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Did the dogs of Mexico City really stop their owners dying rom Covid-19? Isaac Núñez shares an astonishing tale of causal questions, selection bias and confounding

Background Out-of-hospital deaths increased during peak coronavirus disease 2019 (COVID-19) pandemic periods. However, aside from COVID-19 severity, which variables are related to being hospitalized have not been properly studied. We examine the association of several variables with dying at home from COVID-19 as opposed to in a hospital. Methods We used COVID-19 open data from Mexico City from March 2020 until February 2021. A causal model was prespecified to identify variables of interest. Adjusted logistic regressions were performed to calculate ORs for associations between variables of interest and dying out of hospital due to COVID-19. Results Among 61 112 people who died due to COVID-19, 8080 died out of hospital. Older age (OR 3.49, age 90 vs 60 y), male sex (OR 1.18) and higher bed occupancy (OR 2.68, 90% vs 50% occupancy) were positively associated with dying outside of hospital. Conclusion Older age could confer different patient wishes or less ability to look for healthcare. Higher bed occupancy may have prevented hospital admission from people who required in-hospital care.

Case definitions are vital in a pandemic to effectively identify, isolate, and contact trace, particularly where testing is slow, scant, or not available. While case definitions have been developed in the COVID-19 pandemic, their diagnostic properties have not been adequately assessed. This study's objective is to determine the diagnostic properties of local and World Health Organization (WHO) COVID-19 case definitions in the large metropolitan area of Mexico City.We calculated the diagnostic properties of five COVID-19 definitions (three of the Mexican government and two of the WHO) using open data of suspected COVID-19 cases in Mexico City from March 24th, 2020, until May 15th, 2021.All 2,564,782 people included in the analysis met the WHO suspected case definition (sensitivity: 100%, specificity: 0%). The WHO probable case definition was met by 1.2%, while the first and second Mexican suspected case had sensitivities of 61% and specificities of 61% and 67%, respectively. Confirmed case by epidemiological contact had a low sensitivity (32%) but slightly higher specificity (81%).Case definitions should maximize sensitivity, especially in a high-transmission area such as Mexico City. The WHO suspected case definition has the potential for detecting most symptomatic cases. We underline the need for routine evaluation of case definitions as new evidence arises to maximize their usefulness.

The COVID-19 pandemic led to the development and emergency approval of an array of effective vaccines against SARS-CoV-2. Given the relatively small number of patients included in vaccine trials, postapproval epidemiological surveillance is crucial to detect infrequent vaccine-related adverse events. We conducted a nationwide retrospective descriptive study evaluating the incidence of seizures among recipients of SARS-CoV-2 vaccines in Mexico from December 24, 2020 (date of administration of first doses nationwide) to October 29, 2021. Among 81 916 351 doses of any vaccine that were administered, we documented seizures in 53 patients, of which 31 (60%) were new onset seizures. The incidence rate of seizures per million doses was highest for mRNA-1273 (Moderna) with 2.73 per million, followed by BNT162b2 (Pfizer-BioNTech) with 1.02 per million, and Ad5-nCoV (CanSino) with 1.01 per million. Thus, we found that seizures following SARS-CoV-2 vaccination are exceedingly rare events.

Information on Guillain-Barré syndrome (GBS) as an adverse event following immunization (AEFI) against SARS-CoV-2 remains scarce. We aimed to report GBS incidence as an AEFI among adult (≥18 years) recipients of 81,842,426 doses of seven anti-SARS-CoV-2 vaccines between December 24, 2020, and October 29, 2021, in Mexico.Cases were retrospectively collected through passive epidemiological surveillance. The overall observed incidence was calculated according to the total number of administered doses. Vaccines were analyzed individually and by vector as mRNA-based (mRNA-1273 and BNT162b2), adenovirus-vectored (ChAdOx1 nCov-19, rAd26-rAd5, Ad5-nCoV, and Ad26.COV2-S), and inactivated whole-virion-vectored (CoronaVac) vaccines.We identified 97 patients (52 males [53.6%]; median [interquartile range] age 44 [33-60] years), for an overall observed incidence of 1.19/1,000,000 doses (95% confidence interval [CI] 0.97-1.45), with incidence higher among Ad26.COV2-S (3.86/1,000,000 doses, 95% CI 1.50-9.93) and BNT162b2 recipients (1.92/1,00,000 doses, 95% CI 1.36-2.71). The interval (interquartile range) from vaccination to GBS symptom onset was 10 (3-17) days. Preceding diarrhea was reported in 21 patients (21.6%) and mild COVID-19 in four more (4.1%). Only 18 patients were tested for Campylobacter jejuni (positive in 16 [88.9%]). Electrophysiological examinations were performed in 76 patients (78.4%; axonal in 46 [60.5%] and demyelinating in 25 [32.8%]); variants were similar across the platforms. On admission, 91.8% had a GBS disability score ≥3. Seventy-five patients (77.3%) received intravenous immunoglobulin, received seven plasma exchange (7.2%), and 15 (15.5%) were treated conservatively. Ten patients (10.3%) died, and 79.1% of survivors were unable to walk independently.Guillain-Barré syndrome was an extremely infrequent AEFI against SARS-CoV-2. The protection provided by these vaccines outweighs the risk of developing GBS.

Objective Healthcare saturation has been a prominent worry during the COVID-19 pandemic. Increase of hospital beds with mechanical ventilators has been central in Mexico's approach, but it is not known whether this actually improves access to care and the resulting quality of it. This study aimed to determine the impact of healthcare strain and other pre-specified variables on dying from coronavirus disease 2019 (COVID-19) without receiving invasive mechanical ventilation (IMV). Methods A retrospective cohort study was conducted using open data from Mexico City between 8 May 2020 and 5 January 2021. We performed Cox proportional hazards models to identify the strength of the association between proposed variables and the outcomes. Results Of 33 797 hospitalized patients with suspected or confirmed COVID-19, 19 820 (58.6%) did not require IMV and survived, 5414 (16.1%) required IMV and were intubated and 8563 (25.3%) required IMV but died without receiving it. A greater occupation of IMV-capable beds increased the hazard of death without receiving IMV (hazard ratio [HR] 1.56, comparing 90% with 50% occupation). Private healthcare was the most protective factor for death without IMV (HR 0.14). Conclusions Higher hospital bed saturation increased the hazard of dying without being intubated and worsened the outcomes among mechanically ventilated patients. Older age also increased the hazard of the outcomes, while private healthcare dramatically decreased them.

The Golden Gate technique allows the assembly of genetic sequences from libraries of standardized basic components, which are cleaved from their donor vectors and concatenated in the acceptor vector in a defined order. This reaction is subsequently transformed into competent bacterial cells that are grown overnight to reveal positive colonies carrying the correctly assembled vector. The cleavage of the components is done by a TypeIIS restriction enzyme, which recognizes asymmetric DNA sequences and cleaves outside of this site, leaving overhangs in a directional way (Figure 1A). This property allows the creation of ordered assembly positions by defining synthetic syntaxes of these overhangs (Figure 1B). Within the different methodologies and libraries based on Golden Gate, some methods, such as uLoop Assembly (Pollak et al, 2020), allow the cyclic assembly of increasingly large and complex vectors in which the products of a reaction are the substrates of the next level reactions (Figure 2). By this means it is possible to build libraries of easily reusable components to perform assemblies in a combinatorial way of odd and even assembly levels. The standardized DNA components have to be flanked by the enzyme recognition sites and the proper syntax according to the desired position of assembly. These DNA pieces can be obtained from previously created libraries or custom made. The reaction operates cycling between 37°C and 16°C. At the first temperature, the restriction enzyme cuts the DNA pieces. These pieces are concatenated by their overhangs sequence homology and sealed by the catalysis of a ligase enzyme at the second temperature. Once a piece is correctly assembled it cannot be cutted again, and cycle by cycle, the pieces are concatenated in the final assembly product.

Background Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, the causative agent of coronavirus disease 2019 (COVID-19), may lead to severe systemic inflammatory response, pulmonary damage, and even acute respiratory distress syndrome (ARDS). This in turn may result in respiratory failure and in death. Experimentally, acetylcholine (ACh) modulates the acute inflammatory response, a neuro-immune mechanism known as the inflammatory reflex. Recent clinical evidence suggest that electrical and chemical stimulation of the inflammatory reflex may reduce the burden of inflammation in chronic inflammatory diseases. Pyridostigmine (PDG), an ACh-esterase inhibitor (i-ACh-e), increases the half-life of endogenous ACh, therefore mimicking the inflammatory reflex. This clinical trial is aimed at evaluating if add-on of PDG leads to a decrease of invasive mechanical ventilation and death among patients with severe COVID-19. Methods A parallel-group, multicenter, randomized, double-blinded, placebo-controlled, phase 2/3 clinical trial to test the efficacy of pyridostigmine bromide 60 mg/day P.O. to reduce the need for invasive mechanical ventilation and mortality in hospitalized patients with severe COVID-19. Discussion This study will provide preliminary evidence of whether or not -by decreasing systemic inflammation- add-on PDG can improve clinical outcomes in patients with severe COVID-19. Trial registration ClinicalTrials.gov NCT04343963 (registered on April 14, 2020).

Delay in COVID-19 diagnosis due to late real-time reverse transcription-polymerase chain reaction reporting has been described to be an important cause of suboptimal COVID-19 surveillance and outbreak containment.The objective of the study was to determine the duration of diagnostic delay due to test turnaround time and its association with marginalization status.In this observational study using national open data of Mexico and Colombia, we quantified the delay in COVID-19 diagnosis that occurred in both countries. We considered two periods that contributed to the delay in diagnosis: the time from symptom onset until testingAmong 3,696,773 patients from Mexico and Colombia,Diagnostic delay was mostly due to test turnaround time. Marginalization status was an important barrier to diagnostic test access.

Objectives: The aim of this study was to describe the incidence, clinical characteristics, and risk factors of late-onset opportunistic infections (LOI) in people who live with HIV (PWLHA) within the Caribbean, Central and South America network for HIV epidemiology. Methods: We performed a retrospective cohort study including treatment-naive PWLHA enrolled at seven sites (Argentina, Brazil, Chile, Peru, Mexico, and two sites in Honduras). Follow-up began at 6 months after treatment started. Outcomes were LOI, loss to follow-up, and death. We used a Cox proportional hazards model and a competing risks model to evaluate risk factors. Results: A total of 10,583 patients were included. Median follow up was at 5.4 years. LOI occurred in 895 (8.4%) patients. Median time to opportunistic infection was 2.1 years. The most common infections were tuberculosis (39%), esophageal candidiasis (10%), and Pneumocystis jirovecii (P. jirovecii) pneumonia (10%). Death occurred in 576 (5.4%) patients, and 3021 (28.5%) patients were lost to follow-up. A protease inhibitor–based regimen (hazard ratio 1.25), AIDS-defining events during the first 6 months of antiretroviral-treatment (hazard ratio 2.12), starting antiretroviral-treatment in earlier years (hazard ratio 1.52 for 2005 vs 2010), and treatment switch (hazard ratio 1.31) were associated with a higher risk of LOI. Conclusion: LOI occurred in nearly one in 10 patients. People with risk factors could benefit from closer follow-up.

ObjectiveTo compare hypoxemia severity of patients with COVID-19 pneumonia that arrive at an emergency department as classified by three oxygenation indexes.DesignRetrospective analysis of pulse oximeter saturation and arterial blood gas analysis obtained at arrival.SettingTertiary referral hospital in Mexico City converted early in the pandemic to a COVID-19 center.Patients and measurementsA total of 2,960 patients with suspected COVID-19 pneumonia were admitted to the emergency department from April 2020 until March 2021. Pulse oximeter saturation and arterial blood gas analysis was obtained in all of them. Pulse oximeter saturation (SpO2) to inspired oxygen fraction ratio (FiO2), oxygen saturation in arterial blood (SatO2) to FiO2 ratio, and oxygen pressure in arterial blood to FiO2 ratio were calculated for every patient.InterventionsNone.Main ResultsA strong correlation was seen between PaO2/FiO2 & SpO2/FiO2 (rho = 0.6, p < 0.001), and SatO2/FiO2 & SpO2/FiO2 (rho = 0.65, p < 0.001), while a very strong correlation was seen between PaO2/FiO2 & SatO2/FiO2 (rho = 0.88, p < 0.001). When classifying severity by quantiles, considerable cross-over was observed when comparing oxygenation indexes, as only 785 (26.5%) patients were in the same quintile across the three indexes.ConclusionsHypoxemia severity is heterogeneous according to the oxygenation index utilized. This limits their usefulness as sole markers of severity, as inter-observer variability, especially on FiO2 estimation, and different practices limit consistent follow up and treatment decisions.

ObjectiveTo quantify the delay in SARS-CoV-2 real time polymerase chain reaction (RT-PCR) testing and test result reporting in Mexico and Colombia, and to assess the relation between margination status and these delays.MethodsWe quantified time in days from symptom onset until testing (latency one) and delay in test results report (latency two) using freely available country-wide open data from Mexico and Colombia. Directed acyclic graphs were built to determine which associations were appropriate to assess. Stratification by margination status, state and hospitalization status was used to determine corresponding associations.ResultsIn almost all the study period latency two was longer than latency one. Median latency one was 3 (IQR 0-6) days and latency two 7 (IQR 4-11) days in Colombia, while in Mexico they were 3 (IQR 1-5) days and 4 (IQR 3-6) days. In Colombia, worse margination status prolonged latency two. In Mexico, a lower number and percentage of point-of-care (POC) tests in areas with worse margination.ConclusionPOC tests must be used as a widespread means to reduce latency two, and until then should be prioritized in areas with longer latency two. An unequal distribution of this resource should be avoided.

BackgroundPneumonia is the hallmark of severe COVID-19, with supplemental oxygen requirement being the main indication for hospitalization. Refractory hypoxemia in these patients requires invasive mechanical ventilation (IMV) otherwise, death is imminent. In places with a high disease burden, availability of critical care experts, beds, or resources is challenged and many patients could die without receiving them.MethodsWe performed a retrospective cohort study using open databases from Mexico City about suspected or confirmed COVID-19 patients, health system saturation, and deaths between May 8th, 2020, and January 5th, 2021. After building a directed acyclic graph, we performed a binary logistic regression to identify the association between proposed causal variables and dying without receiving IMV (the outcome).ResultsWe included 33 805 hospitalized patients with suspected or confirmed COVID-19, of which 19 820 (58.6%) did not require IMV and survived, 5416 (16.1%) required and received IMV, and 8569 (25.3%) required IMV but died without receiving it. Saturation of IMV-capable beds did not increase the odds of the outcome (odds ratio 1.07, 95% confidence interval 0.94-1.22 of 90%vs50% occupancy), while general bed saturation (2, 1.86-2.14 of 90%vs50% occupancy) and IMV-capable to general bed ratio (1.64, 1.52-1.77 for a ratio of 2vs0.5) did. Private healthcare decreased the odds of the outcome (0.12, 0.08-0.17) and dyspnea increased them (1.33, 1.19-1.9).ConclusionsIn Mexico City, increased general hospital bed saturation and IMV-capable to general bed ratio were associated with a higher risk of dying without receiving IMV. Private healthcare was the most protective factor.Key messagesHospital saturation has been a central feature of public health messaging, but it is not known how outcomes relate to hospital saturation or capacity.In Mexico City, 90% of COVID-19 patients requiring mechanical ventilation died but less than half received it.Higher general bed saturation and an increased ratio of IMV-capable beds to general beds increased the probability of dying without being intubated while receiving private healthcare decreased this probability.Having available beds to intubate patients is possible thanks to the conversion of general beds, however, still yields suboptimal critical care.

BackgroundEpidemiologic case definitions serve a myriad of purposes during a pandemic, including contact tracing and monitoring disease trends. It is unknown how any COVID-19 case definition fares against the current gold standard of molecular or antigen tests.MethodsWe calculated the diagnostic properties of five COVID-19 definitions (three of the Mexican government and two of the WHO) using open data of suspected COVID-19 cases in Mexico City from March 24th 2020 until January 31st 2021.ResultsAll 1,632,420 people included in the analysis met the WHO suspected case definition (sensitivity 100%, specificity 0%). The WHO probable case definition was met by 1.4%, while the first and second Mexican suspected case had sensitivities of 61 and 62% and specificities of 58 and 62%, respectively. Confirmed case by epidemiological contact had a low sensitivity (33%) but slightly higher specificity (77%).ConclusionsCase definitions should maximize sensitivity, especially in a high-transmission area such as Mexico City. The WHO suspected case definition has the potential for detecting most symptomatic cases. We underline the need for routine evaluation of case definitions as new evidence arises to maximize their usefulness.

RT-LAMP (reverse transcription - Loop-mediated isothermal amplification) has gained popularity for the detection of SARS-CoV-2. The high specificity, sensitivity, simple protocols and potential to deliver results without the use of expensive equipment has made it an attractive alternative to RT-PCR. However, the high cost per reaction, the centralized manufacturing of required reagents and their distribution under cold chain shipping limits RT-LAMP’s applicability in low-income settings. The preparation of assays using homebrew enzymes and buffers has emerged worldwide as a response to these limitations and potential shortages. Here, we describe the production of Moloney murine leukemia virus (M-MLV) Reverse Transcriptase and BstLF DNA polymerase for the local implementation of RT-LAMP reactions at low cost. These reagents compared favorably to commercial kits and optimum concentrations were defined in order to reduce time to threshold, increase ON/OFF range and minimize enzyme quantities per reaction. As a validation, we tested the performance of these reagents in the detection of SARS-CoV-2 from RNA extracted from clinical nasopharyngeal samples, obtaining high agreement between RT-LAMP and RT-PCR clinical results. The in-house preparation of these reactions results in an order of magnitude reduction in costs, and thus we provide protocols and DNA to enable the replication of these tests at other locations. These results contribute to the global effort of developing open and low cost diagnostics that enable technological autonomy and distributed capacities in viral surveillance.

SummaryBackgroundHospitalised patients with severe COVID-19 have an increased risk of developing acute respiratory distress syndrome (ARDS) and death from severe systemic inflammatory response. Acetylcholine modulates the acute inflammatory response through a neuro-immune mechanism known as the inflammatory reflex. Pyridostigmine, an acetylcholine-esterase inhibitor, increases the half-life of endogenous ACh, reducing lung and systemic inflammation in murine sepsis. This trial aimed to evaluate whether pyridostigmine could decrease invasive mechanical ventilation (IMV) and death in patients with severe COVID-19.MethodsWe performed a parallel-group, multicentre, double-blinded, placebo-controlled, randomised clinical trial in two COVID-19-designated hospitals in Mexico City, Mexico. Adult (≥ 18-year-old), hospitalised patients with confirmed SARS-CoV-2 infection based on a positive RT-PCR test in a respiratory specimen, a computed tomography compatible with pneumonia, as well as requiring supplementary oxygen were included. Patients were randomly assigned (1:1) to receive oral pyridostigmine (60 mg per day) or placebo for a maximum of 14 days. The intention-to-treat analysis included all the patients who underwent randomisation. The primary endpoint was the composite outcome of initiation of IMV and 28-day all-cause mortality. The trial is registered in ClinicalTrials.gov, NCT04343963.FindingsBetween May 5, 2020, and Jan 29, 2021,188 participants were randomly assigned to placebo (n=94) or pyridostigmine (n=94). The composite outcome occurred in 22 (23·4%) vs. 11 (11·7%) participants, respectively (hazard ratio 0·46, 95% CI 0·22-0·96, p=0·03). The most frequent adverse event was diarrhoea (5 [5·3%] in the pyridostigmine group vs 3 [3·2%] in the placebo group). Most of the adverse events were mild to moderate, with no serious adverse events related to pyridostigmine.InterpretationOur data indicates that the addition of pyridostigmine to standard treatment reduces significantly the fatality rate among patients hospitalized for severe COVID-19.FundingConsejo Nacional de Ciencia y Tecnología, México.

Background: Underestimation of the number of cases during the coronavirus disease 2019 (COVID-19) pandemic has been a constant concern worldwide. Detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA using realtime reverse-transcription polymerase chain reaction (RT-PCR) is the most common method to confirm a case. However, these tests have suboptimal sensitivity. Objective: The objective of the study was to estimate the number of COVID-19 confirmed cases, intensive care unit (ICU) admissions and deaths in Mexico, accounting for the probabilities of false-negative tests. Methods: We used publicly available, national databases of all SARS-CoV-2 tests performed at public laboratories in Mexico between February 27 and October 31, 2020. We used the estimated probabilities of false-negative tests based on the day of clinical sample collection after symptom initiation calculated previously. With the resulting model, we estimated the corrected daily number of cases, ICU admissions, and deaths. Results: Among 2,024,822 people tested in Mexico between February 27 and October 31 with an available result, we estimated 1,248,583 (95% confidence interval 1,094,850-1,572,818) cases, compared to 902,343 cases reported with positive tests. ICU admissions and deaths were 15% and 8% higher than reported, respectively. Conclusion: Accounting for SARS-CoV-2 RT-PCR-based diagnostic tests' precision is a simple way to improve estimations for the true number of COVID-19 cases among tested persons.

COVID-19 can range from asymptomatic to life-threatening. Early identification of patients who will develop severe disease is crucial. A number of scores and indexes have been developed to predict severity. However, most rely on measurements not readily available. We evaluated hematological and biochemical markers taken on admission and determined how predictive they were of development of critical illness or death. We observed that higher values of readily available tests, including neutrophil:lymphocyte ratio; derived neutrophil index; and troponin I were associated with a higher risk of death or critical care admission (P

The coronavirus disease 2019 (COVID-19) pandemic has highlighted bottlenecks in large-scale, frequent testing of populations for infections. Polymerase chain reaction (PCR)-based diagnostic tests are expensive, reliant on centralized labs, can take days to deliver results, and are prone to backlogs and supply shortages. Antigen tests that bind and detect the surface proteins of a virus are rapid and scalable but suffer from high false negative rates. To address this problem, an inexpensive, simple, and robust 60-minute do-it-yourself (DIY) workflow to detect viral RNA from nasal swabs or saliva with high sensitivity (0.1 to 2 viral particles/μL) and specificity (&gt, 97% true negative rate) utilizing reverse transcription loop-mediated isothermal amplification (RT-LAMP) was developed. ALERT (Accessible LAMP-Enabled Rapid Test) incorporates the following features: (1) increased shelf-life and ambient temperature storage, compared to liquid reaction mixes, by using wax layers to isolate enzymes from other reagents, (2) improved specificity compared to other LAMP end-point reporting methods, by using sequence-specific QUASR (quenching of unincorporated amplification signal reporters), (3) increased sensitivity, compared to methods without purification through use of a magnetic wand to enable pipette-free concentration of sample RNA and cell debris removal, (4) quality control with a nasopharyngeal-specific mRNA target, and (5) co-detection of other respiratory viruses, such as influenza B, by multiplexing QUASR-modified RT-LAMP primer sets. The flexible nature of the ALERT workflow allows easy, at-home and point-of-care testing for individuals and higher-throughput processing for labs and hospitals. With minimal effort, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific primer sets can be swapped out for other targets to repurpose ALERT to detect other viruses, microorganisms, or nucleic acid-based markers.

This protocol explains how to prepare dsDNA and ssRNA target positive control for LAMP and RT-LAMP reactions. Particularly it describes the pipeline for SARS-Cov-2 Nucleocapsid (N) gene positive control which is included in the ReClone collection. The general procedure involves amplifying the gene of interest with primers which add T7 promoter and T7 terminator to its sequence followed by T7 in vitro transcription to obtain the ssRNA. Sequence elaboration: Sequence OrfN_5 was designed and synthesized based on IDT SARS-CoV-2 positive control plasmid (CAT#10006625_2019).

This protocol describes the preparation of reaction buffer for homemade LAMP / RT-LAMP reactions which involves the use of home-brewed BstLF and MMLV enzymes. It also contains the instructions to prepare the enzyme storage buffers. Home-brewed enzymes information: BstLF polymerase: expression and purification protocol. expression plasmid sequence. M-MLV reverse transcriptase: expression and purification protocol. expression plasmid sequence. Testing We test the buffers on SARS-CoV-2 LAMP or RT-LAMP reactions with synthetic dsDNA or ssRNA respectively. The general performance of Homemade buffer seems appropriate and no amplification was obtained for non-template controls (NTC). To properly formulate the buffer we tested different KCl concentrations which is known to be a critical factor in the buffer. For the home-brewed BstLF, we found that 50 mM KCl is the best concentration, displaying equal or better performance than the commercial buffer. No amplification was observed for 150 mM KCl formulation. [1] Zhang, Y. et al. Enhancing colorimetric loop-mediated isothermal amplification speed and sensitivity with guanidine chloride. Biotechniques69, (2020).

This protocol collection specifies how to carry out LAMP or RT-LAMP (reverse transcription - Loop-mediated isothermal amplification) reactions with homemade reagents and home-brewed enzymes. It describes how to prepare all the solutions and buffers to perform the reactions. This collection was developed in the context of COVID-19 pandemic to provide access to RT-LAMP tools in accordance with the principles of ReClone community initiative. It is the result of information exchange and reagent sharing with a huge community of collaborators from ReClone, gLAMP and JOGL.

This protocol describes how to perform LAMP / RT-LAMP reactions with home-made reagents, which means home-made buffers and positive controls, together with home-brewed BstLF and MMLV enzymes. Home-brewed enzymes information: BstLF polymerase: expression and purification protocol. expression plasmid sequence. M-MLV reverse transcriptase: expression and purification protocol. expression plasmid sequence. Positive control information Sample preparation protocol here. Testing We test this protocol in LAMP or RT-LAMP reactions with SARS-CoV-2 synthetic dsDNA or ssRNA respectively. As shown in Figure 1, the general performance of reactions with homemade reagents seems appropriate compared with the commercial standard option. No amplification was observed in non-template controls (NTC). [1] Zhang, Y. et al. Enhancing colorimetric loop-mediated isothermal amplification speed and sensitivity with guanidine chloride. Biotechniques69, (2020).

​Background: The development of mRNA vaccines to prevent SARS-CoV-2 has been remarkably successful, with highly effective vaccines available less than one year after confirming the first case. Due to the global burden of COVID-19 on health systems, emergency approval was attained after clinical trials with relatively small sample sizes and short follow-up periods. Limited information exists about the incidence of adverse events following immunisation (AEFI), particularly neurologic ones. Here, we describe the neurologic AEFI reported by recipients of the BNT162b2 mRNA COVID-19 vaccine. Methods: We conducted a prospective observational cohort using de-identified information from a database of all systemic and neurologic AEFI reported to the Mexican Ministry of Health throughout a passive Epidemiological Surveillance System by first-dose vaccine recipients, from December 24, 2020 to February 12, 2021. The cut-off date for this data was February 18, 2021. We performed descriptive analyses on demographics, timing from vaccination to AEFI development, event duration, and current outcome. Findings: Nationwide, 704 003 first-doses were administered; 6536 AEFI were reported. Among those, 4258 (65·1%) had at least one neurologic manifestation. Non-serious neurologic AEFI occurred in 99·6%. Headache (62·2%), transient sensory symptoms (3·5%), and weakness (1%) were the most frequent. Thirty-three serious AEFI were reported, of which 17 (2·4/100 000 doses) were neurologic, seven seizures, four functional syndromes, three Guillain-Barre syndrome (GBS) cases, and two of acute transverse myelitis. All GBS cases were related to a gastrointestinal infection before vaccination, 3/7 seizure episodes were related to poor antiepileptic drug compliance, and 2/7 to anaphylactic reactions. At the time of this report, 16/17 cases of serious neurologic AEFI had been discharged with no observed deaths. Interpretation: Our data suggest that the BNT162b2 mRNA vaccine is effective and safe. Their individual and societal benefits outweigh the low-percentage of serious neurologic and non-neurologic AEFI. Funding: Consejo Nacional de Ciencia y Tecnologia, Mexico. Declaration of Interest: None to declare Ethical Approval: The study was revised and approved by the Ethics and Research Committees of the Instituto Nacional de Ciencias Medicas y Nutricion Salvador Zubiran (Ref. NER-3667-2021) and the Mexican Ministry of Health.

Characterization is fundamental to the design, build, test, learn (DBTL) cycle for engineering synthetic genetic circuits. Components must be described in such a way as to account for their behavior in a range of contexts. Measurements and associated metadata, including part composition, constitute the test phase of the DBTL cycle. These data may consist of measurements of thousands of circuits, measured in hundreds of conditions, in multiple assays potentially performed in different laboratories and using different techniques. In order to inform the learn phase this large volume of data must be filtered, collated, and analyzed. Characterization consists of using this data to parametrize models of component function in different contexts, and combining them to predict behaviors of novel circuits. Tools to store, organize, share, and analyze large volumes of measurement and metadata are therefore essential to linking the test phase to the build and learn phases, closing the loop of the DBTL cycle. Here we present such a system, implemented as a web app with a backend data registry and analysis engine. An interactive frontend provides powerful querying, plotting, and analysis tools, and we provide a REST API and Python package for full integration with external build and learn software. All measurements are associated with circuit part composition via SBOL (Synthetic Biology Open Language). We demonstrate our tool by characterizing a range of genetic components and circuits according to composition and context.

Underestimation of the number of cases during the COVID-19 pandemic has been a constant concern worldwide. Case confirmation is based on identification of SARS-CoV-2 RNA using real time polymerase chain reaction (RT-PCR) in clinical samples. However, these tests have suboptimal sensitivity, especially during the early and late course of infection. Using open data, we estimated that among 1 343 730 people tested in Mexico since February 27th, there were 838 377 (95% CL 734 605 – 1 057 164) cases, compared with 604 376 considering only positive tests. ICU admissions and deaths were around 16% and 9% higher than reported. Thus, we show that accounting for the sensitivity of SARS-Cov-2 RT-PCR diagnostic tests is a simple way to improve estimations for the true number of COVID-19 cases in tested people, particularly in high-prevalence populations. This could aid to better inform public health measures and reopening policies.

This protocol has been optimized for the recombinant expression of a codon-optimized Bst-LF polymerase. The goal of this protocol was to eliminate the use of large volumes for dyalisis through the use of concentrators for buffer exchange before storage conditions. The plasmid encoding the codon-optimized Bst-LF enzyme used here can be found on reclone.org

Standardized type IIS DNA assembly methods are becoming essential for biological engineering and research. These methods are becoming widespread and more accessible due to the proposition of a ‘common syntax’ that enables higher interoperability between DNA libraries. Currently, Golden Gate (GG)-based assembly systems, originally implemented in host-specific vectors, are being made compatible with multiple organisms. We have recently developed the GG-based Loop assembly system for plants, which uses a small library and an intuitive strategy for hierarchical fabrication of large DNA constructs (>30 kb). Here, we describe ‘universal Loop’ (uLoop) assembly, a system based on Loop assembly for use in potentially any organism of choice. This design permits the use of a compact number of plasmids (two sets of four odd and even vectors), which are utilized repeatedly in alternating steps. The elements required for transformation/maintenance in target organisms are also assembled as standardized parts, enabling customization of host-specific plasmids. Decoupling of the Loop assembly logic from the host-specific propagation elements enables universal DNA assembly that retains high efficiency regardless of the final host. As a proof-of-concept, we show the engineering of multigene expression vectors in diatoms, yeast, plants and bacteria. These resources are available through the OpenMTA for unrestricted sharing and open access.

Diverse microbial ecosystems underpin life in the sea. Among these microbes are many unicellular eukaryotes that span the diversity of the eukaryotic tree of life. However, genetic tractability has been limited to a few species, which do not represent eukaryotic diversity or environmentally relevant taxa. Here, we report on the development of genetic tools in a range of protists primarily from marine environments. We present evidence for foreign DNA delivery and expression in 13 species never before transformed and for advancement of tools for eight other species, as well as potential reasons for why transformation of yet another 17 species tested was not achieved. Our resource in genetic manipulation will provide insights into the ancestral eukaryotic lifeforms, general eukaryote cell biology, protein diversification and the evolution of cellular pathways., This collaborative effort was supported by the Gordon and Betty Moore Foundation EMS Program of the Marine Microbiology Initiative (grant nos. GBMF4972 and 4972.01 to F.-Y.B.; GBMF4970 and 4970.01 to M.A. and A.Z.W.; GBMF3788 to A.Z.W.; GBMF 4968 and 4968.01 to H.C.; GBMF4984 to V.H.; GBMF4974 and 4974.01 to C. Brownlee; GBMF4964 to Y. Hirakawa; GBMF4961 to T. Mock; GBMF4958 to P.S.; GBMF4957 to A.T.; GBMF4960 to G.J.S.; GBMF4979 to K.C.; GBMF4982 and 4982.01 to J.L.C.; GBMF4964 to P.J.K.; GBMF4981 to P.v.D.; GBMF5006 to A.E.A.; GBMF4986 to C.M.; GBMF4962 to J.A.F.R.; GBMF4980 and 4980.01 to S.L.; GBMF 4977 and 4977.01 to R.F.W.; GBMF4962.01 to C.H.S.; GBMF4985 to J.M.; GBMF4976 and 4976.01 to C.H.; GBMF4963 and 4963.01 to V.E.; GBMF5007 to C.L.D.; GBMF4983 and 4983.01 to J.L.; GBMF4975 and 4975.01 to A.D.T.; GBMF4973 and 4973.01 to I.R.-T. and GBMF4965 to N.K.), by The Leverhulme Trust (RPG-2017-364) to T. Mock and A. Hopes, and by ERD funds (16_019/0000759) from the Czech Ministry of Education to J.L.

mRNA vaccines against SARS-CoV-2 are remarkably effective. Limited information exists about the incidence of adverse events following immunization (AEFI) with their use. We conducted a prospective observational study including data from 704,003 first-doses recipients; 6536 AEFI were reported, of whom 65.1% had at least one neurologic AEFI (non-serious 99.6%). Thirty-three serious events were reported; 17 (51.5%) were neurologic (observed frequency, 2.4/100,000 doses). At the time of writing this report, 16/17 cases had been discharged without deaths. Our data suggest that the BNT162b2 mRNA COVID-19 vaccine is safe; its individual and societal benefits outweigh the low percentage of serious neurologic AEFI. This information should help to dissipate hesitancy towards this new vaccine platform.

Standardised Type IIS DNA assembly methods are becoming essential for biological engineering and research. Although a ‘common syntax’ has been proposed to enable higher interoperability between DNA libraries, Golden Gate (GG)-based assembly systems remain specific to target organisms. Furthermore, these GG assembly systems become laborious and unnecessarily complicated beyond the assembly of 4 transcriptional units. Here, we describe “universal Loop” (uLoop) assembly, a simple system based on Loop assembly that enables hierarchical fabrication of large DNA constructs (> 30 kb) for any organism of choice. uLoop comprises two sets of four plasmids that are iteratively used as odd and even levels to compile DNA elements in an exponential manner (4n-1). The elements required for transformation/maintenance in target organisms are also assembled as standardised parts, enabling customisation of host-specific plasmids. Thus, this species-agnostic method decouples efficiency of assembly from the stability of vectors in the target organism. As a proof-of-concept, we show the engineering of multi-gene expression vectors in diatoms, yeast, plants and bacteria. These resources will become available through the OpenMTA for unrestricted sharing and open-access.Abstract Figure