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
  2. Middle East respiratory syndrome coronavirus: quantification of the extent of the epidemic, surveillance biases, and transmissibility
  3. Prevalence of IgG Antibody to SARS-Associated Coronavirus in Animal Traders --- Guangdong Province, China, 2003
  4. Viral Load Distribution in
  5. SARS Outbreak