PCR for diagnosis of paracoccidioidomycosis.
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ABSTRACT:
SUBMITTER: Gomes GM
PROVIDER: S-EPMC87412 | biostudies-literature | 2000 Sep
REPOSITORIES: biostudies-literature
Publications
PCR for diagnosis of paracoccidioidomycosis.
Gomes G M GM Cisalpino P S PS Taborda C P CP de Camargo Z P ZP
Journal of clinical microbiology 20000901 9
A PCR assay based on oligonucleotide primers derived from the sequence of the gene coding for the 43,000-Da (gp43) antigen was developed to detect Paracoccidioides brasiliensis DNA in sputa. In the standardized conditions, it could detect 10 cells/ml of sputum, providing sufficient accuracy to be useful for diagnosis of paracoccidioidomycosis. ...[more]
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Project description:Paracoccidioidomycosis (PCM) cannot always be diagnosed by conventional means such as direct examination of histopathology or clinical samples, and serological methods, used as an alternative, still have many cases of cross-reactivity. In this scenario, molecular techniques seem to arise as a rapid approach, specific and direct that could be used in the diagnosis of this mycosis. In this study we analyzed 76 serum samples from patients in southern Bahia suspected of having paracoccidioidomycosis using a conventional PCR with primers for the ITS1 ribosomal DNA of P. brasiliensis. Of these 76 patients, 5 were positive for PCM by double immunodiffusion and/or direct examination and histopathology. To test specificity of PCR, we used human DNA and three isolates of P. lutzii (1578, 01 and ED01). Additionally, we analyzed by serial dilutions of DNA the limit of detection of the assay. The test of PCR proved specific, as only a 144 bp fragment of the three isolates of P. lutzii and no human DNA was amplified. Detection limit was 1.1 pg/µL of DNA. Despite the high detection limit and specificity of PCR none of the 76 serum samples were found positive by PCR, but a biopsy specimen obtained from one of the patients with PCM was positive. These results, albeit limited, show that PCR is not effective in detecting DNA of P. brasiliensis or P. lutzii in serum, but could perhaps be used with other types of clinical samples, especially in those instances in which conventional methods fail.
| S-EPMC3510084 | biostudies-literature
Project description:Paracoccidioidomycosis (PCM) is a systemic granulomatous disease, endemic in Latin America, caused by the thermal dimorphic fungus Paracoccidioides brasiliensis. Although some fungal antigens have already been characterized and used for serological diagnosis, cross-reactions have been frequently observed. Thus, the examination of fungal forms in clinical specimens or isolation of P. brasiliensis by culture is still the most frequent method for the diagnosis of this mycosis. In this study, a random peptide phage display library was used to select mimotopes of P. brasiliensis, which were employed as antigens in an indirect enzyme-linked immunosorbent assay. The protective monoclonal antibody against experimental PCM (anti-gp75) was used as molecular target to screen a phage display library. That approach led to a synthetic peptide named P2, which was synthesized and tested against PCM patients' sera to check whether it was recognized. There was significant recognition of P2 by sera of untreated PCM patients when compared with normal human sera. Sera from treated PCM group, patients with other mycosis or co-infected with HIV had much lower recognition of P2 than untreated patient group. The test showed a sensitivity of 100 and 94.59% of specificity in relation to human sera control. These data indicate a potential use of P2 as diagnostic tool in PCM. Its application for serological diagnosis of PCM may contribute to the development and standardization of simpler, faster and highly reproducible immunodiagnostic tests at low cost.
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Project description:BackgroundSerological tests have long been established as rapid, simple and inexpensive tools for the diagnosis and follow-up of PCM. However, different protocols and antigen preparations are used and the few attempts to standardize the routine serological methods have not succeeded.Methodology/principal findingsWe compared the performance of six Brazilian reference centers for serological diagnosis of PCM. Each center provided 30 sera of PCM patients, with positive high, intermediate and low titers, which were defined as the "reference" titers. Each center then applied its own antigen preparation and serological routine test, either semiquantitative double immunodifusion or counterimmmunoelectrophoresis, in the 150 sera from the other five centers blindly as regard to the "reference" titers. Titers were transformed into scores: 0 (negative), 1 (healing titers), 2 (active disease, low titers) and 3 (active disease, high titers) according to each center's criteria. Major discordances were considered between scores indicating active disease and scores indicating negative or healing titers; such discordance when associated with proper clinical and other laboratorial data, may correspond to different approaches to the patient's treatment. Surprisingly, all centers exhibited a high rate of "major" discordances with a mean of 31 (20%) discordant scores. Alternatively, when the scores given by one center to their own sera were compared with the scores given to their sera by the remaining five other centers, a high rate of major discordances was also found, with a mean number of 14.8 sera in 30 presenting a discordance with at least one other center. The data also suggest that centers that used CIE and pool of isolates for antigen preparation performed better.ConclusionThere are inconsistencies among the laboratories that are strong enough to result in conflicting information regarding the patients' treatment. Renewed efforts should be promoted to improve standardization of the serological diagnosis of PCM.
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Project description:Loop-mediated isothermal amplification (LAMP) methods are now useful for the detection of a specific gene in infectious diseases, genetic diseases, and/or genetic disorders in the large number of medical fields, and it was recently introduced to fungal investigation. It is characterized by the use of four different primers specifically designed to recognize six distinct regions of the target gene, and the reaction process proceeds at a constant temperature using strand displacement reaction. Quickness and simplicity is the advantage of the method. Amplification and detection of gene can be completed in a single step, by incubating the mixture of samples, primers, DNA polymerase with strand displacement activity and substrates at a constant temperature. The method was applied to two fungal infections; paracoccidioidomycosis (PCM), a deep mycosis caused by Paracoccidioides brasiliensis and Ochroconis gallopava infection. For PCM a combination of F3, B3, FIP, and BIP primers designed from the partial sequence of P. brasiliensis gp43 gene was used. The PCR products amplified by the primer set; F3 and B3 showed species specificity for P. brasiliensis and the detection limit of the PCR was 100 fg of fungal genomic DNA. The specific DNA banding pattern of P. brasiliensis was detected in the clinical and nine-banded armadillo derived isolates, paraffin-embedded tissue sample or sputum from PCM patient. LAMP method was used also for the identification of O. gallopava by using species-specific primer sets based on the D1/D2 domain of the LSU rDNA sequence. The method successfully detected the gene from both fungal DNA derived from brains and spleens of experimentally-infected mice with O. gallopava and environmental isolates. In conclusion, LAMP method for PCM and O. gallopava seemed to be useful for identification, diagnosis or retrospective study with advantage in the quickness and simplicity procedure, but require strictly-controlled environments.
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