𝟏 A 🧵 on:
🔸 Reverse Transcription-quantitative Polymerase Chain Reaction (RT-qPCR) currently used to detect #SARSCoV2

🔸Cycle Threshold (CT) & what it means

#scicomm #COVID19
𝟐 RT-qPCR is a common lab technique used to convert RNA to DNA & quantify its amount.

DNA & RNA are two forms of genetic material: DNA is like the mother code, while the RNA is a transcribed copy of (often) a segment of this mother code.
𝟑 #COVID19 virus contains this transcribed form (RNA) wrapped in a protective protein capsule
To detect its presence in cells we collect a swab from a person suspected to be infected w/ the virus.
Note: swab will contain human, viral & other microbes’ particles
𝟒 First, we need to reverse transcribe (reverse copy) whatever RNA we have collected using the swab into DNA. This step is necessary because:
🔸DNA is a lot more stable than RNA to work with in the lab.
🔸Methods we have for amplification of genetic material work on DNA only.
𝟓 The next step is to amplify this reverse transcribed DNA. This allows us to identify whether #SARSCoV2 genome was present in the swab sample & quantify its amount.

But 1st we need to make sure that we’re amplifying only the #SASRCoV2 DNA & nothing else.
𝟔 Using other genetic methods we know the genetic code of #SASRCoV2, this allows us to design stickers (primers) that are so specific they will be able to find and stick to particular segments of the reverse transcribed viral DNA & act as a guide to copy its entire length.
𝟕 The process of using specific stickers to copy the entire length of the viral DNA over and over again is called Polymerase Chain Reaction (PCR).
PCR doubles the amount of DNA in each cycle.
Typically we run the PCR for ~35-40 cycles ➡️ end up with 35-40 billion DNA molecules
𝟖 qPCR is a variation of the PCR, which contains a fluorescent molecule that generates a fluorescent signal at each amplification cycle. The fluorescence signal increases proportionally to the amount of amplified viral DNA ➡️ help us ESTIMATE amount of virus present in the swab.
𝟗 What is Cycle Threshold (Ct) value?
It’s the number of cycles of qPCR amplification required for the fluorescence signal to be detected crossing a threshold, which is above the background signal (a low level signal that is present in the assay regardless of presence of virus)
𝟏𝟎 How is Ct determined?
First, validation experiments are performed using serial dilutions of a sample of known quantity; this helps establish the limit of detection (LOD): an upper and lower range for detection
𝟏𝟏 POSITIVITY cut-off: a Ct value similar to that generated by the lowest copies of target that can be reliably detected (e.g. Ct ≤ 38)

NEGATIVITY cut-off: a Ct value at which target is no longer expected to be detected based on LOD (e.g. Ct ≥40)
𝟏𝟐 Ct is inversely proportional to the log of viral load, BUT their precise relationship depends mostly on sampling variation ➡️ Ct is a snapshot of ONE point in the course of infection & depends on when the sample was collected relative to when infection happened❗️
𝟏𝟑 Furthermore, there are studies that show that distribution of Ct values has changed over the course of the pandemic

medrxiv.org/content/10.110…

medrxiv.org/content/10.110…

What does this mean?
𝟏𝟒 This means that epidemic dynamics ➡️ time since infection distribution ➡️ viral load distribution ➡️ observed Ct values. How these steps link might change depending on for example the instrument used to measure Ct values, but the general principle holds.
𝟏𝟓 In conclusion: Ct values in Quebec will NOT necessarily be the same ones as Germany, France, or even Ontario b/c our pandemic trajectory is different

Please verify the information you read with experts in the field; it is our pleasure & duty to communicate science to you!
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More from @DeNovo_Fatima

13 Oct
1⃣ Following over sensationalized headlines in media about an Australian study (virologyj.biomedcentral.com/articles/10.11…) that looked at how long can #SARSCoV2 last on surfaces (fomites)
Here is a non-sensationalized overview of the data & methods used in this study 🧵👇🏾
2⃣ This is indeed an interesting study & much needed. However the methodologies employed do not really reflect everyday life settings.
The study itself admits in its conclusion that this is under "controlled conditions"
3⃣ Study overview: the authors placed fixed amount of virus on different surface types (in the dark to negate the effect of UV in killing the virus) & then placed these in incubators (controlled temperature & humidity levels) for specific time points
Read 9 tweets
29 Sep
Instituting restrictions shouldn't be taken lightly, neither shld easing them! Until we’ve a vaccine, there's a lot we can learn from other parts of 🌍
I’ll discuss a @TheLancet study () on lessons learned from easing restrictions in other countries
🧵👇🏾
1/
This study, conducted by a group of stellar & multidisciplinary global & public health researchers, compares approaches taken by 9 high-income countries: Hong Kong, Japan, New Zealand, Singapore, South Korea, Germany, Norway, Spain, and the UK
2/
Based on successes/failures of these countries, the authors introduce 5 benchmark to meet prior to easing restrictions
✴️Knowledge of infection status (Epidemiology)
✴️Community engagement
✴️Public Health capacity
✴️Health System capacity
✴️Measures for Border Control
3/
Read 7 tweets
26 Sep
🧵1-A thread for non-scientists on the issue of #COVID19 variant (614G vs. 614D) found most common in Québec (614G) & why it's irresponsible & inaccurate to blame the way pandemic unfolded in Québec on the variant

#scientific evidence shows this variant is NOT more dangerous
2-The phylogenetic analysis of #COVID19 in Québec has shown that majority of viral introductions to the province came from Europe ()
Therefore the variant (version of the virus) that was most common is Europe (614G) is also most common here
3- Wondering what D614G means:
genome is a long code that gets translated to functioning molecules. To keep track of each letter in the code, we assign them a number based on their position, hence 614. D&G refer to 2 molecules that different versions of the code can produce.
Read 9 tweets
26 Sep
🧵In this thread I'll address the INACCURATE claim that the #COVID19 strain found in Québec is somehow more virulent & therefore the reason Québec was the epicentre of the pandemic in🇨🇦
I'll be referring to data from @McGillGenome study & @nextstrain
virological.org/t/genomic-epid…
1/11
1st I'd like to acknowledge @bjesseshapiro @cl_murall & other amazing #scientists whose work & analysis should be relied on by policy makers & media alike.

See my summary of their phylogenetic study of #SARSCoV2 in Québec 👇🏾
2/11
Analyzing samples from 734 Québec residents shows a global introduction of the virus into Québec. However, once the borders were closed & restriction were put in place, certain clades of the virus become more frequent.
What is a clade you ask? good question, keep reading👇🏾
3/11
Read 11 tweets
21 Sep
🧵@McGillGenome @INSPQ have thus far sequenced genomes of 734 Québec residents infected w/ #COVID19 to unravel the genomic epidemiology of the infection (sources of introduction) in Québec

Video of automated sequencing:

Below is summary of findings👇
1/5
Samples were collected between mid-Feb to April 1, 2020
Sequence analysis + travel history showed:
*⃣ 32.7% of infections came from Europe, with France contributing 12.1% of infections
*⃣ 31% from Caribbean/Latin America
*⃣ 23.9% from U.S.
*⃣ 1.2% from Asian, none from China
2/5
Based on the phylogeny of the earliest reported cases, it is possible the first case reached Québec City from the United Kingdom as early as end of January!
This confirms that #COVID19 had spread outside China a lot earlier than originally announced.
3/5
Read 6 tweets
19 May
As Montréal moves to re-open next week, here are my hypotheses on #transmission post lifting of lockdown

A thread ⬇️

1/5
Best possible scenario:
✅We miraculously expand testing criteria & capacity➡️widespread testing + #ContactTracing + isolation

These case-based interventions would allow us to drive transmission down.

We know this is a successful strategy because: S. Korea & Germany
2/5
Reality:
While there has been some progress in building those capacities, we are far from managing our outbreak in this way➡️most communities will remain at more or less steady levels of transmission, with fluctuations which I predict will correlated w/ socioeconomic status

3/5
Read 5 tweets

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