Céline Damiani

A Cluster of Pneumocystis Infections Among Renal Transplant Recipients: Molecular Evidence of Colonized Patients as Potential Infectious Sources of Pneumocystis jirovecii

BACKGROUND Eighteen renal transplant recipients (RTRs) developed Pneumocystis jirovecii infections at the renal transplantation unit of Brest University Hospital (Brest, Brittany, France) from May 2008 through April 2010, whereas no cases of P. jirovecii infection had been diagnosed in this unit since 2002. This outbreak was investigated by identifying P. jirovecii types and analyzing patient encounters. METHODS The identification of P. jirovecii internal transcribed spacer (ITS) types was performed on P. jirovecii isolates from the 18 RTRs (12 patients with Pneumocystis pneumonia [PCP], 6 colonized patients), 22 unlinked control patients (18 patients with PCP, 4 colonized patients), and 69 patients (34 patients with PCP, 35 colonized patients) with contemporaneously diagnosed P. jirovecii infections in the Brest geographic area. A transmission map was drawn up. Its analysis was combined with the results of P. jirovecii typing. RESULTS P. jirovecii ITS type identification was successful in 14 of 18 RTRs, 15 of 22 control patients, and 48 of the 69 patients. Type Eg was the most frequent type in the 3 patient groups. However, its frequency was significantly higher in the first patient group than in the 2 other groups (P < .05 and P < .01, respectively). Fourteen encounters between RTRs who harbored an identical type were observed. Ten patients were considered as possible index patients, of whom 3 were colonized by the fungus, and 7 presented PCP. CONCLUSIONS The results provide to our knowledge the first data on the role of colonized patients as potential sources of P. jirovecii in a context of nosocomial acquisition of the fungus.

Combined quantification of pulmonary Pneumocystis jirovecii DNA and serum (1→3) β-D-glucan for differential diagnosis of Pneumocystis pneumonia and Pneumocystis colonization.

ABSTRACT This study assessed a quantitative PCR (qPCR) assay for Pneumocystis jirovecii quantification in bronchoalveolar lavage (BAL) fluid samples combined with serum (1→3)-β-d-glucan (BG) level detection to distinguish Pneumocystis pneumonia (PCP) from pulmonary colonization with P. jirovecii. Forty-six patients for whom P. jirovecii was initially detected in BAL fluid samples were retrospectively enrolled. Based on clinical data and results of P. jirovecii detection, 17 and 29 patients were diagnosed with PCP and colonization, respectively. BAL fluid samples were reassayed using a qPCR assay targeting the mitochondrial large subunit rRNA gene. qPCR results and serum BG levels (from a Fungitell kit) were analyzed conjointly. P. jirovecii DNA copy numbers were significantly higher in the PCP group than in the colonization group (1.3 × 107 versus 3.4 × 103 copies/μl, P < 0.05). A lower cutoff value (1.6 × 103 copies/μl) achieving 100% sensitivity for PCP diagnosis and an upper cutoff value (2 × 104 copies/μl) achieving 100% specificity were determined. Applying these two values, 13/17 PCP patients and 19/29 colonized patients were correctly assigned to their patient groups. For the remaining 14 patients with P. jirovecii DNA copy numbers between the cutoff values, PCP and colonization could not be distinguished on the basis of qPCR results. Four of these patients who were initially assigned to the PCP group presented BG levels of ≥100 pg/ml. The other 10 patients, who were initially assigned to the colonization group, presented BG levels of <100 pg/ml. These results suggest that the combination of the qPCR assay, applying cutoff values of 1.6 × 103 and 2 × 104 copies/μl, and serum BG detection, applying a 100 pg/ml threshold, can differentiate PCP and colonization diagnoses.

Serum (1→3)-β-D-Glucan Levels in Primary Infection and Pulmonary Colonization with Pneumocystis jirovecii

ABSTRACT This article describes positive (1→3)-β-d-glucan levels in serum from infants with primary Pneumocystis infection and from immunosuppressed patients with Pneumocystis pneumonia (PCP) and negative levels in serum from patients colonized by Pneumocystis jirovecii. Glucan detection is a complementary tool for the diagnosis of the diverse clinical presentations of P. jirovecii infection.

Possible Nosocomial Transmission of Pneumocystis jirovecii

To the Editor: Diversity of genotypes among Pneumocystis jirovecii (human-specific Pneumocystis species) isolates mainly involves internal transcribed spacer (ITS) loci (1). Type Eg, one of the most frequently detected ITS genotypes, has been found worldwide (2). The locus of dihydropteroate synthase (DHPS) is also of interest because DHPS is the target of sulfonamides, the main drugs used to treat Pneumocystis pneumonia (PCP). Studies of the DHPS locus have found mutations at positions 165 and 171, which confer potentially lower sensitivity to sulfonamides to mutant P. jirovecii organisms (3). Airborne transmission of Pneumocystis ssp. has been demonstrated among animals and probably occurs among humans (4). Reports of clusters of PCP cases in hospitals (4,5) provide a rationale for considering the possibility of nosocomial P. jirovecii infections. Moreover, we recently quantified P. jirovecii in the air surrounding patients with PCP (6). Our findings suggested that the fungus is exhaled from infected patients and then spreads into their surrounding air. Because matches of P. jirovecii genotypes between pulmonary and air samples would strengthen these findings, we conducted DHPS and ITS typing of P. jirovecii isolates from PCP patients and from the air in their close environment. We assayed P. jirovecii DNA that we had previously detected in pulmonary samples (bronchoalveolar lavage and induced sputum) from 15 PCP patients and in 15 air samples collected 1 meter from each patient’s head (6). ITS genotyping was based on sequence analysis of ITS 1 and 2 regions after amplification with a nested PCR, cloning, and sequencing, as described (7). ITS alleles were identified by using the typing system by Lee et al. (2). DHPS genotyping was based on a PCR restriction fragment-length polymorphism assay that enables detection of mutations at positions 165 and 171, as described (8). Among the 15 pulmonary samples, ITS genotyping was successful for all 15; among these, 8 ITS genotypes were identified (Table). Type Eg was most frequently identified. Mixed infections, which correspond to detection of >1 genotype in a given sample, were detected in 5 samples. DHPS genotyping was successful for all 15 pulmonary samples. A wild genotype was identified in 9 samples, a 165 mutant genotype in 1 sample, and a 171 mutant genotype in 2 samples. Mixed infections were identified in the 3 remaining samples. Table Genotyping of Pneumocystis jirovecii in pairs of pulmonary and air samples from 15 patients with Pneumocystis pneumonia* Among the 15 room air samples, ITS genotyping was successful for 7; among these, 4 ITS genotypes were identified (Table). Type Eg was again most frequently identified. A mixed infection was detected in 1 of the 7 samples. These results enabled us to compare ITS genotypes for 7 pairs of pulmonary and air samples. A full match was found for 4 (57.1%) pairs of samples, and a partial match, defined as at least 1 common genotype for pulmonary and air samples in mixed infections, was found for 2 (28.6%) pairs. No matches were found for the remaining pair of samples. DHPS genotyping was successful for 6 of the 15 air samples. A wild genotype was identified in 4 samples, a 165 mutant genotype was identified in 1 sample, and a 171 mutant genotype was identified in 1 sample. These results enabled us to compare DHPS genotypes for 6 pairs of samples. A full match was found for these 6 pairs. DHPS and ITS genotype matches were found for 4 pairs. Several lines of evidence suggest that P. jirovecii is exhaled by infected patients and transmitted by the airborne route to susceptible persons (4). In the study reported here, ITS or DHPS genotype matches between pairs of pulmonary and air samples are consistent with the possibility that P. jirovecii organisms in the air originated from patients. DHPS mutants were detected in 6 (40%) of the 15 pulmonary samples; none of the15 patients had received sulfonamide treatment at the time of PCP diagnosis. These results were not unexpected because frequency of finding DHPS mutants in PCP patients in Paris who had no prior sulfonamide treatment is high (8). The exhalation of DHPS mutants from infected patients can spread potentially sulfonamide-resistant organisms. Matches of P. jirovecii genotypes in pairs of pulmonary and room air samples argue in favor of P. jirovecii exhalation by infected patients. The exhalation of P. jirovecii organisms emphasizes the risk for their nosocomial transmission. Our data provide additional arguments in favor of the application of measures to prevent the airborne transmission of P. jirovecii in hospitals.

Pneumocystis jirovecii in the air surrounding patients with Pneumocystis pulmonary colonization

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 1m and 5m from the patients 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 1m and in 5 of the 10 other air samples collected at 5m. 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.

Pneumocystis jirovecii and cystic fibrosis in France

Abstract We retrospectively investigated 76 patients with cystic fibrosis for the presence of Pneumocystis jirovecii, by performing real-time PCR and nested-PCR assays on 146 archival sputum specimens. P. jirovecii was detected in only 1 patient (1.3%) showing that in our region (Brest, France), the fungus is rarely involved in pulmonary colonization in patients with cystic fibrosis.

Circulation of Pneumocystis dihydropteroate synthase mutants in France

Data on the prevalence of Pneumocystis jirovecii (P. jirovecii) dihydropteroate synthase (DHPS) mutants in France are still limited. In this study, mutant prevalence in the Brest region (western France) was determined. Archival pulmonary specimens from 85 patients infected with P. jirovecii and admitted to our institution (University Hospital, Brest) from October 2007 to February 2010 were retrospectively typed at the DHPS locus using a polymerase chain reaction-restriction fragment length polymorphism assay. Type identification was successful in 66 of 85 patients. Sixty-four patients were infected with a wild type, whereas mutants were found in 2 patients (2/66, 3%). Medical chart analysis revealed that these 2 patients usually lived in Paris. Another patient usually lived on the French Riviera, whereas 63 patients were from the city of Brest. Thus, the corrected prevalence of mutants in patients who effectively lived in our geographic area was 0% (0/63). Taking into account that i) Paris is characterized by a high prevalence of mutants from 18.5% to 40%, ii) infection diagnoses were performed in the 2 Parisians during their vacation <30 days, iii) infection incubation is assumed to last about 2 months, the results provide evidence of mutant circulation from Paris to Brest through infected vacationers. The study shows that the usual city of patient residence, rather than the city of infection diagnosis, is a predictor of mutants and that P. jirovecii infections involving mutants do not represent a public health issue in western France.

Usefulness of (1,3) ß-d-glucan detection in bronchoalveolar lavage samples in Pneumocystis pneumonia and Pneumocystis pulmonary colonization

OBJECTIVE OF THE STUDY Recent data demonstrate the usefulness of (1,3) ß-d-glucan (BG) detection in serum samples to distinguish patients developing Pneumocystis pneumonia and patients who are colonized by the fungus. In contrast, data of BG detection in bronchoalveolar lavage (BAL) samples from these patient populations are still rare. PATIENTS In this context, we determined BG levels in BAL samples from 11 Pneumocystis pneumonia (PCP) patients, 10 colonized patients, and 24 Pneumocystis-uninfected patients. MATERIALS AND METHODS BG levels were determined on each BAL sample using the Fungitell(®) kit (Associates of Cape Cod, Inc., Cape Cod, MA, USA) according to the manufacturers instructions applied to serum sample examination. RESULTS The BG levels in BAL samples from the PCP patient group (mean value 20 588 pg/mL) were significantly higher than those in the colonized patient group (mean value 105 pg/mL) (P=0.0001, Mann-Whitney test) and than those in the Pneumocystis-uninfected patient group (mean value 74 pg/mL) (P<0.0001, Mann-Whitney test). The BG levels in BAL samples from the colonized patient group did not differ significantly from those in the Pneumocystis-uninfected patients group (P=0.21). CONCLUSION The results suggest that measurements of BAL BG levels may facilitate the differential diagnosis of PCP and pulmonary colonization with Pneumocystis.

AIDS-related Pneumocystis jirovecii genotypes in French Guiana.

The study described Pneumocystis jirovecii (P. jirovecii) multilocus typing in seven AIDS patients living in French Guiana (Cayenne Hospital) and seven immunosuppressed patients living in Brest, metropolitan France (Brest Hospital). Archival P. jirovecii specimens were examined at the dihydropteroate synthase (DHPS) locus using a PCR-RFLP technique, the internal transcribed spacer (ITS) 1 and ITS 2 and the mitochondrial large subunit rRNA (mtLSUrRNA) gene using PCR and sequencing. Analysis of typing results were combined with an analysis of the literature on P. jirovecii mtLSUrRNA types and ITS haplotypes. A wild DHPS type was identified in six Guianese patients and in seven patients from metropolitan France whereas a DHPS mutant was infected in the remaining Guianese patient. Typing of the two other loci pointed out a high diversity of ITS haplotypes and an average diversity of mtLSUrRNA types in French Guiana with a partial commonality of these haplotypes and types described in metropolitan France and around the world. Combining DHPS, ITS and mtLSU types, 12 different multilocus genotypes (MLGs) were identified, 4 MLGs in Guianese patients and 8 MLGs in Brest patients. MLG analysis allows to discriminate patients in 2 groups according to their geographical origin. Indeed, none of the MLGs identified in the Guianese patients were found in the Brest patients and none of the MLGs identified in the Brest patients were found in the Guianese patients. These results show that in French Guiana (i) PCP involving DHPS mutants occur, (ii) there is a diversity of ITS and mtLSUrRNA types and (iii) although partial type commonality in this territory and metropolitan France can be observed, MLG analysis suggests that P. jirovecii organisms from French Guiana may present specific characteristics.

A misleading false-negative result of Pneumocystis real-time PCR assay due to a rare punctual mutation: A French multicenter study

Abstract This article describes a previously unreported mutation at position 210 (C210T) of the mitochondrial large subunit ribosomal RNA (mtLSUrRNA) gene of Pneumocystis jirovecii, which led to a false‐negative result of a real‐time polymerase chain reaction (PCR) assay. Since the aforementioned real‐time PCR assay is widely used in France, a French multicenter study was conducted to estimate the mutation frequency and its potential impact on the routine diagnosis of Pneumocystis pneumonia (PCP). Through analysis of data obtained from eight centers, the mutation frequency was estimated at 0.28%. This low frequency should not call into question the routine use of this PCR assay. Nonetheless, the occurrence of the false‐negative PCR result provides arguments for maintaining microscopic techniques combined to PCR assays to achieve PCP diagnosis.