Efficient Tracing of the SARS-CoV-2 Omicron Variants in Santa Barbara County Using a Rapid Quantitative Reverse Transcription PCR Assay

The recent emergence of the SARS-CoV-2 Omicron variant is associated with a dramatic surge of cases around the globe in late 2021 and early 2022. The numerous mutations in this variant, particularly in the Spike protein, enhance its transmission, increase immune evasion, and limit treatment with monoclonal antibodies. Identifying a communitys introduction to a novel SARS-CoV-2 variant with new clinical features related to treatment options and infection control needs is imperative to inform decisions by clinicians and public health officials, and traditional sequencing techniques often take weeks to result. Here, we describe a quantitative reverse transcription PCR assay (RT-qPCR) to accurately and precisely detect the presence of the Omicron sublineages BA.1/BA1.1 and BA.2 viral RNA from patient samples in less than four hours. The assay uses primers targeting the BA.1/BA1.1 unique mutations N211del, L212I, and L214 insertion EPE in the Spike protein gene, and the BA.2 specific mutations T19I and L24/P25/P26 deletion in the Spike protein gene. Using this assay, we detected 169 cases of Omicron, 164 BA.1/BA1.1 and 5 BA.2, from 270 residual SARS-CoV-2 positive samples collected for diagnostic purposes from Santa Barbara County (SBC) between December 2021 to February 2022. The RT-qPCR results show concordance with whole viral genome sequencing. Our observations indicate that Omicron was the dominant variant in SB County and is likely responsible for the surge of cases in the area during the sampling period. Using this inexpensive and accurate test, the rapid detection of Omicron in patient samples allowed clinicians to modify treatment strategies and public health officers to enhance contact tracing strategies. This RT-qPCR assay offers an alternative to current variant-specific detection approaches, provides a template for the fast design of similar assays, and allows the rapid, accurate, and inexpensive detection of Omicron variants in patient samples. It can also be readily adapted to new variants as they emerge in the future.

CRISPR-based and RT-qPCR surveillance of SARS-CoV-2 in asymptomatic individuals uncovers a shift in viral prevalence among a university population

The progress of the COVID-19 pandemic profoundly impacts the health of communities around the world, with unique effects on colleges and universities. Here, we examined the prevalence of SARS-CoV-2 in 1808 asymptomatic individuals on a university campus in California, and compared for the first time the performance of CRISPR- and PCR-based assays for large-scale virus surveillance. Our study revealed that there were no COVID-19 cases in our study population in May/June of 2020. Using the same methods, we demonstrated a substantial shift in prevalence approximately one month later, which coincided with changes in community restrictions and public interactions. This increase in prevalence, in a young and asymptomatic population, indicated the leading wave of a local outbreak, and reflected the rising case counts in the surrounding county. Our results substantiate that large, population-level asymptomatic screening using CRISPR- or PCR-based assays is a feasible and instructive aspect of the public health approach within large campus communities.

A Fast and Accessible Method for the Isolation of RNA, DNA, and Protein to Facilitate the Detection of SARS-CoV-2

Management of the COVID-19 pandemic requires widespread SARS-CoV-2 testing. A main limitation for widespread SARS-CoV-2 testing is the global shortage of essential supplies, among these, RNA extraction kits. The need for commercial RNA extraction kits places a bottleneck on tests that detect SARS-CoV-2 genetic material, including PCR-based reference tests. Here we propose an alternative method we call PEARL (Precipitation Enhanced Analyte RetrievaL) that addresses this limitation. PEARL uses a lysis solution that disrupts cell membranes and viral envelopes while simultaneously providing conditions suitable for alcohol-based precipitation of RNA, DNA, and proteins. PEARL is a fast, low-cost, and simple method that uses common laboratory reagents and offers comparable performance to commercial RNA extraction kits. PEARL offers an alternative method to isolate host and pathogen nucleic acids and proteins to streamline the detection of DNA and RNA viruses, including SARS-CoV-2.

A Scalable, Easy-to-Deploy, Protocol for Cas13-Based Detection of SARS-CoV-2 Genetic Material

The COVID-19 pandemic has created massive demand for widespread, distributed tools for detecting SARS-CoV-2 genetic material. The hurdles to scalable testing include reagent and instrument accessibility, availability of highly-trained personnel, and large upfront investment. Here we showcase an orthogonal pipeline we call CREST (Cas13-based, Rugged, Equitable, Scalable Testing) that addresses some of these hurdles. Specifically, CREST pairs commonplace and reliable biochemical methods (PCR) with low-cost instrumentation, without sacrificing detection sensitivity. By taking advantage of simple fluorescence visualizers, CREST allows for a binary interpretation of results. CREST may provide a point- of-care solution to increase the distribution of COVID-19 surveillance.

New mutation in the CASR gene in a family with familial hypocalciuric hypercalcemia (FHH) and neonatal severe hyperparathyroidism (NSHPT).

A loss of calcium-sensing receptor (CASR) function due to inactivating mutations can cause familial hypocalciuric hypercalcemia (FHH) or neonatal severe hyperparathyroidism (NSHPT). NSHPT represents the most severe expression of FHH and courses as a life-threatening condition. The aim of this study was to identify and characterize a CASR mutation in a female infant brought to the health service due to dehydration, apathy, lack of breast feeding and severe hypercalcemia. Molecular analysis was performed on genomic DNA of the index case and her parents. A novel homozygous mutation (p.E519X) in CASR was identified in the proband; both mother and father had the same mutation in heterozygous state, confirming their FHH condition. The mutation results in a truncated and inactive protein due to the lack of transmembrane and intracellular domains. The identification of this novel CASR gene mutation established the basis of hypercalcemia in this family and allowed a genetic counseling.

New mutation in the CASR gene in a family with familial hypocalciuric hypercalcemia (FHH) and neonatal severe hyperparathyroidism (NSHPT) / Nova mutação no gene CASR em uma família com hipercalcemia hipocalciúrica familiar (HHF) e hiperparatireoidismo neonatal grave (HPTNG)

A loss of calcium-sensing receptor (CASR) function due to inactivating mutations can cause familial hypocalciuric hypercalcemia (FHH) or neonatal severe hyperparathyroidism (NSHPT). NSHPT represents the most severe expression of FHH and courses as a life-threatening condition. The aim of this study was to identify and characterize a CASR mutation in a female infant brought to the health service due to dehydration, apathy, lack of breast feeding and severe hypercalcemia. Molecular analysis was performed on genomic DNA of the index case and her parents. A novel homozygous mutation (p.E519X) in CASR was identified in the proband; both mother and father had the same mutation in heterozygous state, confirming their FHH condition. The mutation results in a truncated and inactive protein due to the lack of transmembrane and intracellular domains. The identification of this novel CASR gene mutation established the basis of hypercalcemia in this family and allowed a genetic counseling.

Mutações inativadoras no gene do sensor do cálcio (CASR) podem causar hipercalcemia hipocalciúrica familiar (HHF) ou hiperparatireoidismo neonatal grave (HPTNSG). A HPTNS representa a forma mais grave da HHF cursando com risco de vida. O objetivo deste estudo foi identificar e caracterizar uma mutação no gene CASR de uma criança do sexo feminino levada ao hospital em decorrência de desidratação, apatia, dificuldade para mamar e hipercalcemia grave. A análise molecular foi realizada a partir do DNA genômico do caso índice e de seus pais. Uma nova mutação em homozigose (p.E519X) foi identificada no caso índice; ambos, mãe e pai, apresentaram a mesma mutação em heterozigose, o que os caracteriza como portadores de HHF. Essa alteração resulta em uma proteína truncada e inativa devido à falta dos domínios transmembrana e intracelular. A identificação dessa nova mutação estabeleceu a causa da hipercalcemia na família e permitiu o aconselhamento genético.

Niclosamide prevents the formation of large ubiquitin-containing aggregates caused by proteasome inhibition.

BACKGROUND: Protein aggregation is a hallmark of many neurodegenerative diseases and has been linked to the failure to degrade misfolded and damaged proteins. In the cell, aberrant proteins are degraded by the ubiquitin proteasome system that mainly targets short-lived proteins, or by the lysosomes that mostly clear long-lived and poorly soluble proteins. Both systems are interconnected and, in some instances, autophagy can redirect proteasome substrates to the lysosomes. PRINCIPAL FINDINGS: To better understand the interplay between these two systems, we established a neuroblastoma cell population stably expressing the GFP-ubiquitin fusion protein. We show that inhibition of the proteasome leads to the formation of large ubiquitin-containing inclusions accompanied by lower solubility of the ubiquitin conjugates. Strikingly, the formation of the ubiquitin-containing aggregates does not require ectopic expression of disease-specific proteins. Moreover, formation of these focused inclusions caused by proteasome inhibition requires the lysine 63 (K63) of ubiquitin. We then assessed selected compounds that stimulate autophagy and found that the antihelmintic chemical niclosamide prevents large aggregate formation induced by proteasome inhibition, while the prototypical mTORC1 inhibitor rapamycin had no apparent effect. Niclosamide also precludes the accumulation of poly-ubiquitinated proteins and of p62 upon proteasome inhibition. Moreover, niclosamide induces a change in lysosome distribution in the cell that, in the absence of proteasome activity, may favor the uptake into lysosomes of ubiquitinated proteins before they form large aggregates. CONCLUSIONS: Our results indicate that proteasome inhibition provokes the formation of large ubiquitin containing aggregates in tissue culture cells, even in the absence of disease specific proteins. Furthermore our study suggests that the autophagy-inducing compound niclosamide may promote the selective clearance of ubiquitinated proteins in the absence of proteasome activity.