Enhancing Colorectal Cancer Screening with Droplet Digital PCR (ddPCR) Analysis of Fusobacterium nucleatum in Fecal Immunochemical Test (FIT) Samples.

Fecal immunochemical test (FIT) followed by colonoscopy in positive cases is commonly used for population-based colorectal cancer (CRC) screening. However, specificity of FIT for CRC is not ideal, and has poor performance for advanced adenoma detection. Fecal Fusobacterium nucleatum (Fn) detection has been proposed as a potential non-invasive biomarker for CRC and advanced adenoma detection. We aimed to evaluate the diagnostic performance of Fn detection using droplet digital PCR (ddPCR) in FIT samples from individuals enrolled in a CRC screening program with colorectal adenoma or cancer. We evaluated Fn presence in DNA isolated from FIT leftover material of 300 participants in a CRC Screening Program using ddPCR. The Fn DNA amount was classified as Fn-low/negative and Fn-high, and the association with patients clinicopathological features and accuracy measurements was calculated. Fn high levels were more prevalent in FIT-positive (47.2%n=34 of72) than FIT-negative samples (28.9%, n=66 of 228) (p<0.04). Among FIT-positive samples, high Fn levels were significantly more frequent in cancer patients (CA, n=8) when compared to normal (NT, n=16) (p=0.02), non-advanced adenomas (NAA, n=36) (p=0.01), and advanced adenomas (AA, n=12) (p=0.01). Performance analysis of Fn in FIT-positive samples for colorectal cancer detection yielded an AUC of 0.8203 (CI: 0.6464-0.9942), with high sensitivity (100%) and specificity of 50%%. Concluding, we showed the feasibility of detecting Fn in FIT leftovers using the ultrasensitive ddPCR technique. Furthermore, we highlighted the potential use of Fn levels in fecal samples to ameliorate CRC detection.

Ultraviolet Resistance of Microorganisms Isolated from Uranium-Rich Minerals from Perus, Brazil.

The district of Perus, located in the city of São Paulo, Brazil, is renowned for its weathered granitic-pegmatitic masses, which harbor a significant number of uraniferous minerals that contribute to ionizing radiation levels up to 20 times higher than the background levels. In this study, aseptically collected mineral samples from the area were utilized to isolate 15 microorganisms, which were subjected to pre-screening tests involving UV-C and UV-B radiation. The microorganisms that exhibited the highest resistance to ultraviolet (UV) radiation were selected for the construction of survival curves for UV-C, broad-band UV-B, and solar simulation resistance testing. Subsequently, the four strains that demonstrated superior survival capabilities under UV radiation exposure were chosen for 16S rRNA gene sequencing. Among these, Nocardioides sp. O4R and Nocardioides sp. MA2R demonstrated the most promising outcomes in the UV radiation resistance assessments, showcasing comparable performance to the well-established radioresistant model organism Deinococcus radiodurans. These findings underscore the potential of naturally occurring high-radiation environments as valuable resources for the investigation of UV-resistant microorganisms.

MYCOLICIBACTERIUM FORTUITUM INFECTION IN EX SITU EMERALD TREE BOA (Corallus batesii).

Emerald tree boas (Corallus batesii) are boids that in situ occurs in forested habitats in the Amazon Basin. The mycobacterial species can infect reptiles but the species Mycolicibacterium fortuitum was identified only in feces samples of ex situ Python regius and was isolated from granulomatous lesions of an ex situ Iguana iguana when was still part of the genus Mycobacterium. This article aims to report a mycobacteria infection case in a female Corallus batesii kept under human care. The animal presented apathy and 2 months of anorexia, being found dead. The necropsy revealed presence of tracheal and pulmonary nodules besides multifocal, bacterial, granulomatous pneumonia. After Fite-Faraco histochemical staining, immunohistochemistry, semi-nested polymerase chain reaction (PCR) and genetic sequencing the Mycolicibacterium fortuitum complex was diagnosed with 99.54% of nucleotide similarity. This mycobacterial species was already pointed out as an important nosocomial pathogen and more studies are necessary to explore their zoonotic potential.

Natural mycobacterium tuberculosis complex infection in a brown howler monkey (Alouatta guariba clamitans) in Brazil.

Neotropical primates rarely exhibit active tuberculosis. A brown howler monkey was found injured in an urban area. Histopathology revealed granulomatous inflammation in the lungs, lymph nodes, and liver. Immunohistochemistry and molecular analysis confirmed the presence of Mycobacterium tuberculosis complex. The findings highlight the importance of TB surveillance in nonhuman primates.

Molecular detection of Sarcocystis sp. in a kept under human care black capuchin monkey (Sapajus nigritus., Goldfuss 1809) with necrotizing encephalitis.

A senile male black capuchin monkey (Sapajus nigritus) kept under human care in a Zoo was found dead after 2 weeks presenting signals of weight loss and hyporexia. Histopathological revealed a necrotizing encephalitis. Although it was not observed microscopically, Sarcocystis sp infection was detected in brain tissue from molecular assays. These infections have been rarely described in neotropical primates, particularly associated with tissue lesions.

The Complexity of Tobacco Smoke-Induced Mutagenesis in Head and Neck Cancer.

Tobacco smoke, alone or combined with alcohol, is the predominant cause of head and neck cancer (HNC). Here, we further explore how tobacco exposure contributes to cancer development by mutational signature analysis of 265 whole-genome sequenced HNC from eight countries. Six tobacco-associated mutational signatures were detected, including some not previously reported. Differences in HNC incidence between countries corresponded with differences in mutation burdens of tobacco-associated signatures, consistent with the dominant role of tobacco in HNC causation. Differences were found in the burden of tobacco-associated signatures between anatomical subsites, suggesting that tissue-specific factors modulate mutagenesis. We identified an association between tobacco smoking and three additional alcohol-related signatures indicating synergism between the two exposures. Tobacco smoking was associated with differences in the mutational spectra and repertoire of driver mutations in cancer genes, and in patterns of copy number change. Together, the results demonstrate the multiple pathways by which tobacco smoke can influence the evolution of cancer cell clones.

The Mutographs biorepository: A unique genomic resource to study cancer around the world.

Large-scale biorepositories and databases are essential to generate equitable, effective, and sustainable advances in cancer prevention, early detection, cancer therapy, cancer care, and surveillance. The Mutographs project has created a large genomic dataset and biorepository of over 7,800 cancer cases from 30 countries across five continents with extensive demographic, lifestyle, environmental, and clinical information. Whole-genome sequencing is being finalized for over 4,000 cases, with the primary goal of understanding the causes of cancer at eight anatomic sites. Genomic, exposure, and clinical data will be publicly available through the International Cancer Genome Consortium Accelerating Research in Genomic Oncology platform. The Mutographs sample and metadata biorepository constitutes a legacy resource for new projects and collaborations aiming to increase our current research efforts in cancer genomic epidemiology globally.

Phylogenetic analysis reveals a new introduction of Yellow Fever virus in São Paulo State, Brazil, 2023.

Yellow Fever (YF) is a viral arbovirosis of Public Health importance. In Brazil, surveillance is focused mainly on detecting epizootic events of Platyrrhini. Herein, we compared the detection and phylogenetic analysis of YF virus in two neotropical primates (NTP), a Callithrix detected in the previous epidemic period (2016-2020), and a Callicebus nigrifons, showing a new introduction of YF in 2023. This paper illustrates the importance of joint actions of laboratory and field teams to ensure quick response to Public Health emergencies, such as the intensification of vaccination of susceptible human populations.

Unveiling the role of MGMT and DAPK hypermethylation in response to anti-EGFR agents: Molecular insights for advancing HNSCC treatment.

BACKGROUND: Epidermal growth factor receptor (EGFR) is frequently activated in head and neck squamous cell carcinoma (HNSCC) and serves as a valuable target for therapy. Despite the availability of the EGFR inhibitors Cetuximab, Afatinib, and Allitinib, there are limited predictive markers for their response. Understanding molecular aberrations in HNSCC could facilitate the identification of new strategies for patient clinical and biological classification, offering novel therapeutic avenues. METHODS: We assessed CCNA1, DCC, MGMT, CDKN2A/p16, and DAPK methylation status in HNSCC cell lines and their association with anti-EGFR treatment response. RESULTS: MGMT methylation status displayed high sensitivity and specificity in distinguishing sensitive and resistant HNSCC cell lines to Afatinib (AUC = 0.955) and Allitinib (AUC = 0.935). Moreover, DAPK methylation status predicted response to Allitinib with high accuracy (AUC = 0.852), indicating their putative predictive biomarker roles. CONCLUSION: These findings hold promise for the development of more personalized and effective treatment approaches for HNSCC patients.

Phenotypical Screening of an MMV Open Box Library and Identification of Compounds with Antiviral Activity against St. Louis Encephalitis Virus.

St. Louis encephalitis virus (SLEV) is a neglected mosquito-borne Flavivirus that may cause severe neurological disease in humans and other animals. There are no specific treatments against SLEV infection or disease approved for human use, and drug repurposing may represent an opportunity to accelerate the development of treatments against SLEV. Here we present a scalable, medium-throughput phenotypic cell culture-based screening assay on Vero CCL81 cells to identify bioactive compounds that could be repurposed against SLEV infection. We screened eighty compounds from the Medicines for Malaria Venture (MMV) COVID Box library to identify nine (11%) compounds that protected cell cultures from SLEV-induced cytopathic effects, with low- to mid-micromolar potencies. We validated six hit compounds using viral plaque-forming assays to find that the compounds ABT-239, Amiodarone, Fluphenazine, Posaconazole, Triparanol, and Vidofludimus presented varied levels of antiviral activity and selectivity depending on the mammalian cell type used for testing. Importantly, we identified and validated the antiviral activity of the anti-flavivirus nucleoside analog 7DMA against SLEV. Triparanol and Fluphenazine reduced infectious viral loads in both Vero CCL81 and HBEC-5i cell cultures and, similar to the other validated compounds, are likely to exert antiviral activity through a molecular target in the host.

Detection of herpesviruses in neotropical primates from São Paulo, Brazil.

Transmission of herpesvirus between humans and non-human primates represents a serious potential threat to human health and endangered species conservation. This study aimed to identify herpesvirus genomes in samples of neotropical primates (NTPs) in the state of São Paulo, Brazil. A total of 242 NTPs, including Callithrix sp., Alouatta sp., Sapajus sp., and Callicebus sp., were evaluated by pan-herpesvirus polymerase chain reaction (PCR) and sequencing. Sixty-two (25.6%) samples containing genome segments representative of members of the family Herpesviridae, including 16.1% for Callitrichine gammaherpesvirus 3, 6.1% for Human alphaherpesvirus 1, 2.1% for Alouatta macconnelli cytomegalovirus, and 0.83% for Cebus albifrons lymphocryptovirus 1. No co-infections were detected. The detection of herpesvirus genomes was significantly higher among adult animals (p = 0.033) and those kept under human care (p = 0.008671). These findings confirm the importance of monitoring the occurrence of herpesviruses in NTP populations in epizootic events.

Overall Survival, BRAF, RAS, and MSI Status in Patients Who Underwent Cetuximab After Refractory Chemotherapy for Metastatic Colorectal Cancer.

PURPOSE: Evaluate overall survival (OS), RAS, BRAF, and MSI frequencies in patients with metastatic colorectal cancer (mCRC), refractory to chemotherapy, and finally treated with cetuximab. METHODS: A retrospective cohort study to evaluate 211 mCRC patients with wild-type KRAS treated with cetuximab. BRAF V600E, KRAS, NRAS gene mutations, and MSI status were identified using PCR techniques in a population of pre-treated patients who were refractory to fluoropyrimidines, oxaliplatin, and irinotecan. In addition, we evaluated the mutation frequency of the BRAF and NRAS genes and the MSI status of this population. Uni- and multivariate analyses were performed for independent prognostic factors of OS. RESULTS: The median OS was 10.4 months, 6.6 months for patients with right and 11.5 months for left colon cancers (p = 0.02). The frequencies of mutations were BRAF at 3.9% (median OS of 4.9 months), NRAS at 3.38% (median OS of 6.9 months), and MSI-High status at 3.3% (median OS of 4.6 months). The OS, NRAS, and MSI frequencies were similar to those found in other studies that evaluated cetuximab in poly-treated patients and were associated with lower survival rates in univariate analyses. The frequency of BRAF mutations was lower than that found in previous studies. The only variable that remained significant for OS in the multivariate model was tumour laterality, with patients with right colon cancer presenting a worse prognosis (HR = 2.81). CONCLUSION: Although BRAF, NRAS mutations, and MSI-High status were associated with shorter OS in univariate analyses, only tumour laterality remained an independent prognostic factor in the multivariate analysis.

miR-99a-5p as a biomarker for lymph node metastasis prediction in oral squamous cell carcinoma patients.

BACKGROUND: Metastatic lymph node involvement influences therapy decisions and serves as a prognostic indicator in oral squamous cell carcinoma (OSCC). However, many early-stage patients with clinically negative lymph nodes exhibit no metastasis upon surgical staging. This study aimed to identify differentially expressed miRNAs capable of distinguishing pathologically positive (pN+) from negative (pN0) nodes in OSCC patients without clinical evidence of lymph node metastases (cN0). METHODS: Expression levels of 798 miRNAs were assessed in tumor samples from 10 pN+ and 10 pN0 patients using the Nanostring nCounter platform. Validation was performed in an independent cohort of 15 pN+ and 24 pN0 patients through RT-qPCR. RESULTS: Eight miRNAs exhibited differential expression between pN0 and pN+ patients. Notably, hsa-miR-99a-5p demonstrated high sensitivity and specificity in predicting patients at higher risk of positive lymph nodes. CONCLUSIONS: These findings highlight hsa-miR-99a-5p as a potential biomarker for detecting lymph node metastasis in primary OSCC tumors.

Proteolytic inactivation of CXCL12 in the lungs and circulation of COVID-19 patients.

The human chemokine stromal cell-derived factor-1 (SDF-1) or CXCL12 is involved in several homeostatic processes and pathologies through interaction with its cognate G protein-coupled receptor CXCR4. Recent research has shown that CXCL12 is present in the lungs and circulation of patients with coronavirus disease 2019 (COVID-19). However, the question whether the detected CXCL12 is bioactive was not addressed. Indeed, the activity of CXCL12 is regulated by NH2- and COOH-terminal post-translational proteolysis, which significantly impairs its biological activity. The aim of the present study was to characterize proteolytic processing of CXCL12 in broncho-alveolar lavage (BAL) fluid and blood plasma samples from critically ill COVID-19 patients. Therefore, we optimized immunosorbent tandem mass spectrometry proteoform analysis (ISTAMPA) for detection of CXCL12 proteoforms. In patient samples, this approach uncovered that CXCL12 is rapidly processed by site-specific NH2- and COOH-terminal proteolysis and ultimately degraded. This proteolytic inactivation occurred more rapidly in COVID-19 plasma than in COVID-19 BAL fluids, whereas BAL fluid samples from stable lung transplantation patients and the non-affected lung of lung cancer patients (control groups) hardly induced any processing of CXCL12. In COVID-19 BAL fluids with high proteolytic activity, processing occurred exclusively NH2-terminally and was predominantly mediated by neutrophil elastase. In low proteolytic activity BAL fluid and plasma samples, NH2- and COOH-terminal proteolysis by CD26 and carboxypeptidases were observed. Finally, protease inhibitors already approved for clinical use such as sitagliptin and sivelestat prevented CXCL12 processing and may therefore be of pharmacological interest to prolong CXCL12 half-life and biological activity in vivo.

Characterization of Systemic Disease Development and Paw Inflammation in a Susceptible Mouse Model of Mayaro Virus Infection and Validation Using X-ray Synchrotron Microtomography.

Mayaro virus (MAYV) is an emerging arthropod-borne virus endemic in Latin America and the causative agent of arthritogenic febrile disease. Mayaro fever is poorly understood; thus, we established an in vivo model of infection in susceptible type-I interferon receptor-deficient mice (IFNAR-/-) to characterize the disease. MAYV inoculations in the hind paws of IFNAR-/- mice result in visible paw inflammation, evolve into a disseminated infection and involve the activation of immune responses and inflammation. The histological analysis of inflamed paws indicated edema at the dermis and between muscle fibers and ligaments. Paw edema affected multiple tissues and was associated with MAYV replication, the local production of CXCL1 and the recruitment of granulocytes and mononuclear leukocytes to muscle. We developed a semi-automated X-ray microtomography method to visualize both soft tissue and bone, allowing for the quantification of MAYV-induced paw edema in 3D with a voxel size of 69 µm3. The results confirmed early edema onset and spreading through multiple tissues in inoculated paws. In conclusion, we detailed features of MAYV-induced systemic disease and the manifestation of paw edema in a mouse model extensively used to study infection with alphaviruses. The participation of lymphocytes and neutrophils and expression of CXCL1 are key features in both systemic and local manifestations of MAYV disease.

Caffeic Acid Has Antiviral Activity against Ilhéus Virus In Vitro.

Ilhéus virus (ILHV) is a neglected mosquito-borne flavivirus. ILHV infection may lead to Ilhéus fever, an emerging febrile disease like dengue fever with the potential to evolve into a severe neurological disease characterized by meningoencephalitis; no specific treatments are available for this disease. This study assessed the antiviral properties of caffeic acid, an abundant component of plant-based food products that is also compatible with the socioeconomic limitations associated with this neglected infectious disease. The in vitro activity of caffeic acid on ILHV replication was investigated in Vero and A549 cell lines using plaque assays, quantitative RT-PCR, and immunofluorescence assays. We observed that 500 µM caffeic acid was virucidal against ILHV. Molecular docking indicated that caffeic acid might interact with an allosteric binding site on the envelope protein.

Disseminated Mucor indicus infection in a marmoset (Callithrix sp.).

Mucormycosis is rarely described in Platyrrhines. Herein, we describe the pathologic and molecular features of a gastric and hepatic infection by Mucor indicus in a marmoset (Callithrix sp.).

Canine distemper virus and canine adenovirus type 1 co-infection in a free-living hoary fox (Lycalopex vetulus) from Brazil.

The present case is the first description of a co-infection with canine distemper virus (CDV) and canine adenovirus type 1 (CAdV-1) in a free-living hoary fox pup from Brazil. The animal was found and rescued with poor body condition, dehydration, incoordination, ataxia, excessive vocalization, and "blue eyes" phenomenon. Despite the efforts, euthanasia was elected due to worsening clinical signs and poor prognosis. Pathologic examination revealed a mild, acute, random, necrotizing hepatitis, acute bronchopneumonia, hydrocephalus, corneal edema with epithelium degeneration, and acidophilic intracytoplasmatic inclusion bodies in different epithelial cells types with rare syncytial. Through immunohistochemistry, CDV antigen was observed in the tongue, trachea, lungs, liver, spleen, stomach, intestine and urinary bladder. Adenovirus antigen was identified in the nucleus of scattered hepatocytes. Polymerase chain reaction and sequencing demonstrated high similarity with CAdV-1 and wild-type strain of CDV close related to Brazilian viral lineages isolated from domestic dogs. Disease surveillance in wildlife animals is essential to assess possible conservation threats and consider the implementation of mitigation or control measures.

Diagnosis and successful treatment of Brazillian porcupine poxvirus infection in a free-ranging hairy dwarf porcupine (Coendu spinosus).

Brazilian porcupine poxvirus (BPoPV) is a new poxvirus recently described in porcupines (Coendou prehensilis) from Brazil. Herein, we described a free-ranging adult male Coendou (Sphiggurus) spinosus rescued after being found lethargic on the ground in a rural area. The animal presented crusty, edematous, and suppurative skin lesions on the face, tail, and perineum, and yellowish ocular secretion. The diagnosis was performed by histopathology, transmission electron microscopy (TEM), PCR, and sequencing. Microscopically, proliferative and necrotizing dermatitis, subacute, multifocal with ballooning degeneration, and eosinophilic intracytoplasmic viral inclusion bodies were observed. TEM confirmed large brick-shaped virions inside the keratinocyte cytoplasm, measuring about 200-280 × 120-180 nm. Partial fragment of intracellular mature virion membrane protein gene and putative metalloproteinase gene was successfully amplified and sequenced, and the strain herein denoted IAL/21 V-102 was classified as BPoPV, showing 99.4% of nucleotide identity to the reference strain UFU/USP001. Enrofloxacin 10% (10 mg/kg) was administered every 24 h through intramuscular injection for 10 days, dipyrone/metamizole (25 mg/kg) every 24 h orally (PO) for 3 days, 0.5 ml (mL) of thymomodulin every 24 h PO for 30 days, and each 48 h for another 15 days. The lesions were cleaned and debrided every 15 days. Seventy-five days after the beginning of the treatment, the cutaneous lesions regressed, the animal gained weight, and was clinically stable. After treatment, the skin biopsy showed only mild epidermal acanthosis, intra-cellular edema, and mild lymphoplasmacytic perivascular dermatitis. No viral particles were observed by TEM and no poxviral DNA was amplified by PCR. This study documents the first case of confirmed and treated BPoPV infection in a hairy dwarf porcupine. The implemented therapeutic plan eliminated the infection and improved the general state of the animal.

Somatic targeted mutation profiling of colorectal cancer precursor lesions.

BACKGROUND: Most colorectal cancers (CRC) arise from precursor lesions. This study aimed to characterize the mutation profile of colorectal cancer precursor lesions in a Brazilian population. METHODS: In total, 90 formalin-fixed paraffin-embedded colorectal precursor lesions, including 67 adenomas, 7 sessile serrated lesions, and 16 hyperplastic polyps, were analyzed by next-generation sequencing using a panel of 50 oncogenes and tumor suppressor genes. The genetic ancestry of the patients was estimated. RESULTS: Somatic driver mutations were identified in 66.7% of cases, including alterations in APC (32.2%), TP53 (20.0%), KRAS (18.9%), BRAF (13.3%) and EGFR (7.8%). Adenomas displayed a higher number of mutations, mainly in APC, compared to serrated polyps (73.1% vs. 47.8%, p = 0.026). Advanced adenomas had a significantly higher frequency of mutation in KRAS and a high overall mutation rate than early adenomas (92.9% vs. 59%, p = 0.006). A high degree of ancestry admixture was observed in the population studied, with a predominance of European components (mean of 73%) followed by African (mean of 11.3%). No association between genetic ancestry and type of lesions was found. The mutation profile of Brazilian colorectal precursor lesions exhibits alteration in APC, KRAS, TP53, and BRAF at different frequencies according to lesion type. CONCLUSIONS: These results bestow the knowledge of CRC's biologic history and support the potential of these biomarkers for precursor lesions detection in CRC screening of the Brazilian population.