Diagnostic Microbiology and Infectious Disease xxx (2015) xxx–xxx
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Pneumocystis jirovecii in the air surrounding patients with Pneumocystis pulmonary colonization☆,☆☆,★ Solène Le Gal a,b,⁎, Laurence Pougnet c, Céline Damiani d,e, Emilie Fréalle f, Paul Guéguen g,h, Michèle Virmaux a, Séverine Ansart i,j, Sylvain Jaffuel i, Francis Couturaud k,l, Aurélien Delluc k,l, Jean-Marie Tonnelier m, Philippe Castellant n, Yann Le Meur o,p, Gaétan Le Floch a, Anne Totet d,e,1, Jean Menotti q,r,1, Gilles Nevez a,b,⁎,1 a
University of Brest, LUBEM EA 3882, SFR 148, Brest, France Laboratory of Parasitology and Mycology, Brest University Hospital, Brest, France c Laboratory, Military Teaching Hospital Clermont Tonnerre, Brest, France d University of Picardy-Jules Verne, UMR-I 01, SFR Cap Santé, Amiens, France e Laboratory of Parasitology and Mycology, Amiens University Hospital, Amiens, France f Laboratory of Parasitology and Mycology, Lille University Hospital, Lille, France g Laboratory of Molecular Genetics and Histocompatibility, Brest University Hospital, Brest, France h University of Brest, INSERM U1078, Molecular Genetics and Epidemiological Genetics, SFR 148, Brest, France i Department of Infectious Diseases, Brest University Hospital, Brest, France j University of Brest, INSERM UMR 1101, Laboratory of Medical Information Processing, SFR 148, Brest, France k Department of Internal Medicine and Pneumology, Brest University Hospital, Brest, France l University of Brest, EA3878 (GETBO), CIC INSERM 0502, SFR 148, Brest, France m Medical Intensive Care Unit, Brest University Hospital, Brest, France n Department of Cardiology, Brest University Hospital, Brest, France o Department of Nephrology and Renal Transplantation Unit, Brest University Hospital, Brest, France p University of Brest, EA 2216, SFR 148, Brest, France q Laboratory of Parasitology and Mycology, Saint Louis Hospital APHP, Paris, France r Paris-Diderot University, Paris, France b
a r t i c l e
i n f o
Article history: Received 5 December 2014 Received in revised form 9 January 2015 Accepted 11 January 2015 Available online xxxx Keywords: Pneumocystis jirovecii Colonization Airborne transmission Exhalation
a b s t r a c t In this study, Pneumocystis jirovecii was detected and characterized in the air surrounding patients with Pneumocystis pulmonary colonization. Air samples were collected in the rooms of 10 colonized patients using Coriolis® μ air sampler at 1 m and 5 m from the patient's head. P. jirovecii DNA was amplified and genotyped in pulmonary and air samples at the mitochondrial large subunit ribosomal RNA gene. P. jirovecii DNA was detected in 5 of the 10 air samples collected at 1 m and in 5 of the 10 other air samples collected at 5 m. P. jirovecii genotyping was successful in 4 pairs or triplets of air and pulmonary samples. Full genotype matches were observed in 3 of the 4 pairs or triplets of air and pulmonary samples. These results provide original data supporting P. jirovecii exhalation from colonized patients and emphasize the risk of P. jirovecii nosocomial transmission from this patient population. © 2015 Elsevier Inc. All rights reserved.
1. Introduction Pneumocystis jirovecii is a fungus that causes severe pneumonia in human immunodeficiency virus (HIV)–infected patients and in non– ☆ Financial support: This study was supported by the Agence nationale de sécurité sanitaire de l'alimentation, de l'environnement et du travail (Anses) (Pneumair project; grant number 2011/1/053). ☆☆ Potential conflict of interest: The authors report no conflicts of interest. The authors are responsible for the content and the writing of this paper. ★ The results of this study were presented in part at the 13th International Workshops on Opportunistic Protists, Seville, Spain, 13–15 November 2014, abstract T26. ⁎ Corresponding authors. Tel.: +33-2-98-14-51-02; fax: +33-2-98-14-51-49. E-mail addresses: [email protected] (S. Le Gal), [email protected] (G. Nevez). 1
These authors contributed equally to this work.
HIV-infected patients who receive immunosuppressive therapy (Walzer and Cushion, 2005). Pneumocystis pneumonia (PCP) represents only part of the clinical presentations of Pneumocystis infections, while mild infections, such as pulmonary colonization occurring in patients with diverse levels of immunodeficiency or lung diseases, account for the main part (Morris and Norris, 2012; Nevez et al., 1999). Since Pneumocystis microorganisms infecting each mammalian species are host specific (Stringer et al., 2002), an animal reservoir for P. jirovecii can be excluded. Moreover, as no exosaprophytic form of Pneumocystis spp. has been identified so far, humans harboring P. jirovecii, whatever their clinical presentation, PCP or pulmonary colonization, may represent the fungus reservoir and potential infectious sources for susceptible individuals (Nevez et al., 2003). We recently provided data on the role of colonized patients as potential sources of P. jirovecii in the context of nosocomial acquisition of
http://dx.doi.org/10.1016/j.diagmicrobio.2015.01.004 0732-8893/© 2015 Elsevier Inc. All rights reserved.
Please cite this article as: Le Gal S, et al, Pneumocystis jirovecii in the air surrounding patients with Pneumocystis pulmonary colonization, Diagn Microbiol Infect Dis (2015), http://dx.doi.org/10.1016/j.diagmicrobio.2015.01.004
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S. Le Gal et al. / Diagnostic Microbiology and Infectious Disease xxx (2015) xxx–xxx
the fungus (Le Gal et al., 2012; Nevez et al., 2008). These observations are consistent with that of experimental infections in the course of which colonized Balb/c mice can transmit Pneumocystis murina to susceptible SCID mice that subsequently developed PCP (Dumoulin et al., 2000; Gigliotti et al., 2003). The airborne route of transmission of Pneumocystis spp. has been demonstrated in rodent models (Hughes, 1982; Soulez et al., 1991), and this route for P. jirovecii transmission in humans has been suggested (Bartlett et al., 1997; Choukri et al., 2010; Damiani et al., 2012; Olsson et al., 1996, 1998). Recently, P. jirovecii DNA has been detected and quantified in the air surrounding PCP patients, suggesting the spread of P. jirovecii from these patients within their environment (Choukri et al., 2010). In contrast, the exhalation of P. jirovecii from colonized patients has not been yet investigated.
P. jirovecii detection in the surrounding environment of colonized patients would strengthen their potential role as infectious sources. In this context, we performed the detection, quantification, and characterization of P. jirovecii DNA in air samples from the surrounding environment of 10 colonized patients and 4 PCP patients. 2. Materials and methods 2.1. Patients and diagnoses of P. jirovecii infections The 14 patients diagnosed with Pneumocystis infections in the Brest University Hospital from November 2012 through March 2013 were enrolled in the study (Table 1). Patients' characteristics were extracted
Table 1 Characteristics of 4 patients with PCP and 10 patients with Pneumocystis pulmonary colonization monitored at the University Hospital of Brest, France, for whom Pneumocystis presence in air samples from their surrounding environment was investigated. Date of pulmonary sample retrieval (daymo-year)
Results of P. jirovecii detection in pulmonary samples using microscopy a/PCR b
(1,3) β-D glucan serum level (pg/mL)
No. of days between serum and pulmonary sampling
Clinical presentation of P. jirovecii infection (alternative diagnosis of PCP)
Sputum
27-11-12
ND/+
5024
−1
PCP
BAL
28-11-12
+/+
751
0
PCP
BAL
30-11-12
−/+
25
3
Pulmonary colonization (bacterial pneumonia) Pulmonary colonization (exacerbation of chronic bronchitis) Pulmonary colonization (bronchiolitis obliterans) Pulmonary colonization (pulmonary atelectasis) Pulmonary colonization (bacterial pneumonia) Pulmonary colonization (bacterial pneumonia) Pulmonary colonization (bacterial pneumonia) PCP
Immunosuppressive Pulmonary Patient Sex Age Underlying treatment samples (y) conditions (CD4+ cell counts in blood) examined for P. jirovecii presence 1
M
51
2
F
50
HIV infection (54 × 106/L) Glioblastoma (ND)
3
M
81
COPD (ND)
4
F
70
COPD, pulmonary fibrosis (2928 × 106/L)
Inhaled corticosteroids
BAL
03-12-12
−/+
20
−8
5
M
56
Emphysema and COPD (ND)
Inhaled corticosteroids
BAL
06-12-12
−/+
ND
ND
6
M
31
HBV infection, renal transplant (400 × 106/L)
BAL
18-12-12
−/+
47
1
7
M
71
Emphysema (ND)
BAL
07-01-13
−/+
ND
ND
8
F
73
Lymphoma with leucopenia (ND)
BAL
15-01-13
−/+
71
2
9
F
32
BAL
18-01-13
−/+
ND
ND
10
M
35
Common variable immunodeficiency; bronchiectasis (200 × 106/L) HIV infection (10 × 106/L)
11
M
66
COPD (ND)
Sputum BAL BAL BAL
22-01-13 31-01-13 28-02-13 25-01-13
ND/+ +/+ +/+ −/+
648 ND 502 ND
0 ND −1 ND
12
F
76
BAL
21-02-13
−/+
b8
1
13
M
35
BAL
27-02-13
+/+
1159
0
14
M
29
Antisynthetase syndrome (1045 × 106/L) HIV infection (14 × 106/L) HIV infection (1 × 106/L)
BAL
13-03-13
−/+
46
1
Oral corticotherapy Temozolomide Inhaled corticosteroids
Rituximab Fludarabine Cyclophosphamide
Oral corticotherapy
Pulmonary colonization (bacterial pneumonia) Pulmonary colonization (pulmonary fibrosis) PCP Pulmonary colonization (bacterial pneumonia)
Abbreviations: BAL = bronchoalveolar lavage; COPD = chronic obstructive pulmonary disease; F = female; ICU = intensive care unit; M = male; ND = not determined. a Microscopic detection of P. jirovecii was achieved using Wright–Giemsa and Toluidine Blue O stains and an immunofluorescence assay (MonofluoKit P. jirovecii; Bio-Rad). b PCR detection of P. jirovecii was achieved using a real-time PCR assay targeting the mitochondrial large subunit ribosomal RNA gene (Meliani et al., 2003; Totet et al., 2003).
Please cite this article as: Le Gal S, et al, Pneumocystis jirovecii in the air surrounding patients with Pneumocystis pulmonary colonization, Diagn Microbiol Infect Dis (2015), http://dx.doi.org/10.1016/j.diagmicrobio.2015.01.004
S. Le Gal et al. / Diagnostic Microbiology and Infectious Disease xxx (2015) xxx–xxx
from the Hospital Information System database (Horizon Lab™ Information System; McKesson, San Francisco, CA, USA). Patient median age was 53.5 years (range, 29–81 years); the male/female ratio was 9/5. Biological diagnoses of P. jirovecii infection were initially based on P. jirovecii detection in pulmonary samples by microscopy using Wright–Giemsa and Toluidine Blue O stains, an immunofluorescence assay (Monofluo™ Kit P. jirovecii; Bio-Rad, Marnes-la-Coquette, France), and/or a plus/ minus real time PCR assay with primers PCW3 and PCW4 and a TaqMan® MGB probe specific of the gene encoding the mitochondrial large subunit ribosomal RNA (mtLSUrRNA) (Meliani et al., 2003; Totet et al., 2003) after DNA extraction using NucliSens easyMag system (bioMérieux, Marcy l’Etoile, France). The efficiency of DNA extraction in pulmonary samples was controlled using amplification of a single copy human gene (TaqMan® RNase P Control Reagents Kit; Life Technologies Corporation, Carlsbad, CA, USA). The patients were subjected to 1 pulmonary sample, except patient 10 who was subjected to 3 consecutive pulmonary samples, i.e., 1 sputum sample followed by 2 subsequent BAL samples. P. jirovecii infection diagnoses were also based on (1, 3) β-D-glucan (BG) level determination in serum samples using the Fungitell® kit (Associates of Cape Cod, Cape Cod, MA, USA) with a threshold of 100 pg/mL to distinguish PCP and Pneumocystis pulmonary colonization as we described elsewhere (Damiani et al., 2013). Four patients were diagnosed with PCP, and 10 patients were diagnosed with colonization. The fungus was detected by both microscopic examination and PCR assay in 3 PCP patients, whereas it was only detected by the PCR assay in 1 patient with PCP and in the 10 colonized patients. The diagnosis of PCP for this 1 patient was based on clinical and radiological signs compatible with PCP (CDC, 1992), combined with improvement after specific treatment, absence of alternative diagnoses of PCP, and a BG level N100 pg/mL. The 10 patients were considered to be colonized since they presented risk factors for being infected with
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P. jirovecii but had alternative diagnoses of PCP. Six of them had available BG levels, which were b100 pg/mL (Table 1). DNA extracted from pulmonary samples was stored at −80 °C for further P. jirovecii quantification.
2.2. Air sampling and DNA extraction of air samples After Pneumocystis infection diagnoses, air samples were collected in patient's room using the Coriolis® μ air sampler (Bertin Technologies, Montigny-le-Bretonneux, France). The time interval between the date of pulmonary sampling and the date of air sampling ranged from 0 to 8 days (Table 2). The time interval between treatment initiation and first air sampling ranged from 0 to 8 days (Table 2). Three subsequent air samples were collected in patient 10, i.e., after each pulmonary sampling. Five control air samples were collected: 1 from a terrace of the hospital and 4 in the hospital at a distance from the wards in which patients infected with P. jirovecii could be admitted. Eight other air samples were collected in 8 unoccupied dwellings in the Brest region. Each sample, consisting of 1.5 m3 of air collected at 0.3 m 3/min, was collected into a conic sterile tube containing 15 mL of sterile phosphatebuffered saline and 0.002% Tween 80 (Choukri et al., 2010). In all cases, 1 sample was collected at a distance of 5 m (at the entry door), and another one, at 1 m from the patient's head. For sampling, the Coriolis® μ air sampler was placed 1 m from the floor. The door and windows of the patient's room were kept closed for the 1-m sample. For each sample, the collection liquid was centrifuged at 2500g for 10 min, then the supernatant was carefully removed to leave a 1-mL sediment that was divided into 200-μL aliquots and stored at −20 °C. The removable components of sampler were decontaminated after each sampling with a chemical disinfectant (Aniosurf®; Anios, Lille, France) and autoclaved at 121 °C, 15 min.
Table 2 P. jirovecii detection and quantification in pulmonary and air samples from 4 patients with PCP and 10 patients with Pneumocystis pulmonary colonization monitored at the University Hospital of Brest, France. Patient
1 2 3 4 5 6 7 8 9 10a
11 12 13 14
Hospitalization unit (accommodation)
Infectious diseases unit (single room) Pneumology (single room) Pneumology (double room) Pneumology (single room) Infectious diseases unit (single room) Nephrology (single room) Cardiology (single room) ICU (single room) Pneumology (single room) Infectious diseases unit (single room) Infectious diseases unit (single room) Internal medicine (single room) Infectious diseases unit (single room) Infectious diseases unit (single room)
Clinical presentation of P. jirovecii infection
PCP
No. of days between air sampling and Pulmonary sampling
Treatment or prophylaxis initiation
6
4
PCP
1
Pulmonary colonization
0
P. jirovecii quantification in pulmonary sample, DNA copy number/μL (pulmonary sample)
P. jirovecii quantification in air sample at 1 m and 5 m from patient, DNA copy number/m3 1m
2.4 × 101 (sputum) 5
5m
0
0 4
2.3 × 103
4.0 × 10 (BAL)
1.7 × 10
4
9.0 × 100 (BAL)
6.7 × 102
0
Pulmonary colonization
2
2.0 × 102 (BAL)
1.0 × 103
2.2 × 102
Pulmonary colonization
6
2.8 × 101 (BAL)
0
7.8 × 102
Pulmonary colonization
2
6.0 × 100 (BAL)
0
0
Pulmonary colonization
1
1.1 × 101 (BAL)
2.8 × 102
5.3 × 102
Pulmonary colonization Pulmonary colonization
2 7
4 3
3.5 × 101 (BAL) 2.1 × 102 (BAL)
0 0
0 0
PCP
2 9 41
Pulmonary colonization
2 0 4 4
5.0 2.4 5.1 8.6
0 0 6.7 × 102 6.3 × 103
5.6 × 102 0 2.0 × 103 5.3 × 103
Pulmonary colonization
7
7
1.0 × 101 (BAL)
1.0 × 103
2.3 × 103
PCP
0
0
3.3 × 105 (BAL)
6.3 × 106
5.0 × 106
Pulmonary colonization
8
8
5.0 × 100 (BAL)
0
0
0
× × × ×
102 (sputum) 105 (BAL) 103 (BAL) 101 (BAL)
Abbreviations: BAL = bronchoalveolar lavage; ICU = intensive care unit. a Three pulmonary samples were examined (1 sputum on 22-01-13, 2 BAL samples on 31-01-13 and 28-02-13), and 3 subsequent air samples were collected for patient 10.
Please cite this article as: Le Gal S, et al, Pneumocystis jirovecii in the air surrounding patients with Pneumocystis pulmonary colonization, Diagn Microbiol Infect Dis (2015), http://dx.doi.org/10.1016/j.diagmicrobio.2015.01.004
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S. Le Gal et al. / Diagnostic Microbiology and Infectious Disease xxx (2015) xxx–xxx
2.3. P. jirovecii detection and quantification in air and pulmonary samples
3. Results
DNA extraction of air samples was performed by using the QIAamp DNA Mini Kit (Qiagen, Courtaboeuf, France) according to the recommendations of Choukri et al. (2010). DNA was eluted in 100 μL of elution buffer. DNA samples were stored at −80 °C. The quantitative real-time PCR (qPCR) assay was performed on DNA from pulmonary and air samples, with the specific probe and primers of the gene encoding the mtLSUrRNA, as described above. Plasmid suspensions were used as standards for quantification and positive controls as described elsewhere (Choukri et al., 2010). Each PCR run comprised 8 serial 10-fold dilutions of the plasmid suspension, ranging from 1 × 100 to 1 × 107 copies/μL of extracted DNA, and 2 negative controls (ultrapure water). Plasmid dilutions were used to establish a calibration curve giving the correspondence between cycle threshold values and the number of copies per microliter of extracted DNA. All air and pulmonary samples as well as plasmid dilutions and negative controls were run in triplicate. An internal positive control (TaqMan® Exogenous Internal Positive Control Reagents; Life Technologies Corporation) was used to detect PCR inhibitors. The quantity of P. jirovecii DNA in the samples was determined against the standard curve, and the results were expressed as number of P. jirovecii DNA copies/μL of extracted DNA for pulmonary samples and as number of P. jirovecii DNA copies/m3 for air samples.
3.1. P. jirovecii detection and quantification
2.4. P. jirovecii genotyping at the mtLSUrRNA gene P. jirovecii genotyping was performed using mtLSUrRNA gene sequence analysis. First, the genotyping was performed on air samples for which a positive result for P. jirovecii detection was obtained. Second, it was also performed in pulmonary samples corresponding to air samples for which P. jirovecii genotype identification was successful. The mtLSUrRNA sequences were amplified in pulmonary samples from PCP patients using a single-round PCR assay with the primers pAZ102E and pAZ102-H described by Wakefield et al. (1990) and under PCR conditions described elsewhere (Vargas et al., 2000). For pulmonary samples from colonized patients and for all air samples, a hemi-nested PCR assay was performed with the same primers (first round) and the primers pAZ102-E and pAZ102X (Tsolaki et al., 1998) (second round). The PCR products were purified using the QIAquick PCR purification kit (Qiagen) and sequenced directly from the 2 strands using the BigDye terminator v1.1 cycle sequencing kit on the 3130XL Genetic Analyzer (Applied Biosystems, Foster City, CA, USA). Sequence data were analyzed using the 4Peaks software v1.7.2 (Griekspoor and Groothuis; Cancer Institute, Amsterdam, The Netherlands) and aligned with the reference sequence (Sinclair et al., 1991) using the BioEdit software (version 7.0.0, T.Hall; Ibis Biosciences, Carlsbad, CA, USA) with the Clustal® W program. The genotypes were identified focusing principally on the 2 nucleotide positions 85 and 248 (Beard et al., 2000; Tsolaki et al., 1998; Wakefield, 1998). Polymorphism at these positions renders it possible to identify genotypes C85C248, A85C248, T85C248, C85T248, and A85T248 (Beard et al., 2000; Esteves et al., 2010). For each PCR assay, extraction, reagent preparation, and amplification procedures were performed in 3 separate rooms with different sets of micropipettes and using barrier tips to avoid contamination due to potential environmental amplicons. Extraction and reagent preparations were performed in flow cabinets. To monitor for possible contamination, negative controls (ultrapure water) were included in extraction and PCR round procedures.
2.5. Statistical analysis Comparison of the fungal burdens in pulmonary and air samples between PCP and colonized patients was made using the Mann–Whitney test (significance at P b 0.05).
The control air samples were negative for P. jirovecii DNA detection. Other results are detailed in Table 2 and Figs. 1 and 2. P. jirovecii DNA quantification in pulmonary samples of patients with PCP ranged from 2.4 × 10 1 to 4.0 × 10 5 copies/μL of extracted DNA (median, 1.2 × 10 5). P. jirovecii DNA quantification in pulmonary samples of colonized patients ranged from 5.0 × 10 0 to 2.1 × 10 2 copies/μL of extracted DNA (median, 1.9 × 10 1). Fungal burdens in pulmonary samples (sputum samples and BAL samples taken together or BAL samples taken alone) were significantly higher in PCP patients than in colonized patients (P = 0.005 or P = 0.002). At 5 m, P. jirovecii DNA detection was positive in 4 of the 6 air samples collected from the 4 PCP patients. Patient 1 was negative, whereas patients 2, 10, and 13 were positive. Of the 3 air samples from patient 10, 2 were positive. P. jirovecii DNA quantification in the 4 positive samples from the PCP patients ranged from 5.6 × 10 2 to 5.0 × 10 6 copies/m 3 (median, 2.2 × 10 3). At 5 m again, P. jirovecii DNA detection was positive in 5 of the 10 air samples collected from colonized patients. Patients 3, 6, 8, 9, and 14 were negative, whereas patients 4, 5, 7, 11, and 12 were positive. P. jirovecii DNA quantities in these 5 samples ranged from 2.2 × 102 to 5.3 × 103 copies/m3 (mean, 7.8 × 102). Thus, fungal burdens in air samples at 5 m from PCP patients were higher than those from colonized patients at the same distance. Nonetheless, this difference did not appear to be significant (P = 0.5). At 1 m, P. jirovecii DNA detection was positive in 3 of the 6 air samples collected from the 4 PCP patients. Patient 1 was negative at 1 m. Patients 2, 10, and 13 were positive. Three air samples from patient 10 were available; only the third one was positive. P. jirovecii DNA quantities in the 3 positive air samples from the 4 PCP patients ranged from 6.7 × 10 2 to 6.3 × 10 6 copies/m 3 (median, 1.7 × 10 4). At 1 m again, P. jirovecii DNA detection was positive in 5 of the 10 air samples collected from the colonized patients. Patients 5, 6, 8, 9, and 14 were negative, whereas patients 3, 4, 7, 11, and 12 were positive. P. jirovecii DNA quantities in these 5 positive samples ranged from 2.8 × 102 to 6.3 × 103 copies/m3 (median, 1.0 × 103). Thus, fungal burdens in 1-m air samples from PCP patients were higher than those at the same distance from colonized patients. Nonetheless, this difference did not appear to be significant (P = 0.29).
Fig. 1. Pneumocystis DNA quantification in pulmonary samples (DNA copy number per microliter of extracted DNA) from patients with PCP or with Pneumocystis pulmonary colonization (colonization). Median values are represented by horizontal bars. #Fungal burdens in pulmonary samples (sputum samples and BAL samples) were significantly higher in patients with PCP than in patients with Pneumocystis pulmonary colonization (P = 0.005; Mann–Whitney test). mtLSUrRNA = mitochondrial large sub unit ribosomal RNA gene.
Please cite this article as: Le Gal S, et al, Pneumocystis jirovecii in the air surrounding patients with Pneumocystis pulmonary colonization, Diagn Microbiol Infect Dis (2015), http://dx.doi.org/10.1016/j.diagmicrobio.2015.01.004
S. Le Gal et al. / Diagnostic Microbiology and Infectious Disease xxx (2015) xxx–xxx
Fig. 2. Pneumocystis DNA quantification in air samples (DNA copy number per cubic meter of air) collected at 1 m and 5 m from patients with PCP or with Pneumocystis pulmonary colonization (colonization). Median values are represented by horizontal bars. mtLSUrRNA = mitochondrial large sub unit ribosomal RNA gene.
3.2. mtLSUrRNA genotyping results Positive results of P. jirovecii genotyping were obtained in 7 air samples collected at 1 m, in 6 air samples collected at 5 m, and in the 8 corresponding pulmonary samples (Table 3). Four different genotypes were identified, C85C248, A85C 248, T85C 248, and C85T 248. The first 3 genotypes were observed in both patient populations, whereas genotype C85T 248 was observed only in the colonized patients. The genotyping was successful for the pulmonary and air samples in 3 PCP patients (patients 2, 10, and 13) and in 4 colonized patients (patients 3, 4, 11, and 12) (Table 3). A full or partial match of genotypes was observed between 3 pulmonary samples and the corresponding air samples in the 3 PCP patients. A full match of genotypes was observed between 3 pulmonary samples and the corresponding air samples in 3 of the 4 colonized patients. 4. Discussion This study provides the first data on P. jirovecii detection and quantification in air samples collected in the environment of colonized patients using the Coriolis® μ air sampler and additional data on P. jirovecii detection and quantification in air samples collected in the environment of PCP patients using this device, as previously reported by Choukri et al. (2010). Negative results of P. jirovecii DNA detection in control air samples, specifically those out of the hospital, provide additional data on the absence of putative exosaprophytic form of P. jirovecii.
5
A plus/minus real-time PCR assay amplifying the mtLSUrRNA gene has been routinely used to perform diagnoses of P. jirovecii infections in our laboratory for many years. For this reason, this gene was also chosen as the target of the qPCR assay (Choukri et al., 2010). This multicopy gene provides a highly sensitive amplification, which is required for detection of low fungal burden in pulmonary samples specifically in colonized patients, and in air samples. P. jirovecii DNA quantities in BAL samples from PCP patients appeared to be higher than those in BAL samples from colonized patients. Likewise, P. jirovecii DNA quantities in air samples at 1 m and 5 m from PCP patients appeared to be higher than in air samples at the same distances from colonized patients. These results may be consistent with a relationship between fungal burdens in the lungs and fungal burdens in patient environment. However, at present, this relationship cannot be affirmed because of the absence of statistical significance due to the small number of patients enrolled in the study. In 2 patients (1 PCP patient and 1 colonized patient), P. jirovecii DNA was not detected in air samples at 1 m, whereas it was detected at 5 m. These observations seem to be inconsistent with the results by Choukri et al. (2010) who observed that fungal burdens in air samples decreased with the distance from the patient. Our results may be explained by the fact that we did not strictly follow the method used by this author (Choukri et al., 2010). Air samples at 5 m were collected first, and air samples at 1 m were collected second. We collected air samples at 5 m with the door open, in contrast to the study by Choukri et al. (2010), during which air samples at 5 m were collected with the door closed. The opening of the door may have provoked air movement from inside to outside the room. This air movement may have increased the P. jirovecii air burden at 5 m, rendering it easier to detect the fungus at this distance. Conversely, the P. jirovecii air burden at 1 m may have contemporarily decreased, limiting the detection at this distance. This is still a hypothesis since in 4 other patients, 2 PCP patients and 2 colonized patients, P. jirovecii DNA quantities in air samples at 5 m were effectively lower than in air samples at 1 m, consistent with results by Choukri et al. (2010). P. jirovecii DNA was not detected in the air surrounding 2 PCP patients: patient 1 and patient 10 (air samples at 1 m and 5 m corresponding to the second pulmonary sample of this patient 10). The results may be explained by the time interval between air sampling and the start of treatment: 4 days for patient 1 and 9 days for patient 10. During these respective periods of 4 and 9 days, specific treatment for P. jirovecii may have reduced i) P. jirovecii pulmonary burdens; ii) P. jirovecii exhalation; and, consequently, iii) fungal burdens in the corresponding air samples. Again, identical reasons may explain the fact that P. jirovecii DNA quantities in air samples from PCP patient 10 who was treated for P. jirovecii were lower than those from colonized patients 11 and 12. Nonetheless, data on the reduction of P. jirovecii burdens in the lungs and on the kinetics of the fungal exhalation in the course of treatment are still unavailable. P. jirovecii DNA was not detected in the air surrounding 4
Table 3 P. jirovecii genotyping at the mitochondrial large subunit ribosomal RNA gene in pulmonary and air samples from patients for whom P. jirovecii DNA was detected in air samples. Patient
Clinical presentation of P. jirovecii infection
mtLSUrRNA genotype Pulmonary sample
1-m air sample
5-m air sample
2 3 4 5 7 10a
PCP Pulmonary colonization Pulmonary colonization Pulmonary colonization Pulmonary colonization PCP
C85C248; A85C248; T85C248 T85C248 A85C248
A85C248; T85C248
11 12 13
Pulmonary colonization Pulmonary colonization PCP
C85C248; A85C248; T85C248 T85C248 A85C248 ND ND A85C248 A85C248 C85C248; C85T248 A85C248 C85C248
UD A85C248; C85C248 C85C248; C85T248 C85C248; C85T248 C85C248
UD UD A85C248 C85C248 C85C248; C85T248 C85C248 C85C248
Abbreviations: ND = not determined; UD = unavailable data (the genotyping was not successful). a For this patient, 3 pulmonary specimens were examined (1 sputum on 22-01-13, 2 BAL samples on 31-01-13 and 28-02-13), and 3 subsequent air samples were collected. P. jirovecii DNA was detected in air samples corresponding to the sputum sample and the second BAL sample.
Please cite this article as: Le Gal S, et al, Pneumocystis jirovecii in the air surrounding patients with Pneumocystis pulmonary colonization, Diagn Microbiol Infect Dis (2015), http://dx.doi.org/10.1016/j.diagmicrobio.2015.01.004
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S. Le Gal et al. / Diagnostic Microbiology and Infectious Disease xxx (2015) xxx–xxx
colonized patients. These results were expected since, by definition, colonized patients are characterized by having low fungal pulmonary burdens and, consequently, exhaling low and potentially undetectable quantities of P. jirovecii. Risk factors for being a colonized patient exhaling P. jirovecii or not exhaling P. jirovecii were not identified. No matches of genotypes were observed in 2 pairs of air and pulmonary samples (patients 10 and 12). To explain the discrepancy observed in patient 10, we hypothesized that P. jirovecii identified in the air sample taken at 5 m on 04-03-13 (genotype C 85C 248) was not exhaled by this patient and came from another source. Indeed, it is noteworthy that patient 13, who developed PCP and who was infected by genotype C85C248, was hospitalized in the same unit at the same period. Nonetheless, encounters between patients 10 and 13 as well as between other patients that may identify potential human infectious sources were not objectivized. In contrast, the reason for the absence of genotype matches in colonized patient 12 remains unclear. Thus, P. jirovecii DNA was detected in the air surrounding 3 PCP patients and 6 colonized patients with genotype matches in 3 pairs (or triplets) of air and pulmonary samples in the first patient group and in 3 pairs (or triplets) of air and pulmonary samples in the second patient group. These results provide additional data concerning P. jirovecii diffusion from PCP patients and original data regarding this diffusion from colonized patients. The CDC recommends applying standard precautions and avoiding placement of a patient developing PCP in the same room with an immunocompromised patient (Siegel et al., 2007). Our present findings combined with our previous data on the role of colonized patients as potential infectious sources (Le Gal et al., 2012) and with those of Pneumocystis transmission from colonized rodent models (Dumoulin et al., 2000; Gigliotti et al., 2003) currently seem to be sufficient to extend these measures to colonized patients. Finally, our observations emphasize the risk of nosocomial transmission of P. jirovecii via the airborne route from diverse patients harboring the fungus, PCP patients or colonized patients. Acknowledgments The authors would like to thank Dr D. Quinio and Mrs C. Carrou, P. Lecordier, D. Roué, V. Abiven, C. Cam, C. Le Guen, K. Quinaou, and C. Roger for performing biological diagnoses of Pneumocystis infections. The authors would like to thank Pr EM Aliouat who has contributed to the Pneumair project. References Bartlett MS, Vermund SH, Jacobs R, Durant PJ, Shaw MM, Smith JW, et al. Detection of Pneumocystis carinii DNA in air samples: likely environmental risk to susceptible persons. J Clin Microbiol 1997;35:2511–3. Beard CB, Carter JL, Keely SP, Huang L, Pieniazek NJ, Moura IN, et al. Genetic variation in Pneumocystis carinii isolates from different geographic regions: implications for transmission. Emerg Infect Dis 2000;6:265–72. Centers for Disease Control and Prevention (CDC). Revised classification system for HIV infection and expanded surveillance case definition for AIDS among adolescents and adults. MMWR Recomm Rep 1992;41:1–19.
Choukri F, Menotti J, Sarfati C, Lucet JC, Nevez G, Garin YJ, et al. Quantification and spread of Pneumocystis jirovecii in the surrounding air of patients with Pneumocystis pneumonia. Clin Infect Dis 2010;51:259–65. Damiani C, Choukri F, Le Gal S, Menotti J, Sarfati C, Nevez G, et al. Possible nosocomial transmission of Pneumocystis jirovecii. Emerg Infect Dis 2012;18:877–8. Damiani C, Le Gal S, Da Costa C, Virmaux M, Nevez G, Totet A. Combined quantification of pulmonary Pneumocystis jirovecii DNA and serum (1-N3) beta-D-glucan for differential diagnosis of Pneumocystis pneumonia and Pneumocystis colonization. J Clin Microbiol 2013;51:3380–8. Dumoulin A, Mazars E, Seguy N, Gargallo-Viola D, Vargas S, Cailliez JC, et al. Transmission of Pneumocystis carinii disease from immunocompetent contacts of infected hosts to susceptible hosts. Eur J Clin Microbiol Infect Dis 2000;19:671–8. Esteves F, Gaspar J, Marques T, Leite R, Antunes F, Mansinho K, et al. Identification of relevant single-nucleotide polymorphisms in Pneumocystis jirovecii: relationship with clinical data. Clin Microbiol Infect 2010;16:878–84. Gigliotti F, Harmsen AG, Wright TW. Characterization of transmission of Pneumocystis carinii f. sp. muris through immunocompetent BALB/c mice. Infect Immun 2003;71: 3852–6. Hughes WT. Natural mode of acquisition for de novo infection with Pneumocystis carinii. J Infect Dis 1982;145:842–8. Le Gal S, Damiani C, Rouille A, Grall A, Treguer L, Virmaux M, et al. A cluster of Pneumocystis infections among renal transplant recipients: molecular evidence of colonized patients as potential infectious sources of Pneumocystis jirovecii. Clin Infect Dis 2012;54:e62–71. Meliani L, Develoux M, Marteau-Miltgen M, Magne D, Barbu V, Poirot JL, et al. Real time quantitative PCR assay for Pneumocystis jirovecii detection. J Eukaryot Microbiol 2003;50(Suppl):651. Morris A, Norris KA. Colonization by Pneumocystis jirovecii and its role in disease. Clin Microbiol Rev 2012;25:297–317. Nevez G, Chabe M, Rabodonirina M, Virmaux M, Dei-Cas E, Hauser PM, et al. Nosocomial Pneumocystis jirovecii infections. Parasite 2008;15:359–65. Nevez G, Raccurt C, Vincent P, Jounieaux V, Dei-Cas E. Pulmonary colonization with Pneumocystis carinii in human immunodeficiency virus-negative patients: assessing risk with blood CD4+ T cell counts. Clin Infect Dis 1999;29:1331–2. Nevez G, Totet A, Jounieaux V, Schmit JL, Dei-Cas E, Raccurt C. Pneumocystis jirovecii internal transcribed spacer types in patients colonized by the fungus and in patients with pneumocystosis from the same French geographic region. J Clin Microbiol 2003;41:181–6. Olsson M, Lidman C, Latouche S, Bjorkman A, Roux P, Linder E, et al. Identification of Pneumocystis carinii f. sp. hominis gene sequences in filtered air in hospital environments. J Clin Microbiol 1998;36:1737–40. Olsson M, Sukura A, Lindberg LA, Linder E. Detection of Pneumocystis carinii DNA by filtration of air. Scand J Infect Dis 1996;28:279–82. Siegel JD, Rhinehart E, Jackson M, Chiarello L. Guideline for isolation precautions: preventing transmission of infectious agents in health care settings. Am J Infect Control 2007;35:S65–S164. Sinclair K, Wakefield AE, Banerji S, Hopkin JM. Pneumocystis carinii organisms derived from rat and human hosts are genetically distinct. Mol Biochem Parasitol 1991;45: 183–4. Soulez B, Palluault F, Cesbron JY, Dei-Cas E, Capron A, Camus D. Introduction of Pneumocystis carinii in a colony of SCID mice. J Protozool 1991;38:123S–5S. Stringer JR, Beard CB, Miller RF, Wakefield AE. A new name (Pneumocystis jirovecii) for Pneumocystis from humans. Emerg Infect Dis 2002;8:891–6. Totet A, Meliani L, Lacube P, Pautard JC, Raccurt C, Roux P, et al. Immunocompetent infants as a human reservoir for Pneumocystis jirovecii: rapid screening by noninvasive sampling and real-time PCR at the mitochondrial large subunit rRNA gene. J Eukaryot Microbiol 2003;50(Suppl):668–9. Tsolaki AG, Beckers P, Wakefield AE. Pre-AIDS era isolates of Pneumocystis carinii f. sp. hominis: high genotype similarity with contemporary isolates. J Clin Microbiol 1998;36:90–3. Vargas SL, Ponce CA, Gigliotti F, Ulloa AV, Prieto S, Munoz MP, et al. Transmission of Pneumocystis carinii DNA from a patient with P. carinii pneumonia to immunocompetent contact health care workers. J Clin Microbiol 2000;38:1536–8. Wakefield AE. Genetic heterogeneity in human-derived Pneumocystis carinii. FEMS Immunol Med Microbiol 1998;22:59–65. Wakefield AE, Pixley FJ, Banerji S, Sinclair K, Miller RF, Moxon ER, et al. Detection of Pneumocystis carinii with DNA amplification. Lancet 1990;336:451–3. Walzer PD, Cushion MT. Pneumocystis pneumonia. New York: Marcel Dekker; 2005.
Please cite this article as: Le Gal S, et al, Pneumocystis jirovecii in the air surrounding patients with Pneumocystis pulmonary colonization, Diagn Microbiol Infect Dis (2015), http://dx.doi.org/10.1016/j.diagmicrobio.2015.01.004
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Pneumocystis jirovecii in the air surrounding patients with Pneumocystis pulmonary colonization☆,☆☆,★ Solène Le Gal a,b,⁎, Laurence Pougnet c, Céline Damiani d,e, Emilie Fréalle f, Paul Guéguen g,h, Michèle Virmaux a, Séverine Ansart i,j, Sylvain Jaffuel i, Francis Couturaud k,l, Aurélien Delluc k,l, Jean-Marie Tonnelier m, Philippe Castellant n, Yann Le Meur o,p, Gaétan Le Floch a, Anne Totet d,e,1, Jean Menotti q,r,1, Gilles Nevez a,b,⁎,1 a
University of Brest, LUBEM EA 3882, SFR 148, Brest, France Laboratory of Parasitology and Mycology, Brest University Hospital, Brest, France c Laboratory, Military Teaching Hospital Clermont Tonnerre, Brest, France d University of Picardy-Jules Verne, UMR-I 01, SFR Cap Santé, Amiens, France e Laboratory of Parasitology and Mycology, Amiens University Hospital, Amiens, France f Laboratory of Parasitology and Mycology, Lille University Hospital, Lille, France g Laboratory of Molecular Genetics and Histocompatibility, Brest University Hospital, Brest, France h University of Brest, INSERM U1078, Molecular Genetics and Epidemiological Genetics, SFR 148, Brest, France i Department of Infectious Diseases, Brest University Hospital, Brest, France j University of Brest, INSERM UMR 1101, Laboratory of Medical Information Processing, SFR 148, Brest, France k Department of Internal Medicine and Pneumology, Brest University Hospital, Brest, France l University of Brest, EA3878 (GETBO), CIC INSERM 0502, SFR 148, Brest, France m Medical Intensive Care Unit, Brest University Hospital, Brest, France n Department of Cardiology, Brest University Hospital, Brest, France o Department of Nephrology and Renal Transplantation Unit, Brest University Hospital, Brest, France p University of Brest, EA 2216, SFR 148, Brest, France q Laboratory of Parasitology and Mycology, Saint Louis Hospital APHP, Paris, France r Paris-Diderot University, Paris, France b
a r t i c l e
i n f o
Article history: Received 5 December 2014 Received in revised form 9 January 2015 Accepted 11 January 2015 Available online xxxx Keywords: Pneumocystis jirovecii Colonization Airborne transmission Exhalation
a b s t r a c t In this study, Pneumocystis jirovecii was detected and characterized in the air surrounding patients with Pneumocystis pulmonary colonization. Air samples were collected in the rooms of 10 colonized patients using Coriolis® μ air sampler at 1 m and 5 m from the patient's head. P. jirovecii DNA was amplified and genotyped in pulmonary and air samples at the mitochondrial large subunit ribosomal RNA gene. P. jirovecii DNA was detected in 5 of the 10 air samples collected at 1 m and in 5 of the 10 other air samples collected at 5 m. P. jirovecii genotyping was successful in 4 pairs or triplets of air and pulmonary samples. Full genotype matches were observed in 3 of the 4 pairs or triplets of air and pulmonary samples. These results provide original data supporting P. jirovecii exhalation from colonized patients and emphasize the risk of P. jirovecii nosocomial transmission from this patient population. © 2015 Elsevier Inc. All rights reserved.
1. Introduction Pneumocystis jirovecii is a fungus that causes severe pneumonia in human immunodeficiency virus (HIV)–infected patients and in non– ☆ Financial support: This study was supported by the Agence nationale de sécurité sanitaire de l'alimentation, de l'environnement et du travail (Anses) (Pneumair project; grant number 2011/1/053). ☆☆ Potential conflict of interest: The authors report no conflicts of interest. The authors are responsible for the content and the writing of this paper. ★ The results of this study were presented in part at the 13th International Workshops on Opportunistic Protists, Seville, Spain, 13–15 November 2014, abstract T26. ⁎ Corresponding authors. Tel.: +33-2-98-14-51-02; fax: +33-2-98-14-51-49. E-mail addresses: [email protected] (S. Le Gal), [email protected] (G. Nevez). 1
These authors contributed equally to this work.
HIV-infected patients who receive immunosuppressive therapy (Walzer and Cushion, 2005). Pneumocystis pneumonia (PCP) represents only part of the clinical presentations of Pneumocystis infections, while mild infections, such as pulmonary colonization occurring in patients with diverse levels of immunodeficiency or lung diseases, account for the main part (Morris and Norris, 2012; Nevez et al., 1999). Since Pneumocystis microorganisms infecting each mammalian species are host specific (Stringer et al., 2002), an animal reservoir for P. jirovecii can be excluded. Moreover, as no exosaprophytic form of Pneumocystis spp. has been identified so far, humans harboring P. jirovecii, whatever their clinical presentation, PCP or pulmonary colonization, may represent the fungus reservoir and potential infectious sources for susceptible individuals (Nevez et al., 2003). We recently provided data on the role of colonized patients as potential sources of P. jirovecii in the context of nosocomial acquisition of
http://dx.doi.org/10.1016/j.diagmicrobio.2015.01.004 0732-8893/© 2015 Elsevier Inc. All rights reserved.
Please cite this article as: Le Gal S, et al, Pneumocystis jirovecii in the air surrounding patients with Pneumocystis pulmonary colonization, Diagn Microbiol Infect Dis (2015), http://dx.doi.org/10.1016/j.diagmicrobio.2015.01.004
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S. Le Gal et al. / Diagnostic Microbiology and Infectious Disease xxx (2015) xxx–xxx
the fungus (Le Gal et al., 2012; Nevez et al., 2008). These observations are consistent with that of experimental infections in the course of which colonized Balb/c mice can transmit Pneumocystis murina to susceptible SCID mice that subsequently developed PCP (Dumoulin et al., 2000; Gigliotti et al., 2003). The airborne route of transmission of Pneumocystis spp. has been demonstrated in rodent models (Hughes, 1982; Soulez et al., 1991), and this route for P. jirovecii transmission in humans has been suggested (Bartlett et al., 1997; Choukri et al., 2010; Damiani et al., 2012; Olsson et al., 1996, 1998). Recently, P. jirovecii DNA has been detected and quantified in the air surrounding PCP patients, suggesting the spread of P. jirovecii from these patients within their environment (Choukri et al., 2010). In contrast, the exhalation of P. jirovecii from colonized patients has not been yet investigated.
P. jirovecii detection in the surrounding environment of colonized patients would strengthen their potential role as infectious sources. In this context, we performed the detection, quantification, and characterization of P. jirovecii DNA in air samples from the surrounding environment of 10 colonized patients and 4 PCP patients. 2. Materials and methods 2.1. Patients and diagnoses of P. jirovecii infections The 14 patients diagnosed with Pneumocystis infections in the Brest University Hospital from November 2012 through March 2013 were enrolled in the study (Table 1). Patients' characteristics were extracted
Table 1 Characteristics of 4 patients with PCP and 10 patients with Pneumocystis pulmonary colonization monitored at the University Hospital of Brest, France, for whom Pneumocystis presence in air samples from their surrounding environment was investigated. Date of pulmonary sample retrieval (daymo-year)
Results of P. jirovecii detection in pulmonary samples using microscopy a/PCR b
(1,3) β-D glucan serum level (pg/mL)
No. of days between serum and pulmonary sampling
Clinical presentation of P. jirovecii infection (alternative diagnosis of PCP)
Sputum
27-11-12
ND/+
5024
−1
PCP
BAL
28-11-12
+/+
751
0
PCP
BAL
30-11-12
−/+
25
3
Pulmonary colonization (bacterial pneumonia) Pulmonary colonization (exacerbation of chronic bronchitis) Pulmonary colonization (bronchiolitis obliterans) Pulmonary colonization (pulmonary atelectasis) Pulmonary colonization (bacterial pneumonia) Pulmonary colonization (bacterial pneumonia) Pulmonary colonization (bacterial pneumonia) PCP
Immunosuppressive Pulmonary Patient Sex Age Underlying treatment samples (y) conditions (CD4+ cell counts in blood) examined for P. jirovecii presence 1
M
51
2
F
50
HIV infection (54 × 106/L) Glioblastoma (ND)
3
M
81
COPD (ND)
4
F
70
COPD, pulmonary fibrosis (2928 × 106/L)
Inhaled corticosteroids
BAL
03-12-12
−/+
20
−8
5
M
56
Emphysema and COPD (ND)
Inhaled corticosteroids
BAL
06-12-12
−/+
ND
ND
6
M
31
HBV infection, renal transplant (400 × 106/L)
BAL
18-12-12
−/+
47
1
7
M
71
Emphysema (ND)
BAL
07-01-13
−/+
ND
ND
8
F
73
Lymphoma with leucopenia (ND)
BAL
15-01-13
−/+
71
2
9
F
32
BAL
18-01-13
−/+
ND
ND
10
M
35
Common variable immunodeficiency; bronchiectasis (200 × 106/L) HIV infection (10 × 106/L)
11
M
66
COPD (ND)
Sputum BAL BAL BAL
22-01-13 31-01-13 28-02-13 25-01-13
ND/+ +/+ +/+ −/+
648 ND 502 ND
0 ND −1 ND
12
F
76
BAL
21-02-13
−/+
b8
1
13
M
35
BAL
27-02-13
+/+
1159
0
14
M
29
Antisynthetase syndrome (1045 × 106/L) HIV infection (14 × 106/L) HIV infection (1 × 106/L)
BAL
13-03-13
−/+
46
1
Oral corticotherapy Temozolomide Inhaled corticosteroids
Rituximab Fludarabine Cyclophosphamide
Oral corticotherapy
Pulmonary colonization (bacterial pneumonia) Pulmonary colonization (pulmonary fibrosis) PCP Pulmonary colonization (bacterial pneumonia)
Abbreviations: BAL = bronchoalveolar lavage; COPD = chronic obstructive pulmonary disease; F = female; ICU = intensive care unit; M = male; ND = not determined. a Microscopic detection of P. jirovecii was achieved using Wright–Giemsa and Toluidine Blue O stains and an immunofluorescence assay (MonofluoKit P. jirovecii; Bio-Rad). b PCR detection of P. jirovecii was achieved using a real-time PCR assay targeting the mitochondrial large subunit ribosomal RNA gene (Meliani et al., 2003; Totet et al., 2003).
Please cite this article as: Le Gal S, et al, Pneumocystis jirovecii in the air surrounding patients with Pneumocystis pulmonary colonization, Diagn Microbiol Infect Dis (2015), http://dx.doi.org/10.1016/j.diagmicrobio.2015.01.004
S. Le Gal et al. / Diagnostic Microbiology and Infectious Disease xxx (2015) xxx–xxx
from the Hospital Information System database (Horizon Lab™ Information System; McKesson, San Francisco, CA, USA). Patient median age was 53.5 years (range, 29–81 years); the male/female ratio was 9/5. Biological diagnoses of P. jirovecii infection were initially based on P. jirovecii detection in pulmonary samples by microscopy using Wright–Giemsa and Toluidine Blue O stains, an immunofluorescence assay (Monofluo™ Kit P. jirovecii; Bio-Rad, Marnes-la-Coquette, France), and/or a plus/ minus real time PCR assay with primers PCW3 and PCW4 and a TaqMan® MGB probe specific of the gene encoding the mitochondrial large subunit ribosomal RNA (mtLSUrRNA) (Meliani et al., 2003; Totet et al., 2003) after DNA extraction using NucliSens easyMag system (bioMérieux, Marcy l’Etoile, France). The efficiency of DNA extraction in pulmonary samples was controlled using amplification of a single copy human gene (TaqMan® RNase P Control Reagents Kit; Life Technologies Corporation, Carlsbad, CA, USA). The patients were subjected to 1 pulmonary sample, except patient 10 who was subjected to 3 consecutive pulmonary samples, i.e., 1 sputum sample followed by 2 subsequent BAL samples. P. jirovecii infection diagnoses were also based on (1, 3) β-D-glucan (BG) level determination in serum samples using the Fungitell® kit (Associates of Cape Cod, Cape Cod, MA, USA) with a threshold of 100 pg/mL to distinguish PCP and Pneumocystis pulmonary colonization as we described elsewhere (Damiani et al., 2013). Four patients were diagnosed with PCP, and 10 patients were diagnosed with colonization. The fungus was detected by both microscopic examination and PCR assay in 3 PCP patients, whereas it was only detected by the PCR assay in 1 patient with PCP and in the 10 colonized patients. The diagnosis of PCP for this 1 patient was based on clinical and radiological signs compatible with PCP (CDC, 1992), combined with improvement after specific treatment, absence of alternative diagnoses of PCP, and a BG level N100 pg/mL. The 10 patients were considered to be colonized since they presented risk factors for being infected with
3
P. jirovecii but had alternative diagnoses of PCP. Six of them had available BG levels, which were b100 pg/mL (Table 1). DNA extracted from pulmonary samples was stored at −80 °C for further P. jirovecii quantification.
2.2. Air sampling and DNA extraction of air samples After Pneumocystis infection diagnoses, air samples were collected in patient's room using the Coriolis® μ air sampler (Bertin Technologies, Montigny-le-Bretonneux, France). The time interval between the date of pulmonary sampling and the date of air sampling ranged from 0 to 8 days (Table 2). The time interval between treatment initiation and first air sampling ranged from 0 to 8 days (Table 2). Three subsequent air samples were collected in patient 10, i.e., after each pulmonary sampling. Five control air samples were collected: 1 from a terrace of the hospital and 4 in the hospital at a distance from the wards in which patients infected with P. jirovecii could be admitted. Eight other air samples were collected in 8 unoccupied dwellings in the Brest region. Each sample, consisting of 1.5 m3 of air collected at 0.3 m 3/min, was collected into a conic sterile tube containing 15 mL of sterile phosphatebuffered saline and 0.002% Tween 80 (Choukri et al., 2010). In all cases, 1 sample was collected at a distance of 5 m (at the entry door), and another one, at 1 m from the patient's head. For sampling, the Coriolis® μ air sampler was placed 1 m from the floor. The door and windows of the patient's room were kept closed for the 1-m sample. For each sample, the collection liquid was centrifuged at 2500g for 10 min, then the supernatant was carefully removed to leave a 1-mL sediment that was divided into 200-μL aliquots and stored at −20 °C. The removable components of sampler were decontaminated after each sampling with a chemical disinfectant (Aniosurf®; Anios, Lille, France) and autoclaved at 121 °C, 15 min.
Table 2 P. jirovecii detection and quantification in pulmonary and air samples from 4 patients with PCP and 10 patients with Pneumocystis pulmonary colonization monitored at the University Hospital of Brest, France. Patient
1 2 3 4 5 6 7 8 9 10a
11 12 13 14
Hospitalization unit (accommodation)
Infectious diseases unit (single room) Pneumology (single room) Pneumology (double room) Pneumology (single room) Infectious diseases unit (single room) Nephrology (single room) Cardiology (single room) ICU (single room) Pneumology (single room) Infectious diseases unit (single room) Infectious diseases unit (single room) Internal medicine (single room) Infectious diseases unit (single room) Infectious diseases unit (single room)
Clinical presentation of P. jirovecii infection
PCP
No. of days between air sampling and Pulmonary sampling
Treatment or prophylaxis initiation
6
4
PCP
1
Pulmonary colonization
0
P. jirovecii quantification in pulmonary sample, DNA copy number/μL (pulmonary sample)
P. jirovecii quantification in air sample at 1 m and 5 m from patient, DNA copy number/m3 1m
2.4 × 101 (sputum) 5
5m
0
0 4
2.3 × 103
4.0 × 10 (BAL)
1.7 × 10
4
9.0 × 100 (BAL)
6.7 × 102
0
Pulmonary colonization
2
2.0 × 102 (BAL)
1.0 × 103
2.2 × 102
Pulmonary colonization
6
2.8 × 101 (BAL)
0
7.8 × 102
Pulmonary colonization
2
6.0 × 100 (BAL)
0
0
Pulmonary colonization
1
1.1 × 101 (BAL)
2.8 × 102
5.3 × 102
Pulmonary colonization Pulmonary colonization
2 7
4 3
3.5 × 101 (BAL) 2.1 × 102 (BAL)
0 0
0 0
PCP
2 9 41
Pulmonary colonization
2 0 4 4
5.0 2.4 5.1 8.6
0 0 6.7 × 102 6.3 × 103
5.6 × 102 0 2.0 × 103 5.3 × 103
Pulmonary colonization
7
7
1.0 × 101 (BAL)
1.0 × 103
2.3 × 103
PCP
0
0
3.3 × 105 (BAL)
6.3 × 106
5.0 × 106
Pulmonary colonization
8
8
5.0 × 100 (BAL)
0
0
0
× × × ×
102 (sputum) 105 (BAL) 103 (BAL) 101 (BAL)
Abbreviations: BAL = bronchoalveolar lavage; ICU = intensive care unit. a Three pulmonary samples were examined (1 sputum on 22-01-13, 2 BAL samples on 31-01-13 and 28-02-13), and 3 subsequent air samples were collected for patient 10.
Please cite this article as: Le Gal S, et al, Pneumocystis jirovecii in the air surrounding patients with Pneumocystis pulmonary colonization, Diagn Microbiol Infect Dis (2015), http://dx.doi.org/10.1016/j.diagmicrobio.2015.01.004
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S. Le Gal et al. / Diagnostic Microbiology and Infectious Disease xxx (2015) xxx–xxx
2.3. P. jirovecii detection and quantification in air and pulmonary samples
3. Results
DNA extraction of air samples was performed by using the QIAamp DNA Mini Kit (Qiagen, Courtaboeuf, France) according to the recommendations of Choukri et al. (2010). DNA was eluted in 100 μL of elution buffer. DNA samples were stored at −80 °C. The quantitative real-time PCR (qPCR) assay was performed on DNA from pulmonary and air samples, with the specific probe and primers of the gene encoding the mtLSUrRNA, as described above. Plasmid suspensions were used as standards for quantification and positive controls as described elsewhere (Choukri et al., 2010). Each PCR run comprised 8 serial 10-fold dilutions of the plasmid suspension, ranging from 1 × 100 to 1 × 107 copies/μL of extracted DNA, and 2 negative controls (ultrapure water). Plasmid dilutions were used to establish a calibration curve giving the correspondence between cycle threshold values and the number of copies per microliter of extracted DNA. All air and pulmonary samples as well as plasmid dilutions and negative controls were run in triplicate. An internal positive control (TaqMan® Exogenous Internal Positive Control Reagents; Life Technologies Corporation) was used to detect PCR inhibitors. The quantity of P. jirovecii DNA in the samples was determined against the standard curve, and the results were expressed as number of P. jirovecii DNA copies/μL of extracted DNA for pulmonary samples and as number of P. jirovecii DNA copies/m3 for air samples.
3.1. P. jirovecii detection and quantification
2.4. P. jirovecii genotyping at the mtLSUrRNA gene P. jirovecii genotyping was performed using mtLSUrRNA gene sequence analysis. First, the genotyping was performed on air samples for which a positive result for P. jirovecii detection was obtained. Second, it was also performed in pulmonary samples corresponding to air samples for which P. jirovecii genotype identification was successful. The mtLSUrRNA sequences were amplified in pulmonary samples from PCP patients using a single-round PCR assay with the primers pAZ102E and pAZ102-H described by Wakefield et al. (1990) and under PCR conditions described elsewhere (Vargas et al., 2000). For pulmonary samples from colonized patients and for all air samples, a hemi-nested PCR assay was performed with the same primers (first round) and the primers pAZ102-E and pAZ102X (Tsolaki et al., 1998) (second round). The PCR products were purified using the QIAquick PCR purification kit (Qiagen) and sequenced directly from the 2 strands using the BigDye terminator v1.1 cycle sequencing kit on the 3130XL Genetic Analyzer (Applied Biosystems, Foster City, CA, USA). Sequence data were analyzed using the 4Peaks software v1.7.2 (Griekspoor and Groothuis; Cancer Institute, Amsterdam, The Netherlands) and aligned with the reference sequence (Sinclair et al., 1991) using the BioEdit software (version 7.0.0, T.Hall; Ibis Biosciences, Carlsbad, CA, USA) with the Clustal® W program. The genotypes were identified focusing principally on the 2 nucleotide positions 85 and 248 (Beard et al., 2000; Tsolaki et al., 1998; Wakefield, 1998). Polymorphism at these positions renders it possible to identify genotypes C85C248, A85C248, T85C248, C85T248, and A85T248 (Beard et al., 2000; Esteves et al., 2010). For each PCR assay, extraction, reagent preparation, and amplification procedures were performed in 3 separate rooms with different sets of micropipettes and using barrier tips to avoid contamination due to potential environmental amplicons. Extraction and reagent preparations were performed in flow cabinets. To monitor for possible contamination, negative controls (ultrapure water) were included in extraction and PCR round procedures.
2.5. Statistical analysis Comparison of the fungal burdens in pulmonary and air samples between PCP and colonized patients was made using the Mann–Whitney test (significance at P b 0.05).
The control air samples were negative for P. jirovecii DNA detection. Other results are detailed in Table 2 and Figs. 1 and 2. P. jirovecii DNA quantification in pulmonary samples of patients with PCP ranged from 2.4 × 10 1 to 4.0 × 10 5 copies/μL of extracted DNA (median, 1.2 × 10 5). P. jirovecii DNA quantification in pulmonary samples of colonized patients ranged from 5.0 × 10 0 to 2.1 × 10 2 copies/μL of extracted DNA (median, 1.9 × 10 1). Fungal burdens in pulmonary samples (sputum samples and BAL samples taken together or BAL samples taken alone) were significantly higher in PCP patients than in colonized patients (P = 0.005 or P = 0.002). At 5 m, P. jirovecii DNA detection was positive in 4 of the 6 air samples collected from the 4 PCP patients. Patient 1 was negative, whereas patients 2, 10, and 13 were positive. Of the 3 air samples from patient 10, 2 were positive. P. jirovecii DNA quantification in the 4 positive samples from the PCP patients ranged from 5.6 × 10 2 to 5.0 × 10 6 copies/m 3 (median, 2.2 × 10 3). At 5 m again, P. jirovecii DNA detection was positive in 5 of the 10 air samples collected from colonized patients. Patients 3, 6, 8, 9, and 14 were negative, whereas patients 4, 5, 7, 11, and 12 were positive. P. jirovecii DNA quantities in these 5 samples ranged from 2.2 × 102 to 5.3 × 103 copies/m3 (mean, 7.8 × 102). Thus, fungal burdens in air samples at 5 m from PCP patients were higher than those from colonized patients at the same distance. Nonetheless, this difference did not appear to be significant (P = 0.5). At 1 m, P. jirovecii DNA detection was positive in 3 of the 6 air samples collected from the 4 PCP patients. Patient 1 was negative at 1 m. Patients 2, 10, and 13 were positive. Three air samples from patient 10 were available; only the third one was positive. P. jirovecii DNA quantities in the 3 positive air samples from the 4 PCP patients ranged from 6.7 × 10 2 to 6.3 × 10 6 copies/m 3 (median, 1.7 × 10 4). At 1 m again, P. jirovecii DNA detection was positive in 5 of the 10 air samples collected from the colonized patients. Patients 5, 6, 8, 9, and 14 were negative, whereas patients 3, 4, 7, 11, and 12 were positive. P. jirovecii DNA quantities in these 5 positive samples ranged from 2.8 × 102 to 6.3 × 103 copies/m3 (median, 1.0 × 103). Thus, fungal burdens in 1-m air samples from PCP patients were higher than those at the same distance from colonized patients. Nonetheless, this difference did not appear to be significant (P = 0.29).
Fig. 1. Pneumocystis DNA quantification in pulmonary samples (DNA copy number per microliter of extracted DNA) from patients with PCP or with Pneumocystis pulmonary colonization (colonization). Median values are represented by horizontal bars. #Fungal burdens in pulmonary samples (sputum samples and BAL samples) were significantly higher in patients with PCP than in patients with Pneumocystis pulmonary colonization (P = 0.005; Mann–Whitney test). mtLSUrRNA = mitochondrial large sub unit ribosomal RNA gene.
Please cite this article as: Le Gal S, et al, Pneumocystis jirovecii in the air surrounding patients with Pneumocystis pulmonary colonization, Diagn Microbiol Infect Dis (2015), http://dx.doi.org/10.1016/j.diagmicrobio.2015.01.004
S. Le Gal et al. / Diagnostic Microbiology and Infectious Disease xxx (2015) xxx–xxx
Fig. 2. Pneumocystis DNA quantification in air samples (DNA copy number per cubic meter of air) collected at 1 m and 5 m from patients with PCP or with Pneumocystis pulmonary colonization (colonization). Median values are represented by horizontal bars. mtLSUrRNA = mitochondrial large sub unit ribosomal RNA gene.
3.2. mtLSUrRNA genotyping results Positive results of P. jirovecii genotyping were obtained in 7 air samples collected at 1 m, in 6 air samples collected at 5 m, and in the 8 corresponding pulmonary samples (Table 3). Four different genotypes were identified, C85C248, A85C 248, T85C 248, and C85T 248. The first 3 genotypes were observed in both patient populations, whereas genotype C85T 248 was observed only in the colonized patients. The genotyping was successful for the pulmonary and air samples in 3 PCP patients (patients 2, 10, and 13) and in 4 colonized patients (patients 3, 4, 11, and 12) (Table 3). A full or partial match of genotypes was observed between 3 pulmonary samples and the corresponding air samples in the 3 PCP patients. A full match of genotypes was observed between 3 pulmonary samples and the corresponding air samples in 3 of the 4 colonized patients. 4. Discussion This study provides the first data on P. jirovecii detection and quantification in air samples collected in the environment of colonized patients using the Coriolis® μ air sampler and additional data on P. jirovecii detection and quantification in air samples collected in the environment of PCP patients using this device, as previously reported by Choukri et al. (2010). Negative results of P. jirovecii DNA detection in control air samples, specifically those out of the hospital, provide additional data on the absence of putative exosaprophytic form of P. jirovecii.
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A plus/minus real-time PCR assay amplifying the mtLSUrRNA gene has been routinely used to perform diagnoses of P. jirovecii infections in our laboratory for many years. For this reason, this gene was also chosen as the target of the qPCR assay (Choukri et al., 2010). This multicopy gene provides a highly sensitive amplification, which is required for detection of low fungal burden in pulmonary samples specifically in colonized patients, and in air samples. P. jirovecii DNA quantities in BAL samples from PCP patients appeared to be higher than those in BAL samples from colonized patients. Likewise, P. jirovecii DNA quantities in air samples at 1 m and 5 m from PCP patients appeared to be higher than in air samples at the same distances from colonized patients. These results may be consistent with a relationship between fungal burdens in the lungs and fungal burdens in patient environment. However, at present, this relationship cannot be affirmed because of the absence of statistical significance due to the small number of patients enrolled in the study. In 2 patients (1 PCP patient and 1 colonized patient), P. jirovecii DNA was not detected in air samples at 1 m, whereas it was detected at 5 m. These observations seem to be inconsistent with the results by Choukri et al. (2010) who observed that fungal burdens in air samples decreased with the distance from the patient. Our results may be explained by the fact that we did not strictly follow the method used by this author (Choukri et al., 2010). Air samples at 5 m were collected first, and air samples at 1 m were collected second. We collected air samples at 5 m with the door open, in contrast to the study by Choukri et al. (2010), during which air samples at 5 m were collected with the door closed. The opening of the door may have provoked air movement from inside to outside the room. This air movement may have increased the P. jirovecii air burden at 5 m, rendering it easier to detect the fungus at this distance. Conversely, the P. jirovecii air burden at 1 m may have contemporarily decreased, limiting the detection at this distance. This is still a hypothesis since in 4 other patients, 2 PCP patients and 2 colonized patients, P. jirovecii DNA quantities in air samples at 5 m were effectively lower than in air samples at 1 m, consistent with results by Choukri et al. (2010). P. jirovecii DNA was not detected in the air surrounding 2 PCP patients: patient 1 and patient 10 (air samples at 1 m and 5 m corresponding to the second pulmonary sample of this patient 10). The results may be explained by the time interval between air sampling and the start of treatment: 4 days for patient 1 and 9 days for patient 10. During these respective periods of 4 and 9 days, specific treatment for P. jirovecii may have reduced i) P. jirovecii pulmonary burdens; ii) P. jirovecii exhalation; and, consequently, iii) fungal burdens in the corresponding air samples. Again, identical reasons may explain the fact that P. jirovecii DNA quantities in air samples from PCP patient 10 who was treated for P. jirovecii were lower than those from colonized patients 11 and 12. Nonetheless, data on the reduction of P. jirovecii burdens in the lungs and on the kinetics of the fungal exhalation in the course of treatment are still unavailable. P. jirovecii DNA was not detected in the air surrounding 4
Table 3 P. jirovecii genotyping at the mitochondrial large subunit ribosomal RNA gene in pulmonary and air samples from patients for whom P. jirovecii DNA was detected in air samples. Patient
Clinical presentation of P. jirovecii infection
mtLSUrRNA genotype Pulmonary sample
1-m air sample
5-m air sample
2 3 4 5 7 10a
PCP Pulmonary colonization Pulmonary colonization Pulmonary colonization Pulmonary colonization PCP
C85C248; A85C248; T85C248 T85C248 A85C248
A85C248; T85C248
11 12 13
Pulmonary colonization Pulmonary colonization PCP
C85C248; A85C248; T85C248 T85C248 A85C248 ND ND A85C248 A85C248 C85C248; C85T248 A85C248 C85C248
UD A85C248; C85C248 C85C248; C85T248 C85C248; C85T248 C85C248
UD UD A85C248 C85C248 C85C248; C85T248 C85C248 C85C248
Abbreviations: ND = not determined; UD = unavailable data (the genotyping was not successful). a For this patient, 3 pulmonary specimens were examined (1 sputum on 22-01-13, 2 BAL samples on 31-01-13 and 28-02-13), and 3 subsequent air samples were collected. P. jirovecii DNA was detected in air samples corresponding to the sputum sample and the second BAL sample.
Please cite this article as: Le Gal S, et al, Pneumocystis jirovecii in the air surrounding patients with Pneumocystis pulmonary colonization, Diagn Microbiol Infect Dis (2015), http://dx.doi.org/10.1016/j.diagmicrobio.2015.01.004
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S. Le Gal et al. / Diagnostic Microbiology and Infectious Disease xxx (2015) xxx–xxx
colonized patients. These results were expected since, by definition, colonized patients are characterized by having low fungal pulmonary burdens and, consequently, exhaling low and potentially undetectable quantities of P. jirovecii. Risk factors for being a colonized patient exhaling P. jirovecii or not exhaling P. jirovecii were not identified. No matches of genotypes were observed in 2 pairs of air and pulmonary samples (patients 10 and 12). To explain the discrepancy observed in patient 10, we hypothesized that P. jirovecii identified in the air sample taken at 5 m on 04-03-13 (genotype C 85C 248) was not exhaled by this patient and came from another source. Indeed, it is noteworthy that patient 13, who developed PCP and who was infected by genotype C85C248, was hospitalized in the same unit at the same period. Nonetheless, encounters between patients 10 and 13 as well as between other patients that may identify potential human infectious sources were not objectivized. In contrast, the reason for the absence of genotype matches in colonized patient 12 remains unclear. Thus, P. jirovecii DNA was detected in the air surrounding 3 PCP patients and 6 colonized patients with genotype matches in 3 pairs (or triplets) of air and pulmonary samples in the first patient group and in 3 pairs (or triplets) of air and pulmonary samples in the second patient group. These results provide additional data concerning P. jirovecii diffusion from PCP patients and original data regarding this diffusion from colonized patients. The CDC recommends applying standard precautions and avoiding placement of a patient developing PCP in the same room with an immunocompromised patient (Siegel et al., 2007). Our present findings combined with our previous data on the role of colonized patients as potential infectious sources (Le Gal et al., 2012) and with those of Pneumocystis transmission from colonized rodent models (Dumoulin et al., 2000; Gigliotti et al., 2003) currently seem to be sufficient to extend these measures to colonized patients. Finally, our observations emphasize the risk of nosocomial transmission of P. jirovecii via the airborne route from diverse patients harboring the fungus, PCP patients or colonized patients. Acknowledgments The authors would like to thank Dr D. Quinio and Mrs C. Carrou, P. Lecordier, D. Roué, V. Abiven, C. Cam, C. Le Guen, K. Quinaou, and C. Roger for performing biological diagnoses of Pneumocystis infections. The authors would like to thank Pr EM Aliouat who has contributed to the Pneumair project. References Bartlett MS, Vermund SH, Jacobs R, Durant PJ, Shaw MM, Smith JW, et al. Detection of Pneumocystis carinii DNA in air samples: likely environmental risk to susceptible persons. J Clin Microbiol 1997;35:2511–3. Beard CB, Carter JL, Keely SP, Huang L, Pieniazek NJ, Moura IN, et al. Genetic variation in Pneumocystis carinii isolates from different geographic regions: implications for transmission. Emerg Infect Dis 2000;6:265–72. Centers for Disease Control and Prevention (CDC). Revised classification system for HIV infection and expanded surveillance case definition for AIDS among adolescents and adults. MMWR Recomm Rep 1992;41:1–19.
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Please cite this article as: Le Gal S, et al, Pneumocystis jirovecii in the air surrounding patients with Pneumocystis pulmonary colonization, Diagn Microbiol Infect Dis (2015), http://dx.doi.org/10.1016/j.diagmicrobio.2015.01.004
