ABSTRACT
Aspergillus species can colonize and infect immunocompetent and immunocompromised hosts. Conventional fungal identification depends on microscopic analysis and microorganism medium growth. Other diagnostic methods, non-growth dependent, to invasive fungal infections, are the biomarkers that detect circulating polysaccharides, for example, 1-3-ß-d-Glucan and galactomannan. Both are polysaccharides present on the external layer of fungi cell wall and can be detected in clinical samples during the growth of the fungus in the patient. This study aimed to compare the galactomannan detection of Lateral Flow Assay and Enzyme Immunoassay methods in Bronchoalveolar Lavage Fluid. The galactomannan antigen in Bronchoalveolar Lavage Fluid was measured using Enzyme Immunoassay according to the manufacturer's instructions (PLATELIA ASPERGILLUS™ BioRad) and, using a Lateral Flow Assay according to the manufacturer's instructions (Galactomannan LFA IMMY©). The 71 samples were Bronchoalveolar Lavage Fluid of patients hospitalized at Unicamp Clinical Hospital between 2019 and 2021; of these samples 12/71 (16.9 %) resulted in positive Galactomannan-Lateral Flow Assay. In contrast, Galactomannan-Enzyme Immunoassay resulted as positive in 9/71 (12.6 %) samples, a difference that showed not significant statistically (p-value = 0.36) Comparing both assays' results identified 8 divergences between them, about 11 % of the total sample. The Sensitivity (73.3 %), Specificity (92.35 %), Positive Predictive Value (62.85 %) and Negative Predictive Value (95.15 %) of Lateral Flow Assay were calculated using the Galactomannan Enzyme Immunoassay as standard. The Lateral Flow Assay demonstrated good results when compared with the Enzyme Immunoassay.
Subject(s)
Aspergillus , Bronchoalveolar Lavage Fluid , Galactose , Immunoenzyme Techniques , Mannans , Sensitivity and Specificity , Mannans/analysis , Galactose/analogs & derivatives , Humans , Bronchoalveolar Lavage Fluid/microbiology , Bronchoalveolar Lavage Fluid/chemistry , Aspergillus/immunology , Aspergillus/isolation & purification , Immunoenzyme Techniques/methods , Aspergillosis/diagnosis , Aspergillosis/microbiology , Biomarkers/analysis , Antigens, Fungal/analysis , Reproducibility of ResultsABSTRACT
BACKGROUND: Aspergillus fumigatus is an important concern for immunocompromised individuals, often resulting in severe infections. With the emergence of resistance to azoles, which has been the therapeutic choice for Aspergillus infections, monitoring the resistance of these microorganisms becomes important, including the search for mutations in the cyp51A gene, which is the gene responsible for the mechanism of action of azoles. We conducted a retrospective analysis covering 478 A. fumigatus isolates. METHODS: This comprehensive dataset comprised 415 clinical isolates and 63 isolates from hospital environmental sources. For clinical isolates, they were evaluated in two different periods, from 1998 to 2004 and 2014 to 2021; for environmental strains, one strain was isolated in 1998, and 62 isolates were evaluated in 2015. Our primary objectives were to assess the epidemiological antifungal susceptibility profile; trace the evolution of resistance to azoles, Amphotericin B (AMB), and echinocandins; and monitor cyp51A mutations in resistant strains. We utilized the broth microdilution assay for susceptibility testing, coupled with cyp51A gene sequencing and microsatellite genotyping to evaluate genetic variability among resistant strains. RESULTS: Our findings reveal a progressive increase in Minimum Inhibitory Concentrations (MICs) for azoles and AMB over time. Notably, a discernible trend in cyp51A gene mutations emerged in clinical isolates starting in 2014. Moreover, our study marks a significant discovery as we detected, for the first time, an A. fumigatus isolate carrying the recently identified TR46/F495I mutation within a sample obtained from a hospital environment. The observed cyp51A mutations underscore the ongoing necessity for surveillance, particularly as MICs for various antifungal classes continue to rise. CONCLUSIONS: By conducting resistance surveillance within our institution's culture collection, we successfully identified a novel TR46/F495I mutation in an isolate retrieved from the hospital environment which had been preserved since 1998. Moreover, clinical isolates were found to exhibit TR34/L98H/S297T/F495I mutations. In addition, we observed an increase in MIC patterns for Amphotericin B and azoles, signaling a change in the resistance pattern, emphasizing the urgent need for the development of new antifungal drugs. Our study highlights the importance of continued monitoring and research in understanding the evolving challenges in managing A. fumigatus infections.
ABSTRACT
Candidiasis is the most common fungal infection affecting hospitalized patients, especially immunocompromised and critical patients. Limitations regarding the assertive diagnosis of both Candidemia and Candidiasis not only impairs the introduction of effective treatments but also lays a heavy financial burden over the health system. Furthermore, it is still challenging to ascertain whether diagnostic methods are accurate and whether treatment is effective for patients with Candidemia. These constraints come from the uncertainty of the pathophysiological mechanism by which the pathogen establishes the opportunistic infection. Additionally, it is the reason why some patients present positive blood culture results, and others do not, and why it is very difficult during clinical routines to prove Candidemia or invasive candidiasis. Taking into account the current situation, this contribution proposes two markers that may help to understand the mechanisms of infection by the pathogen: Leukotriene F4 and 5,6-dihydroxy-eicosatetraenoic. These two lipids putatively modulate the host's immune response, and the initial data presented in this contribution suggest that these lipids allow the opportunistic infection to be installed. The study was carried out using an omics-based platform using direct-infusion high-resolution mass spectrometry and allied with bioinformatics tools to provide accurate and reliable results for biomarker candidates screening.
Subject(s)
Candidemia , Candidiasis , Opportunistic Infections , Antifungal Agents/therapeutic use , Candida , Candidemia/diagnosis , Candidiasis/diagnosis , Candidiasis/drug therapy , Humans , LeukotrienesABSTRACT
From 2006 to 2013, an increasing incidence of fusariosis was observed in the hematologic patients of our University Hospital. We suspected of an environmental source, and the indoor hospital air was investigated as a potential source of the fungemia. Air samplings were performed in the hematology and bone marrow transplant (BMT) wards using an air sampler with pre-defined air volumes. To study the molecular relationship among environmental and clinical isolates, 18 Fusarium spp. recovered from blood cultures were included in the study. DNA sequencing of a partial portion of TEF1α gene was performed for molecular identification. Molecular typing was carried out by multi-locus sequence typing (MLST) using a four-gene scheme: TEF1α, rDNA, RPB1 and RPB2. One hundred four isolates were recovered from the air of the hematology (n = 76) and the BMT (n = 28) wards. Fusarium isolates from the air were from five species complexes: Fusarium fujikuroi (FFSC, n = 56), Fusarium incarnatum-equiseti (FIESC, n = 24), Fusarium solani (FSSC, n = 13), Fusarium chlamydosporum (FCSC, n = 10), and Fusarium oxysporum (FOSC, n = 1). Fifteen Fusarium isolates recovered from blood belonged to FSSC, and three to FFSC. MLST identified the same sequence type (ST) in clinical and environmental isolates. ST1 was found in 5 isolates from blood and in 7 from the air, both identified as FSSC (Fusarium petroliphilum). STn1 was found in one isolate from blood and in one from the air, both identified as FFSC (Fusarium napiforme). F. napiforme was isolated from the air of the hospital room of the patient with fungemia due to F. napiforme. These findings suggested a possible clonal origin of the Fusarium spp. recovered from air and bloodcultures. In conclusion, our study found a diversity of Fusarium species in the air of our hospital, and a possible role of the air as source of systemic fusariosis in our immunocompromised patients.
Subject(s)
Fusariosis/diagnosis , Fusarium/genetics , Hematologic Neoplasms/pathology , Bone Marrow Transplantation , DNA, Fungal/chemistry , DNA, Fungal/genetics , DNA, Fungal/metabolism , Fungal Proteins/chemistry , Fungal Proteins/genetics , Fungal Proteins/metabolism , Fusariosis/complications , Fusariosis/microbiology , Fusarium/classification , Fusarium/isolation & purification , Hematologic Neoplasms/complications , Hematologic Neoplasms/therapy , Humans , Immunocompromised Host , Multilocus Sequence Typing , Peptide Elongation Factor 1/chemistry , Peptide Elongation Factor 1/genetics , Peptide Elongation Factor 1/metabolism , PhylogenyABSTRACT
A DNA microarray platform, based on the nucleotide sequences of the internal transcribed spacer regions (ITS1 and ITS2) of the rRNA gene, was developed to identify 32 fungal pathogens at the species level. The probe sequences were spotted onto polycarbonate slides with a mini-microarray printer, and after the hybridization, the results were visible with the naked eye. The performance of the microarray platform was evaluated against the commercial automated systems (Vitek 2 and BD Phoenix systems) and DNA sequencing (gold standard). A total of 461 blood culture bottles were tested: 127 positive for fungi, 302 positive for bacteria, and 32 that were negative. Once the microorganisms were identified by automated systems, fungal DNA was extracted directly from the blood culture bottles. The DNA products were tested using the microarray platform, and DNA sequencing was performed. The results of the microarray and DNA sequencing were concordant in 96.7% of cases, and the results from the automated systems and DNA sequencing were concordant in 98.4%. Of all the nucleotide sequences contained in the microarray platform, the microarray failed to identify four fungal isolates (one Candida parapsilosis, two Candida tropicalis, and one Cryptococcus neoformans). Of note, the microarray detected Candida krusei DNA in two blood cultures from the same patient, whereas the automated system was only positive for Enterococcus faecium Our microarray system provided reliable and fast fungal identification compared to that from DNA sequencing and the automated systems. The simplicity of reading the results by the naked eye made this DNA platform a suitable method for fungal molecular diagnosis.
Subject(s)
Fungi/classification , Fungi/genetics , Molecular Diagnostic Techniques/methods , Mycoses/diagnosis , Oligonucleotide Array Sequence Analysis , Blood Culture , DNA, Fungal/genetics , DNA, Ribosomal Spacer/genetics , Fungi/isolation & purification , Humans , Molecular Diagnostic Techniques/instrumentation , Molecular Diagnostic Techniques/standards , Mycoses/microbiology , Oligonucleotide Array Sequence Analysis/instrumentationABSTRACT
A Plasmodium falciparum circumsporozoite protein (CSP)-based recombinant fusion vaccine is the first malaria vaccine to reach phase III clinical trials. Resistance to infection correlated with the production of antibodies to the immunodominant central repeat region of the CSP. In contrast to P. falciparum, vaccine development against the CSP of Plasmodium vivax malaria is far behind. Based on this gap in our knowledge, we generated a recombinant chimeric protein containing the immunodominant central repeat regions of the P. vivax CSP fused to Salmonella enterica serovar Typhimurium-derived flagellin (FliC) to activate the innate immune system. The recombinant proteins that were generated contained repeat regions derived from each of the 3 different allelic variants of the P. vivax CSP or a fusion of regions derived from each of the 3 allelic forms. Mice were subcutaneously immunized with the fusion proteins alone or in combination with the Toll-like receptor 3 (TLR-3) agonist poly(I·C), and the anti-CSP serum IgG response was measured. Immunization with a mixture of the 3 recombinant proteins, each containing immunodominant epitopes derived from a single allelic variant, rather than a single recombinant protein carrying a fusion of regions derived from each of 3 allelic forms elicited a stronger immune response. This response was independent of TLR-4 but required TLR-5/MyD88 activation. Antibody titers significantly increased when poly(I·C) was used as an adjuvant with a mixture of the 3 recombinant proteins. These recombinant fusion proteins are novel candidates for the development of an effective malaria vaccine against P. vivax.
Subject(s)
Adjuvants, Immunologic/pharmacology , Flagellin/pharmacology , Malaria Vaccines/administration & dosage , Malaria Vaccines/immunology , Plasmodium vivax/immunology , Protozoan Proteins/immunology , Salmonella typhimurium/immunology , Adjuvants, Immunologic/administration & dosage , Adjuvants, Immunologic/genetics , Animals , Antibodies, Protozoan/blood , Epitopes/genetics , Epitopes/immunology , Female , Flagellin/genetics , Injections, Subcutaneous , Malaria Vaccines/genetics , Mice , Plasmodium vivax/genetics , Poly I-C/administration & dosage , Poly I-C/pharmacology , Protozoan Proteins/genetics , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/immunology , Salmonella typhimurium/genetics , Vaccines, Synthetic/administration & dosage , Vaccines, Synthetic/genetics , Vaccines, Synthetic/immunologyABSTRACT
A esquistossomose é uma doença importante em saúde pública. Dos genes selecionados como diferencialmente expressos em esquistossômulos a partir do transcriptoma do S. mansoni, 56% foram confirmados por RT-PCR em tempo real. Entre eles, a proteína Ly6.5, está presente no tegumento de esquistossômulos e vermes adultos por âncoras de GPI. Não foi detectada a função de inibir o sistema complemento, mas pode estar envolvido na manutenção do tegumento. O gene SmVal7 revelou transcritos nas glândulas esofágicas de vermes adultos por hibridização in situ, enquanto a localização da proteína não está definida. Anexina está associada ao tegumento de esquistossômulos e vermes adultos, de maneira dependente de cálcio. A supressão do gene por RNAi não resultou em alteração fenotípica significativa em esquistossômulos in vitro. Foi observada atividade parcial de inibição de coagulação e potencial atividade de endocitose de anticorpos ligados à superfície. A imunização com rLy6.5, rSmVal7, rAneI-II ou rAneII-III não levou a redução da carga parasitária após desafio.
Schistosomiasis is an important disease in public health. Genes selected from the S. mansoni transcriptome, 56% of them were confirmed as differentially expressed in schistosomula by real time RT-PCR. Among them, the protein Ly6.5 is present in the tegument of schistosomula and adult worms by GPI anchors. The function of inhibiting the complement system was not detected, but it may be involved in maintenance of the tegument. The gene SmVal7 revealed transcripts in the esophageal glands of adult worms by in situ hybridization, while the localization of the protein is not defined. Annexin is associated with the membranes of the schistosomula and adult worms tegument in a calcium-dependent manner. The suppression of the gene by RNAi did not resulted in a significant phenotypic change in schistosomula in vitro. Parcial inhibition of the coagulation activity and potential function of endocytosis of membrane-bound antibodies were observed. Immunization with the rLy6.5, rSmVal7, rAneI or rAneII-II-III did not show reduction in worm burden recovery after challenge.
Subject(s)
Schistosomiasis mansoni/immunology , Gene Expression/genetics , Gene Expression/immunology , Polymerase Chain Reaction/methods , Suppression, Genetic/genetics , Suppression, Genetic/immunologyABSTRACT
We here describe the cloning and characterization of the Schistosoma mansoni Annexin 2, previously identified in the tegument by proteomic studies, and as an up-regulated gene in schistosomulum stage by microarray data. In silico analysis predicts a conserved core containing four repeat domains of Annexin (ANX) and a variable N-terminal region similar to that described for mammalian isoforms. Real-time RT-PCR and Western blot analysis determined that S. mansoni Annexin 2 is significantly up-regulated in the transition from free-living cercaria to schistosomulum and adult worm parasitic stages. Immunolocalization experiments and tegument membrane preparations confirmed Annexin 2 as a protein mainly localized in the tegument of schistosomula and adult worms. Furthermore, it binds to the tegument surface membranes in a calcium-dependent manner. These results suggest that S. mansoni Annexin 2 is closely associated to the tegument arrangement, being a potential target for immune intervention.
Subject(s)
Annexin A2/genetics , Schistosoma mansoni/chemistry , Amino Acid Sequence , Animals , Annexin A2/analysis , Annexin A2/chemistry , Annexin A2/immunology , Antibodies, Helminth/biosynthesis , Blotting, Western , Cloning, Molecular , Cricetinae , DNA, Complementary/chemistry , DNA, Helminth/chemistry , Electrophoresis, Polyacrylamide Gel , Exons , Female , Fluorescent Antibody Technique, Indirect , Gene Expression , Life Cycle Stages/genetics , Male , Mice , Microscopy, Confocal/methods , Molecular Sequence Data , Reverse Transcriptase Polymerase Chain Reaction , Schistosoma mansoni/genetics , Schistosoma mansoni/growth & development , Sequence AlignmentABSTRACT
We here describe the cloning and characterization of the Schistosoma mansoni Annexin 2, previously identifiedin the tegument by proteomic studies, and as an up-regulated gene in schistosomulum stage by microarray data. In silico analysis predicts a conserved core containing four repeat domains of Annexin (ANX) and a variable N-terminal region similar to that described for mammalian isoforms. Real-time RT-PCR and Western blot analysis determined that S. mansoni Annexin 2 is significantly up-regulatedin the transition from free-living cercaria to schistosomulum and adult worm parasitic stages. Immunolocalization experiments and tegument membrane preparations confirmed Annexin 2 as a protein mainlylocalized in the tegument of schistosomula and adult worms. Furthermore, it binds to the tegument surface membranes in a calcium-dependent manner. These results suggest that S. mansoni Annexin 2 is closely associated to the tegument arrangement, being a potential target for immune intervention.
Subject(s)
Humans , Schistosoma mansoni/immunology , Vaccines , Annexins , Fluorescent Antibody Technique/methods , Microscopy, Confocal/methods , Integumentary SystemABSTRACT
The promoting activity of the herbicide Diuron was evaluated in a medium-term rat liver carcinogenesis bioassay that uses as endpoint immunohistochemically identified glutathione S-transferase positive (GST-P+) foci. Male Wistar rats were allocated to the following groups: G1 to G6 were initiated for liver carcinogenesis by a single dose of diethylnitrosamine (DEN, 200 mg/kg) while groups G7 and G8 received only 0.9% NaCl (DEN vehicle). From the 2nd week animals were fed a basal diet (G1 and G7) or a diet added with Diuron at 125, 500, 1250, 2500 and 2500 ppm (G2 to G5 and G8, respectively) or 200 ppm Hexaclorobenzene (HCB; G6). The animals were submitted to 70% partial hepatectomy at the 3rd week and sacrificed at the 8th week. The herbicide did not alter ALT or creatinine serum levels. No conspicuous GST-P+ foci development was registered in non-initiated rats fed Diuron at 2500 ppm. While DEN-initiated animals fed Diuron at 1250 or 2500 ppm developed mild centrilobular hypertrophy, DEN-initiated HCB-fed animals showed severe liver centrilobular hypertrophy and significant GST-P+ foci development. These findings indicate that the medium-term assay adopted in this study does not reveal any liver carcinogenesis initiating or promoting potential of Diuron in the rat.