Saturday, 30 November 2013

Parechovirus infections in babies in New South Wales, Australia

UPDATE #1. 10AUG2016
The parechoviruses (HPeVs; par -echo-virus) don't make the headlines too often. 

I'm not sure why but I expect it is mostly because they are (a) not frequently sought and (b) not frequently found. That last point is a matter of perspective though as we see in a report on the ABC news site today. 

NSW health reports 20 babies (<16-weeks of age) as being positive for an HPeV (?typed). What this cluster's symptoms are is unclear but the HPeVs have often been found in people (often notably in young children) with fever, cough, acute gastroenteritis and rash as well as liver and central nervous system symptoms. They have been linked with nosocomial (within hospital) infection acquisitions.


Click on image to enlarge.
Genomes drawn to scale using Genbank sequences. 

These are viruses might be important in presentations
or outbreaks of paediatric fever of unknown origin.
Feel free to use the graphic, just cite this website
and Assoc.Prof Ian M. Mackay.
The human parechoviruses comprise 16 members of the family Picornaviridae, genus Parechovirus, species Human parechovirus. They are not a "new virus" as the ABC news report stated, but have been known of since the 1950s when they were thought of as enteroviruses called echovirus 22 and 23 (ECHO-enteric, cytopathogenic, orphan ; now HPeV-1 and HPeV-22). The first complete polyprotein sequence of HPeV-1 was lodged on GenBank in 1992 and from 1991 it was noted that a redefinition of these viruses into a separate genus may be more realistic3,9,11 because of a range of genetic and biological features that differentiated them from other enteroviruses.

A small research grant I had some years back funded a study which also detected and reported on parechoviruses (HPeVs) in a retrospective, hospital-based PCR screening study investigating mostly nasopharyngeal aspirate specimens collected in 2003 from around South East Queensland, Australia. We reported this in 2006. We found 2 instances among 315 patient specimens (prevalence of 0.6%). We didn't type the HPeVs at that stage. Our energies were directed toward some weird rhinoviruses we'd found (later to be known as RV-C); we were trying to work out just how weird they were. Both HPeVs were co-detected in samples from female children (an 8-month and a 1-year, 7-month old) along with a bocavirus; 1 also had another picornavirus, human rhinovirus (3 viruses in total) detected and both had cough on their admission slip.
The HPeVs are ubiquitous, more prevalent during the summer and they usually infect children under 3-years of age. All can be detected using real-time RT-PCR methods and can be grown in Vero cell culture (I haven't had much luck myself!). There is still a lot of room to define causal links between detection and disease, among other things.

As ever, hand-washing, especially by kids,  plays a major role in blocking the transmission chain.

Most cases of infection resolve within days - but if those days are spent with your feverish baby lying in hospital bed and a lab diagnosis not available to you or your Doctor....those can be the longest days of your life...and the nights are made much darker by the possibilities that churn through your mind.

So it's great to see these extended molecular (PCR-based) testing panels being used by labs to seek out the causes of disease.

h/t to FluTrackers for noting this story and the page they have on it at http://www.flutrackers.com/forum/showthread.php?p=515648&posted=1#post515648/ Also thanks to my excellent student, Donna McNeale, for pointing out an error (now corrected) about the date of early mentions of the need for a new genus.

Other reading on parechoviruses...
    1. Reference to the genus: http://vir.sgmjournals.org/content/79/4/649.long
    2. [First ICTV inclusion of genus] King, A. M. Q., F. Brown, P. Christian, T. Hovi, T. Hyypia¨, N. J. Knowles, S. M. Lemon, P. D. Minor, A. C. Palmenberg, T. Skern, and G. Stanway. 1999. Picornaviridae, p. 996. In M. H. V. Van Regenmortel, C. M. Fauquet, D. H. L. Bishop, C. H. Calisher, E. B. Carsten, M. K. Estes, S. M. Lemon, J. Maniloff, M. A. Mayo, D. J. McGeoch, C. R. Pringle, and R. B. Wickner (ed.), Virus taxonomy. Seventh Report of the International Committee for the Taxonomy of Viruses. Academic Press, New York, N.Y. 
    3. Harvala H, Simmonds P. Human parechoviruses: biology, epidemiology and clinical significance. J Clin Virol 2009 May;45(1):1-9. 
    4. Tapia G, Cinek O, Witso E, Kulich M, Rasmussen T, Grinde B et al. Longitudinal observation of parechovirus in stool samples from Norwegian infants. J Med Virol 2008 October;80(10):1835-42.
    5. Al-Sunaidi M, Williams CH, Hughes PJ, Schnurr DP, Stanway G. Analysis of a new human parechovirus allows the definition of parechovirus types and the identification of RNA structural domains. J Virol 2007;81(2):1013-21.
    6. Benschop KSM, Schinkel J, Luken ME, van der Broek PJM, Beersma MFC, Menelik N et al. Fourth human parechovirus serotype. Emerg Infect Dis 2006;12(10):1572-5.
    7. Nix WA, Maher K, Johansson ES, Niklasson B, Lindberg AM, Pallansch MA et al. Detection of all known parechoviruses by real-time PCR. J Clin Microbiol 2008 August;46(8):2519-24.
    8. Sedmak G, Nix WA, Jentzen J, Haupt TE, Davis JP, Bhattacharyya S et al. Infant deaths associated with human parechovirus infection in wisconsin. Clin Infect Dis 2010 February 1;50(3):357-61.
    9. M. Steven Oberste, Kaija Maher, Mark A. Pallansch. Complete sequence of echovirus 23 and its relationship to echovirus 22 and other human enteroviruses. Vir Res 1998 56(2): 217-23
    10. Arden KE, McErlean P, Nissen MD, Sloots TP, Mackay IM. Frequent detection of human rhinoviruses, paramyxoviruses, coronaviruses, and bocavirus during acute respiratory tract infections. J Med Virol. 2006 Sep;78(9):1232-40.
    11. B-A. G. Coller, S.M. Tracy, D. Etchison. Cap-Binding Complex Protein p220 Is Not Cleaved during Echovirus 22 Replication in HeLa Cells. J Virol 1991 65(7):3903-05
      http://jvi.asm.org/content/65/7/3903.long
    12. T Hyypia, C Horsnell, M Maaronen, M Khan, N Kaljkkinen, P Auvinen, L Kinnunen, G Stanway. A distinct picornavirus group identified by sequence analysis. PNAS. 1992 89:8847-51
      http://www.pnas.org/content/89/18/8847.long
    Updates...

    1. Amended to note that one Echovirus 23 (CT86-6760) is not HPeV-2, but a misclassified HPeV-5 - Al-Sunaidi et al. J Virol 81;2. 1013-1021

    Friday, 29 November 2013

    Rhinovirus (RV) transmission by aerosol: does it happen or is transmission solely by hand-contact and self-inoculation?

    I'll be writing a few posts over the coming weeks based in the papers I've found on this topic of RV transmission. How applicable these study results are to transmission of other respiratory viruses is unknown.

    The focus will be on answering the question of "Do rhinoviruses transmit by an aerosol route?" The endpoint is usually the development of a clinical upper respiratory tract infection (URTI) or "common cold". 

    A lot of volunteer human infection experiments have been conducted using RVs since their identification 60-years ago. This is likely because RVs were seen to cause only mild illness, reducing the health risk for human volunteers. Less common were influenza studies of this sort (correct me if I'm wrong though). It's also worth noting that adults rather than children were included, so the true spectrum of RV disease was not observed. 
    From Elliot Dick et al, the Journal of
    Infectious Diseases
     

    Author: Elliot Dick et al
    Journal:  J Infect Dis 156(3):442-448
    Year: 1987
    RV type used: RV-A16
    RV receptor type: major group; ICAM-I

    This study set out to see whether RV was transmitted by aerosol, indirect contact, or both.

    Key features of the study layout..

    • 27-34 males >18-years of age were inoculated intranasally with 56-2,500 TCID50 of safety tested1 RV-A16 on 2 successive days.
    • 8-days after inoculation, the 8 cases with the most severe URTIs played cards with 12 RV-B16 neutralizing antibody-free males for ~12-hours in a room containing 4 tables spaced 1.4m apart.
    • Each table seated 2 "donors" and 3 "recipients" and the recipients moved locations each hour. Donors were replaced with fresh donors if their URTIs waned
    • Coughs, sneezes, nose blows and hand-to-face movements were monitored
    • Acquisition of a separate infection during meal times was eliminated by staggering their egress and entry into the card-playing room and by seating recipients 50ft (15m) apart in a well-ventilated room
    • 4 experiments, A-D, were performed.
      • Experiment A-C tested aerosol transmission.
        • 6/12 males used cloth handkerchiefs; the remaining 6 were restrained from any hand-to-head movements
        • In experiment A, a 3ft (1m) plastic collar was worn around the neck
        • In experiment B and C, arm restraints were used
      • Experiment D utilised Experiment C's contaminated furniture and card playing equipment, moving it all into a 2nd card playing room. 
        • 12 new recipients were immediately introduced to the room for 12-hours of poker with exaggerated hand-to-face movements
        • Card-playing equipment was exchanged between rooms each hour to keep the contaminant levels high
        • All meals were eaten in the experiment room to avoid contact with any donors
    • After the 12-hour game, recipients returned to the laboratory each day for 2-weeks to provide nasal washings and record symptoms. If they were symptomatic they were taken to a separate laboratory for sampling.
    • Exaggerated exposures of "sentinel" recipients consisted of recipients present during the donor's nasal wash collections or undertaking nasal washing alongside symptomatic recipients
    • Nasal washing were collected into Hanks balanced sslt solution (HBSS) medium with 0.5% gelatin and inoculated onto WI-38, Hep-2 and primary rhesus monkey kidney cells within 4-hours after collection
    Key results included...
    • Experiment A: 11/2 recipients were infected, 5 by aerosol alone
    • Experiment B: 6/12 infected, 1 by aerosol alone
    • Experiment C: 5/12 infected, 4/5 in the restrained recipients
    • 12/18 (67%) control recipients (could be infected by any route) were infected versus 10/18 (56%) restrained recipients
      • infected recipients were symptomatic and shed virus for ≥1-day 
    • Experiment D: no infections but 5/8 donor hands yielded culturable RV-A16 virus while none of the recipient's hands did
    • No sentinel recipients became symptomatic
    The authors concluded...
    • Aerosol transmission was the most important  mechanism of natural spread of RV in adults in this study
    • Aerosol transmission was nearly as efficient as transmission by combined aerosol/direct contact/indirect contact
    • RV-A16 load declined rapidly to near zero on the journey between donor and the nose of the recipient.
    • Virus shedding in a recipient was usually first detected 3-days after proximity to the donor

    The authors raised some interesting points...
    • Some previous studies to defining that RV transmission was primarily due to fomite and droplet contact may have failed to detect a small and large aerosol modality because recipient exposure was too short or to too small a viral inoculum
    • In a previous study by these authors, the donor had to have a mild to moderate URTI, in which they shed ≥103 TCID50/ml, before transmission reached the desired endpoint
    • Brief, casual exposures to an infected RV case infrequently results in adequate transmission as measured by occurrence of a symptomatic episode
    •  Exposure by direct inoculation with fresh nasal secretions is practically unlikely
    Further reading and references...
    1. Safety testing of RV preparations.
      D'Alessio et al. J Infect Dis. 1976;133:28-36.

    Stuff from the literature: very SARS-like coronavirus in Chinese horsehoe bats...

    The smoking bat for SARS-CoV?
    Xing-Yi Ge and colleagues from China, USA, Australia and Singapore described some new severe acute respiratory syndrome (SARS)-like coronaviruses (SL-CoVs) in bats, publishing in Nature last month.

    These discoveries were especially notable (not that any new virus discovery isn't) because they displayed more "SARS-like" properties than many earlier so-called SARS-like CoVs. One could grow in the same line of lab cells and also in human cells, it could be visualized by electron microscopy and it could use the same receptor as the SARS-CoV (angiotensin converting enzyme II; ACE2) . Plus, they were genetically very similar.

    The bat species was confirmed by gene sequencing to be Rhinolophus sinicus, family Rhinolophidae; the Chinese rufous horseshoe bat.

    Throat and faecal samples (anal swabs and faeces) were screened using RT-PCR with primers towards the conserved RNA-dependent RNA polymerase region (RdRp) and new primers were designed to detect other regions of any discoveries. 27 of 117 samples were CoV POS and had sequences determined.

    Two novel (and 5 previously identified) SL-CoVs, each with a 29,787+ base pair RNA genome and sharing 95% nucleotide identity with the Tor2 strain of the SARS-CoV which is higher than previous SL-CoVs from China. The receptor binding domain (RBD) of the new CoVs shared 85-96% amino acid identify with the SARS-CoV. These were called:

    1. RsSHC014
    2. Rs3367

    Vero cells were used to attempt growth of SL-CoV virions that were first concentrated from samples. This succeeded for one sample, a variant of Rs2267 (99.9% nucleotide identity with Rs3367) and they named this isolate SL-CoV-WIV1. This success is something that hasn't been achieved with the majority of recently identified bat CoVs.

    WIV1 also grew, although less efficiently, in:

    • human alveolar basal epithelial (A549) cells
    • pig kidney (PK-15) cells
    • R.sinicus kidney (RSKT) cells
    ...but not in...

    • Human cervix (HeLa) cells
    • Syrian golden hamster kidney (BHK21) cells
    • Myotis davidii kidney (BK) cells
    • Myotis chinensis kidney (MCKT) cells
    • Rousettus leschenaulti kidney (RLK) cells
    • Pteropus alecto kidney (PaKi) cells
    So we have much more convincing evidence that the SARS-CoV is likely to have originated from a bat.

    h/t to @MERS_inSAUDI

    Thursday, 28 November 2013

    Dutch researchers in collaboration with Qatar are at work sequencing MERS-CoV from camels...

    And from the WHO comes confirmation of some of my earlier bits and pieces about the MERS-CoV in camels story from earlier....


    Further, some very interesting titbits from a Twitter exchange this evening.

    Firstly Prof. Marion Koopmans, Head of Virology at the Laboratory for Infectious Diseases of the National Institute of Public Health in the Netherlands confirmed that this was the MERS-CoV and not something requiring lengthy sentences filled with "probable" and "MERS-CoV-like"...


    ..and that for the most useful conclusions to be drawn from any sequencing being undertaken..


    ..but that despite all sorts of great leaps in technology, not to mention in distance-spanning scientific collaborations, things don't just happen overnight. 

    We should all be mindful that there are many steps between taking a (hopefully adequate) sample(s) from a human or animal, and reaching any useful conclusion about how the molecularly characterized virus might have travelled (human to dromedary, vice versa or via some other vector or intermediate)...


    As Prof Andrew Rambaut, Institute of Evolutionary Biology, University of Edinburgh, noted...


    And on the subject of whether the new sequences will lead to an indication of which direction this particular cluster of infections is travelling i.e. from human-to-camel or camel-to-human, Prof. Rambaut had this thought on following the viral genome's sequence variations (polymorphisms)...


    This is all really great to watch. A fast and fruitful collaboration between sample holders and laboratory researchers, expert in their fields.
    Click on image to enlarge.
    Those POS for a fragment of MERS-CoV or
    MERS-CoV-like virus sequence are highlighted
    in red. Whether there are other intermediates
    remains to be confirmed.

    At this point, I believe (and it is just a belief) that the camel is looking good for a source of MERS-CoV acquisition by humans. Is it an endemic camel virus? Well, we still have the knowledge that bats seem to harbour a lot of CoVs, and there is that pesky Taphozus perforatus sequence discovered from earlier in the year. It looked an awful lot like a fragment of the MERS-CoV genome. Baboons - I'm holding out for them to be the link between bats and camels...but that is a hope in the absence of any data whatsoever!

    Today's confirmation of a cluster of 3 POS camels among 14 represents 21% of the animals POS in a single area. 

    If we consider this to be human-to-camel transmission, then this would be a much steeper proportion of positives than we normally see when we look at studies of close contacts of human MERS cases. Camels must be very susceptible to MERS-CoV infection because human contact testing just does not show this level of onward transmission. More susceptible to humans? No, I think we're getting closer to confirming that it's a camel-to-human thing...but we are not there yet.


    Work continues, but today was a significant day and one in which I give thanks for the ability of people from all over the world to work together towards common goals in preventing human disease. 

    New influenza A(H7N9) virus case in

    Chinanews reports a new infection by H7N9 today.

    A 57-year old male was confirmed as positive 27-Nov and is in hospital in Hangzhou, the largest city in Zhejiang province. Zhejiang province was the hot spot for H7N9 earlier in the year.

    This is the 141st case of H7N9 which emerged in 2013 in south east China. Most of the recent cases have been in Zhejiang.

    Clustered camel coronavirus cases...

    Adding significant weight to the camel-as-vector hypothesis, stories here,  and here are being reported of a collaborative study between the Qatari Supreme Council of Health, the Qatari Ministry of Environment, the Netherlands’ Health Ministry’s National Public Health Institute, the Erasmus Medical College and the World Health Organization (WHO) that have identified 3 camels that are positive for the MERS-CoV.

    The camels were part of a a farm herd of 14 asymptotic animals that have been linked to 2 previous MERS-CoV cases in Qatar. 

    h/t @Crof, @MERS_inSAUDI, @HelenBranswell

    Wednesday, 27 November 2013

    MERS by the numbers: monthly MERS

    Click on image to enlarge.Cases (numbers on the y-axis; left) including
    deaths (green) and fatal cases (red) are
    shown for each month (x-axis, bottom) of
    2012 and 2013. This includes 68 deaths
    and 161 total cases. The Hajj occurred
    in October of each year.

    These two charts show the number of cases (including deaths) and the number of deaths by month, split between the 2-years we've known the Middle East respiratory syndrome coronavirus (MERS-CoV) to have existed.
    The charts are only as good as the public data they are based on but they give a good idea of what's been happening and what is happening. Cases are not declining [a slow moving epidemic ;) ]

    Numbers are ridiculously small in 2012 (overall really) to conclude anything much but it does look like more deaths happen toward April while more cases occurs around September. I Doubt that would be statistically significant though - just something to watch over time.

    MERS-CoV by the numbers: recent weekly case activity...

    Click on image to enlarge.
    Confirmed MERS-CoV cases including 
    fatal infections (green) and deaths (red) 
    each week. Case numbers are listed on the 
    y-axis (side), days of each week  along 
    the x-axis (bottom). Case dates are 
    derived from announced date of onset 
    but if absent, on the date of reporting. 
    Deaths are listed by date of death.
    This follows on from my previous post (you can track links to earlier weekly charts) about lab-confirmed Middle East respiratory syndrome coronavirus (MERS-CoV) cases, plotted by week.

    Approximately 85% of all cases have come from the Kingdom of Saudi Arabia. Around 63% of all cases with sex data are male (1:1.73, M:F). Among the fatal cases with data it's 75% male (1:3.06, M:F).

    I've added in some previous charts because as the new cases and case details appear, so do the placement of the cases alter slightly. Hopefully, with WHO doing such a good job in providing details, these graphs will solidify and we can move on in the next post. 

    I've marked in the Hajj week and the 14-day outer limit of the incubation period. Nothing much to see from that; no spike in cases, even after time has passed to allow the case data to catch up.

    My tally suggests 161 cases (still awaiting the Spanish case to be confirmed or not) with 68 deaths, a proportion of fatal cases sitting at 42%.
     
    A couple of things stand out to me from these charts...
    1. What is the lag between illness onset and MERS-CoV case announcement like? For example the recent 37-year old man who died was reported on 20-Nov, but became ill 9-Nov. Obviously there is time required to reach hospital (13-Nov) and then be tested and re-tested [confirmed] but this case died 18-Nov and was not reported as MERS-CoV case for 2-days. The case before that, a 65-year old man became ill 4-Nov, was in hospital 14-Nov and was reported 19-Nov. Before that the 73-year old woman became ill 13-Nov, hospitalized 14-Nov, died 19-Nov and was also reported 19-Nov.
      I presume that this indicates there is no active MERS-CoV PCR screening of influenza-like illness, but rather for  in the Kingdom of Saudi Arabia?
    2. There have been 5-8 fatal cases per month since June and 16-25 cases per month in total. However, with that lag, there may be more to come from November. 

    In particular, point #2 makes me wonder if the KSA is settling in to stable (albeit very small numbers of total cases) transmission or acquisitions of MERS-CoV?

    The MERS-CoV case slience has fallen lifted thanks to the WHO

    Well, apart from a blatant Dr Who references, this post is dedicated to providing a huge portion of back-patting for the great job the World Health Organisation (WHO) have done on their recent Disease Outbreak News reports (see yesterday's here). I now have a new colour code in my Excel sheet that indicates "confirmed by WHO" - because its become worthwhile doing that. 

    The current approach to detailed MERS-CoV News posts continue to hold the sort of detail I'd hoped for. 

    Also, congratulations must go to the Kingdom of Saudi Arabia's Ministry of Health (KSA MOH; and at other times, other MOHs from the region) for providing the WHO with these details. As well as a global tally, which may still lag a little behind Ministry or media case announcements because of the time it takes to officially collate and centralize the data from multiple sites (I presume), we now seem to be regularly getting:
    1. Sex
    2. Age
    3. Occurrence of animal exposure
    4. Presence of comorbidities
    5. Date of illness onset
    6. Date of hospitalization
    7. Date of death if a fatal case
    8. Region the case occurred in (still a bit patchy)
    On September 11th I wrote a specific wishlist, revised from an earlier version and from that of Crawford Kilian's memo to the Ministry somewhat to account for patient confidentiality, that included 16 items. The WHO's efforts address most of the items on that list. Well done and keep up the good work!

    My full wishlist, with some amendments, is below. I still feel these extra few bits (in blue) of information would be useful, especially the unique code to globally track cases and some detail on what may have worked to help support the infected patients course. 
    1. A unique, continuous identifying code of KSA cases
    2. Sex of case
    3. Age of case
    4. Possible exposures to animals and other human contacts
    5. Occurrence of comorbidities
    6. Date of illness onset
    7. Date of hospitalization
    8. Date and type of laboratory testing
    9. Date of death if a fatal case
    10. Region the case occurred in 
    11. Date of release from hospital
    12. Treatments or management
    In the meantime, FluTrackers curates the world's best, and most rigorously checked, MERS-CoV case key. Such a stable Rosetta stone of MERS-CoV cases is essential. It provides the world with a solid, unchanging and reliable set point for each case around which our discussions and ideas can revolve. Not the kind of stone out of which a Weeping Angel is made - one that shifts menacingly every time you blink or look away - but one with the dependability of a Dalek's determination to "Exterminate", or of a Sontaran's desire for a good fight.

    Research papers come and go and their conclusions change like a shape-shifting Zygon, but a permanent list of public information on MERS-CoV cases over time is the gold standard against which they can be given important context. 

    The more information we can rely on during times of emerging viral outbreaks (or slow-moving epidemics), the better prepared we are to get in front of them, contain them and not be unnecessarily scared by them.

    Monday, 25 November 2013

    No symptoms but still shedding virus?

    Click on image to enlarge.
    A stylized trace of the temperatures during a PCR cycle.
    D-denaturation, when primers and double-stranded
    DNA (dsDNA) are reverted to single strands of DNA;
    A-annealing, when primers bind to their complementary
    target and DNA re anneals to form dsDNA; E-extension,
    when the DNA-dependent DNA polymerase enzyme
    finds a primer, binds to it attached to a strand of
    template  and makes the complementary strand.
    Feel free to use. Please cite this website and
    Dr I M Mackay as illustrator.
    One of the many questions that remain unresolved for MERS-CoV is whether a human who is PCR-positive for the virus, but does not show signs or symptoms of being sick, can spread that infection on to other humans - or animals for that matter.

    Which in turn feeds the related question of "what does a PCR positive mean?"

    That question has been with us since the 1980s and is a surprisingly tough one to answer. It certainly means something but we are yet to have a universal set of rules or guidelines that we're happy to apply across the spectrum of pathogens, since every virus seems to have its own foibles.

    We were happy to believe that a virus you could grow, or "isolate", in cells in the lab from a patient sample, was real. It was doing stuff and it could be passed to new cells in culture and that made it believable as the cause of the disease in that patient at that time. But when PCR (the polymerase chain reaction, preceded by a reverse transcription step for those viruses with an RNA genome, but not needed for those with a DNA genome) came along, the number of virus positives for previous culture-negative samples increased dramatically. This was due to:
    • Inability to isolate some viruses using the cells of the day
    • Viruses present in very small amounts could not be grown by poorly sensitive cell culture
    • Culture was just not reproducible enough
    • Samples weren't transported carefully enough to keep virus alive for culture
    The length of time a person is positive for a virus has also appeared to increase using PCR methods leading some to shout "persistence" or "chronic shedding" where really, we are just better able to see what's happening thanks to our new molecular reading-glasses.


    Click on image to enlarge.
    Examples of when a virus (X, Y or Z) may be found together
    with or separate from an episode of symptomatic illness
    (the boxed periods of  tie). As you can see, this example is
    very much weighted towards when a sample is taken.
    3 testing scenarios are shown. (a) 1 sample at the beginning 

    and end of a study, (b) sampling only at the beginning of the 
    symptomatic periods and (c) regular sampling1. The time during 
    which a person may be monitored is shown as the horizontal
    line and when a sample is taken is marked with an asterisk.
    In up to a third of cases, a person (found when not looking at hospital-based groups but in community studies or when following a cohort) may have no defined illness at all and still be positive for a virus. Heresy!!

    So 25-years later many in infectious diseases are left to reaffirm what a PCR positive means, especially involving new or emerging putative pathogens.

    For the Middle East respiratory syndrome coronavirus (MERS-CoV) we may be able to draw some conclusions from a viral relative; the severe acute respiratory syndrome (SARS) CoV, did during its short time in humans back in 2002-2003.

    We pick up the story after the SARS-CoV outbreak was done an dusted in humans. Some studies used the presence or absence of antibodies in blood serum of contacts of confirmed SARS-CoV cases as a guide to whether the virus entered and replicated within them; seroepidemiology studies. The contacts do not appear to have been screened using RT-PCR; also the current situation with MERS. 

    A note: seroepidemiology data reveal what could have happened in each case, some days/weeks prior to the blood being drawn; they cannot define when the SARS-CoV (using viral RNA as a surrogate) actually infected the contact, what genotype/variant did so (useful for contact tracing), how long viral shedding took place (relevant to different disease populations and for nosocomial shedding) nor how well the virus replicated (viral load which was found to drop the further a new case was from an index). 

    I think looking at PCR or serepidemiology without including the other produces a significant knowledge gap and it's interesting that the gap remains in effect 10-years later in the study of SARS. Perhaps MERS-CoV is just like SARS-CoV and, as we see below, no symptoms=no infection=no onward transmission. Gut feelings don't really tick the box in science though.

    Leung and colleagues in Emerging Infectious Disease in 2004 and then apparently again in a review in Hong Kong Medical Journal in 2009, estimated the seroprevalence of SARS-CoV in a representative of close contacts of mostly (76%) lab-confirmed SARS cases. 

    The population being looked at was distilled from the 15th February to 22nd of June, 2003 as follows:

    • 3612 close contacts of  samples 
    • 505 were diagnosed with SARS
    • Of the remaining 3107, 2337 were contacted and 1776 were interviewed
    • 1068 blood samples were analysed for SARS-CoV IgG antibody
    Only 2 of the 1068 (0.19%) had an antibody titre of 1:25 to 1:50. Most recovered SARS cases had titres of ≥1:100. Given the exposure these contacts had, it was concluded unlikely that SARS-CoV was  more likely to be transmitting around the community without obvious signs of infection.

    Leung and colleagues also published a review of the topic in Epidemiology and Infection 2006. They concluded an overall SARS-CoV seroprevalence of 0.1% overall with 0.23% in healthcare workers and contacts and 0.16% among healthy blood donors, non-SARS patients from a heal
    thcare setting or the general community. Other interesting bits of information from this review include:
    • 16 studies were examined
    • Asymptomatic infection was <3%, excepting wild animal handlers and market workers
    • In live bird markets, 15% of workers had prior exposure to SARS-CoV (or closely related virus) without significant signs and symptoms
    • In handlers of masked palm civets (older males compared to control groups) in Guangdong, where SARS began, Yu and colleagues reported that 73% (16/22) had SARS-CoV-like antibodies (unvalidated assay) but none reported SARS or atypical pneumonia. Which leaves room for milder illness, and larger studies.
    • Prevailing SARS-CoV strains almost always led to symptomatic illness

    So what has been done for MERS-CoV? We have some camel seroepidemiology studies which I've previously described here and here. Human studies?

    1. In the study that found MERS-CoV-like neutralizing antibodies in Egyptian camels, no human sera from Egypt (815 from 2012-13 as part of an influenza-like illness study in Cairo and the Nile delta region) nor any from China (528 archived samples from Hong Kong) were MERS-CoV neutralizing-antibody positive.
    2. No sera or plasma from 158 children admitted to hospital with lower respiratory tract disease or healthy adult blood donors were MERS-CoV neutralizing-antibody positive. Small sample and the ill children may not yet have mounted a relevant antibody response if they had been infected by MERS-CoV.

    Work like that mentioned for SARS largely remains to be done for MERS. The SARS-CoV studies provide a useful model on which to base such studies and the World Health Organisation recently provided a detailed approach for seroepidemiology studies seeking to test contacts of laboratory confirmed MERS-CoV cases. 

    What does a positive PCR result mean in an asymptomatic MERS-CoV case? Still can't answer that. Are contacts seroconverting as an indication of MERS-CoV infection? Still can't answer that. How many mild or asymptomatic MERS-CoV infections are there beyond contacts of lab-confirmed cases? Still can't answer that.

    Once we can rule out occult community transmission - we can tick another concern off the MERS-list.

    Further reading...


    1. Observational Research in Childhood Infectious Diseases (ORChID): a dynamic birth cohort study
      http://bmjopen.bmj.com/cgi/pmidlookup?view=long&pmid=23117571
    2. Middle East respiratory syndrome coronavirus: quantification of the extent of the epidemic, surveillance biases, and transmissibility
      http://www.thelancet.com/journals/laninf/article/PIIS1473-3099(13)70304-
      9/abstract
    3. Prevalence of IgG Antibody to SARS-Associated Coronavirus in Animal Traders --- Guangdong Province, China, 2003
      http://www.cdc.gov/mmwr/preview/mmwrhtml/mm5241a2.htm
    4. Viral Load Distribution in
    5. SARS Outbreak
    6. http://wwwnc.cdc.gov/eid/article/11/12/pdfs/04-0949.pdf

    Tuesday, 19 November 2013

    H7N9 vaccine progresses through Phase I trials...

    Back in August I wrote about Novavax entering Phase I clinical trials with its virus-like particle vaccine (VLP) to prevent influenza A(H7N9) virus disease. It is based on the A/Anhui/1/2013 strain.

    Novavax, A United States company, has now reported in the New England Journal of Medicine that 80% of people may be protected by the generation of anti-H7N9 antibodies in response to 2x 5μg injections in the presence of 60 units of CSL's Iscomatrix adjuvant (see more on adjuvants in my August piece). 284 people were enrolled in a trial in Australia to determine these "very preliminary" results. Increased reactions were seen among the immunized at the injection site, but few were severe.

    The move away from the egg-based vaccine manufacturing system is likely to allow vaccines to be produced in much shorter periods; 12-weeks after an outbreak starts, with 50,000,000 doses potentially available in 4-months.

    You may ask, why then is it precisely 9-months after the 1st H7N9 case was retrospectively identified, and Novavax is still only at Phase I trials? I think, and I'm no expert in this area, that the process will increase in speed once the 'backbone' (the VLPs being used here which are based on a baculovirus, all produced in insect cells) in combination with this adjuvant etc, have been through the entire clinical trial process the first time. A successful backbone can be leveraged for other vaccines too.

    You can see a little more of the process of making the VLPs, in this case for respiratory syncytial virus, here.

    So, big changes lie not-too-far ahead for influenza vaccines....assuming the course through clicnial trials is smooth sailing of course!


    For those hypersensitive to hyperlinks...

    Monday, 18 November 2013

    MERS-CoV tally....



    Click on image to enlarge.
    The global MERS-CoV map as of 18-11-2013.
    Kuwait is currently depicted as having imported, 
    rather than locally transmitted or acquired cases.
    The WHO tally for Middle East respiratory syndrome coronavirus (MERS-CoV) lab-confirmed cases now stands at 157, of which 66 have died. 

    The 2 latest cases, with lots of relevant WHO details are from Kuwait but are reportedly not contacts.


    • FT#158. 47-year old male, ill on 30-Oct, hospitalized 7-Nov. He is critically ill. Travel outside of Kuwait, within a time-frame that might suggest MERS-CoV acquisition, has not been noted so far so I am marking this in red on the map to indicate a local acquisition for now.
    • FT#159. 52-year old male, ill on 7-Nov, hospitalized 10-Nov. He recently travelled overseas and there is possible exposure to camels (WHO tweet without specific detail, 16-Nov). Also critically ill.

    My tally lists another case, that of the case imported into Spain (61-year old female). However, that case has not yet been confirmed to WHO standards which may require a change to the map if the case, like the 2 from Italy in September, are classified as "probable" rather than confirmed cases. 
    Thus the proportion of fatal cases stands at 42%.

    RSV retreated, flu fading, parainfluenza picking up: Queensland respiratory virus numbers up to Week 45, 2013

    If you like to keep track of influenza cases in Queensland, Australia, then the Queensland Government's Queensland Health (QH) influenza data website is for you.

    It's a great place to drop by and check out the comings and goings of influenza viruses and many of the other traditional respiratory viruses including adenoviruses (AdVs), parainfluenzaviruses (PIVs) 1, 2 and 3, human metapneumovirus (MPV) and respiratory syncytial virus (RSV) - the "Big8". Testing is not routinely conducted for the rhinoviruses (RVs).

    The snippet below is from data that are publicly reported on the QH website. These images cover to the week beginning 3rd of November (up to Sunday, Nov 10th, 2013).

    The charts highlight that
     the 2013 flu season is winding down in Australia, also reflected by the WHO global updates. This year flu followed on from what seemed to have been a large RSV season. Unfortunately I couldn't find data for this same time period last year to compare RSV prevalence.

    In the wake of influenzavirus season, the parainfluenzaviruses are now on the rise in the lead up to summer. I expect the RVs (and enteroviruses) are also climbing, but in greater numbers.

    Click to enlarge. 
    A snippet from the Queensland Health Statewide Weekly Influenza Surveillance Report for 01.01.2013-10.11.2013
    My thanks to the team at the Communicable Diseases Unit, Queensland Health.

    The source of these data  can be read in full..

    Thursday, 14 November 2013

    The book of MERS has several chapters yet to write

    Epidemic is a big word, and while it generally means "a rise in the number of cases above what you'd expect", you can see from the definitions below that there are many ways to spin the meaning. For the public at large, it generally means "bad scary stuff" and so it's important that we use this word sparingly.

    An epidemic is defined by Oxford Dictionaries as:


    a widespread occurrence of an infectious disease in a community at a particular time

    ..or more applicably..



    a sudden, widespread occurrence of an undesirable phenomenon

    ...from Merriam Webster online...


    affecting or tending to affect a disproportionately large number of individuals within a population, community, or region at the same time

    ...from Wikipedia...


    In epidemiology, an epidemic (from επί (epi), meaning "upon or above" and δήμος (demos), meaning "people") occurs when new cases of a certain disease, in a given human population, and during a given period, substantially exceed what is expected based on recent experience.

    The Middle East respiratory syndrome (MERS) was so-named back in May 2013, and prior to March 2012, there had been no known cases of the coronavirus (CoV) named for the disease it was associated with.

    Yesterday we saw a detailed publication by Cauchemez and colleagues in the Lancet Infectious Diseases (LID). Accompanying that was an excellent piece in the Canadian press written by Helen Branswell which included some comments from the authors.

    Click to enlarge. 
    Accumulation of MERS-CoV lab detections by week (blue
    mountain, 
    left y-axis) and the accumulating deaths
    (red line, left y-axis). The proportion of fatal cases is slowly
    declining as fewer cases have died recently (ratio; black line,
    right y-axis). No data exist for ~3 or so deaths and I include
    the unconfirmed 2nd case in Kuwait for now.

    Feel free to use, just cite me and here.

    The key phrase slowly-growing epidemic, used by both, has been not-so-slowly appearing everywhere since then. Does that phrase accurately represent MERS to the world?

    Yes, it does. If you have a look at the chart above, its been a steady increase ("blue mountain"), but despite the apparent steep slope of new cases, the steepest part of the mountain, extracted and plotted below, is in fact very linear. A steady but slow growth in cases. No exponential take off. No major deviations. So yes, there is an epidemic. And yes, it is slow. 156 (157 if 2nd Kuwaiti instance is confirmed) cases over 87 weeks in a country of 20,000,000+; a country that just hosted the biggest human gathering of the year (the Hajj) and a country which provides a launch point for around 18,000,000 travelers and a destination for almost as many



    Click to enlarge.
    A slowly growing outbreak of an emerging  coronavirus.
    Cases have been accumulating worldwide but at a
    linear rate since the week beginning April 7th.
    Why the spike from this week? I'm not sure.
    Feel free to use, just cite me and here.
    But I think we need to be careful when throwing around the "E" word. An outbreak of an emerging virus may still be the best term to describe this chapter in the book of MERS. When someone asks on Twitter "to panic or not to panic"? (this was in reference to the latest MERS-map I posted) then I wonder if the correct message is being conveyed.
    Another central message of the new LID paper was a no-brainer; well it was to me but perhaps I'm just too close to it all - in which case take this with a grain of salt.


    I thought it was as obvious as the hump on a camel that where 1 case of a respiratory virus infection was detected, others were there to be found. After all, a virus needs us to survive - no us (which means no us actually harbouring infections, acting as a living incubator) then no more cases of the virus). Perhaps that's not obvious at all. Perhaps there is a lack of general understanding that our pathology laboratory systems do not test everyone with illness for even the "standard" endemic human respiratory viruses; that only those presenting to the right place, with the appropriate signs and symptoms, get a sample collected and get tested. This is apparently also true for MERS-CoV-which is by no means a standard virus. Do you go to your doctor if you feel mildly crook? Of course not - you go to work. What if you just have a fleeting headache, a stiff neck, feel a bit hot? Still going to work? Still going shopping? Still packing the kids off to school? Of course you are because we have these all the time and we have an immune system that does a wonderful job keeping it all mostly under control. Life goes on.
    But you may be positive for a virus and you are a key part of the transmission chain. You are an incubator. A host.


    So if routine testing is not geared towards finding out this extra information how do we find out what's going on in those who are not presenting with kidney failure or pneumonia; a relative small sliver of the population? Someone has to run a research study in which you enrol or get permission from people who are not very ill and sample them. Then you know something new about how widely the virus you are interested in is spread, for how long a person sheds it (if you sample the same person a few times during a month) and even how many other people get it (because all of a sudden your "contacts" become those of a less ill person and the numbers go up and you capture more of a picture of what's happening). So where are the research studies doing this?

    When the illness is just some fleeting thing its no real problem. Especially when it's due to a virus we know all about and don't track for public health reasons (we track influenza virus positives, but the reality is you have to be sick enough to be tested in order to add to that pool of data). 



    Click to enlarge.
    A very exaggerated example of how failing to test mildly ill or asymptomatic
    cases of infection in the community may confound our ability to make a link
    between cases of severe illness leaving knowledge gaps. These gaps prevent our ability
    to track spread of the pathogen and thus interrupt spread of disease.
    Feel free to use, just cite me and here.

    But if that virus is not yet in a textbook, not yet understood, not yet weighed and measured against the viruses we are more familiar with, emerges from an unknown place, is not considered endemic and is often notifiable, then not knowing this basic stuff becomes a major hole in our knowledge and our ability to respond appropriately. This is where we (still) are, 87-weeks after the first known MERS-CoV positive. Guessing (however educated) at what's happening by extrapolation and modelling.

    I guess not everyone knows that for every time there is a noticeably ill person infected with a "respiratory virus", it's fair to assume that there will be at least 1 or other who gave it to them, got it from them or got it from the one who gave it to them and who are not as sick or even considered sick at all. For MERS-CoV, they are missed and thus we have no idea how the virus is spreading. Just models. But we can make mathematically supported guesses to back up gut instinct, fair assumptions and logic.


    The hallmark of, and big problem with, the MERS outbreak (an epidemic mostly for the Kingdom of Saudi Arabia [KSA]), is that testing has been LIMITED to those who have pneumonia, or another severe disease, and their close contacts. Back in August Memish noted that surveillance was focused on those with pneumonia which was again noted by a WHO representative yesterday.


    Why, why oh why not test more people? Why?! Is it because "it's too costly to prospectively test people by RT-PCR unless they are (very) ill"? It might be for some nations, but the KSA is not one of those. 


    If you don't test others then you see these modelling publications arise. Idle hands and all that. Yes, it is great to have a model to support what many of us think to be true. And as Fisman and Tuite note in their editorial accompanying the LID article..



    ..inferences based on the best available data, even if those data are imperfect, allow decision makers to follow optimum courses of action based on what is known at a given point in time.

    The question is, can decision-makers sign off on any actions if they don't have actual data? If those data are not forthcoming, how can we ever test the validity of the MERS models?

    For now at least, I think we can agree that there is just too little testing to know enough to write more than a few chapters of the MERS-CoV textbook. A book for which we do have a table of contents. Many viruses have emerged before this one and they have each taught us what pages to skip ahead to. Unfortunately, we seem to have a recalcitrant author for 1 or 2 chapters.