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.

Soft-metal(loid)s induce protein aggregation in Escherichia coli.

Metal(loid) salts were used to treat infectious diseases in the past due to their exceptional biocidal properties at low concentrations. However, the mechanism of their toxicity has yet to be fully elucidated. The production of reactive oxygen species (ROS) has been linked to the toxicity of soft metal(loid)s such as Ag(I), Au(III), As(III), Cd(II), Hg(II), and Te(IV). Nevertheless, few reports have described the direct, or ROS-independent, effects of some of these soft-metal(loid)s on bacteria, including the dismantling of iron-sulfur clusters [4Fe-4S] and the accumulation of porphyrin IX. Here, we used genome-wide genetic, proteomic, and biochemical approaches under anaerobic conditions to evaluate the direct mechanisms of toxicity of these metal(loid)s in Escherichia coli. We found that certain soft-metal(loid)s promote protein aggregation in a ROS-independent manner. This aggregation occurs during translation in the presence of Ag(I), Au(III), Hg(II), or Te(IV) and post-translationally in cells exposed to Cd(II) or As(III). We determined that aggregated proteins were involved in several essential biological processes that could lead to cell death. For instance, several enzymes involved in amino acid biosynthesis were aggregated after soft-metal(loid) exposure, disrupting intracellular amino acid concentration. We also propose a possible mechanism to explain how soft-metal(loid)s act as proteotoxic agents.

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.

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.

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.

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.

Anaerobiosis favors biosynthesis of single and multi-element nanostructures.

Herein we report the use of an environmental multimetal(loid)-resistant strain, MF05, to biosynthesize single- or multi-element nanostructures under anaerobic conditions. Inorganic nanostructure synthesis typically requires methodologies and conditions that are harsh and environmentally hazardous. Thus, green/eco-friendly procedures are desirable, where the use of microorganisms and their extracts as bionanofactories is a reliable strategy. First, MF05 was entirely sequenced and identified as an Escherichia coli-related strain with some genetic differences from the traditional BW25113. Secondly, we compared the CdS nanostructure biosynthesis by whole-cell in a design defined minimal culture medium containing sulfite as the only sulfur source to obtain sulfide reduction from a low-cost chalcogen reactant. Under anaerobic conditions, this process was greatly favored, and irregular CdS (ex. 370 nm; em. 520-530 nm) was obtained. When other chalcogenites were tested (selenite and tellurite), only spherical Se0 and elongated Te0 nanostructures were observed by TEM and analyzed by SEM-EDX. In addition, enzymatic-mediated chalcogenite (sulfite, selenite, and tellurite) reduction was assessed by using MF05 crude extracts in anaerobiosis; similar results for nanostructures were obtained; however Se0 and Te0 formation were more regular in shape and cleaner (with less background). Finally, the in vitro nanostructure biosynthesis was assessed with salts of Ag, Au, Cd, and Li alone or in combination with chalcogenites. Several single or binary nanostructures were detected. Our results showed that MF05 is a versatile anaerobic bionanofactory for different types of inorganic NS. synthesis.

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.

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.

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.

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.

Accumulation of heme biosynthetic intermediates contributes to the antibacterial action of the metalloid tellurite.

The metalloid tellurite is highly toxic to microorganisms. Several mechanisms of action have been proposed, including thiol depletion and generation of hydrogen peroxide and superoxide, but none of them can fully explain its toxicity. Here we use a combination of directed evolution and chemical and biochemical approaches to demonstrate that tellurite inhibits heme biosynthesis, leading to the accumulation of intermediates of this pathway and hydroxyl radical. Unexpectedly, the development of tellurite resistance is accompanied by increased susceptibility to hydrogen peroxide. Furthermore, we show that the heme precursor 5-aminolevulinic acid, which is used as an antimicrobial agent in photodynamic therapy, potentiates tellurite toxicity. Our results define a mechanism of tellurite toxicity and warrant further research on the potential use of the combination of tellurite and 5-aminolevulinic acid in antimicrobial therapy.

Hydrogen peroxide and hypochlorous acid influx through the major S. Typhimurium porin OmpD is affected by substitution of key residues of the channel.

OmpD is the major Salmonella enterica serovar Typhimurium (S. Typhimurium) porin and mediates hydrogen peroxide (H2O2) influx. The results described herein extend this finding to hypochlorous acid (HOCl), another reactive oxygen species that is also part of the oxidative burst generated by the phagosome. S. Typhimurium cells lacking OmpD show decreased HOCl influx, and OmpD-reconstituted proteoliposomes show an increase in the uptake of the toxic compound. To understand this physiologically relevant process, we investigated the role of key OmpD residues in H2O2 and NaOCl transport. Using a theoretical approach, residue K16 was defined as a major contributor to the channel electrostatic properties, and E111 was shown to directly participate in the size-exclusion limit of the channel. Together, we provide theoretical, genetic, and biochemical evidence that OmpD mediates H2O2 and NaOCl uptake, and that key residues of the channel are implicated in this process.

Participation of the Salmonella OmpD porin in the infection of RAW264.7 macrophages and BALB/c mice.

Salmonella Typhimurium is the etiological agent of gastroenteritis in humans and enteric fever in mice. Inside these hosts, Salmonella must overcome hostile conditions to develop a successful infection, a process in which the levels of porins may be critical. Herein, the role of the Salmonella Typhimurium porin OmpD in the infection process was assessed for adherence, invasion and proliferation in RAW264.7 mouse macrophages and in BALB/c mice. In cultured macrophages, a ΔompD strain exhibited increased invasion and proliferation phenotypes as compared to its parental strain. In contrast, overexpression of ompD caused a reduction in bacterial proliferation but did not affect adherence or invasion. In the murine model, the ΔompD strain showed increased ability to survive and replicate in target organs of infection. The ompD transcript levels showed a down-regulation when Salmonella resided within cultured macrophages and when it colonized target organs in infected mice. Additionally, cultured macrophages infected with the ΔompD strain produced lower levels of reactive oxygen species, suggesting that down-regulation of ompD could favor replication of Salmonella inside macrophages and the subsequent systemic dissemination, by limiting the reactive oxygen species response of the host.

Probing the ArcA regulon under aerobic/ROS conditions in Salmonella enterica serovar Typhimurium.

BACKGROUND: Hydrogen peroxide (H2O2) is a reactive oxygen species (ROS), which is part of the oxidative burst encountered upon internalization of Salmonella enterica serovar Typhimurium (S. Typhimurium) by phagocytic cells. It has previously been established that, the ArcAB two-component system plays a critical role in ROS resistance, but the genes regulated by the system remained undetermined to date. We therefore investigated the ArcA regulon in aerobically growing S. Typhimurium before and after exposure to H2O2 by querying gene expression and other physiological changes in wild type and ΔarcA strains. RESULTS: In the ΔarcA strain, expression of 292 genes showed direct or indirect regulation by ArcA in response to H2O2, of which 141were also regulated in aerobiosis, but in the opposite direction. Gene set enrichment analysis (GSEA) of the expression data from WT and ΔarcA strains, revealed that, in response to H2O2 challenge in aerobically grown cells, ArcA down regulated multiple PEP-PTS and ABC transporters, while up regulating genes involved in glutathione and glycerolipid metabolism and nucleotide transport. Further biochemical analysis guided by GSEA results showed that deletion of arcA during aerobic growth lead to increased reactive oxygen species (ROS) production which was concomitant with an increased NADH/NAD+ ratio. In absence of ArcA under aerobic conditions, H2O2 exposure resulted in lower levels of glutathione reductase activity, leading to a decreased GSH (reduced glutathione)/GSSG (oxidized glutathione) ratio. CONCLUSION: The ArcA regulon was defined in 2 conditions, aerobic growth and the combination of peroxide treatment and aerobic growth in S. Typhimurium. ArcA coordinates a response that involves multiple aspects of the carbon flux through central metabolism, which ultimately modulates the reducing potential of the cell.