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

UPDATE #1: 01FEB2016 AEST
UPDATE #2: 05FEB2016 AEST

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...

Q: The US averages 20-120 microcephaly births per 100,000 per year. Brazil about 5 pre-#Zika.
Why higher in US?
ping: @who @DrJudyStone
1/2

— Ian M Mackay, PhD (@MackayIM) January 22, 2016

Refs...
US: https://t.co/rG1jDQJqnv
Brazil: https://t.co/DrppzKOWEh
2/2 #Zika

— Ian M Mackay, PhD (@MackayIM) January 22, 2016

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.

References...

1. Possible Association Between Zika Virus Infection and Microcephaly — Brazil, 2015
   http://www.cdc.gov/mmwr/volumes/65/wr/pdfs/mm6503e2.pdf
2. Dengue, chikungunya and Zika co-infection in a patient from Colombia
   http://www.jiph.org/article/S1876-0341(15)00221-X/abstract
3. https://smartraveller.gov.au/countries/brazil
4. http://www.nature.com/news/zika-virus-brazil-s-surge-in-small-headed-babies-questioned-by-report-1.19259
5. http://www.wsj.com/articles/the-brazilian-doctors-who-sounded-alarm-on-zika-and-microcephaly-1454109620
6. http://crofsblogs.typepad.com/h5n1/2016/01/is-microcephaly-surging-in-brazil-or-just-efforts-to-find-it.html
7. http://northshorejournal.org/microcephaly-and-zika
8. http://northshorejournal.org/guillain-barre-syndrome-and-zika-viral-illnesses
9. http://www.ncbi.nlm.nih.gov/pubmed/26837723

Updates..

1. Added my 2nd tweet with references; added links to Charles Simmin's blogs 
2. Added reference 9

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]

References...

1. http://jtm.oxfordjournals.org/content/jtm/23/1/tav011.full.pdf
2. http://www.eurosurveillance.org/images/dynamic/EE/V19N04/art20683.pdf
3. http://wwwnc.cdc.gov/eid/article/21/1/pdfs/14-0894.pdf
4. http://www.sciencedirect.com/science/article/pii/S138665321500133X

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.
   http://www.ncbi.nlm.nih.gov/pubmed/20981798
2. Viral etiology of community-acquired pneumonia among adolescents and adults with mild or moderate severity and its relation to age and severity
   http://www.ncbi.nlm.nih.gov/pubmed/25812108
3. Etiology and clinical outcomes of acute respiratory virus infection in hospitalized adults.
   http://www.ncbi.nlm.nih.gov/pubmed/25024868
4. Outbreak of Human Metapneumovirus Infection in a Severe Motor-and-Intellectual Disabilities Ward in Japan
   http://www.medsci.cn/Sci/show_paper.asp?id=6ded8759301
5. An outbreak of human metapneumovirus in a rehabilitation center for alcoholics in Tampere, Finland.
   http://www.ncbi.nlm.nih.gov/pubmed/25761612
6. An outbreak of severe respiratory tract infection due to human metapneumovirus in a long-term care facility
   http://www.ncbi.nlm.nih.gov/pubmed/17407031
7. An outbreak of severe respiratory tract infection due to human metapneumovirus in a long-term care facility for the elderly in Oregon.
   http://www.ncbi.nlm.nih.gov/pubmed/22078146
8. Ten years of human metapneumovirus research.
   http://www.ncbi.nlm.nih.gov/pubmed/22074934
9. Longitudinal course of human metapneumovirus antibody titers and reinfection in healthy adults.
   http://www.ncbi.nlm.nih.gov/pubmed/20981798

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. 
doi:10.1007/978-3-7643-9912-2_4

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.

References...

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
   http://www.nature.com/index.html?file=/nm/journal/v7/n6/abs/nm0601_719.html&dynoptions=doi1020034962
2. Evidence of human metapneumovirus in Australian childrenMichael D Nissen, Ian M Mackay, Stephen J Withers, David J Siebert and Theo P Slootshttps://www.mja.com.au/journal/2002/176/4/evidence-human-metapneumovirus-australian-children
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.
   http://www.ncbi.nlm.nih.gov/pubmed/15200856
4. Respiratory virus RNA is detectable in airborne and droplet particles.
   Gralton J1, Tovey ER, McLaws ML, Rawlinson WD.
   http://www.ncbi.nlm.nih.gov/pubmed/23959825
5. http://www.ictvonline.org/virusTaxonomy.asp

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...

Oh-last thing @UQ_News - don't get bogged down in how many followers you have. Build it & they will come. Then go. Then others turn up. Etc

— Ian M Mackay, PhD (@MackayIM) January 19, 2016

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.

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. 

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. 

References...

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
   http://onlinelibrary.wiley.com/doi/10.1002/uog.15831/abstract
2. http://www.eurosurveillance.org/ViewArticle.aspx?ArticleId=20751
3. https://www.google.com.au/url?sa=t&rct=j&q=&esrc=s&source=web&cd=1&cad=rja&uact=8&ved=0ahUKEwiI47n55ZvKAhVHlKYKHb5LAQYQFggbMAA&url=http%3A%2F%2Fwww.paho.org%2Fhq%2Findex.php%3Foption%3Dcom_docman%26task%3Ddoc_download%26Itemid%3D%26gid%3D32405%26lang%3Den&usg=AFQjCNGl3MMwrJYjsLvy4eC4tll7yzthEw&sig2=SpTsu1DtQGhIp21MWLJ8mA&bvm=bv.111396085,d.dGY
4. http://portalsaude.saude.gov.br/index.php/cidadao/principal/agencia-saude/21459-saude-divulga-dados-atualizados-de-microcefalia

Research on MERS in South Korea seems fractured...

I would be remiss if I didn't point out that the Kingdom of Saudi Arabia (KSA) is not the only country that can be seen as slow to initiate local research on local cases of Middle East respiratory syndrome (MERS), MERS-coronavirus (MERS-CoV) or the epidemiology thereof.

The outbreak in South Korea began when an incubating person flew in from Qatar, arriving 4th May. He became ill while in South Korea, with symptoms beginning on the 11th May 2015. This was to become the largest MERS-CoV outbreak outside of the Arabian Peninsula and it is still to be investigated as the overarching single event that it really was. Scientific papers, letters, media and case reports all look at small groups of specimens, this or that specific hospital, a particular slice in time or an interesting group of cases and they reach a range of conclusions. But there appears to be no over-arching all-encompassing study of the outbreak. We did eventually see such a report from the KSA in its description of four dozen early MERS cases - 13 months after the first case became ill.[4] South Korea only had 185 cases in total (exporting one to southern China) so this should not be a great task.

A recent article in The Korea Times noted that "while the initial tests were conducted on a limited number of patients, research is underway to screen clinical specimens from 32 people that will help shed better light on the matter."[1] Well, perhaps a part of the matter. [1]
The article focusses on Spike gene mutations that were found in the MERS-CoV variants from 8 human cases - which is not a remarkable discovery and has been happening since we first saw sequenced MERS-CoV from humans (see "Tracking MERS-CoV through time: a spikey problem" [2]). 

The article also goes on to say that "There is a need to focus the country’s research capability on finding the reason for the change." Those will be dollars wasted since one cannot "find the reason" for a virus mutating because that is akin to asking why we exist. 

Scientific studies can determine that the variants have mutated at certain points and they can seek out whether those changes have any effect on the efficiency of viral replication, transmission or on our immune response to infection. This will be achieved by comparison to other non-mutated MERS-CoV variant viruses and will need more than one older variant to be included so as to encompass all of the different Spike gene mutations that have been determined to date. But these studies cannot find the reason for these mutations. Yes, that is a pedantic point.
We are about 6 months out from when the final MERS case first became ill in the South Korean outbreak. This media report sadly highlights that the world is still far away from seeing comparative experimental data defining whether or not there was anything special about the MERS-CoV variants in the outbreak. Strangely though, it has already been concluded that the rapid spread of MERS-CoV in South Korea was not due to viral changes but instead because of the poor control of transmission in healthcare settings.[3] That makes this report and its message to the public both a little stale and perhaps even unnecessarily alarming.

Clearly, the KSA was not alone in needing to improve its collaboration, communication and organization of research in response to the appearance of a new virus. 

References...

1. http://www.koreatimesus.com/mutation-detected-in-s-korean-mers-virus-govt/
2. http://virologydownunder.blogspot.com.au/2013/08/tracking-mers-cov-through-time-spikey.html
3. http://www.who.int/csr/disease/coronavirus_infections/situation-assessment/update-15-06-2015/en/
4. http://www.ncbi.nlm.nih.gov/pubmed/23891402

Zika virus disease (ZVD): 14 cases until 2007 then Yap island, Micronesia...

Update #1: 17JAN2016

Between the discovery of Zika virus (ZIKV) in 1947 and today's review subject, an outbreak on Yap Island,[1] there had only been 14 cases of Zika virus disease (ZVD) described. The keyword there is detected, or actively sought.

Some opinions on ZIKV...

In my opinion - and as the following study supports for this outbreak it describes - the confirmed ZIKV cases we heard of during 2014 are likely the tip of a vastly bigger iceberg of human infections. Many of the countries from which we have recent reports of "first" autochthonous (local) ZIKV transmission may well have had this virus in their midst for much (much, much, much?) longer since it often causes mild illness. Unless carefully looked for, ZVD is clinically very similar to other mosquito-borne virus infections. 

I hope that on the list of things to do to understand ZIKV - probably topped by investigating the suspected link between ZIKV infection and microcephaly) - is go back to stored human sera and look for traces of ZIKV or antibodies against ZIKV in these newly announced transmission zones. This will give us an idea of how long this virus has been around.

Yap Island ZVD outbreak in 2007...

In April and May of 2007, an outbreak of rash, conjunctivitis, subjective (not measured) fever, arthralgia, and arthritis occurred on an island group (the 4 Yap Islands; population ~7,400 in 2000) that comprise Yap State in the Federated States of Micronesia. Three initial patients tested positive for IgM antibodies that suggested recent dengue virus (DENV) infection (although IgM can be non-specifically triggered as a result of another infection). However. the doctors believed the symptoms were not due to DENV infection.

Samples were sent away to the United States Centers for Disease Control and Prevention and ZIKV RNA was detected in the sera of 10 of 71 (14.1%) symptomatic people using a specific reverse transcription polymerase chain reaction (RT-PCR) assay. Other viruses were considered but could not be detected using specific assays. These viruses included dengue, chikungunya, o’nyongnyong, Ross River, Barmah Forest, and Sindbis virus. The finding implicated ZIKV as the likely cause of the outbreak, and ZVD as the disease.

The result triggered a bigger investigation during which 185 suspected cases (including 49 confirmed and 59 probable) were further investigated. 66% of the confirmed and probable casess were female and these 108 people had a median age of 36y. The earliest infections occurred 15th April. Among 31 of the confirmed cases who reported symptoms, rash lasted for a median of 6d (2-14d) and arthralgia for 3.5d (1-14d). None had travelled outside of Yap.

Enzyme-linked immunosorbent assay (ELISA), an antibody detection method, was used to look for IgM antibodies indicating recent ZIKV or DENV infections. The identification of very specific infection-neutralizing antibody to these viruses was also sought and the level determined by using a plaque reduction neutralization test with a threshold value of 90% (PRNT₉₀). Antibodies to other flaviviruses were not sought in this study.

A case definition was created:

• A patient with suspected ZVD had acute onset of generalized macular or papular rash, arthritis, arthralgia, or nonpurulent conjunctivitis. 
• Blood samples were requested from acute phase ( within 10d after the symptom onset) and convalescent phase (14d later)
• A patient with probable ZVD had:
• IgM antibody against ZIKV
• ZIKV PRNT₉₀ that was still detectable at a dilution of (titer) at least 1:20
• A ratio of ZIKV PRNT₉₀ titre to DENV PRNT₉₀ titre was less than 4
• Either no ZIKV RNA detected by RT-PCR or a sample which was inadequate for the performance of RT-PCR
• A patient with confirmed Zika virus disease had:
• ZIKV RNA detected in their serum or 
• IgM antibody against ZIKV (detected by ELISA) and 
• A ZIKV PRNT₉₀ titre of at least 20 and 
• A ratio of ZIKV PRNT₉₀ titre to DENV PRNT₉₀ titre of at least 4. 

173 of 200 randomly selected households were surveyed yielding 557 blood samples of which 414 (74%) had IgM anti-ZIKV antibody detected and 156 of these recalled a relevant recent illness. Among those without any IgM detected, 27 also recalled a relevant illness. Extrapolation of these figures resulted in the authors estimating that 73% of residents were infected during this outbreak with 18% of those infected having a clinical illness likely due to ZIKV infection.

An examination of 1,366 containers holding water, frequently (43%) found that they also contained 9 species of mosquito larvae/pupae and such containers were present in 87% of households. [A simple intervention method is to tip these out and ensure they cannot refill with water]. Three other mosquito species were identified to as adult mosquitoes. Aedes hensilli was the most frequently identified mosquito species overall. 

No mosquitoes tested contained detectable infectious ZIKV (examined by cell culture methods) or ZIKV RNA (RT-PCR).

So, jumping forward from the 1940s to the naughties (2007), we see a major shift in the diagnostic arsenal which supported this study. It allowed us to see just how effectively a mosquito-borne virus can apparently consume an entire island community. I wonder what IgG results - generally interpreted to indicate older earlier infection by an agent - on these same sera would yield? Would they further support the author's conclusions that a viraemic human (infected individual with ZIKV in their blood) or a virus-infected mosquito had only recently arrived from somewhere else?

Could something have changed in the levels of the vector population/species resulting in a sudden surge in virus? 

The authors note that there had not been any previous disease outbreaks of this sort reported on Yap-but were isolated cases occurring earlier? Had the virus been slowly smoldering in rare transmissions from mosquito to human/other animal to mosquito etc, for a while before building up speed and manifesting as an outbreak?  Why did none of the tested mosquitoes contain traces of ZIKV virus? 

It is also worth noting that many ZVD "cases" described in more recent months may be better defined as "suspect" and not yet confirmed. But I stand to be corrected on that.

References...

1. Zika Virus Outbreak on Yap Island, Federated States of Micronesia
   Duffy MR, Chen TH, Hancock WT, Powers AM, Kool JL, Lanciotti RS, Pretrick M, Marfel M, Holzbauer S, Dubray C, Guillaumot L, Griggs A, Bel M, Lambert AJ, Laven J, Kosoy O, Panella A, Biggerstaff BJ, Fischer M, Hayes EB.
   N Engl J Med. 2009 Jun 11;360(24):2536-43. doi: 10.1056/NEJMoa0805715.
   http://www.nejm.org/doi/full/10.1056/NEJMoa0805715

Updates...

1. Added 18% figure to clinical illness from population extrapolations in this paper

Zika virus, mosquitoes and a monkey on a platform...

UPDATED: 07JAN2016 AEST

As I noted yesterday the first published description of the isolation of Zika virus was from a study set up in the Zika forest in Uganda. It was described in print by Dr George Dick and colleagues in the September 1952 issue of Transactions of the Royal Society of Tropical Medicine and Hygiene.[1]

Rhesus macaque (Macaca mulatta) [2]

In the first study, which began April 1947, six platforms were set up in the forest canopy and upon each, a caged rhesus monkey was placed. 

The first Zika virus was identified from a monkey...

This was to be a Yellow fever virus (YFV) study since Zika forest was known to harbour a lot of previously YFV infected, antibody-positive monkeys-but stuff happens on the road to completing a good plan.

By the way: placing or penning an animal in a location known to, or under suspicion of, harbouring an infectious agent - a virus in this case - is the precess of creating a sentinel animal.

On 18th April, the daily temperature recording for "Rhesus 766" had increased to 39.7'C, rising to 40'C the next day. It was taken to the lab at Entebbe, a blood sample collected and then the primate was observed for the next month. The only sign or symptom of illness in 766 was the fever.

The blood was injected intraperitoneally (no signs of illness) or intracerebrally (became ill from day 10 post-injection) into mice and underneath the skin of Rhesus 771 (which did not develop any signs of illness).

A filterable "agent" (filters were used to remove everything but very small viruses) was recovered from the brains of the ill mice and also from the serum of 766 - it was later named Zika virus (766 strain). The virus's growth could be hampered by antibodies which developed in the serum of 766 a month later, identifying the that the Zika virus was capable of triggering a specific immune response, despite a mild illness. The more lab-savvy of you may have realised by now, that this all happened before cell culture was being used. The only way to "grow" or amplify more virus was to infect anew a susceptible host animal-which could also reveal whether the agent was capable of causing disease, so long as the experiments were suitably controlled.

A second Zika virus was found among mosquitoes...

A second virus (later called strain E/1) was acquired during a different study, but also set up for YFV. The E/1 was identified from a ground up, unfiltered preparation of Aedes africanus mosquitoes in saline which had been caught 11-12th January 1948 using a variation of the platform system. . The preparation was injected intracerebrally into 6 mice and subcutaneously into Rhesus 758 via 9 inoculations, across 2 weeks. 

Diagnosis of Zika virus by infection of animals...

Primate 758 showed no signs of illness, but the mice had - at the 7th day - so at day 8, 9 and 10 after 758's injection, blood was collected and serum injected intracerebrally into groups of 6 new mice. 

• From the first primate sample (day 8), 2 injected mice died and virus could be passed on to additional mice from filtered preparations of their infected brain tissue
• From the second sample (day 9), 1 mouse died (and its brain tissue filtrate could also produce new infections in mice). A second mouse was paralysed but another virus, called Theiler's mouse encephalitis virus [TMEV], a picornavirus, was identified upon further infections 
• No mice died from the 3rd 758 inoculation of serum
• The serum from 758 was shown to block infection of animals by Zika virus after it was first preincubated with preparations of:
• the agent isolated from 758 serum after inoculation with the A. africanus preparation or
• the E/1 Zika virus strain or
• the 766 Zika virus strain

The authors concluded Zika virus was a new virus and that it triggered a specific antibody response which did not cross-react with YFV, dengue virus or the TMEV found in the paralysed mouse. 

It was also notable that disease was mild or went unnoticed in primates. Primates are usually considered to be close animal substitutes for humans in studies of disease progression. 

No studies of pregnant primates were conducted which does raise the question of whether we should consider a check-list of things to research for each and every virus capable of replicating in us.

References...

1. Zika virus. I. Isolations and serological specificity.
   DICK GW, KITCHEN SF, HADDOW AJ.
   Trans R Soc Trop Med Hyg. 1952 Sep;46(5):509-20.
   http://www.ncbi.nlm.nih.gov/pubmed/12995440.
2. https://www.flickr.com/photos/wild_speedy/4185543087/

Updates..

1. Added some links and worked out some typos and layout issues.

Zika virus briefly...

Update #1. 05JAN2016 2030 AEST
Update #2. 30JAN2016 1430 AEST

Zika (pronounced [zēk′ ə; 1] or zeek-a) virus has been in the news in recent months as it has seemingly spread very quickly from country to country, seemingly at the same time as a rise in cases of an otherwise rare human disease, microcephaly. 

The virus was first grown in the laboratory from samples from a naturally infected sentinel rhesus macaque (monkey) which were placed in cages on variously elevated platforms in the Zika ("overgrown") forest in Uganda in 1947.[18] The virus was not descried in the literature until 1952 although many others from the discoveries in this region were.[18]

I'm new to Zika virus and the study of its spread and disease so I'm on a mission to read up on it. I like to start from the beginning thus I have some of the earliest papers and will gradually read them and share with you any summaries I write up. For now, a general overview of some key bits.

Zika virus causes Zika virus disease (ZVD) or Zika fever - it's that virus versus disease thing that we have for almost all infectious diseases. Zika virus is often abbreviated to ZIKV.

From ViralZone's (www.expasy.org/viralzone,

SIB Swiss Institute of Bioinformatics) excellent

Flaviviridae page at 

http://viralzone.expasy.org/all_by_species/43.html.[2]

ZIKV is a mosquito-borne virus that has an RNA genome (positive sense) and is enveloped by a lipid membrane (with exposed viral bits embedded-see adjacent image) surrounding an icosahedral capsid. Inside the capsid is the genome. This is the same basic structure as that found among other viruses of the family Flaviviridae of which ZIKV is a member; it falls into the genus Flavivirus of the family Flaviviridae to me a bit more precise.

ZIKV replicates in the infected host cell's cytoplasm and first makes a single protein (a polyprotein) which is subsequently cleaved up into different functional peptides.[5,8]

Aedes aegypti mosquito. One of the genus Aedes of 

mosquitoes found to host ZIKV. Other mosquitoes 

include Aedes aegypti, Aedes africanus, 

Aedes apicoargenteus, Aedes furcifer, Aedes hensilli, 

Aedes luteocephalus and Aedes vitattus.

Image from CDC via Wikipedia.[9]

Some flaviviruses are borne to us (and other animals) by other arthropods - ticks apart from mosquitoes - infecting us via a virus-laden puncture/bite/injection during which virus is introduced. These viruses are all lumped together under the umbrella term of arthropod-borne viruses or arboviruses. 

Arboviruses replicate (grow) within the cells of the particular arthropod host, where they are amplified to higher viral loads; for example cells lining the mosquito gut in the case of Dengue virus.[10,11] Some human and animal hosts, as far as we know, cannot amplify the virus enough for it to be sucked back out by and infect the next arthropod that might come along and feed on us - these are called incidental or "dead-end" hosts.[4]

Some flaviviruses have not yet been linked to an insect host. Wikipedia maintains a great long list of flaviviruses, arthropod hosts and their mammalian and avian incidental hosts, as well as those viruses not yet linked to a human or other animal hosts.[4]

Other flaviviruses (members of that genus) you may have heard of include Yellow fever virus (YFV; the prototype virus and from where "flavi", which derives from flavus, meaning yellow/blond/golden in Latin, comes from), Dengue virus (DENV), Japanese encephalitis virus (JEV), Tick-borne encephalitis virus (TBEV), West Nile virus (WNV), St. louis encephalitis virus and Murray valley encephalitis virus (MVEV). But there are 53 species listed in the genus by the International Committee on Taxonomy of Viruses (ICTV) as of 2014 - many of which you won't have heard anything about.[3]

Generally, ZIKV causes a relatively mild illness in a proportion of those infected.[13] Signs and symptoms can include fever, rash, joint (arthralgia) and muscle (myalgia) pain, conjunctivitis, headache and jaundice but with its recent rapid spread - or more rigorous detection - a link is being investigated to a parallel rise in cases of a rare disease, detected at birth or thereafter, called microcephaly. This has been reported in some countries with ZIKV cases, but not from all. At least to date that has been the case - it may change with the new attention this disease and this virus have now attracted. 

Countries and territories with autochthonous transmission.
Epidemiological Week 51 - 2015. PAHO & WHO.[23]

Microcephaly manifests, as the name suggests, as markedly smaller than normal head size and is linked to reduced brain growth in utero or brain development after birth.[6] While a link between microcephaly and viruses is not new, a link (statistical, supported with data) has yet to be found to ZIKV infection.[7] That is not to say a link will not be found, but it is awaiting the required studies. Three instances report ZIKV in amniotic fluid or in newborn tissues and one case of sexual transmission tentatively described.[19,21] Thousands of ZVD cases have reportedly been accruing on a weekly basis in Colombia alone which found its first local (autochthonous) transmission October 2015.[20] The first autochthonous reports of ZIKV infection in the Americas were confirmed in February 2014 on Easter Island, Chile.[22] In May 2015, Brazil reported discovery of its first autochthonous cases and November 2015 saw the first autochthonous circulation reports by El Salvador, Guatemala, Mexico, Paraguay, Suriname, and Venezuela.[22]

Laboratory confirmation of a suspected or probable case can be by detection of ZIKV RNA using RT-PCR in samples from an acutely infected case and by the finding of IgM antibodies 5 or more days after illness onset.[12,13] ZIKV antibody studies must be considered alongside studies of other flaviviruses which may cross-react or non-specifically flare up during infection by another related, or unrelated, virus.[14,15,16,17] The time during which virus remains in the blood may only be 3 to 5 days.[13]

But other infections can look just like ZVD including:[12]

• dengue viruses
• leptospirosis
• malaria
• rickettsia
• group A streptococcus
• rubella virus
• measles virus
• parvovirus
• enterovirus
• adenovirus
• Chikungunya virus
• Mayaro virus
• Ross River virus
• Barmah Forest virus
• O’nyong-nyong
• Sindbis viruses

So you can see that a lot of work, time and money is required for a lab asked to confirm the disease, rather than a specific viral infection.

No specific antiviral or vaccine exists for ZIKV infection - or most other viral infections.

References...

1. http://wwwnc.cdc.gov/eid/article/20/6/et-2006_article
2. http://viralzone.expasy.org/all_by_species/43.html
3. http://www.ictvonline.org/taxonomyHistory.asp?taxnode_id=20141996&taxa_name=Flavivirus
4. https://en.wikipedia.org/wiki/Flavivirus
5. http://web.stanford.edu/group/virus/flavi/2008/flavi.html
6. http://www.mayoclinic.org/diseases-conditions/microcephaly/basics/definition/con-20034823
7. http://www.childrenshospital.org/conditions-and-treatments/conditions/microcephaly/symptoms-and-causes
8. http://viralzone.expasy.org/all_by_protein/24.html
9. https://commons.wikimedia.org/wiki/File:Aedes_aegypti_CDC-Gathany.jpg
10. http://www.sciencedirect.com/science/article/pii/S0042682207006642
11. http://journals.plos.org/plosntds/article?id=10.1371/journal.pntd.0001385
12. http://www.cdc.gov/zika/hc-providers/clinicalevaluation.html
13. http://ecdc.europa.eu/en/healthtopics/zika_virus_infection/factsheet-health-professionals/Pages/factsheet_health_professionals.aspx
14. http://www.cdc.gov/westnile/healthcareproviders/healthcareproviders-diagnostic.html
15. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2732478/
16. https://books.google.com.au/books?id=73pYBAAAQBAJ&pg=PA1158&lpg=PA1158&dq=flavivirus+infection+triggers+heterologous+antibody&source=bl&ots=UYgmkBSso3&sig=CV1uAEb5NZTGfhD-hd0v6EZ7P4g&hl=en&sa=X&ved=0ahUKEwi8idCd0ZHKAhUMkZQKHXMnBNgQ6AEIOzAD#v=onepage&q=flavivirus%20infection%20triggers%20heterologous%20antibody&f=false
17. https://books.google.com.au/books?id=BseNCgAAQBAJ&pg=PA1898&lpg=PA1898&dq=flavivirus+infection+triggers+heterologous+antibody&source=bl&ots=0JKxv695TR&sig=hvvhStq7BABzIKhzecMhFRW50cg&hl=en&sa=X&ved=0ahUKEwi8idCd0ZHKAhUMkZQKHXMnBNgQ6AEINjAC#v=onepage&q=flavivirus%20infection%20triggers%20heterologous%20antibody&f=false
18. Zika virus. I. Isolations and serological specificity.
    DICK GW, KITCHEN SF, HADDOW AJ.
    Trans R Soc Trop Med Hyg. 1952 Sep;46(5):509-20.
    http://www.ncbi.nlm.nih.gov/pubmed/12995440
19. http://www.forbes.com/sites/judystone/2016/01/04/zika-coming-to-america-through-mosquitoes-travel-and-sex/
20. http://outbreaknewstoday.com/colombia-averaging-more-than-1000-zika-cases-weekly-for-the-past-month-54367/
21. http://wwwnc.cdc.gov/eid/article/21/2/14-1363_article
22. http://www.paho.org/Hq/index.php?option=com_docman&task=doc_download&Itemid=&gid=32405&lang=en
23. http://www.paho.org/hq/images/stories/AD/HSD/IR/Viral_Diseases/Zika-Virus/2015-cha-autoch-human-cases-zika-virus-ew-51.jpg

Updates...

1. Grammatical reference and additional location details
2. Fixed some reference errors