Metodo LAMP como alternativa diagnostica para la deteccion del virus SARS-CoV-2

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.

Molecular Diagnostics on Smartphone

During the COVID-19 pandemic, point-of-care genetic testing (POCT) devices were used for on-time and on-site detection of the virus, which helped to prevent and control the spread of the pandemic. Smartphones, which are widely used electronic devices with many functions, have the potential to be used as a molecular diagnostic platform for universal healthcare monitoring. Several integrated diagnostics platforms for the real-time and end-point detection of COVID-19 were developed using the functions of smartphones, such as the operating system, power, sound, camera, data storage, and display. These platforms use the 5V output power of smartphones, which can be amplified to power a micro-capillary electrophoresis system or a thin-film heater, and the CMOS camera of smartphones can capture the color change during a colorimetric loop-mediated isothermal amplification test and detect fluorescence signals. Smartphones can also be used with self-written web-based apps to enable automatic and remote pathogen analysis on POCT platforms. Our lab developed a handheld micro-capillary electrophoresis device for end-point detection of SARS-CoV-2, as well as an integrated smartphone-based genetic analyzer for the qualitative and quantitative colorimetric detection of foodborne pathogens with the help of a custom mobile app. © 2023 SPIE.

'CoviSwiftTM': A point-of-care RT-PCR device for SARS-CoV-2 and its variant detection.

We present the point-of-care (POC) molecular diagnostic solution, evaluated during COVID-19 pandemic caused by SARS-CoV-2 (Dec 2021). The POC comprised of a complete platform from self-sampling to RT-PCR testing of SARS-CoV-2 and its variants on portable Compact Q Real time polymerase chain reaction system. The multiplex assay was designed to target S, ORF1, and N genes of SARS-CoV-2 genome in a single tube with RNaseP as endogenous internal control. The present POC enables high accuracy (>95%) and high-throughput testing with a turnaround time of 45 min. It provides a unique platform from self-sample collection to report generation with rapid protocol, pipette and expert-free operation, long shelf-life stability and room temperature storage which enable to increase the efficiency of molecular testing. This novel test named "CoviSwift™ COVID-19 S PLUS RAPID PCR KIT" has been approved by CDSCO, Indian National Regulatory Authority, India, and is in use for clinical settings in India.

An Old Acquaintance: Could Adenoviruses Be Our Next Pandemic Threat?

Human adenoviruses (HAdV) are one of the most important pathogens detected in acute respiratory diseases in pediatrics and immunocompromised patients. In 1953, Wallace Rowe described it for the first time in oropharyngeal lymphatic tissue. To date, more than 110 types of HAdV have been described, with different cellular tropisms. They can cause respiratory and gastrointestinal symptoms, even urinary tract inflammation, although most infections are asymptomatic. However, there is a population at risk that can develop serious and even lethal conditions. These viruses have a double-stranded DNA genome, 25-48 kbp, 90 nm in diameter, without a mantle, are stable in the environment, and resistant to fat-soluble detergents. Currently the diagnosis is made with lateral flow immunochromatography or molecular biology through a polymerase chain reaction. This review aimed to highlight the HAdV variability and the pandemic potential that a HAdV3 and 7 recombinant could have considering the aggressive outbreaks produced in health facilities. Herein, we described the characteristics of HAdV, from the infection to treatment, vaccine development, and the evaluation of the social determinants of health associated with HAdV, suggesting the necessary measures for future sanitary control to prevent disasters such as the SARS-CoV-2 pandemic, with an emphasis on the use of recombinant AdV vaccines to control other potential pandemics.

Towards Point of Care CRISPR-Based Diagnostics: From Method to Device.

Rapid, accurate, and portable on-site detection is critical in the face of public health emergencies. Infectious disease control and public health emergency policymaking can both be aided by effective and trustworthy point of care tests (POCT). A very promising POCT method appears to be the clustered regularly interspaced short palindromic repeats and associated protein (CRISPR/Cas)-based molecular diagnosis. For on-site detection, CRISPR/Cas-based detection can be combined with multiple signal sensing methods and integrated into smart devices. In this review, sensing methods for CRISPR/Cas-based diagnostics are introduced and the advanced strategies and recent advances in CRISPR/Cas-based POCT are reviewed. Finally, the future perspectives of CRISPR and POCT are summarized and prospected.

Advances in molecular diagnostic tests for community-acquired pneumonia

The use of a timely and appropriate antibiotic therapy, which requires early and accurate microorganisms' detection in pneumonia. Currently, the identification of microorganisms in pneumonia is limited by the low sensitivity and long response time of standard culture-based diagnostic tools. For this reason, treatment in pneumonia is empirical. An inadequate empirical treatment is related to poor outcomes in patients with pneumonia. The microbiological diagnosis is key to improve the outcomes in patient with pneumonia. Over the past years there was a significant advance in the molecular diagnosis of infectious diseases including pneumonia. Also the impact of the COVID-19 pandemic has impacted the development and application of these new molecular techniques. This review summarizes the advances in molecular diagnosis of community-acquired pneumonia.Copyright © 2022 EDIZIONI MINERVA MEDICA.

The application of nano-biosensors in the detection of 2019-nCoV.

The pandemic of 2019 novel coronavirus (2019-nCoV) infection since 2020 caused Coronavirus Disease 2019 (COVID-19) leads the serious threaten to global public health. It is urgent to diagnose COVID-19, guide epidemiological measures, control the infection rates, research/develop the antiviral treatment and promote the vaccine research. The application of nano-material based biosensors (the nano-biosensors) has achieved the high-performance detection of a variety of biomarkers due to their small device size, label free detection, high sensitivity, good specificity, short detection time, and has been considered as great potential to become a point-of-care testing tool for detecting 2019-nCoV. Therefore, by summarizing the working principle and classification of nano-biosensors, and focusing on the research progress of nano-biosensors in the detection of 2019-nCoV reported in the recent years, our review provides the challenges and future development prospects of the nano-biosensor in clinical laboratory.Copyright © 2022 Chin J Lab Med. All rights reserved.

Rapid detection of mpox virus using recombinase aided amplification assay.

A recent, unprecedented outbreak of human mpox virus infection has led to cases in non-African nations, and the number of confirmed or suspected cases outside of Africa has exceeded 1,000 within 5 weeks. Mpox may pose a double threat to public health in the context of the ongoing COVID-19 pandemic. It is difficult to distinguish mpox virus infection from other diseases in the early stages, and patients are contagious from the onset of nonspecific symptoms; therefore, it is crucial to develop rapid and specific diagnostic methods. The diagnosis of mpox relies on real-time polymerase chain reaction, a time-consuming method that requires a highly sophisticated thermal cycler, which makes it unsuitable for widespread use in underdeveloped areas, where the outbreak is still severe. In this study, we developed a recombinase-aided amplification (RAA) assay that can detect mpox virus within 5-10 minutes. The conserved regions of the A27L gene and F3L gene were selected as targets, as they amplify well from different mpox virus clades with no cross-reaction from other pathogens. The sensitivity of this RAA assay is 10 copies/reaction for the A27L gene and 102 copies/reaction for the F3L gene. When applied to simulated clinical samples, both targets showed 100% specificity, and the detection limits were consistent with the sensitivity results. Moreover, through clinical blinded sample detection, RAA exhibits the same detection power as RT-PCR. In summary, the RAA mpox assay described here exhibits rapid detection, high sensitivity and specificity, and low operational difficulty, making it suitable for mpox virus detection in less developed countries and regions.

Genotype-Specific Detection of Ferret Coronavirus by Conventional and Real-Time Reverse Transcription Polymerase Chain Reaction

Ferret coronavirus is associated with two disease presentations in ferrets, namely, epizootic catarrhal enteritis and a feline infectious peritonitis (FIP)-like systemic disease. In this chapter, we describe conventional and real-time one-step reverse transcription polymerase chain reaction assays that are routinely used in our laboratory to detect either genotype 1 or genotype 2 ferret coronavirus in clinical specimens. These assays were designed based upon the conserved spike gene sequence difference found between three strains of ferret systemic coronavirus and three strains of ferret enteric coronavirus. Recent literature evidence indicates that pathotype is not associated with a specific genotype, and therefore, it is important to test for both genotypes either in enteric or systemic disease. Copyright © 2016 Springer Science+Business Media New York.

Head-to-Head Comparison of Nasopharyngeal, Oropharyngeal and Nasal Swabs for SARS-CoV-2 Molecular Testing.

Nasopharyngeal swabs (NPS) are considered the gold standard for SARS-CoV-2 testing but are technically challenging to perform and associated with discomfort. Alternative specimens for viral testing, such as oropharyngeal swabs (OPS) and nasal swabs, may be preferable, but strong evidence regarding their diagnostic sensitivity for SARS-CoV-2 testing is still missing. We conducted a head-to-head prospective study to compare the sensitivity of NPS, OPS and nasal swabs specimens for SARS-CoV-2 molecular testing. Adults with an initial positive SARS-CoV-2 test were invited to participate. All participants had OPS, NPS and nasal swab performed by an otorhinolaryngologist. We included 51 confirmed SARS-CoV-2-positive participants in the study. The sensitivity was highest for OPS at 94.1% (95% CI, 87 to 100%) compared to NPS at 92.5% (95% CI, 85 to 99%) (p = 1.00) and lowest for nasal swabs at 82.4% (95% CI, 72 to 93%) (p = 0.07). Combined OPS/NPS was detected in 100% of cases, while the combined OPS/nasal swab increased the sensitivity significantly to 96.1% (95% CI, 90 to 100%) compared to that of the nasal swab alone (p = 0.03). The mean Ct value for NPS was 24.98 compared to 26.63 for OPS (p = 0.084) and 30.60 for nasal swab (p = 0.002). OPS achieved a sensitivity comparable to NPS and should be considered an equivalent alternative for SARS-CoV-2 testing.

Contribution of PEPFAR-Supported HIV and TB Molecular Diagnostic Networks to COVID-19 Testing Preparedness in 16 Countries.

The US President's Emergency Plan for AIDS Relief (PEPFAR) supports molecular HIV and tuberculosis diagnostic networks and information management systems in low- and middle-income countries. We describe how national programs leveraged these PEPFAR-supported laboratory resources for SARS-CoV-2 testing during the COVID-19 pandemic. We sent a spreadsheet template consisting of 46 indicators for assessing the use of PEPFAR-supported diagnostic networks for COVID-19 pandemic response activities during April 1, 2020, to March 31, 2021, to 27 PEPFAR-supported countries or regions. A total of 109 PEPFAR-supported centralized HIV viral load and early infant diagnosis laboratories and 138 decentralized HIV and TB sites reported performing SARS-CoV-2 testing in 16 countries. Together, these sites contributed to >3.4 million SARS-CoV-2 tests during the 1-year period. Our findings illustrate that PEPFAR-supported diagnostic networks provided a wide range of resources to respond to emergency COVID-19 diagnostic testing in 16 low- and middle-income countries.

Novel Lateral Flow-Based Assay for Simple and Visual Detection of SARS-CoV-2 Mutations.

Identification of the main SARS-CoV-2 variants in real time is of interest to control the virus and to rapidly devise appropriate public health responses. The RT-qPCR is currently considered to be the reference method to screen SARS-CoV-2 mutations, but it has some limitations. The multiplexing capability is limited when the number of markers to detect increases. Moreover, the performance of this allele-specific method may be impacted in the presence of new mutations. Herein, we present a proof-of-concept study of a simple molecular assay to detect key SARS-CoV-2 mutations. The innovative features of the assay are the multiplex asymmetric one-step RT-PCR amplification covering different regions of SARS-CoV-2 S gene and the visual detection of mutations on a lateral flow DNA microarray. Three kits (Kit 1: N501Y, E484K; Kit 2: L452R, E484K/Q; Kit 3: K417N, L452R, E484K/Q/A) were developed to match recommendations for surveillance of SARS-CoV-2 variants between January and December 2021. The clinical performance was assessed using RNA extracts from 113 SARS-CoV-2-positive samples with cycle thresholds <30, and results demonstrated that our assay allows specific and sensitive detection of mutations, with a performance comparable to that of RT-qPCR. The VAR-CoV assay detected four SARS-CoV-2 targets and achieved specific and sensitive screening of spike mutations associated with the main variants of concern, with a performance comparable to that of RT-qPCR. With well-defined virus sequences, this assay can be rapidly adapted to other emerging mutations; it is a promising tool for variant surveillance.

Rapid Implementation of Real-Time Reverse-Transcription Polymerase Chain Reaction (Real-Time Rt-Pcr) Assay for the Detection of Sars-Cov-2 in a Moroccan Hospital

Background: The main challenge faced in the African countries was to implement efficient molecular diagnostic facilities and start Covid-19 diagnosis as fast as possible to handle the rapid and unpredictable rise of cases. Materials, Methods and Results: We describe our experience in implementing a molecular biology unit at Sheikh Zaid International University Hospital in Rabat, with a delay as short as one week, and starting real-time RT-PCR assay for the detection of SARS-Cov-2 infection, since the outbreak widened in Morocco in mid-March, 2020. Conclusion(s): The challenges encountered in the first period of Covid-19 pandemic are still present. This work aims to give an example of a rapid and adaptive response in order to maintain our diagnosis ability for Covid-19 and for other pathogens. Copyright © 2022, African Traditional, Herbal Medicine Supporters Initiative. All rights reserved.

The application of nano-biosensors in the detection of 2019-nCoV

The pandemic of 2019 novel coronavirus (2019-nCoV) infection since 2020 caused Coronavirus Disease 2019 (COVID-19) leads the serious threaten to global public health. It is urgent to diagnose COVID-19, guide epidemiological measures, control the infection rates, research/develop the antiviral treatment and promote the vaccine research. The application of nano-material based biosensors (the nano-biosensors) has achieved the high-performance detection of a variety of biomarkers due to their small device size, label free detection, high sensitivity, good specificity, short detection time, and has been considered as great potential to become a point-of-care testing tool for detecting 2019-nCoV. Therefore, by summarizing the working principle and classification of nano-biosensors, and focusing on the research progress of nano-biosensors in the detection of 2019-nCoV reported in the recent years, our review provides the challenges and future development prospects of the nano-biosensor in clinical laboratory. © 2022 Chin J Lab Med. All rights reserved.

Performance of Non-nasopharyngeal Sample Types for Molecular Detection of SARS-CoV-2.

Nasopharyngeal swabs have historically been considered the preferred specimen type for the detection of respiratory viruses, including SARS-CoV-2. However, in response to a global pandemic with shortages of swabs and specimen transport media, limited access to qualified health care personnel, and needs for large-scale testing in nonmedical settings, alternative sample types have been validated for COVID-19 diagnosis. The purpose of this review is to highlight the diagnostic accuracy and clinical utility of non-nasopharyngeal respiratory samples for SARS-CoV-2 molecular diagnostic testing.

Clinical validation of the isothermal rt-lamp test for the rapid diagnosis of SARS-CoV-2

Introduction: Since the first report in Wuhan (China) in 2019, the SARS-CoV 2 virus has spread throughout the world, with a significant impact in public health. To contain the spread of SARS-CoV-2, the WHO has encouraged the development of rapid, simple, sensitive and specific tests that complement the gold standard RT-qPCR. Loop-mediated isothermal amplification (LAMP) has shown a good yield to detect SARS-CoV2 in different fluids. Objective: To validate the colorimetric RT-LAMP technique using two sets of oligonucleotides aimed at identifying the N gene of SARS-CoV-2 in 117 nasopharyngeal swab samples, previously confirmed by RT-qPCR using the Charite/Berlin protocol. Material and methods: 153 nasopharyngeal swab samples from individuals with suspected Covid-19 were subjected to qRT-PCR and RT-LAMP using a commercial colorimetric kit (NEB, Germany). RT-LAMP was run using both extracted RNA samples and raw samples without prior RNA extraction, and the result was assessed by a simple color change in the reaction. Results: RT-LAMP sensibility and specificity for gen N SARS-CoV-2 detection using one primers set previosly reported got values 0.97 (0.85, 1.00) and 0.81 (0.65, 0.92) respectively, for CI 95%. The other set primer used in this paper also reported previosly had 0.96 (0.78,1.00) sensibility and 0.77 (0.55,0.92) specificity to RT-LAMP. Without RNA extraction we found sensibility value of 0.95 (0.74, 1.00) and specificity 0.88 (0.64, 0.99). Conclusion: Taking together, the obtained results show RT-LAMP technique could be considered a rapid diagnostic test, easy to perform, free of sophisticated equipment, sensitive and specific to diagnose SARS-CoV-2 in nasopharyngeal swabs with and without prior RNA extraction, which that allows scaling its portability to places with scarce sources of resources.

A pandemic guided by the SARS-CoV-2 PCR test: What should the clinician know?

Amidst an ever-evolving pandemic, the demand for timely and accurate diagnosis of coronavirus disease 2019 (COVID-19) continues to increase. Critically, managing and containing the spread of the disease requires expedient testing of infected individuals. Presently, the gold standard for the diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection remains the polymerase chain reaction (PCR) test. Potential vulnerabilities of this testing methodology can range from preanalytical variables to laboratory-related analytical factors and, ultimately, to the interpretation of results.

Performance Comparison of a Duplex Implementation of the CDC EUA 2019-nCoV Assay with the Seegene Allplex-SARS-CoV-2 Assay for the Detection of SARS-CoV-2 in Nasopharyngeal Swab Samples.

RT-PCR tests have become the gold standard for detecting the SARS-CoV-2 virus in the context of the COVID-19 pandemic. Because of the extreme number of cases in periodic waves of infection, there is a severe financial and logistical strain on diagnostic laboratories. For this reason, alternative implementations and validations of academic protocols that employ the lowest cost and the most widely available equipment and reagents found in different regions are essential. In this study, we report an alternative implementation of the EUA 2019-nCoV CDC assay which uses a previously characterized duplex PCR reaction for the N1 and RNAse P target regions and an additional uniplex reaction for the N2 target region. Taking advantage of the Abbott m2000 Sample Preparation System and NEB Luna Universal Probe One-Step RT-qPCR kit, some of the most widely available and inexpensive nucleic acid extraction and amplification platforms, this modified test shows state-of-the-art analytical and clinical sensitivities and specificities when compared with the Seegene Allplex-SARS-CoV-2 assay. This implementation has the potential to be verified and implemented by diagnostic laboratories around the world to guarantee low-cost RT-PCR tests that can take advantage of widely available equipment and reagents.