA) were not included

This paper has a nice central finding which goes something like: if you don't have contact with camels, you don't get infected by MERS-CoV. If you do regularly have contact with camels - you are much more likely to get infected as determined by you having developed antibodies to that virus; you were infected but you fought off the infection. A similar finding came out of the larger serosurvey from the Kingdom of Saudi Arabia.[3] 

I do wonder about the reactive sheep slaughterers though (Cohort A2) - where did those infections come from?  

The authors also addressed why other serologic studies of humans with occupational exposures have not found reactive sera-those studies hardly ever documented infected camels at the workplaces and there may not have been any (for some significant period of time presumably). More infected camels may be associated with more human infections. No surprise. The authors had found, outside this publication, that 60% of camels at the CAM and slaughterhouse were shedding MERS-CoV. This discrepancy has been a question of mine for a long while - and I like this answer.

Interestingly, the participants with antibodies don't recall being seriously sick. So you may get infected and just think you have the flu, or a cold, or nothing at all. This result may further confuse camel-deniers who do not have any background in the wide spectrum of outcomes one can expect after infection by any virus. Nonetheless, such apparently unnoticeable infections add more weight to the story that the current proportion of fatal cases is an exaggeration. So we learned yesterday that MERS (the disease) is rare, that camel contact makes up only a proportion of the likely sources of infection and now we see that you may not even get sick if you do get infected. A few things to digest there.

Also very interesting to me is that the neutralizing antibody titres were lower than had been found elsewhere. The authors suggest this may be due to these infections producing only mild disease. Without a prospective study though, it's very hard to be sure about the true disease severity - recall bias can be a pest. This is an area that needs a more focussed study; do our antibody tools detect mild and asymptomatic cases as reliably as severe MERS cases, for how long and in all cases of infection?

Its feels like its getting pretty hard to mount any realistic case for why we should ignore the role of camels in infecting us with MERS-CoV - even if they do so rarely, and perhaps often without serious complications.

References...

1. http://wwwnc.cdc.gov/eid/article/21/8/15-0481_article
2. http://virologydownunder.blogspot.com.au/2015/10/kenyan-camel-coronaviruses.html
3. http://www.thelancet.com/journals/laninf/article/PIIS1473-3099(15)70090-3/abstract
4. http://www.thelancet.com/journals/laninf/article/PIIS1473-3099(13)70164-6/abstract

Bats in a tree...

Meliandou and the burnt tree that
once housed a bat colony (from Fig 3, [1]).

While not snakes on a plane, I'm fairly sure the level of swearing has at times been at least as bad among those suffering from and dealing with the possible fall-out from these bats - if in fact they were the source for the biggest Ebola virus disease (EVD) epidemic on record.

A recent animal counting, trapping and testing study in Guinea included sampling in and around the village of Meliandou.[1] This village is, to the best of our knowledge, the site of the first animal-to-human, or zoonotic, transmission of the Ebola virus variant called Makona.[2]

The study team, made up of researchers affiliated with Germany, Sweden, Core d'Ivoire and Canada, did not find any decline in numbers of usually susceptible larger mammals around the index village; a sign during other outbreaks, of active local ebolavirus "activity". The team also found that primate hunting was not a big thing in this region, which is rather devoid of these and other Ebola virus mammalian host animals (including few of the Duiker, or forest antelope). Fruit bat hunting was common though.

The team captured 169 bats representing at least 13 different species and 6 families. But in the house of the 2-year old boy considered the epidemic's index case, fruits bats were not eaten and no bat hunters resided there. No Ebola virus RNA was detected in any bats and antibody screening results from bat blood were inconclusive. 

These findings led the authors to study Meliandou, resulting in an hypothesis that a nearby hollow tree that once housed a large colony of free-tailed bats [locally described as lolibelo - small and smelly bats - otherwise known to belong to the species of insectivorous bat, Mops condylurus of the family Molossidea; [3], may have been the source of  infection. Why only one child was infected this way when the tree was a site of frequent play by many children is not known. The tree was burned out in March 2014 which caused many bat deaths, some of which were collected for consumption. Sequencing of a PCR-amplified mitochondrial DNA segment found that in 5 of 11 ash and soil samples from around the tree, contained traces of Mops condylurus genetic material. So that species was at least there.

So, this is all quite far from a conclusive link between the 2-year old boy and these bats. But it does read as though every avenue has been tested in this village, perhaps apart from better animal antibody testing (serology), and some serology on the blood of those villagers who remain alive in Meliandou. 

Serology testing is going to be very important for answering many questions around EVD and this outbreak and epidemic. 

Of course this will raise the usual question of whether we cull all bats to prevent this from ever happening again. Don't be ignorant! Bats have very important roles in pollinating and thus in keeping our ecosystem going. Should we kill all bees because they sting us? I'm pretty sure I've been stung by a bee more times than I've had Ebola/Hendra/SARS/Nipah/MERS/Lyssavirus or any other bat-hosted virus infection. Killing off everything to prevent a very rare zoonotic event when better knowledge can resolve the problem is just a typically short-sighted and knee-jerk human reaction (not a fan-can you guess?).

One question that does still remain, and one that is of extreme interest to me, is how often mild disease results from an Ebola virus infection? Good, robust serology methods to the rescue.


References...

1. Investigating the zoonotic origin of the West African Ebola epidemic. EMBO Molecular Medicine(2014). http://embomolmed.embopress.org/content/embomm/early/2014/12/29/emmm.201404792.full.pdf
2. Nomenclature- and Database-Compatible Names for the Two Ebola Virus Variants that Emerged in Guinea and the Democratic Republic of the Congo in 2014. Viruses 2014, 6(11), 4760-4799.
   http://www.mdpi.com/1999-4915/6/11/4760
3. Mops condylurus via the IUCN Red List of threatened species (listed as of least concern)
   http://www.iucnredlist.org/details/full/13838/0

MERS-CoV detections: The April wave recedes...

So welcome to the 114th Week of MERS-CoV among us. That week numbering may change shortly. Stay tuned if week numbering is your thing.

We currently have a tally of 649 detections of MERS-CoV or viral antibodies in humans. I don't list camel numbers. My count says 192 fatalities among infected people, resulting in a proportion of fatal cases of 29.6%. That seems high. Because, until very recently, the Kingdom of Saudi Arabia's Ministry of Health did not regularly report deaths alongside their date of illness onset, it has been an interesting hobby to try and link them. The number is solid so along as the MOH has not been doubling up in the reporting or coming back later to re-report deaths. You'll be familiar with these issues if you follow me on Twitter.

I made a point of saying antibodies earlier because I am going to be including these sorts of laboratory data in my tally when produced by trustworthy laboratories who have described their methods and shown some validation data and an understanding of what the cross-reaction issues are when dealing with MERS-CoV serology. This will be despite the current WHO MERS case definition not allowing for inclusion of people who only have antibody but no virus or viral RNA detected in their samples. There may be some hiccups with MERS-CoV antibody testing along the way, but we need these data in humans and it's good to see the wheels rolling on this at last.

[One of those hiccups occurred 28-May-2014, when the test result from an Illinois man who had originally tested positive in an Ab test, was retracted.]

In my estimation though, serology (the testing of human sera for antibodies against a virus here, the main target being IgG which takes a couple of weeks to become detectable after infection) is a much more reliable way of defining an infection by MERS-CoV virus than by relying on patient recall bias of symptoms 2-weeks ago, or from directly observing signs and symptoms that are nondescript and difficult to distinguish, alone. The latter approach has been the mainstay of identifying cases of human infection for a very long time; still is. This approach is especially important during times of outbreak and pandemic when labs are swamped by testing requests and it must be assumed that cases are due to the bug of interest; if it looks like a camel, slobbers like a duck and walks like a duck, then it is a MERS-CoV infection yeah? No. If you can clinically characterise and laboratory test then you will more often know the virus the patient has/had than if you don't test. But I'm sure that's clear to everyone anyway.

For MERS, as for H1N1pdm09 influenza and perhaps SARS, finding a reliable pathognomonic set of signs or symptoms capable of reliably distinguishing a respiratory virus of interest from another virus capable of the same disease is not possible. These viruses cause a spectrum of illness. Testing is paramount if you want to know what's there and to address other aspects relevant to public health during an infectious disease cluster/outbreak/pandemic. There are a couple of issues here (at least!)...

From a patient management perspective, who really cares what is making my patient very ill anyway? It really doesn't matter right now if it's this respiratory virus or that one; there are few vaccines and I don't have an antiviral for most of them anyway. I and my healthcare team are already taking respiratory infection precautions and I just want to direct my supportive therapy and resources to the problems they have, right? I'll be (well...you, experienced medical types of which I am not one) doing that before many lab results show up anyway. 

From the perspective of interrupting and understanding viral transmission however, nondescript signs and symptoms are a nightmare. And in the early days of a new virus where we seem to know very little about what path(s) transmission is taking (and perhaps we're also learning some more about those possibilities in general), any infection by whatever method it is empirically determined should, I believe, be recorded as an infection in order to provide the biggest picture possible; a process we have seen unfolding in the United States with its 2 3 detections (1 locally transmitted) of MERS-CoV or its spiky little footprints.

THIS RESULT WAS RETRACTED 28-May-2014 FOLLOWING A NEGATIVE NEUTRALIZING ANTIBODY TESTING.

Given that many viruses cannot be distinguished by signs and symptoms alone, a clinical diagnosis to define a case is less reliable than any pathogen-specific laboratory test. I hope the WHO alters their case definition in the near future. Infectious disease is always teaching us - seems we learned a heap from SARS but even the relatively a few cases of MERS are presenting interesting issues and testing us in new ways. 

[While the US antibody-positive result above has since been retracted, I stand by these comments-Ab testing requires rigor, but that can be provided using several assays and applying a good understanding of Ab technologies and limitations to produce reliable results]

Anyhooooo...been stewing on that for a few days apparently. Let's move on and have a look at the 3 updated charts below. 

We are definitely through to the other side of the Jeddah outbreak (see weeklies chart). While cases do keep accruing each and every day (see dailies chart from 20-March), the downward trend of smaller numbers of illness onsets each day also continues. 

Weekly MERS-CoV detections.
Click to enlarge.

Daily MERS-CoV detections from 20-March.

Click to enlarge.

For perspective on the size and the influence of what 1 hospital cluster can turn into and how that can influence how a virus "looks", take a gander at the extent of the April outbreak. Case are still falling out into April as we get more data. If you look at the monthlies chart at the bottom, I've readjusted that y-axis scale again such that it's maximum value is now 10x higher (350 vs 35) than the scale used for 2012 or 2013's charts. May's tally is currently 4x greater than any month from 2012 or 2013. 

What does MERS-CoV hold for us in the coming months? 

Daily detections of MERS-CoV, 2012-current.

Click to enlarge. 

Monthly detection of MERS-CoV 2012-current.
Click to enlarge.

Neither market nor farm poultry all that positive for H7N9; songbirds the culprit...?

Following on from yesterday's post, "If not poultry then what?", I thought it worth noting the impressive numbers from the Chinese Ministry of Agriculture.

From 2013:

• 1,630,000 poultry and environmental samples tested
• 88 POS; all from live bird markets
• None from poultry farms

From 2014, to date:

• 33,400 poultry and environmental samples
• 8 H7N9 POS; all from live bird markets
• None from poultry farms

The other alternative to answer the question in my heading; the testing methods are at fault. 

No detail of what approach has been used to obtain these numbers in the links below. Viral culture and serology with some PCR have been noted before. I'd wager culture yields chicken scratchings compared to PCR for detecting virus in he wild; but serology has successfully been a pillar upon which animal testing rests. So that's why the numbers above are such a quandary for the epidemiologist who reads about the high frequency of links between human disease and exposure to poultry.

It would be nice to see some technical papers on antibody test testing (development and validation) at some point. If only to reassure everyone that the testing methods are doing what testing methods should be doing.

See #3 below for influenza PCR discussion at WHO.

Sources..

1. http://english.agri.gov.cn/news/dqnf/201304/t20130427_19537.htm
2. http://english.cntv.cn/program/china24/20140130/100709.shtml
3. http://www.who.int/influenza/gisrs_laboratory/report_2012pcrwg5thmeeting.pdf

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

MERS case-control study during the Hajj

Dr Ziad Memish, Deputy Minister of Health, Kingdom of Saudi Arabia, has made a welcome comment about some analysis of ill cases that went on during the Hajj. In the Saudi Gazette..

He added that in addition to detailed investigations of every suspected case, case-control studies for index cases and intensive follow-up of contacts with serological testing to improve understanding of the critical features of MERS-CoV infection were carried out.

I'm not clear on whether that indicates there were MERS-CoV cases during the Hajj, or if he is referring to probable cases that were not confirmed (no contacts then?) or to respiratory illnesses in general. He unfortunately wasn't quoted as saying whether any of those results were positive for MERS-CoV infection. 

Given that 997,3709 pilgrims apparently partook in some degree of medical healthcare service while in the KSA for Hajj, this study should provide some very useful information about what MERS-CoV was doing both in the ill and the healthy in mid-October. I might even be able to stop whingeing about lack of testing of all but those who are severely ill (or their contacts)!

The case-control study protocol is likely to follow that defined by the WHO in July - which can be found here.

The controls (best if >1 per case) will be randomly selected people of equal age (leeway varies with age band) and sex ("matched"), living in the same neighbourhood (to ensure try and capture the same environmental exposures; difficult for visiting pilgrims so general are of pilgrimage might suffice) that are not presenting with the same illness as the confirmed "case" at the time of sampling. Sampling (described in the lab testing WHO document here) which is recommended to include material from the lower respiratory tract - which may prove difficult from otherwise well controls. Informed consent is recommended as part of the (any such) study so controls will know what they are in for ahead do time.

Interestingly the WHO document comments that...

Currently, circulation of this virus in the community is thought to be nonexistent or minimal at most and the numbers of infections low. For that reason, prospective controls who have not had recent respiratory illness can be enrolled without laboratory

This study will address whether this is an accurate premise.

A summary of Influenza A(H7N9) virus findings in birds and humans [UPDATED, AMENDED FIGURE]

An article from Bloomberg news highlights some interesting studies, how they present opposing conclusions and why we can expect to see more H7N9 activity, perhaps peaking at Chinese New Year.

Click on image to enlarge.
H7N9-positive birds and humans (see MOA report) in 
April 2013. 17x more humans were virus-positive 
than humans were PCR/symptom positive. Based on 
Li et al's April 24th New England Journal of Medicine 
article from a similar time period which uses observation 
for signs of disease among 1,251 followed contacts of 81 cases and
sentinel surveillance PCR data from 5,551 humans to
identify H7N9 cases).

The authors (Khan and Loo) remind us that earlier in the year, China's Ministry of Agriculture reported 46 positive poultry samples among 68,060 tested positive using viral culture, for H7N9 (0.07% or about 1:1,500). 

In a more detailed report from MOA from 30th May 2013, 88 of 899,758 [0.009%] duck, pigeon, chicken (722,380 or 80% of all the samples tested), wild bird, pig, geese, "other" animal or environmental samples were virus [197,389 of the samples tested this way] &/or antibody [702,369 of the samples] positive (chicken, duck and pigeons were the positives; 3 were positive for both). The report presented by Zhang Zhongqiu does not make clear how many swabs and bloods were tested per animal so I'll just talk about sample numbers. The report notes that there were no clinical cases reported from 44 million farming households and no positives from 51,876 samples of 746,212 samples (?chickens) sent to Hong Kong; monitored by the General Administration of Quality Supervision, Inspection and Quarantine, China) nor among the 120/samples being tested per day in Hong Kong. In 1,874 samples collected from Henan and Jiangxi provinces, none were positive. Transmission among chickens was possible but was not efficient among ducks.

• Lam and colleagues (previously reviewed) identified 8 avian H7N9 strains from 1,308 (0.6%) chickens (95% of samples), ducks, pigeon and geese samples collected from live bird markets (LBMs) in Rizhao, Shandong province (about 9 times more than the 1st MOA study above, if they can be compared directly). 
• Yang and colleagues (previously reviewed) found H7N9 antibodies in 25 (6%) of 396 humans poultry workers (none prior to 2013) but only 9 of 1,129 (0.8%) members of the general community showed some weak sign of past exposure (or cross-reaction with another influenza). No viral RNA was found in these poultry workers.
• Wang and colleagues, writing in the Journal of Infectious diseases,  recently traced the source of some cases in the Hangzhou region of Zhejiang, to LBMs. 95 samples from chickens (n=47 samples), ducks (n=9), quails (n=2), pigeons (n=3) and poultry handlers and 4 from water were inoculated into eggs and were tested by real-time RT-PCR, within the first 2-weeks of April 2013. H7N9 RNA was found in 41/85 (48%) of samples. 40% of the chicken samples, 89% of the duck samples and a third of the pigeon samples. No human or environmental samples were positive. The authors concluded that migratory birds would continue the spread of H7N9 viruses and that their findings highlight LBMs as the major source of infection an as such control measures are needed.
• Shi and colleagues reached a similar conclusion in April in the Chinese Science Bulletin. "Strong measures" were needed to control the spread of H7N9 in order to prevent more infections. This followed the testing of 970 samples of drinking water, soil, cloacal and tracheal swabs from LBM poultry in Shanghai and Anhui province using egg inoculation. All 20 (10 from chickens) of the H7N9 isolates came from LBMs in Shanghai, confirming high genetic homology across the H7N9 genome from human H7N9 cases.

Today's Bloomberg article quotes researchers' concerns that the cooler weather will drive the re-appearance of H7N9, since influenza usually reaches epidemic levels during cooler months. In other words they believe this particular strain of H7N9 (the one infecting humans) was never removed from the ecosystem.

Re-opening of the LBMs has been ongoing since June in Shanghai municipality and Zhejiang and Jiangsu provinces, albeit in a more regulated fashion. The cleansing of the markets after culling more than 560,000 poultry from LBMs as of May 2013 combined to precede the precipitous decline in what had been an alarming rate of new cases in those regions. Is testing of these markets an ongoing process?

With the markets refilling from farms located in rural regions with exposure to mobile wild bird populations that may (albeit infrequently) carry H7N9 (and many other influenza viruses including its components), the risk of fresh outbreaks among humans is also growing. 

It's a numbers game. 

Even 1 human case, like the one we saw infected this week could signal an even wider level of circulation of H7N9. Let's hope testing will make sure our number's not up this time around.

Editor's Note - the figure was altered 01.02.14 to correct an error in the proportions and to adjust down the number of contacts since not all had been followed.

More MERS molecular masterfulness: Egyptian camels contain lots of anti-MERS-CoV antibodies [AMENDED]

UPDATE #1: 23NOV2015

Perera and colleagues from China, Japan, Egypt and the United States report in Eurosurveillance that they
have found a high prevalence of Egyptian camels which have antibodies to a piece of the Middle East respiratory syndrome coronavirus (MERS-CoV) spike protein.

The group looked at 1,343 human sera (815 from Cairo as part of influenza study; 528 archived from Hong Kong) and 625 animal sera (from goats, sheep, water buffalo, cows, camels, pigs and birds) from Egypt and China (no MERS reported there, so this acts as control population; unfortunately no camels tested from there either). 

A positive control serum was provided by Prof Drosten from an earlier MERS case.

The novel assay does not need to be used in a biosafety level 3 containment environment (BSL2 is fine) because it does not use whole/live MERS-CoV to capture the human/animal antibodies, rather it uses just a piece of the virus; the spike protein, which is known to attract the lion's share of antibody attention. 

The spike protein was merged with HIV (non-infectious) proteins to create a pseudoparticle of proteins that could enter cells, and also bind to MERS-CoV antibody which would act to prevent such entry by the virus-like particle. If there was no antibody, the pseudoparticles could enter cells and this entry could be measured using a marker enzyme reaction. This novel assay (pseudoparticle neutralization test; ppNT) was run in parallel with a more standard micro-neutralization (MNT) test; the two tests agreed well but the standard MNT test needed to be carried out in a BSL3 laboratory).

The more traditional MNT test pre-incubated infectious MERS-CoV with serum and then measured whether the live MERS-CoV could still infect and damage cells. If there was antibody in the serum, it blocked infection (as it presumably can in us during a second or third infection). 

Pseudoparticle/virus will be more or less blocked from cell entry if more or less antibody is present in the serum. One can determine how much antibody was in the serum sample too. These type of tests measure the ability of antibody in a patient/animal to block virus entry; it's neutralizing ability. 

A high proportion (103/110; 93.6%) of dromedary camels from Egypt had antibodies that could block cell culture infection by a lab strain of the MERS-CoV (MERS-CoV/EMC from Erasmus University Medical Center). 

No humans had antibodies. No animal sera from China (Hong Kong) were positive - this included pigs (n=260) and wild birds (n=204). 

Numbers were sometimes very low but no goats (n=13), sheep (n=5), water buffalo (n=8) or cows (n=25) were antibody-positive in Egypt. Only the camels.

The authors specify that this does not exclude cross-reactivity with a closely related, but non-MERS, CoV that has at some previous point infected the camels. If only there was some sort of plan to do this sort of virus hunting in mammals.

Click to enlarge.

So how does this fit in with the human acquisition model proposed 1-week ago? Pretty well. It supports previous findings from Omani camels too. 

Camels could be part of the chain of infection. It says nothing about how they may then go on to be a source of human infections that start off as a respiratory illness, presumably requiring, at least in most cases anyway, inhalation or self-inoculation (I'm looking at you, nose-pickers). For that we'd need to know anything about survival of MERS-CoV in excreta and on surfaces. I'd also like to see some more animal test results. 

Nonetheless, it looks as though we have some very useful antibody detection assays shaping up, and they can do just that.

Here's hoping for a KSA study next.

UPDATE #1: Fixed some typos and grammar.

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? 

Camels carry signs of coronavirus contagion

Reusken and a European collaborative team have this morning described the first study looking for evidence of prior infection with the MERS-CoV, in animals. This evidence take the form of antibodies (immunoglobulin G or IgG ) made after the animal's immune system recognizes and then defends against future infection by that invader. 

The study used a very specific piece of the MERS-CoV Spike (S) protein. S is the bit of a CoV that sticks out and gives it the characteristic crown-like appearance under electron microscopy. The small piece of S acts as bait to detect the antibodies in serum samples and this interaction is identified by a fluorescent signal in the protein microarray system they used. 

These antibodies were found in the sera of 50 of 50 retired racing dromedary camels from Oman and from 1 in 7 Spanish (14 of 105) dromedary camels from the Canary islands. 

No antibodies were found in 80 cattle, 40 sheep, 40 goats or 34 other camelids.

The antibodies retained an ability to stop MERS-CoV infection in test that diluted the sera between 1:320-1:2560 for the Omani camels, and 1:20-1:320 for the Spanish camels 

Camels also had some signs of antibody reactivity to bovine coronavirus (BCoV), but the authors, after additional testing, concluded that the MERS-CoV reactivity was specific to that virus and not to BCoV. Sera from 2 human cases of infection by with the BCoV relative (both betacoronaviruses), HC0V-OC43, did not stop MERS-CoV from infecting cells - infection was not neutralized by the patient's HCoV-OC43 antibodies.

Camels were implicated earlier during the outbreak, in the death from MERS-CoV (then the "novel coronavirus" or nCoV) of a 73-year old male from Abu Dhabi, capital of the United Arab Emirates. 

"The patient owned racing camels. One of them got ill and was very weak; the patient was in close contact with that camel, and on the evening the camel got very sick, the patient developed flu-like symptoms. Three days later, he was in a medical unit in Abu Dhabi. There is another family member who also had close contact with the camel; he also got ill, but we could not follow up with that gentleman."

So this article points a finger at camels as some sort of host, possibly as an intermediate host between Pipistrellus spp and Rousettus aegypticus bats and humans. Perhaps the MERS-CoV story is akin to the Hendra virus story - bats contaminate horses and from there, close contact with horses can, on occasion  result in disease in humans. 

At the very least - there may be other animals involved yet and we still don't have viral RNA or a viral isolate from within a camel  - we now have a specific animal contact to track and trace for each human case. Perhaps specific risk avoidance measures can also be implemented, and the hotzones can communicate to their populations that close, perhaps any, contact with camels carries with it some risk of MERS-CoV infection  especially to be if you are in a category that places you at higher risk of severe outcomes from a MERS-CoV infection-older male, underlying conditions. 

Housing camels away from bats and areas known to be bat flyovers or frequented by feeding or birthing bats, or keeping camels under cover may all be helpful reduce transmission of the virus between these animals.

Jennifer Yang and Helen Branswell have breakdowns of this story as well.