covid: infectivity of variants determined by culture not PCR

 A recent Swiss article compared SARS-CoV-2 viral load by PCR versus the quantitative measure of infectious virus by cell culture, finding among other things a very poor correlation between the two, in this preprint, pre-peer reviewed article (see covid PCR viral load vs culture medrxiv2022 in dropbox, or https://www.medrxiv.org/content/10.1101/2022.01.10.22269010v1.full.pdf). thanks to Jon Pincus for bringing this to my attention

 

Details:

-- 384 patients had nasopharyngeal swabs for SARS-CoV-2 daily during the first five symptomatic days of Covid-19. All had mildly symptomatic Covid infections

    -- 118 unvaccinated individuals infected with pre-VOC SARS-CoV-2 (pre-Variant-of-Concern, i.e. basically the original, wild type virus)

    -- 248 patients had the delta variant, 127 of whom were unvaccinated/121 had had two vaccinations at least 14 days prior and subsequent breakthrough infections

    -- 18 had breakthrough infections with omicron (i.e. all vaccinated)

-- essentially all vaccinated patients had an mRNA vaccine: median time between the second dose and breakthrough infection was 79.5 days for delta and 136 for omicron

 

-- on each day, PCR was done to assess the cycle threshold, Ct, which is basically the inverse of viral load. Only those with Ct values below 27 were included in the study (this is an accepted threshold for a high enough viral load to be reliably associated with true infection and transmissibility)

-- additionally, quantitative infectious viral titers, IVT, were also assessed using a technique to quantify cell cultures, which reflect viable virus

 

Results: 

-- correlation between PCR Ct and IVT: very low correlation (R²=0.119) for the pre-VOC samples; though the correlation was slightly higher (but still low) from samples from unvaccinated and vaccinated delta-infected patients (R²=0.312)

-- no correlation between IVT and age or sex in patients

-- delta virus infection during the first 5 symptomatic days in unvaccinated patients, as compared to pre-VOC virus (all unvaccinated):

    -- higher titers by PCR testing in pre-VOC virus (log 0.653, p<0.0001)

        -- BUT, IVT were significantly higher in delta-infected patients ( 0.343 log, p=0.373)

    -- timing of difference: 

        -- PCR copies for pre-VOC virus were higher at 1 to 2 days post onset of symptoms (dpos), but were similar to delta on days 0, 3, 4, and 5 dpos

        -- but,  IVT viral shedding was higher for delta at 3-5 dpos, but similar at 0-2 dpos

        -- and, genome copies by PCR were stable until 5 dpos, though they declined more steadily with IVT analysis

    -- by age and sex: 84 patients with delta variant were matched with those with pre-VOC infection, finding higher IVT in the delta-infected group, 0.51 log, p=0.001

-- vaccinated versus unvaccinated patients:

    -- Delta variant, in 1^st 5 dpos:

        -- PCR genome copies did not differ significantly between vaccinated and unvaccinated patients

        -- BUT, IVT was significantly decreased in vaccinated patients (0.68 log, p<0.0001)

    -- timing of viral loads: IVT was substantially lower in vaccinated patients at all dpos, with PCR-based viral loads starting to decline at 4 dpos; IVT in vaccinated patients had the biggest decrease observed at 3-5 dpos

    -- BUT, at 5 dpos infectious virus was detectable in 7/13 (54%) unvaccinated and 11/13 (80%) of unvaccinated patients

    -- 67 unvaccinated patients with delta were matched with vaccinated patients with breakthrough delta infections regarding age, sex, and dpos, finding IVT was markedly elevated in unvaccinated patients (0.97 log, p<0.0001)

    -- and they found no significant correlation between IVT and the time interval since administration of the last vaccine dose

-- omicron variant:

    -- comparing nasopharyngeal viral loads in 18 omicron and 17 delta-infected patients, all vaccinated: comparable PCR genome copies, but modestly lower IVT in those with omicron versus delta, though not statistically significant (0.69 log, p=0.10); similar results after matching patients are age, sex, and dpos

 

Commentary: 

-- higher viral load is an important predictor of viral transmissibility overall, though there are many other components to viral transmission, including mitigation strategies (use of masks, distancing, ventilation), housing (concentration of people living in the housing unit, size of the housing unit), high risk comorbidities (obesity, diabetes, immunocompromise), etc

-- in prior PCR-based studies, viral shedding occurs on average 2 days prior to the beginning of symptoms, gradually declining over the 8 days after the onset of symptoms. Further detection of virus by PCR in immunocompetent hosts after that time period usually is from non-infectious virus (PCR picks up nonviable virus as well)

-- some prior studies have found that successful virus isolation by culture drops significantly when the RNA viral loads are below 6 log10 copies/mL, or collected later than 8 days after symptom onset

 

-- this study found several very important things:

    -- one important clinical issue is to determine the best predictor of secondary transmission of SARS-CoV-2. This study suggested that even a very low Ct PCR (high viral load) does not correlate with the amount of culturable live virus.  Other studies have found that culturable virus is a successful proxy for transmissibility

        -- this finding also informs our understanding of transmissibility for breakthrough infections from those vaccinated: they have a lower viral load by IVT, which might help explain the decreased secondary transmissibility as found in some studies. For example, a Danish study found that vaccination of the primary person halved the likelihood of secondary transmission: see http://gmodestmedblogs.blogspot.com/2022/01/covid-omicron-increased-transmissibility.html

    -- unvaccinated delta-infected people had higher IVT than unvaccinated people with pre-VOC viral infection, consistent with the finding of increased transmissibility in unvaccinated people of the delta variant over prior SARS-CoV-2 infections

    -- in vaccinated versus unvaccinated people with delta variant, there was no difference between RNA genome copies (by PCR), but vaccinated people had significantly lower IVTs suggesting their lower transmissibility, and they cleared the virus more quickly

        -- of note, the finding of 3-10-fold higher viral copies by PCR in unvaccinated patients with delta versus pre-VOC infected people was not found in this study using cell culture (in part because prior studies did not fully control for the supposed symptom onset, age, or sex). However, a study comparing delta to alpha variants found that the delta one had more infectious virus on cell culture than alpha, both in unvaccinated and vaccinated people (see https://pubmed.ncbi.nlm.nih.gov/34462756/ )

    -- though small numbers, vaccinated individuals with omicron infection had comparable IVTs to Delta breakthrough infections

    -- and, one big flag in this study is that they were able to detect infectious viral particles in ½ the patients at 5 dpos (though small numbers of patients assessed), suggesting that the new CDC recommendations of shortening the time of quarantine in infected people to 5 dpos (and without even an antigen test being done) may not be so great…  (for these updated CDC findings from 12/27/21, see https://www.cdc.gov/media/releases/2021/s1227-isolation-quarantine-guidance.html#:~:text=Stay%20home%20for%205%20days,others%20for%205%20additional%20days.

 

Limitations:

-- cell cultures of virus do reflect viable virus, though it still should be remembered that this is a laboratory technique, and SARS-CoV-2 may grow more or less well on artificial viral culture media versus the human upper respiratory tract, adipose tissue, etc. Studies have found cell cultures to be a reasonable proxy for infectiousness, though the exact relationship between quantitative viral cell culture and what happens in the body may well be approximate (ie, not a 1:1 relationship)

-- they only assessed up to 5 days after symptom onset, limiting data on how long viable virus remains and is potentially transmissible

-- they limited their study to those with high viral loads per PCR (Ct<27) and finding limited correlation with live virus that was culturable, limiting our understanding of IVT for those with higher Ct values, or even what cutpoint of IVT quantization is associated with transmissibility

-- no info about asymptomatic people with Covid infection: how often do they have quantifiable IVTs?

-- All of these patients had nasopharyngeal swabs, which may be less sensitive for the omicron variant and more sensitive for delta (see http://gmodestmedblogs.blogspot.com/2022/01/covid-rapid-test-less-accurate-for.html )

-- limited number of omicron patients to draw definitive conclusions, and all were vaccinated. Since omicron is so new to the world, we will need to wait some time before we have more definitive epidemiology. And this study had no info on unvaccinated people with omicron infection

-- not clear what the results would be for those vaccinated with other than mRNA vaccines

-- using quantitative cell cultures, i would imagine, is much more labor-intensive than PCR and i assume not scalable for wide-spread use. For us in primary care, this is really no different from PCR, since we only get qualitative, no quantitative results. But it does seem that quantitative cultures are important in studies to inform our practice (as in this study). And, if this study were replicated, this technique would be useful in assessing the time course of infectivity of the different variants (including future ones), and in more detailed studies in the epidemiology in those at higher risk by immunocompromise, age, diabetes, obesity

 

-- This is an important study for several reasons:

    -- it includes pretty up-to-date data, including the omicron variant

    -- they support a fundamental change in how we look at viral transmissibility, moving away from looking at the cycle threshold on quantitative PCR as the marker of infectivity and more to quantification of viral culture

    -- it documents that vaccination does lower viral transmissibility and increases the speed of viral clearance, including for the much more transmissible delta variant, and likely with the omicron variant (need more data)

-- and, perhaps the CDC recommendation of stopping isolation 5 days after symptom onset should be reconsidered…

geoff

 

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