ABSTRACT
Loop-mediated isothermal amplification (LAMP) has been recently studied as an alternative method for cost-effective diagnostics in the context of the current COVID-19 pandemic. Recent reports document that LAMP-based diagnostic methods have a comparable sensitivity and specificity to that of RT-qPCR. We report the use of a portable Arduino-based LAMP-based amplification system assisted by pH microelectrodes for the accurate and reliable diagnosis of SARS-CoV-2 during the first 3 minutes of the amplification reaction. We show that this simple system enables a straightforward discrimination between samples containing or not containing artificial SARS-CoV-2 genetic material in the range of 10 to 10,000 copies per 50 {micro}L of reaction mix. We also spiked saliva samples with SARS-CoV-2 synthetic material and corroborated that the LAMP reaction can be successfully monitored in real time using microelectrodes in saliva samples as well. These results may have profound implications for the design of real-time and portable quantitative systems for the reliable detection of viral pathogens including SARS-CoV-2.
ABSTRACT
This study is the first focused on the presence of SARS-CoV-2 in different freshwater environments in an urban setting. Groundwater and surface water reservoirs for drinking water as well as water from receiving rivers of the Monterrey Metropolitan Area were sampled repeatedly during a SARS-CoV-2 peak phase between October 2020 and January 2021, and viral RNA was measured by quantitative reverse transcription polymerase chain reaction. Forty-four percent of the groundwater samples had detectable viral loads between 2.6 and 38.3 copies/ml. A significant correlation between viral load and sucralose concentration in groundwater reaffirmed the hypothesis of leaching and infiltrating effluent from surface and/or failing sewage pipes and emphasized the importance of water disinfection. Twelve percent of the surface water dam samples tested positive for viral RNA, with values varying between 3.3 and 3.8 copies/ml. Finally, 13% of the river samples were positive for viral RNA, with concentrations ranging from 2.5 to 7.0 copies/ml. Untreated wastewater samples taken in the same period showed viral loads of up to 3535 copies/ml, demonstrating a dilution effect and/or wastewater facilities efficiency of three orders of magnitude. Variations in the viral loads in the groundwater and surface water over time and at the submetropolitan level generally reflected the reported trends in infection cases for Monterrey. The viral loads in the freshwater environments of Monterrey represent a low risk for recreational activities according to a preliminary risk assessment model. However, this result should not be taken lightly due to uncertainty regarding data and model constraints and the possibility of situations where the infection risk may increase considerably.
ABSTRACT
We have investigated the importance of the rate of vaccination to contain COVID-19 in urban areas. We used an extremely simple epidemiological model that is amenable to implementation in an Excel spreadsheet and includes the demographics of social distancing, efficacy of massive testing and quarantine, and coverage and rate of vaccination as the main parameters to model the progression of COVID-19 pandemics in densely populated urban areas. Our model predicts that effective containment of pandemic progression in densely populated cities would be more effectively achieved by vaccination campaigns that consider the fast distribution and application of vaccines (i.e., 50% coverage in 6 months) while social distancing measures are still in place. Our results suggest that the rate of vaccination is more important than the overall vaccination coverage for containing COVID-19. In addition, our modeling indicates that widespread testing and quarantining of infected subjects would greatly benefit the success of vaccination campaigns. We envision this simple model as a friendly, readily accessible, and cost-effective tool for assisting health officials and local governments in the rational design/planning of vaccination strategies.
ABSTRACT
Massive worldwide serological testing for SARS-CoV-2 is needed to determine the extent of virus exposure in a particular region, the ratio of symptomatic to asymptomatic infected persons, and the duration and extent of immunity after infection. To achieve this aim, the development and production of reliable and cost-effective SARS-CoV-2 antigens is critical. Here, we report the bacterial production of the peptide S-RBDN318-V510, which contains the receptor binding domain of the SARS-CoV-2 spike protein. We purified this peptide using a straightforward approach involving bacterial lysis, his-tag mediated affinity chromatography, and imidazole-assisted refolding. The antigen performances of S RBDN318 V510 and a commercial full-length spike protein were compared in two distinct ELISAs. In direct ELISAs, where the antigen was directly bound to the ELISA surface, both antigens discriminated sera from non-exposed and exposed individuals. However, the discriminating resolution was better in ELISAs that used the full-spike antigen than the S-RBDN318-V510. Attachment of the antigens to the ELISA surface using a layer of anti-histidine antibodies gave equivalent resolution for both S-RBDN318-V510 and the full length spike protein. Our results demonstrate that ELISA-functional SARS-CoV-2 antigens can be produced in bacterial cultures. S-RBDN318-V510 is amenable to massive production and may represent a cost-effective alternative to the use of structurally more complex antigens in serological COVID-19 testing.
ABSTRACT
We present a simple epidemiological model that includes demographic density, social distancing, and efficacy of massive testing and quarantine as the main parameters to model the progression of COVID-19 pandemics in densely populated urban areas (i.e., above 5,000 inhabitants km2). Our model demonstrates that effective containment of pandemic progression in densely populated cities is achieved only by combining social distancing and widespread testing for quarantining of infected subjects. Our results suggest that extreme social distancing without intensive testing is ineffective in extinguishing COVID-19. This finding has profound epidemiological significance and sheds light on the controversy regarding the relative effectiveness of widespread testing and social distancing. Our simple epidemiological simulator is also useful for assessing the efficacy of governmental/societal responses to an outbreak. This study also has relevant implications for the concept of smart cities, as densely populated areas are hotspots that are highly vulnerable to epidemic crises.
ABSTRACT
By the third week of June 2020, more than 8,500,000 positive cases of COVID-19 and more than 450,000 deaths had been officially reported worldwide. The COVID-19 pandemic arrived in Latin America, India, and Africa--territories in which the mounted infrastructure for diagnosis is greatly underdeveloped. Here, we demonstrate the combined use of a three-dimensional (3D)-printed incubation chamber for commercial Eppendorf PCR tubes, and a colorimetric embodiment of a loop-mediated isothermal amplification (LAMP) reaction scheme for the detection of SARS-CoV-2 nucleic acids. We used this strategy to detect and amplify SARS-CoV-2 genetic sequences using a set of in-house designed initiators that target regions encoding the N protein. We were able to detect and amplify SARS-CoV-2 nucleic acids in the range of 62 to 2 x 105 DNA copies by this straightforward method. Using synthetic SARS-CoV-2 samples and a limited number of RNA extracts from patients, we also demonstrate that colorimetric LAMP is a quantitative method comparable in diagnostic performance to RT-qPCR. We envision that LAMP may greatly enhance the capabilities for COVID-19 testing in situations where RT-qPCR is not feasible or is unavailable. Moreover, the portability, ease of use, and reproducibility of this strategy make it a reliable alternative for deployment of point-of-care SARS-CoV-2 detection efforts during the pandemics.
ABSTRACT
The coronavirus disease 2019 (COVID-19) pandemic has crudely demonstrated the need for massive and rapid diagnostics. By the first week of July, more than 10,000,000 positive cases of COVID-19 have been reported worldwide, although this number could be greatly underestimated. In the case of an epidemic emergency, the first line of response should be based on commercially available and validated resources. Here, we demonstrate the use of the miniPCR, a commercial compact and portable PCR device recently available on the market, in combination with a commercial well-plate reader as a diagnostic system for detecting genetic material of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causal agent of COVID-19. We used the miniPCR to detect and amplify SARS-CoV-2 DNA sequences using the sets of initiators recommended by the World Health Organization (WHO) for targeting three different regions that encode for the N protein. Prior to amplification, samples were combined with a DNA intercalating reagent (i.e., EvaGreen Dye). Sample fluorescence after amplification was then read using a commercial 96-well plate reader. This straightforward method allows the detection and amplification of SARS-CoV-2 nucleic acids in the range of [~]625 to 2x105 DNA copies. The accuracy and simplicity of this diagnostics strategy may provide a cost-efficient and reliable alternative for COVID-19 pandemic testing, particularly in underdeveloped regions where RT-QPCR instrument availability may be limited. The portability, ease of use, and reproducibility of the miniPCR makes it a reliable alternative for deployment in point-of-care SARS-CoV-2 detection efforts during pandemics.
ABSTRACT
COVID-19, the first pandemic of this decade and the second in less than 15 years, has harshly taught us that viral diseases do not recognize boundaries; however, they truly do discriminate between aggressive and mediocre containment responses. We present a simple epidemiological model that is amenable to implementation in Excel spreadsheets and sufficiently accurate to reproduce observed data on the evolution of the COVID-19 pandemics in different regions (i.e., Italy, Spain, and New York City (NYC)). We also show that the model can be adapted to closely follow the evolution of COVID-19 in any large city by simply adjusting two parameters related to (a) population density and (b) aggressiveness of the response from a society/government to epidemics. Moreover, we show that this simple epidemiological simulator can be used to assess the efficacy of the response of a government/society to an outbreak. The simplicity and accuracy of this model will greatly contribute to democratizing the availability of knowledge in societies regarding the extent of an epidemic event and the efficacy of a governmental response.
