Pathogens

Secondary bacterial and fungal infections are associated with respiratory viral infections and invasive mechanical ventilation. In Coronavirus disease 2019 (COVID-19), lung injury by SARS-CoV-2 and impaired immune response can provide a favorable environment for microorganism growth and colonization in hospitalized individuals. Recent studies suggest that secondary bacterial pneumonia is a risk factor associated with COVID-19. In Brazil, knowledge about microbiota present in COVID-19 patients is incipient. This work describes the microbiota of 21 COVID-19 patients admitted to intensive care units from two Brazilian centers. We identified respiratory, nosocomial and bacterial pathogens as prevalent microorganisms. Other bacterial opportunistic and commensal species are also represented. Virulence factors of these pathogenic species, metabolic pathways used to evade and modulate immunological processes and the interconnection between bacterial presence and virulence in COVID-19 progression are discussed. Article Summary LineWe identified respiratory, nosocomial and bacterial pathogens as prevalent microorganisms in 21 Brazilian COVID-19 patients admitted to Intensive Care Units. Pathogen virulence factors and immune response evasion metabolic pathways are correlated to COVID-19 severity.

Even after two decades since the identification of the first giant virus, the Acanthamoeba polyphaga mimivirus (APMV), it still elude scientists. Their gigantic size and genome are unique in the whole virosphere, and many aspects of their biology are still unknown, including their possible hosts. They are cultivated in laboratories using Acanthamoeba cells as hosts, but little is known about the infectivity of these giant viruses in vertebrate cells. However, there is evidence of the possible involvement of APMV in pneumonia and activation of inflammatory pathways. Among the hundreds of prospected giant viruses members is Tupanvirus, isolated in Brazil. Its particles have a characteristically large size varying between 1.2 to 2 m and are covered by fibrils. In the present work, we aim to study the consequences of the incubation of APMV and Tupanvirus with mammalian cells. These cells express Toll-like receptors (TLR) that are capable of recognizing lipopolysaccharides, favoring the internalization of the antigen and activation of the inflammatory system. We used a lineage of human lung adenocarcinoma cells (A549) to evaluate possible effects of TLR activation by the giant viruses and if we could detect the probable cause of the said giant-virus dependent pneumonia. Our results show that APMV and Tupanvirus (TPV) activate cellular receptors related to the Toll-like 4 type-induced inflammatory response and that the A549 cells are capable of internalizing the latter virus. Therefore, this study brings new insights into the possible interactions established between mimiviruses (here represented by APMV and Tupanvirus) and members of the innate cellular immune response.

BackgroundRecurrence is a hallmark of ocular toxoplasmosis (OT), and conditions that influence its occurrence remain a challenge. Natural killer cells (NK) are effectors cells whose primary function is the cytotoxic activity against many parasites, including Toxoplasma gondii. Among the NK cell receptors, immunoglobulin-like receptors (KIR) deserve attention due to their high polymorphism. This study aimed to analyze the influence of KIR gene polymorphism in the course of OT infection and its association with recurrences after an active episode. MethodsNinety-six patients from the Ophthalmologic Clinic of the National Institute of Infectology Evandro Chagas (INI/Fiocruz/RJ, Brazil) were followed for up to five years. After DNA extraction, genotyping of the patients was performed by PCR-SSO utilizing Luminex equipment for reading. During follow-up, 57.4% had a recurrence. ResultsWe identified 25 KIR genotypes and found a higher frequency of genotypes 1 (31.7%) with worldwide distribution. We note that the KIR2DL2 inhibitor gene and the gene activator KIR2DS2 were more frequent in patients without recurrence (P = 0.03 and P = 0.02, respectively). Additionally, we observed one activating gene, KIR2DS1, associated with more than four times faster progression to the development of recurrent ocular toxoplasmosis than individuals without this gene (aRR = 4.6, P = 0.04). ConclusionThe KIR2DL2 and KIR2DS2 are associated as possible protection markers and the KIR2DS1 acting as a possible susceptibility marker. Additionally, the lower proportion of activating genes observed in individuals with recurrence corroborating with the hypothesis that these individuals are more susceptible to ocular toxoplasmosis recurrence (OTR).

Endogenous endophthalmitis caused by Gram-negative bacteria is an intra-ocular infection that can rapidly progress to irreversible loss of vision. While most endophthalmitis isolates are susceptible to antibiotic therapy, the emergence of resistant bacteria necessitates alternative approaches to combat intraocular bacterial proliferation. In this study the ability of predatory bacteria to limit intraocular growth of Pseudomonas aeruginosa, Serratia marcescens, and Staphylococcus aureus was evaluated in a New Zealand White rabbit endophthalmitis prevention model. Predatory bacteria Bdellovibrio bacteriovorus and Micavibrio aeruginosavorus were able to reduce proliferation of keratitis isolates of P. aeruginosa and S. marcescens. However, it was not able to significantly reduce S. aureus, which is not a productive prey for these predatory bacteria, suggesting that the inhibitory effect on P. aeruginosa requires active predation rather than an antimicrobial immune response. Similarly, UV-inactivated B. bacteriovorus were unable to prevent proliferation of P. aeruginosa. Together, these data suggest in vivo predation of Gram-negative bacteria within the intra-ocular environment.

We present spread parameters for first and second waves of the COVID-19 pandemy for USA states, and third wave for 32 regions (19 countries and 13 states of the USA) detected beginning of August 2020. USA first/second wave spreads increase/decrease with population density, are uncorrelated with temperature and median population age. Pooling all 32 regions, third wave spread is slower than for first wave, similar to second wave, and increases with mean altitude (second wave slopes decrease above 900m). Apparently, viruses adapted in spring (second wave) to high temperatures and infecting the young, and in summer (third) waves for spread at altitudes above 1000m. Third wave slopes are not correlated to temperature, so patterns with elevation presumably indicate resistance to relatively high UV regimes. Environmental trends of the COVID-19 pandemy change at incredible rates, making predictions based on classical epidemiological knowledge particularly uncertain.

Viral pandemics have taken a significant toll on humanity and the world now is contending with the SARS-CoV-2 epidemic. Readily available economical preventive measures should be immediately explored. Xylitol has been reported to reduce the severity of viral infections as well as the severity of pneumonia, and increase the survivability of animal subjects. Since pneumonia and acute respiratory distress syndrome are potentially fatal complications of COVID-19, the present study tested the in vitro effectiveness of xylitol against SARS-CoV-2. Virus titers and LRV of SARS-CoV-2, were incubated with a single concentration of nasal spray. Toxicity was observed in the top dilution (1/10). Virus was seen below that dilution so it did not affect calculations of virus titer or LRV. After a 25-minute contact time, the nasal spray (11% Pure Xylitol, 0.85%NaCL (Saline), and 0.20% grapefruit seed extract) reduced virus from 4.2 to 1.7 log10 CCID50 per 0.1 mL, a statistically significant reduction (P<0.001) of 2.5 log10 CCID50. STEM Images obtained at the BIoCryo Laboratory revealed virus contained on the cell wall but none intra-cellular, possibly due to D-xylose (xylitol) production of glycoaminoglycans decoy targets. Xylitol and grapefruit seed extract are not exotic nor expensive rare high technology answers to viral epidemics. The potential in saving lives and the economies of the world by using X-GSE combination therapy should inspire large clinical trials, especially in those nations whereas the healthcare system would be dangerously compromised by the adoption of less effective and significantly more financially demanding therapies. Because there are no risk factors in using the X/GSE combination therapy, and the nasal spray is over the counter available without a prescription, and the spray allows for comfortable long term mask-wearing, adoption of this preventive anti-viral therapy should be encouraged.

BackgroundCandida auris (C. auris) can cause nosocomial transmission in susceptible hosts. This study aimed to use the Loop-Mediated Isothermal Amplification (LAMP) technique as a novel detection technique for C. auris and compare the efficacy with PCR MethodsThis study was performed on C. auris fungal isolates as positive samples and five different standard Candida species as negative control samples. The sensitivity and specificity were determined using different C. auris DNA concentrations. LAMP products were identified by electrophoresis, turbidity, and colorimetric methods. Finally, the products were sequenced ResultsThe LOD (limit of detection) of the LAMP was 10 times lower than the PCR reaction. LAMP and PCR correctly identified C. auris with 100% specificity. The nBLAST results showed the similarity between the sequences of the PCR products and Gene Bank sequences, confirming the specific amplification of target gene in C. auris genome by LAMP and PCR methods. ConclusionThe reproducibility and reliability of the LAMP reaction in differentiating C. auris from closely related species were confirmed. The developed LAMP technique holds promise for testing the clinical samples for quick detection of C. auris with excellent accuracy.

BackgroundThe study of retroviruses dates back to the early 1900s during investigations on neoplastic diseases in chickens. Subsequently, Robert Gallo reported the first human retrovirus HLTV in 1980. What we report here is not an archetypal retrovirus, but the discovery of an oncogenic giant microbial agent with a mega-genome, where the transforming retroviral nature co-exists with multiple archaeal oncogenes. MethodsAfter their isolation from human T cells Leukaemia, these organisms were examined at electron microscopy, tested for reverse transcriptase activity, fully sequenced, used for transformation tests on NIH-3T3 cells in vitro and tumours formation in mice. Same type of particles were also isolated from Canine Transmissible Venereal Tumour (CTVT), the oldest contagious cancer in nature. ResultsEM showed the presence of giant viral particles displaying retroviral antigens. These microbial entities harbour in their mega-genome a transforming retroviral kinase, cell-based oncogenes and have reverse transcriptase activity. The purified viral particles transformed NIH-3T3 cells and induced metastatic tumours in nude mice, three weeks post infection. Ruling out the possible presence of filterable retroviruses, a filtered supernatant did not display RT activity and did not transforms. ConclusionsWe discovered an ancestral microbial agent, acutely transforming. For its giant dimensions, the ability to retain the Gram stain, the presence of a mega-genome and its retroviral nature, we tentatively named the agent Retro-Giant-Virus (RGV). However, distinct from amoeba giant Mimiviruses, this transforming human agent has a different nature and does not require for its isolation amoeba co-culture, since amoeba is not its natural host. The morphology, biology and genetic features allocate this mammalian giant microbe halfway in between a classic oncogenic virus and an infectious cancer cell. Its transforming nature goes with its constant ability to induce tumours formation in mice.

PurposePseudomonas aeruginosa keratitis is a severe ocular infection that can lead to perforation of the cornea. In this study we evaluated the role of bacterial quorum sensing in generating corneal perforation and bacterial proliferation and tested whether co-injection of the predatory bacteria Bdellovibrio bacteriovorus could alter the clinical outcome. P. aeruginosa with lasR mutations were observed among keratitis isolates from a study collecting samples from India, so an isogenic lasR mutant strain of P. aeruginosa was included. MethodsRabbit corneas were intracorneally infected with P. aeruginosa strain PA14 or an isogenic {Delta}lasR mutant and co-injected with PBS or B. bacteriovorus. After 24 h, eyes were evaluated for clinical signs of infection. Samples were analyzed by scanning electron microscopy, optical coherence tomography, sectioned for histology, and corneas were homogenized for CFU enumeration and for inflammatory cytokines. ResultsWe observed that 54% of corneas infected by wild-type PA14 presented with a corneal perforation (n=24), whereas only 4% of PA14 infected corneas that were co-infected with B. bacteriovorus perforate (n=25). Wild-type P. aeruginosa proliferation was reduced 7-fold in the predatory bacteria treated eyes. The {Delta}lasR mutant was less able to proliferate compared to the wild-type, but was largely unaffected by B. bacteriovorus. ConclusionThese studies indicate a role for bacterial quorum sensing in the ability of P. aeruginosa to proliferate and cause perforation of the rabbit cornea. Additionally, this study suggests that predatory bacteria can reduce the virulence of P. aeruginosa in an ocular prophylaxis model.

Lyme disease, caused by infection with members of the Lyme borreliosis group of Borrelia spirochete bacteria, is increasing in frequency and distribution worldwide. This spread is driven by the expansion of ticks, vectors of these pathogens. Epigenetics may be involved in the interaction between mammalian host, tick, and bacterial pathogen, but is still poorly understood. Next-generation sequencing (NGS) allows the study of host response at the transcriptomic and methylomic scale. We tested the effect of the Borrelia burgdorferi strain B31 on a human primary cell line (HUVEC) and an immortalized cell line (HEK-293) for 72 h, a time consistent with the duration of tick feeding and host cell exposure to B. burgdorferi. RNA and DNA were extracted from cells and used for RNA-seq and Enzymatic-Methyl-seq (EM-seq). Differential expression and Reactome pathway enrichment analysis were performed. More significantly differentially expressed genes (a total of 69) were found in HUVECs compared to HEK-293 (a total of 8). Borrelia burgdorferi exposure significantly induced genes in the interferon, cytokine, and other immune response signaling in HUVECs. In HEK-293, pre-NOTCH processing in Golgi was identified as a significant pathway. Other significant genes suggest extracellular matrix binding and interaction in HEK-293. Data comparison with other transcriptomic studies of human cells exposed to B. burgdorferi revealed a small overlap in genes identified in HUVEC, but no overlap for HEK-293. No significant methylation changes were detected in HUVECs or HEK-293 exposed to B. burgdorferi. However, two long non-coding RNAs and a pseudogene were deregulated in response to B. burgdorferi in HUVEC suggesting that other epigenetic mechanisms may be initiated by infection. This is the first study examining the transcriptome and methylome of human cells exposed to B. burgdorferi for 72 h and so contributes to the description of early-stage B. burgdorferi infection at the cellular level.

The genomic analysis of the 556 viral sequences from Jordan uncovered three dominant genetic SARS-CoV-2 lineages that are currently circulating in Jordan: B.1.1.312 (76%), B.1.36.10 (11%), and B.1.1.7 (6%), replacing the genetic strains that were dominant before sustained community transmission in Jordan. This raises speculations about these new genetic lineages and their relationship to the severity of observed disease symptoms in Jordan.

We present an optimised method for the recovery of laboratory generated SARS-CoV-2 virus by plaque assay. This method allows easy incorporation into existing standard operating procedures of biological containment level 3 (BCL3) laboratories.

The upper respiratory tract (URT) virome is crucial in respiratory health and response to pathogens. While common respiratory viruses are well-studied, the presence and potential impact of giant DNA viruses, such as Mimiviridae, in the human URT remain underexplored. This study employed a whole genome metagenomics approach to profile the URT virome of 48 SARS-CoV-2-positive patients from central India. Mimiviridae reads were detected in two elderly male patients with severe acute respiratory infection (SARI) or influenza-like illness (ILI), contributing to 24% and 44% of the total virome in their samples. The dominant species were Acanthamoeba polyphaga mimivirus and Moumouvirus. Although Mimiviridae are not traditionally associated with human respiratory infections, their presence in SARS-CoV-2 patients raises questions about their potential role in co-infections and disease severity, particularly in individuals with ongoing respiratory infections. These findings underscore the need to investigate further giant viruses clinical significance, transmission, and pathogenicity in humans. Future research should focus on their epidemiology and the development of improved diagnostic tools to assess their contribution to human health.

Carbapenem-resistant Acinetobacter baumannii (CRAB) is a leading cause of nosocomial infections worldwide, and the occurrence of extensively drug-resistant (XDR) lineages among them is increasing. Most of A. baumannii pandemic lineages, known as International clones, are represented by MDR/XDR CRAB strains. The IC2 is considered one of the most successful and widespread pandemic clones, however, it is rare in South America, where IC1, IC4 and IC5 are prevalent. In Brazil, besides sporadic reports, an IC2 outbreak was reported only once in Sao Paulo city during the COVID-19 pandemics. This study characterized an outbreak caused by IC2 strains (n=16) in a hospital in Rio de Janeiro in 2022. MLST (MLST Pasteur scheme) analysis revealed that all strains recovered from nosocomial infections belonged to ST2 and corresponded to CRAB presenting the XDR phenotype. In general, this broad resistance spectrum was explained by the presence of several antibiotic resistance genes (ARGs) (armA, blaTEM, blaOXA-23, blaOXA-66, and aacA4-catB8-aadA1-qacE{Delta}1/sul1 carried in class 1 integron). Interestingly, the strains characterized here presented a broader resistance spectrum compared to those of the unique other and contemporary IC2 outbreak in Brazil, although they shared most of the ARGs. This study stressed the possibility of the successful establishment of IC2 in Brazilian clinical settings during and after the COVID-19 pandemics in response to a series of events, such as the overuse of antibiotics, during that period.

There are overlapping world distributions of the mosquito-borne dengue viruses (DENVs) and water-borne bacterial disease leptospirosis which cause large numbers of human infections and fatalities. As such, early differential diagnosis is required for appropriate early leptospirosis antibiotic therapy or DENV patient supportive care, but co-infections have also been reported. From 200 paired (S1 and S2) serum samples collected from suspected DENV infected patients, 70 (35%) were confirmed as on-going infections by demonstrating > 4-fold S1 to S2 sample increased anti-DENV IgG and/or IgM ELISA titers. Of those, 8.57% (6/70) also displayed > 4-fold increased S1 to S2 sample titers in the gold standard Leptospira spp. microscopic agglutination test (MAT) and paraformaldehyde (cell-membrane-impermeable fixative) treated leptospires in immunofluorescence assays (IFAs) due to cross-reactions with 68-72 and 38-42 KDa outer surface membrane antigens present on all Leptospira spp. serovars tested. While DENV-1, -2 or -3 serotypes were isolated from their S1 sera: a) Leptospira spp. could not be isolated from them, b) their S1 sera were all PCR-negative using a Leptospira spp.-specific gene target, and c) their S2 sera were all negative using a commercial anti-Leptospira spp. IgM ELISA. As such, we believe this is the first report of DENVs causing false positive reactions in the gold standard Leptospira spp. MAT, IFAs, and immunoblot assays and which needs further assessments using patients samples which were possibly falsely reported as DENV-Leptospira spp. co-infections in other studies, and to identify these 68-72 and 38-42 KDa Leptospira spp. outer surface membrane antigens by proteomic analyses.

Porcine reproductive and respiratory syndrome (PRRS) is a globally distributed disease caused by Betaarterivirus europensis (PRRSV-1) and Betaarterivirus americense (PRRSV-2). Its clinical presentation ranges from subclinical infection to severe disease, depending on viral evolution and the emergence of novel variants. The aim of this study was to characterize the genetic diversity and identify recombination events in the ORF7 (nucleocapsid, N) gene of ten PRRSV-2 strains circulating in pig farms in Lima, Peru. Bioinformatic analyses were performed using DNAMAN v10.0, MEGA 6, BepiPred-2.0, DnaSP v6, and RDP v4.101. Phylogenetic analysis revealed two well-defined lineages: eight strains clustered within lineage 1A (NADC34-like), and two within lineage 5A (VR2332-like), demonstrating the co-circulation of genetically distinct variants in the region. Comparative sequence analysis identified significant amino acid substitutions in eight strains (15, 16, 17, 20, 21, 22, 23, and 24), with strain 24 being the most divergent, accumulating multiple substitutions, including T81I, R109S, I115F, R116S, and A119K within the C-terminal region encompassing antigenic domains I-V. B-cell epitope prediction using BepiPred-2.0 identified six epitope patterns (A-F) comprising nine potential B-cell epitope regions (positions 5-19, 33-72, 33-73, 84-85, 87-98, 84-98, 87-97, 84, and 119). Patterns B, E, and F exhibited four to five predicted epitope sites and corresponded to strains 21, 22, 23, and 24. Recombination analysis using RDP v4.101 detected a statistically robust recombination event in strain 18_montana2020-R (lineage 5A), with strain 24_montana2020-WT (lineage 1A) identified as the putative major parent (100% similarity) and the vaccine-like VR2332 strain (lineage 5A) as the minor parent (99.3% similarity). Secondary evidence of the same recombination event was observed in strain 19_montana2020-R. Genetic diversity analysis of the ORF7 gene identified 50 polymorphic nucleotide sites and 52 mutations. Overall, these findings demonstrate substantial genetic variability in the ORF7 gene of PRRSV-2 circulating in Lima, Peru, characterized by lineage co-circulation and inter-lineage recombination. Continuous molecular surveillance is warranted to monitor viral evolution, assess potential antigenic implications, and support effective PRRS control strategies in the Peruvian swine industry.

The eukaryotic protozoan Entamoeba gingivalis (E.g.) is strongly associated with inflamed periodontal pockets. Unlike other obligate anaerobic Entamoeba species, it is considered to not have a life cycle of actively dividing trophozoites and dormant cysts. Accordingly, it has been regarded as non-infectious. To investigate if E.g is capable of encystation in response to adverse environmental conditions, we cultivated clinical isolates of E.g. collected from inflamed periodontal pockets in antibiotics for 8 days. The cytomorphological and ultrastructure forms of the amoeba were investigated by transmission and scanning electron microscopy to reveal cyst formation. We observed exocysts and the encapsulated trophozoids separated by an intra-cystic space, a dense poorly vesiculated cytoplasma and polygonal surface areas of cysts. The cysts walls were composed of chitin. Cysts were conspicuously smaller compared to trophozoids and lacked pseudo- and filipodia. We did not observe multi-nucleated trophozoids after antibiotic induces encystation. Cyst formation in E.g may explain why established treatment approaches often do not stop periodontal tissue destruction during periodontitis and periimplantitis.

Rapid identification of SARS-CoV-2 infected individuals through viral RNA detection followed by effective personal isolation remains the most effective way to prevent the spread of this virus. Large-scale RNA detection involves mass specimen collection and transportation. For biosafety reasons, denaturing viral transport medium has been extensively used during the pandemic. But the high concentrations of guanidinium isothiocyanate (GITC) in such media have raised issues around sufficient GITC supply and laboratory safety. Here, we tested whether supplementing media containing low concentrations of GITC with ammonium sulfate (AS) would affect the throat-swab detection of SARS-CoV-2 pseudovirus or a viral inactivation assay targeting both enveloped and non-enveloped viruses. Adding AS to the denaturing transport media reduced the need for high levels of GITC and improved SARS-COV-2 RNA detection without compromising virus inactivation.

Microbial keratitis is a sight-threatening corneal infection with varying etiological agents, primarily bacteria and fungi. Assessing and contrasting the virulence factors of microorganisms isolated from a non-contact lens-associated keratitis (NCLAK) and contact lens-associated keratitis (CLAK) is the goal of the current investigation. Samples were collected from over 60 patients and analysed using standard microbiological techniques, including culture, Gram staining, KOH mount, biochemical tests, antimicrobial susceptibility testing, and biofilm assays. The results demonstrated that CLAK isolates were predominantly bacterial, especially Pseudomonas aeruginosa, known for strong biofilm production and high multidrug resistance. In contrast, NCLAK showed a higher incidence of fungal infections, particularly Candida albicans. The results highlight the significance of early diagnosis, tailored and improved awareness regarding contact lens hygiene to prevent complications associated with keratitis.

This study compares the collection dates and locations of the Omicron BA.1 lineage and other major SARS-CoV- 2 mutants registered in NCBI GenBank and provides a detailed analysis of the emergence patterns of pure reverse mutants, which contain only reverse mutations and no other mutations in the surface glycoprotein. The results indicate that Omicron BA.1.1 and its pure reverse mutants were widely distributed throughout the United States from the early days of their emergence, showing a statistically significant difference compared to other major variants, which spread from a small number of sources. The peak emergence of BA.1.1 and BA.1.1.18 pure reverse mutants occurred a few weeks before the peak of all collected samples, whereas the peak of pure reverse mutants in major BA.1 variants and BA.2 coincides with the overall sampling peak. Although the peaks of BA.1.x collections are not all synchronous, the peaks of pure reverse mutants in the BA.1 lineage completely overlap, with the number of such mutants declining abruptly after the peak. These regional and temporal anomalies in the Omicron BA.1 lineage, especially in the BA.1.1 lineage, are virtually impossible to explain by current theories of natural mutation and spread by human-to-human infection.