Rapid, Multiplexed, and Enzyme-Free Nucleic Acid Detection Using Programmable Aptamer-Based RNA Switches.

Rapid, simple, and low-cost diagnostic technologies are crucial tools for combatting infectious disease. We describe a class of aptamer-based RNA switches or aptaswitches that recognize target nucleic acid molecules and initiate folding of a reporter aptamer. Aptaswitches can detect virtually any sequence and provide an intense fluorescent readout without intervening enzymes, generating signals in as little as 5 minutes and enabling detection by eye with minimal equipment. Aptaswitches can be used to regulate folding of seven fluorogenic aptamers, providing a general means of controlling aptamers and an array of multiplexable reporter colors. Coupling isothermal amplification reactions with aptaswitches, we reach sensitivities down to 1 RNA copy/µL in one-pot reactions. Application of multiplexed all-in-one reactions against RNA from clinical saliva samples yields an overall accuracy of 96.67% for detection of SARS-CoV-2 in 30 minutes. Aptaswitches are thus versatile tools for nucleic acid detection that are readily integrated into rapid diagnostic assays.

COVID-19 reinfections in Mexico City: implications for public health.

Background: Since its appearance, COVID-19 has immensely impacted our society. Public health measures, from the initial lockdowns to vaccination campaigns, have mitigated the crisis. However, SARS-CoV-2's persistence and evolving variants continue to pose global threats, increasing the risk of reinfections. Despite vaccination progress, understanding reinfections remains crucial for informed public health responses. Methods: We collected available data on clinical and genomic information for SARS-CoV-2 samples from patients treated in Mexico City from 2020 epidemiological week 10 to 2023 epidemiological week 06 encompassing the whole public health emergency's period. To identify clinical data we utilized the SISVER (Respiratory Disease Epidemiological Surveillance System) database for SARS-CoV-2 patients who received medical attention in Mexico City. For genomic surveillance we analyzed genomic data previously uploaded to GISAID generated by Mexican institutions. We used these data sources to generate descriptors of case number, hospitalization, death and reinfection rates, and viral variant prevalence throughout the pandemic period. Findings: The fraction of reinfected individuals in the COVID-19 infected population steadily increased as the pandemic progressed in Mexico City. Most reinfections occurred during the fifth wave (40%). This wave was characterized by the coexistence of multiple variants exceeding 80% prevalence; whereas all other waves showed a unique characteristic dominant variant (prevalence >95%). Shifts in symptom patient care type and severity were observed, 2.53% transitioned from hospitalized to ambulatory care type during reinfection and 0.597% showed the opposite behavior; also 7.23% showed a reduction in severity of symptoms and 6.05% displayed an increase in severity. Unvaccinated individuals accounted for the highest percentage of reinfections (41.6%), followed by vaccinated individuals (31.9%). Most reinfections occurred after the fourth wave, dominated by the Omicron variant; and after the vaccination campaign was already underway. Interpretation: Our analysis suggests reduced infection severity in reinfections, evident through shifts in symptom severity and care patterns. Unvaccinated individuals accounted for most reinfections. While our study centers on Mexico City, its findings may hold implications for broader regions, contributing insights into reinfection dynamics.

Rapid and Multiplexed Nucleic Acid Detection using Programmable Aptamer-Based RNA Switches.

Rapid, simple, and low-cost diagnostic technologies are crucial tools for combatting infectious disease. Here, we describe a class of aptamer-based RNA switches called aptaswitches that recognize specific target nucleic acid molecules and respond by initiating folding of a reporter aptamer. Aptaswitches can detect virtually any sequence and provide a fast and intense fluorescent readout, generating signals in as little as 5 minutes and enabling detection by eye with minimal equipment. We demonstrate that aptaswitches can be used to regulate folding of six different fluorescent aptamer/fluorogen pairs, providing a general means of controlling aptamer activity and an array of different reporter colors for multiplexing. By coupling isothermal amplification reactions with aptaswitches, we reach sensitivities down to 1 RNA copy/µL in one-pot reactions. Application of multiplexed one-pot reactions against RNA extracted from clinical saliva samples yields an overall accuracy of 96.67% for detection of SARS-CoV-2 in 30 minutes. Aptaswitches are thus versatile tools for nucleic acid detection that can be readily integrated into rapid diagnostic assays.

Seroconversion dynamic and SARS-CoV-2 seropositivity in unvaccinated population during the first and second outbreaks in Mexico.

Serosurveillance helps establish reopening guidelines and determine the immunity levels in different populations to reach herd immunity. Then, there is an urgent need to estimate seroprevalence population wide. In Mexico, information about COVID-19 cases and related deaths is scarce. Also, there is no official serosurveillance, limiting our knowledge of the impact of the SARS-CoV-2 pandemic. Here, we report the prevalence of anti-SARS-CoV-2 antibodies in 522,690 unvaccinated people from July 5th to December 31st, 2020. The overall seroprevalence was 32.8% and highest in adults aged 30-39 years (38.5%) than people under 20 years (33.0%) or older (28.9%). Moreover, in a cohort of 1655 individuals confirmed COVID-19 by PCR, we found that symptomatic people (HR = 2.56) increased seroconversion than presymptomatic. Also, we identified that the most discriminative symptoms for COVID-19 that could predict seroconversion were anosmia and ageusia (HR = 1.70), fever, myalgia/arthralgia, and cough (HR = 1.75). Finally, we found that obese people had lower seroconversion (HR = 0.53) than healthy people, but the opposite happens in diabetic people (HR = 1.39). These findings reveal that around one-third of Mexican outpatients had anti-SARS-CoV-2 antibodies before vaccination. Also, some symptoms improve empirically COVID-19 diagnosis and seroconversion. This information could help fine-tune vaccination schemes and the reopening and back-to-work algorithms.

Multi-arm RNA junctions encoding molecular logic unconstrained by input sequence for versatile cell-free diagnostics.

Applications of RNA-based molecular logic have been hampered by sequence constraints imposed on the input and output of the circuits. Here we show that the sequence constraints can be substantially reduced by appropriately encoded multi-arm junctions of single-stranded RNA structures. To conditionally activate RNA translation, we integrated multi-arm junctions, self-assembled upstream of a regulated gene and designed to unfold sequentially in response to different RNA inputs, with motifs of loop-initiated RNA activators that function independently of the sequence of the input RNAs and that reduce interference with the output gene. We used the integrated RNA system and sequence-independent input RNAs to execute two-input and three-input OR and AND logic in Escherichia coli, and designed paper-based cell-free colourimetric assays that accurately identified two human immunodeficiency virus (HIV) subtypes (by executing OR logic) in amplified synthetic HIV RNA as well as severe acute respiratory syndrome coronavirus-2 (via two-input AND logic) in amplified RNA from saliva samples. The sequence-independent molecular logic enabled by the integration of multi-arm junction RNAs with motifs for loop-initiated RNA activators may be broadly applicable in biotechnology.

End-point RT-PCR based on a conservation landscape for SARS-COV-2 detection.

End-point RT-PCR is a suitable alternative diagnostic technique since it is cheaper than RT-qPCR tests and can be implemented on a massive scale in low- and middle-income countries. In this work, a bioinformatic approach to guide the design of PCR primers was developed, and an alternative diagnostic test based on end-point PCR was designed. End-point PCR primers were designed through conservation analysis based on kmer frequency in SARS-CoV-2 and human respiratory pathogen genomes. Highly conserved regions were identified for primer design, and the resulting PCR primers were used to amplify 871 nasopharyngeal human samples with a previous RT-qPCR based SARS-CoV-2 diagnosis. The diagnostic test showed high accuracy in identifying SARS-CoV-2-positive samples including B.1.1.7, P.1, B.1.427/B.1.429 and B.1.617.2/ AY samples with a detection limit of 7.2 viral copies/µL. In addition, this test could discern SARS-CoV-2 infection from other viral infections with COVID-19-like symptomatology. The designed end-point PCR diagnostic test to detect SARS-CoV-2 is a suitable alternative to RT-qPCR. Since the proposed bioinformatic approach can be easily applied in thousands of viral genomes and over highly divergent strains, it can be used as a PCR design tool as new SARS-CoV-2 variants emerge. Therefore, this end-point PCR test could be employed in epidemiological surveillance to detect new SARS-CoV-2 variants as they emerge and propagate.

Epidemiological and clinical characteristics of pregnant women and neonates with COVID-19 in Northwest Mexico.

INTRODUCTION: The SARS-CoV-2 virus, which causes COVID-19, has spread quickly worldwide, causing millions of cases and thousands of deaths. Some risk factors in the general population are related to the development of severe COVID-19 or death, but in pregnant women and neonates, the information is limited. OBJECTIVE: To determine the epidemiological and clinical characteristics of pregnant women and neonates diagnosed with COVID-19 by RT-PCR and serological tests, and analyze the relationship between the influenza vaccination and COVID-19 symptoms in infected pregnant women in Sinaloa state. METHODS: We collected samples from 116 pregnant women and 84 neonates from the Women´s Hospital of Sinaloa. They were diagnosed with COVID-19 by RT-PCR and serological tests (IgG), and sociodemographic, clinical and laboratory parameters were recorded. RESULTS: A total of 11.2% (13/116) of the pregnant women were RT-PCR+, 25% (29/116) were IgG+ and 4.3% (5/116) were positive for both tests. Symptoms such as rhinorrhea (P = .04), cough (P = .02) and polypnea (P = .04) in pregnant women were related to COVID-19, also leukocyte index was higher in pregnant women with COVID-19 (P = .03), but the associations were lost after the Bonferroni correction. No laboratory parameters or underlying diseases were associated with COVID-19, and most infected pregnant women had mild cases. We found an association between the influenza vaccine and less common COVID-19 symptoms in pregnant women who were infected (P = .01). A total of 7.2% (6/84) of neonates were RT-PCR+, 35.7% (30/84) were IgG+, and there were no symptoms or underlying diseases associated with neonates who were infected. In conclusion, this work demonstrated that some symptoms were related to COVID-19, most pregnant women and neonates had mild cases, and the influenza vaccine could decrease the severity of COVID-19 cases in pregnant women.

Early Genomic, Epidemiological, and Clinical Description of the SARS-CoV-2 Omicron Variant in Mexico City.

Omicron is the most mutated SARS-CoV-2 variant-a factor that can affect transmissibility, disease severity, and immune evasiveness. Its genomic surveillance is important in cities with millions of inhabitants and an economic center, such as Mexico City. Results. From 16 November to 31 December 2021, we observed an increase of 88% in Omicron prevalence in Mexico City. We explored the R346K substitution, prevalent in 42% of Omicron variants, known to be associated with immune escape by monoclonal antibodies. In a phylogenetic analysis, we found several independent exchanges between Mexico and the world, and there was an event followed by local transmission that gave rise to most of the Omicron diversity in Mexico City. A haplotype analysis revealed that there was no association between haplotype and vaccination status. Among the 66% of patients who have been vaccinated, no reported comorbidities were associated with Omicron; the presence of odynophagia and the absence of dysgeusia were significant predictor symptoms for Omicron, and the RT-qPCR Ct values were lower for Omicron. Conclusions. Genomic surveillance is key to detecting the emergence and spread of SARS-CoV-2 variants in a timely manner, even weeks before the onset of an infection wave, and can inform public health decisions and detect the spread of any mutation that may affect therapeutic efficacy.

Control and Prevention of SARS-CoV-2 Outbreaks among Healthcare Workers from 129 Healthcare Facilities in Mexico.

Few reports have shared the workflows to reduce SARS-CoV-2 infections among risk groups, including healthcare workers (HCWs). This study describes an occupational health program implemented to reduce the incidence of COVID-19 and establishes a back-to-work algorithm in HCWs of 129 Salud Digna outpatient care clinics in Mexico. This program was composed of training plans, screening SARS-CoV-2 infections, the containment of infections, follow-up COVID-19 cases, and continuing supervision in addition to the steady supply and training for the correct use of PPE. From 16 April 2020 to 15 April 2021, 7376 individuals were enrolled, of which 423 were excluded because they did not meet the inclusion criteria or refused the follow-up. In the cohort studied, we found a COVID-19 incidence of 35.4% (2610 individuals), lower hospitalization (0.11%), ICU (0.04%) and lethality rate (0.04%). Additionally, 85.9% of COVID-19 cases tested negative for SARS-CoV-2 after 14 days of the first positive test with an average isolation time of 26-33 days. Finally, 99% of people received personal protective equipment and adequate training to use it. Our results show that the program implemented reduced the hospitalization ICU admission and lethality in HCWs; we consider this workflow to help other workplaces offer safe conditions for HCWs and patients.

Association of FTO, ABCA1, ADRB3, and PPARG variants with obesity, type 2 diabetes, and metabolic syndrome in a Northwest Mexican adult population.

AIM: To identify associations among allelic variants of the genes FTO, ABCA1, ADRB3, and PPARG with anthropometric and biochemical traits, metabolic diseases (obesity, T2D or metabolic syndrome) in an adult population from Northwest Mexico. METHODS: Blood samples were collected from 846 subjects including 266 normal weight subjects, 285 with obesity, and 295 with T2D. Of the 846 persons in the study, 365 presented metabolic syndrome diagnostic criteria. Anthropometric and biochemical traits were recorded and 4 single nucleotide polymorphisms (SNPs): FTO rs9939609 A-allele, ABCA1 rs9282541 A-allele, ADRB3 rs4994 G-allele, and PPARG rs1801282 G-allele were genotyped by real-time PCR. RESULTS: FTO rs9939609 A-allele was significantly associated with obesity (p: 8.3 × 10-4), and metabolic syndrome (p: 0.001), but no individual SNPs were significantly associated with T2D. Finally, the cumulative risk of the four SNPs was significantly associated with obesity (p: 1.95 × 10-4). CONCLUSION: Associations in FTO, ABCA, ADRB3, and PPARG SNPs presented in this study with obesity and metabolic syndrome could represent a risk for developing metabolic diseases in Northwest Mexican adult subjects.

Analysis of Epidemiological and Clinical Characteristics of COVID-19 in Northwest Mexico and the Relationship Between the Influenza Vaccine and the Survival of Infected Patients.

The first cases of unexplained pneumonia were reported in Wuhan, China, in December of 2019. Later, a novel coronavirus (SARS-CoV-2) was identified as the causal agent of pneumonia. This virus has since spread to more than 180 countries and has been declared a pandemic by the World Health Organization. Herein, we aimed to determine the epidemiological and clinical characteristics of symptomatic patients with coronavirus disease 2019 (COVID-19) and the relationship between the influenza vaccine with a lower risk of severe COVID-19 infection in the state of Sinaloa. We collected demographic and clinical data of 4,040 patients with acute respiratory infections across Sinaloa state hospitals from February 28 to May 15, 2020. The prevalence of COVID-19 among hospitalized patients with respiratory symptoms in Sinaloa showed 45.2% of men were more affected than women (p < 0.001), and people aged 40-49 years were the most affected. The main symptoms of COVID-19 infection were cough and fever (p < 0.001), while hypertension, obesity, and type 2 diabetes were the chronic diseases associated with COVID-19 than non-COVID-19 (p < 0.003). Healthcare workers were most likely to be infected compared to other occupations (p < 0.001). The general lethality rate was 14.1%, and males >62 years were the ones who had a higher lethality rate (p < 0.001); the aforementioned chronic diseases were related to higher lethality of COVID-19 (p < 0.001). Likewise, higher lethality was seen in housewives and patient retirees/pensioners compared with other occupations (p < 0.001). Finally, we found there was a relationship between influenza vaccination and a lower risk of severe COVID-19 infection and mortality (p < 0.001). These findings showed that healthcare workers, men >62 years with chronic diseases, and retired people were most affected. Furthermore, the influenza vaccine could decrease the severeness of COVID-19 cases.

Epidemiology of COVID-19 in Mexico: Symptomatic profiles and presymptomatic people.

OBJECTIVES: The COVID-19 diagnosis is difficult and ambiguous due to nonspecific symptoms. Further, data from Mexico arehospitable population-based without signs and symptoms information. Thus, this work aims to provide epidemiology information about the burden of COVID-19 in Mexican outpatients and to identify symptomatic COVID-19 profiles that could help in the early diagnosis of the disease. METHODS: From June to September, epidemiological, clinical, and demographic data of 482,413 individuals diagnosed by RT-PCR test for SARS-CoV-2 in Salud Digna clinics were collected. RESULTS: We observed a 41% incidence of SARS-CoV-2 infections with a mean age of 36 years and with young adults (20-40 years) being the most affected. Among occupations, delivery persons (OR 1.38) or informal traders (OR 1.33) had a higher risk of COVID-19. Moreover, 13% of SARS-CoV-2 infections were in presymptomatic patients. Finally, we identified three different symptomatic profiles (common, respiratory, and gastrointestinal) associated with COVID-19. CONCLUSION: The incidence of SARS-CoV-2 was high among outpatients with a significant proportion of presymptomatic carriers, and thus it is necessary to increase testing and continue SARS-CoV-2 surveillance with a better description of signs and symptoms; in this regard, we identified three symptomatic profiles that could help in the diagnosis of COVID-19.

The Evolutionary Landscape of SARS-CoV-2 Variant B.1.1.519 and Its Clinical Impact in Mexico City.

The SARS-CoV-2 pandemic is one of the most concerning health problems around the globe. We reported the emergence of SARS-CoV-2 variant B.1.1.519 in Mexico City. We reported the effective reproduction number (Rt) of B.1.1.519 and presented evidence of its geographical origin based on phylogenetic analysis. We also studied its evolution via haplotype analysis and identified the most recurrent haplotypes. Finally, we studied the clinical impact of B.1.1.519. The B.1.1.519 variant was predominant between November 2020 and May 2021, reaching 90% of all cases sequenced in February 2021. It is characterized by three amino acid changes in the spike protein: T478K, P681H, and T732A. Its Rt varies between 0.5 and 2.9. Its geographical origin remain to be investigated. Patients infected with variant B.1.1.519 showed a highly significant adjusted odds ratio (aOR) increase of 1.85 over non-B.1.1.519 patients for developing a severe/critical outcome (p = 0.000296, 1.33-2.6 95% CI) and a 2.35-fold increase for hospitalization (p = 0.005, 1.32-4.34 95% CI). The continuous monitoring of this and other variants will be required to control the ongoing pandemic as it evolves.

The burden of 14 hr-HPV genotypes in women attending routine cervical cancer screening in 20 states of Mexico: a cross-sectional study.

In Mexico, HPV vaccines available immunize against genotypes 16/18 and 16/18/6/11; however, there is limited surveillance about carcinogenic subtypes in different states of the country that allow evaluating the effectiveness of vaccination and cervical cancer screening programs. Here, we report the regional and age-specific prevalence of 14 hr-HPV genotypes as well as their prevalence in abnormal cytology (from ASCUS to cervical cancer) among Mexican women which were undergoing from cervical cancer screening in the Salud Digna clinics in 20 states of the country. This study includes women with social security from the majority of public health institutions (IMSS, ISSSTE, SEMAR, and PEMEX), and women without social security. For cervical cancer screening, we used the SurePath liquid-based cytology and the BD Onclarity HPV Assay. From December 1, 2016, to August 2, 2018, the hr-HPV prevalence among 60,135 women was 24.78%, the most prevalent types were HPV 16 (4.13%), HPV 31 (4.12%) and HPV 51 (3.39%), while HPV 18 (1.70%) was less prevalent among infected women. Interestingly, the genotypes not covered by current vaccines in Mexico were commonly found in precancerous lesions, evidencing their carcinogenic potential, so it is necessary to increase their surveillance and inclusion in cervical cancer screening triage.

Blood Groups Distribution and Gene Diversity of the ABO and Rh (D) Loci in the Mexican Population.

OBJECTIVE: To determine the frequency and distribution of ABO and Rh (D) antigens and, additionally, investigate gene diversity and the structure of Mexican populations. MATERIALS AND METHODS: Blood groups were tested in 271,164 subjects from 2014 to 2016. The ABO blood group was determined by agglutination using the antibodies anti-A, Anti-B, and Anti-D for the Rh factor, respectively. RESULTS: The overall distribution of ABO and Rh (D) groups in the population studied was as follows: O: 61.82%; A: 27.44%; B: 8.93%; and AB: 1.81%. For the Rh group, 95.58% of people were Rh (D), and 4.42% were Rh (d). Different distributions of blood groups across regions were found; additionally, genetic analysis revealed that the IO and ID allele showed an increasing trend from the north to the center, while the IA and Id allele tended to increase from the center to the north. Also, we found more gene diversity in both loci in the north compared with the center, suggesting population structure in Mexico. CONCLUSION: This work could help health institutions to identify where they can obtain blood products necessary for medical interventions. Moreover, this piece of information contributes to the knowledge of the genetic structure of the Mexican populations which could have significant implications in different fields of biomedicine.

[Cost/benefit and cost/effectiveness study: Anahuac telemedicine]. / Análisis costo-beneficio. Telemedicina Anáhuac.

BACKGROUND: We undertook this study to make an accurate cost/benefit and cost/effectiveness assessment of the "Telemedicina Anáhuac" project, which provides virtual satellite medical care via fixed teleconsultations and movable units in rural/marginal areas. METHODS: The basis of the study was a bibliographical analysis of health areas. RESULTS: When making a monetary analysis and observing fair profits, accessibility and increase in coverage in these populations, it is easier to determine whether or not the project is viable and whether or not it accomplishes cost/benefit and cost/effectiveness conditions. It has not been possible to accurately evaluate epidemiological situations or their changes and impact on health because of the short duration of the project in each community. The most valuable benefit of "Telemedicina Anáhuac" is the appropriate medical referral of surgical patients. CONCLUSIONS: From the results obtained, we are aware that this project offers to our country a great opportunity to resolve health problems in marginalized areas. It offers several benefits to the population, to the Health Ministry, to the personnel and to the social service medical students who operate it. In the surgical area, better diagnoses are obtained, and the project helps to decongest the second level of medical care in Mexico.

Transformation From a Conventional Clinical Microbiology Laboratory to Full Automation.

BACKGROUND: To validate the performance, reproducibility, and reliability of BD automated instruments in order to establish a fully automated clinical microbiology laboratory. METHODS: We used control strains and clinical samples to assess the accuracy, reproducibility, and reliability of the BD Kiestra WCA, the BD Phoenix, and BD Bruker MALDI-Biotyper instruments and compared them to previously established conventional methods. The following processes were evaluated: sample inoculation and spreading, colony counts, sorting of cultures, antibiotic susceptibility test, and microbial identification. RESULTS: The BD Kiestra recovered single colonies in less time than conventional methods (e.g. E. coli, 7h vs 10h, respectively) and agreement between both methodologies was excellent for colony counts (κ=0.824) and sorting cultures (κ=0.821). Antibiotic susceptibility tests performed with BD Phoenix and disk diffusion demonstrated 96.3% agreement with both methods. Finally, we compared microbial identification in BD Phoenix and Bruker MALDI-Biotyper and observed perfect agreement (κ=1) and identification at a species level for control strains. Together these instruments allow us to process clinical urine samples in 36h (effective time). CONCLUSION: The BD automated technologies have improved performance compared with conventional methods, and are suitable for its implementation in very busy microbiology laboratories.

Refractive errors among children, adolescents and adults attending eye clinics in Mexico.

AIM: To assess the proportion of refractive errors in the Mexican population that visited primary care optometry clinics in fourteen states of Mexico. METHODS: Refractive data from 676 856 patients aged 6 to 90y were collected from optometry clinics in fourteen states of Mexico between 2014 and 2015. The refractive errors were classified by the spherical equivalent (SE), as follows: sphere+½ cylinder. Myopia (SE>-0.50 D), hyperopia (SE>+0.50 D), emmetropia (-0.50≤SE≤+0.50), and astigmatism alone (cylinder≥-0.25 D). A negative cylinder was selected as a notation. RESULTS: The proportion (95% confidence interval) among all of the subjects was hyperopia 21.0% (20.9-21.0), emmetropia 40.7% (40.5-40.8), myopia 24.8% (24.7-24.9) and astigmatism alone 13.5% (13.4-13.5). Myopia was the most common refractive error and frequency seemed to increase among the young population (10 to 29 years old), however, hyperopia increased among the aging population (40 to 79 years old), and astigmatism alone showed a decreasing trend with age (6 to 90y; from 19.7% to 10.8%). There was a relationship between age and all refractive errors (approximately 60%, aged 50 and older). The proportion of any clinically important refractive error was higher in males (61.2%) than in females (58.3%; P<0.0001). From fourteen states that collected information, the proportion of refractive error showed variability in different geographical areas of Mexico. CONCLUSION: Myopia is the most common refractive error in the population studied. This study provides the first data on refractive error in Mexico. Further programs and studies must be developed to address the refractive errors needs of the Mexican population.