Gustavo Wissmann

Universidade Federal do Rio Grande do Sul

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Luciano Z. Goldani

Hospital de Clínicas de Porto Alegre

Enrique J. Calderón

Instituto de Biomedicina de Sevilla

André Luis Aquino Müller

Hospital de Clínicas de Porto Alegre

Vicente Friaza

Universidad de Sevilla

Andréia Spanamberg

Universidade Federal do Rio Grande do Sul

Yaxsier de Armas

Instituto de Medicina Tropical “Pedro Kourí”

Laerte Ferreiro

Universidade Federal do Rio Grande do Sul

João Carlos Prolla

Irmandade da Santa Casa de Misericórdia de São Paulo

J.M. Varela

Hospital Universitario Virgen del Rocío

Edna Maria Cavallini Sanches

Universidade Federal do Rio Grande do Sul

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Universidade Federal do Rio Grande do Sul

Hospital de Clínicas de Porto Alegre

Hospital Universitario Virgen del Rocío

Universidad de Sevilla

Instituto de Biomedicina de Sevilla

Instituto de Salud Carlos III

Universitat de Barcelona

Consorci Institut D'Investigacions Biomediques August Pi I Sunyer

Hospital Clínic de Barcelona

Centro de Investigación Biomédica en Red

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Real-time pcr and nested-pcr assays for detection of Pneumocystis sp. in lung tissues of bats

Edna Maria Cavallini Sanches Laerte Ferreiro Caroline Pinto de Andrade Susi Missel Pacheco Jânio Morais Santúrio

Background: Pneumocystis constitutes a highly diversifi ed biological group, with numerous species, which are strongly host-specifi c and well adapted to live inside the lungs of a diverse range of mammals. The detection of DNA from Pneumocystis in clinical specimens by PCR assays is leading to important advances in pneumonia caused by Pneumocystis and its epidemiology. The aim of this study was to analyze two different diagnostic methods, real-time PCR (qPCR) using primers based in the Major Surface Glycoprotein (MSG) of Pneumocystis sp. and conventional nested PCR using primers designed to the small subunit of mitochondrial rRNA (mtSSU rRNA) for detection of Pneumocystis DNA in lung tissue from bats. Materials, Methods & Results: Bats (195 samples) were captured (2007-2009) in caves, forests, and urban areas, were obtained from the Program of Rabies Control of two states in Brazil: Mato Grosso and Rio Grande do Sul, located respectively in the Mid-Western and Southern regions of the country approximately 2000 km apart. Lung tissue (250 mg) was fi nely minced, homogenized with crushing and DNA extraction was carried out with commercial kit. DNA samples the lung tissue of bats were analyzed by nested PCR, using oligonucleotide primers designed for the gene encoding the mitochondrial small subunit r RNA (mtSSU rRNA) and Taqman probe and primers for qPCR were selected based on the Major Surface Glycoprotein (MSG) of Pneumocystis sp. Chi-square (P < 0.001 was considered signifi cant) and the McNemar’s test was used to analyze nested PCR and qPCR as methods of detection of Pneumocystis sp. and the Kappa was calculated by Win Episcope 2.0. To assess the sensitivity and specifi city of the qPCR assay, a nested PCR assay was considered as the reference method. The positivity was 36.4% in the nested PCR and 24.1% using the qPCR. Concordance was obtained in 68.2% of the samples (133/195). It was demonstrated that there was no statistically signifi cant difference between the techniques used and, both tests proved to be specifi c for the detection of Pneumocystis species. Specifi city was 71% for the nested PCR and 84.6% for the qPCR. Pneumocystis was detected (71/195) by the nested PCR assay in 14 species:. Tadarida brasiliensis, Histiotus velatus, Desmodus rotundus, Molossus molossus, Glossophaga soricina, Nyctinomops laticaudatus, Promops nasutus, Artibeus sp., Eptesocus furinalus, Lasurus blossevillii, Molossus currentium, Molossus rufus, Myotis levis and Nyctinomops macrotis. Discussion: This study detected the DNA from Pneumocystis through the nested PCR and qPCR assays, and the frequency found is comparable to that obtained in a previous study, which used the nested PCR in Central American, South American and European countries. Pneumocystis sp. was observed in a high number of different bat species (14) in two Brazilian States (RS and MT). The qPCR showed a higher specifi city in comparison to the nested PCR. The literature has similar fi ndings to the results obtained by this research, employing the same tests and genes. The nested PCR and qPCR assays are indicated in the diagnosis of Pneumocystis sp. in bats and it is important to highlight that a better diagnostic precision is achieved with the association of both tests. Additionally, this study was the fi rst to detect Pneumocystis sp. in the lungs of bats using qPCR.

TaqMan

Pneumocystis pneumonia

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Pneumocystisand Pneumocystosis: first meeting of experts from Latin-American and Portuguese-speaking countries – a mini-review

Expert Review of Anti-infective Therapy (2014)

Francisco Esteves Francisco J. Medrano Yaxsier de Armas Gustavo Wissmann Enrique J. Calderón

The Pneumocystis and Pneumocystosis: first meeting of experts from Latin-American and Portuguese-speaking countries was held in Lisbon, Portugal, on 24-26 October 2013. A total of 20 speakers from Latin America, Africa and Europe participated in the meeting. The epidemiological studies presented in this meeting begin to change the misconception that since the AIDS epidemic, Pneumocystis pneumonia (PcP) has become an infrequent disease, showing that today PcP remains a major opportunistic infection in HIV-infected patients in both developed and developing countries and an emerging problem in immunocompromised patients without HIV infection worldwide. PcP management remains a challenge. Right now, the combination of caspofungin and trimethoprim-sulfamethoxazole (TMP-SMX) is a promising therapeutic approach that needs to be assessed in controlled clinical trials.

Pneumocystosis

Pneumocystis pneumonia

Pentamidine

10.1586/14787210.2014.894883

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Estudo da colonização pelo Pneumocystis jirovecii na comunidade

Gustavo Wissmann Rosicler Luzia Brackmann Mendes André Luis Aquino Müller Franciane Brackmann Mendes Humberto Butzke da Motta

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Relationship between intracranial hypertension and antifungal agent levels in the CSF of patients with cryptococcal meningitis

Open Forum Infectious Diseases (2016)

Fernanda Wirth Carmen Pilla Valério Rodrigues Aquino Gustavo Wissmann Luciano Z. Goldani

Cryptococcal meningitis

10.1093/ofid/ofw172.1344

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El ser humano como reservorio de Pneumocystis

Enfermedades Infecciosas y Microbiología Clínica (2009)

Gustavo Wissmann Rubén Morilla Vicente Friaza Enrique J. Calderón J.M. Varela

10.1016/j.eimc.2008.07.008

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Perfil dos pacientes do HCPA com Fusariose Invasiva

Priscila Dallé da Rosa Carolina dos Santos Corrêa Rafael J. Borges Gustavo Wissmann Valério Rodrigues Aquino

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High prevalence ofPneumocystis jiroveciicolonization in Brazilian cystic fibrosis patients

Medical Mycology (2012)

Marco Aurélio Pederiva Gustavo Wissmann Vicente Friaza Rubem Morilla Carmen de la Horra

A high rate of Pneumocystis jirovecii colonization was observed in Brazilian cystic fibrosis (CF) patients (13 out of 34; 38.2%) who underwent bronchoscopy between March 2006 and August 2009 at the Hospital de Clinicas de Porto Alegre, Brazil. Bronchoalveolar lavage samples were collected from these patients and studied by nested PCR amplification of the mitochondrial gene coding for the large subunit ribosomal RNA (mtLSUrDNA). The observed rate of colonization was higher than that reported in European populations. Genotypic characterization of the mtLSUrDNA locus revealed a predominance of the polymorphisms 85C/248C (genotype 1) and 85T/248C (genotype 3), with all samples possessing the wild-type genotype of dihydropteroate synthase. These findings suggest that cystic fibrosis patients could be an important reservoir and source of P. jirovecii infection. Further studies are required to elucidate the role of this common fungal colonization in the evolution of CF patients.

10.3109/13693786.2011.645892

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Detecção de T. Pallidum em líquor de pacientes com neurossífilis e HIV através da reação em cadeia da polimerase

Marcelo Simi Czykiel Daniela Duarte de Fraga André Luis Aquino Müller Gustavo Wissmann Luciano Z. Goldani

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Pneumocystis sp. in bats evaluated by qPCR

Journal de Mycologie Médicale (2013)

Edna Maria Cavallini Sanches Laerte Ferreiro Caroline Pinto de Andrade Susi Missel Pacheco Laura Lopes de Almeida

Pneumocystis pneumonia

Desmodus rotundus

10.1016/j.mycmed.2012.11.002

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High prevalence of Pneumocystis jirovecii colonization among HIV-positive patients in southern Brazil

Medical Mycology (2014)

Robson Martins Pereira André Luis Aquino Müller Ricardo Ariel Zimerman Denise B. Antunes Vítor Freitas Zinn

A high prevalence of Pneumocystis jirovecii colonization was observed in patients positive for the human immunodeficiency virus (HIV) admitted to a tertiary hospital in southern Brazil between August 2012 and December 2012. Amplification of the mitochondrial large subunit ribosomal RNA gene in oropharyngeal samples through nested polymerase chain reaction identified P. jirovecii colonization in 26 of 58 (44.8%) HIV-positive patients admitted for causes other than Pneumocystis pneumonia. Colonization was more frequent among patients with an absolute CD4 count ≤200 cells/μl. These findings suggest that the HIV-infected population is a major reservoir and source of P. jirovecii infection and that identification of such individuals may contribute to future strategies for improving management of HIV-infected patients.

Pneumocystis pneumonia

10.1093/mmy/myu059

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Citations (8)