Are MERS cases in Saudi Arabia and the UAE linked to camel imports?

Special Guest writer: @influenza_bio

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Looking at the history of MERS coronavirus infections to date, many puzzling questions come to mind.

Evidence of MERS infection has been detected in dromedary camels from Spain to Egypt to the Arabian Peninsula. Why have we seen human cases arise only in a handful of countries in the Arabian Peninsula?

Why have nearly all MERS cases originated in Saudi Arabia (KSA)?

As of June 22, 2014, 718 cases are thought to have been contracted in KSA. The UAE, a distant second, has had 69 cases. Jordan has had 17; Qatar has had 9 (although 1 had a travel history to KSA), Kuwait 3, Oman 2 and Yemen 1. All of these countries have a lot of camels.

• Has KSA had more cases just because its population is larger? Have other countries had just as many cases, per capita?

No.

Figure 1. MERS cases per capita (2012 data)

• Is it just an accident that cases have appeared primarily in KSA, followed by the UAE?
  
  It could be.

• Is it an artifact of surveillance that most cases have appeared in KSA, followed by the UAE? Are KSA and the UAE just better at detecting MERS cases?
  
  This would seem unlikely. It's certainly possible that all countries with MERS have missed some to a lot of cases, especially milder ones, and that some countries are better at detecting MERS cases than others. However, the fact that all cases detected outside the Arabian Peninsula also follow the same general pattern of country of origin suggests that we probably have the big picture at least close to right.

• Have KSA and the UAE had more cases because they have more camels or more camels per person?
  No. First of all, these countries don't have the biggest domestic camel stocks in the region. Second, when you look at the number of MERS cases as a function of the size of domestic camel stocks (not including imported camels, which are typically slaughtered), there is no relationship.
  
  

  

  Figure 2. MERS cases as a function of the size of domestic camel stocks, not including imported camels (2012 FAO data[1])

  
  Alternatively, when you look at the number of MERS cases per capita as a function of the number of camels per capita (again, considering only domestic camel stocks, not imported camels), there is also no relationship. KSA and the UAE also do not have the greatest numbers of camels per person in the region.

Figure 3. MERS cases per capita (2012 data) as a function of the number of 
camels per capita (domestic camel stocks only; 2012 FAO data[1])

• Are there more camels with MERS in KSA and, to a lesser extent, the UAE?
  
  We have no idea, as there has been almost no camel surveillance. But hopefully new surveillance and studies underway in KSA and the UAE will help us to understand the situation in camels in these countries better.

• Is the large number of cases in KSA due only to nosocomial transmission (i.e., transmission in health care settings)?
  
  Not really, even though a very large proportion of its cases have been nosocomial. Many of the cases that KSA has had are from April and May 2014 and were in Jeddah, and the WHO has published an estimate that 75% of the cases arising in KSA in April-May 2014 may have resulted from nosocomial transmission.[2] Subsequently, the WHO wrote that, "Approximately one-third of these Jeddah cases are considered to be primary cases, although investigations are currently ongoing to determine whether these patients had contact with another confirmed case."[3] In other words, the WHO may end up revising the 75% nosocomial estimate upwards. Looking at graphs of MERS cases over time[4] suggests that cases may dip a bit in the winter and surge a bit in the spring, although MERS hasn't been around long enough in humans to know this for certain. Depending on the final WHO estimate for the rate of nosocomial transmission in April and May, anywhere from the "expected" number of zoonotic cases (i.e., the average monthly rate before April 2014) to almost double this number (if 75% of cases were nosocomial) may have appeared in April and May. However, even if it turns out that zoonotic cases have appeared in KSA at a more or less steady rate over time, this large amount of nosocomial transmission still wouldn't explain why most zoonotic cases are in KSA. Moreover, it would not seem likely that the effectiveness of infection control and prevention is vastly different across the Arabian Peninsula, and, in fact, there is evidence of nosocomial transmission in KSA, the UAE and Jordan, the countries with the most cases. However, it is puzzling that nosocomial transmission has seemed to peak in the spring of both 2013 and 2014 in many different settings; some environmental factor, analogous to winter for the flu, may be at work here.

• Are there environmental factors in KSA more conducive to infection with MERS, especially around April and May (when we have seen a large number of infections this year, and to a far lesser extent, last year)?
  
  As mentioned above, there may be something special about spring. It is also possible that something about KSA and the UAE are particularly conducive to human infection with MERS.

• Is anything else going on?
  
  There are probably many other factors at work that we don't know about. For one thing, we still do not know how MERS is transmitted to humans � whether it is transmitted by camel kisses, milk, urine or meat, among other possibilities, for instance. Once we know the routes of transmission and have some estimates of how much each route contributes to the numbers of infections, then we may have a better chance of teasing out even more differences across countries. Yet other factors may be at work, as well.

Is the number of MERS cases in a country related to the number of camels it imports?

At this point I would like to introduce a very speculative hypothesis: that the number of MERS cases in countries of the Arabian Peninsula is related to the number of camels that are imported into those countries. Significant oil wealth in some of these countries has fueled a greater demand for meat over time, and in particular, camel meat. Over recent years, this demand has been met by slaughtering primarily imported camels. 

• KSA and the UAE are the dominant importers in the region.
  
  KSA imports the most camels in the region, followed by the UAE. No other country in the region comes close.

Figure 4. Camel imports in 2011 (FAO data[5])

• Camel imports to KSA and the UAE have been increasing dramatically over the past few years, and the dominant sources of these imports are ports in the Horn of Africa.
  
  It is difficult to find hard numbers on the numbers of camels imported into KSA or the UAE broken down by country of origin. Also, the most recent FAO import data is from 2011. However, in the absence of access to recent export and import data from a variety of governments, a combination of FAO import data, FAO export data and news reports over the past few years paints a picture of vast and vastly increasing camel exports from northeastern Africa into primarily Saudi Arabia.
  
  First of all, it should be noted that, according to the FAO,[1] Somalia had 7,000,000 camels in 2012 and Ethiopia had 915,518, while KSA had 260,000. Sudan also had 4,571,000 camels.
  
  On November 25, 2013, the Financial Times reported in an article entitled, "Somali meat exports to Saudi Arabia soar,"[6] that, 

"Including goats, cattle and camels, total livestock exports from the civil war afflicted territories rose to 4.8m in what is the world�s largest on-the-hoof movement in the live animal trade. 

�There�s no single time that they ever exported such large numbers before,� says Ernest Njoroge, livestock expert at the EU's Somalia Unit. �If the ports in Berbera, Bosaso and Mogadishu become very, very efficient, then that will even increase.

�A lot of Ethiopian livestock is also coming through Berbera. It�s a very big market for us and it�s time for us to increase the capacity of Berbera port,� says Mr Yonis, who hopes planned investments will deliver a terminal dedicated to livestock for export to the Middle East."

A recent article by National Geographic[7] examines the huge and growing livestock trade from, in particular, Somaliland, and notes that, "While livestock�including cattle and camels�are exported year-round from Somaliland, the seasonal Hajj is the busiest time of year." A photo is shown of animals being herded onto a ship bound for Jeddah, KSA. Another photo shows animals in quarantine at the port of Berbera. During both quarantine and transport, animals are packed so closely together that it is hard not to imagine any diseases in the herds spreading like wildfire.

A news article[8] in March, 2011 discussed an announcement by KSA that the Kingdom planned "to increase livestock imports from the Horn of Africa two-fold by 2012," to close to 2 million heads of livestock, composed of sheep, goats, camels and cattle. Perhaps ironically, the article states that, "The decision to increase imports follows after Saudi Arabia's quarantine officials at the Port of Jeddah declared animals from the Horn were disease free, great in quality and strong demand in the local market." The article also notes that, "In October 2009, Saudi Arabia relaxed a eleven-year ban on Somaliland livestock and Somaliland animals have been steadily on high demand in the Saudi Kingdom ever since�. The main markets for livestock from Somaliland are Saudi Arabia, UAE, Oman and Yemen." FAO import data for 2000-2011, however, shows Yemen not importing any camels and Oman importing between 0 and 8,114 camels (which is very few camels).... Perhaps they don't import many camels.

Another news article,[9] in October, 2012, discusses the livestock export trade from Somaliland, and quotes a local official as saying, "Saudi Arabia sells barrels of oil to the world. We make our money selling livestock to Saudi Arabia." This article also mentions animals being shipped to Jeddah, KSA.

Camels from Somalia that are slaughtered for meat have typically been >5 years old.[11]

In addition to importing camels for meat, the UAE may also import some camels from Central Asia for milk production.[10]

Examining the relevant FAO camel import and export statistics for the Horn of Africa and the Arabian Peninsula, it becomes clear that most, and potentially nearly all, camels have been imported into the Arabian Peninsula from the Horn of Africa, meaning largely from Somalia, to an unknown extent from Ethiopia and to a lesser extent from Djibouti; that these imports have been primarily for slaughter, for meat; and that these imports have been increasing rapidly in recent years.

• The dominant port for animal imports into Saudi Arabia is Jeddah.
  
  "Jeddah Islamic Port is the largest port in the world of handling of livestock."[12]
  
  Jeddah is also the second largest city in KSA, but is it a coincidence that the largest number of MERS cases this year have been in Jeddah, just as camel imports have been surging over the past few years?

• Three camels imported into Egypt from Ethiopia and Sudan were found to be infected with MERS. MERS is in African camel populations.[13]
  
  One study examined 110 nasal swabs from apparently healthy camels, >6 years of age, in abattoirs in Egypt in June-December, 2013. Serum samples from 52 of these camels were also examined. Forty-eight of the serum samples were positive for MERS antibodies. However, when the nasal swabs were tested for the presence of the MERS virus, it was found that "The animals positive for either MERS-CoV or BCoV-like virus were all imported from Sudan or Ethiopia for slaughter." One of these virus samples was sequenced, and it was found that this virus was of a different lineage than the other MERS virus sequences known to date, from the Arabian Peninsula.
  
  Some amount of surveillance for human MERS cases is occurring in Sudan. The WHO has stated that, "health authorities are in active search for any suspected MERS-CoV cases by strengthening surveillance in the state general hospital, private clinics and hospitals."[14] I have seen no reports of surveillance in Ethiopia, Djibouti or Somalia; I hope that at least some exists.

• A fairly close relative of the MERS virus also circulates in bats, and a close bat relative to MERS CoV viruses isolated from humans has been found in South Africa.[15]
  
  A portion (816 nt) of the genome of a virus from a bat in South Africa was sequenced, and the protein version of this viral fragment was found to differ from human MERS by only 1 amino acid (0.3% difference). The protein versions of two other, shorter fragments of the genome differed by 10.9% and 14%. Other, related viruses have been found in bats from around the world; this virus, from South Africa, was found to be closer to human MERS than any other virus known at the time this work was published. The authors of this study wrote that the "relatedness [between human MERS and the South African bat fragment] was as close as that of SARS-CoV and the most closely related bat coronavirus known." They also write that their finding "enables speculations of an African origin for bat reservoir hosts of MERS-CoV ancestors."
  
  Subsequently, a short fragment (182 nt) of a virus was isolated from a bat in KSA, and that fragment had 100% identity with the corresponding fragment of human MERS.[16]
  
  Together, these findings suggest that MERS may have originated in bats in the Arabian Peninsula or Africa. Further sampling in bats in both regions, along with sequencing greater portions of viral genomes, as they become available, would help us to understand the evolution of this virus in bats.
  
  

• The number of MERS cases in each country may be related to the number of camels that are imported.
  The figure below shows that a potentially strong relationship may exist between the number of MERS cases in a country and the number of camels that that country imports. The more camels that are imported into a country, the more people that have been infected with the MERS virus. However, the numbers of data points are small; only KSA and the UAE stand clearly away from zero imports and zero cases. Thus, this graph is what I would call intriguing, but not completely convincing.

Figure 5. MERS cases as a function of number of camels imported (FAO 2011 data[5])

• The number of MERS cases per capita in each country may be related to the number of camels that are imported per capita.
  
  If we "normalize" the data examined above to the population size of each country, a relationship between the number of camels imported and the number of MERS cases still holds.

Figure 6. MERS cases per capita (2012 data) as a function of the number of camels imported (2011 FAO data[5]) per person

• The number of MERS cases in each country is not strongly related to the total number of camels (domestic stocks plus imported camels) in each country.
  If we compare the number of MERS cases in each country to the total number of camels that set hoof in that country in a given year (approximately), there is a possible suggestion of a relationship. Yemen is problematic, though, because it has more camels than any other country in the region but has had only one recorded human MERS case. Thus, there aren't more MERS infections in KSA and the UAE because they have the most camels in the region; they don't have the most camels. 
  
  

  

  Figure 7. MERS cases as a function of the total number of camels (domestic stocks [2012 FAO data] plus imported camels [2011 FAO data[5]])

• The number of MERS cases per capita in each country is not related to the total number of camels (domestic stocks plus imported camels) per capita in each country.
  
  The UAE does have the most camels per person in the region, but KSA is very far from having the most camels per person. Thus, there is no apparent relationship between the total number of camels setting hoof in a country per capita and the number of MERS cases per capita in that country.

Figure 8. MERS cases per capita (2012 data) as a function of the total number of 
camels (domestic stocks [2012 FAO data[1]] plus imported camels [2011 
FAO data[5]]) per capita

Taken together, this data supports the hypothesis that the number of human MERS cases in a country may be driven by the number of camels imported into that country, and perhaps that what matters is how many camels are imported from the Horn of Africa. Correlation is not causality, of course; this is only a hypothesis, not a fact.

In May, KSA stopped importing camels from Somalia, Ethiopia and Sudan.[17] So, I'm clearly not the first person to have had this idea. It's not clear whether camels currently being imported into KSA are being tested for MERS, though; one recent Arab News article[18] discusses how "all livestock" in KSA are now going to be tested for MERS, but it was not entirely clear to me whether imported camels would be tested. Subsequent echoes[19] of this news article, however, stated that, "Imported camels would also be tested for MERS and quarantined, Arab News reported him [the Agriculture Minister] as saying;" in reality, the Arab News article made no such direct statement. It would be nice to get total clarity on this issue.

The UAE, on the other hand, has not suspended any camel imports.[20] However, the UAE is now testing camels being imported from Saudi Arabia, Qatar, Oman, Kuwait and Bahrain.[21]

It should also be noted that MERS appears to circulate in domestic camel stocks in at least some countries in the Arabian Peninsula at at least some times; I am not by any means suggesting that MERS is likely to be only in imported camels.

For further information on animal husbandry practices for camels in northeastern Africa, see here.[22] For further information on the Livestock Trade in the Djibouti, Somali and Ethiopian Borderlands, see here.[23]

How can we know whether camel imports are actually a driving force behind human MERS infections?

This part is a little bit complicated. As a first step, it would be important to determine whether MERS is circulating in areas exporting camels to the Arabian Peninsula, and if so, whether the MERS virus in these regions is genetically the "same" as (i.e., extremely similar to) MERS in the Arabian Peninsula.

Ideally, we would need to try to find camels with active MERS infections on the verge of being exported to the UAE, and we would need to sequence their MERS genomes. Such sampling would probably best be done in the ports of Mogadishu, Bosaso and Berbera in February, March and April. It would also be great to see sampling done in the UAE in, say, February through May. If the sequences from the camels on the verge of export were to match the sequences seen in camels or people in the UAE, then this would suggest that MERS is circulating between the Horn of Africa and the Arabian Peninsula. It might be possible from such sequence data to determine which direction the virus is traveling, i.e., whether the virus is traveling from Africa to the Arabian Peninsula or from the Arabian Peninsula to Africa. The fact that the animals are traveling primarily, if not exclusively, in one direction, though, would suggest that the virus would be moving primarily from Africa to the Arabian Peninsula.

On the other hand, if MERS sequences from export ports in the Horn of Africa would turn out to be of lineages different from those of MERS sequences seen in the UAE at the same time, then we could safely conclude that MERS is not being imported from Africa.

Is there any other way that imported camels could be driving MERS infections in the Arabian Peninsula besides by bringing more MERS virus with them? Possibly. For instance, it's possible that imported camels provide a large, susceptible pool for new MERS virus infections in their destination country. Travel may make such camels more susceptible than usual, as well. Depending on how long these camels are alive in their destination countries and on how MERS is transmitted to humans, a role for new infections of imported camels in their destination countries could turn out to be more or less relevant. For instance, if it is found that most human MERS infections develop after contact with camel meat, then new MERS infections in imported camels would be important. If no such link of this sort could be found to imported camels, then this apparent correlation would be likely to be spurious, i.e., correlated with MERS infections but for no real, underlying reason.

Suppose that camel imports do drive human MERS infections. What could this tell us? What questions would this raise?

Suppose, for the moment, that some causal relationship does exist between the number of MERS cases in a country and the number of camels imported into that country. This would raise many questions, including the following:

If infected camels are being imported�

• Are there human MERS cases in the Horn of Africa?
• How big of a problem are human MERS cases now?
• How many years back can we trace human infections?
• Are imported camels infecting people in the Arabian Peninsula?
• How?
• Do people becoming infected through eating camel meat?
• Is meat a more important source of human infections than other forms of camel contact (direct, milk, urine, �)?
• How can we prevent imported camels from infecting people?
• Are imported camels infecting camels in the Arabian Peninsula, who then infect humans in the Arabian Peninsula?
• How are imported camels infecting domestic camel stocks?
• How are domestic camels infecting people?
• How can we prevent imported camels from infecting domestic camels?
• How can we prevent infected camels from being imported?
• Does the import process increase the incidence of MERS among imported camels?
• Does contact with other camels at the sites of export and/or import increase the incidence?
• Does quarantine at the sites of export and/or import increase the incidence?
• Does shipping increase the incidence?
• What are the dynamics of MERS transmission in camels in the Horn of Africa and the Arabian Peninsula?
• Have we been looking for MERS in the light under the lamppost?

If infected camels are not being imported�

• Are imported camels becoming infected in their destination countries?
• Are infected imported camels infecting people?
• Are infected imported camels infecting domestic camels?

And finally, what will happen over the coming year, if KSA continues to ban camel imports from Somalia, Ethiopia and Sudan, while the UAE does not? Will KSA find other sources of camels? New human cases continue to arise in KSA; MERS is clearly still in KSA. But without camels imported from the Horn of Africa, will human cases die down, and will we see no surge next spring? Time will tell.

In the meantime�

We don't know whether imported camels affect MERS transmission in the Arabian Peninsula. We don't know whether any camels exported to the Arabian Peninsula are infected with MERS. We don't know for absolute fact whether camels give MERS to people.

In short, many more studies are needed to discover where people and camels are becoming infected with MERS, to learn how MERS is transmitted to people and to understand local and global MERS transmission dynamics in camels.

It would be good to do some surveillance in Somali ports, though.

References

1. http://faostat3.fao.org/faostat-gateway/go/to/download/Q/QA/E
2. http://www.who.int/csr/disease/coronavirus_infections/MERS_CoV_RA_20140424.pdf?ua=1
3. http://www.who.int/csr/disease/coronavirus_infections/MERS_CoV_Update_09_May_2014.pdf?ua=1
4. http://newsmedicalnet.blogspot.com.au/2014/06/snapdate-mers-by-month.html 
5. http://faostat3.fao.org/faostat-gateway/go/to/download/T/TA/E
6. http://www.ft.com/cms/s/0/a5c38622-37db-11e3-8668-00144feab7de.html#ixzz35TEHV5sD
7. http://proof.nationalgeographic.com/2014/06/16/robin-hammond-the-largest-trade-on-the-hoof/
8. http://somalilandpress.com/horn-of-africa-to-double-livestock-exports-to-saudi-arabia-21259
9. http://somalilandpress.com/somalilandbustling-livestock-trade-boosts-somalias-economy-36695
10. http://www.uaeinteract.com/docs/UAE_set_to_import_camels/45348.htm
11. http://www.researchgate.net/publication/256297268_Improving_mature_camel-meat_quality_characteristics_with_calcium_chloride_injection
12. http://www.jeg.org.sa/data/modules/contents/uploads/infopdf/1090.pdf
13. http://wwwnc.cdc.gov/eid/article/20/6/14-0299_article
14. http://www.emro.who.int/sdn/sudan-news/sudan-dengue-outbreak2014.html
15. http://wwwnc.cdc.gov/eid/article/19/10/13-0946_article
16. http://wwwnc.cdc.gov/eid/article/19/11/pdfs/13-1172.pdf
17. http://www.albayan.ae/one-world/arabs/2014-05-06-1.2116311
18. http://www.arabnews.com/node/582041
19. http://www.reuters.com/article/2014/06/05/us-health-mers-saudi-camels-idUSKBN0EG1FT20140605
20. http://www.khaleejtimes.com/kt-article-display-1.asp?xfile=data/government/2014/May/government_May17.xml&section=government
21. http://www.uaeinteract.com/docs/Camel_shipments_from_GCC_to_undergo_strict_screening_for_MERS-CoV_MoEW/61573.htm
22. http://www.mbali.info/doc199.htm
23. http://www.fao.org/fileadmin/user_upload/drought/docs/chatham%20house%20majid%20djibouti%20livestock.pdf

NOTE: I (Ian M. Mackay, VDU Editor) did not have a hand in writing this post and thus take no credit for it. This was entirely the work of the Guest Writer. 

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Watching zoonoses evolve...

Special guest writer: @influenza_bio

For the first time in human history, we are watching diseases jump from animals to humans on a large scale. We've seen diseases appear for the first time in humans before; that's not new. We've seen HIV and several new strains of influenza emerge over the past century or so, for example. What is new is that we can now watch this process as it happens. We are able to watch animal diseases trickle case by case into humans, and we wonder whether any of these diseases might some day become human diseases. We wonder whether we might be watching pandemics develop in real time.

A disease that jumps from a non-human animal to a human (or the other way around) is called a "zoonotic" disease or a "zoonosis." Individual cases are called "zoonotic" cases. When a zoonotic disease is trying to make the jump to us permanent, we call this disease an "emerging infectious disease."

We have certainly been watching a lot of zoonotic MERS coronavirus and bird flu (e.g., H7N9 and H5N1) cases develop in people lately, along with Ebola virus cases. Zoonotic cases of other diseases, including infections with various strains of bird and swine flu, occasionally develop, as well, and are watched closely.

When the 2009 H1N1 flu pandemic started, we had no clue much beforehand that it was on its way.  We didn't even have surveillance data about swine flu strains that were even particularly close to the strain that emerged in us. A large animal flu surveillance gap blindsided us that year.

And we will undoubtedly be blindsided again by other emerging infectious diseases that we won't even see coming, although people are doing their best to see what's out there.

When an emerging infectious disease jumps to humans, it can cause either a relatively local outbreak or a worldwide outbreak, called a "pandemic." If a disease becomes a pandemic, that just means that it's spreading worldwide; the word "pandemic" doesn't imply anything about how bad the disease might or might not be. In some sense, the worst case can be when a disease jumps to humans and becomes "endemic" in humans, meaning that it gets established in people and regularly infects people, year after year. Endemic diseases can circulate worldwide (e.g., influenza) or in more restricted geographical regions (e.g., malaria).

Our knowledge and resources have grown to the extent that we are currently able to monitor some significant zoonotic outbreaks of disease. We are currently watching the MERS coronavirus and the influenza A(H7N9) virus both try to become human viruses.

Will either one succeed? We can't say. We've never watched this process happen before. We don't know how long such a process "usually" takes, or whether there even is a "usual" amount of time that it takes. We don't know how long it might take, or how quickly it has happened before. We doknow that the process is "stochastic," meaning that it involves a lot of chance. A pathogen that in one situation might cause a pandemic might just die out in another situation. Everything depends on the specific changes in a pathogen that get a chance to develop and on whether those changes end up getting passed on. We don't know how often pathogens "fail" when they "try" to make the jump to humans.

A lot of us have watched the recent surge in MERS coronavirus cases with some amount of concern. As of April 19, 2014, there are two large clusters of cases in the Middle East, and at least one of them is still growing. One cluster, in Jeddah, Saudi Arabia, now has 60 cases; 7 cases were added to this cluster today, and 6 were added yesterday. There are perhaps over a dozen cases in another cluster in the UAE. One patient who became ill with MERS in Jeddah at the end of March flew to his home country of Malaysia while ill and subsequently died in Malaysia; 79 of his contacts are now being watched closely in Malaysia. Test results are starting to come in for a number of these contacts, and thankfully all are negative for MERS so far. An asymptomatically infected Filipino health care worker traveled on an airplane back to the Philippines a few days ago. Yesterday, a MERS case was announced in Greece; a Greek man who had been living in Saudi Arabia was recently in Jeddah and presumably became infected there before flying back to Greece. He arrived in Greece with a fever; his contacts are now being monitored. In other words, MERS case numbers are growing quickly right now, at least in part through human-to-human transmission, and infected � and potentially infectious � patients are getting on airplanes to travel around the globe.

Does what we're seeing now represent changes in the virus that are making it more transmissible among humans? Or are we seeing a random fluctuation in the numbers of cases? Or, are we seeing more cases simply as a result of improved surveillance? I would argue that what we're seeing likely reflects one or more changes in the virus, simply because

1. We've been seeing so many more symptomatic cases recently, 
2. We've been seeing significantly larger clusters than we've ever observed before,
3. A greater number of health care workers appear to be getting infected than ever before, and
4. A greater proportion of cases are in health care workers than ever before. 

It's not that we've been seeing a rise only in the number of asymptomatic cases detected, which could suggest that we're only seeing the effects of improved surveillance. Moreover, while surveillance does seem to be picking up more mild and asymptomatic cases, it is difficult to know whether we are seeing more of these cases because of improved surveillance or because there simply are more such cases now. A lot of variables are being changed at the same time, and we don't have perfect information.

Nonetheless, the sheer numbers of recent cases suggest to me, at least, that the virus is changing and becoming more transmissible among humans. Until recently, we rarely saw evidence for human-to-human transmission of MERS; most cases may have been zoonotic. Now, however, large clusters involving roughly 1 to 4 dozen people are being seen, with single infected individuals infecting possibly up to a dozen or more other people. This is new. I don't think that we're seeing these clusters just as a result of improved surveillance, although I would be very happy to be wrong.

What does the future hold for MERS? We can't know. We might be watching MERS become a pandemic, and we might not. We might be watching the current relatively small MERS outbreak develop into a larger outbreak that eventually gets contained, as was seen with SARS. Or, the whole outbreak might all just simmer down or go away. Even if the virus were currently changing to become more transmissible, the current spate of cases could still simmer down or go away, just stochastically, just through sheer chance.

Prudence would dictate that we remain concerned and vigilant, however, especially as symptomatic MERS cases have had an approximately 40% case fatality rate (CFR). If MERS did cause one or more wider outbreaks in humans, that CFR might or might not change. Even if the CFR dropped to 10% of what it is now, it would still be on the same scale as the CFR for the 1918-19 influenza pandemic.

As a global society, we have an obligation to do everything in our power to prevent the MERS coronavirus from causing larger disease outbreaks in humans. We need more surveillance in affected countries, including much more genetic sequence data. And in countries of the Arabian Peninsula that are currently detecting MERS cases, infection control procedures need to be improved to the point where nosocomial cases in health care workers and patients are prevented. Health care workers in other countries should be educated about the possibility of MERS patients arriving from afar and about how to treat such patients safely. If this virus becomes more transmissible, we should not be caught unprepared. We can see this one coming.

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Can we believe every H7N9 seroprevalence study we see?

Special Guest writer: @influenza_bio

A little over a year ago, the first known human patient got sick with avian influenza virus(H7N9). The number of H7N9 cases rose and fell in the spring of 2013, and a total of 134 people were known to have contracted H7N9 before June, 2013. Since then, sporadic cases appeared in the summer and fall, and by the end of December, 2013, new cases started to pick up again. We have now seen a second wave rise and fall, although several new cases still being reported each week. As of the time of this writing, just over 400 people are known to have been infected with H7N9. The case fatality rate (CFR) � roughly speaking, the percentage of people infected with H7N9 who die from it � for these known cases is almost 40%.

One question that is on a lot of people's minds is, how many other H7N9 cases are out there that we don't know about? How many mild cases are there that never get tested? How many asymptomatic cases are there that are missed? If there were a lot of undetected cases out there, that would mean that H7N9 is a lot less fatal than the known cases would make us think. On the other hand, if we were somehow miraculously seeing every single actual case, then the CFR would be as bad as all of these cases make it out to be. (And imagine what the CFR would be like without hospitals, ventilators and oseltamivir!)

How do we find out if there are cases that we're missing? One way is to do what is called a seroprevalence study. This means collecting blood samples from as wide a swath of a population as possible and testing to see how many of these samples have antibodies to H7N9. Antibodies are molecules that are made by cells of the immune system and that stick to specific pathogens to help our bodies to rid themselves of these pathogens. If someone gets sick with H7N9 influenza, his or her body would most likely continue to produce a significant amount of antibodies specifically against that strain for at least a good number of months after infection and possibly much longer. In general, people who are infected with influenza but who do not develop symptoms will also produce such antibodies, but their bodies will make fewer of them, and, on average, they won't make as many of them for as long. We don't know exactly what the pattern of antibody production is for people who are infected with H7N9 but don't develop symptoms, though, because researchers haven't identified enough of these individuals to study.

It is very important that we get these seroprevalence studies right. If they're done wrong and we miss a lot of cases, then we will simultaneously underestimate how common H7N9 cases are and overestimate how deadly the strain is. On the other hand, if seroprevalence studies are done wrong and we think a lot of people were infected with H7N9 when they weren't, then we will overestimate how common H7N9 cases are but underestimate how deadly the strain is. Facts can help us to respond to H7N9, and if we get the facts wrong, then we can't respond properly. For example, if we come to think mild H7N9 cases are far more numerous than the severe ones that actually get diagnosed, then we might not worry as much about H7N9 as we should.

What I'd like to talk about here are some of the important ways that seroprevalence studies can go wrong. To answer my title question, no, we cannot always believe the conclusions of every seroprevalence study we see. Scientists make mistakes, just like everyone else, and sometimes things just go wrong, too. I'd like for you to understand just how some of these mistakes can arise, so that you can better judge for yourself whether a study is likely to be reliable or not, or so that you can at least know that there are things out there that can go wrong.

How are seroprevalence studies done?

There are 2 types of laboratory assays (tests) that are usually used in seroprevalence studies (although there are others): hemagglutination inhibition (HI) assays¹and microneutralization (MN) assays.² (For more information about the HI assay in general, see a nice description by Dr. Racaniello.³) MN assays are considered better (more sensitive and specific) than HI assays, but they are harder to do. MN assays require a significant amount of extra work at the end that HI assays don't. But, more importantly for H7N9 studies, HI assays can be done with either "killed," modified or "live" virus, whereas MN assays require "live" H7N9 virus. In other words, HI assays can be done in almost any lab, but MN assays require a BSL-3 lab. A neutralization assay⁴ has been developed that uses a "pseudovirus" instead of live H7N9 and is therefore far less hazardous to work with, but formal WHO diagnostic criteria still require standard HI and/or MN assays.

First, blood samples are collected. Each blood sample is drawn into a tube, and after 15-30 minutes, the tube is centrifuged to separate clotted red blood cells from the rest of the blood. The red blood cells are discarded; what's left is called serum, and that's what's studied. The serum samples should then be put in a refrigerator if they'll be studied within a few days; if they'll be studied later, they should be frozen. Once a researcher is ready to study the serum samples, the serum samples are thawed. Virus is also used for the assay, so one or more tubes of virus are thawed, too. Different types of mammalian or bird cells are prepared: typically horse, turkey or chicken red blood cells for HI assays, or a special type of dog kidney cells ("MDCK" cells) for MN assays. Various solutions are prepared. Serum samples, virus preparations and cells are diluted as needed, and everything is transferred into little wells in a plastic "plate" in just the right way. In the HI assay, the plate then sits at room temperature for 1 hour, after which it is "read" by eye. In the MN assay, the plate then sits at 37�C (body temperature) for 19-21 hours, after which it is read by a machine (an "ELISA reader"). The assay is done. The results of the assay are then written down and analyzed, and voil�, a paper appears in the scientific literature.

What could possibly go wrong with these blood tests?

Let's start with some things that can go wrong with the lab work:

1. If blood samples are left sitting around for a long time without being centrifuged, the red blood cells will start to break apart, and enzymes released from the red blood cells will start to destroy antibodies (and everything else) in the blood samples. This happens even faster if blood samples are not refrigerated.
2. If serum samples are left in the fridge too long, things can start to deteriorate, just like food in your fridge would. The antibodies that you would like to measure start to be broken down. (Sometimes, for many different kinds of studies, people study serum samples left over after patients' blood tests at hospitals. Those samples sometimes sit around in a fridge for quite a while. Some of them can even be green from stuff growing in them while they're sitting around. Yuck.)
3. If plasma (what's left in blood after unclotted red blood cells are removed) is used instead of serum (what's left in blood after clotted red blood cells are removed), then the assay can read artificially high. Serum should always be used, not plasma.
4. Every time serum is frozen and thawed, some of the antibodies are effectively destroyed. This should not be done over and over. Serum samples should be put into the right size tubes that the researcher will want to use, so that the samples are put through only 1-2 "freeze-thaw cycles" before they are tested. And all serum samples should go through the same number of freeze-thaw cycles.
5. The same thing is true for virus samples used in MN assays. A single freeze-thaw cycle can reduce virus infectivity by a factor of 10. Virus samples also need to be kept on ice when they're being worked with.
6. The plate can be read wrong. It's hard to imagine reading an HI assay plate wrong, but a special procedure (ELISA) and special equipment (ELISA plate reader) are used in the MN assay, and ELISA assays can go wrong.

But, hopefully all of that was done right. Not all researchers, students and technicians are created equal, but hopefully the lab "PI" (Principal Investigator; the person running the lab) is competent and ensures that everyone is doing things correctly.

What could go wrong with the data analysis?

What else could go wrong? The data analysis might not be done correctly. And it's here where perfectly good data can be ruined and where you have to look at seroprevalence studies most closely.

Suppose you've measured your antibody amounts ("titers") in your serum samples. How do you decide which titers mean the sample came from someone who was infected with H7N9, and which titers mean they didn't? Do you just pick a number out of thin air? If you don't have data to tell you which titers mean what, then all you are doing is measuring antibody levels in a population, and you can make no interpretation about what those levels mean. You can't say that they mean any people have or have not been infected with H7N9 at all.

Instead, you need actual measurements using serum samples from people who are known to have been infected with H7N9 to tell you what your titers mean. Someone has to study a number of patients to see what their actual H7N9 antibody titers are, and then a mathematical analysis of that data is done to come up with a threshold titer value, above which serum samples can be said to have come from people infected with H7N9 with some large degree of certainty, and below which they are thought to have come from people who were not infected. We've seen almost no asymptomatic cases (cases with no symptoms), so we really can't say much about them. So we have to go with data from H7N9 patients who have had symptoms. Here's a great graph showing antibody titers, as measured using the HI assay, in serum samples from H7N9 patients:⁵

Figure 1. H7N9 HI
Euro Surveill. 2013 Dec 12;18(50):20657

As you can see in the graph above (Figure 1), by around 3 weeks after infection onset, all samples from patients whose HI titer was measured had titers =40.

The graph below (Figure 2), from a different study,⁴ shows that the HI titer for all H7N9 samples studied by this set of authors was also =40. In addition, this graph shows titers from "control" samples (i.e., samples from people who did not have H7N9 infections); all control samples had titers that were <40.

Figure 2: H7N9 IC50 HI4
Emerg Infect Dis. 2013 Oct;19(10):1685-7

Finally, below (Figure 3) is another nice graph, from a third study,⁶ showing anti-H7N9 antibody levels ("IgG"), "HI" assay results and MN assay ("NAb") results for several H7N9 patients, again showing that all samples from the H7N9 patients studied had HI titers =40. This graph also shows that all H7N9 patient serum samples had an MN titer of =20, if samples were taken after enough time had elapsed since their infections had started.

Figure 3. H7N9 IgG HI NAb.
Emerg Infect Dis. 2014 Feb;20(2):192-200

In other words, if an individual's anti-H7N9 antibody titer is =40 by the HI assay or =20 by the MN assay, these data suggest that we could pretty safely say that he or she has had a symptomatic H7N9 infection within the past few months, and if the HI or MN titers are below those cutoffs, then the individual probably hasn't had a symptomatic H7N9 infection. We don't know to what extent asymptomatic H7N9 infections will be captured by these cutoffs, but it is likely that some asymptomatic cases would be missed using these cutoffs. It is also possible that some mild infections could be missed using these cutoffs. However, it would be a great step forward just to get estimates of what percentages of any regional population or occupational group of people have had any kind of H7N9 infection. A comparison of antibody titers for asymptomatically infected and symptomatically infected H5N1 cases may be instructive when thinking about H7N9.⁷

WHO guidelines are even stricter than the cutoffs discussed in the paragraph above. WHO guidelines say that, using the HI assay, only single samples with titers of =160 can be considered "seropositive": "Paired sera (acute and convalescent sera) with a 4-fold rise in HI titer or single sera collected in convalescent phase with HI titer of =160 could be considered as H7N9 HI antibody positive. Sera with HI titer of 20-80 should be confirmed by MN or WB assay."¹ For the MN assay, however, the WHO does not give specific cutoffs: "With single-serum samples, care must be taken in interpreting low titers such as 20 and 40. Generally, knowledge of the antibody titers in an age-matched control population is needed to determine the minimum titer that is indicative of a specific antibody response to the virus used in the assay."²

Now, it should be noted that WHO assay instructions recommend the use of horse red blood cells for the HI assay, and not everyone uses horse red blood cells. Some people use chicken, turkey, guinea pig or other kinds of red blood cells. That starts making comparisons between different groups' assays difficult. Horse red blood cells are better to use than turkey red blood cells for H7N9 because they have more a2,3-linked ("bird") sialic acids (influenza receptors); HI results are more sensitive with horse red blood cells. In other words, it may take less antibody in the assays to get the same result using horse red blood cells than it would using turkey red blood cells. This would translate into a higher number, when discussing H7N9 patient titers, for HI assays using horse red blood cells, compared to assays using turkey red blood cells. I have not seen direct comparisons of titers obtained using different types of red blood cells in HI assays specifically for H7N9, but the situation is probably similar to that for H5N1.⁸

Figures 1 and 3 above were made with HI data obtained using horse red blood cells. Figure 2 used guinea pig red blood cells. Are they completely comparable? No. Are they pretty comparable? Yes.

Are you getting a feeling for how complicated it is to interpret a seroprevalence paper? And for how difficult it is to compare results across studies?

Why does all of this matter?

It matters because some seroprevalence studies don't use appropriate cutoffs. And because it can be hard to determine even what an appropriate cutoff is when red blood cells from different species are used in an HI assay. This is where the reader has to be really careful. Cutoffs for seropositivity have been a big issue⁹ with H5N1 seroprevalence studies; some researchers have used cutoffs that were too low, and hence they have almost certainly overestimated how common H5N1-specific antibodies were in the populations studied.

So far, only one H7N9 serology paper published to date has reported probable seropositive samples, and this paper simply reported HI titers without using any specific threshold for seropositivity. Only one used study horse red blood cells in HI assays. The one paper that used an MN assay did use appropriate cutoffs. It should be noted that the new WHO HI guidelines were only published in December, 2013, after a couple of these papers were already published.

Here are the studies that have been published so far (I hope I haven't left any out):

1. Bai et al.¹⁰ looked at serum samples collected before November, 2012 from poultry workers in eastern China and found no H7N9-positive samples. The study used HI and MN assays. Turkey red blood cells were used in the HI assay. Appropriate cutoffs were used for the MN assay.
2. Hsieh et al.¹¹ studied 14 close contacts of the first H7N9 case in Taiwan. The authors took blood samples within 18-28 days after the contacts' earliest exposures. The authors used an HI assay but not an MN assay. They used turkey red blood cells for the HI assay. They found all contacts to have an HI titer �10, and declared all to be seronegative. The HI titer for the H7N9 patient in their study was 1:80. These conclusions seem very sound.
3. Yang et al.¹² looked at serum samples from 1129 people from regions of China in which H7N9 cases had been seen, and from 396 poultry workers from 10 districts in which H7N9 cases had been seen. None of the samples from the general population was found to be seropositive, whereas >6% of the poultry workers were found to be seropositive. The authors also examined serum samples from several H7N9 patients. The study used an HI assay but not an MN assay. The authors used a cutoff of =80, along with turkey red blood cells, for the HI assay. Because the authors examined serum samples from H7N9 patients using their methods and got results that are reasonably similar to other results, their cutoffs are most likely reasonable, and their conclusions are probably quite sound. The authors report:
• "Of the 1129 serum samples collected from individuals (age range, 1�88 years) in the general population, 9 (0.8%) had an HI titer of=40 to in?uenza A(H7N9), but no serum samples with an HI titer of=80 were found (Table 1). In contrast, among poultry workers, 13.9% (55/396) and 6.3% (25/396) had in?uenza A(H7N9) antibody titers of =40 and =80 (20 had an HI titer of 80, and 5 had an HI titer of 160), respectively."
  
  It is hard to imagine that an HI titer of 160 can be a spurious finding ("non-specific," to the initiated). Thus, these data strongly suggest that at least some H7N9 cases have been going undetected among poultry workers. Suppose we consider only the poultry workers with HI titer =80, or 6.3% of the poultry workers. If we then consider how many poultry workers there are, total, in districts from which H7N9 cases have emerged, then this study suggests that it's possible that quite a large number of poultry workers have been exposed to H7N9. Still, this study examined only a very small number of people, and we should be cautious about reading too much into these results.
4. Qiu et al.¹³ looked at 3 H7N9 patients and 3 close household contacts of the patients who were exposed before infection control practices were put in place. The authors looked for viral RNA using a sensitive test (PCR) and examined serum samples drawn 15-26 days post-exposure using both an HI assay and a pseudovirus-based neutralization assay. They found no contacts to be seropositive. The H7N9 patients had HI titers that reached 160-640 during this time, and the patient contacts all had titers <10. The authors used horse red blood cells for the HI assay. These findings also seem sound.

To summarize, the conclusions from all of these papers do seem sound. But, it would be wise to keep all of these issues in mind as subsequent studies appear over time.

An additional study¹⁴looked at antibody titers in 1723 serum samples collected in Vietnam using a very different kind of assay (a protein microarray). Because seropositivity cutoff levels had not been determined with authors' assay methods using actual H7N9 patient samples, these authors were appropriately very careful not to attempt to draw any conclusions about H7N9 seroprevalence from their data:

"Because titers calculated from our assay are not directly comparable to HI or microneutralization tests, no cutoff is chosen to represent positivity or clinical protection. It is not possible to associate these titers with past exposure or past infection, as serological assays have not yet been validated for H7N9."

For the future

So, as new H7N9 serology studies gradually come out, you be the judge. Figure out whether they're believable or not. Ask yourself the following:

1. What assay(s) were used? Did the authors use an MN assay? They get bonus points if they did. 
• If only an HI assay was used, then the conclusions are slightly less certain than if an MN assay was used.
2. If the authors used an HI assay, what species were the red blood cells from?
• If horse red blood cells weren't used, then HI titer cutoffs lower than 160 are probably appropriate, but there is also more uncertainty about what an appropriate cutoff would be.
3. What cutoff(s) did they use for seropositivity in their assay(s)? Do these cutoffs mesh with WHO guidelines? Do they mesh with what we know about H7N9 patient HI and MN antibody titers?

References

1. http://www.who.int/influenza/gisrs_laboratory/cnic_serological_diagnosis_hai_a_h7n9_20131220.pdf
2. http://www.who.int/influenza/gisrs_laboratory/cnic_serological_diagnosis_microneutralization_a_h7n9.pdf
3. http://www.virology.ws/2009/05/27/influenza-hemagglutination-inhibition-assay/
4. Qiu C, Huang Y, Zhang A, Tian D, Wan Y, Zhang X, Zhang W, Zhang Z, Yuan Z, Hu Y, Zhang X, Xu J. Safe pseudovirus-based assay for neutralization antibodies against influenza A(H7N9) virus. Emerg Infect Dis. 2013 Oct;19(10):1685-7
5. Zhang A, Huang Y, Tian D, Lau EH, Wan Y, Liu X, Dong Y, Song Z, Zhang X, Zhang J, Bao M, Zhou M, Yuan S, Sun J, Zhu Z, Hu Y, Chen L, Leung CY, Wu JT, Zhang Z, Zhang X, Peiris JS, Xu J. Kinetics of serological responses in influenza A(H7N9)-infected patients correlate with clinical outcome in China, 2013. Euro Surveill. 2013 Dec 12;18(50):20657 
6. Guo L, Zhang X, Ren L, Yu X, Chen L, Zhou H, Gao X, Teng Z, Li J, Hu J, Wu C, Xiao X, Zhu Y, Wang Q, Pang X, Jin Q, Wu F, Wang J. Human antibody responses to avian influenza A(H7N9) virus, 2013. Emerg Infect Dis. 2014 Feb;20(2):192-200
7. Buchy P et al., PLoS One. 2010 May 27;5(5):e10864
8. See, e.g., Table 4 in Pawar SD et al., Virol J. 2012 Oct 30;9:251
9. Osterholm MT and Kelley NS, MBio. 2012 Feb 24;3(2):e00045-12
10. Bai T et al., N Engl J Med. 2013 Jun 13;368(24):2339-40
11. Hsieh SM et al., J Infect. 2013 Nov;67(5):494-5
12. Yang S et al., J Infect Dis. 2014 Jan 15;209(2):265-9
13. Qiu C et al., J Clin Virol. 2014 Feb;59(2):129-31
14. Boni MF et al., J Infect Dis. 2013 Aug 15;208(4):554-8

NOTE: I did not have a hand in writing this post and thus take no credit for it. This was entirely the work of the Guest Writer. 

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