Thursday, 11 February 2016

Not testing for the virus you are trying to associate with a disease is really dumb...

I quickly skimmed over two new papers last Friday (AEST) and was appalled.[1,2] 

In their current format, these papers really just should not have been published at all. 

Any respectable peer review would have sent them back for additional results, to be completely re-written as a letter or more simply - and what I would have recommended to the Editor - that they be rejected outright. 

I was asked if this was just a lost-in-translation error, but complete absence of laboratory data is not that. And that's what these lack - any testing for the virus they name - Zika virus. 

How can you publish something even hinting that a disease is caused by a virus infection, when you present no data to show that person was ever infected by that virus? How can you seriously have that conversation at all?

And using "presumable"and "clinically diagnosed" doesn't get you out of jail either. 

You need some evidence that the virus is or was in the tissue(s) in question (viral genetic material or infectious virus isolated in culture), or was present at some point in the past (antibodies or cells tat respond to the virus when challenged by it in the laboratory) in the person. Neither of the papers here do that. Which astounds me.

Sadly today, there seems to be a journal willing to take any manuscript so that even had the authors been rejected, they most likely would have had their paper appeared elsewhere. 

Or perhaps they had been rejected - and this is that "elsewhere".

I think this sort of publishing sets a hugely dangerous precedent. If the "scientific and medical" literature cannot be relied upon to present its results accurately and with necessary context, how do we expect the community to be able to accurately judge risk and respond reasonably, or our public health experts to find the extra time to sort the signal from the background noise, or the media to get the story straight? 

The Zika virus epidemic of 2015/16 has made much more background noise than signal to date. I hope that changes soon because its hugely disappointing to see so many responding to noise as if it were fact, and fact as if it was hiding conspiracy.

  1. Ophthalmological findings in infants with microcephaly and presumable intra-uterus Zika virus infection.
    Arquivos Brasileiros de Oftalmologia
  2. Zika virus-related hypertensive iridocyclitis
    Arquivos Brasileiros de Oftalmologia

Zika virus detected in the brain of a terminated foetus with severe brain injury...

In a new paper in the New England Journal of Medicine, Mlakar and colleagues present some compelling evidence for the presence of Zika virus (ZIKV) in brain tissue of a foetus, probably infected by the mother (vertical transmission), that was medically terminated because of severe brain disease, microcephaly and a poor prognosis.[1] 

This study has a very strong title: Zika Virus Associated with Microcephaly

...and it can make that claim because of several robust findings.

  1. Presence of flavivirus-like particles in foetal brain as imaged using electron microscopy, which the authors suggest may reflect persistence in an immune-privileged site. They don't have data to support that though but presumably infection would need to persist for long enough to cause the structural damage seen in these diagnoses - whether that must begin within the first trimester has not been shown, just hypothesized. We do know that virus in the blood usually clears in a week, with virus detectable in the urine for a little longer
  2. Detection of viral RNA in the brain tissue at levels, or 'viral load', higher than usually reported in adult ZIKV patients
  3. Detection of antibodies in the mother's serum indicating past ZIKV, and also Dengue virus, infections
  4. Determination of a complete, full-length genome of the Asian clade of ZIKV, from the foetal brain tissue (KU527068) using an Ion Torrent deep sequencing approach
  5. Absence of ZIKV RNA in many other tissues
  6. Absence of finding other viral genetic material which were sought and included dengue viruses, chikungunya virus, cytomegalovirus, herpesviruses, enteroviruses, rubella virus and others
This very tightly focussed located finding of ZIKV in brain tissues but not any other tissues, (including the spinal cord?), suggests a strong liking for brain tissue.  

An accompanying Editorial [2] notes that this is not proof of cause, but it definitely adds more and better information building a a stronger case for ZIKV doing something in the brain that we had seen just once before, in the French Polynesia outbreak of 2014.[3]

Virus was not cultured - but the finding of a complete viral genomes is good evidence of intact virus present. There have not yet been any studies to identify whether there are babies with ZIKV in their brain tissues but no brain injury disease (babies who died of other, unrelated causes perhaps?) nor is there any gauge of whether every case of comparatively similar brain injury in the region has ZIKV in their brains. We also don't know if ZIKV may simply be a passenger in the brain, or if whether brain tissue is a last, transient site of an infection that is being dispelled by the body after being acquired my the mum and passed to the foetus during an epidemic of that virus. 

Much work lies ahead, but this is stronger evidence towards an association than we have seen so far.


Saturday, 6 February 2016

Urine found to contain infectious Zika virus...again...

In what is becoming a trend of "part-of-the-story" science releases from Brazil's Zika virus (ZIKV) epidemic, we saw an overnight media report of the "first" finding of infections ZIKV in urine and saliva samples.[1,2]

It was not the first in urine. 

There was an earlier report from 2014 that described RNA and infectious virus in the urine of a Canadian traveller who had returned from Thailand, where she presumably acquired her ZIKV infection.[3]

Rushing out appallingly weak stories into scientific journals that exhibit very little peer review [4] and poorly worded or  thought out statements to describe ZIKV findings is not helping the situation in Brazil. In particular, it is doing nothing to investigate the link between ZIKV infection and congenital malformations or Guillain Barre syndrome. At all. 

I urge that everyone take a deep breath and perhaps involve a lot more more peer review - even for media releases.

And on the use of urine, just because previous studies have not sought infectious ZIKV (or other viruses) using culture techniques, or have not found it when they did look,[5,6] is not reason not rule out urine or any fluid as possibly contained infectious virus. When RNA is detected but RT-PCR, there is a real risk of infectious virus being present and there is a decent chance that culture will fail to detect that because it isn't a hugely sensitive technique at times and because of the many infectivity-degrading specimen handling steps between collection and culture. 

Whether that RNA detection, or this infectious virus detection for that matter, equates with an "infectious dose" of virus (enough virus to start an actual infection before the body's defences contain the virus) is the next issue to consider. Also, would mucous membranes onto which virus-contaminated saliva might be propelled, have the receptors to bind virus and the cells to support ZIKV replication? More questions.

Seriously though, has Ebola taught research studies nothing? Look everywhere and expect everything.


  3. First case of Zika virus infection in a returning Canadian traveler.
  4. Ophthalmological findings in infants with microcephaly and presumable intra-uterus Zika virus infection
  5. Detection of Zika virus in urine.
  6. Value of Routine Dengue Diagnostic Tests in Urine and Saliva Specimens
  7. Detection of Zika virus in saliva.

Saturday, 30 January 2016

Microcephaly in Brazil: is it occurring in greater numbers than normal or not?

A paper came out yesterday (AEST) from Morbidity and Mortality Weekly Report (MMWR) with the heading...

Possible Association Between Zika Virus Infection and Microcephaly — Brazil, 2015

Reads as though some great data may finally show us a hint of an association between Zika virus (ZIKV) infection and microcephaly disease. Right? 

Nope. There are none. At least none that could approach satisfying that title which highlights that it is not just the popular media who can generate misleading titles (headlines in their case).

In fact there are no Zika virus testing results in this study of 35 infants with microcephaly (defined using a useful protocol described detailed within). At all.

The closest we get to identifying a role for ZIKV in this disease are the statements...
  1. "Therefore a mother’s report of a rash illness during pregnancy was used as a proxy indicator of potential Zika virus infection."
    ZIKV is not the only agent capable of causing a rash-we've seen evidence for concurrent circulation of other rash-causing arboviruses in print from Brazil including dengue virus and chikungunya virus.[2-aklthough serology is not a reliable way of diagnosing arbovirus coinfection because of anamnestic responses [9] ] There is also malaria, filariasis, leishmaniasis and yellow fever to consider in this region.[3]
  2. [cerebrospinal fluid] "CSF samples from all infants enrolled in the cohort were sent to a reference laboratory in Brazil for Zika virus testing; the results are not yet available."
    Why publish these data ahead of these CSF results I wonder? It does not take long to perform RT-PCR for ZIKV. Certainly not long enough to hold up a paper for more than a day or two at most
Oh and there's this..

"because Zika virus infection was not laboratory confirmed in infants or their mothers, the history of a nonspecific rash illness during pregnancy is subject to recall bias and might have resulted in misclassification of potential Zika virus exposure

Recall bias being that thing you get when you don't really remember what happened a while ago; the details get a little hazy. I imagine that might be a factor in at least some instances here. But clearly not all.

The authors also note that the case definition they describe is unlikely to have been used prior to November 2015. For example, head measurement was not being routinely recorded thus milder cases may not have been reported prior to the ZIKV epidemic/pandemic/global epidemic/multi-country outbreak (or whatever it is being called-my choice underlined). Again, this doesn't negate the rise nor the issue, but it may reduce the total a little. This idea was given more oxygen earlier in the week in an article by Declan Butler in Nature.[4] Although not all agree, some even suggesting the numbers are underestimates.[5]

A little over a week ago I wondered why the United States had a higher averaged number of microcephaly cases than Brazil...

Flu-blog-eyah's ProfCrof has just penned a very nice analysis of this issue.[6]

He looks more deeply than I did and folks, this is why we should demand and ensure public health infectious disease data are accessible to the public-not all useful ideas come from, or are communicated by, a paid position. 

The wash-up of Crof's analysis is that perhaps the rise in microcephaly cases is not a rise at all, but simply a more successful effort to collect data, and that the total number of microcephalic babies may not be too far from the norm (also considering those issues above).[6]

UPDATE 1: See Charles Simmins analysis for microcephaly [7] and also Guillain-Barre syndrome.[8]

The MMWR study did look for, and found no evidence of the following infections of diseases...
  • syphilis
  • toxoplasmosis
  • rubella
  • cytomegalovirus
  • herpes simplex virus
But no mention was made of seeking signs of those other agents listed in #1 above. And what about toxicology?

So the MMWR paper is essentially providing a useful definition of microcephaly and informing us that a cohort has been started/is still recruiting (?) For me, its title would have been better written as 

"Case definition of microcephaly epidemic in Brazil"or perhaps, 

">insert name< : a birth cohort study into ZIKV infection and congenital disease"...or something.

The search for a cause for Brazil's reported spike in microcephaly cases will go on. If it is a true spike then it must, but even that is not clear right now. As is much at all about ZIKV. This event in the Americas really serves a s reminder that we must keep our eyes open to all possibilities - we can do, and think, about more than one thing at a time. If it is not a true spike, then an awful lot of resources are currently moving towards defeating ZIKV that probably, sadly, would not be moving if microcephaly was not a front page story. Right now it seems we're not very clear on even the most basic facts.

It is great to see some more papers coming out because there have not been many to date (see graph above). Bravo. It would be greater still if they had more relevant data and came out faster. 

As always, don't just believe the headline. Read further. And maybe do your own analysis, like the Crof did.

  1. Possible Association Between Zika Virus Infection and Microcephaly — Brazil, 2015
  2. Dengue, chikungunya and Zika co-infection in a patient from Colombia
  1. Added my 2nd tweet with references; added links to Charles Simmin's blogs 
  2. Added reference 9

Friday, 29 January 2016

Zika virus disease samples...don't pass urine (by)..

It is becoming anecdotal in the Tweet'verse that Zika virus (ZIKV) viraemia (virus in the blood) can only be detected for 5 days - presumably after illness onset  - I need to chase that down.

But blood is not the only sample to test. 

In fact it may even be one of the less informative samples to test if trying to detect signs of a current or very recent infection. A lot of that being done in the Americas, and as a result, in laboratories worldwide, right now.

Urine has been shown to be positive for ZIKV RNA beyond 5 days, sometimes when blood is completely negative by highly sensitive polymerase chain reaction (PCR) methods.[1,2,3] Saliva has also been of use,[4] but urine seems to outperform it for PCR purposes.

In particular - beyond 10 days in the study by Gourinat et al.[3]
From Emerging Infectious Diseases article,Vol. 21, No. 1,
January 2015 Detection of Zika Virus in Urine.[3]

Thursday, 28 January 2016

Human metapneumovirus (HMPV) in adults....

Just a random quick and dirty list of some publications in the human metapneumovirus (hMPV) space...

HMPV is just one of a spectrum of human respiratory viruses than can cause a spectrum of illness in a spectrum of age groups across a spectrum of time...and reinfect you later.
  1. Longitudinal course of human metapneumovirus antibody titers and reinfection in healthy adults.
  2. Viral etiology of community-acquired pneumonia among adolescents and adults with mild or moderate severity and its relation to age and severity
  3. Etiology and clinical outcomes of acute respiratory virus infection in hospitalized adults.
  4. Outbreak of Human Metapneumovirus Infection in a Severe Motor-and-Intellectual Disabilities Ward in Japan
  5. An outbreak of human metapneumovirus in a rehabilitation center for alcoholics in Tampere, Finland.
  6. An outbreak of severe respiratory tract infection due to human metapneumovirus in a long-term care facility
  7. An outbreak of severe respiratory tract infection due to human metapneumovirus in a long-term care facility for the elderly in Oregon.
  8. Ten years of human metapneumovirus research.
  9. Longitudinal course of human metapneumovirus antibody titers and reinfection in healthy adults.

Tuesday, 26 January 2016

A primer on human metapneumovirus (hMPV)...

Human metapneumovirus (hMPV) was first described by van den Hoogen et al in 2001.[1] It had been isolated from young children with respiratory tract disease and it was found, by studying old sera, that hMPV had been present in the European community for more than 50 years. The presence of hMPV was next reported in the Australian paediatric population.[2] It is now well known that hMPV is one of many endemic and global human respiratory viruses.

The first reported detection of hMPV outside of the Netherlands was in Queensland,
Australia. I used an in-house conventional reverse-transcriptase polymerase chain reaction to
screen samples suspected of infection by a respiratory  virus, but negative for the "usual suspects". 

Yellow star indicates the positive specimen extract
HMPV infection can result in signs and symptoms that are impossible to differentiate from those due to infection with any other common respiratory virus. An infected individual may remain asymptomatic or show clinical features including rhinorrhoea, cough, shortness of breath, wheeze, vomiting, pharyngitis, chest wheeze with crackles, bilateral lung cellular infiltrates and bronchiolitis.
HMPV genotypes. Aligned in Geneious v8.1.
Neighbor-Joining tree, drawn to scale 

using MEGA v6; 500 bootstraps

This is a member for the Order Mononegavirales,(single strand of negative sense genetic material) Family Paramyxoviridae, Subfamily Pneumonia, genus Metapneumovirus, species Human metapneumovirus.[5] The closest genetic relative remains the avian pneumovirus (APV). HMPV variants exist as two serotypes (reflected by two genetic lineages) called A and B.[3]
Viruses like these have their genetic material, RNA, covered in a protein which, together with other material, is contained within a lipid envelope. The exceptions are the viral proteins which protrude from the envelope. HMPV RNA has been detected in large and small droplets expelled from infected individuals.[4] As with any respiratory virus, containing its spread is difficult once it has become established in health care settings, homes or other close-quarter environments.

Data from Queensland. Figure adapted with permission, 
from Mackay, Ian M., Arden, Katherine E. and Lambert, 
Stephen B. (2009). Epidemiology. In Ronald Eccles and 
Olaf Weber (Ed.), Common Cold 
(pp. 77-106). Basel Switzerland: Birkhäuser Basel. 

HMPV causes initial disease in a similar age-group to respiratory syncytial virus (RSV; a well known childhood respiratory virus pathogen)- the majority of children are infected with hMPV by five years of age. In addition, the virus can iHmpact severely upon the elderly and the immunocompromised.

HMPV infections peak with a seasonality in temperate climates. The spring and autumn periods tend to show more activity, but virus can circulate all year around, as is the case for all respiratory viruses.

Scale comparison of HMPV, APV and RSV genomes.
  1. A newly discovered human pneumovirus isolated from young children with respiratory tract disease
    Bernadette G. van den Hoogen, Jan C. de Jong, Jan Groen, Thijs Kuiken, Ronald de Groot, Ron A.M. Fouchier & Albert D.M.E. Osterhaus
  2. Evidence of human metapneumovirus in Australian childrenMichael D Nissen, Ian M Mackay, Stephen J Withers, David J Siebert and Theo P Sloots
  3. Antigenic and genetic variability of human metapneumoviruses.
    van den Hoogen BG, Herfst S, Sprong L, Cane PA, Forleo-Neto E, de Swart RL, Osterhaus AD, Fouchier RA.
  4. Respiratory virus RNA is detectable in airborne and droplet particles.
    Gralton J1, Tovey ER, McLaws ML, Rawlinson WD.

Tuesday, 19 January 2016

Editor's Note #24 : Tweepidemiology Update #3...

I was involved with @UQ_News and others today in a discussion about use of social media for researchers today. I made the point that...
Which reminded me.

I haven't looked at #VirolDU's Tweepidemiology for over 6 months! 

So here's how the followers have been accruing since my lsat Tweepidemiology update.

These are data generated in response to a blogger who tweets and writes about a sliver of the viral infectious disease outbreaks that affect the world...

Followers of my @MackayIM Twitter account (and this blog which gets
promoted through it) since I started tweeting.
This shows the cumulative rises, pauses and dips of followers and the relationship between the rate of that rise and some active periods of infectious disease outbreaks..
Click on the graph to enlarge
Because everything generates data and every load of data looks better when graphical'd.

Thursday, 14 January 2016

Ebola virus disease is not known to be occurring in humans anywhere in the world right now...but the virus is probably everywhere it was before..

"Ebola" is not "extinct"

Ebola virus is not kicked out of West Africa.

West Africa is not "Ebola free".

It is just wrong to say there is no more Ebola virus in Liberia, Guinea or Sierra Leone.

But - as of right now - there are no known human cases of Ebola virus disease (EVD) in Guinea, Liberia, Sierra Leone, or anywhere else in the world for that matter.

That does not mean there are not cases we don't know about. It certainly does not mean that Ebola virus is not still to be found in one or more species of animal reservoir in the forests throughout Africa.

Of course we still don't know every way in which Ebola virus can re-occur in and from humans. We hardly know all the details of what it has done during the past 2 years thanks to slow and old fashioned ideas about publishing important clicnial data during times of wide need. 

Do we have reliable antivirals to use after this epidemic? We do not. We have a pretty good looking vaccine - and lots of other potential things. We did show what the world can do when it works together in trying to get a vaccine to people. But we started much too late, in every aspect of the response. Everyone did. Except those on the ground already.

Is Ebola virus persisting in an unknown number of EVD survivors? Undoubtedly. Sorry - but stigmatisation aside - that is a fact. Sticking our heads in the sand and not looking closely enough at events are two of the many reasons EVD was able to sow the destruction it did in West Africa. 

Let's face the facts. Not hide from them or fail to fully explore them. 

For the 900 health workers, of whom >500 died; for the >28,000 EVD case and >11,000 deaths; for the many families and friends, villages and communities who lost someone dear to them - we are all so glad it is nearly over. There will be more challenges in the coming month and years, but the constant Ebola-driven fear, sickness and death is behind you now. 

One way or another. 

For the rest of us, it is our responsibility to do honour to those who have and still are suffering by never again being so slow, so arrogant and so limited in our imagination as to let this happen - to any country - again.

But of course, we will. 

We do. 

We are. 

Saturday, 9 January 2016

Zika virus in amniotic fluid...but is that enough?

A report published this month provides background of an investigation of two women described as being part of the 'microcephaly cluster' in Brazil.

The study describes ZIKV detection in the amniotic fluids of the two women; the first laboratory confirmed detections of vertical transmission (from mother to embryo/foetus/baby) of Zika virus (ZIKV; genotyped as "Asian" strains).[1] Transmission around the time of birth (perinatal transmission) has been reported before.[2]

Interestingly, neither mother had ZIKV RNA detected by RT-PCR suggesting they were not acutely infected at the time of the investigations. The authors were not clear whether viral culture was attempted nor whether antibody studies were conducted on the mothers. There is no real time frame for when the mothers were infected beyond a comment that they had relevant symptoms. The authors also do not describe investigations for any other infectious agents or environmental factors nor anything about the deliveries.

Investigation #1...

Foetal ultrasound was conducted at 30.1 weeks' gestation and revealed anomalies including:
  • unusually small head circumference
  • low weight
  • brain atrophy
  • coarse calcifications of the frontal lobe

Investigation #2...

Foetal ultrasound 29.2 weeks' gestation and revealed anomalies including:
  • unusually small head circumference
  • low weight
  • a range of brain anomalies
  • some calcifications
  • cataracts in both eyes and one eye was smaller than the other
Around the same time in the same State (Paraiba)....
  • 6 children borne with small head circumference were diagnosed with ZIKV (lab confirmed?) and born (without complication?) to mothers reporting symptoms during pregnancy
The authors noted that the lack of cases of vertical ZIKV transmission found in over 65 preceding years may be because cases have not been sought, not been reported or were just rare until now. This lack of data may also relate to levels of local immunity which may act to moderate ZIKV infection among people in areas of endemic transmission, or perhaps because the virus has changed and this is a new trick it has acquired..because of the M word. In short, we don;t know if microcephaly and central nervous system developmental interference is a new or an old-but-unknown feature of Zika virus disease (ZVD). Or if it is even related to ZIKV at all.

The authors conclude by warning that if investigations in other states of Brazil find people with ZIKV antibodies, that this is a severe health threat. However they make no mention of the importance of seeking other culprits - likely or not - in the search for understanding of why cases of microcephaly in Brazil have risen so rapidly. Differential diagnosis can be a complex beast in the mosquito-borne virus world.

In a 1 December Pan American Health Organization (PAHO)/World Health Organization (WHO) epidemiological alert,[3] data up to 30 November 2015 listed 1,248 cases of microcephaly (99.7 / 100,000 live births), including 7 deaths, in 14 states of Brazil. By comparison, in 2000, the prevalence was 5.5 cases / 100,000 live births and 5.7 cases / 100,000 live births in 2010.[3] 

More recently the Brazil Ministry of Health tabulated all microcephaly cases. From an average of 156 cases in each of the preceding 5 years, the number has jumped to 3,174 cases in 2015

Both data sets describe a 20-fold increase in cases in just a year.[4]

Whether ZIKV is to blame, or something else, the urgency with which this outbreak of microcephaly is now being investigated in Brazil is clearly warranted. 


  1. Zika virus intrauterine infection causes fetal brain abnormality and microcephaly: tip of the iceberg?
    A. S. Oliveira Melo, G. Malinger, R. Ximenes, P. O. Szejnfeld, S. Alves Sampaio and A. M. Bispo de Filippis