Evaluation of SARS-CoV-2 ORF7a Deletions from COVID-19-Positive Individuals and Its Impact on Virus Spread in Cell Culture.

The spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), causing the COVID-19 outbreak, posed a primary concern of public health worldwide. The most common changes in SARS-CoV-2 are single nucleotide substitutions, also reported insertions and deletions. This work investigates the presence of SARS-CoV-2 ORF7a deletions identified in COVID-19-positive individuals. Sequencing of SARS-CoV-2 complete genomes showed three different ORF7a size deletions (190-nt, 339-nt and 365-nt). Deletions were confirmed through Sanger sequencing. The ORF7a∆190 was detected in a group of five relatives with mild symptoms of COVID-19, and the ORF7a∆339 and ORF7a∆365 in a couple of co-workers. These deletions did not affect subgenomic RNAs (sgRNA) production downstream of ORF7a. Still, fragments associated with sgRNA of genes upstream of ORF7a showed a decrease in size when corresponding to samples with deletions. In silico analysis suggests that the deletions impair protein proper function; however, isolated viruses with partial deletion of ORF7a can replicate in culture cells similarly to wild-type viruses at 24 hpi, but with less infectious particles after 48 hpi. These findings on deleted ORF7a accessory protein gene, contribute to understanding SARS-CoV-2 phenotypes such as replication, immune evasion and evolutionary fitness as well insights into the role of SARS-CoV-2_ORF7a in the mechanism of virus-host interactions.

Different Profiles of Cytokines, Chemokines and Coagulation Mediators Associated with Severity in Brazilian Patients Infected with Dengue Virus.

The incidence of dengue in Latin America has increased dramatically during the last decade. Understanding the pathogenic mechanisms in dengue is crucial for the identification of biomarkers for the triage of patients. We aimed to characterize the profile of cytokines (IFN-γ, TNF-α, IL-1ß, IL-6, IL-18 and IL-10), chemokines (CXCL8/IL-8, CCL2/MCP-1 and CXCL10/IP-10) and coagulation mediators (Fibrinogen, D-dimer, Tissue factor-TF, Tissue factor pathway inhibitor-TFPI and Thrombomodulin) during the dengue-4 epidemic in Brazil. Laboratory-confirmed dengue cases had higher levels of TNF-α (p < 0.001), IL-6 (p = 0.005), IL-10 (p < 0.001), IL-18 (p = 0.001), CXCL8/IL-8 (p < 0.001), CCL2/MCP-1 (p < 0.001), CXCL10/IP-10 (p = 0.001), fibrinogen (p = 0.037), D-dimer (p = 0.01) and TFPI (p = 0.042) and lower levels of TF (p = 0.042) compared to healthy controls. A principal component analysis (PCA) distinguished between two profiles of mediators of inflammation and coagulation: protective (TNF-α, IL-1ß and CXCL8/IL-8) and pathological (IL-6, TF and TFPI). Lastly, multivariate logistic regression analysis identified high aspartate aminotransferase-to-platelet ratio index (APRI) as independent risk factors associated with severity (adjusted OR: 1.33; 95% CI 1.03-1.71; p = 0.027), the area under the receiver operating characteristics curve (AUC) was 0.775 (95% CI 0.681-0.869) and an optimal cutoff value was 1.4 (sensitivity: 76%; specificity: 79%), so it could be a useful marker for the triage of patients attending primary care centers.

SARS-CoV-2 diagnostic diary: from rumors to the first case. Early reports of molecular tests from the military research and diagnostic institute of Rio de Janeiro.

Corona virus disease (COVID-19) presents a serious threat to global health. A historical timeline of early molecular diagnostics from government alert (January 22) (D) was presented. After in silico analysis, Brazilian Army Institute of Biology (IBEx-RJ) tested samples in house using real-time reverse transcriptase polymerase chain reaction (RT-PCR) (fast mode) based on Centers for Disease Control and Prevention (CDC) recommendations. First cases from Brazil, Rio de Janeiro, IBEx, and diagnosis team were reported in D36, D44, D66, and D74 respectively. Therefore, after 1300 tests, we recommend N1/N2 primer sets (CDC) for preliminary and Charité protocol confirmation in case of positive results. Moreover, every professional should be tested before starting work, in addition to weekly tests for everyone involved.

SARS-CoV-2 diagnostic diary: from rumors to the first case. Early reports of molecular tests from the military research and diagnostic institute of Rio de Janeiro

Corona virus disease (COVID-19) presents a serious threat to global health. A historical timeline of early molecular diagnostics from government alert (January 22) (D) was presented. After in silico analysis, Brazilian Army Institute of Biology (IBEx-RJ) tested samples in house using real-time reverse transcriptase polymerase chain reaction (RT-PCR) (fast mode) based on Centers for Disease Control and Prevention (CDC) recommendations. First cases from Brazil, Rio de Janeiro, IBEx, and diagnosis team were reported in D36, D44, D66, and D74 respectively. Therefore, after 1300 tests, we recommend N1/N2 primer sets (CDC) for preliminary and Charité protocol confirmation in case of positive results. Moreover, every professional should be tested before starting work, in addition to weekly tests for everyone involved.

A Multidimensional Approach to Assessing Anthropometric and Aerobic Fitness Profiles of Elite Brazilian Endurance Athletes and Military Personnel.

INTRODUCTION: Military personnel must remain physically active to meet operational requirements. Military physical training not only provides the performance capabilities required for performing occupational tasks but also fosters the development of sport. Thus, Armed Forces across the world have historically invested in developing elite- and Olympic-level athletes. This study aimed to assess the anthropometric and physiological differences among groups of Brazilian military athletes (MA), non-military athletes (A), and military non-athletes (M). MATERIALS AND METHODS: Seventy-five individuals participated in the study: 17 MA (23.7 ± 4.8 years), 27 A (24.7 ± 5.3 years), and 31 M (26.9 ± 3.3 years). MA and A individuals specialized in endurance sports, and had a mean weekly training volume of (100.0 ± 34.8 and 106.3 ± 40.5 Km; F = 0.894, p = 0.6), respectively. Anthropometric measures and maximal oxygen uptake (V̇O2máx) were assessed in all participants. Ergospirometry and anthropometry variables were analyzed with one-way analysis of variance (ANOVA) for independent measures. Comparisons of weekly training volume (km) and training experience (years) were performed only between the A and MA using the Student's t-test for independent samples. For a multidimensional approach, Partial least squares discriminant analysis (PLS-DA) was performed for all variables using the online tool MetaboAnalyst. RESULTS: We found no differences in anthropometric and physiological profiles between A and MA, but significant differences between M and MA/A in body mass index (kg/m2) (BMI), body fat percentage, fat mass (kg), waist circumference (cm) (WC), somatotype, and V̇O2máx (mL min-1 kg-1). CONCLUSION: In conclusion, military endurance athletes have similar anthropometric and physiological profiles to non-military athletes and superior levels to non-athlete military. These findings indicate that the Brazilian Armed Forces scouting system has been successful in identifying endurance athletic talent in line with their historic role of developing sport in Brazil.

Trichosporon asahii secretes a 30-kDa aspartic peptidase.

Trichosporon asahii is a fungal opportunistic pathogen that causes superficial and deep-seated infections presenting high mortality. Very little is known about the virulence attributes produced by this fungus. Herein, aspartic peptidase production was identified in Brazilian clinical isolates of T. asahii by different methodologies. Initially, T. asahii strain 250 (from skin lesion) was inoculated in both liquid and solid culture media containing bovine serum albumin (BSA) as the sole nitrogenous source. A translucent halo around the fungal colony was observed from the 5th day of culture. The cell-free culture supernatant revealed that soluble BSA was hydrolyzed along the growth, generating low molecular mass polypeptides as observed by electrophoresis. Subsequently, the secretions from four clinical strains of T. asahii were analyzed by BSA-SDS-PAGE and a single proteolytic band of 30-kDa was detected under acidic pH at 37°C. The secreted aspartic peptidase of T. asahii efficiently cleaved the cathepsin D peptide substrate, but not the substrates with specificity to HIV-1 peptidase and rennin. The capability to cleave either cathepsin D substrate in a fluorogenic assay or BSA immobilized within a gel matrix varied according to the T. asahii isolate. T. asahii extracellular peptidase activity was strongly inhibited by pepstatin A and HIV peptidase inhibitors, classifying it as an aspartic-type peptidase. Human serum albumin, mucin, non-immune immunoglobulin G and gelatin induced, in different levels, the secretion of this aspartic peptidase. With these results, T. asahii must be included in the list of many human fungal opportunistic pathogens able to secrete an aspartic-type peptidase.

Booster dose after 10 years is recommended following 17DD-YF primary vaccination.

A single vaccination of Yellow Fever vaccines is believed to confer life-long protection. In this study, results of vaccinees who received a single dose of 17DD-YF immunization followed over 10 y challenge this premise. YF-neutralizing antibodies, subsets of memory T and B cells as well as cytokine-producing lymphocytes were evaluated in groups of adults before (NVday0) and after (PVday30-45, PVyear1-4, PVyear5-9, PVyear10-11, PVyear12-13) 17DD-YF primary vaccination. YF-neutralizing antibodies decrease significantly from PVyear1-4 to PVyear12-13 as compared to PVday30-45, and the seropositivity rates (PRNT≥2.9Log10mIU/mL) become critical (lower than 90%) beyond PVyear5-9. YF-specific memory phenotypes (effector T-cells and classical B-cells) significantly increase at PVday30-45 as compared to naïve baseline. Moreover, these phenotypes tend to decrease at PVyear10-11 as compared to PVday30-45. Decreasing levels of TNF-α(+) and IFN-γ(+) produced by CD4(+) and CD8(+) T-cells along with increasing levels of IL-10(+)CD4(+)T-cells were characteristic of anti-YF response over time. Systems biology profiling represented by hierarchic networks revealed that while the naïve baseline is characterized by independent micro-nets, primary vaccinees displayed an imbricate network with essential role of central and effector CD8(+) memory T-cell responses. Any putative limitations of this cross-sectional study will certainly be answered by the ongoing longitudinal population-based investigation. Overall, our data support the current Brazilian national immunization policy guidelines that recommend one booster dose 10 y after primary 17DD-YF vaccination.

Candida haemulonii complex: species identification and antifungal susceptibility profiles of clinical isolates from Brazil.

OBJECTIVES: The emerging fungal pathogens comprising the Candida haemulonii complex (Candida haemulonii, Candida haemulonii var. vulnera and Candida duobushaemulonii) are notable for their antifungal resistance. Twelve isolates with phenotypic similarity to C. haemulonii were recovered from patients in Brazilian hospitals. Here we aimed to identify these isolates by a molecular approach, using the current classification of this fungal complex, and to evaluate their antifungal susceptibility profiles. METHODS: The fungal isolates were rechecked to certify their authentication by mycology methodologies and then characterized by ITS1-5.8S-ITS2 gene sequencing. A susceptibility assay was performed using the broth microdilution method published by CLSI (M27-A3/M27-S3). RESULTS: Based on biochemical tests, all Brazilian isolates were identified as C. haemulonii. After employing ITS sequencing, five isolates were identified as C. haemulonii, four as C. duobushaemulonii and three as C. haemulonii var. vulnera. All 12 clinical isolates were resistant to amphotericin B (MICs ranged from 2 to >16 mg/L) and fluconazole (MICs ≥ 64 mg/L). One isolate of C. haemulonii var. vulnera and two isolates of C. duobushaemulonii were susceptible-dose dependent to itraconazole, while the remaining isolates (75%) were resistant to this antifungal. Eight out of 12 isolates (66.7%) were resistant to voriconazole (MICs ≥ 16 mg/L), while all isolates were susceptible to caspofungin (MICs ≤ 0.5 mg/L). CONCLUSIONS: Our results reinforce the importance of molecular identification in differentiating species of the C. haemulonii complex. Moreover, the antifungal multiresistant profile of clinical isolates of the C. haemulonii complex represents a challenge to the treatment of such infections.

Cellular characterisation of Candida tropicalis presenting fluconazole-related trailing growth.

We assessed fluconazole susceptibility in 52 Candida tropicalis clinical strains using seven antifungal susceptibility methods, including broth microdilution (BMD) [standard M27 A3 (with neutral and acid pH), ATB Fungus 3, Vitek 2 system and flow cytometric analysis] and agar-based methods (disk diffusion and E-test). Trailing growth, detection of cell-associated secreted aspartic proteases (Saps) and morphological and ultrastructural traits of these clinical strains were also examined. The ranges of fluconazole 24 h-minimum inhibitory concentration (MIC) values were similar among all methods. The essential agreement among the methods used for MIC determinations was excellent and all methods categorised all strains as susceptible, except for one strain that showed a minor error. The presence of the trailing effect was assessed by six methods. Trailing positivity was observed for 86.5-100% of the strains. The exception was the BMD-Ac method where trailing growth was not observed. Morphological and ultrastructural alterations were detected in C. tropicalis trailing cells, including mitochondrial swelling and cell walls with irregular shapes. We tested the production of Saps in 13 C. tropicalis strains expressing trailing growth through flow cytometry. Our results showed that all of the C. tropicalis strains up-regulated surface Sap expression after 24 h or 48 h of exposure to fluconazole, which was not observed in untreated yeast strains. We concluded that C. tropicalis strains expressing trailing growth presented some particular features on both biological and ultrastructural levels.

Cellular characterisation of Candida tropicalis presenting fluconazole-related trailing growth

We assessed fluconazole susceptibility in 52 Candida tropicalis clinical strains using seven antifungal susceptibility methods, including broth microdilution (BMD) [standard M27 A3 (with neutral and acid pH), ATB Fungus 3, Vitek 2 system and flow cytometric analysis] and agar-based methods (disk diffusion and E-test). Trailing growth, detection of cell-associated secreted aspartic proteases (Saps) and morphological and ultrastructural traits of these clinical strains were also examined. The ranges of fluconazole 24 h-minimum inhibitory concentration (MIC) values were similar among all methods. The essential agreement among the methods used for MIC determinations was excellent and all methods categorised all strains as susceptible, except for one strain that showed a minor error. The presence of the trailing effect was assessed by six methods. Trailing positivity was observed for 86.5-100 percent of the strains. The exception was the BMD-Ac method where trailing growth was not observed. Morphological and ultrastructural alterations were detected in C. tropicalis trailing cells, including mitochondrial swelling and cell walls with irregular shapes. We tested the production of Saps in 13 C. tropicalis strains expressing trailing growth through flow cytometry. Our results showed that all of the C. tropicalis strains up-regulated surface Sap expression after 24 h or 48 h of exposure to fluconazole, which was not observed in untreated yeast strains. We concluded that C. tropicalis strains expressing trailing growth presented some particular features on both biological and ultrastructural levels.

Identification, antifungal susceptibility and scanning electron microscopy of a keratinolytic strain of Rhodotorula mucilaginosa: a primary causative agent of onychomycosis.

Onychomycosis is a dermatological problem of high prevalence that mainly affects the hallux toenail. Onychomycosis caused by the yeast Rhodotorula mucilaginosa was identified using colony morphology, light microscopy, urease and carbohydrate metabolism in a 57-year-old immunocompetent patient from Rio de Janeiro, Brazil. High-resolution scanning electron microscopy of nail fragments, processed by a noncoating method, led to the observation with fine detail of the structures of both nail and fungus involved in the infection. Yeasts were mainly found inside grooves in the nail. Budding yeasts presented a spiral pattern of growth and blastoconidia were found in the nail groove region. Keratinase assays and keratin enzymography revealed that this isolate was highly capable of degrading keratin. Antifungal susceptibility tests showed that the fungus was susceptible to low concentrations of amphotericin B and 5-flucytosine and resistant to high concentrations of fluconazole, itraconazole, voriconazole and terbinafine. These findings showed data for the first time concerning the interaction of R. mucilaginosa in toenail infection and suggest that this emerging yeast should also be considered an opportunistic primary causative agent of onychomycosis.

Peptidase profiles from non-albicans Candida spp. isolated from the blood of a patient with chronic myeloid leukemia and another with sickle cell disease.

Candida lipolytica and Candida rugosa were isolated from blood samples from a patient with chronic myeloid leukemia (31 years old) and a patient with sickle cell disease (1-year-old), respectively. Isolates were grown for 48 h at 37 degrees C in either Sabouraud or tryptone soy broth (TSB). Peptidases were tested for using substrate sodium dodecyl sulfate-polyacrylamide gels with gelatin, casein, bovine serum albumin (BSA) or hemoglobin. Enzymography analyses were made on the following substrates: human albumin, IgG and human fibrinogen, which had been incubated with the concentrated supernatants. For C. lipolytica, a approximately 60-kDa gelatin-degrading serine proteolytic activity was found in the TSB supernantant as well as a metallopeptidase activity capable of hydrolysing human albumin, IgG and human fibrinogen. With C. rugosa, albumin, IgG and human fibrinogen substrates were degraded by an aspartyl-like peptidase activity. Supernatants from C. rugosa also showed three serine proteolytic activities towards gelatin (approximately 50 kDa, TSB), casein ( approximately 94 kDa, TSB) and BSA ( approximately 120-kDa, Sabouraud), in addition to a metallopeptidase capable of degrading casein ( approximately 110 kDa, Sabouraud). Little is known about peptidases of C. rugosa and C. lipolytica and this preliminary data may prove useful for future work on host-parasite relationship and antifungal agents.