Eur J Clin Microbiol Infect Dis (2010) 29:1147–1152 DOI 10.1007/s10096-010-0980-5 ARTICLE A new method for the detection of Pneumocystis jirovecii using flow cytometry J. Barbosa & C. Bragada & S. Costa-de-Oliveira & E. Ricardo & A. G. Rodrigues & C. Pina-Vaz Received: 7 August 2009 /Accepted: 22 May 2010 /Published online: 16 June 2010 # Springer-Verlag 2010 Abstract Pneumocystis jirovecii is an opportunistic patho- reactions to bacteria or fungi. All positive cases detected by gen responsible for severe pneumonia in immunocompro- IFS were positive by FC; however, FC classified eight mised patients. Its diagnosis has been based upon direct samples to be positive which were classified as negative by microscopy either by classic staining (Gomori Grocott) or routine technique. These samples were obtained from by epifluorescence microscopy (immunofluorescence stain- patients with respiratory symptoms who responded favour- ing, IFS), both of which are time-consuming and low on ably to Pneumocystis-specific therapy and were subsequently sensitivity. Our aim was to develop a flow cytometric (FC) considered to be true-positives. Using clinical diagnosis as a protocol for the detection of P. jirovecii on respiratory reference method, FC showed 100% sensitivity and speci- samples. In our study, 420 respiratory samples were analysed ficity, whereas IFS showed 90.9% sensitivity and 100% in parallel by IFS and FC, and compared from clinical specificity. According to our results, a new diagnostic diagnosis to its resolution upon specific anti-Pneumocystis approach is now available to detect P. jirovecii in respiratory therapy. The optimum specific antibody concentration for FC samples. analysis was determined to be 10 µg/ml, without any cross- Introduction * : : : : J. Barbosa ( ) :C. Bragada S. Costa-de-Oliveira E. Ricardo A. G. Rodrigues C. Pina-Vaz Pneumocystis jirovecii (previously named Pneumocystis Department of Microbiology, Porto Faculty of Medicine, carinii University of Porto, ) is an atypical fungus. For 80 years, it was Al. Prof. Hernâni Monteiro, considered to be a protozoan, since its diagnostic form is 4200-319 Porto, Portugal a cyst, but ribosomal RNA and DNA studies demonstrated e-mail: [email protected] similarities to fungus [1]. In 2001, this microorganism was J. Barbosa officially reclassified as a fungus belonging to the class Escola Superior de Saúde Jean Piaget, Archiascomycetes [1] and was renamed P. jirovecii [2]. In Vila Nova de Gaia, Portugal humans, this pathogen is responsible for an opportunistic infection at the lower respiratory tract, but it can lead to A. G. Rodrigues Pneumocystis Burn Unit, Department of Plastic and Reconstructive Surgery, severe pneumonia (PcP) in immunocompro- Hospital S. João, mised patients, particularly in those with AIDS [3, 4]. Prior Porto, Portugal to the AIDS epidemic, P. jirovecii had been sporadically reported as a cause of death in malnourished infants and C. Pina-Vaz Department of Microbiology, Hospital S. João, responsible for epidemics in institutions like nursing homes Porto, Portugal and hospitals [5]. Currently, it represents the most frequent : : : AIDS opportunistic pathogen, although PcP incidence has S. Costa-de-Oliveira E. Ricardo A. G. Rodrigues C. Pina-Vaz decreased significantly due to the extensive use of highly Cardiovascular Research and Development Unit, Faculty of Medicine, University of Porto, active anti-retroviral therapy (HAART) and prophylactic Porto, Portugal treatment [4–6]. PcP may also represent a small fraction of 1148 Eur J Clin Microbiol Infect Dis (2010) 29:1147–1152 respiratory infections in patients with pulmonary diseases patients, 130 were HIV-positive. Samples were kindly such as chronic obstructive pulmonary disease (COPD) or provided by the Microbiology Laboratories of several displaying immunity conditions like malignancy [7, 8]. Portuguese hospitals, namely, Hospital São João (HSJ, Porto, Over the past 15 years, P. jirovecii carriage in healthy Portugal), Instituto Português de Oncologia Francisco Gentil subjects has been widely demonstrated through the pres- (IPOFG, Porto, Portugal), Hospital Joaquim Urbano (HJU, ence of anti-P. jirovecii antibodies in their serum, with Porto, Portugal) and Centro Hospitalar de Coimbra (CHC, serologic analysis being used simply for the assessment of Coimbra, Portugal). A maximum of 30 mL of BAL or BW, the infection epidemiology [9]. PcP, however, is considered and 2 to 4 mL of BS samples were placed in a sterile container to result from de novo infection rather than from the and frozen at −70°C for further processing for immunofluo- reactivation of a latent infection [6]. It is assumed that rescence staining (IFS) and FC analysis. human transmission of P. jirovecii occurs through the airborne route, as suggested by studies performed in rodent Sample preparation models [6]. Nonetheless, inter-individual transmission has been suggested due to the occurrence of PcP case clusters For IFS, clinical samples were mixed with mucolytic agent in hospitals, namely, in paediatrics, hematology–oncology, N-acetyl-L-cysteine (Merck®) and incubated in a water intensive care, transplantation unit and infectious diseases bath at 37°C for 20 min or until complete dissolution, units [10]. P. jirovecii may be exhaled to the environment followed by centrifugation at 3,000g for 15 min. Subse- from infected patients, as demonstrated from cyst DNA quently, the samples were washed twice with sterile water detection in air filters from the hospital rooms of patients (H2O), centrifuged as above and the sediment re-suspended who developed PcP [10]. in a small volume of water which was divided into two The detection of P. jirovecii in an infected patient is portions; one portion was placed on a glass slide, air dried based upon the direct visualisation of organisms (cysts, and fixed, while the other portion was used for FC analysis. sporocysts or trophozoites) in clinical specimens, such as bronchoalveolar lavage (BAL), induced sputum (IS) or lung Immunofluorescence staining biopsy, which is the gold-standard specimen [6, 11]. Although specific fluorescent staining techniques were Smears were stained according to the manufacturer’sinstruc- developed for assessment under fluorescent microscopy, tions with the Detect IF™ kit Pneumocystis carinii such methods are very time-consuming, too cumbersome (Axis-Shield Diagnostics Limited, United Kingdom) and and subject to human error, especially when samples yield a analysed under an epifluorescence microscope Leitz Labor- low number of P. jirovecii organisms [11]. Molecular lux K (Leica, NY, USA). Smears were considered to be studies such as polymerase chain reaction (PCR) presented positive for P. jirovecii whenever two or more green cysts a higher sensitivity and variable specificity when compared were visualised, whether isolated or in a group. to the microscopic detection of P. jirovecii in BAL [12, 13]. However, PCR remains unavailable in most clinical Optimisation of a flow cytometry protocol microbiology laboratories and is an expensive technique [14, 15]. More so, a variable specificity has been described For the optimisation of the FC protocol, a mix of four ranging from 62.5 to 100%, depending on the type of positive samples and a mix of four negative samples specimen [14, 15]. Distinct applications of flow cytometry (evaluated by IFS) were prepared. Samples were divided (FC) in microbiology have been developed by our group in into two equal aliquots and one of them was passed through order to increase the diagnostic sensitivity in clinical a filter with a 30-μm pore size (Partec CellTrics®). Twenty samples [16–18]. In the present study, we have developed microlitres of specific enzyme from Detect IF™ kit and optimised a specific FC protocol for the detection of P. Pneumocystis carinii (Axis-Shield Diagnostics Limited, jirovecii on respiratory samples. United Kingdom) were added to 100 µl of the respiratory sample and incubated at 37°C for 30 min; 500 µl of sterile H2O were then added and a subsequent centrifugation was Materials and methods performed at 3,000g for 5 min. One hundred microlitres of centrifuged sample were Clinical specimens stained with serial concentrations (0, 5.0, 10.0, 15.0 and 20.0 μl) of specific P. jirovecii mouse monoclonal antibody A total of 420 samples, including 380 BAL and bronchial (Axis-Shield Diagnostics Limited, United Kingdom), washing (BW) specimens, and 40 bronchial secretion (BS) followed by dark incubation at 37°C for 15 min. Subse- aspirates, were prospectively collected from patients with quently, 500 µl of sterile H2O were added and centrifuga- different immunological conditions. From those 420 tion at 3,000g for 5 min was performed; the supernatant Eur J Clin Microbiol Infect Dis (2010) 29:1147–1152 1149 was discarded and the pellet re-suspended in 100 μlof Data comparison sterile H2O, vortexed for 30 s, transferred to a propylene tube and analysed by FC. The results of IFS were compared to those obtained by FC. Clinical diagnosis was used as the standard. When results Flow cytometry analysis were discrepant, clinical symptoms and signs were consid- ered; patients with oxygen level PaO2 <70 mmHg, an X-ray The optical characteristics of the cysts suspensions were depicting a diffuse bilateral infiltrate and with a favourable evaluated on a FACSCalibur flow cytometer (BD Bioscien- outcome upon specific therapy were considered as true- ces, Sydney , Australia) standard model equipped with three positive cases for PcP. photomultipliers (PMTs), standard filters (FL1: BP 530/30 nm; FL2: BP 585/42 nm; FL3: LP 650 nm), a 15-mW 488-nm argon laser and with CellQuest Pro Software Results (version 4.0.2, BD Biosciences, Sydney, Australia). Operating conditions included log scales on all detectors (forward scatter Respiratory samples were considered to be positive for P. [FSC], side scatter [SSC] and fluorescence detectors [FL1]). jirovecii when two or more green round formations, Acquisition settings were defined using a non-stained sample namely, cysts, were observed on epifluorescence micros- (autofluorescence) and adjusting the PMTs’ voltage to the first copy. By IFS, 340 samples were negative and 80 samples logarithmic (log) decade.