Evaluation of self-collected nasal, urine, and saliva samples for molecular detection of SARS-CoV-2 using an EUA approved RT-PCR assay and a laboratory developed LAMP SARS-CoV-2 test.

As the SARS-CoV-2 virus spread throughout the world, millions of positive cases of COVID-19 were registered and, even though there are millions of people already vaccinated against SARS-CoV-2, a large part of the global population remains vulnerable to contracting the virus. Massive nasopharyngeal sample collection in Puerto Rico at the beginning of the pandemic was limited by the scarcity of trained personnel and testing sites. To increase SARS-CoV-2 molecular testing availability, we evaluated the diagnostic accuracy of self-collected nasal, saliva, and urine samples using the TaqPath reverse transcription polymerase chain reaction (RT-PCR) COVID-19 kit to detect SARS-CoV-2. We also created a colorimetric loop-mediated isothermal amplification (LAMP) laboratory developed test (LDT) to detect SARS-CoV-2, as another strategy to increase the availability of molecular testing in community-based laboratories. Automated RNA extraction was performed in the KingFisher Flex instrument, followed by PCR quantification of SARS-CoV-2 on the 7500 Fast Dx RT-PCR using the TaqPath RT-PCR COVID-19 molecular test. Data was interpreted by the COVID-19 Interpretive Software from Applied Biosystems and statistically analyzed with Cohen's kappa coefficient (k). Cohen's kappa coefficient (k) for paired nasal and saliva samples showed moderate agreement (0.52). Saliva samples exhibited a higher viral load. We also observed 90% concordance between LifeGene-Biomarks' SARS-CoV-2 Rapid Colorimetric LAMP LDT and the TaqPath RT-PCR COVID-19 test. Our results suggest that self-collected saliva is superior to nasal and urine samples for COVID-19 testing. The results also suggest that the colorimetric LAMP LDT is a rapid alternative to RT-PCR tests for the detection of SARS-CoV-2. This test can be easily implemented in clinics, hospitals, the workplace, and at home; optimizing the surveillance and collection process, which helps mitigate global public health and socioeconomic upheaval caused by airborne pandemics.

Promoter DNA methylation patterns in oral, laryngeal and oropharyngeal anatomical regions are associated with tumor differentiation, nodal involvement and survival.

Differentially methylated regions (DMRs) can be used as head and neck squamous cell carcinoma (HNSCC) diagnostic, prognostic and therapeutic targets in precision medicine workflows. DNA from 21 HNSCC and 10 healthy oral tissue samples was hybridized to a genome-wide tiling array to identify DMRs in a discovery cohort. Downstream analyses identified differences in promoter DNA methylation patterns in oral, laryngeal and oropharyngeal anatomical regions associated with tumor differentiation, nodal involvement and survival. Genome-wide DMR analysis showed 2,565 DMRs common to the three subsites. A total of 738 DMRs were unique to laryngeal cancer (n=7), 889 DMRs were unique to oral cavity cancer (n=10) and 363 DMRs were unique to pharyngeal cancer (n=6). Based on the genome-wide analysis and a Gene Ontology analysis, 10 candidate genes were selected to test for prognostic value and association with clinicopathological features. TIMP3 was associated with tumor differentiation in oral cavity cancer (P=0.039), DAPK1 was associated with nodal involvement in pharyngeal cancer (P=0.017) and PAX1 was associated with tumor differentiation in laryngeal cancer (P=0.040). A total of five candidate genes were selected, DAPK1, CDH1, PAX1, CALCA and TIMP3, for a prevalence study in a larger validation cohort: Oral cavity cancer samples (n=42), pharyngeal cancer tissues (n=25) and laryngeal cancer samples (n=52). PAX1 hypermethylation differed across HNSCC anatomic subsites (P=0.029), and was predominantly detected in laryngeal cancer. Kaplan-Meier survival analysis (P=0.043) and Cox regression analysis of overall survival (P=0.001) showed that DAPK1 methylation is associated with better prognosis in HNSCC. The findings of the present study showed that the HNSCC subsites oral cavity, pharynx and larynx display substantial differences in aberrant DNA methylation patterns, which may serve as prognostic biomarkers and therapeutic targets.

Evaluation of silica spin­column and magnetic bead formats for rapid DNA methylation analysis in clinical and point­of­care settings.

Late-stage cancers lack effective treatment, underscoring the need for early diagnosis to improve prognosis and decrease mortality rates. Molecular markers, such as DNA methylation, offer promise in early cancer detection. The present study compared commercial kits for analyzing DNA from cervical liquid cytology samples in cancer screening. Rapid bisulfite conversion kits using silica spin-columns and magnetic beads were assessed against standard DNA extraction and bisulfite conversion methods for profiling DNA methylation using quantitative methylation-specific PCR. ß-actin amplification indicated the suitability of small sample volumes for methylation studies using either the pellet or supernatant (cell-free DNA) parts. Comparison of Bisulfite Conversion Kit-Whole Cell (Abcam), Methylamp Bisulfite Modification (Epigentek), EpiTect Fast LyseAll Bisulfite Kit (Qiagen GmbH) and EZ DNA Methylation-Direct Kit (Zymo Research Corp.) showed no significant differences in ß-actin cycle threshold values. EZ-96 DNA Methylation-Lightning MagPrep (Zymo Research Corp.), a hybrid kit in a 96-well plate format, exhibited swift turnaround time and similar amplification efficiency. Automation with magnetic bead kits increased throughput without compromising amplification efficiency in open PCR systems. Cost analysis favored direct kits over the gold standard manual protocol. This comparison aids in selecting cost-effective DNA methylation diagnostic tests. The present study confirmed comparable kit performance in methylation-based analysis, highlighting the adequacy of cytology samples and the potential of bodily fluids as alternatives for liquid biopsy.