The current time is unprecedented. We haven’t seen anything like this in the last ~100 years and (hopefully) won’t see in the next 100 years. But, as a Ph.D. student in Molecular Genetics, it is moving to answer curious questions that people from non-scientific backgrounds might have regarding how coronavirus works and what can be done to slow it down.
In addition, I was fortunate enough to participate in the COVID-19 testing facility at the Weizmann Institute of Science, Israel. As the number of infections in Israel is going up, the facility at the Israel National Center for Personalized Medicine was commissioned to ramp the testing numbers. The center is one of the most sophisticated, top-of-the-line facilities, which can run ~4000 tests a day at its fullest capacity, while presently only 2500 tests are performed a day in Israel for a population of ~9 million. The idea of this article is to show how a COVID-19 testing facility looks like and take a step-by-step look at the operational pipeline.
Step 1 at Station A: The nasopharyngeal swab samples are received from hospitals/paramedical service in plastic tubes along with a document containing the patient information. Upon reception, each sample is cleaned and disinfected thoroughly with 70% ethanol and packed in a cooler box for internal transportation.
Step 2 at Station B: Here, hundreds of tubes are prepared, each containing a special kind of solution, called lysis/shield buffer. The genetic information of the SARS-Cov-2 virus (nCoV-2) is encoded by a molecule called RNA, which is a rather unstable molecule. The solution can stabilize the RNA molecule. It also contains a detergent that can inactivate the viral particles. Each of the tubes carries a unique barcode.
Step 3 at Station C: The swab samples from Station A and the buffer solutions from Station B are brought here. Station C is a biosafe room with biological hoods. These hoods are extremely sterile chambers, free from any biological contamination so that the technicians have minimal chance to come in contact with the virus. Each swab sample is manually inspected and added to the lysis buffer solution inside the hood. The barcode and the sample document are uploaded to an internal tracking software. The viral particles are inactivated from now on and can be handled with less stringency. The test tubes with the samples are then arranged in racks. The remaining swab sample from patients is returned to a fridge in order to be stored for at least 48 hours, in case a repetition of the test becomes necessary.
Step 4 at Station D: The racks (containing the sample in lysis buffer) from Station C are brought to Station D where an automated system can take a small volume containing the patient swab and put into a 96-well plate format. Such 96-well plates are routinely used in molecular biology approaches to detect nucleic acids (DNA/RNA).
Step 5 at Station E: In this station, a robotic liquid handler can assemble all the ingredients required for the subsequent chemical reactions. In the first reaction, the RNA from the virus is converted to its complementary DNA (cDNA) by an enzyme called reverse-transcriptase (co-incidentally, also first discovered in a virus). In the next reaction, the cDNA is acted upon by an enzyme (called DNA-polymerase that can work at high temperature) to produce multiple copies of a part of the cDNA by a process called polymerase chain reaction (PCR). The choice of the part of the cDNA is critical as it gives specificity to the detection of SARS-CoV-2, vis-a-vis other coronaviruses. The output of the test is typically in the form of a number (called, Ct) between 5-40, which is inversely related to the viral load in the patient. Thus, the lower the Ct, the more likely the patient is positive.
To increase the confidence in the test, two such regions of the nCoV-2 cDNA (N1 and N2) are chosen. For a test to be called positive, both N1 and N2 have to return a number below 40. More typically, the number hovers between 30 and 40 for positive cases. For negative cases, the numbers are above 40.
|Ct for N1
|Ct for N2
Step 6: In the last step, the data and corresponding patient ID are uploaded to the internal software and the final results are sent to the Ministry of Health.
All the stations are staffed with teams of 3-4 technicians while the entire operation is managed by a control center overlooking all stations and ensuring a smooth relay of materials and information between teams.
Accuracy: RT-PCR based testing for the nCoV-2 virus is an extremely accurate test, with about a 3% chance of being falsely negative. Other than the operational steps, the false-negatives can arise from the presence of an extremely low amount of viral particles in the tested swab, below the detection limit of PCR. According to the US CDC, nasopharyngeal swabs are likely to yield the best results compared to swabs from other parts (nasal, oral, etc,)
What about scaling up?
In the present framework, all actions from Station D onwards (involving inactivated virus) are handled by automation, so it is relatively easy to scale up. However, all activities from receiving the samples to inactivating them are done manually under extreme care by trained professionals so as to minimize contamination of samples or spillage. Thus, it becomes one of the most time- and effort- consuming parts of the operation. Additionally, the collection of swabs is also done by trained front-line workers one-by-one, adding to the effective testing time. Thus, the rate-limiting step of the entire process becomes the collection and pre-processing of the samples, instead of the actual tests. The current end-to-end time (from the reception of the sample to delivery of results) is ~24 hours.
- US-CDC Manual on COVID-19 testing: https://www.fda.gov/media/134922/download
- Reverse Transcription and Polymerase Chain Reaction: https://www.youtube.com/watch?v=gubLAtn2o4s
- How to make sense of RT-PCR data: https://www.youtube.com/watch?v=ZhXDIDRB70I
- More about COVID-19 testing at Weizmann Institute: http://www.weizmann.ac.il/WeizmannCompass/sections/briefs/coronavirus-testing-begins-at-weizmann?