Friday 29 April 2016

Zika virus (ZIKV) disease in Brazil (not just microcephaly) gets its own report...kinda

UPDATE #1: 05MAY2016
I admit I was hoping for more.

Brazil's Ministry of Health announced its first report containing dedicated Zika virus (ZIKV) numbers this week.[1] Not just the 194 detection made among diagnoses of microcephaly and central nervous system disorders,[2] but among the population as a whole. 

I was hoping for something like the Colombian data.[3]

We learned that there have been 91,387 probable cases and 3 deaths in Brazil, nationwide, up until the 2nd April 2016 and that autochthonous (local) transmission was first detected in April 2015.[1]

The report itself is a bit hard to translate though.[4] But it states that 31,616 probable cases have been confirmed. [UPDATE#1 PAHOWWHO lists 1,034 confirmed infections-a more believable figure; sad it was not clearly defined here. I infer from this that "confirmed means probable and "probable" means suspected] It does not say whether this is our old friend "clinically confirmed", or if an actual sample was collected and put through an actual test to see that there was any specific trace of ZIKV or its actual antibody produced in response to infection. This report would be greatly helped by a lot of definitions.

Geographically, Most ZIKV probable cases (39%) were in the south-east followed closely by 33% in the north-east, from where the majority of the world's temporally related M&CD diagnoses have been reported. After that there is a big step down to 19% of probable cases in the west, 7% in the north and 2% in the south. 

The highest incidence (cases per 100,000 population) occurred in the mid-west (113.4), followed by the north-east (53.5), south-east (41.4) north (36.0) and south (6.1). It would be very interesting to see this breakdown over time to observe whether ZIKV has moved during this reporting period and if so, in what direction, when and at what speed. 
From MMWR article.[5]

Overall, Brazil reports an incidence of 44.7 ZIKV cases per 100,000 people. These are only symptomatic people though, and it remains an open question as to whether the Yap Island ~80% asymptomatic rate that is so widely used and quoted,[6] holds in the Americas epidemic. As recent study of travellers who moved to or returned back to the United States after travel to ZIKV-affected countries,  found few ZIKV positives except among those with symptoms - and relevant ones at that (Fever, rash, arthralgia, or conjunctivitis).[5]
From PAHO-WHO report.

We also learned that here had been 802,429 probable cases of Dengue and 140 associated deaths as well as 39,017 cases of Chikungunya and 15 deaths - over the same period. With so many similar diseases around, I continue to boggle at how "clinically confirmed" Zika virus disease can carry much weight at all - no matter how distinctive the rash looks. Just look at the overlap in the latest PAHO-WHO report's graph above. How do you untangle prevalence let alone causality, without more lab testing?

So, with that, I look forward to Colombia's epidemiology report this weekend.

  1. Added PAHO-WHO data on actual confirmed ZIKV infections - 1,034 from new reference, [8]
  2. .

Brazil's microcephaly and CNS disorder (M&CD) monitoring: Report No. 23, 2016-Week No. 16...

These graphs are made using data obtained from Week 16's Brazil Ministry of Health microcephaly and foetal and infant microcephaly and central nervous system (CNS) disorders (M&CD) report [1] and media report.[2]

Suspected M&CD cases...

The total number of suspected M&CD cases increased by 78 to 7,228 this week - the smallest rise reported by Brazil to date. This should not be confused with comments in the latest World Health Organization's Zika virus (ZIKV) situation report which states...

 "At this stage, based on the evidence available, WHO does not see an overall decline in the outbreak." 

That statement refers to current data whereas Brazil's M&CD report #23 is describing events that may have occurred approximately 20 to 40 weeks ago.[3] I picked that range because foetal disorders with preceding ZIKV detection have been reported to occur very rapidly, between 17 and 20 weeks in one case study.[4]

The chart above reports the number of newly suspected M&CD diagnoses in Brazil up to 23-April-2016. The cumulative curve (yellow dots; left hand axis) is steady and growing, but slowly steadily. This was the lowest weekly rise (orange bars; right-hand axis) on record.

Confirmed and discarded M&CD diagnoses...

M&CD cases under investigation decreased by 31 to 3,710 this week - the fifth consecutive decrease.

In the graph above, we can see that 79 (blue bars; right hand axis) suspected M&CD diagnoses were discarded upon closer investigation.The rate of these resolved diagnoses (line with blue dots, left-hand axis) is slowing but still seems to be outpacing the rate of confirmed M&CD diagnoses (red dots, left-hand axis). The cumulative number of M&CD diagnoses does continue its climb this week (+30), but this is the smallest addition of new diagnoses on record (red bars; right-hand axis).

The number of these M&CD diagnoses to be confirmed with a ZIKV infection also grows (green dots; left-hand axis) but by just 2 new detections to 194 this week (green bars; right-hand axis). That's the smallest rise on record. Those confirmed ZIKV infections represent 16% of all confirmed diagnoses and 3% of all suspect M&CD cases - but these are not fair comparisons for a range of reasons I won't go on about here.

Why are M&CD diagnoses slowing...?

It's not precisely known why the numbers of M&CD diagnoses are slowing in Brazil. Whatever their cause(s), the trigger(s) for the anomalies occurred in the past - as far as 9 months or as close as perhaps 20 weeks. One suggestion which ties in with the timing of arbovirus epidemics in Brazil, is the weather and its impact on the mosquito breeding cycle. (Thanks Luis F. B. Correia) Mosquitoes are assumed to be the major vector for transmitting ZIKV, wherever in the world it may be. That's a safe bet as far as we know. Undoubtedly, reporting on mosquito detections is not a priority. Only Mexico has reported infected wild-caught mosquitoes to date.[5] There have been more monkeys identified as ZIKV-infected than mosquitoes![5,6] For now at least.

The rainy season begins in the north-east (where M&CD has mostly been diagnosed) in May. Infections are likely to pick up from then - if we crudely add 6-9 months to May 2015 we get to October-2015 - January-2016. 

August saw a rise in microcephaly diagnoses in Pernambuco State in the north-east.[6] 

November is when the Brazil ministry declared a link between M&CD and ZIKV.[7] 

It all kinda fits. 

None of these numbers are precise though because when and where ZIKV was becoming established in Brazil is not known. It is predicted to have been as early as May-December 2013 based on analysis of viral genome sequences to hand,[9] but local transmission was not detected (looked for?) until April/May 2015.[9,10] It may have been there earlier.

Solid data and answers continue to evade trapping. As do ZIKV positive mosquitoes apparently.


Thursday 28 April 2016

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

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

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

From [1].
ZIKV RNA was not detected in any baby's CSF or serum samples - nor was Dengue virus or Chikungunya virus. No word on other viruses such as cytomegalovirus, rubella virus, enteroviruses etc. No unbiased investigation for viruses were described nor was the serostatus of the partner at conception or birth. This was a "first past the post" diagnostic conclusion.

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

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

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

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

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



Wednesday 27 April 2016

Three monkey species, Zika virus and some labs...

From Wikipedia page on primates.[6]
Thought I'd better follow up on a comment about primates that I made in my recent post about Zika virus (ZIKV) mosquitoes (Biting assumptions; [1]). 

The latest Pan American Health Organization (PAHO) and the World Health Organization (WHO) Zika Epidemiological Update (21APR2016) talked about the finding of the current ZIKV Asian lineage variant for the first time in wild caught Aedes albopictus mosquitoes.[2] 

Also in that report's "Significant Findings" section was the description of the discovery of ZIKV in the sera anad oral swabs of 7 of 24 animals from two monkey species - marmosets (Callithrix jacchus) and capuchin monkeys (Sapajus libidinosus).[2] Slightly more detail went online at BioRxiv 20-April-2016 in a nicely collaborative Brazil-based publication including state health and university stakeholders.[3] Prof Racaniello has also covered this paper in a recent post on his blog.[4]    

In the preceding week's PAHO-WHO Zika Epidemiological Update (14APR2016), there was also mention of finding ZIKV in the heart and spleen tissues collected from a dead howler monkey (genus Alouatta), reported 9th March 2016, by Ecuador.[5] 

This finding seems to have been part of an investigation into three dozen howler monkey deaths which occurred in the first 2 weeks of February 2016 (1st to 10th). Only one monkey is reported as having tested positive for ZIKV and there is no other information about the general cause of the howler monkey deaths. 

I can't help but wonder what our response to assigning causality would be if 1 of 39 deceased humans tested positive for ZIKV in today's climate?

Both studies found virus genetic material (RNA) using RT-PCR. No culture of infectious virus was described.

The Brazil detections are a fantastic example of how data being generated during a public health emergency, or even just an outbreak, can be released quickly for wide dissemination - and use. Okay - the monkey's had been captured in July and November 2015 - so there is still a lot of time in getting to the point of publication. Nonetheless, on the publication side, despite not yet having been peer-reviewed, it ticks the boxes of being...

  • Well formatted
  • Clear
  • Descriptive/detailed
  • Freely accessible
  • Collaborative
These findings support that ZIKV is in the Americas to stay. This is evidence of a reservoir of virus so transmission cycles can include non-human primates: mosquito>primate>mosquito with human cases being predominantly (we presume) acquired by mosquito bites. Another couple of pieces of good data to add to the learning knowledge gaps of ZIKV in the Americas.


  3. First detection of Zika virus in neotropical primates in Brazil: a possible new reservoir.

Monday 25 April 2016

The many faces of Colombian Zika virus data...

I've written before about an apparent lag in the lab results from Colombia, a guess based on there being nothing obviously appearing on their weekly epidemiology reports' graph (red bars=lab confirmed Zika virus& detections)....for example the one adjacent.

But, when one plots the numbers from the reports oneself - they look like the green bars below..

Well it looks like it was just an error in reporting after all (option No.3). If you take a look at recent PAHO-WHO data for Colombia..the most recent laboratory confirmed cases (dark orange) seem to have appeared where they should be.

I imagine nothing is easy when there is so much pressure and so many watching. Here's hoping the Weekly epidemiological reports catch up next week.


Biting assumptions...

This is not Aedes albopictus.
But it is a mosquito.
The Pan American Health Organization (PAHO) and the World Health Organization (WHO) Zika Epidemiological Update (status as of 21 April 2016) had some nice Zika virus (ZIKV) graphs from different - that is, not Brazil and Colombia - ZIKV affected countries.[1] 

It also had a very interesting snippet - that the Mexico Epidemiological Surveillance System (SINAVE) has reported detection of ZIKV in wild caught Aedes albopictus mosquitoes in Mexico.[1] The were identified using real-time RT-PCR and confirmed 1st April 2016.

That's a first for Mexico and also a first for the Americas in terms of detecting the variant of the Asian lineage of ZIKV that has been running rampant across the region, in a presumed vector. To date, all the detection have been from (many) humans, a howler monkey and a marmoset.[1,4,]

The 1966 Malaysian Ae.aegypti
ZIKV isolate is HQ234499 at the top
of the Asian lineage branch
The only (that I know of - please let me know if there are others) other reported detection of ZIKV from wild-caught (not lab infected) Aedes aegypti mosquitoes was a divergent virus in 1966 in Malaysia.[2,3] In that study, culture of infectious virus was also achieved. In Mexico - only ZIKV RNA was detected. It is also possible that the virus sucked up ZIKV from an infected animal or human but that these particular Ae.albopictus might not able to effectively infect other animals or humans. We'd need to know whether the mosquito testing was of whole mosquitoes or mosquito midguts, thoraces, heads, legs, saliva or salivary glands (this being the money part of the mozzie as it shows the virus infected the mosquito, left the midgut and spread, ending up in the part that will produce the infectious spit that is vomited while feeding on the animal). 

Detection in midgut only indicates ingestion whereas finding virus in the legs for example, indicates disseminated infection of the mozzies (ingested, infected, whole body spread). Whole squashed mozzies alone = more work needed. Nonetheless - this is a very valuable finding and its great to see someone is pursuing this and succeeding. And talking about it.

Up until this point we'd been assuming mosquitoes were the major transmitter because they had been in the past - as far as we knew. Now we have a little hard data.


  3. Genetic Characterization of Zika Virus Strains: Geographic Expansion of the Asian Lineage

Did Zika virus detections peak in Colombia during Week #3 2016?

Does the graph below, taken from the Colombian Ministry of Health's latest (Week No. 15) epidemiological report, signal that Zika virus detections (red bars; laboratory confirmed) peaked in Week No.3 of January-2016?

Excerpted from [1].
If it does, then we should add to the hypothetical list [2] of reasons why there is not yet any surge of microcephaly and central nervous system disorder diagnoses (M&CD) in Colombia - the country with the second biggest reported number of Zika virus detections - that we may have to wait for at least 184 days from Week No.3 (give-or-take, obviously). 

In other words, around Week No.29 we may see an equivalent surge in M&CD diagnoses in Colombia....all other things being equal between the two countries (which they likley are not) and if Zika virus is the driving cause of M&CD. This graph comes from Week No.15.

Why 184 days? Only because that was the length of time between when Brazil first reported local (autochthonous) transmission of Zika virus and its first report of the possibly linked M&CD surge (in which it bundling 141 cases up in one announcement).[3] 

Yeah, it's a flexible number to be sure but it is what it is, and it's more solid than some of the data flying around.

Worth watching anyway.

Sunday 24 April 2016

Colombia Zika virus report, Week No. 15...

The latest epidemiological report, which includes data on Zika virus disease (ZVD; 10APR2016-16APR2016), has been produced by the Colombian National Institute for Health team.[1]

Graph No.1. The cumulative curve of confirmed ZVD cases 
(green circles, left-hand axis) and the change in confirmed ZVD case 
numbers when compared to the preceding week's total 
(green bars, right-hand axis). Data from [1]. 
Click on graph to enlarge.

Graph No. 1 shows that zero new laboratory confirmed cases of ZVD reported this week. The total holds steady at 3,292 or 5% of all the clinically suspected Zika virus (ZIKV) detections. I wonder if this is further support for a theory that there is a lag in laboratory reporting in Colombia, or was there was no testing conducted (seems unlikely)?
Graph No.2. The cumulative curve of suspectedZVD cases
(pink circles, left-hand axis) and the change in suspected ZVD case
numbers when compared to the preceding week's total 
(red bars, right-hand axis). Data from [1]. 
Click on graph to enlarge.
Graph No. 2 shows the change in suspected cases. Most of these are not laboratory confirmed, but (I'm presuming here) include those that were from Graph No. 1. The suspected ZVD cases keep accumulating - another 3,059.
Graph No.3. The cumulative curve of confirmed ZIKV infections 
(lilac circles, left-hand axis) and the change in confirmed ZIKV infection 
numbers when compared to the preceding week's total 
(purple bars, right-hand axis). Now added the reported umber of microcephaly cases 
confirmed as ZIKV infected (yellow bars, right-hand axis). To account for adjustments 
that take cases away when there is no weekly case growth, a negative 
value - the y-axes now allow for negative values. Data from [1]. 
Click on graph to enlarge.
Graph No. 3 shows that to Week No. 15, 11,099 suspected and 1,703 confirmed ZIKV infections have been identified in pregnant women. Last week there were 1,706 confirmed diagnoses so the purple bar for this week strays into negative territory (-3 compared to last week).

As of this report, 4 (increased by 2 from last week) live births have been diagnosed with microcephaly/central nervous system disorders and were reported as being ZIKV positive; 22 (up from 15) other microcephaly diagnoses are under investigation.[1]


Saturday 23 April 2016

On Zika and microcephaly: causality, consensus and checklists....

Over the past month the World Health Organization (WHO) and then the United States Centers for Disease Control and Prevention (CDC) have associated infection of by Zika virus (ZIKV) with cases of microcephaly and central nervous system disease (M&CD).[1]
The first WHO statement cited a scientific consensus identified at a meeting called to examine the evidence linking ZIKV infection with foetal malformations and neurological disorders.[1] Later in March the WHO pointed to case studies as the origin for that scientific consensus.[2] I've talked about my views on gaps in some of these studies before - for example here and here.

The CDC based their much stronger comments on a review of the literature and its application to address historically robust checklists - Shepard's criteria and the Bradford Hill criteria - that have heretofore proven themselves useful to identify teratogens (causes of embryonic malformation).[3] The CDC authors of this paper note that there is no "smoking gun" at this point and Dr Tom Frieden, CDC Director said it could take years before answers to other questions are found.[12] But not about ZIKV causing microcephaly. That question is answered according to two of the world's leading public health agencies. 

Checking the checklists.

The checklists CDC used were applied to resolve the questions around the cause of an apparent surge of M&CD diagnoses - the so-called congenital Zika syndrome.[4,5] To date this has been almost exclusively occurring in north east Brazil. Some instances have been reported in other countries with current - or past - ZIKV epidemics, but the numbers are small enough for questions to linger about whether these represent part of "normal" M&CD figures, perhaps brought to attention because of the enhanced focus on congenital deformities in 2015 and 2016 or ZIKV-caused disease.

Shepard noted in 1994 that..

"the rare malformation/rare exposure 
or case report method is far easier, 
less expensive, and more common than 
full epidemiologic studies."

He described "Examples of this rare defect/rare exposure "proof" (or better stated strong association)"[sic] including the virus driven congenital rubella. I'm not sure about rare in terms of ZIKV infection tough. While data are near on-existent from Brazil, we've been told that over a million people have been infected with ZIKV.[19] That seems to be a much less stringent use of Shepard's criteria than that of the CDC's "no longer any doubt".[12] Shepard's criteria have gone on to be used in legal definitions [7] which also take a broader view on causality in the study of teratology..

"causation is demonstrated between an 
exposure and an outcome if the outcome 
would not have occurred but for the exposure. 
The but-for test is typically modified by a 
substantial factor test, that is, the 
exposure was a substantial factor in 
bringing about the outcome, or by 
consideration of the exposure as a 
contributory cause"

The other checklist was the tabulated criteria of Bradford Hill, described in 1965 in his occupational medicine-focussed paper, The Environment and Disease: Association or Causation? which sought to relate sickness, injury and conditions of work.[18] I find Bradford Hill's comments in the Experiment section of his criteria interesting as they discuss whether a preventative action in fact reduces the frequency of the event...

"Here the strongest support for the
causation hypothesis may be revealed."

The CDC interpret this wholly in relation to an animal model.[3] Perhaps this meaning has evolved in subsequent analyses that I admittedly haven't read, but I see this criterion differently. To me it is addressing the need to wait and watch for any impact on reduced mosquito breeding and presence either because of seasonal variation or human interventions; a long wait.

In a statement similar to Koch's about his postulates, Bradford Hill stresses that none of these criteria can be used as if they were set in stone and none bring indisputable evidence for or against a cause-and-effect hypothesis. They are intended to..

"help us to make up or mins on the fundamental 
question - is there any other way of 
explaining the set of facts before us, is 
there any other answer equally, or more, 
likely than cause and effect?"

This seems - to me at least - at odds with the CDC's strident use of these criteria to define causality here.

Neither checklist necessarily hits the mark perfectly for what we're seeing in Brazil but, as with Koch's original postulates, these have been "matured" and tuned over time to fit the need of the moment-presence of a common virus infection causing a rare syndrome. 

Is the strong language essential to a response?

But let's back quietly out of the courtroom and return to the world of science, research and causality. We don't have a smoking gun but that has not prevented some important triggers being pulled. Principal among these was that the WHO called a Public Health Emergency of International Concern (PHEIC) on the 1st of February 2016.[8] Well before the need to use stronger language in March 2016, the PHEIC generated recommendations [9] for...
  • To interrupt ZIKV transmission using enhanced surveillance, diagnostics, risk communication, vector control measures, counselling and more
  • research and develop vaccines, therapeutics and diagnostics and increases  relevant services in affected areas
  • provide uptodate advice on travel to affected areas, disinfection of aircraft but do not restrict travel or trade
  • ensure rapid and timely reporting and sharing of information of public health importance relevant to the PHEIC
Obviously those things are expensive. We know from recent experience that the WHO struggled to get the pledged funds they'd requested to mount for an effective Ebola virus disease reposes fast enough and to match the requested spend. Perhaps stronger language is intended to free up the purse strings.[10,11] The main sigh of relief outcome from making such a strong statement by the CDC was...

"Now that we've determined the causal the 
relationship, we can use this information to 
redouble our efforts to prevent Zika, more 
narrowly focus our research and communicate
 even more directly about the risks of Zika."

And herein lies one of my concerns. Narrowed research, by definition, could miss things that have contributed to congenital Zika syndrome. Things that might include...
  • other viruses - rubella and cytomegalovirus are teratogenic viruses that are sometimes sought and not often found but that search can use a hodgepodge of methods. But what about new viruses and new variants of existing viruses? 
  • the impact of chemicals or toxins - the pesticide issue has not gone away [13]
  • the very complex immune responses that to date have mostly been a topic for discussion as a problem for antibody detection in the lab, but may be a part of the process [14,16] although did not seem to play a role in Guillain-Barre syndrome [15]
But all those things may still be included in a narrowed research focus. Those things aside we do know that ZIKV loves to grow in epithelial cell-derived neural stem cells; there is a lot of IgM antibody to ZIKV in babies born with microcephaly [20] and ZIKV has been found in the brain tissues of foetuses with disease.[21]

Devil's advocate - what other things might we consider?

We have not yet addressed whether ZIKV is just as harmless as we used to think it was and whether it is found in these tissues as a passenger and not a pathogen. 

Might it also be in the brain tissues of ZIKV-infected foetuses who do not develop any congenital anomalies? There has been little or no exploration of controls in most papers to date. Mostly - this would be unethical, but there might be other reasons for related tissues to be sampled which could then be leveraged fro important testing. Its important yet missing information.

Excerpted from WHO Zika virus microcephaly
and Guillain-Barre syndrome situation report. [17]
There is also the Colombian elephant in the room. 

This week saw the number of M&CD diagnoses in Colombia double...okay, from 2 to 4... having decreased the week before. This might be normal and part of the 140 annual cases reported in Colombia annually-that's an average of 2.6 per week. 

More time has now passed in Colombia than had in Brazil before Brazil reported its first concerns - and 141 M&CD diagnoses - over ZIKV and M&CD, after it identified local ZIKV transmission. As I've discussed previously, there could be many reasons for that difference - and a rise in cases in Colombia this week may herald that the starting line has been crossed indicating the beginning of some important supporting evidence for ZIKV causing M&CD. Or we might just be seeing normal levels of M&CD that also happen to be ZIKV infected-during an epidemic of ZIKV.

To my mind, the studies used to check off the lists leave some important things unanswered. Patience might have been advisable since the previous announcement of a PHEIC mean that there was no obvious need for the issue to be forced. Or perhaps this is more about the politics of finding a better way to secure the funding needed to address congenital Zika syndrome, and avoid the pitfalls of funding gaps dug during the fight to contain Ebola virus syndrome.

Only time will tell whether such strong WHO and CDC language was needed, helped or even hindered the response and understanding of congenital Zika syndrome. 


  1. WHO Director-General briefs the media on the Zika situation
  2. Zika situation report 31-March 2016
  3. Zika Virus and Birth Defects — Reviewing the Evidence for Causality
  6. "Proof" of Human Teratogenicity
  7. Causation in Teratology-Related Litigation
  18. The Environment and Disease: Association or Causation?