11 Sept Rapid tests cultivability and infectiousness

Sat, 09/12/2020 - 12:01

COVID-ology Episode 73: Progress in rapid diagnosis; reflections on reinfection and evolution.

Dear colleagues,

My contribution of today will be a bit short, since we will take advantage of the (first?) Indian Summer.

  1. Three papers on rapid detection:
    1. Simplexa is a modified RT-PCR, with a “all in one” reaction mix, making time consuming extraction obsolete.  The authors claim a gain of 80 % in time (from 5 to 1 hour), without significant loss in sensitivity in regular naso- or oro- pharyngeal (NP or  OP) samples.  They even see a better sensitivity in broncho-alveolar samples. Remains to be independently evaluated.
    2. The Biosensor paper in ACS Nano proposes a method, based on direct hybridization of RNA.  The signal (but not the RNA itself) is amplified by a combination of plasmonic photothermal effect and localized surface plasmon resonance. Very complicated and certainly not ready-to-use, but interesting as it is very sensitive (in the picomolar range) and thus might allow direct detection of viral RNA without any PCR amplification. Remains to be validated against RT-PCR.
    3. The BD Veritor is a chromatographic immunoassay for nucleocapsid antigen as a point-of-care test.  As compared to RT-PCR, the assay has an excellent specificity, but a lower sensitivity, but still acceptable (over 80 %).  Veritor was also compared with the Sofia antigen from a competitor and showed to be slightly superior….

The obvious limitation remains that these tests lose sensitivity at a lower viral load and longer time after the onset of symptoms.  Clearly, all the tests were done on samples from symptomatic patients and the question how they perform with asymptomatic SARS-CoV-2 subjects remains open. However, since asymptomatics presumably have lower viral loads, the antigen tests may be not sensitive enough for contact tracing…?     


  1. Infectivity: There is an ongoing discussion about the relation between a positive PCR and true infectivity of a patient.  Intuitively, we presume that infectivity may decline with increasing time after symptoms onset and decreasing viral load.  We also presume that respiratory virus might be more infectious than fecal virus.  The next two papers addressed this question by using classical culture on Vero cells:
    1. The first paper is a very rigorous study on 9 mildly symptomatic young patients from an early cluster (end of January) in Munich (mit Deutsche Gründlichkeit).  It is very rich and unfortunately I could nor retrieve the pdf… Some observations:
      1. Viral load in sputum tended to be higher than oro- or nasopharyngeal swab (NP and OP are equivalent), VL in stools was also rather high, without much GI symptoms (Fig 2).   
      2. Late lung symptoms (only 2 pt) were associated with rise in sputum VL.
      3. Whereas the virus was readily isolated (on Vero cells) during the first week of symptoms from a considerable fraction of samples (16.66% of swabs and 83.33% of sputum samples), no isolates were obtained from samples taken after day 8 in spite of ongoing high viral loads. And….no virus was isolated from stools. (Fig 1d)
      4. The success of virus isolation also depended on viral load: samples that contained <106 copies per ml (or copies per sample) never yielded an isolate.
      5.  In vivo evidence of viral replication (i.e. subgenomic RNA) was mainly seen in sputum, only early on in throat and very rare in stools (Fig 1h)
      6. There was no clear correlation of “cultivability” with seroconversion: no cultivated virus after day 7, but only 50 % seroconversion.


In their conclusions, the authors point to various interesting points:

  • It seems that sputum might be longer infectious than throat samples. The signs of replicating virus in throat could be related to “anosmy and ageusia” (loss of taste and smell).  There is even evidence of independency of virus in throat and lung, as in one patient the throat virus had a different genetic signature from the lung virus.  
  • There are interesting differences between the viral dynamics in SARS-CoV-1 and -2: the VL in the “old” SARS only rises gradually AFTER symptom onset, whereas it can be already maximal in COVID at the time of presentation. Hence the difference in transmission: COVID is transmitted in pre- or a-symptomatic patients, whereas most probably SARS was only transmittable after the onset of symptoms.


    1. The paper by Bullard et al investigates 10 times more and many unrelated patients, but they were all symptomatic and PCR (+) on nasopharyngeal or endotracheal samples, of which only 26 (30 %) were positive in culture.  The analysis largely confirms what was shown in the Nature paper in that samples with low VL (Ct > 24) or more than 8 days after symptoms onset (SO) were consistently negative.  As can be seen in Fig 3, however, the culture could also be negative at < 8 days SO and/or with higher VL.   One confounder, not discussed by the authors, is the time that the samples were kept at 4 ° before freezing at -80 (24-72 H).  It is also not clear whether there was any trending difference between the NP and endotracheal samples, the latter more similar  to sputum.   


Interpretation and Implications

Obviously, it is also not clear what the exact relationship is between “in vitro cultivability” and in vivo infectivity.   Many samples that one would presume to be infectious (rather high VL, early after symptoms onset) were negative in culture.  A more sensitive technique has been described in a VERO line with enhanced expression of the “spike-activating” human protease TMPRSS2. It is being used at ITM, I understand from Kevin.

It would be interesting therefore to investigate the relationship between a reasonably sensitive  antigen detection (such as Veritor or SOFIA  diagnostic tool), cultivability (in an enhanced assay) and (clinical/epidemiological) evidence of transmissibility. If indeed, it can be shown that  PCR+ but antigen (-) samples are poorly or not transmittable, then such type of  antigen tests could be reliably used for screening and epidemiological purposes at population level. Although it will still not exclude infection at individual level, but the “missed” cases will presumably have a mild clinical course, with low  chances on further transmission.    


  1. The Nature paper “Making sense of mutations” of yesterday reflects on the general evolutionary characteristics of SARS-CoV-2: mutation rates are two times slower than Influenza and 4 times lower than HIV.  In my opinion, that is still considerable. Yet, most of the comments in this paper seem to say that this rate is certainly not alarming and rapid evolution towards higher fitness or immune escape is not to be expected, although caution should be taken (e.g. by combining 2 different monoclonals in therapy).  A lot of the comments focus on the famous D614G mutation, which has “taken over” the epidemic (Fig p. 2) and indeed might have resulted in a more “open” confirmation of the spike, resulting in easier viral entry (Fig on p.3).  Yet, most comments also tend to rather downplay the real importance of this mutation. 


  1. The “three questions about reinfection” also in yesterday’s Nature are:
    1. How common: until now exceptional, but the true frequency will become more clear if we really enter a “second wave”
    2. How severe: in the two described cased, the first was milder and the second more severe than the primary infection.  Of course, there is anxiety about possible antibody (or T cell)- mediated enhancement by low affinity antibodies or T cells.
    3. The implication for vaccines?  If vaccine-induced antibodies resemble the naturally induced ones, the same questions can be asked as in a and b.  Obviously, boosting or second generation vaccines, inducing longer lasting immunity may offer solutions.        

Best wishes,