Heterogeneity of hypoxemia severity according to oxygenation index in COVID-19 pneumonia (preprint)

ABSTRACT Objective To compare hypoxemia severity of patients with COVID-19 pneumonia that arrive at an emergency department as classified by three oxygenation indexes. Design Retrospective analysis of pulse oximeter saturation and arterial blood gas analysis obtained at arrival. Setting Tertiary referral hospital in Mexico City converted early in the pandemic to a COVID-19 center. Patients and measurements A 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. Interventions None. Main Results A 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. Conclusions Hypoxemia 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.

Delay of molecular SARS-CoV-2 testing and turnaround time in Mexico and Colombia (preprint)

Objective: To 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. Methods: We 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. Results: In 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. Conclusion: POC 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.

Hospital saturation and risk of death without receiving mechanical ventilation in hospitalized COVID-19 patients: a city-wide analysis (preprint)

Background Pneumonia 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. Methods We 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 8 th , 2020, and January 5 th , 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). Results We 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). Conclusions In 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 messages Hospital 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.

Homebrew reagents for low cost RT-LAMP (preprint)

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-LAMPs 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.

Pyridostigmine in adults with severe SARS-CoV-2 infection: the PISCO trial (preprint)

Background: Hospitalized patients with severe COVID-19 have an increased risk of developing severe systemic inflammatory response, pulmonary damage, and acute respiratory distress syndrome (ARDS), resulting in end-organ damage and death. 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, chemically stimulating the inflammatory reflex. This trial aimed to evaluate whether pyridostigmine could decrease invasive mechanical ventilation (IMV) and death in patients with severe COVID-19. Methods: We performed a parallel-group, multicenter, double-blinded, placebo-controlled, randomized clinical trial to evaluate if add-on pyridostigmine to standard treatment reduced the composite outcome of initiation of IMV and 28-day all-cause mortality among hospitalized patients with severe COVID-19. Results: 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% confidence interval 0.22-0.96, p=0.03). Most of the adverse events were mild to moderate, with no serious adverse events related to pyridostigmine; discontinuation of the study drugs was similar in both groups. Conclusions: We provide evidence indicating that the addition of pyridostigmine to standard treatment resulted in a clinically significant reduction in the composite outcome (IMV/death) among patients hospitalized for severe COVID-19. (Funded by Consejo Nacional de Ciencia y Tecnologia, Mexico; ClinicalTrials.gov number: NCT04343963).

Systemic and Neurologic Adverse Events Among 704,003 First-Dose Recipients of the Pfizer-Biontech (BNT162b2) mRNA COVID-19 Vaccine in Mexico (preprint)

​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-Barré 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 Tecnología, México.Declaration of Interest: None to declareEthical Approval: The study was revised and approved by the Ethics and Research Committees of the Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán (Ref. NER-3667-2021) and the Mexican Ministry of Health.

Accessible LAMP-Enabled Rapid Test (ALERT) for detecting SARS-CoV-2 (preprint)

The COVID-19 pandemic has highlighted bottlenecks in large-scale, frequent testing of populations for infections. PCR-based diagnostic tests are expensive, reliant on expensive 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 inexpensive but suffer from high false negative rates. To address this problem, we have created 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/{micro}l) and specificity (>97% True Negative Rate) utilizing reverse transcription loop-mediated isothermal amplification (RT-LAMP). Our workflow, ALERT (Accessible LAMP-Enabled Rapid Test), incorporates the following features: 1) Increased shelf-life and ambient temperature storage by using wax layers to isolate enzymes from reaction, 2) Improved specificity by using sequence-specific QUASR reporters, 3) Increased sensitivity 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 duplexing 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 centralized labs and hospitals. With minimal effort, 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.

Corrected Estimation of COVID-19 Cases in Mexico: A Nation-Wide Open Data Modelling Study (preprint)

Background: 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. We estimated the corrected number of COVID-19 confirmed cases, ICU admissions and deaths in Mexico accounting for the probabilities of false-negative tests. Methods: We used a publicly available, national database of all SARS-CoV-2 tests performed at public laboratories in Mexico between February 27th and August 31st, 2020. We used the estimated probabilities of false negatives tests based on the day of clinical sample collection after symptom initiation previously calculated by Kucirca et al. Applying results derived from the false negative estimation model on the government official dataset, we estimated the daily corrected number of cases, ICU admissions and deaths at the National level and for each of the 32 States. Results: There were 1 343 730 people tested between February 27th and August 31st, 2020. We included 1 280 910 patients with available results. There were 604 376 confirmed cases, 13 038 ICU admissions and 64 360 deaths due to COVID-19. We estimated a total of 838 377 (95% CL 734 605 – 1 057 164) positive cases (39% higher than confirmed cases); 15 085 ICU admissions and 69 835 deaths (about 16% and 9% more than reported). In our corrected estimates, 50 000 cases were reached by May 11th, while the official count reached that number at May 18th. The magnitude of difference between official counts and corrected estimates varied between states . Interpretation: 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 in tested people, particularly in high-prevalence populations. This could aid to better inform public health measures and reopening policies. Funding: The authors received no funding for this study.Declaration of Interests: All authors state they have no conflict of interests.Ethics Approval Statement: The ethics committee of the Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán reviewed and approved the study.