A retrospective study suggests 55 days of persistence of SARS-CoV-2 during the first wave of the pandemic in Santiago de Chile.

Background: As the COVID-19 pandemic persists, infections continue to surge globally. Presently, the most effective strategies to curb the disease and prevent outbreaks involve fostering immunity, promptly identifying positive cases, and ensuring their timely isolation. Notably, there are instances where the SARS-CoV-2 virus remains infectious even after patients have completed their quarantine. Objective: Understanding viral persistence post-quarantine is crucial as it could account for localized infection outbreaks. Therefore, studying and documenting such instances is vital for shaping future public health policies. Design: This study delves into a unique case of SARS-CoV-2 persistence in a 60-year-old female healthcare worker with a medical history of hypertension and hypothyroidism. The research spans 55 days, marking the duration between her initial and subsequent diagnosis during Chile's first COVID-19 wave, with the analysis conducted using RT-qPCR. Results: Genomic sequencing-based phylogenetic analysis revealed that the SARS-CoV-2 detected in both Nasopharyngeal swab samples (NPSs) was consistent with the 20B clade of the Nextstrain classification, even after a 55-day interval. Conclusion: This research underscores the need for heightened vigilance concerning cases of viral persistence. Such instances, albeit rare, might be pivotal in understanding sporadic infection outbreaks that occur post-quarantine.

P2X7 Receptor in Dendritic Cells and Macrophages: Implications in Antigen Presentation and T Lymphocyte Activation.

The P2X7 receptor, a member of the P2X purinergic receptor family, is a non-selective ion channel. Over the years, it has been associated with various biological functions, from modulating to regulating inflammation. However, its emerging role in antigen presentation has captured the scientific community's attention. This function is essential for the immune system to identify and respond to external threats, such as pathogens and tumor cells, through T lymphocytes. New studies show that the P2X7 receptor is crucial for controlling how antigens are presented and how T cells are activated. These studies focus on antigen-presenting cells, like dendritic cells and macrophages. This review examines how the P2X7 receptor interferes with effective antigen presentation and activates T cells and discusses the fundamental mechanisms that can affect the immune response. Understanding these P2X7-mediated processes in great detail opens up exciting opportunities to create new immunological therapies.

An ecological study on reinfection rates using a large dataset of RT-qPCR tests for SARS-CoV-2 in Santiago of Chile.

Introduction: As the SARS-CoV-2 continues to evolve, new variants pose a significant threat by potentially overriding the immunity conferred by vaccination and natural infection. This scenario can lead to an upswing in reinfections, amplified baseline epidemic activity, and localized outbreaks. In various global regions, estimates of breakthrough cases associated with the currently circulating viral variants, such as Omicron, have been reported. Nonetheless, specific data on the reinfection rate in Chile still needs to be included. Methods: Our study has focused on estimating COVID-19 reinfections per wave based on a sample of 578,670 RT-qPCR tests conducted at the University of Santiago of Chile (USACH) from April 2020 to July 2022, encompassing 345,997 individuals. Results: The analysis reveals that the highest rate of reinfections transpired during the fourth and fifth COVID-19 waves, primarily driven by the Omicron variant. These findings hold despite 80% of the Chilean population receiving complete vaccination under the primary scheme and 60% receiving at least one booster dose. On average, the interval between initial infection and reinfection was found to be 372 days. Interestingly, reinfection incidence was higher in women aged between 30 and 55. Additionally, the viral load during the second infection episode was lower, likely attributed to Chile's high vaccination rate. Discussion: This study demonstrates that the Omicron variant is behind Chile's highest number of reinfection cases, underscoring its potential for immune evasion. This vital epidemiological information contributes to developing and implementing effective public health policies.

Genomic Evidence Suggests Viral Persistence of SARS-CoV-2 for 386 Days in Health Worker: A Case Report from Santiago of Chile.

The COVID-19 pandemic continues to affect several countries. One of the best ways to control its spread is the timely identification of infected patients for isolation and quarantine. While an episode of infection lasts an average of 8-10 days from the onset of symptoms, there is literature describing long-lasting viral persistence events. Here, we report a case of persistence of SARS-CoV-2 for 386 days in a health worker from Santiago de Chile. Our study could be one of the longest reported viral persistence events. RNA sequencing analyses indicated that the first positive diagnosis (8 June 2020) corresponded to a SARS-CoV-2 variant belonging to Clade Nextstrain 20A. Three hundred eighty-six days later (23 September 2021), the second positive result reached the same viral variant (Clade 20A) but without presence or circulation in Chile since May 2021. Both sequencing coverages showed an identity of 99.21%, with some mutations related to the severity of the disease (ORF1b:P314L) and more infectivity (S:D614G). This work reinforces the idea of implementing an RT-qPCR or rapid antigen test once the quarantine is fulfilled to ensure viral absence, identify potential persistence, and, consequently, minimize the risk of local outbreaks of SARS-CoV-2 infection.

Evaluation and comparison of the sensitivity of three commercial RT-qPCR kits used for the detection of SARS-CoV-2 in Santiago, Chile.

Introduction: The COVID-19 pandemic is still in force, causing global public health challenges and threats. Although vaccination and herd immunity have proven to be the most efficient way to control the pandemic, massive and early testing of patients using the RT-qPCR technique is crucial for constant genomic surveillance. The appearance of variants of SARS-CoV-2 with new mutations can reduce the efficiency of diagnostic detection. In this sense, several commercial RT-qPCR kits have been the target of extensive analysis because low assay performance could lead to false-negative diagnoses. Methods: In this study, we evaluated the performance of three commercial RT-qPCR kits; Thermo Fisher (TaqMan 2019-nCoV Assay Kit v1), BGI and Roche (LightCycler® Multiplex RNA Virus Master) used for the diagnosis of COVID-19 throughout the pandemic in Santiago de Chile. Results: Under our best assay conditions, we found significant differences in Cq amplification values for control and viral probes, against the same nasopharyngeal swab samples (NPSs). In addition, in some cases, the sensitivity of the RT-qPCR kits decreased against viral variants. Conclusion: Our study suggests evaluating the RT-qPCR kits used to detect SARS-CoV-2 because variants such as Omicron, which has several mutations, can compromise their detection and underestimate viral circulation.

Sensitivity analysis of rapid antigen tests for the Omicron SARS-CoV-2 variant detection from nasopharyngeal swab samples collected in Santiago of Chile.

The COVID-19 pandemic continues to be a concern and keeps global health authorities on alert. The RT-PCR technique has been the gold-standard assay for detecting the SARS-CoV-2 virus. However, rapid antigen tests (RATs) have been widely used to increase the number of tests faster and more efficiently in the population. Nevertheless, the appearance of new viral variants, with genomic mutations associated with greater contagiousness and immune evasion, highlights the need to evaluate the sensitivity of these RATs. This report evaluates the sensitivity of SD Biosensor-Roche, Panbio™, and Clinitest® RATs widely used in Santiago de Chile in the detection of the Omicron variant from Nasopharyngeal samples (NPSs), the most predominant SARS-CoV-2 variant in Chile and the world. SD Biosensor-Roche shows a detection sensitivity of 95.7% in the viral amplification range of 20 ≤ Cq < 25, while Panbio™ and Clinitest® show 100% and 91.3%, respectively. In the viral amplification ranges of 25 ≤ Cq < 30, the detection sensitivity decreased to 28% for SD Biosensor-Roche, 32% for Panbio™, and 72% for Clinitest®. This study indicates that the tested RATs have high sensitivity in detecting the Omicron variant of concern (VOC) at high viral loads. By contrast, its sensitivity decreases at low viral loads. Therefore, it is suggested to limit the use of RATs as an active search method, considering that infections in patients are increasingly associated with lower viral loads of SARS-CoV-2. These antecedents could prevent contagion outbreaks and reduce the underestimation of the current Omicron variant circulation at the local level.

Epidemiological characteristics of Omicron and Delta SARS-CoV-2 variant infection in Santiago, Chile.

The variant of concern (VOC) SARS-CoV-2 Omicron (B.1.1529) has been described as a highly contagious variant but less virulent than the current variant being monitored (VBM) Delta (B.1.617.2), causing fewer cases of hospitalizations, symptomatology, and deaths associated with COVID-19 disease. Although the epidemiological comparison of both variants has been previously reported in other countries, no report indicates their behavior and severity of infection in Chile. In this work, we report for the first time the effect of the Omicron and Delta variants in a cohort of 588 patients from the Hospital de Urgencia Asistencia pública (HUAP), a high-complexity health center in Santiago, Chile. This report is framed at the beginning of Chile's third wave of the COVID-19 pandemic, with a marked increase in the Omicron variant and a decrease in the circulating Delta variant. Our results indicated a similar proportion of patients with a complete vaccination schedule for both variants. However, the Delta variant was associated with a higher prevalence of hospitalization and more significant symptomatology associated with respiratory distress. On the other hand, our data suggest that vaccination is less effective in preventing infection by the Omicron variant. This antecedent, with a low severity but high contagiousness, suggests that the Omicron variant could even collapse the primary health care service due to the high demand for health care.

Comparison of the First and Second Wave of Infections by SARS-CoV-2: A Retrospective and Longitudinal Study From a Primary Health Care Center in Santiago of Chile.

The current COVID-19 pandemic is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Many countries have reported the experience of at least two contagion waves, describing associated mortality rates and population behavior. The analysis of the effect of this pandemic in different localities can provide valuable information on the key factors to consider in the face of future massive infectious diseases. This work describes the first retrospective and comparative study about behavior during the first and second waves of the COVID-19 pandemic in Chile from a primary Healthcare Center. From 19,313 real-time quantitative PCR (RT-qPCR) tests assessed, the selected 1,694 positive diagnostics showed a decrease in mortality rate in the second wave (0.6%) compared with the first (4.6%). In addition, we observed that infections in the second wave were mainly in young patients with reduced comorbidities. The population with a complete vaccination schedule shows a decrease in the duration of symptoms related to the disease, and patients with more comorbidities tend to develop severe illness. This report provides evidence to partially understand the behavior and critical factors in the severity of the COVID-19 pandemic in the population of Santiago of Chile.

The Comparative Analysis of Two RT-qPCR Kits for Detecting SARS-CoV-2 Reveals a Higher Risk of False-Negative Diagnosis in Samples with High Quantification Cycles for Viral and Internal Genes.

The early detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) using the real-time quantitative polymerase chain reaction (RT-qPCR) as a gold-standard molecular tool has allowed to test and trace the viral spread and the isolation of COVID-19-infected patients. The detection capacity of viral and internal genes is an essential parameter to consider and analyze during the assay. In this study, we analyze the performance of the two commercial RT-qPCR kits used in Chile, TaqMan™ 2019-nCoV Control Kit v1 (Thermo Fisher) and MaxCov19 (TAAG Genetics), for the COVID-19 diagnosis from nasopharyngeal swab samples (NPSs). Our results show a lower sensitivity of the TAAG kit compared to the Thermo Fisher kit, even in the detection of SARS-CoV-2 mutations associated with its variants. This study reinforces the relevance of evaluating the performance of RT-qPCR kits before being used massively since those with lower sensitivity can generate false negatives and produce outbreaks of local infections.

First Identification of Reinfection by a Genetically Different Variant of SARS-CoV-2 in a Homeless Person from the Metropolitan Area of Santiago, Chile.

The identification and tracking of SARS-CoV-2 infected patients in the general population are essential components of the global strategy to limit the COVID-19 viral spread, specifically for maintaining traceability and suppressing the resurgence of local outbreaks. Public health programs that include continuous RT-qPCR testing for COVID-19 in the general population, viral sequencing, and genomic surveillance for highly contagious forms of the virus have allowed for the identification of SARS-CoV-2 infections and reinfections. This work identified SARS-CoV-2 reinfection in a homeless person, which occurred 58 days after the first COVID-19 diagnosis. Genomic sequencing identified a different Nextstrain classification clade (20A and 20B) and PANGO lineage, with a divergence of 4 single nucleotide variants (SNVs) in S and ORF1ab genes, suggesting reinfection by different viral variants. This study is the first from the great metropolitan area of Santiago, Chile, one of the top ten countries in the world to live during the COVID-19 pandemic. We support the importance of performing intensive genomic surveillance programs in the whole population and high-risk groups, such as homeless people, nearly 20 thousand people in Chile, and have limited access to health care services and poor viral traceability.

Description of Symptoms Caused by the Infection of the SARS-CoV-2 B.1.621 (Mu) Variant in Patients With Complete CoronaVac Vaccination Scheme: First Case Report From Santiago of Chile.

Vaccine administration is one of the most efficient ways to control the current coronavirus disease 2019 (COVID-19) pandemic. However, the appearance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants can avoid the immunity generated by vaccines. Thus, in patients with a complete vaccine schedule, the infection by SARS-CoV-2 may cause severe, mild, and asymptomatic manifestations of the disease. In this case report, we describe for the first time the clinical symptoms of four patients (three symptomatic; one asymptomatic) from Santiago of Chile, with a complete vaccination schedule with two doses of CoronaVac (Sinovac Life Science) infected with the variant of interest (VOI) B.1.621 (Mu). They were compared with four unvaccinated patients, who had a higher prevalence of symptoms after infection compared to vaccinated patients. In the CoronaVac-vaccinated group, an 80-year-old patient who registered various comorbidities required Invasive mechanical ventilation for 28 days with current home medical recovery discharge. By contrast, in the unvaccinated group, a 71-year-old presented more symptoms with more than 45 days of Invasive mechanical ventilation, which continues to date, presenting greater lung damage than the vaccinated hospitalized patient. This first report evidence differences in the clinical symptomatology of patients vaccinated and non-vaccinated infected with the VOI B.1.621 (Mu) and suggest the protective effects of CoronaVac against this variant.

Analysis by real-time PCR of five transport and conservation mediums of nasopharyngeal swab samples to COVID-19 diagnosis in Santiago of Chile.

Due to the COVID-19 pandemic, many transport kits have been manufactured to preserve and transport nasopharyngeal swab samples (NPSs) from patients. However, there is no information on the performance of the different virus transport media (VTM) used in COVID-19 diagnosis in the population of Santiago de Chile. We compared the RT-qPCR amplification profile of five different viral transport kit mediums, including DNA/RNA Shield™, NAT, VTM-N, Ezmedlab™, and phosphate-buffered saline (PBS), for NPSs from Central Metropolitan Health Service, Santiago, Chile. The DNA/RNA Shield™ medium showed a better performance in terms of Cq and RFU values for the internal reference RNase P and viral ORF1ab probes. By contrast, the PBS transport medium registered higher Cq values for the viral and reference gene, compared to the other VTM. DNA/RNA Shield™ shows higher relative fluorescence units (RFUs) and lower Cq values for the reference gene. Collectively, our results suggest that the PBS medium could compromise the sample diagnosis because of its lower RT-qPCR performance. The NAT, Ezmedlab and VTM-N, and DNA/RNA Shield™ media show acceptable RT-qPCR parameters and, consequently, seem suitable for use in COVID-19 diagnosis.

P2X7 receptor is essential for cross-dressing of bone marrow-derived dendritic cells.

T cell activation requires the processing and presentation of antigenic peptides in the context of a major histocompatibility complex (MHC complex). Cross-dressing is a non-conventional antigen presentation mechanism, involving the transfer of preformed peptide/MHC complexes from whole cells, such as apoptotic cells (ACs) to the cell membrane of professional antigen-presenting cells (APCs), such as dendritic cells (DCs). This is an essential mechanism for the induction of immune response against viral antigens, tumors, and graft rejection, which until now has not been clarified. Here we show for first time that the P2X7 receptor (P2X7R) is crucial to induce cross-dressing between ACs and Bone-Marrow DCs (BMDCs). In controlled ex vivo assays, we found that the P2X7R in both ACs and BMDCs is required to induce membrane and fully functional peptide/MHC complex transfer to BMDCs. These findings show that acquisition of ACs-derived preformed antigen/MHC-I complexes by BMDCs requires P2X7R expression.

In Vivo Antitumor Effect against Murine Cells of CT26 Colon Cancer and EL4 Lymphoma by Autologous Whole Tumor Dead Cells.

Active immunotherapy against cancer is based on immune system stimulation, triggering efficient and long-lasting antigen-specific immune responses. Immunization strategies using whole dead cells from tumor tissue, containing specific antigens inside, have become a promising approach, providing efficient lymphocyte activation through dendritic cells (DCs). In this work, we generate whole dead tumor cells from CT26, E.G7, and EL4 live tumor cells as antigen sources, which termed immunogenic cell bodies (ICBs), generated by a simple and cost-efficient starvation-protocol, in order to determine whether are capable of inducing a transversal anticancer response regardless of the tumor type, in a similar way to what we describe previously with B16 melanoma. We evaluated the anticancer effects of immunization with doses of ICBs in syngeneic murine tumor models. Our results showed that mice's immunization with ICBs-E.G7 and ICBs-CT26 generate 18% and 25% of tumor-free animals, respectively. On the other hand, all carrying tumor-animals and immunized with ICBs, including ICBs-EL4, showed a significant delay in their growth compared to not immunized animals. These effects relate to DCs maturation, cytokine production, increase in CD4+T-bet+ and CD4+ROR-γt+ population, and decrease of T regulatory lymphocytes in the spleen. Altogether, our data suggest that whole dead tumor cell-based cancer immunotherapy generated by a simple starvation protocol is a promising way to develop complementary, innovative, and affordable antitumor therapies in a broad spectrum of tumors.

Adenosine triphosphate, polymyxin B and B16 cell-derived immunization induce anticancer response.

Aim: Whole dead tumor cells can be used as antigen source and the induction of protective immune response could be enhanced by damage-associated molecular patterns. Materials & methods: We generated whole dead tumor cells called B16-immunogenic cell bodies (ICBs) from B16 melanoma cells by nutrient starvation and evaluated the in vivo antitumor effect of B16-ICBs plus ATP and polymyxin B (PMB). Results: The subcutaneous immunization with B16-ICBs + PMB + ATP a 50% of tumor-free animals and induced a significant delay in tumor growth in a prophylactic approach. These results correlated with maturation of bone marrow-derived dendritic cells and activation of T CD8+ lymphocytes in vitro. Conclusion: Altogether, ICB + ATP + PMB is efficient in inducing the antitumor efficacy of the whole dead tumor cells vaccine.

Evaluation of the Immune Response Induced by CoronaVac 28-Day Schedule Vaccination in a Healthy Population Group.

CoronaVac vaccine from Sinovac Life Science is currently being used in several countries. In Chile, the effectiveness of preventing hospitalization is higher than 80% with a vaccination schedule. However, to date, there are no data about immune response induction or specific memory. For this reason, we recruited 15 volunteers without previous suspected/diagnosed COVID-19 and with negative PCR over time to evaluate the immune response to CoronaVac 28 and 90 days after the second immunization (dpi). The CoronaVac administration induces total and neutralizing anti-spike antibodies in all vaccinated volunteers at 28 and 90 dpi. Furthermore, using ELISpot analysis to assay cellular immune responses against SARS-CoV-2 spike protein, we found an increase in IFN-gamma- and Granzyme B-producing cells in vaccinated volunteers at 28 and 90 dpi. Together, our results indicate that CoronaVac induces a robust humoral immune response and cellular immune memory of at least 90 dpi.

The Rapid Antigen Detection Test for SARS-CoV-2 Underestimates the Identification of COVID-19 Positive Cases and Compromises the Diagnosis of the SARS-CoV-2 (K417N/T, E484K, and N501Y) Variants.

Timely detection of severe acute respiratory syndrome due to coronavirus 2 (SARS-CoV-2) by reverse transcription quantitative polymerase chain reaction (RT-qPCR) has been the gold- strategy for identifying positive cases during the current pandemic. However, faster and less expensive methodologies are also applied for the massive diagnosis of COVID-19. In this way, the rapid antigen test (RAT) is widely used. However, it is necessary to evaluate its detection efficiency considering the current pandemic context with the circulation of new viral variants. In this study, we evaluated the sensitivity and specificity of RAT (SD BIOSENSOR, South Korea), widely used for testing and SARS-CoV-2 diagnosis in Santiago of Chile. The RAT showed a 90% (amplification range of 20 ≤ Cq <25) and 10% (amplification range of 25 ≤ Cq <30) of positive SARS-CoV-2 cases identified previously by RT-qPCR. Importantly, a 0% detection was obtained for samples within a Cq value>30. In SARS-CoV-2 variant detection, RAT had a 42.8% detection sensitivity in samples with RT-qPCR amplification range 20 ≤ Cq <25 containing the single nucleotide polymorphisms (SNP) K417N/T, N501Y and E484K, associated with beta or gamma SARS-CoV-2 variants. This study alerts for the special attention that must be paid for the use of RAT at a massive diagnosis level, especially in the current scenario of appearance of several new SARS-CoV-2 variants which could generate false negatives and the compromise of possible viral outbreaks.

P2X7 Receptor at the Crossroads of T Cell Fate.

The P2X7 receptor is a ligand-gated, cation-selective channel whose main physiological ligand is ATP. P2X7 receptor activation may also be triggered by ARTC2.2-dependent ADP ribosylation in the presence of extracellular NAD. Upon activation, this receptor induces several responses, including the influx of calcium and sodium ions, phosphatidylserine externalization, the formation of a non-selective membrane pore, and ultimately cell death. P2X7 receptor activation depends on the availability of extracellular nucleotides, whose concentrations are regulated by the action of extracellular nucleotidases such as CD39 and CD38. The P2X7 receptor has been extensively studied in the context of the immune response, and it has been reported to be involved in inflammasome activation, cytokine production, and the migration of different innate immune cells in response to ATP. In adaptive immune responses, the P2X7 receptor has been linked to T cell activation, differentiation, and apoptosis induction. In this review, we will discuss the evidence of the role of the P2X7 receptor on T cell differentiation and in the control of T cell responses in inflammatory conditions.

Chitosan-Based Delivery of Avian Reovirus Fusogenic Protein p10 Gene: In Vitro and In Vivo Studies towards a New Vaccine against Melanoma.

Reovirus is known to have an anticancer effect in both the preclinical and clinical assays. Current evidence suggests that the reovirus-mediated impact on tumor growth depends on the activation of specific antitumor immune responses. A feasible explanation for the oncolytic effects and immune system activation is through the expression of the fusogenic reovirus protein. In this work, we evaluated the in vivo antitumor effects of the expression of fusogenic protein p10 of avian reovirus (ARV-p10). We used chitosan nanoparticles (CH-NPs) as a vehicle for the ARV-p10 DNA in murine B16 melanoma models both in vitro and in vivo. We confirmed that ARV-p10 delivery through a chitosan-based formulation (ARV-p10 CH-NPs) was capable of inducing cell fusion in cultured melanoma cells, showing a mild cytotoxic effect. Interestingly, intratumor injection of ARV-p10 CH-NPs delayed tumor growth, without changing lymphoid populations in the tumor tissue and spleen. The injection of chitosan nanoparticles (CH-NPs) also delayed tumor growth, suggesting the nanoparticle itself would attack tumor cells. In conclusion, we proved that in vitro ARV-p10 protein expression using CH-NPs in murine melanoma cells induces a cytotoxic effect associated with its cell fusion. Further studies are necessary for establishing a protocol for efficient in vivo DNA delivery of fusion proteins to produce an antitumoral effect.

Chitosan-Based Nanoparticles for Intracellular Delivery of ISAV Fusion Protein cDNA into Melanoma Cells: A Path to Develop Oncolytic Anticancer Therapies.

Oncolytic virus therapy has been tested against cancer in preclinical models and clinical assays. Current evidence shows that viruses induce cytopathic effects associated with fusogenic protein-mediated syncytium formation and immunogenic cell death of eukaryotic cells. We have previously demonstrated that tumor cell bodies generated from cells expressing the fusogenic protein of the infectious salmon anemia virus (ISAV-F) enhance crosspriming and display prophylactic antitumor activity against melanoma tumors. In this work, we evaluated the effects of the expression of ISAV-F on the B16 melanoma model, both in vitro and in vivo, using chitosan nanoparticles as transfection vehicle. We confirmed that the transfection of B16 tumor cells with chitosan nanoparticles (NP-ISAV) allows the expression of a fusogenically active ISAV-F protein and decreases cell viability because of syncytium formation in vitro. However, the in vivo transfection induces a delay in tumor growth, without inducing changes on the lymphoid populations in the tumor and the spleen. Altogether, our observations show that expression of ISAV fusion protein using chitosan nanoparticles induces cell fusion in melanoma cells and slight antitumor response.