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Summary Introduction: The SARS-CoV-2 coronavirus, that causes the COVID-19 disease, has become a global public health problem that requires the implementation of rapid and sensitive diagnostic tests. Aim(s): To evaluate and compare the sensitivity of LAMP assay to a standard method and use RT-LAMP for the diagnosis of SARS-CoV-2 in clinical samples from Colombian patients. Method(s): A descriptive and cross-sectional study was conducted. A total of 25 nasopharyngeal swab samples including negative and positive samples for SARS-CoV-2 were analyzed, through the RT-LAMP method compared to the RT-qPCR assay. Result(s): LAMP method detected ~18 copies of the N gene, in 30 min, evidenced a detection limit similar to the standard method, in a shorter time and a concordance in RT-LAMP of 100% with the results. Conclusion(s): RT-LAMP is a sensitive, specific, and rapid method that can be used as a diagnostic aid of COVID-19 disease.Copyright © 2021. All Rights Reserved.
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Colorimetric RT-LAMP Assay is a diagnostic method that has attracted much attention because of its rapidity, simplicity, and accuracy compared to other disease diagnosis methods. Despite having many advantages, the RT-LAMP Colorimetric Assay has disadvantages, especially for kits that use phenol red as an indicator. The disadvantage derives from the input RNA/DNA samples containing high buffer levels, which causes no color change and false-negative results. This study aimed to develop and optimize the colorimetric RT-LAMP method on high-buffered SARS-CoV-2 RNA samples. We found that a temperature of 69°C for 50 minutes with the addition of post-treatment in the form of heating at 80°C for 10 minutes is an optimal condition for high-buffered SARS-CoV-2 samples. The condition proved effective in changing the result's color from red (negative) to yellow (positive). We also classified the analysis results based on the correlation between the Cycle threshold (Ct) value of SARS-CoV-2 viruses and the Optical Density (OD) value, which was quantified using a spectrophotometer at 415 nm (with a correlation value of-0.9084), where yellow color indicated Ct below 20, amber color indicated Ct between 20 and 30, orange color indicated Ct between 30 and 35, and red indicated Ct more than 35 (negative). In conclusion, this study successfully detects the SARS-CoV-2 virus in high-buffered samples using Phenol Red Colorimetric RT-LAMP Assay, with a sensitivity of 85% for Ct Cutoff 40. © 2023, Bogor Agricultural University. All rights reserved.
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Objectives: Globally, cataract and glaucoma are the predominant causes of blindness. Screening glaucoma in patients referred for cataract surgery is a convenient tool for detecting glaucoma cases in rural population. The COVID period has adversely affected eye care as the routine screening and follow-ups at hospital were substantially reduced owing to pandemic restrictions. We aim to study the impact of COVID on detection of glaucoma in patients with cataract. Method(s): It was a retrospective study conducted to compare the prevalence of glaucoma in rural patients presenting with cataract pre- and post-COVID. Details of 975 consecutive patients each were taken prior to March 2020 (pre-COVID) and after October 2021 (post-COVID) from hospital database and patient case files. Result(s): The prevalence of glaucoma was higher during the pre-COVID time (3.8%) as compared to pre-COVID (3.8%), but the result was not statistically significant. In both the groups, primary open-angle glaucoma was the pre-dominant form of glaucoma, with prevalence being 1.5% and 2.2% in the pre-COVID and post-COVID groups, respectively. The mean intraocular pressure and mean VCDR values were higher in the post-COVID group as compared to the pre-COVID group, and the result was statistically significant. Conclusion(s): This was the first study to compare the prevalence of glaucoma in patients with cataract in rural population in the pre-COVID and post-COVID periods. In the aftermath of the pandemic, the present study emphasizes the role of screening and follow-ups in glaucoma management to prevent irreversible loss of vision.Copyright © 2023 The Authors.
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A 40-year-old male patient, an office worker, is a hospital staff. He applied with the complaint of sudden onset of blurred vision in the right eye. He stated that he had the first dose of inactivated covid vaccine (sinovac) 4 days ago in his story. In his examination, his vision was 0.5 in the right eye, 1.0 in the left eye, and his intraocular pressure was in both eyes. It was at the level of 15 mmHg. Biomicroscopically, the anterior segment looked natural. CSC was diagnosed in the FFA and OCT examinations.Treatment with oral acetazolamide (2x250 mg), topical nepafanac (4x1) was started. On the 13th day of the treatment, there was insufficient improvement in clinical findings, and oral acetazolamide was used. The dose was reduced (2x125 mg), oral epleronone (50 mg) was added. On the 70th day of the treatment, the vision in the right eye increased to full level in the control examination and it was observed that the retina returned to its normal appearance in the OCT examination.Copyright © 2023 Gazi Eye Foundation. All rights reserved.
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Introduction: The use of smartphones to provide specialist ophthalmology services is becoming a more commonly used method to support patients with eye pathologies. During the COVID-19 pandemic, demand for telehealth services such as tele-ophthalmology, is increasing rapidly.Methods: In 2019, the agreement between diagnostic tests was investigated by comparing the diagnostic performance for eye posterior pole pathologies of the images obtained by a smartphone coupled to a medical device known as open retinoscope (OR), handled by a nurse and subsequently assessed by an ophthalmologist versus the images obtained by an ophthalmologist using a slit lamp associated to a 76 diopter indirect ophthalmic lens (Volk Super FieldVR ) (SL-IOL) at the outpatient department of a hospital. The OR used in this study worked with a 28 diopter indirect lens.Results: An examination of 151 dilated eyes (79 adult patients, mean age of 66.7 years, 59.5% women) was conducted. Sensitivity was 98.9%, specificity was 89.8%, the positive predictive value was 93.8% and the negative predictive value was 98.2%. The kappa index between both tests was 0.90 (95% CI: 0.83-0.97) in basic diagnosis, 0.81 (95% CI: 0.74-0.89) in syndromic diagnosis (13 categories) and 0.70 (95% CI: 0.62-0.77) in advanced diagnosis (23 categories).Discussion: Images obtained by a nurse using a smartphone coupled to the OR and subsequently assessed by an ophthalmologist showed a high diagnostic performance for eye posterior pole pathologies, which could pave the way for remote ophthalmology systems for this patient group.
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Airborne-mediated microbial diseases represent one of the major challenges to public health. Ultraviolet C radiation (UVC) is among the different sanitation techniques useful to reduce the risk of infection in healthcare facilities. Previous studies about the germicidal activity of UVC were mainly performed in artificial settings or in vitro models. This study aimed to assess the sanitizing effectiveness of a UVC device (SanificaAria 200, Beghelli, Valsamoggia, Bologna, Italy) in 'real-life' conditions by evaluating its ability to reduce microbial loads in several hospital settings during routine daily activities. The efficacy of the UVC lamp in reducing the bacterial component was evaluated by microbial culture through the collection of air samples in different healthcare settings at different times (30 min-24 h) after turning on the device. To assess the anti-viral activity, air samplings were carried out in a room where a SARS-CoV-2-positive subject was present. The UVC device showed good antibacterial properties against a wide range of microbial species after 6 h of activity. It was effective against possible multi-drug resistant microorganisms (e.g., Pseudomonas spp., Acinetobacter spp.) and spore-forming bacteria (e.g., Bacillus spp.). In addition, the UVC lamp was able to inactivate SARS-CoV-2 in just one hour. Thanks to its effectiveness and safety, SanificaAria 200 could be useful to inactivate airborne pathogens and reduce health risks.
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Objectives: To establish a point-of-care test for coronavirus disease 2019 (COVID-19), we developed a dry loop-mediated isothermal amplification (LAMP) method to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA. Methods: We carried out reverse transcription (RT)-LAMP using the Loopamp SARS-CoV-2 Detection kit (Eiken Chemical, Tokyo, Japan). The entire mixture, except for the primers, is dried and immobilized inside the tube lid. Results: To determine the specificity of the kit, 22 viruses associated with respiratory infections, including SARS-CoV-2, were tested. The sensitivity of this assay, determined by either a real-time turbidity assay or colorimetric change of the reaction mixture, as evaluated by the naked eye or under illumination with ultraviolet light, was 10 copies/reaction. No LAMP product was detected in reactions performed with RNA from any pathogens other than SARS-CoV-2. After completing an initial validation analysis, we analyzed 24 nasopharyngeal swab specimens collected from patients suspected to have COVID-19. Of the 24 samples, 19 (79.2%) were determined by real-time RT-PCR analysis as being positive for SARS-CoV-2 RNA. Using the Loopamp SARS-CoV-2 Detection kit, we detected SARS-CoV-2 RNA in 15 (62.5%) of the 24 samples. Thus, the sensitivity, specificity, positive predictive value, and negative predictive values of the Loopamp 2019-CoV-2 detection reagent kit were 78.9%, 100%, 100%, and 55.6%, respectively. Conclusions: The dry LAMP method for detecting SARS-CoV-2 RNA is fast and easy to use, and its reagents can be stored at 4°C, solving the cold chain problem; thus, it represents a promising tool for COVID-19 diagnosis in developing countries.
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The emergence of SARS-CoV-2 has caused worldwide pandemic of COVID-19. Infection is difficult to diagnose early as some patients remain asymptomatic and may carry this virus to other people. Currently, qRT-PCR is the widely accepted mode for detection. However, the need for sophisticated instrument and trained personnel may hinder its application, especially in remote and facility-lacking areas. Reverse transcription loop-mediated isothermal amplification (RT-LAMP) may serve as a potential approach for detection of SARS-CoV-2 as the resources needed in its application is far less complex than those of qRT-PCR. Herein, we evaluated RT-LAMP based analytical method (COVIDNow), relative to qRT-PCR, in detecting SARS-CoV-2 by using 63 clinical respiratory samples. Based on our finding, COVIDNow exhibited sensitivity and specificity values of 87.5% and 80.6%, respectively. Taken together, RT-LAMP based detection of SARS-CoV-2 by utilizing COVIDNow might serves as a valuable diagnostic tool in the management of global COVID-19 pandemic condition.
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With the rise of the COVID-19 pandemic across the globe, people have come to understand the requirement of sanitation. However, other than the personal hygiene, sanitization of all the appliances (like mobile phone, wristwatch, wallet, eye wear, etc.) has become very important. Therefore, the requirement of germicides is being increased to sanitize all the appliances. A few germicides comprise chemical sanitization mechanisms and others devices are based on high radiant UV (ultraviolet) light. From the available literature, UV-based germicides are more efficient and effective in killing the harmful microorganisms. However, in the existing system the rate of disinfection is less, which makes this system lag. In general, the UV-based germicides use UV rays for the sanitization process. The UV rays are one of the forms of electromagnetic radiation with the wave lengths from 10 to 400nm. Typically, there are three types of UV rays, viz., ultraviolet A (UVA), ultraviolet B (UVB), and ultraviolet C (UVC) Moreover, UVA are longer waves, used as a black light through which microorganisms may be noticeable, while UVC are the shorter waves that kill the harmful microorganisms directly by destructing their DNA. Further, we have developed a germicidal system with greater rate of disinfection that is comparatively faster than the existing system, and it also includes the sanitization of our working environment too to disinfect the airborne organisms with greater accuracy. In the present invention, an IoT device that provides surface sanitization through microcontroller-based UV germicide is developed to reduce the disinfection time and air sterilization as well. © 2023 Elsevier Ltd. All rights reserved.
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Molecular detection of SARS-CoV-2 in gargle and saliva complements the standard analysis of nasopharyngeal swabs (NPS) specimens. Although gargle and saliva specimens can be readily obtained non-invasively, appropriate collection and processing of gargle and saliva specimens are critical to the accuracy and sensitivity of the overall analytical method. This review highlights challenges and recent advances in the treatment of gargle and saliva samples for subsequent analysis using reverse transcription polymerase chain reaction (RT-PCR) and isothermal amplification techniques. Important considerations include appropriate collection of gargle and saliva samples, on-site inactivation of viruses in the sample, preservation of viral RNA, extraction and concentration of viral RNA, removal of substances that inhibit nucleic acid amplification reactions, and the compatibility of sample treatment protocols with the subsequent nucleic acid amplification and detection techniques. The principles and approaches discussed in this review are applicable to molecular detection of other microbial pathogens.
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BACKGROUND: COVID-19 has become a major public health problem after the outbreak caused by SARS-CoV-2 virus. Great efforts to contain COVID-19 transmission have been applied worldwide. In this context, accurate and fast diagnosis is essential. METHODS: In this prospective study, we evaluated the clinical performance of three different RNA-based molecular tests - RT-qPCR (Charité protocol), RT-qPCR (CDC (USA) protocol) and RT-LAMP - and one rapid test for detecting anti-SARS-CoV-2 IgM and IgG antibodies. RESULTS: Our results demonstrate that RT-qPCR using the CDC (USA) protocol is the most accurate diagnostic test among those evaluated, while oro-nasopharyngeal swabs are the most appropriate biological sample. RT-LAMP was the RNA-based molecular test with lowest sensitivity while the serological test presented the lowest sensitivity among all evaluated tests, indicating that the latter test is not a good predictor of disease in the first days after symptoms onset. Additionally, we observed higher viral load in individuals who reported more than 3 symptoms at the baseline. Nevertheless, viral load had not impacted the probability of testing positive for SARS-CoV-2. CONCLUSION: Our data indicates that RT-qPCR using the CDC (USA) protocol in oro-nasopharyngeal swabs samples should be the method of choice to diagnosis COVID-19.
Subject(s)
COVID-19 , SARS-CoV-2 , Humans , SARS-CoV-2/genetics , COVID-19/diagnosis , COVID-19 Testing , Prospective Studies , Brazil/epidemiology , Clinical Laboratory Techniques/methods , Health Personnel , RNA , Immunoglobulin G , Immunoglobulin M , Sensitivity and SpecificityABSTRACT
Rapid point-of-care diagnostics, essential in settings such as airport on-site testing and home-based screening, displayed important implications for infectious disease control during the SARS-CoV-2 outbreak. However, the deployment of simple and sensitive assays in real-life scenarios still faces the concern of aerosol contamination. Here, we report an amplicon-depleting CRISPR-based one-pot loop-mediated isothermal amplification (CoLAMP) assay for point-of-care diagnosis of SARS-CoV-2 RNA. In this work, AapCas12b sgRNA is designed to recognize the activator sequence sited in the loop region of the LAMP product, which is crucial for exponential amplification. By destroying the aerosol-prone amplifiable products at the end of each amplification reaction, our design can significantly reduce the amplicons contamination that causes false positive results in point-of-care diagnostics. For at-home self-testing, we designed a low-cost sample-to-result device for fluorescence-based visual interpretation. As well, a commercial portable electrochemical platform was deployed as a proof-of-concept of ready-to-use point-of-care diagnostic systems. The field deployable CoLAMP assay can detect as low as 0.5 copies/µL of SARS-CoV-2 RNA in clinical nasopharyngeal swab samples within 40 min without the need for specialists for its operation.
Subject(s)
Biosensing Techniques , COVID-19 , Humans , SARS-CoV-2/genetics , COVID-19/diagnosis , RNA, Viral/genetics , Biosensing Techniques/methods , Molecular Diagnostic Techniques/methods , Nucleic Acid Amplification Techniques/methods , Sensitivity and Specificity , COVID-19 TestingABSTRACT
Loop-mediated isothermal amplification (LAMP), as the rank one alternative to a polymerase chain reaction (PCR), has been widely applied in point-of-care testing (POCT) due to its rapid, simple, and cost-effective characteristics. However, it is difficult to achieve real-time monitoring and multiplex detection with the traditional LAMP method. In addition, these approaches that use turbidimetry, sequence-independent intercalating dyes, or pH-sensitive indicators to indirectly reflect amplification can result in false-positive results if non-specific amplification occurs. To fulfill the needs of specific target detection and one-pot multiplex detection, a variety of probe-based LAMP assays have been developed. This review focuses on the principles of these assays, summarizes their applications in pathogen detection, and discusses their features and advantages over the traditional LAMP methods.
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CRISPR-Cas-based diagnostics have the potential to elevate nucleic acid detection. CRISPR-Cas systems can be combined with a pre-amplification step in a one-pot reaction to simplify the workflow and reduce carryover contamination. Here, we report an engineered Cas12b with improved thermostability that falls within the optimal temperature range (60°C-65°C) of reverse transcription-loop-mediated isothermal amplification (RT-LAMP). Using de novo structural analyses, we introduce mutations to wild-type BrCas12b to tighten its hydrophobic cores, thereby enhancing thermostability. The one-pot detection assay utilizing the engineered BrCas12b, called SPLENDID (single-pot LAMP-mediated engineered BrCas12b for nucleic acid detection of infectious diseases), exhibits robust trans-cleavage activity up to 67°C in a one-pot setting. We validate SPLENDID clinically in 80 serum samples for hepatitis C virus (HCV) and 66 saliva samples for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) with high specificity and accuracy. We obtain results in as little as 20 min, and with the extraction process, the entire assay can be performed within an hour.
Subject(s)
COVID-19 , Nucleic Acids , Humans , SARS-CoV-2/genetics , COVID-19/diagnosis , COVID-19/genetics , Nucleic Acids/genetics , COVID-19 Testing , CRISPR-Cas Systems/geneticsABSTRACT
Porcine epidemic diarrhea virus (PEDV) and porcine circovirus type 2 (PCV2) are both important global pathogenic viruses which have a significant impact on the swine industry. In this study, a duplex loop-mediated isothermal amplification (duplex LAMP) method was developed in combination with lateral flow dipstick (LFD) for simultaneous detection of PEDV and PCV2 using specific sets of primers and probes designed based on the conserved regions of a spike gene (KF272920) and an ORF gene (EF493839), respectively. The limit of detection (LOD) values of the duplex LAMP-LFD for the detection of PEDV and PCV2 were 0.1 ng/µL and 0.246 ng/µL, respectively. The LOD of duplex LAMP-LFD was 10-times more sensitive than conventional PCR and RT-PCR-agarose gel-electrophoresis (PCR-AGE and RT-PCR-AGE). No cross-reaction to each other and to other pathogenic viruses that can infect pigs were observed according to analytical specificity tests. The duplex LAMP-LFD method for the simultaneous detection of PEDV and PCV2 co-infection could be completed within approximately 1.5 h, and only a simple heating block was required for isothermal amplification. The preliminary validation using 50 swine clinical samples with positive and negative PEDV and/or PCV2 revealed that the sensitivity, specificity, and accuracy of duplex LAMP-LFD were all 100% in comparison to conventional PCR and RT-PCR. Hence, this study suggests that duplex LAMP-LFD is a promising tool for the early detection and initial screening of PEDV and PCV2, which could be beneficial for prevention, planning, and epidemiological surveys of these diseases.
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3D PRINTING OF MAGNETIC SEPARATOR: AN AFFORDABLE APPROACH TO SAMPLE PREPARATION IN THE COVID-19 DIAGNOSIS. This report describes the fabrication of a low-cost magnetic separator holder combining 3D printing and compact neodymium blocks for allowing magnetic extraction and purification of RNA from samples collected by nasopharyngeal swab from patients infected by SARS-CoV-2. The device was designed to contain 24 entrances for plastic microtubes in an arrangement like a commercial device. The proof of concept of the proposed device was successfully demonstrated through the sample extraction and purification of swab samples collected from eight patients suspected of SARS-CoV-2 infection. The sample preparation protocol was performed using a commercial kit containing magnetic beads and different solutions. The performance of the printed device was compared to a commercial magnetic separator, usually employed in the golden standard techniques. The fabrication of the 3D printed magnetic separator was completed under optimized printing conditions within 6 h at cost of 4 USD per unit. The RNA extracted from samples using both devices was analyzed by reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) and the achieved results have indicated no statistical different at confidence level of 95%. Based on the achievements, the use of 3D printing and neodymium blocks have demonstrated an alternative route to be used in routing analysis associated to COVID-19 diagnosis.
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COVID-19 outbreak caused by the newly discovered SARS-CoV-2 has become a major public health threat around the world and has create a tremendous effect on the global economy. Hence, there is a high demand for rapid and accurate diagnosis to contain the spread of the disease. The Reverse-Transcription Polymerase Chain Reaction (RT-PCR), the current standard for diagnosis of COVID-19 however possesses certain drawbacks that limits its application to meet the high demand of the continually increasing COVID-19 cases. Conversely, Loop-Mediated Isothermal Amplification (LAMP) is another nucleic acid amplification method that shows a great potential as an alternative tool in rapid diagnosis of COVID-19 due to its simplicity and rapidity. This review summarized the recent published research articles related to the application and modification of RT-LAMP assay for the rapid detection of COVID-19 in comparison with other available diagnostic methods. © 2023 Authors. All rights reserved.
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Background: Safe hospital access needs rapid testing for SARS-CoV-2 to enable rationale use of limited resources. The current standard method for Coronavirus detection is the RT-qPCR. This study aimed to determine the diagnostic performance of the new rapid RT-LAMP test, compared to RT-qPCR, and his efficiency for rapid hospital access through the Emergency Department (E.D.). Method(s): 1576 UTM nasopharyngeal swabs, collected in E.D., have been tested for SARS-CoV-2 infection, using a kit RTLAMP. The same samples were also analyzed with a traditional RT-qPCR assay and the results have been compared in terms of sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV). Result(s): The assay has demonstrated a sensitivity of 73.3% (95%CI: 62.4/82.0) and specificity of 87.1% (95%CI: 85.3/88.7), PPV 22.1%, NPV 98.5%. Conclusion(s): ICGENE RNA RT-LAMP kit (ICGENEHEALTH;Enbiotech, Angri, Salerno, Italy) efficiently exclude the presence of infection and reliably detects infectious patients (with Ct<30). RNA RT-LAMP could replace rRTPCR where there is the need to rapidly identify potentially contagious individuals, but its low PPV suggests that positive results should be confirmed by a reference method.Copyright © 2022 EDIZIONI MINERVA MEDICA.
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The coronavirus disease 2019 (COVID-19) pandemic caused unparalleled global threat in terms of public health and economic loss. To date, there is no effective way of treating this disease, and the only way to control this disease is the extensive diagnosis of COVID-19 symptomatic patients and isolate them from the healthy population and treat them with appropriate medicine. There is a requirement of a global standard diagnosis method to quickly control this pandemic disease, which should be specific, easy to use, and inexpensive and requires least instrumentation at point-of-care testing (POCT). Serology-based tests are popular, inexpensive, and easy to use to diagnose COVID-19 patients, but they lack sensitivity at lower inoculum concentrations and may indicate false negatives which possess a major threat in spreading this pandemic disease. To avoid this issue, nucleic acid-based tests are more specific and sensitive to diagnose COVID-19 patients. However, it has some limitations such as a low sample throughput, expensive reagents, an extensive time, and requirement of costly quantitative reverse transcription–polymerase chain reaction instruments. To overcome this limitation, the latest CRISPR-based detection methods coupled with allied isothermal nucleic acid amplification methods such as loop-mediated isothermal amplification would provide inexpensive, quick, accurate, and easy ways of diagnosing a large number of populations at POCT. Here, we discuss some of the promising CRISPR-based assays which have the potential to transform COVID-19 diagnosis globally and curb this pandemic disease in the shortest possible time. © 2023 Elsevier Inc. All rights reserved.