Prevalence of symptoms, comorbidities, and reinfections in individuals infected with Wild-Type SARS-CoV-2, Delta, or Omicron variants: a comparative study in western Mexico.

Introduction: The variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been classified into variants of interest (VOIs) or concern (VOCs) to prioritize global monitoring and research on variants with potential risks to public health. The SARS-CoV-2 high-rate mutation can directly impact the clinical disease progression, epidemiological behavior, immune evasion, vaccine efficacy, and transmission rates. Therefore, epidemiological surveillance is crucial for controlling the COVID-19 pandemic. In the present study, we aimed to describe the prevalence of wild-type (WT) SARS-CoV-2 and Delta and Omicron variants in Jalisco State, Mexico, from 2021 to 2022, and evaluate the possible association of these variants with clinical manifestations of COVID-19. Methods: Four thousand and ninety-eight patients diagnosed with COVID-19 by real-time PCR (COVIFLU, Genes2Life, Mexico) from nasopharyngeal samples from January 2021 to January 2022 were included. Variant identification was performed by the RT-qPCR Master Mut Kit (Genes2Life, Mexico). A study population follow-up was performed to identify patients who had experienced reinfection after being vaccinated. Results and Discussion: Samples were grouped into variants according to the identified mutations: 46.3% were Omicron, 27.9% were Delta, and 25.8% were WT. The proportions of dry cough, fatigue, headache, muscle pain, conjunctivitis, fast breathing, diarrhea, anosmia, and dysgeusia were significantly different among the abovementioned groups (p < 0.001). Anosmia and dysgeusia were mainly found in WT-infected patients, while rhinorrhea and sore throat were more prevalent in patients infected with the Omicron variant. For the reinfection follow-up, 836 patients answered, from which 85 cases of reinfection were identified (9.6%); Omicron was the VOC that caused all reported reinfection cases. In this study, we demonstrate that the Omicron variant caused the biggest outbreak in Jalisco during the pandemic from late December 2021 to mid-February 2022 but with a less severe form than the one demonstrated by Delta and WT. The co-analysis of mutations and clinical outcomes is a public health strategy with the potential to infer mutations or variants that could increase disease severity and even be an indicator of long-term sequelae of COVID-19.

Beyond SARS-CoV-2: epidemiological surveillance of respiratory viruses in Jalisco, Mexico.

Introduction: Respiratory viral infections represent a significant global health burden. Historically, influenza, rhinovirus, respiratory syncytial virus, and adenovirus have been the prevalent viruses; however, the landscape shifted with the widespread emergence of SARS-CoV-2. The aim of this study is to present a comprehensive epidemiological analysis of viral respiratory infections in Jalisco, Mexico. Methods: Data encompassing individuals with flu-like symptoms from July 2021 to February 2023 was scrutinized for viral diagnosis through PCR multiplex. The effect of social mobility on the increase in respiratory viral diagnosis infection was considered to estimate its impact. Additionally, sequences of respiratory viruses stored in public databases were retrieved to ascertain the phylogenetic classification of previously reported viruses in Mexico. Results: SARS-CoV-2 was the most detected virus (n = 5,703; 92.2%), followed by influenza (n = 479; 7.78%). These viruses were also found as the most common co-infection (n = 11; 50%), and for those with influenza, a higher incidence of severe disease was reported (n = 122; 90.4%; p < 0.001). Regarding comorbidities and unhealthy habits, smoking was found to be a risk factor for influenza infection but a protective factor for SARS-CoV-2 (OR = 2.62; IC 95%: 1.66-4.13; OR = 0.65; IC 95%: 0.45-0.94), respectively. Furthermore, our findings revealed a direct correlation between mobility and the prevalence of influenza infection (0.214; p < 0.001). Discussion: The study presents evidence of respiratory virus reemergence and prevalence during the social reactivation, facilitating future preventive measures.

An Upgrade on the Surveillance System of SARS-CoV-2: Deployment of New Methods for Genetic Inspection.

SARS-CoV-2 variants surveillance is a worldwide task that has been approached with techniques such as Next Generation Sequencing (NGS); however, this technology is not widely available in developing countries because of the lack of equipment and limited funding in science. An option is to deploy a RT-qPCR screening test which aids in the analysis of a higher number of samples, in a shorter time and at a lower cost. In this study, variants present in samples positive for SARS-CoV-2 were identified with a RT-qPCR mutation screening kit and were later confirmed by NGS. A sample with an abnormal result was found with the screening test, suggesting the simultaneous presence of two viral populations with different mutations. The DRAGEN Lineage analysis identified the Delta variant, but there was no information about the other three mutations previously detected. When the sequenced data was deeply analyzed, there were reads with differential mutation patterns, that could be identified and classified in terms of relative abundance, whereas only the dominant population was reported by DRAGEN software. Since most of the software developed to analyze SARS-CoV-2 sequences was aimed at obtaining the consensus sequence quickly, the information about viral populations within a sample is scarce. Here, we present a faster and deeper SARS-CoV-2 surveillance method, from RT-qPCR screening to NGS analysis.

Phylogenomics and population genomics of SARS-CoV-2 in Mexico during the pre-vaccination stage reveals variants of interest B.1.1.28.4 and B.1.1.222 or B.1.1.519 and the nucleocapsid mutation S194L associated with symptoms.

Understanding the evolution of the SARS-CoV-2 virus in various regions of the world during the Covid-19 pandemic is essential to help mitigate the effects of this devastating disease. We describe the phylogenomic and population genetic patterns of the virus in Mexico during the pre-vaccination stage, including asymptomatic carriers. A real-time quantitative PCR screening and phylogenomic reconstructions directed at sequence/structure analysis of the spike glycoprotein revealed mutation of concern E484K in genomes from central Mexico, in addition to the nationwide prevalence of the imported variant 20C/S:452R (B.1.427/9). Overall, the detected variants in Mexico show spike protein mutations in the N-terminal domain (i.e. R190M), in the receptor-binding motif (i.e. T478K, E484K), within the S1-S2 subdomains (i.e. P681R/H, T732A), and at the basis of the protein, V1176F, raising concerns about the lack of phenotypic and clinical data available for the variants of interest we postulate: 20B/478K.V1 (B.1.1.222 or B.1.1.519) and 20B/P.4 (B.1.1.28.4). Moreover, the population patterns of single nucleotide variants from symptomatic and asymptomatic carriers obtained with a self-sampling scheme confirmed the presence of several fixed variants, and differences in allelic frequencies among localities. We identified the mutation N:S194L of the nucleocapsid protein associated with symptomatic patients. Phylogenetically, this mutation is frequent in Mexican sub-clades. Our results highlight the dual and complementary role of spike and nucleocapsid proteins in adaptive evolution of SARS-CoV-2 to their hosts and provide a baseline for specific follow-up of mutations of concern during the vaccination stage.

RT-qPCR Assays for Rapid Detection of the N501Y, 69-70del, K417N, and E484K SARS-CoV-2 Mutations: A Screening Strategy to Identify Variants With Clinical Impact.

Background: Several variants of the SARS-CoV-2 have been documented globally during the current COVID-19 pandemic. The N501Y, 69-70del, K417N, and E484K SARS-CoV-2 mutations have been documented among the most relevant due to their potential pathogenic biological effects. This study aimed to design, validate, and propose a fast real-time RT-qPCR assay to detect SARS-CoV-2 mutations with possible clinical and epidemiological relevance in the Mexican population. Methods: Targeting spike (S) gene mutations of SARS-CoV-2 (N501Y, 69-70del, K417N, and E484K), specific primers, and probes for three specific quantitative reverse transcription PCR (RT-qPCR) assays were designed, and validated using Sanger sequencing. These assays were applied in clinical samples of 1060 COVID-19 patients from Jalisco Mexico. Results: In silico analyzes showed high specificity of the three assays. Amplicons of samples were confirmed through sequencing. The screening of samples of COVID-19 patients allowed the identification of the E484K mutation in nine individuals and the identification of P.2 Brazilian variant in Mexico. Conclusion: This work provides low-cost RT-qPCR assays for rapid screening and molecular surveillance of mutations with potential clinical impact. This strategy allowed the detection of E484K mutation and P.2 variant for the first time in samples from the Mexican population.

Effect of HPV16 L1 virus-like particles on the aggregation of non-functionalized gold nanoparticles.

Colorimetric assays based on gold nanoparticles (GNPs) are of considerable interest for diagnostics because of their simplicity and low-cost. Nevertheless, a deep understanding of the interaction between the GNPs and the intended molecular target is critical for the development of reliable detection technologies. The present report describes the spontaneous interaction between HPV16 L1 virus-like particles (VLPs) and non-functionalized GNPs (nfGNPs) resulting in the inhibition of nfGNPs salt-induced aggregation and the stabilization of purified VLPs. Ionic-competition experiments suggested that the nature of nfGNPs-VLPs interaction is non-covalent. Adsorption of an RNA aptamer on nfGNPs surface showed an additive aggregation-inhibitory effect. The use of mutant VLPs confirmed that the interaction nfGNPs-VLPs is not mediated by the opposing superficial electrostatic charges, suggesting that non-electrostatic forces participate in the arrangement of nfGNPs on the VLPs surface. Competition experiments using increasing ethanol concentrations on nfGNPs-VLPs complexes suggested hydrophobic interactions as the main stabilizing force. Therefore, the nfGNPs-VLPs interaction described here should facilitate the development of adsorption assays based on nfGNPs for HPV detection and cervical cancer prevention.

Identification of influenza A pandemic (H1N1) 2009 variants during the first 2009 influenza outbreak in Mexico City.

BACKGROUND: In March 2009, public health surveillance detected increased numbers of influenza-like illness presenting to hospitals in Mexico City. The aetiological agent was subsequently determined to be a novel influenza A (H1N1) triple reassortant, which has spread worldwide. As a consequence the World Health Organisation has declared the first Influenza pandemic of the 21st century. OBJECTIVES: To describe clinically and molecularly the first outbreak of influenza A pH1N1 (2009) during 1-5 May to establish a baseline of epidemiological data for pH1N1. Also, to monitor for the emergence of antiviral resistance, and mutations affecting virulence and transmissibility. STUDY DESIGN: Samples were collected from 751 patients with influenza-like symptoms throughout Mexico City and were tested for influenza A pH1N1 (2009) using real-time PCR. In the samples that were positive for influenza A pH1N1 (2009) fragments from the haemagglutinin (H1) and neuraminidase (N1) genes were sequenced. RESULTS: A total of 203/751 (27%) patients were positive for the pandemic H1N1 (2009) virus (53% male and 47% female). The 0-12-year-old group was the most affected 85/751 (42%). Sequence analysis showed five new variants of the pandemic H1N1 (2009) virus for NA: G249E (GQ292900), M269I (GQ292892), Y274H (GQ292913), T332A (GQ292933), N344K (GQ292882), and four variants for HA: N461K (GQ293006), K505R (GQ292989), I435V (GQ292995), I527N (GQ292997). CONCLUSIONS: We have provided a baseline of epidemiological data from the first outbreak of influenza A pH1N1 (2009) during 1-5 May in Mexico City. The sequencing of partial fragments of the HA and NA genes did not show the presence of previously described mutations affecting known sites of antiviral resistance in seasonal influenza A such as the H275Y (oseltamivir resistance), R293 or N295 etc.

Two or more enteropathogens are associated with diarrhoea in Mexican children.

BACKGROUND: Diarrhoeal diseases constitute a major public health problem, particularly in the developing world, where the rate of mortality and morbidity is very high. The purpose of this study was to conduct a 2 years and 3 months study in order to determine the prevalence of five enteropathogen diarrheogenic agents in Mexico City. METHODS: Faecal samples were obtained from 300 Mexican children diagnosed as positive for diarrhoea, aged > 2 to < 12 years old, and from 80 children matched for age but with no symptoms of the disease (control group). Two multiplex PCR were used to detect Escherichia coli, Salmonella spp., and Shigella spp. In addition, the two protozoan parasites Entamoeba histolytica/Entamoeba dispar and Giardia intestinalis were detected by conventional methods. RESULTS: All diarrhoeal samples were positive for one or more enteropathogens. The most common enteropathogens in diarrhoeal samples were E. histolytica/E. dispar (70.3%), Salmonella (ohio 28.3%; typhimurium 16.3%; infantis 8%; anatum 0.6%; Newport 0.3%), G. intestinalis (33%), E. coli (ETEC 13.3%; EPEC 9.3%; VTEC 8.6%; EIEC 1%) and Shigella spp. (flexneri 1.6%, sonnei 1%). Infections by two (24%) three (16%) and four (12%) pathogens were observed. CONCLUSION: This study revealed that 52% of the patients were infected by more than one enteropathogen, notably E. histolitica/E. dispar and Salmonella ohio. These results are useful for clinicians to improve the empiric treatment used in such cases.

Membrane vesicles released by Avibacterium paragallinarum contain putative virulence factors.

Avibacterium paragallinarum, the causative agent of infectious coryza, releases extracellular membrane vesicles (MVs), containing immunogenic proteins, proteases, putative RTX proteins, haemagglutinin, and nucleic acids, into the medium. MVs ranging 50-300 nm in diameter were observed by electron microscopy. They contained immunogenic proteins in the range of 20-160 kDa, detected using vaccinated or experimentally infected chicken sera raised against Av. paragallinarum, but not in pooled sera from specific pathogen-free chickens. Proteolytic activity was not detected in MVs through zymograms; however, immune recognition of high molecular mass bands was observed by Western blotting using an antiprotease serum against Actinobacillus pleuropneumoniae serotype 1 purified protease, suggesting its presence. MVs agglutinated glutaraldehyde-fixed chicken red blood cells indicating the presence of haemagglutinating antigens. Nucleic acids were also detected inside MVs. Avibacterium paragallinarum releases MVs containing putative virulence factors, which could be important in the pathogenesis of infectious coryza.

Actinobacillus pleuropneumoniae metalloprotease: cloning and in vivo expression.

The complete amino acid and nucleotide sequence of a secreted metalloprotease produced by Actinobacillus pleuropneumoniae serotype 1 is reported. A clone showing proteolytic activity in cell-free culture media was selected from a genomic library of A. pleuropneumoniae serotype 1 in pUC 19. The sequence obtained contained an open reading frame encoding a protein with 869 amino acids. This protein was identified as a zinc neutral-metalloprotease belonging to the aminopeptidase family, with a predicted molecular weight of approximately 101 kDa. This sequence showed high homology with other predicted or sequenced aminopeptidases reported for different Gram-negative bacteria. Expression of the protease was observed in lung tissue from pigs that died of porcine pleuropneumonia suggesting a role in pathogenesis.

Antigenic secreted proteins from Haemophilus paragallinarum. A 110-kDa putative RTX protein.

Haemophilus paragallinarum is the causal agent of infectious coryza, an economically important disease for the poultry industry. This bacterium secreted proteins of 25-110 kDa during its growth in brain heart infusion, tryptic soy broth, or Luria-Bertani glucose phosphate media, all lacking serum. Some of these proteins were recognized by sera from chickens experimentally infected with H. paragallinarum. A 110-kDa protein was recognized by a serum pool from convalescent-phase pigs naturally infected with Actinobacillus pleuropneumoniae, and also by a rabbit polyclonal serum against Apx I as well as a rabbit serum against Mannheimia haemolytica leukotoxin, suggesting the presence of an RTX-like protein in H. paragallinarum. H. paragallinarum secreted proteins could be important immunogens in the control of infectious coryza.