Monday, 15 February 2016

Zika virus: pathology, postulates and questions...

Zika virus (ZIKV [1]) infection usually results in a mild outcome for the mosquito-bitten human host. 

Only 18% of probable infections had related symptoms in a previous outbreak.[24] Despite 82% having no obvious disease, virus replicates happily enough in us for there to be enough of it circulating in our blood (a viraemia; vy-ree-me-yah) that from around a week later (3-12 days), another protein-seeking female mosquito can consume some of it and become infected herself. And thus the cycle continues.

There may be some much less frequent possible methods of human-to-human infection. These may include sexual transmission,[2,16,17,18] perinatal infection [3] and blood transfusion.[4] Recently, viral RNA had been detected from the amniotic fluids of two pregnant mothers [3] and from the brain of at least 5 foetuses as well as the placental tissue of 2 of those.[13,14]

Bigger outbreaks meant more chance to see rare diseases...

While usually mild - a feature entirely based on relatively few confirmed cases up until 2013 - understanding and awareness about what constitutes ZIKV disease (ZVD) begun to change after this. At this time there were concomitant outbreaks of ZIKV infection and Guillain-Barr√© syndrome in French Polynesia,[5,8] first noted in October 2013.[7] This outbreak was estimated to have involved 19,000 cases, making it the largest outbreak recorded to that point; 294/584 patient sera (50%) contained ZIKV RNA.[7]

Size does matter here because when there are only a few or even a few hundred cases, less frequent disease complications may not show up. This point is very relevant to the complication that is today being "linked" and "associated" with the epidemic of ZIKV in Brazil; a diagnosis of microcephaly.[1] These two separate, or perhaps linked, events have been co-occurring in Brazil during 2015 and there are now data to suggest that microcephaly diagnoses of 150 prior to 2015 were an underestimate, with diagnoses occurring in much greater numbers, well before Brazilian health authorities declared an emergency.[9,10,25] 

In fairness, ZIKV may also have been present well before 2015 - but laboratories may not have tested for it. This is also a question for other countries reporting their "first" local transmission of ZIKV. Phylogeography estimates suggest that ZIKV may have arrived in Brazil around 2013-2015,[11,12] but these are based on the sequence data to hand and could be affected by the discovery of genomes from months or years earlier from stored sample material for example. Also, some existing gene and genome sequences come from viruses that have been passaged though cell culture and animals so many times that they may not accurately reflect the virus that comes out of a mosquito.

Associating infection with disease means more than the dictionary definition..

In the olllld days (ya know, before smartphones) a thing called Koch's postulates was used as a guide to whether a bacterial infection (these weren't developed with viruses in the picture) was the cause of disease in an infected person. A 1976 summary by Alfred S. Evans [15] noted that these postulates were a check-list requiring that...
  1. The parasite occurs in every case of the disease in question and under circumstances which can account for the pathological changes and clinical course of the disease.
  2. The parasite occurs in no other disease as a fortuitous and non-pathogenic parasite.
  3. After being fully isolated from the body and repeatedly grown in pure culture, the parasite can induce the disease anew.
A few obvious problems may jump out to some of you - for virus infections at least. For example..
  1. Detection methods today are mostly based upon the detection of viral genetic material (polymerase chain reaction [PCR]-based techniques) which can detect virus at levels which could never be detected using culture-based methods. But these are not detection of infectious virus. They are detections of bits of genetic material. Also, many viral pathogens discovered in recent times have not grown, or not grown easily, in the cell culture and other culture-based techniques used. On top of that, virus isolation in cell and tissue culture has become a dying art form, with broad speciality left mostly in reference laboratories
  2. We now know that the many viruses may cause a spectrum of disease ranging from severe disease states through to infection that does not yield any obvious or measurable sign or symptom of disease at all
  3. We simply cannot replicate disease using humans, and often it is hard to find an animal model that accurately represents the disease course experienced in a human. This can mean that the results we get from infecting an animal are misleading or just plain irrelevant
Once viruses were discovered and more became known about our immune system, then that original check-list of Koch's became more of a wishlist.[19]

Moving to the molecular..

Some "Molecular" postulate variants emerged from the late 80's onwards (still in the ollllld days!). In 1988, Stanley Falkow highlighted that Koch's postulates were not meant to be taken as dogma and liberally applied to everything, rather they were intended as a starting point for broader conversation, to encourage more rigorous thinking and evaluation and to convince the sceptics of the day that bacteria caused disease at all.[20,21] For example this version to which I've added some comments about how well this has been addressed for the proposed ZIKV/microcephaly...

  1. A nucleic acid sequence belonging to a putative pathogen should be present in most cases of an infectious disease. Microbial nucleic acids should be found preferentially in those organs or gross anatomic sites known to be diseased (i.e., with anatomic, histologic, chemical, or clinical evidence of pathology) and not in those organs that lack pathology.
    We're nowhere on the first part-too little testing, but in a very few instances we have seen evidence f ZIKV in brain tissue - the site of damage in microcephaly diagnoses 
  2. Fewer, or no, copy numbers of pathogen-associated nucleic acid sequences should occur in hosts or tissues without disease.
    Fail. 80% of infection are without noticeable disease if it couple of studies are to be believed
  3. With resolution of disease (for example, with clinically effective treatment), the copy number of pathogen-associated nucleic acid sequences should decrease or become undetectable. With clinical relapse, the opposite should occur.
    There is no resolution of microcephaly disease as the major structural changes are permanent. In "normal" ZVD, viraemia resolves in a week, urine clears in 20 days and while semen may harbour ZIKV genetic sequences for 62 days or more, there does not seem to be accompanying disease.[26,16] Relapse has not yet been documented.
  4. When sequence detection predates disease, or sequence copy number correlates with severity of disease or pathology, the sequence-disease association is more likely to be a causal relationship.
    Perhaps - it is usually the case that higher viral load correlates with overt disease but more work is needed to investigate these correlations for ZIKV. There was at least as much viral RNA in brain tissue from a terminated foetus, as is usually found in blood.[13]
  5. The nature of the microorganism inferred from the available sequence should be consistent with the known biological characteristics of that group of organisms. When phenotypes (e.g., pathology, microbial morphology, and clinical features) are predicted by sequence-based phylogenetic relationships, the meaningfulness of the sequence is enhanced.
    Little is known about ZIKV lineages and whether they impart change in virulence or clinical course so this is not able to be ticked off either.
  6. Tissue-sequence correlates should be sought at the cellular level: efforts should be made to demonstrate specific in situ hybridization of microbial sequence to areas of tissue pathology and to visible micro-organisms or to areas where micro-organisms are presumed to be located.
    Not been done yet.
  7. These sequence-based forms of evidence for microbial causation should be reproducible.
    As described above, only a few cases have been thoroughly examined - and even they have significant gaps in testing. There has not been any study controlled by testing of a population of non-disease individuals to date.
Postulates can help discussion but have they found an association...?

Sadly, neither the original nor the modified postulates help us find the path to proving ZIKV can, or cannot, cause microcephaly. This is because we have seen little confirmatory testing data from Brazil, we most often detect ZIKV in mild or asymptomatic people, we have yet to develop a clinically relevant animal model and there is no way a pure culture of ZIKV is going to be injected into any mums-to-be to replicate any chance of seeing central nervous system disease in their foetus/infant. Plus those reason in red.

Finding answers will not be an easy journey, but in the meantime a lot of important responses addressing the ZIKV threat - if one exists - have been mobilized. How much work is being done by and within Brazil remains to be communicated more clearly. There is not a lot of information coming out to fill in the blanks and of course that leave idle the tinfoil-encompassed minds of some. Voids get filled by whatever fits. 

Questions remain...

There remain a few of voids around the ZIKV/microcephaly topic that I can and have listed below...
  • How long has ZIKV been in Brazil and in what numbers?
  • What are the real (not affected by extra attention/poor reporting) rates of microcephaly in Brazil?
  • Do real rates of microcephaly diagnoses vary by region/state?
  • What are the real rates of microcephaly in countries other than Brazil?
  • How many microcephaly diagnoses have been infected with ZIKV? How many infections by other viruses including Dengue virus (DENV)?
  • Is microcephaly a seasonal diagnosis? What other possible causes of microcephaly also occur with a seasonal pattern? Are DENV and Chikungunya virus (CHIKV) and other mosquito-borne virus infections seasonal? Is ZIKV seasonal?
  • Is mosquito treatment and/or mosquito breeding in this region seasonal?
  • Apart from Pyriproxyfen, what other pesticides are in use and are they all equally safe?
  • What has been excluded from having a role in causing a GBS or microcephaly diagnosis so far? What are the data supporting this?
  • What about other chemicals that have been associated with microcephaly, for example Isoretinoids in cosmetics [22]
  • What is the tissue distribution of the ZIKV receptor(s) and are we happy we know what that receptor(s) is/are [23]?
  • Does ZIKV cross the blood-brain and placental barriers in the absence of microcephaly?
  • What does ZIKV do in foetal, or infant/child/adult, brain tissue? Is it destructive or inflammatory and does it constantly produce new virus or does it enter some type of latency?
  • How long does ZIKV persist in foetal tissues?
  • Are there long-term disease consequences from ZIKV infection of brain tissue that stretch into adulthood?
  • Does ZIKV persist in the adult central nervous system or anywhere else apart from semen (and what is the upper limit of persistence in semen)? Do DENV, CHIKV, JEV etc persist?
  • Are the mutations observed between ZIKV lineages and within lineages important for viral virulence, transmissibility or for the clinical course of disease?
  • Is ZIKV present in and/or able to persist in the eye (during or after conjunctivitis) or in other organs?
  • Are there mums with evidence of past ZIKV infection who have delivered completely healthy babies? Do these babies have any other issues?
  • How has the study of ZIKV infection in mums been done - by unbiased selection and testing or testing only those with symptoms?
  • When would we expect to see signs of microcephaly among pregnant mothers in Colombia and other countries with large outbreaks of ZIKV?
I'm sure you have your own questions as well.

  1. Some text colour and formatting corrections