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      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 * Translated title: 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

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          Abstract

          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.

          Translated abstract

          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.

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          Histoplasmosis: a Clinical and Laboratory Update

          Infection with Histoplasma capsulatum occurs commonly in areas in the Midwestern United States and Central America, but symptomatic disease requiring medical care is manifest in very few patients. The extent of disease depends on the number of conidia inhaled and the function of the host's cellular immune system. Pulmonary infection is the primary manifestation of histoplasmosis, varying from mild pneumonitis to severe acute respiratory distress syndrome. In those with emphysema, a chronic progressive form of histoplasmosis can ensue. Dissemination of H. capsulatum within macrophages is common and becomes symptomatic primarily in patients with defects in cellular immunity. The spectrum of disseminated infection includes acute, severe, life-threatening sepsis and chronic, slowly progressive infection. Diagnostic accuracy has improved greatly with the use of an assay for Histoplasma antigen in the urine; serology remains useful for certain forms of histoplasmosis, and culture is the ultimate confirming diagnostic test. Classically, histoplasmosis has been treated with long courses of amphotericin B. Today, amphotericin B is rarely used except for severe infection and then only for a few weeks, followed by azole therapy. Itraconazole is the azole of choice following initial amphotericin B treatment and for primary treatment of mild to moderate histoplasmosis.
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            Multiplex PCR using internal transcribed spacer 1 and 2 regions for rapid detection and identification of yeast strains.

            Multiplex PCR amplification followed by either agarose gel electrophoresis (PCR-AGE) or microchip electrophoresis (PCR-ME) was used to test a total of 120 fungal strains. The internal transcribed spacer 1 (ITS1) and ITS2 regions and the 5.8S ribosomal DNA (rDNA) region of the fungi were amplified by using universal primers ITS1 and ITS4. The ITS2 region was simultaneously amplified by using universal primers ITS3 and ITS4. Since Trichosporon asahi and T. asteroides showed similar lengths for two amplicons, 29 different gel patterns were demonstrated for 30 yeast species tested on the basis of differences in the lengths of one or two amplicons. Of 75 yeast isolates from clinical materials, 5 isolates (6.8%) which were incompletely identified or not identified by the phenotypic method were identified with our PCR-based method (2 isolates as Candida guilliermondii, 2 as C. krusei, and 1 as C. zeylanoides). No differences in discriminating power or sensitivity were observed between the PCR-AGE method and the PCR-ME method. These methods, prospectively applied to 24 yeast-positive blood culture bottles (16 patients), resulted in the correct detection of 24 yeast strains. In conclusion, multiplex PCR followed by electrophoresis seems to be a promising tool for the rapid identification of common and uncommon yeast strains from culture colonies and from yeast-positive blood culture bottles (5.5 h for the PCR-AGE method and 3 h for the PCR-ME method).
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              Evaluation of two nested PCR assays for detection of Histoplasma capsulatum DNA in human tissue.

              In order to evaluate the diagnostic relevance of two nested PCR assays for diagnosis of histoplasmosis in clinical specimens, 100 paraffin-embedded biopsy specimens were examined. Upon microscopy of tissue, 50 biopsy specimens were histoplasma positive and 50 were negative. Due to destruction by formalin fixation, successful extraction of amplifiable human DNA was limited to 29 and 33 samples, respectively. A product of the Histoplasma capsulatum nested PCR assay targeting the gene encoding the unique fungal 100-kDa-like protein was detected in 20 histopathologically positive biopsy specimens but in none of the microscopically negative samples. Sequencing revealed that all 20 products of 210 bp were identical to the sequence of H. capsulatum in the GenBank database. In contrast, the nested PCR assay targeting the fungal 18S rRNA genes amplified products in 26 histopathologically positive but also in 18 microscopically negative biopsy specimens. However, sequencing revealed that only 20 of these 44 PCR products (231 bp) were identical to the sequence of H. capsulatum. The remaining 24 sequences were homologous to those of several Euascomycetes. These PCR products were detected only in tissues possibly colonized by nonpathogenic fungi, possibly causing these nonspecific amplifications. The detection limit of both H. capsulatum nested PCR assays was 1 to 5 fungal cells per sample. The two assays were similarly sensitive in identifying H. capsulatum. In this preliminary study, the novel 100-kDa-like-protein gene nested PCR revealed a specificity of 100% without requiring sequencing, which was necessary for identification of the 18S ribosomal DNA nested PCR products in order to avoid a high rate of false-positive results.
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                Author and article information

                Journal
                An Bras Dermatol
                An Bras Dermatol
                An. bras. dermatol.
                Anais brasileiros de dermatologia
                Sociedade Brasileira de Dermatologia
                0365-0596
                1806-4841
                Jan-Feb 2013
                : 88
                : 1
                : 141-143
                Affiliations
                [1 ] Pharmacist. Doctor of Science awarded by the Faculty of Medicine of the University of São Paulo (FMUSP) - researcher in the Faculty of Medicine of the University of São Paulo (FMUSP) – São Paulo (SP), Brazil.
                [2 ] Master of Science awarded by the Faculty of Medicine of the University of São Paulo (FMUSP)- Researcher in the LIM 53 of the Hospital das Clínicas, Faculty of Medicine, University of São Paulo (HCFMUSP) – São Paulo (SP), Brazil.
                [3 ] Doctor of Microbiology and Immunology, degree awarded by the Federal University of São Paulo (UNIFESP) - Adolfo Lutz Institute – State Health Secretariat (IAL) – São Paulo (SP), Brazil.
                [4 ] Doctor, degree awarded by the Faculty of Medicine of the University of São Paulo (FMUSP) – Infectologist of the Emílio Ribas Infectology Institute – São Paulo (SP), Brazil.
                [5 ] Post-doctorate - Professor of the Dermatology Department of the Faculty of Medicine of the University of São Paulo (FMUSP) – São Paulo (SP), Brazil.
                [6 ] Post-doctorate - Professor in the stricto sensu Postgraduate Course of the Faculty of Medicine, University of São Paulo (FMUSP) and at the Medical Welfare Insitute for State Public Employees (IAMSPE) – São Paulo (SP), Brazil.
                [7 ] Doctor of Science (Dermatology) awarded by the Faculty of Medicine of the University of São Paulo (FMUSP) - Doctor in the Dermatology Division of the Hospital das Clínicas, Faculty of Medicine of the University of São Paulo (HCFMUSP) – São Paulo (SP), Brazil.
                Author notes
                Mailing address: Paulo Ricardo Criado Divisão de Dermatologia -Instituto Central Av. Dr. Enéas de Carvalho Aguiar, 255 Cerqueira César 05403 000 São Paulo, SP. Brazil E-mail: prcriado@ 123456uol.com.br
                Article
                10.1590/S0365-05962013000100025
                3699932
                23539023
                b58e7239-3c4d-41b2-8e5b-84a0430525b3
                ©2013 by Anais Brasileiros de Dermatologia

                This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License, which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

                History
                : 29 May 2012
                : 04 June 2012
                Funding
                Funded by: FAPESP
                Award ID: 2009/50362-0
                Financial funding: FAPESP funding No. 2009/50362-0.
                Categories
                Communication

                aids-related opportunistic infections,histoplasma,hiv seropositivity,molecular diagnostic techniques,polymerase chain reaction

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