The COVID-19 pandemic
has underscored the shortcomings in the deployment
of state-of-the-art diagnostics platforms. Although several polymerase
chain reaction (PCR)-based techniques have been rapidly developed
to meet the growing testing needs, such techniques often need samples
collected through a swab, the use of RNA extraction kits, and expensive
thermocyclers in order to successfully perform the test. Isothermal
amplification-based approaches have also been recently demonstrated
for rapid severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)
detection by minimizing sample preparation while also reducing the
instrumentation and reaction complexity. In addition, there are limited
reports of saliva as the sample source, and some of these indicate
inferior sensitivity when comparing reverse transcription loop-mediated
isothermal amplification (RT-LAMP) with PCR-based techniques. In this
paper, we demonstrate an improved sensitivity assay from saliva using
a two-step RT-LAMP assay, where a short 10 min RT step is performed
with only B3 and backward inner primers before the final reaction.
We show that while the one-step RT-LAMP demonstrates satisfactory
results, the optimized two-step approach allows detection of only
few molecules per reaction and performs significantly better than
the one-step RT-LAMP and conventional two-step RT-LAMP approaches
with all primers included in the RT step. We show control measurements
with RT-PCR, and importantly, we demonstrate RNA extraction-free RT-LAMP-based
assays for detection of SARS-CoV-2 from viral transport media and
saliva clinical samples.
The COVID-19 pandemic has underscored the shortcomings in the deployment of state-of-the-art diagnostic platforms. Although several PCR-based techniques have been rapidly developed to meet the growing testing needs, such techniques often need samples collected through a swab, the use of RNA extraction kits, and expensive thermocyclers in order to successfully perform the test. Isothermal amplification-based approaches have also been recently demonstrated for rapid SARS-CoV-2 detection by minimizing sample preparation while also reducing the instrumentation and reaction complexity. There are limited reports of saliva as the sample source and some of these indicate inferior sensitivity when comparing RT-LAMP with PCR-based techniques. In this paper, we demonstrate an improved sensitivity assay to test saliva using a 2-step RT-LAMP assay, where a short 10-minute RT step is performed with only B3 and BIP primers before the final reaction. We show that while the 1-step RT-LAMP demonstrate satisfactory results, the optimized 2-step approach allows for single molecule sensitivity per reaction and performs significantly better than the 1-step RT-LAMP and conventional 2-step RT-LAMP approaches with all primers included in the RT Step. Importantly, we demonstrate RNA extraction-free RT-LAMP based assays for detection of SARS-CoV-2 from VTM and saliva clinical samples.
A 53-year-old male presented to the emergency room with chest pain, shortness of breath, and back pain. He had recently recovered from COVID-19 infection and returned home on room air. Chest imaging showed bilateral hydropneumothoraces that were not present on the imaging performed during his prior admission three weeks ago. The patient was treated with bilateral chest tube drainage and oxygen support and responded well to treatment. This case represents a unique occurrence of spontaneous loculated bilateral hydropneumothoraces in the context of recent clinical recovery from COVID-19 infection requiring inpatient treatment. This case highlights the importance of an awareness of a potential sequela of COVID-19 that may occur even after presumed clinical recovery.
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