scholarly journals Detection of Paracoccidioides brasiliensis in Tissue Samples by a Nested PCR Assay

2000 ◽  
Vol 38 (8) ◽  
pp. 2940-2942 ◽  
Author(s):  
Ralf Bialek ◽  
Aida Ibricevic ◽  
Christian Aepinus ◽  
Laura K. Najvar ◽  
Annette W. Fothergill ◽  
...  
Keyword(s):  
Nested Pcr ◽  
Histoplasma Capsulatum ◽  
Paracoccidioides Brasiliensis ◽  
High Sensitivity ◽  
Dimorphic Fungus ◽  
Pcr Assay ◽  
Tissue Samples ◽  
Negative Results ◽  
Pcr Product ◽  
Atcc Strain

A nested PCR assay for the detection of Paracoccidioides brasiliensis DNA was evaluated, using a sequence of the immunogenic gp43 gene as a target. This gene encodes an outer membrane protein unique to this dimorphic fungus. DNA from six clinical isolates and the ATCC strain 60885 of P. brasiliensis, as well as DNA from closely related fungi, was examined to determine detection limits and cross-reactions. PCR was done on DNA extracts of lung homogenates from 23 experimentallyP. brasiliensis-infected and two uninfected BALB/c mice and from 20 Histoplasma capsulatum-infected ICR mice. The results were compared to quantitative cultures. A detection limit of 0.5 fg of specific DNA was determined using cloned plasmid DNA. In all seven P. brasiliensis isolates, the expected 196-bp nested PCR product was found. Their sequences were 100% identical to thegp43 gene sequence in GenBank. DNA extracts of all other, related fungi were negative. The PCR assay was positive in 21 out of 23 culture-positive lung homogenates with concentrations of 1 × 103 to 1.3 × 107 CFU of P. brasiliensis per g of lung. Uninfected BALB/c mice and H. capsulatum-infected mice samples gave negative results. The high sensitivity and specificity of this new nested PCR assay for the detection of P. brasiliensis in tissue samples were demonstrated. The assay may be useful for diagnosis in human tissue samples.

scholarly journals A semi-nested PCR assay for molecular detection of Paracoccidioides brasiliensis in tissue samples

2010 ◽  
Vol 43 (6) ◽  
pp. 728-730 ◽  
Author(s):  
Andrea Cristine Koishi ◽  
Débora Fonseca Vituri ◽  
Pedro Sebastião Raimundo Dionízio Filho ◽  
Alexandre Augusto Sasaki ◽  
Maria Sueli Soares Felipe ◽  
...  
Keyword(s):  
Nested Pcr ◽  
Molecular Detection ◽  
Paracoccidioides Brasiliensis ◽  
Systemic Infection ◽  
Oral Lesions ◽  
Pcr Assay ◽  
Tissue Samples

INTRODUCTION: Paracoccidioidomycosis is a systemic infection caused by Paracoccidioides brasiliensis. METHODS: In this study, a semi-nested PCR for paracoccidioidomycosis diagnosis was developed. The primers ITS1 and ITS4 were used in the first reaction, while the primers MJ03 and ITS1 primer were used in the second reaction. The semi-nested PCR was used to investigate biopsies of five patients with oral lesions that resembled paracoccidioidomycosis. RESULTS: The semi-nested PCR was positive for four samples and negative for a sample from a patient later diagnosed with leishmaniasis. CONCLUSIONS: The new semi-nested PCR describe is useful for paracoccidioidomycosis diagnosis.

scholarly journals Validation and Clinical Application of a Molecular Method for Identification of Histoplasma capsulatum in Human Specimens in Colombia, South America

2009 ◽  
Vol 17 (1) ◽  
pp. 62-67 ◽  
Author(s):  
Cesar Muñoz ◽  
Beatriz L. Gómez ◽  
Angela Tobón ◽  
Karen Arango ◽  
Angela Restrepo ◽  
...  
Keyword(s):  
Nested Pcr ◽  
Predictive Value ◽  
Histoplasma Capsulatum ◽  
Clinical Samples ◽  
Healthy Individuals ◽  
Molecular Method ◽  
Pcr Assay ◽  
Molecular Assays ◽  
Panfungal Pcr ◽  
Negative Controls

ABSTRACT The conventional means of diagnosis of histoplasmosis presents difficulties because of the delay to the time that the diagnosis is made, indicating the need for the implementation of molecular assays. We evaluated 146 clinical samples from 135 patients suspected of having histoplasmosis using a previously reported nested PCR assay for the H istoplasma capsulatum-specific 100-kDa protein (the Hc100 PCR). In order to determine the specificity of this molecular test, we also used samples from healthy individuals (n = 20), patients suspected of having respiratory disease with negative fungal cultures (n = 29), and patients with other proven infections (n = 60). Additionally, a sizable collection of DNA from cultures of H. capsulatum and other medically relevant pathogens was studied. A panfungal PCR assay that amplified the internal transcribed spacer 2 region was also used to identify all fungal DNAs. All PCR-amplified products were sequenced. Of the 146 clinical samples, 67 (45.9%) were positive by culture and PCR, while 9 samples negative by culture were positive by PCR. All the sequences corresponding to the 76 amplified products presented ≥98% identity with H. capsulatum. The Hc100 PCR exhibited a sensitivity of 100% and specificities of 92.4% and 95.2% when the results were compared to those for the negative controls and samples from other proven clinical entities, respectively; the positive predictive value was 83% and the negative predictive value was 100%; the positive and negative likelihood rates were 25 and 0, respectively. These results suggest that the Hc100 nested PCR assay for the detection of H. capsulatum DNA is a useful test in areas where mycosis caused by this organism is endemic.

scholarly journals Small Subunit Ribosomal DNA Sequence ShowsParacoccidioides brasiliensis Closely Related toBlastomyces dermatitidis

2000 ◽  
Vol 38 (9) ◽  
pp. 3190-3193 ◽  
Author(s):  
Ralf Bialek ◽  
Aida Ibricevic ◽  
Annette Fothergill ◽  
Dominik Begerow
Keyword(s):  
Histoplasma Capsulatum ◽  
Paracoccidioides Brasiliensis ◽  
Large Subunit ◽  
Close Relation ◽  
Small Subunit ◽  
Molecular Diagnostic ◽  
Diagnostic Tools ◽  
Rrna Gene ◽  
Dimorphic Fungus ◽  
The Family

The similarities of paracoccidioidomycosis and blastomycosis are highly suggestive of a close relation of the two etiological agents. Whereas the agent of the first disease is exclusively endemic in Latin America, the agent of the latter one is endemic in North America and Africa. In symptomatic travelers visiting both areas of endemicity, differentiation of the diseases might be impossible, even though therapy and prognosis for these two diseases differ significantly. In order to identify differences in the 18S rRNA gene (rDNA) for use as molecular diagnostic tools, we sequenced this gene from five isolates of Paracoccidioides brasiliensis and compared them to known sequences of other fungi. Neighbor-joining, maximum parsimony, and maximum likelihood analyses and, finally, the Kishino-Hasegawa test revealed that P. brasiliensis, Blastomyces dermatitidis, and Emmonsia parva are more closely related than Histoplasma capsulatum and B. dermatitidis, whose teleomorphic forms belong to one genus,Ajellomyces. In accordance with the work of other investigators who have used internal transcribed spacer and large subunit rDNA sequences, our small subunit rDNA data show that the dimorphic fungus P. brasiliensis must be grouped within the order Onygenales and is closely related to members of the family Onygenaceae. There are hints in the molecular phylogenetic analysis that the family Onygenaceae might be further divided into two families. The subgroup that includes P. brasiliensis comprises all zoopathogenic species. The differences in the 18S rDNAs appear to be too small to allow species identification of the members of the family Onygenaceae pathogenic for humans by use of target sequences within this gene.

scholarly journals Capacity of Histoplasma capsulatum to Survive the Composting Process

2019 ◽  
Vol 2019 ◽  
pp. 1-9
Author(s):  
Luisa Fernanda Gómez Londoño ◽  
Laura Carolina Pérez León ◽  
Juan Guillermo McEwen Ochoa ◽  
Alejandra Zuluaga Rodriguez ◽  
Carlos Alberto Peláez Jaramillo ◽  
...  
Keyword(s):  
Nested Pcr ◽  
Phosphorus Content ◽  
Histoplasma Capsulatum ◽  
Organic Fertilizer ◽  
Raw Material ◽  
Chicken Manure ◽  
High Nitrogen ◽  
Dimorphic Fungus ◽  
Nitrogen And Phosphorus ◽  
Composting Process

Histoplasma capsulatum (H. capsulatum) is a thermal-dimorphic fungus, the causal agent of histoplasmosis. Its presence in the environment is related with chicken manure due to their high nitrogen and phosphorus content. In Colombia, chicken manure is the most used raw material in the composting process; however, there is no information about the capacity of H. capsulatum to survive and remain viable in a composted organic fertilizer. To address this question, this study shows three assays based on microbiological culture and the Hc100 nested PCR. First, a composting reactor system was designed to transform organic material under laboratory conditions, and the raw material was inoculated with the fungus. From these reactors, the fungus was not isolated, but its DNA was detected. In the second assay, samples from factories where the DNA of the fungus was previously detected by PCR were analyzed. In the raw material samples, 3 colonies of H. capsulatum were isolated and its DNA was detected. However, after the composting process, neither the fungus was recovered by culture nor DNA was detected. In the third assay, sterilized and nonsterilized organic composted samples were inoculated with H. capsulatum and then evaluated monthly during a year. In both types of samples, the fungus DNA was detected by Hc100 nested PCR during the whole year, but the fungus only grew from sterile samples during the first two months evaluated. In general, the results of the assays showed that H. capsulatum is not able to survive a well-done composting process.

scholarly journals The Pathogenic Fungus Paracoccidioides brasiliensis Exports Extracellular Vesicles Containing Highly Immunogenic α-Galactosyl Epitopes

2011 ◽  
Vol 10 (3) ◽  
pp. 343-351 ◽  
Author(s):  
Milene C. Vallejo ◽  
Alisson L. Matsuo ◽  
Luciane Ganiko ◽  
Lia C. Soares Medeiros ◽  
Kildare Miranda ◽  
...  
Keyword(s):  
Extracellular Vesicles ◽  
Fungal Pathogens ◽  
Histoplasma Capsulatum ◽  
Paracoccidioides Brasiliensis ◽  
Pathogenic Fungus ◽  
Yeast Cells ◽  
Enzyme Linked Immunosorbent Assay ◽  
Dimorphic Fungus ◽  
Content Type

ABSTRACTExosome-like vesicles containing virulence factors, enzymes, and antigens have recently been characterized in fungal pathogens, such asCryptococcus neoformansandHistoplasma capsulatum. Here, we describe extracellular vesicles carrying highly immunogenic α-linked galactopyranosyl (α-Gal) epitopes inParacoccidioides brasiliensis. P. brasiliensisis a dimorphic fungus that causes human paracoccidioidomycosis (PCM). For vesicle preparations, cell-free supernatant fluids from yeast cells cultivated in Ham's defined medium-glucose were concentrated in an Amicon ultrafiltration system and ultracentrifuged at 100,000 ×g. P. brasiliensisantigens were present in preparations from phylogenetically distinct isolates Pb18 and Pb3, as observed in immunoblots revealed with sera from PCM patients. In an enzyme-linked immunosorbent assay (ELISA), vesicle components containing α-Gal epitopes reacted strongly with anti-α-Gal antibodies isolated from both Chagas' disease and PCM patients, withMarasmius oreadesagglutinin (MOA) (a lectin that recognizes terminal α-Gal), but only faintly with natural anti-α-Gal. Reactivity was inhibited after treatment with α-galactosidase. Vesicle preparations analyzed by electron microscopy showed vesicular structures of 20 to 200 nm that were labeled both on the surface and in the lumen with MOA. InP. brasiliensiscells, components carrying α-Gal epitopes were found distributed on the cell wall, following a punctuated confocal pattern, and inside large intracellular vacuoles. Lipid-free vesicle fractions reacted with anti-α-Gal in ELISA only when not digested with α-galactosidase, while reactivity with glycoproteins was reduced after β-elimination, which is indicative of partial O-linked chain localization. Our findings open new areas to explore in terms of host-parasite relationships in PCM and the role playedin vivoby vesicle components and α-galactosyl epitopes.

scholarly journals Detection of Aspergillus DNA by a nested PCR assay is able to improve the diagnosis of invasive aspergillosis in paediatric patients

2009 ◽  
Vol 58 (10) ◽  
pp. 1291-1297 ◽  
Author(s):  
Margit Hummel ◽  
Birgit Spiess ◽  
Julia Roder ◽  
Gregor von Komorowski ◽  
Matthias Dürken ◽  
...  
Keyword(s):  
Fungal Infection ◽  
Invasive Aspergillosis ◽  
Nested Pcr ◽  
Fungal Infections ◽  
High Sensitivity ◽  
Clinical Findings ◽  
Clinical Samples ◽  
Pcr Assay ◽  
Imaging Results ◽  
Adolescent Patients

Fungal infections are a leading cause of morbidity and mortality in severely immunocompromised patients and have been increasing in incidence in recent years. Invasive aspergillosis (IA) is the most common filamentous fungal infection and is, in adults as well as in children, difficult to diagnose. Several PCR assays to detect Aspergillus DNA have been established, but so far, studies on molecular tools for the diagnosis of IA in children are few. We evaluated the results of a nested PCR assay to detect Aspergillus DNA in clinical samples from paediatric and adolescent patients with suspected IA. Blood and non-blood samples from immunocompromised paediatric and adolescent patients with suspected invasive fungal infection were sent for processing Aspergillus PCR to our laboratory. PCR results from consecutive patients from three university children's hospitals investigated between November 2000 and January 2007 were evaluated. Fungal infections were classified according to the EORTC classification on the grounds of clinical findings, microbiology and radio-imaging results. Two hundred and ninety-one samples from 71 patients were investigated for the presence of Aspergillus DNA by our previously described nested PCR assay. Two, 3 and 34 patients had proven, probable and possible IA, respectively. Sensitivity (calculated from proven and probable patients, n=5) and specificity (calculated from patients without IA, n=32) rates of the PCR assay were 80 and 81 %, respectively. Our nested PCR assay was able to detect Aspergillus DNA in blood, cerebrospinal fluid and bronchoalveolar lavage samples from paediatric and adolescent patients with IA with high sensitivity and specificity rates.

scholarly journals Evaluation of Different PCR Assay Formats for Sensitive and Specific Detection of SARS-CoV-2 RNA

2020 ◽  
Author(s):  
Jeremy Ratcliff ◽  
Dung Nguyen ◽  
Monique Andersson ◽  
Peter Simmonds
Keyword(s):  
Real Time ◽  
Quantitative Pcr ◽  
Nested Pcr ◽  
High Sensitivity ◽  
Clinical Samples ◽  
Pcr Assay ◽  
Copy Detection ◽  
Accurate Identification ◽  
Laboratory Equipment ◽  
Rna Transcripts

ABSTRACTAccurate identification of individuals infected with SARS-CoV-2 is crucial for efforts to control the ongoing COVID-19 pandemic. Polymerase chain reaction (PCR)-based assays are the gold standard for detecting viral RNA in patient samples and are used extensively in clinical settings. Most currently used quantitative PCR (RT-qPCRs) rely upon real-time detection of PCR product using specialized laboratory equipment. To enable the application of PCR in resource-poor or non-specialist laboratories, we have developed and evaluated a nested PCR method for SARS-CoV-2 RNA using simple agarose gel electrophoresis for product detection. Using clinical samples tested by conventional qPCR methods and RNA transcripts of defined RNA copy number, the nested PCR based on the RdRP gene demonstrated high sensitivity and specificity for SARS-CoV-2 RNA detection in clinical samples, but showed variable and transcript length-dependent sensitivity for RNA transcripts. Samples and transcripts were further evaluated in an additional N protein real-time quantitative PCR assay. As determined by 50% endpoint detection, the sensitivities of three RT-qPCRs and nested PCR methods varied substantially depending on the transcript target with no method approaching single copy detection. Overall, these findings highlight the need for assay validation and optimization and demonstrate the inability to precisely compare viral quantification from different PCR methodologies without calibration.

A high sensitivity-nested PCR assay for BHV-1 detection in semen of naturally infected bulls

1998 ◽  
Vol 63 (1) ◽  
pp. 1-11 ◽  
Author(s):  
M.A Rocha ◽  
E.F Barbosa ◽  
S.E.F Guimarães ◽  
E Dias Neto ◽  
A.M.G Gouveia
Keyword(s):  
Nested Pcr ◽  
High Sensitivity ◽  
Pcr Assay

scholarly journals Mucocutaneous Histoplasmosis in immunocompetent adult patients

2019 ◽  
Vol 19 (1) ◽  
pp. 94-97
Author(s):  
Mallika Sengupta ◽  
Anita Mitra Nandi ◽  
Himansu Roy ◽  
Soma Sarkar ◽  
Manideepa Sengupta
Keyword(s):  
Histoplasma Capsulatum ◽  
Tertiary Care ◽  
Medical Science ◽  
The Body ◽  
Dimorphic Fungus ◽  
Tertiary Care Centre ◽  
Tissue Samples ◽  
Pulmonary Histoplasmosis ◽  
Disseminated Disease ◽  
Excised Tissue

Introduction: Histoplasmosis is a fungal infection caused by the agent Histoplasma capsulatum,a dimorphic fungus. The spectrum of illness ranges from subclinical infection to progressive disseminated disease. The major bulk of histoplasma infections are asymptomatic or pulmonary histoplasmosis. In immune compromised patients it can cause disseminated infections involving different organs of the body. In immune competent individuals it may cause isolated adrenal histoplasmosis. Material and methods: A retrospective study was done to look for mucocutaneous histoplasmosis. The excised tissue samples were cultured and only culture confirmed cases were included in the study. Result: Here, we present five cases of primary mucocutaneous histoplasmosis in immune competent individuals in a tertiary care centre in India. These patients had no other co-morbidities and had only isolated lesions in oral cavity or penis.The tissue on excision was cultured and showed growth of Histoplasma capsulatum. All the patients improved with treatment. There are limited cases of mucocutaneous ulcerated lesions caused by Histoplasma capsulatum in immune competent people in published literature. Conclusion: This study emphasizes the necessity of a vigilant look out and clinical suspicion of fungal causes like histoplasma in chronic non healing ulcers which should be confirmed by the laboratory investigations. Accurate diagnosis helps in specific management of these cases. Bangladesh Journal of Medical Science Vol.19(1) 2020 p.94-97

scholarly journals An optimized one-tube, semi-nested PCR assay for Paracoccidioides brasiliensis detection

2013 ◽  
Vol 46 (6) ◽  
pp. 783-785 ◽  
Author(s):  
Amanda de Faveri Pitz ◽  
Andrea Cristine Koishi ◽  
Eliandro Reis Tavares ◽  
Fabio Goulart de Andrade ◽  
Eduardo Alexandre Loth ◽  
...  
Keyword(s):  
Nested Pcr ◽  
Paracoccidioides Brasiliensis ◽  
Pcr Assay
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