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Quantification and Spread of Pneumocystis jirovecii in the Surrounding Air of Patients with Pneumocystis Pneumonia

  1. Firas Choukri1,
  2. Jean Menotti1,
  3. Claudine Sarfati1,
  4. Jean-Christophe Lucet2,
  5. Gilles Nevez3,
  6. Yves J. F. Garin1, and
  7. Anne Totet4⇓
  1. 1Saint Louis Hospital APHP, Paris-Diderot University, Paris, France
  2. 2Bichat-Claude Bernard Hospital APHP, Paris-Diderot University, Paris, France
  3. 3Université de Brest, CHU de Brest, Brest, France
  4. 4University of Picardy and University Hospital, Amiens, France
  1. Reprints or correspondence: Dr Anne Totet, Laboratoire de Parasitologie-Mycologie, CHU, Centre Hospitalier Sud, 1, ave René Laennec, 80054 Amiens, France (totet.anne{at}chu-amiens.fr).
 
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Abstract

Background. Airborne transmission of Pneumocystis has been demonstrated in animal models and is highly probable in humans. However, information concerning burdens of Pneumocystis jirovecii (human-derived Pneumocystis) in exhaled air from infected patients is lacking. Our objective is to evaluate P. jirovecii air diffusion in patients with Pneumocystis pneumonia.

Methods. Patients admitted with Pneumocystis pneumonia were prospectively enrolled from 9 January 2008 to 21 July 2009. Air samples (1.5 m3) were collected on liquid medium with a commercial sampler at 1-, 3-, 5-, and 8-m distances from patients' heads. Air control samples were collected away from Pneumocystis pneumonia patient wards and outdoors. Samples were examined for P. jirovecii detection and quantification using a real-time polymerase chain reaction assay targeting the mitochondrial large subunit ribosomal RNA gene.

Results. Forty patients were diagnosed as having Pneumocystis pneumonia. Air sampling was performed in the environment for 19 of them. At a 1-m distance from patients' heads, P. jirovecii DNA was detected in 15 (79.8%) of 19 patients, with fungal burdens ranging from 7.5 × 103 to 4.5 × 106 gene copies/m3. These levels decreased with distance from the patients ( P < .002). Nevertheless, 4 (33.3%) of the 12 samples taken at 8 m, in the corridor adjacent to their room, were still positive. Forty control samples were collected and remained negative.

Conclusion. This study provides the first quantitative data on the spread of P. jirovecii in exhaled air from infected patients. It sustains the risk of P. jirovecii direct transmission in close contact with patients with Pneumocystis pneumonia and leads the way for initiating a quantitative risk assessment for airborne transmission of P. jirovecii.

Pneumocystis jirovecii, the causative agent of Pneumocystis pneumonia in humans, is an important cause of morbidity and mortality in patients infected with human immunodeficiency virus but also in other patients receiving immunosuppressive therapy for transplantation, malignant tumors, and connective tissue diseases [1,2]. For many years, Pneumocystis pneumonia was thought to be due to reactivation of latent infection, but several lines of evidence are now in favor of a de novo acquisition of the fungus from an exogenous source. An animal reservoir for P. jirovecii is improbable because Pneumocystis organisms infecting each mammalian species are host specific. These data strongly suggest that Pneumocystis infection in humans is an anthroponosis with humans as a reservoir for P. jirovecii [3].

Host-to-host transmission of the fungus has been demonstrated in rodent models [4] and is highly suggested in humans. The results of genotypic studies of P. jirovecii isolates collected in Pneumocystis pneumonia case clusters in hospitals and within households strongly support this transmission [5–13]. It was clearly established in animal models that host-to-host transmission occurs via the airborne route [14,15]. These experiments, combined with the fact that P. jirovecii has a high tropism for the human lungs, support the hypothesis that acquisition of P. jirovecii organisms by humans also takes place via the airborne route. Because Pneumocystis organisms multiply at the alveolar surface, they are exhaled during ventilation by infected patients. This was supported by the presence of P. jirovecii DNA in air filters placed in the hospital rooms of patients with Pneumocystis pneumonia [16] and subsequent genotypic matching with patients isolates [17,18]. Similarly, it was also shown for a patient with Pneumocystis pneumonia receiving ventilatory support because P. jirovecii DNA was detected in the air collected from the intubation system used [19]. Furthermore, using reverse-transcriptase polymerase chain reaction (PCR), P. jirovecii complementary DNA has been evidenced in air samples from rooms of patients with Pneumocystis pneumonia, suggesting that the detected airborne stage of P. jirovecii was still viable and therefore potentially infectious [20,21]. Although convincing for the airborne excretion of P. jirovecii in the patient environment, these studies were not quantitative because they used conventional or nested PCR for Pneumocystis detection. Therefore, information on the diffusion of P. jirovecii in air is still lacking. Such determination is of major importance to estimating the potential exposure and risk for susceptible contact patients but is hampered by the lack of quantitative methods adapted for sampling large volumes of air to obtain concentrated samples and then sensitive detection of P. jirovecii. Until now, filtration techniques have been largely used in which airborne P. jirovecii organisms are trapped on filters from which DNA is extracted for PCR analysis. More recently, air sampling with a portable bioimpactor has been proposed [22], but the method requires 24-h collection for a 1-m3 sample. These technical limitations, due to either the difficulties of extraction from a solid support or the lengthy conditions of sampling, led us to consider alternative methods that would be suitable for both sampling and quantifying P. jirovecii in air.

In the present study, we developed a method for P. jirovecii detection and quantification in air samples from the environment of Pneumocystis pneumonia patients using a new liquid impactor air sampling device (Coriolis µ; Bertin Technologies) combined with a quantitative real-time PCR assay. We show that this method is efficient at quantifying fungal burdens in air and estimating its diffusion in the environment of patients with Pneumocystis pneumonia. These results make it possible to estimate hazard exposure and open the field for Pneumocystis pneumonia microbial quantitative risk assessment (MQRA).

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Materials and Methods

Patients and diagnosis of Pneumocystis pneumonia. This prospective study was conducted between 9 January 2008 and 21 July 2009 and included consecutive patients with Pneumocystis pneumonia admitted to 2 Parisian hospitals (hospitals A and B). Pneumocystis pneumonia diagnosis was established by microscopic visualization of cysts and trophic forms in bronchoalveolar lavage or induced sputum specimens stained with Giemsa stain and using an immunofluorescence assay (MonofluoKit Pneumocystis; Bio-Rad). Part of the specimen was centrifuged and then pellets were stored at 4°C until quantitative PCR assay was performed on the sample, which took place within 72 h after sampling.

Air sampling. After microscopic diagnosis of Pneumocystis pneumonia and as soon as possible depending on availability of the air sampling device, the operator, and the patient's condition, air samples were collected in each patient room using the Coriolis µ air sampler. Each sample, consisting of 1.5 m3 of air collected at 0.3 m3/min, was taken into a conic sterile tube containing 15 mL of sterile phosphate-buffered saline and 0.002% Tween 80. In all cases, 1 sample was collected at a distance of 1 m from the patient' head, followed by additional air samples collected inside the room (3 m from the patient) and outside the room (at the door entrance, 5 m from the patient, and in the corridor next to the room, 8 m from the patient) when possible. For sampling, the Coriolis µ air sampler was placed 1 m from the floor and the door and the windows of patient's room were kept closed. Controls consisted of 1.5m3 air samples far from the patient ward and outdoors. For each sample, the collection liquid was centrifuged at 2500g for 10 min then the supernatant was carefully removed to leave a 1-mL pellet that was subsequently stored at 4°C until quantitative PCR assay was performed, which took place within 72 h after sampling.

DNA extraction and P. jirovecii detection and quantification. Ten units of lyticase (Sigma) were first added to 200 µL of ea. ch of the pulmonary and air sample pellets. After mixing, the samples were incubated for 30 min at 37°C. DNA was then extracted by using the QIAamp DNA Mini Kit (QIAGEN) according to the manufacturer's recommendations.

A real-time quantitative PCR assay targeting the mitochondrial large subunit (mtLSU) ribosomal RNA (rRNA) gene of P. jirovecii was used, as described by Meliani et al [23]. Thermocycling and fluorescence detection were performed on a PCR system (7500 PCR system, Applied Biosystems) in a final volume of 25 µL by using the TaqMan Gene EXpression Master Mix (Applied Biosystems), with a 0.2-µmol/L concentration of each primer, a 0.1-µmol/L concentration of the probe, and 5 µL of extracted DNA. After 2 min at 50°C and 10 min at 95°C, amplification consisted of 45 cycles with 15 s of denaturation at 95°C followed by 1 min of annealing and extension at 60°C. Plasmid suspensions were used as standards for quantification and positive controls. They were prepared by cloning the mtLSU rRNA insert into the plasmid vector pGEM-T (pGEM-T Easy Vector System II; Promega). After propagation and purification of the plasmids, the concentration of mtLSU rRNA gene copies (number of copies per microliter) was derived from the A260 optical density measurement and the plasmid molecular weight. Each PCR run comprised 10 serial 10-fold dilutions of the plasmid suspension, ranging from 7.0 × 10−2 to 7.0 × 107 copies/μL of extracted DNA, and 2 negative controls (ultrapure water). The 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. By interpolation, this allowed the expression of the results in number of copies per microliter of extracted DNA. The detection limit was estimated at 7 copies/μL of extracted DNA, corresponding to a cycle threshold value of 43. All pulmonary and air samples, as well as plasmid dilutions and negative controls, were run in duplicate, with 1 tube containing an internal positive control (TaqMan Exogenous Internal Positive Control Reagents; Applied Biosystems) to detect PCR inhibitors.

Statistical analysis. Comparison of the fungal burdens in air samples was made using the Kruskal-Wallis nonparametric test, followed by the Dunn posttests using Graphpad Prism, version 5.01, for Windows (Graphpad Software; http://www.graphpad.com).

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Results

In the study period, 40 consecutive patients were diagnosed as having Pneumocystis pneumonia. Thirty-two and 8 patients were admitted to hospital A and hospital B, respectively. Air sampling was performed in the environment for 19 of them (15 patients in hospital A and 4 patients in hospital B). Characteristics of these 19 patients are given in Table 1. Patients were admitted to the departments of internal medicine, infectious diseases, pneumology, dermatology, and hematology and the intensive care unit (ICU) of hospital A and the department of internal medicine of hospital B. Except for the ICU, which is an open ward, all patient rooms are conventional rooms (ie, with no negative pressure or laminar flow). The male-female sex ratio was 17:2; the mean age of the patients was 45 years. Fifteen patients were infected with human immunodeficiency virus, including 1 patient with non-Hodgkin lymphoma, 2 who had undergone hematopoietic stem cell transplantation for an underlying hematologic disease (1 myeloma and 1 Hodgkin disease), 1 who had chronic lymphoid leukemia, and 1 who was a renal transplant recipient. All had typical clinical and radiographic presentations of Pneumocystis pneumonia. The diagnosis of Pneumocystis pneumonia was assessed by the microscopic detection of P. jirovecii in bronchoalveolar lavage samples of 11 patients and in induced sputum samples of the 8 other patients. Most patients were treated within hours after diagnosis or had been empirically treated because of a high clinical or radiologic suspicion of Pneumocystis pneumonia, so that 12 of the 19 patients had already received Pneumocystis pneumonia treatment 1–9 days before air samples could be collected.

Figure 1
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Figure 1

Number of Pneumocystis jirovecii gene copies per cubic meter of air sampled inside (filled squares) and outside (open squares) the room of patients with Pneumocystis pneumonia. For each series (of distance from patient), median values are represented by horizontal bars. *P < .05 (Dunn posttest); mtLSU, mitochondrial large subunit.

Table 1
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Table 1

Characteristics of Patients Diagnosed as Having Pneumocystis Pneumonia at 2 Parisian Hospitals

For all 19 patients with Pneumocystis pneumonia, air samples could be collected at a distance of 1 m from the patients' heads. In 13 patients, additional samples could be collected inside the rooms at 3 m, and for 12 patients, samples were also collected outside the room at 5-m and 8-m distances from the patients. Forty control air samples were collected. Nineteen were indoor samples collected at a distance from the wards in which Pneumocystis pneumonia patients could be admitted, and 21 were collected outdoors in the hospital yard.

Results of P. jirovecii detection are given in Table 2. All pulmonary specimens were PCR positive, with gene copy numbers ranging from 1.0 × 10 3 to 2.1 × 108 copies/μL of extracted DNA (mean, 1.7 × 107 copies/μL), and all control air samples were negative. By contrast, PCR was positive in 33 of the 56 air samples collected in the environment of patients with Pneumocystis pneumonia. At the closest distance to the patient (1 m), P. jirovecii DNA was found in 15 (78.9%) of the 19 samples, with copy numbers ranging from 7.5 × 10 3 to 4.5 × 106 copies/m3. Ten of these 15 positive air samples were collected in rooms of patients for whom Pneumocystis pneumonia treatment was started <2 days before air sampling. For the 5 remaining air samples, the duration of treatment ranged from 2 to 9 days. At a 3-m distance, 9 (69.2%) of the 13 air samples were positive (range, 1.2 × 10 3−6.5 × 105 copies/m3). This proportion decreased to 5 (41.7%) of the 12 samples at 5 m (range, 5.5 × 10 3−1.6 × 105 copies/m3) and 4 (33.3%) of the 12 samples at 8 m (range, 1 × 104−7.9 × 104 copies/m3). Overall, fungal burdens in air samples regularly decreased with the distance from the patient (Kruskal-Wallis test, P < .002), and a significant correlation was found between the decreasing rate and the distance (Spearman correlation coefficient r 2 =.−0.643; P < .001 ). Pair comparison with posttests showed a significant difference between fungal burdens collected at 1 m and those collected at 5 m and 8 m (Dunn posttest, P < .05) (Figure 1). We also found a trend to correlation between fungal burdens in the patient's pulmonary samples and the corresponding air samples collected at 1 m of patients' heads ( r 2 = 0.188 ; P = .06).

Table 2
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Table 2

Pneumocystis jirovecii Quantification in Clinical and Room Air Samples of Patients with Pneumocystis Pneumonia at 2 Parisian Hospitals (January 2008–July 2009)

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Discussion

This study provides original quantitative data concerning P. jirovecii air levels in the proximity of Pneumocystis pneumonia patients and extends the results of previous studies on P. jirovecii presence in the surrounding air of these patients [16–19]. These results were obtained using a high-throughput air sampler in conjunction with real-time PCR amplification of P. jirovecii DNA. In particular, the Coriolis μ cyclonic air sampler used offered the possibility to sample large volumes of air during a short period and subsequently concentrate the captured airborne microorganisms into a small volume of liquid [24]. This was crucial for efficient capture and detection of Pneumocystis in the present case because DNA can be more readily extracted from the liquid collection medium than from filters and microorganisms are better preserved in the liquid medium. The absolute recovery efficiency of Pneumocystis using the Coriolis μ sampler is not known because it was impossible to perform experiments with controlled doses of purified Pneumocystis in the air. However, a reasonably high recovery rate is expected on the basis of the reported performance of this sampler for bacteria, pollen, and fungal spores, compared with other bioimpactors or membrane filter samplers [24]. We choose to perform a real-time PCR assay targeting the mtLSU rRNA gene, which is a multicopy gene that ensures a high sensitivity of this assay.

No Pneumocystis DNA was detected in indoor or outdoor air samples collected in locations at the hospital away from wards that admit patients with Pneumocystis pneumonia. In contrast, P. jirovecii was detected and quantified in 79% of air samples collected at 1 m of patients' heads and to a lesser extend in patient rooms and adjacent corridors. However, no P. jirovecii DNA was detected in the close environment of 4 patients. Several hypotheses can be raised to explain these negative results. First, the severity of the disease, and consequently the fungal burden in the lungs and in air, probably differs among patients, and it is likely that patients with moderate Pneumocystis pneumonia are low excretors. Second, negative results could also be related to administration of a specific treatment before air sampling. Although it is assumed that P. jirovecii can remain detectable in pulmonary samples up to 4 weeks under treatment [25], the decrease of fungal burden in the lungs, and thereby fungal excretion, has not been quantified. Third, sampling conditions could also account for negative results, especially in open wards (ICU) and when door and windows of the patient room were kept opened before sampling.

Our study also provides new information on the diffusion of P. jirovecii from the patient source. Incidence of P. jirovecii in air samples decreased with distance from the patient, with a concurring decrease in air fungal burdens. These trends show that the diffusion of P. jirovecii is mainly confined to the patient room. Nonetheless, some diffusion to the corridor is observed that could represent a risk for immunocompromised patients and also for health care workers. Several studies and case reports have shown that human-to-human transmission can occur in the hospital setting, resulting in Pneumocystis pneumonia for immunocompromised patients or colonization for health care workers [26–28]. Indeed, these results strongly support the Centers for Disease Control and Prevention recommendation to avoid placement of an immunocompromised patient in the same room with a patient with Pneumocystis pneumonia [29].

Extending these observations toward initiation of an MQRA for airborne transmission of Pneumocystis is an important objective, and this study provides a first set of data on hazard identification and exposure assessment. The further steps of MQRA would be the viability assessment of the microorganisms in air samples and the estimation of the dose-infection relationships. The infective dose for P. jirovecii is unknown in humans, but it has been estimated at <10 P. carinii organisms in rodent models [30]. In our study, we attempted to make the conversion of gene copies in microorganism number by establishing a correlation between quantitative PCR on the mtLSU rRNA multicopy gene and the dihydropteroate synthase single copy gene (data not shown). Results from these experiments led to the estimation that 1 P. jirovecii organism contained 10 copies of the mtLSU rRNA gene and that P. jirovecii burdens in air ranged from 7 × 102 to5 × 105 organisms/m3 at 1-m distance from patients with Pneumocystis pneumonia. This is consistent with a risk for patients and health care workers in close contact with Pneumocystis pneumonia patients and supports preventive measures, such as patient isolation and even supplemental air treatment.

In conclusion, the results obtained with the combination of a high-throughput air sampling procedure and a sensitive PCR quantification of P. jirovecii open diverse fields of investigation. One field is to better estimate the risk of airborne transmission of P. jirovecii, with the possibility to initiate a risk assessment analysis; another is to provide a sensitive tool for assessing preventive measures.

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Acknowledgments

We thank physicians from the departments of infectious diseases, pneumology, internal medicine, dermatology, and hematology and the ICU of Saint Louis, Bichat-Claude Bernard, and Lariboisiere hospitals for addressing clinical specimens for Pneumocystis pneumonia diagnosis in our laboratories and for allowing air sampling in the wards. We thank S. Houzé for her assistance in laboratory diagnosis and Vance Bergeron for the English revision of the manuscript. Patients are greatly acknowledged for their participation, as well as Bertin Technologies for providing the Coriolis µ air sampler.

Financial support. This study was supported by the Agence Francçaise de la Sécurité Sanitaire de l'Environnement et du Travail (conventions EST/ 2006/1/41 and 07-CRD-29).

Potential conflict of interest. All authors: no conflicts.

  • Received November 26, 2009.
  • Accepted March 4, 2010.
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  1. Clin Infect Dis. (2010) 51 (3): 259-265. doi: 10.1086/653933
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