ABSTRACT
A major bottleneck in scaling-up COVID-19 testing is the need for sophisticated instruments and well-trained healthcare professionals, which are already overwhelmed due to the pandemic. Moreover, the high-sensitive SARS-CoV-2 diagnostics are contingent on an RNA extraction step, which, in turn, is restricted by constraints in the supply chain. Here, we present CASSPIT (Cas13 Assisted Saliva-based & Smartphone Integrated Testing), which will allow direct use of saliva samples without the need for an extra RNA extraction step for SARS-CoV-2 detection. CASSPIT utilizes CRISPR-Cas13a based SARS-CoV-2 RNA detection, and lateral-flow assay (LFA) readout of the test results. The sample preparation workflow includes an optimized chemical treatment and heat inactivation method, which, when applied to COVID-19 clinical samples, showed a 97% positive agreement with the RNA extraction method. With CASSPIT, LFA based visual limit of detection (LoD) for a given SARS-CoV-2 RNA spiked into the saliva samples was ~200 copies; image analysis-based quantification further improved the analytical sensitivity to ~100 copies. Upon validation of clinical sensitivity on RNA extraction-free saliva samples (n = 76), a 98% agreement between the lateral-flow readout and RT-qPCR data was found (Ct<35). To enable user-friendly test results with provision for data storage and online consultation, we subsequently integrated lateral-flow strips with a smartphone application. We believe CASSPIT will eliminate our reliance on RT-qPCR by providing comparable sensitivity and will be a step toward establishing nucleic acid-based point-of-care (POC) testing for COVID-19.
Subject(s)
COVID-19 Testing/methods , COVID-19/diagnosis , CRISPR-Cas Systems , RNA, Viral/isolation & purification , SARS-CoV-2/isolation & purification , Saliva/chemistry , Humans , Molecular Diagnostic Techniques/methods , Point-of-Care Testing , Real-Time Polymerase Chain Reaction , SARS-CoV-2/genetics , Sensitivity and Specificity , Smartphone , Specimen Handling/methods , WorkflowABSTRACT
BACKGROUND AND OBJECTIVES: Biofilms are colonies of microbial cells encased in a self-produced organic polymeric matrix. The biofilm production is more important for nonalbicans Candida (NAC); as C. albicans possess many other mechanisms to establish infections. Correct identification of Candida species has gained importance due to persistent rise in infections caused by NAC. We sought to isolate, identify Candida species in clinical isolates and study biofilm formation. MATERIALS AND METHODS: Modified microtiter plate method was performed to study biofilm formation by isolates in Sabouraud's dextrose broth. It was then quantitatively assessed using a spectrophotometer. Biofilm formation was graded as negative, +1, +2, +3 and + 4 on the basis of percentage absorbance. RESULTS: Biofilm formation was observed in 16 of 40 (40.0%) isolates of C. albicans as compared to 39 of 78 (50.0%) of isolates of NAC. Strong (+4) biofilm production was seen in maximum biofilm producers in C. tropicalis (12 of 27) followed by C. albicans (8 of 16). Total biofilm producers were significantly more among high vaginal swab isolates 63.2% (12 of 19) and urine isolates 59.2% (29 of 49), when compared to blood isolates 34.2% (13 of 38) as well as other isolates 27.5% (11 of 40). INTERPRETATION AND CONCLUSIONS: NAC species are qualitatively and quantitatively superior biofilm producers than C. albicans. Biofilm production is the most important virulence factor of NAC species and compared to other lesions, it is more significantly associated with luminal infections.
