72 Matching Annotations
  1. Sep 2024
    1. Tests performed: Haemostatic tests ristocetin-induced platelet aggregation plasma VWF antigen VWF risocetin cofactor VWF FVIII-binding capacity VWF multimers and FVIII activity DDAVP test Concentration timecourses

      sequencing from genomic DNA performed and reported in 2011

      cDNA analysis

      Real-time-PCR analysis

      long PCR

      Ellman assay (quantifying sulfhydryls)

      Dynamic light scattering measurements

      circular dichroism

      ADAMTS13 proteolysis assay

      ADAMTS13-VWF binding assay

    1. we found that the clouds can become more compact and produce substantially less rain upon: (i) reducing the ramp rate to 1 °C/sec in every PCR step (Fig. 10a), (ii) increasing the annealing/extension time to 2 minutes (up from 1 minute; Fig. 10b), and (iii) increasing the number of cycles to 50 (Fig. 10c)28
  2. Aug 2024
  3. May 2024
    1. DNA fragmentation by restriction digestion prior to dropletgeneration enables optimal accuracy by separating tandemgene copies, reducing sample viscosity, and improving templateaccessibility for input samples >66 ng per well

      NEB says

      Digestion is recommended whenever DNA input is greater than 75 ng Source

      NEB says that biorad recommends these enzymes: AluI, CviQI, HaeIII, HindIII-HF, MseI

      More guidelines

      • Assemble ddPCR reactions at room temperature, this allows the restriction enzyme to digest the gDNA during the reaction set-up period
      • Prepare reaction mixes as you would for a standard ddPCR reaction. Add 0.5–1 μL of each restriction enzyme (5–20 units, depending on enzyme concentration) to the reaction mixture
      • After set-up, simply continue droplet generation as normal
      • Restriction enzyme will be inactivated during first PCR denaturation step
  4. Oct 2023
    1. bird banding

      Bird banding is one technique used to monitor migratory flock patterns and given the infection rate of HPAIV A/H5N1 is near 100% the virus can be understood as endemic in all migratory waterfowl so tracking their movements is an effective method for tracking the transmission dynamics of the virus itself.

  5. Feb 2023
  6. Nov 2022
    1. Fundamentally, all real-time thermal cyclers are engineered to detect fluorescein (FAM) first and foremost, so dyes with longer wavelength emission may be detected less sensitively
    2. For those shorter than 25 bases, a standard (dual labeled) BHQ™ Probe may be perfectly satisfactory.

      Nova might be better to have for 1-step ddPCR applications where the BHQ quenchers are unstable due to reduction of the N=N azo bond by the DTT additive

  7. Aug 2022
    1. Pritchard, E., Matthews, P. C., Stoesser, N., Eyre, D. W., Gethings, O., Vihta, K.-D., Jones, J., House, T., VanSteenHouse, H., Bell, I., Bell, J. I., Newton, J. N., Farrar, J., Diamond, I., Rourke, E., Studley, R., Crook, D., Peto, T. E. A., Walker, A. S., & Pouwels, K. B. (2021). Impact of vaccination on new SARS-CoV-2 infections in the United Kingdom. Nature Medicine, 1–9. https://doi.org/10.1038/s41591-021-01410-w

  8. Apr 2022
  9. Mar 2022
    1. Mia Malan. (2021, November 25). [Thread] What is the potential impact of the new B.1.1.529 #COVID19 variant? @rjlessells: 1. It’s relatively simple to detect some B.1.1.529 cases, as it’s possible to use PCR tests to do this in some cases 2. B.1.1.529 = has many mutations across different parts of the virus https://t.co/ytktqLzJUi [Tweet]. @miamalan. https://twitter.com/miamalan/status/1463846528578109444

  10. Feb 2022
  11. Jan 2022
  12. Dec 2021
    1. Arieh Kovler. (2021, November 26). Israel has identified four cases of the B.1.1.529 variant, all recent travellers. One case, a 32-year-old woman returning from South Africa, was triple vaccinated with Pfizer and had her 3rd dose just two months ago. [Tweet]. @ariehkovler. https://twitter.com/ariehkovler/status/1464190991204859919

    1. Tom Moultrie. (2021, December 17). A 1-figure Gauteng update, bringing in data through Wednesday 15/12 (PCR only; by date of collection). The turn continues. On similar metrics (not shown) ALL northern provinces (NW, GT, MP, LP) seem to have now turned. Https://t.co/6Bh3kZsooK [Tweet]. @tomtom_m. https://twitter.com/tomtom_m/status/1471723711287996416

    1. Jay Varma. (2021, December 16). Um, we’ve never seen this before in #NYC. Test positivity doubling in three days 12/9—3.9% 12/10—4.2% 12/11—6.4% 12/12—7.8% Note: Test % is only for PCR & NYC does more per capita daily than most places ~67K PCR/day + 19K [reported] antigen over past few days (1/2) https://t.co/PhxsZq55jn [Tweet]. @DrJayVarma. https://twitter.com/DrJayVarma/status/1471485885447389186

  13. Nov 2021
  14. Oct 2021
  15. Sep 2021
  16. Jul 2021
    1. Kraemer, M. U. G., Hill, V., Ruis, C., Dellicour, S., Bajaj, S., McCrone, J. T., Baele, G., Parag, K. V., Battle, A. L., Gutierrez, B., Jackson, B., Colquhoun, R., O’Toole, Á., Klein, B., Vespignani, A., Consortium‡, T. C.-19 G. U. (CoG-U., Volz, E., Faria, N. R., Aanensen, D., … Pybus, O. G. (2021). Spatiotemporal invasion dynamics of SARS-CoV-2 lineage B.1.1.7 emergence. Science. https://doi.org/10.1126/science.abj0113

  17. May 2021
  18. Mar 2021
    1. de Oliveira T, Lutucuta S, Nkengasong J, Morais J, Paixao JP, Neto Z, Afonso P, Miranda J, David K, Ingles L, Amilton P A P R R C, Freitas H R, Mufinda F, Tessema K S , Tegally H, San E J, Wilkinson E, Giandhari J, Pillay S, Giovanetti M, Naidoo Y, Katzourakis A, Ghafari M, Singh L, Tshiabuila D, Martin D, Lessells R. (2021) A Novel Variant of Interest of SARS-CoV-2 with Multiple Spike Mutations Detected through Travel Surveillance in Africa. medRxiv. https://www.krisp.org.za/publications.php?pubid=330. Accessed 26 March 2021.

  19. Feb 2021
  20. Dec 2020
    1. Most rRT-PCRs are qualitative (i.e. target is detectedor not detected with no target copy number reported), not quantitative (target copy number per unit volume of specimen matrix or per reaction is reported).

      There is no way to know how many times a target has been copied.

    2. In some commercial assays, the cut-off is determined individually for each run based on the amplification curve and Ct value of the positive control.

      This seems like it would more accurate than just about any other test.

  21. Nov 2020
    1. In most clinical settings (including the one I work in), all the doctor is provided with is a positive or negative result. No mention is made of the number of cycles used to produce the positive result.

      [[Z: The number of PCR cycles has not been standardized, and is usually not even mentioned to the doctor]]

    2. If you get a positive PCR test and you want to be sure that what you’re finding is a true positive, then you have to perform a viral culture. What this means is that you take the sample, add it to respiratory cells in a petri dish, and see if you can get those cells to start producing new virus particles. If they do, then you know you have a true positive result. For this reason, viral culture is considered the “gold standard” method for diagnosis of viral infections. However, this method is rarely used in clinical practice, which means that in reality, a diagnosis is often made based entirely on the PCR test.

      [[Z: A positive PCR should be followed by a viral culture test to see if you're dealing with a live infection]]

      After a positive PCR test, you don't know if the virus is alive or not. To find this out you can add it to respiratory cells (in the case of a respiratory virus) and see if they start producing virus particles).

      [[Z: Viral culture tests are rarely used in clinical practice]]

      Positive diagnoses of COVID-19 are done base on PCR only.

    3. One thing that’s important to understand at this point is that PCR is only detecting sequences of the viral genome, it is not able to detect whole viral particles, so it is not able to tell you whether what you are finding is live virus, or just non-infectious fragments of viral genome.

      PCR only tells you if you're detecting sequences of the viral genome. It doesn't tell you that what you're finding is live virus or not.

  22. Oct 2020
  23. Sep 2020
    1. He added that while it would not be possible to check every test to see whether there was active virus, the likelihood of false positive results could be reduced if scientists could work out where the cut-off point should be.

      Take Away: This is an incorrect usage of the term "false positive." A positive PCR test result from a recovered infection is a valid and true positive.

      Claim: PCR tests for SARS-CoV-2 give false positive results when there is no active virus.

      Evidence: The diagnostic PCR tests currently in widespread use are designed to detect the presence of the SARS-CoV-2 viral RNA in a clinical sample. The RNA is only a part of the complete virus and is not infectious on its own. Research has shown that viral RNA can be detected in some samples up to 12 weeks after onset of symptoms (1). In other words, this is like testing if an oven is warmer than the room temperature - it could be hot even after it has been turned off.

      By definition, in the context of SARS-CoV-2 PCR tests, a "false positive" means that a test result is deemed positive when in reality there was no viral RNA in the sample. If a person is recovering from an infection, gets tested, and then is given a positive test result, that is a true positive regardless of whether they are infectious or not.

      Sources: 1) https://www.cdc.gov/coronavirus/2019-ncov/hcp/duration-isolation.html

    1. Your Coronavirus Test Is Positive. Maybe It Shouldn’t Be.

      Take Away: Diagnostic tests are most useful when they are both sensitive and rapid. The sensitivity of SARS-CoV-2 PCR tests is not the issue, but rather the time it takes to get a result. Additionally, the "90%" statistic is likely misleading due to the data source and not generalisable to all testing results.

      The Claim: The usual PCR diagnostic tests may be too sensitive and too slow, with up to 90% of positive cases due to trace amounts of virus.

      The Evidence: Polymerase Chain Reaction (PCR)-based tests, which are currently in the most widespread use for detection of SARS-CoV-2 RNA, involves a molecular process that amplifies target DNA sequences in repeated temperature-dependent cycles. The amount of target DNA is measured after each cycle and the number of the cycle when the target can be reliably detected is often referred to as the cycle threshold (Ct). The Ct value is proportional to the amount of starting DNA in the sample and can be used to estimate the viral load of a patient. In some ways this is like a teacher making photocopies of a chapter from a textbook until they have enough for all their students.

      However, Ct values are relative measurements and need to be directly compared to controls for every sample - a Ct value taken alone can be meaningless. For instance, consider an infected patient who is tested twice: the first time they are gently swabbed and the sample is relatively dilute, the second time they are vigorously swabbed and the sample is relatively concentrated. The resulting Ct values could be drastically different. Therefore, Ct values need to be considered carefully in the proper context for making medical or policy decisions. The FDA also recommends that a PCR result alone should not be used to determine infection status.

      Positive results are indicative of the presence of SARS-CoV-2 RNA; clinical correlation with patient history and other diagnostic information is necessary to determine patient infection status. (1)

      Current PCR test results are generally given as a binary positive/negative based on a cutoff value for Ct. The cutoff needs to be determined based on the performance of each individually developed SARS-CoV-2 test, of which there are currently over 160 that have been granted emergency use authorization by the FDA (2). Based on unpublished data from the CDC, setting a stringent Ct cutoff of 30 could return negative results in patients who are both infected and potentially infectious (3 Fig 5). Furthermore, a 30 cycle cutoff would return invalid results for samples which are too diluted. Based on the same CDC data, up to 30% of potentially infectious patients would get invalid results and need to be re-swabbed, thereby extending the time between getting infected and getting a positive result.

      The period of time when RNA from SARS-CoV-2 can be detected (and a positive PCR test result returned) may extend up to 12 weeks after recovery, with Ct values trending higher over time (3,4). According to The New York Times article, they looked at Ct values from people who tested positive in Massachusetts in July and found 85-90% of results had Ct values greater than 30. The epidemiology of COVID-19 is highly time and region dependent. Massachusetts had a peak in COVID-19 hospitalizations on April 21 (5), which is 9-12 weeks prior to the testing data analyzed by The NY Times. Therefore, the detection of a large proportion of people with lingering viral RNA is not surprising. These results are likely not universal and can not be applied to other regions, especially where community spread is still significant.

      Sources:

      (1) https://www.fda.gov/media/135900/download

      (2) https://www.fda.gov/medical-devices/coronavirus-disease-2019-covid-19-emergency-use-authorizations-medical-devices/vitro-diagnostics-euas

      (3) https://www.cdc.gov/coronavirus/2019-ncov/hcp/duration-isolation.html

      (4) Li N, Wang X, Lv T. Prolonged SARS-CoV-2 RNA Shedding: Not a Rare Phenomenon. J Med Virol 2020 Apr 29. doi: 10.1002/jmv.25952.

      (5) https://www.bostonherald.com/2020/05/22/massachusetts-finally-seeing-downward-coronavirus-trends/

    1. Detection of viruses using Polymerase Chain Reaction (PCR) is helpful so long as its accuracy can be understood: it offers the capacity to detect RNA in minute quantities, but whether that RNA represents infectious virus is another matter. RT-PCR uses enzymes called reverse transcriptase to change a specific piece of genetic material called RNA into a matching piece of genetic DNA. The test then amplifies this DNA exponentially; millions of copies of DNA can be made from a single viral RNA strand.

      Take away: The claim that virus can be detected for a long time but is not infectious needs further clarification. This claim was based on a Lancet article (1). Within the Lancet article, some of the studies cited detected RNA in stool/blood/seminal fluid samples instead of nasal swabs. Other studies cited did not test infectious nature of virus detected by PCR. It is several logic steps to travel from detecting virus in stool/blood/seminal fluid in Lancet article to concluding that PCR of nasal swabs for COVID-19 results in large numbers of false positives.

      The claim: RNA from coronavirus is present and can be detected for a long time but may not be infectious.

      The evidence: The Spectator article links to the article "SARS-CoV-2 shedding and infectivity" in the Lancet (1). This article cites seven articles to support the statement that RNA persists long after virus is not infectious. Of these articles, only one reports that virus was detected at ~30 days but could not be cultured beyond three weeks (2). This article also states that detection was easier in stool samples than nasal samples after the first five days. Several articles cited by source 1 did not report infectivity of virus detected (3, 4, 5, and 7). Of the two remaining articles, virus was detected in serum/blood (6 and 8). In the serum study, 58% of tested specimens were infectious (6).

      Source:

      1 https://www.thelancet.com/journals/lancet/article/PIIS0140-6736(20)30868-0/fulltext

      2 https://www.thelancet.com/journals/lancet/article/PIIS0140-6736(03)13412-5/fulltext

      3 https://pubmed.ncbi.nlm.nih.gov/15030700/

      4 https://pubmed.ncbi.nlm.nih.gov/27682053/

      5 https://pubmed.ncbi.nlm.nih.gov/29648602/

      6 https://www.thelancet.com/journals/langlo/article/PIIS2214-109X(16)30243-1/fulltext

      7 https://pubmed.ncbi.nlm.nih.gov/28195756/

      8 https://pubmed.ncbi.nlm.nih.gov/22872860/

  24. Jun 2020
  25. May 2020
    1. Mei, X., Lee, H.-C., Diao, K., Huang, M., Lin, B., Liu, C., Xie, Z., Ma, Y., Robson, P. M., Chung, M., Bernheim, A., Mani, V., Calcagno, C., Li, K., Li, S., Shan, H., Lv, J., Zhao, T., Xia, J., … Yang, Y. (2020). Artificial intelligence for rapid identification of the coronavirus disease 2019 (COVID-19). MedRxiv, 2020.04.12.20062661. https://doi.org/10.1101/2020.04.12.20062661

    1. Shweta, F., Murugadoss, K., Awasthi, S., Venkatakrishnan, A., Puranik, A., Kang, M., Pickering, B. W., O’Horo, J. C., Bauer, P. R., Razonable, R. R., Vergidis, P., Temesgen, Z., Rizza, S., Mahmood, M., Wilson, W. R., Challener, D., Anand, P., Liebers, M., Doctor, Z., … Badley, A. D. (2020). Augmented Curation of Unstructured Clinical Notes from a Massive EHR System Reveals Specific Phenotypic Signature of Impending COVID-19 Diagnosis [Preprint]. Infectious Diseases (except HIV/AIDS). https://doi.org/10.1101/2020.04.19.20067660

  26. Jan 2020
  27. Apr 2019
    1. using a commercially-available buffer that also requires a 15-minute incubation period prior to direct PCR