Comparative analysis of diagnostic methods for the detection of Cryptococcus neoformans meningitis.

BACKGROUND: Cryptococcosis is a devastating opportunistic infection in immunocompromised individuals, primarily in people living with HIV/AIDS. This study evaluated a protocol for the early diagnosis of meningitis due to C. neoformans, utilizing established molecular techniques from serum and CSF samples. METHODS: The 18S and 5.8S (rDNA-ITS) sequence-specific nested PCR assays were compared with direct India ink staining and the latex agglutination test for detection of C. neoformans in serum and cerebrospinal fluid (CSF) from 49 Brazilian suspected meningitis patients. Results were validated with samples obtained from 10 patients negative for cryptococcosis and HIV, and by analysis of standard C. neoformans strains. PRINCIPAL FINDINGS: The 5.8S DNA-ITS PCR was more sensitive (89-100%) and specific (100%) than the 18S rDNA PCR and conventional tests (India ink staining and latex agglutination) for identification of C. neoformans. While the 18S PCR exhibited a sensitivity (72%) similar to that of the latex agglutination assay in serum samples, it was superior to the latex agglutination assay when testing CSF, with a sensitivity of 84%. However, the latex agglutination was superior to the 18SrDNA PCR in specificity in CSF (92%). The 5.8S DNA-ITS PCR yielded the highest levels of accuracy (96-100%) of any test for detection (serological and mycological) of C. neoformans in both serum and CSF. CONCLUSION: Use of the nested 5.8S PCR was superior to other techniques for the diagnosis of cryptococcosis. The possibility of using serum, a non-invasively collected material, in a targeted 5.8S PCR analysis to identify Cryptococcus spp. is recommended, especially in immunosuppressed patients. Our results indicate that nested 5.8S PCR can increase the diagnostic capability of cryptococcosis, and we suggest its use to monitor patients in the future.

Airborne microplastics and SARS-CoV-2 in total suspended particles in the area surrounding the largest medical centre in Latin America.

Microplastics (MPs) have been reported in the outdoor/indoor air of urban centres, raising health concerns due to the potential for human exposure. Since aerosols are considered one of the routes of Coronavirus disease 2019 (COVID-19) transmission and may bind to the surface of airborne MPs, we hypothesize that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) could be associated with the levels of MPs in the air. Our goal was to quantify the SARS-CoV-2 RNA and MPs present in the total suspended particles (TSP) collected in the area surrounding the largest medical centre in Latin America and to elucidate a possible association among weather variables, MPs, and SARS-CoV-2 in the air. TSP were sampled from three outdoor locations in the areas surrounding a medical centre. MPs were quantified and measured under a fluorescence microscope, and their polymeric composition was characterized by Fourier transform infrared (FT-IR) microspectroscopy coupled with attenuated total reflectance (ATR). The viral load of SARS-CoV-2 was quantified by an in-house real-time PCR assay. A generalized linear model (GzLM) was employed to evaluate the effect of the SARS-CoV-2 quantification on MPs and weather variables. TSP samples tested positive for SARS-CoV-2 in 22 out of 38 samples at the three sites. Polyester was the most frequent polymer (80%) found in the samples. The total amount of MPs was positively associated with the quantification of SARS-CoV-2 envelope genes and negatively associated with weather variables (temperature and relative humidity). Our findings show that SARS-CoV-2 aerosols may bind to TSP, such as MPs, and facilitate virus entry into the human body.

Diffuse alveolar damage patterns reflect the immunological and molecular heterogeneity in fatal COVID-19.

BACKGROUND: Severe COVID-19 lung disease exhibits a high degree of spatial and temporal heterogeneity, with different histological features coexisting within a single individual. It is important to capture the disease complexity to support patient management and treatment strategies. We provide spatially decoded analyses on the immunopathology of diffuse alveolar damage (DAD) patterns and factors that modulate immune and structural changes in fatal COVID-19. METHODS: We spatially quantified the immune and structural cells in exudative, intermediate, and advanced DAD through multiplex immunohistochemistry in autopsy lung tissue of 18 COVID-19 patients. Cytokine profiling, viral, bacteria, and fungi detection, and transcriptome analyses were performed. FINDINGS: Spatial DAD progression was associated with expansion of immune cells, macrophages, CD8+ T cells, fibroblasts, and (lymph)angiogenesis. Viral load correlated positively with exudative DAD and negatively with disease/hospital length. In all cases, enteric bacteria were isolated, and Candida parapsilosis in eight cases. Cytokines correlated mainly with macrophages and CD8+T cells. Pro-coagulation and acute repair were enriched pathways in exudative DAD whereas intermediate/advanced DAD had a molecular profile of elevated humoral and innate immune responses and extracellular matrix production. INTERPRETATION: Unraveling the spatial and molecular immunopathology of COVID-19 cases exposes the responses to SARS-CoV-2-induced exudative DAD and subsequent immune-modulatory and remodeling changes in proliferative/advanced DAD that occur side-by-side together with secondary infections in the lungs. These complex features have important implications for disease management and the development of novel treatments. FUNDING: CNPq, Bill and Melinda Gates Foundation, HC-Convida, FAPESP, Regeneron Pharmaceuticals, and the Swedish Heart & Lung Foundation.

A single-centre, retrospective study of the incidence of invasive fungal infections during 85 years of autopsy service in Brazil.

Autopsy continues to play an essential role in monitoring opportunistic fungal infections. However, few studies have analysed the historical trends of fungal infections in autopsies. Here, we analyse available data on fungal infections obtained from autopsy reports during 85 years of autopsies performed by the largest autopsy service in Brazil. All invasive fungal infections presented in autopsy reports between 1930 and 2015 were included. Of the 158,404 autopsy reports analysed, 1096 involved invasive fungal infections. In general, paracoccidioidomycosis (24%) was the most frequent infection, followed by candidiasis (18%), pneumocystosis (11.7%), cryptococcosis (11%), aspergillosis (11%) and histoplasmosis (3.8%). Paracoccidioidomycosis decreased after the 1950s, whereas opportunistic fungal infections increased steadily after the 1980s during the peak of the AIDS pandemic. The lung was the most frequently affected organ (73%). Disseminated infection was present in 64.5% of cases. In 26% of the 513 cases for which clinical charts were available for review, the diagnosis of opportunistic fungal infections was performed only at autopsy. Our unique 85-year history of autopsies showed a transition from endemic to opportunistic fungal infections in São Paulo, Brazil, reflecting increased urbanization, the appearance of novel diseases, such as AIDS in the 1980s, and advances in medical care over time.

Quantification of airborne SARS-CoV-2 genomic particles in different hospital settings.

We quantified the presence of SARS-CoV-2 RNA in the air of different hospital settings and the autopsy room of the largest medical centre in Sao Paulo, Brazil. Real-time reverse-transcription PCR was used to determine the presence of the envelope protein of SARS-CoV-2 and the nucleocapsid protein genes. The E-gene was detected in 5 out of 6 samples at the ICU-COVID-19 ward and in 5 out of 7 samples at the ward-COVID-19. Similarly, in the non-dedicated facilities, the E-gene was detected in 5 out of 6 samples collected in the ICU and 4 out of 7 samples in the ward. In the necropsy room, 6 out of 7 samples were positive for the E-gene. When both wards were compared, the non-COVID ward presented a significantly higher concentration of the E-gene than in the COVID-19 ward (p = 0.003). There was no significant difference in E-gene concentration between the ICU-COVID-19 and the ICU (p = 0.548). Likewise, there was no significant difference among E-gene concentrations found in the autopsy room versus the ICUs and wards (dedicated or not) (p = 0.245). Our results show the widespread presence of aerosol contamination in different hospital units.

Comparison of diagnostic methods to detect Histoplasma capsulatum in serum and blood samples from AIDS patients.

BACKGROUND: Although early and rapid detection of histoplasmosis is essential to prevent morbidity and mortality, few diagnostic tools are available in resource-limited areas, especially where it is endemic and HIV/AIDS is also epidemic. Thus, we compared conventional and molecular methods to detect Histoplasma capsulatum in sera and blood from HIV/AIDS patients. METHODOLOGY: We collected a total of 40 samples from control volunteers and patients suspected of histoplasmosis, some of whom were also infected with other pathogens. Samples were then analyzed by mycological, serological, and molecular methods, and stratified as histoplasmostic with (group I) or without AIDS (group II), uninfected (group III), and infected with HIV and other pathogens only (group IV). All patients were receiving treatment for histoplasmosis and other infections at the time of sample collection. RESULTS: Comparison of conventional methods with nested PCR using primers against H. capsulatum 18S rRNA (HC18S), 5.8S rRNA ITS (HC5.8S-ITS), and a 100 kDa protein (HC100) revealed that sensitivity against sera was highest for PCR with HC5.8S-ITS, followed by immunoblotting, double immunodiffusion, PCR with HC18S, and PCR with HC100. Specificity was equally high for double immunodiffusion, immunoblotting and PCR with HC100, followed for PCR with HC18S and HC5.8-ITS. Against blood, sensitivity was highest for PCR with HC5.8S-ITS, followed by PCR with HC18S, Giemsa staining, and PCR with HC100. Specificity was highest for Giemsa staining and PCR with HC100, followed by PCR with HC18S and HC5.8S-ITS. PCR was less efficient in patients with immunodeficiency due to HIV/AIDS and/or related diseases. CONCLUSION: Molecular techniques may detect histoplasmosis even in cases with negative serology and mycology, potentially enabling early diagnosis.

Analysis of the genetic polymorphism of Paracoccidioides brasiliensis and Paracoccidioides cerebriformis "Moore" by random amplified polymorphic DNA (RAPD) and 28S ribosomal DNA sequencing--Paracoccidioides cerebriformis revisited.

Our purpose was to compare the genetic polymorphism of six samples of P. brasiliensis (113, 339, BAT, T1F1, T3B6, T5LN1), with four samples of P. cerebriformis (735, 741, 750, 361) from the Mycological Laboratory of the Instituto de Medicina Tropical de São Paulo, using Random Amplified Polymorphic DNA Analysis (RAPD). RAPD profiles clearly segregated P. brasiliensis and P. cerebriformis isolates. However, the variation on band patterns among P. cerebriformis isolates was high. Sequencing of the 28S rDNA gene showed nucleotide conservancy among P. cerebriformis isolates, providing basis for taxonomical grouping, and disclosing high divergence to P. brasiliensis supporting that they are in fact two distinct species. Moreover, DNA sequence suggests that P. cerebriformis belongs in fact to the Aspergillus genus.

Importance of the association of molecular and immunological diagnosis in immunocompetent patient with Histoplasma capsulatum and Cryptoccocus neoformans infection: a case report.

This case reports an immunocompetent 29-year-old woman with suspected pneumonia, suggestive of fungal infection. Immunoblotting analysis reactivity against Histoplasma capsulatum and Paracoccidioides brasiliensis were observed. Nested-PCR in blood employing species-specific primers was positive for H. capsulatum and Cryptococcus neoformans. The evaluation of paucisymptomatic patients with positive results for H. capsulatum and C. neoformans could be relevant for the prevention as well as the possible evaluation of the reactivated quiescent foci. In conclusion, the associated methodology may have contributed to the monitoring endogenous reactivation of these diseases.

The use of nested Polymerase Chain Reaction (nested PCR) for the early diagnosis of Histoplasma capsulatum infection in serum and whole blood of HIV-positive patients.

The aim of the study was to detect the rDNA sequences and their regions in Histoplasma capsulatum, which could be considered species-specific and used as a molecular method for this diagnosis by the technique of nested polymerase chain reaction (nested PCR), employing specific sequences (primers) for H. capsulatum: 18S rDNA region (HC18), 100 kDa (HC100) and the sequence 5.8 S-ITS rDNA (HC5.8). The PCR sequences HC18, HC100 and HC5.8 resulted in a specificity of 100%. The molecular assays may increase the specificity, sensitivity and speed in the diagnosis of Histoplasmosis.

Superficial mycosis and the immune response elements.

Superficial mycoses are prevalent worldwide. They are often caused by dermatophytes and restricted to the stratum corneum. The host's immune response against infections caused by dermatophytes basically depends on the host's defense against metabolites of the fungi, virulence of the infecting strain or species and anatomical site of the infection. We will review some of the factors of the host's immune defense that influence the efficacy of the immune response. We will particularly review the role of pattern recognition receptors (PRRs), such as toll-like receptors or lectin receptors (DCSIGN and Dectin 2), which participate in the innate immune response, bringing specificity to the immune response and setting its pattern. The predominance of a cellular or humoral immune response determines the clinical manifestations and the prognosis of the infection, leading to healing or chronicity.

Paracoccidioides brasiliensis causing a rib lesion in an adult AIDS patient.

Paracoccidioidomycosis is a systemic mycosis with a geographic distribution that is limited to Central and South America; Brazil has the highest number of cases. Severe disseminated disease caused by paracoccidioidomycosis was observed in acquired immunodeficiency syndrome patients who live or have resided in endemic paracoccidioidomycosis areas. Here we describe a male patient admitted to a large public hospital with diffuse nodular infiltrates observed in chest radiographs and with erosion at the second rib near the sternum. Blood tests showed anti-human immunodeficiency virus antibodies, a human immunodeficiency virus viral load of 59,700 (4.8 log), and CD4 144/mm(3), with negative serology result for fungal infections. Aspirate of the rib lesion showed cells with a typical morphology of Paracoccidioides brasiliensis, aside from benign inflammatory cells. The histology of the rib biopsy showed typical granulomas and immunostained fungal cells. Although there was no growth in the Sabouraud cultures, Paracoccidioides brasiliensis gp43 and rDNA genes were detected in the aspirate by polymerase chain reaction. Therapy with amphotericin resulted in complete recovery. This type of bone lesion is rare and has been described primarily in the juvenile form of paracoccidioidomycosis; it must be included in the differential diagnosis of bone lesions in adult acquired immunodeficiency syndrome patients of endemic areas.

Importance of the association of molecular and immunological diagnosis in immunocompetent patient with Histoplasma capsulatum and Cryptoccocus neoformans infection: a case report

This case reports an immunocompetent 29-year-old woman with suspected pneumonia, suggestive of fungal infection. Immunoblotting analysis reactivity against Histoplasma capsulatum and Paracoccidioides brasiliensis were observed. Nested-PCR in blood employing species-specific primers was positive for H. capsulatum and Cryptococcus neoformans. The evaluation of paucisymptomatic patients with positive results for H. capsulatum and C. neoformans could be relevant for the prevention as well as the possible evaluation of the reactivated quiescent foci. In conclusion, the associated methodology may have contributed to the monitoring endogenous reactivation of these diseases.

Importance of the association of molecular and immunological diagnosis in immunocompetent patient with Histoplasma capsulatum and Cryptoccocus neoformans infection: a case report

This case reports an immunocompetent 29-year-old woman with suspected pneumonia, suggestive of fungal infection. Immunoblotting analysis reactivity againstHistoplasma capsulatum and Paracoccidioides brasiliensis were observed. Nested-PCR in blood employing species-specific primers was positive for H. capsulatum andCryptococcus neoformans. The evaluation of paucisymptomatic patients with positive results for H. capsulatum and C. neoformans could be relevant for the prevention as well as the possible evaluation of the reactivated quiescent foci. In conclusion, the associated methodology may have contributed to the monitoring endogenous reactivation of these diseases.(AU)

The use of nested Polymerase Chain Reaction (nested PCR) for the early diagnosis of Histoplasma capsulatum infection in serum and whole blood of HIV-positive patients* / Uso da Reação em Cadeia da Polimerase aninhada (PCR aninhada) para o diagnóstico precoce da infecção pelo Histoplasma capsulatum no soro e no sangue total de pacientes HIV positivos

The aim of the study was to detect the rDNA sequences and their regions in Histoplasma capsulatum, which could be considered species-specific and used as a molecular method for this diagnosis by the technique of nested polymerase chain reaction (nested PCR), employing specific sequences (primers) for H. capsulatum: 18S rDNA region (HC18), 100 kDa (HC100) and the sequence 5.8 S-ITS rDNA (HC5.8). The PCR sequences HC18, HC100 and HC5.8 resulted in a specificity of 100%. The molecular assays may increase the specificity, sensitivity and speed in the diagnosis of Histoplasmosis.

O objetivo do estudo consistiu em detectar seqüências no ADNr e as suas regiões no Histoplasma capsulatum, que pudessem ser consideradas espécie-específicas e usadas como método molecular para o diagnóstico pela técnica da reação em cadeia da polimerase aninhada ("nested PCR") com seqüências específicas ("primers") para H. capsulatum: regiões 18S ADNr (HC18), 100kDa (HC100) e a seqüência 5.8 S ADNr-ITS (HC5.8). A "nested PCR" com as seqüências HC18, HC100 e HC5.8 resultaram em 100% de especificidade. Os ensaios moleculares podem aumentar a especificidade, sensibilidade e rapidez na diagnose da Histoplasmose.

Evaluation of phenotypic and genotypic alterations induced by long periods of subculturing of Cryptococcus neoformans strains.

Cryptococcus neoformans is an encapsulated fungal organism that can cause disease in apparently immunocompetent, as well as immunocompromised, hosts. Since 1930, successive subculture has been used to preserve C. neoformans isolates in our Fungus Collection. In the 1970s, some of these Fungus Collection samples were selected to be subjected to a different methods of maintenance--that of lyophilized. Our objective was to analyze C. neoformans isolates in order to make a comparative evaluation between these two methods of preservation. The overall aim of this study was to qualify the preservation technique used in our mycology laboratory since the technique used might affect the survival, stability and purity of the primary isolates in culture. The samples were analyzed using classical mycology methods and using the randomly amplified polymorphic DNA technique In the analysis of phenotypes and genotypes, the typical characteristics of C. neoformans were found to differ in relation to the different methods of preservation employed. The aim of this study was to demonstrate the importance of selecting the appropriate method of preservation for fungus collections. This selection can affect the survival and purity of the cultures, and preserve the stability of their physiological, biochemical, and genetic characteristics.

Micoses superficiais e os elementos da resposta imune / Superficial mycosis and the immune response elements

As micoses superficiais são prevalentes em todo o mundo, geralmente ocasionadas por dermatófitos e restritas à camada córnea. A resposta imunológica do hospedeiro às infecções dos fungos dermatófitos depende basicamente das defesas do hospedeiro a metabólitos do fungo, da virulência da cepa ou da espécie infectante e da localização anatômica da infecção. Serão revistos alguns dos fatores da defesa imunológica do hospedeiro que influenciam na eficácia da resposta imune. Em especial, a participação dos receptores de padrão de reconhecimento (PRRs), tais como os receptores toll-like ou os da família lectina (DC-SIGN e dectin-2), que participam da resposta imune inata, conferindo-lhe especificidade e definindo o padrão da resposta imune como um todo. O predomínio celular ou humoral da resposta imune definirá o quadro clínico e o prognóstico da infecção, levando à cura ou cronicidade.

Superficial mycoses are prevalent worldwide. They are often caused by dermatophytes and restricted to the stratum corneum. The host's immune response against infections caused by dermatophytes basically depends on the host's defense against metabolites of the fungi, virulence of the infecting strain or species and anatomical site of the infection. We will review some of the factors of the host's immune defense that influence the efficacy of the immune response. We will particularly review the role of pattern recognition receptors (PRRs), such as toll-like receptors or lectin receptors (DCSIGN and Dectin 2), which participate in the innate immune response, bringing specificity to the immune response and setting its pattern. The predominance of a cellular or humoral immune response determines the clinical manifestations and the prognosis of the infection, leading to healing or chronicity.

Evaluation of phenotypic and genotypic alterations induced by long periods of subculturing of Cryptococcus neoformans strains

Cryptococcus neoformans is an encapsulated fungal organism that can cause disease in apparently immunocompetent, as well as immunocompromised, hosts. Since 1930, successive subculture has been used to preserve C. neoformans isolates in our Fungus Collection. In the 1970s, some of these Fungus Collection samples were selected to be subjected to a different methods of maintenance - that of lyophilized. Our objective was to analyze C. neoformans isolates in order to make a comparative evaluation between these two methods of preservation. The overall aim of this study was to qualify the preservation technique used in our mycology laboratory since the technique used might affect the survival, stability and purity of the primary isolates in culture. The samples were analyzed using classical mycology methods and using the randomly amplified polymorphic DNA technique In the analysis of phenotypes and genotypes, the typical characteristics of C. neoformans were found to differ in relation to the different methods of preservation employed. The aim of this study was to demonstrate the importance of selecting the appropriate method of preservation for fungus collections. This selection can affect the survival and purity of the cultures, and preserve the stability of their physiological, biochemical, and genetic characteristics.

Study of activity transcription factors C/EBPalpha in region - 53 to - 33 of promoter apolipoprotein B gene

Apolipoprotein B (ApoB) plays a major role in the regulation of cellular cholesterol homeostasis and pathogenesis of atherosclerosis. This protein acts as a ligand for the cellular recognition and catabolism of low density lipoprotein (LDL) by the LDL receptor. Previous studies have shown that the expression of apoB in hepatic cells is regulated by the interaction of factors binding to enhancer elements in intron 2 and three elements designated III, IV and V. These elements lie within regions respectively -86 to -62, -72 to -53 and -53 to -33 from the ApoB promoter. In this study, we have suggested that transcription factor C/EBPalpha, which binds to the -53 to -33 region of the apoB, interacts with the HNF-4 synergistic complex and C/EBPalpha factors within -86 to -53 and may contribute to increase transcription of the ApoB gene.

A apolipoproteina B (apoB) tem um importante papel na regulação na homeostasia celular, do colesterol e na patogênese da aterosclerose. Esta proteína age como ligante para o reconhecimento e catabolismo lipoproteínas de baixa densidade (LBD) através do receptor de LDL. Estudos anteriores mostraram que a expressão do gene da apolipoproteína B (APOB) em células hepáticas é regulada pela interação de fatores ligados ao elemento enhancer no intron 2, e em 3 elementos denominados de III, IV e V localizados nas regiões -86 a -62, -72 a -53 e -53 a -33 , respectivamente, do promotor do gene da APOB. Neste trabalho, nós sugerimos que o fator de transcrição C/EBPalfa ligado a região -53 a -33 da APOB interage com o complexo HNF-4 e C/EBPalfa localizado dentro da região -86 a -53 do APO B e contribui para aumentar a transcrição do gene APOB.

Analysis of the genetic polymorphism of Paracoccidioides brasiliensis and Paracoccidioides cerebriformis "Moore" by random amplified polymorphic DNA (RAPD) and 28S ribosomal DNA sequencing: Paracoccidioides cerebriformis revisited

Nosso propósito foi comparar o polimorfismo genético de seis amostras de P. brasiliensis (113, 339, BAT, T1F1, T3B6, T5LN1), com quatro amostras de P. cerebriformis (735, 741, 750, 361) do laboratório de micologia do Instituto de Medicina Tropical de São Paulo, utilizando a técnica de Amplificação Aleatória do Polimorfismo de DNA (RAPD). O perfil de bandas do RAPD diferenciou claramente os isolados de P. brasiliensis de P. cerebriformis. Entretanto, ocorreu uma variação significativa no padrão de bandas das amostras de P. cerebriformis. O sequenciamento do gene ribossomal 28S revelou seqüências de nucleotídeos bastante conservadas entre os isolados de P. cerebriformis, fornecendo subsídio para o agrupamento taxonômico destas amostras, diferenciando estas de P. brasiliensis e mostrando que de fato são espécies distintas. A seqüência de DNA sugere que P. cerebriformis pertence ao gênero Aspergillus.