Clinical characteristics and risk factors associated with Pneumocystis jirovecii infection in patients with solid tumors: study of thirteen-year medical records of a large cancer center.

BACKGROUND: Pneumocystis jirovecii pneumonia (PCP)-related risk factors among patients with solid tumors are not completely defined. Thus, we aimed to characterize PCP cases with underlying solid tumors, to highlight the factors contributing to its development besides the prolonged use of moderate-to-high dose corticosteroids. METHODS: We retrospectively reviewed the medical records of patients with solid tumors diagnosed with PCP between 2006 and 2018 at a cancer center in Tokyo, Japan. Demographic and clinical data were collected, which included malignancy types, total lymphocyte count, coexisting pulmonary disease, chemotherapy, radiation therapy, corticosteroid use, and PCP-attributable mortality. RESULTS: Twenty cases of PCP with solid tumors were documented in 151,718 patients and 788,914 patient-years. Lung cancer (n = 6, 30%) was the most common underlying tumor, followed by breast cancer (n = 3, 15%). Only six (30%) patients were taking a dosage of ≥20 mg prednisone equivalents daily for ≥4 weeks from the onset of PCP. Among the remaining 14 patients, seven (50%) had coexisting pulmonary diseases, 10 (71%) had received chemotherapy within 90 days prior to PCP diagnosis, seven (50%) had undergone chest radiation therapy before PCP diagnosis, seven (50%) had received only intermittent corticosteroids, and one (7%) received no corticosteroids. Mortality attributable to PCP was 40%. CONCLUSIONS: More than half of the patients were not taking a dosage of ≥20 mg prednisone equivalents daily for ≥4 weeks. Multiple other factors (e.g., lymphocytopenia, radiation to chest) may have potentially contributed to PCP in patients with solid tumors in a composite manner. We need to establish a method for estimating the likelihood of PCP taking multiple factors into account in this patient population.

Quantitative Pneumocystis jirovecii real-time PCR to differentiate disease from colonisation.

We studied a Pneumocystis jirovecii quantitative polymerase chain reaction (qPCR) for distinguishing P. jirovecii disease from colonisation. Eighty-two respiratory samples from 65 patients with qPCR results were analysed against a gold standard clinical diagnosis of Pneumocystis pneumonia. High inter-assay reproducibility using recombinant and clinical material was observed. Contemporaneous samples from the same patient displayed high variability (median difference 2.6 log10 copies/mL, IQR 2.1-3.1 log10 copies/mL). Despite this, area under the receiver operator characteristic curve was 0.8. An optimum cut-off of 2.8 log10 copies/mL (equivalent to CT of 34.0 cycles) had 59% sensitivity and 92% specificity. The median P. jirovecii load was 7.3 log10 copies/mL in HIV patients compared to 2.6 log10 copies/mL in non-HIV patients. Specificity was 100% in non-HIV patients with qPCR of >3.8 log10 copies/mL. qPCR was useful for distinguishing P. jirovecii disease from colonisation. A quantitative standard, standardisation of definitions and methods are required to improve the generalisability of results.

Revisiting the Pneumocystis host specificity paradigm and transmission ecology in wild Southeast Asian rodents.

Pneumocystis fungi are opportunistic parasites of mammalian lungs whose evolution, ecology and host specificity in natural host populations remain poorly understood and controversial. Using an extensive collection of 731 lung samples from 27 rodent species sampled in five Southeast Asian countries, and nested PCR amplification of mitochondrial and nuclear genes, we investigated the host specificity and genetic structure of Pneumocystis lineages infecting wild rodents. We also identified the rodent species playing a central role in the transmission of these parasites using network analysis and centrality measurement and we characterized the environmental conditions allowing Pneumocystis infection in Southeast Asia using generalized linear mixed models. Building upon an unprecedented Pneumocystis sampling from numerous rodent species belonging to closely related genera, our findings provide compelling evidence that the host specificity of Pneumocystis lineages infecting rodents is not restricted to a single host species or genus as often presented in the literature but it encompasses much higher taxonomic levels and more distantly related rodent host species. The phylogenetic species status at both mitochondrial and nuclear genetic markers of at least three new Pneumocystis lineages, highly divergent from Pneumocystis species currently described, is also suggested by our data. Our models show that the probability of Pneumocystis infection in rodent hosts is positively correlated to environmental variables reflecting habitat fragmentation and landscape patchiness. Synanthropic and habitat-generalist rodents belonging to the Rattus, Sundamys and Bandicota genera played a role of bridge host species for Pneumocystis spreading in these heterogeneous habitats, where they can reach high population densities. These are critical findings improving our understanding of the ecology of these enigmatic parasites and the role played by cospeciation and host switches in their evolution. Our results also confirmed the role of land-use change and habitat fragmentation in parasite amplification and spillover in rodents.

Pneumocystis Mating-Type Locus and Sexual Cycle during Infection.

Pneumocystis species colonize mammalian lungs and cause deadly pneumonia if the immune system of the host weakens. Each species presents a specificity for a single mammalian host species. Pneumocystis jirovecii infects humans and provokes pneumonia, which is among the most frequent invasive fungal infections. The lack of in vitro culture methods for these fungi complicates their study. Recently, high-throughput sequencing technologies followed by comparative genomics have allowed a better understanding of the mechanisms involved in the sexuality of Pneumocystis organisms. The structure of their mating-type locus corresponding to a fusion of two loci, Plus and Minus, and the concomitant expression of the three mating-type genes revealed that their mode of sexual reproduction is primarily homothallism. This mode is favored by microbial pathogens and involves a single self-compatible mating type that can enter into the sexual cycle on its own. Pneumocystis sexuality is obligatory within the host's lungs during pneumonia in adults, primary infection in children, and possibly colonization. This sexuality participates in cell proliferation, airborne transmission to new hosts, and probably antigenic variation, processes that are crucial to ensure the survival of the fungus. Thus, sexuality is central in the Pneumocystis life cycle. The obligate biotrophic parasitism with obligate sexuality of Pneumocystis is unique among fungi pathogenic to humans. Pneumocystis organisms are similar to the plant fungal obligate biotrophs that complete their entire life cycle within their hosts, including sex, and that are also difficult to grow in vitro.

[Pneumocystis jirovecii and quantitative PCR: Pneumonia or colonization?] / Pneumocytis jirovecii et PCR quantitative : pneumonie ou colonisation à Pneumocystis jirovecii ?

BACKGROUND: Quantitative PCR to detect Pneumocystis jirovecii (Pj) is a new tool for the diagnosis of Pneumocystis jirovecii pneumonia (PJP). The yield of this technique, in cases of low fungal burden, when the standard technique using immunofluorescence (IF) is negative, needs to be evaluated. METHODS: We retrospectively reviewed the charts of all patients with a positive PCR but negative IF test (PCR+/IF-) in bronchoalveolar lavage (BAL) fluid performed over one year. We used an algorithm based on underlying immunosuppression, clinical picture, thoracic CT scan appearances, existence of an alternative diagnosis and the patient's outcome on treatment. Using this, each case was classified as probable PJP, possible PJP or colonization. RESULTS: Among the 416 BAL performed, 48 (12%) were PCR+/IF- and 43 patients were analyzed. Patients were mostly male (56%) with a median age of 60 years. Thirty-five (84%) were immunocompromised: 4 (9%) HIV-infected patients, 26 (60%) with hematologic or solid organ cancer, 3 (7%) were renal transplant recipients. Seven (16%) were classified as probable PPJ and 9 (21%) as possible PJP. Patients with a probable or possible PJP were more frequently admitted to the ICU (P<0.02) and had higher risk of death (P<0.01) when compared to those with colonization. Median PCR levels were very low and were not different between PJP or colonized patients (P=0.23). CONCLUSIONS: Among patients with a positive Pj PCR in BAL but with negative IF, only 37% had probable or possible PJP and PCR could not discriminate PJP from colonization.

Genotyping of Pneumocystis jirovecii isolates obtained from clinical samples by multilocus sequencing: a molecular epidemiology study conducted in Turkey.

Pneumocystis jirovecii is an opportunistic respiratory pathogen causing Pneumocystis pneumonia (PcP) in immunocompromised patients. The aim of this study was to investigate the genetic diversity of P. jirovecii isolates (n: 84) obtained from PcP patients using multilocus sequencing method based on mt26S, SOD, and CYB loci. Among the 84 clinical samples that were positive for P. jirovecii DNA, 31 (36.90%) of them were genotyped using at least one locus. Of the 31 clinical samples, 26 of them were successfully genotyped using all loci whereas three samples were genotyped using either mt26S/CYB loci or mt26S/SOD loci. Additionally, there were two more clinical samples that were genotyped using CYB or SOD locus. Using mt26S locus, genotypes 2, 3, 7, and 8 were detected. Frequencies of genotype 7 and 8 were higher and both of them were found in 11 (n: 29; 37.93%) clinical samples. Using SOD locus, SOD 1, 2, and 4 genotypes were detected. SOD 1 was the predominant genotype (20/28; 71.42%). During the analyses of CYB locus, CYB 1, 2, 5, 6, and 7 as well as a new CYB genotype were detected. CYB 1 (16/29; 55.17%) and 2 (10/29; 34.48%) were the predominant genotypes. Overall, according to the multilocus sequencing results E, F, M, N, P, and V multilocus genotypes were detected among the PcP patients. In addition, SOD 1 was the predominant genotype and CYB had a more polymorphic locus.

Myeloid C-type lectin receptors that recognize fungal mannans interact with Pneumocystis organisms and major surface glycoprotein.

Myeloid C-type lectin receptors (CLRs) are innate immune recognition molecules that bind to microorganisms via their carbohydrate recognition domains. In this study, we utilized a library of CLRs that recognize fungal mannans. We used this library to screen against Pneumocystis carinii (Pc) homogenates or purified Pc major surface glycoprotein (Msg) present on Pneumocystis. The results demonstrated that all of the mammalian CLR hFc-fusions tested displayed significant interaction/binding with Pc organisms, and furthermore to isolated Msg. Highest Pc organism and Msg binding activities were with CLR members Mincle, Dectin-2, DC-SIGN and MCL. An immunofluorescence assay with the respective CLR hFc-fusions against whole Pc life forms corroborated these findings. Although some of these CLRs have been implicated previously as important for Pneumocystis pathogenesis (Dectin-1/Dectin-2/Mincle), this is the first analysis of head-to-head comparison of known fungal mannan binding CLR-hFc fusions with Pc. Lastly, heat treatment resulted in reducted CLR binding.

Inhibition of polymerase chain reaction: Pathogen-specific controls are better than human gene amplification.

PCR inhibition is frequent in medical microbiology routine practice and may lead to false-negative results; however there is no consensus on how to detect it. Pathogen-specific and human gene amplifications are widely used to detect PCR inhibition. We aimed at comparing the value of PCR inhibitor detection using these two methods. We analysed Cp shifts (ΔCp) obtained from qPCRs targeting either the albumin gene or the pathogen-specific sequence used in two laboratory-developed microbiological qPCR assays. 3152 samples including various matrixes were included. Pathogen-specific amplification and albumin qPCR identified 62/3152 samples (2.0%), and 409/3152 (13.0%) samples, respectively, as inhibited. Only 16 samples were detected using both methods. In addition, the use of the Youden's index failed to determine adequate Cp thresholds for albumin qPCR, even when we distinguished among the different sample matrixes. qPCR targeting the albumin gene therefore appears not adequate to identify the presence of PCR inhibitors in microbiological PCR assays. Our data may be extrapolated to other heterologous targets and should discourage their use to assess the presence of PCR inhibition in microbiological PCR assays.

Colonization by Pneumocystis jirovecii in patients with chronic obstructive pulmonary disease: association with exacerbations and lung function status.

Exposure to Pneumocystis jirovecii (P. jirovecii) can lead to a wide variety of presenting features ranging from colonization in immunocompetent patients with lung disease, to invasive infections in immunocompromised hosts. Colonization by this fungus in patients with chronic obstructive pulmonary disease (COPD) could be associated with higher rates of exacerbations and impaired lung function in these patients. Our objective was to determine whether colonization by P. jirovecii in patients with COPD is associated with increased exacerbations and deterioration of lung function. This was a prospective cohort study on patients with COPD. All participants meeting selection criteria underwent clinical and microbiological assessments and were then classified as colonized vs. non-colonized patients. Chi-squared tests were performed and multivariate logistic models were fitted in order to obtain risk ratios (RR) with 95% confidence intervals (CI). We documented a frequency of colonization by P. jirovecii of 32.3%. Most patients were categorized as having GOLD B and D COPD. The history of significant exacerbations in the last year, health status impairment (COPD Assesment Tool ≥10), airflow limitation (percent of post-bronchodilator FEV1), and BODEx score (≥5) were similar between groups. After a 52-week follow-up period, the rate of adjusted significant exacerbations did not differ between groups. However, a decrease in FEVI was found in both groups.

Surfactant protein-D modulation of pulmonary macrophage phenotype is controlled by S-nitrosylation.

Surfactant protein-D (SP-D) is a regulator of pulmonary innate immunity whose oligomeric state can be altered through S-nitrosylation to regulate its signaling function in macrophages. Here, we examined how nitrosylation of SP-D alters the phenotypic response of macrophages to stimuli both in vivo and in vitro. Bronchoalveolar lavage (BAL) from C57BL6/J and SP-D-overexpressing (SP-D OE) mice was incubated with RAW264.7 cells ± LPS. LPS induces the expression of the inflammatory genes Il1b and Nos2, which is reduced 10-fold by SP-D OE-BAL. S-nitrosylation of the SP-D OE-BAL (SNO-SP-D OE-BAL) abrogated this inhibition. SNO-SP-D OE-BAL alone induced Il1b and Nos2 expression. PCR array analysis of macrophages incubated with SP-D OE-BAL (±LPS) shows increased expression of repair genes, Ccl20, Cxcl1, and Vcam1, that was accentuated by LPS. LPS increases inflammatory gene expression, Il1a, Nos2, Tnf, and Ptgs2, which was accentuated by SNO-SP-D OE-BAL but inhibited by SP-D OE-BAL. The transcription factor NF-κB was identified as a target for SNO-SP-D by IPA, which was confirmed by Trans-AM ELISA in vitro. In vivo, SP-D overexpression increases the burden of infection in a Pneumocystis model while increasing cellular recruitment. Expression of iNOS and the production of NO metabolites were significantly reduced in SP-D OE mice relative to C57BL6/J. Inflammatory gene expression was increased in infected C57BL6/J mice but decreased in SP-D OE. SP-D oligomeric structure was disrupted in C57BL6/J infected mice but unaltered within SP-D OE. Thus SP-D modulates macrophage phenotype and the balance of multimeric to trimeric SP-D is critical to this regulation.

Primary infection by Pneumocystis induces Notch-independent Clara cell mucin production in rat distal airways.

Clara cells are the main airway secretory cells able to regenerate epithelium in the distal airways through transdifferentiating into goblet cells, a process under negative regulation of the Notch pathway. Pneumocystis is a highly prevalent fungus in humans occurring between 2 and 5 months of age, a period when airways are still developing and respiratory morbidity typically increases. Pneumocystis induces mucus hyperproduction in immunocompetent host airways and whether it can stimulate Clara cells is unknown. Markers of Clara cell secretion and Notch1 activation were investigated in lungs of immunocompetent rats at 40, 60, and 80 days of age during Pneumocystis primary infection with and without Valproic acid (VPA), a Notch inducer. The proportion of rats expressing mucin increased in Pneumocystis-infected rats respect to controls at 60 and 80 days of age. Frequency of distal airways Clara cells was maintained while mRNA levels for the mucin-encoding genes Muc5B and Muc5ac in lung homogenates increased 1.9 and 3.9 times at 60 days of infection (P. = 0.1609 and P. = 0.0001, respectively) and protein levels of the Clara cell marker CC10 decreased in the Pneumocystis-infected rats at 60 and 80 days of age (P. = 0.0118 & P. = 0.0388). CC10 and Muc5b co-localized in distal airway epithelium of Pneumocystis-infected rats at day 60. Co-localization of Muc5b and Ki67 as marker of mitosis in distal airways was not observed suggesting that Muc5b production by Clara cells was independent of mitosis. Notch levels remained similar and no transnucleation of activated Notch associated to Pneumocystis infection was detected. Unexpectedly, mucus was greatly increased at day 80 in Pneumocystis-infected rats receiving VPA suggesting that a Notch-independent mechanism was triggered. Overall, data suggests a Clara to goblet cell transdifferentiation mechanism induced by Pneumocystis and independent of Notch.

Genotyping of Pneumocystis jirovecii by Use of a New Simplified Nomenclature System Based on the Internal Transcribed Spacer Regions and 5.8S rRNA Gene of the rRNA Operon.

Genotyping based on internal transcribed spacer 1 (ITS1) and ITS2 of the rRNA operon has played an important role in understanding the transmission and epidemiology of Pneumocystis jirovecii, one of the major opportunistic pathogens in individuals with AIDS and other immunocompromised individuals. The widespread use of this typing system has resulted in several problems, including inconsistent genotype nomenclatures, difficult data transferability, and complicated interpretation of the length variation in multiple homopolymeric tracts. The aim of this study was to establish a new, simplified genotype nomenclature system for P. jirovecii based on the ITS1 and ITS2 sequences. We first analyzed the complete ITS1, 5.8S rRNA gene, and ITS2 sequences (termed ITS1-5.8S-ITS2) in 27 recent P. jirovecii isolates from China and identified 18 unique genotypes. Subsequently, we performed a comprehensive classification of more than 400 ITS1- and ITS2-related sequences from GenBank and an in-depth evaluation of the length variation of multiple homopolymeric tracts within ITS1-5.8S-ITS2. Integration of the results from these analyses led to a new, simplified genotype nomenclature system including 62 unique ITS1-5.8S-ITS2 genotypes, simply designated types 1 through 62. This new system offers several advantages over traditional ITS1- and ITS2-based typing systems, including a simpler analysis and interpretation process, a higher discriminative power, and no limitation in assigning potential new genotypes. This new system is expected to facilitate the standardization of P. jirovecii genotyping and easy data exchanges across different laboratories.

Novel dihydropteroate synthase gene mutation in Pneumocystis jirovecii among HIV-infected patients in India: Putative association with drug resistance and mortality.

OBJECTIVES: Pneumocystis pneumonia (PCP) remains a debilitating cause of death among HIV-infected patients. The combination trimethoprim/sulfamethoxazole (SXT) is the most effective anti-Pneumocystis treatment and prophylaxis. However, long-term use of this combination has raised alarms about the emergence of resistant organisms. This study was performed to investigate mutations in the dihydropteroate synthase (DHPS) gene and their clinical consequences in HIV-infected patients with PCP. METHODS: A total of 76 clinically suspected cases of PCP among HIV-seropositive adult patients from March 2014 to March 2017 were included. Clinical samples (bronchoalveolar lavage fluid and sputum) were investigated for the detection of Pneumocystis jirovecii using both microscopy and nested PCR. DHPS genotyping and mutational analyses were performed and the data were correlated with clinical characteristics. RESULTS: Among the 76 enrolled HIV-positive patients, only 17 (22.4%) were positive for P. jirovecii. DHPS gene sequencing showed a novel nucleotide substitution at position 288 (Val96Ile) in three patients (3/12; 25.0%). Patients infected with the mutant P. jirovecii genotype had severe episodes of PCP, did not respond to SXT and had a fatal outcome (P=0.005). All three patients had a CD4+ T-cell count <100 cells/µL, and two also had co-infections. CONCLUSION: This study suggests that the emergence of a mutant P. jirovecii genotype is probably associated with drug resistance and mortality. The data also suggest that DHPS mutational analyses should be performed in HIV-seropositive patients to avoid treatment failure and death due to PCP. However, the role of underlying disease severity and co-morbidities should not be underestimated.

Comparative Evaluation Between the RealStar Pneumocystis jirovecii PCR Kit and the AmpliSens Pneumocystis jirovecii (carinii)-FRT PCR Kit for Detecting P. jirovecii in Non-HIV Immunocompromised Patients.

BACKGROUND: Real-time PCR is more sensitive than microscopic examination for detecting Pneumocystis jirovecii. We compared the performance of two assays for detecting P. jirovecii DNA: the RealStar Pneumocystis jirovecii PCR Kit 1.0 CE (Altona Diagnostics, Hamburg, Germany) and the AmpliSens Pneumocystis jirovecii (carinii)-FRT PCR kit (InterLabService Ltd., Moscow, Russia). METHODS: We used 159 samples from the lower respiratory tract (112 bronchoalveolar lavage [BAL] fluid, 37 sputum, and 10 endotracheal aspirate [ETA] samples) of non-HIV immunocompromised patients. Nested PCR and sequencing were used to resolve discordant results. The performance of the two assays was evaluated according to clinical categories (clinical Pneumocystis pneumonia [PCP], possible PCP, or unlikely PCP) based on clinical and radiological observations. RESULTS: The positive and negative percent agreement values were 100% (95% confidence interval [CI], 85.4-100%) and 96.6% (95% CI, 90.9-98.9%), respectively, and kappa was 0.92 (95% CI, 0.84-0.99). P. jirovecii DNA load was significantly higher in the clinical PCP group than in the other groups (P<0.05). When stratified by sample type, the positive rate for BAL fluids from the clinical PCP group was 100% using either assay, whereas the positive rate for sputum/ETA samples was only 20%. CONCLUSIONS: The two assays showed similar diagnostic performance and detected low P. jirovecii burden in BAL fluids. Both assays may be useful as routine methods for detecting P. jirovecii DNA in a clinical laboratory setting, though their results should be interpreted considering sample type.

The clinical impact of pneumocystis and viral PCR testing on bronchoalveolar lavage in immunosuppressed patients.

INTRODUCTION: Pulmonary infiltrates in immunosuppressed patients are common. Yields from bronchoscopy with bronchoalveolar lavage (BAL) has been reported to be between 31 and 65%. The clinical impact of pneumocystis and viral Polymerase chain reaction (PCR) testing on BAL has not been extensively evaluated in a mixed immunosuppressed patient population. METHODS: We performed a retrospective chart review of immunosuppressed adults with pulmonary infiltrates who underwent BAL at the University of Rochester Medical Center. Only one BAL per patient was included. We compared the rate of positive PCR testing to conventional testing. We then investigated factors associated with positive PCR testing. Finally, we assessed for changes in antimicrobial therapy after bronchoscopy. RESULTS: Three hundred and fifty-nine patients underwent BAL with 249 patients having pneumocystis PCR testing and 142 having viral PCR testing. Pneumocystis identification occurred in 43 patients and viral species identification occurred in 56 patients. PCR testing increased pneumocystis identification compared to microscopy, 14% vs. 5%, p = 0.01, and viral identification compared to culture, 25% vs. 6%, p = 0.0001. Of the patients with positive pneumocystis PCR testing 49% had antibiotics stopped, 66% were started on anti-pneumocystis therapy, and only 6% did not receive treatment. There was no difference in the number of patients with antibiotics stopped based on viral PCR testing results. DISCUSSION: PCR testing increases BAL yield in immunosuppressed patients compared to conventional testing. Pneumocystis identified by PCR only may cause a self-limited infection and may not require antimicrobial therapy. PCR testing should be included in the evaluation of pulmonary infiltrates in immunosuppressed patients.

Intrinsic Programming of Alveolar Macrophages for Protective Antifungal Innate Immunity Against Pneumocystis Infection.

Invasive fungal infections, including Pneumocystis Pneumonia (PcP), remain frequent life-threatening conditions of patients with adaptive immune defects. While innate immunity helps control pathogen growth early during infection, it is typically not sufficient for complete protection against Pneumocystis and other human fungal pathogens. Alveolar macrophages (AM) possess pattern recognition molecules capable of recognizing antigenic and structural determinants of Pneumocystis. However, this pathogen effectively evades innate immunity to infect both immunocompetent and immunosuppressed hosts, albeit with differing outcomes. During our studies of mouse models of PcP, the FVB/N strain was identified as unique because of its ability to mount a protective innate immune response against Pneumocystis infection. In contrast to other immunocompetent strains, which become transiently infected prior to the onset of adaptive immunity, FVB/N mice rapidly eradicated Pneumocystis before an adaptive immune response was triggered. Furthermore, FVB/N mice remained highly resistant to infection even in the absence of functional T cells. The effector mechanism of innate protection required the action of functional alveolar macrophages, and the adoptive transfer of resistant FVB/N AMs, but not susceptible CB.17 AMs, conferred protection to immunodeficient mice. Macrophage IFNγ receptor signaling was not required for innate resistance, and FVB/N macrophages were found to display markers of alternative activation. IFNγ reprogrammed resistant FVB/N macrophages to a permissive M1 biased phenotype through a mechanism that required direct activation of the macrophage IFNγR. These results demonstrate that appropriately programmed macrophages provide protective innate immunity against this opportunistic fungal pathogen, and suggest that modulating macrophage function may represent a feasible therapeutic strategy to enhance antifungal host defense. The identification of resistant and susceptible macrophages provides a novel platform to study not only the mechanisms of macrophage-mediated antifungal defense, but also the mechanisms by which Pneumocystis evades innate immunity.

Site-Directed Mutagenesis of the 1,3-ß-Glucan Synthase Catalytic Subunit of Pneumocystis jirovecii and Susceptibility Assays Suggest Its Sensitivity to Caspofungin.

The echinocandin caspofungin inhibits the catalytic subunit Gsc1 of the enzymatic complex synthesizing 1,3-ß-glucan, an essential compound of the fungal wall. Studies with rodents showed that caspofungin is effective against Pneumocystis asci. However, its efficacy against asci of Pneumocystis jirovecii, the species infecting exclusively humans, remains controversial. The aim of this study was to assess the sensitivity to caspofungin of the P. jirovecii Gsc1 subunit, as well as of those of Pneumocystis carinii and Pneumocystis murina infecting, respectively, rats and mice. In the absence of an established in vitro culture method for Pneumocystis species, we used functional complementation of the Saccharomyces cerevisiae gsc1 deletant. In the fungal pathogen Candida albicans, mutations leading to amino acid substitutions in Gsc1 confer resistance to caspofungin. We introduced the corresponding mutations into the Pneumocystis gsc1 genes using site-directed mutagenesis. In spot dilution tests, the sensitivity to caspofungin of the complemented strains decreased with the number of mutations introduced, suggesting that the wild-type enzymes are sensitive. The MICs of caspofungin determined by Etest and YeastOne for strains complemented with Pneumocystis enzymes (respectively, 0.125 and 0.12 µg/ml) were identical to those upon complementation with the enzyme of C. albicans, for which caspofungin presents low MICs. However, they were lower than the MICs upon complementation with the enzyme of the resistant species Candida parapsilosis (0.19 and 0.25 µg/ml). Sensitivity levels of Gsc1 enzymes of the three Pneumocystis species were similar. Our results suggest that P. jirovecii is sensitive to caspofungin during infections, as are P. carinii and P. murina.

Real-time PCR assay for screening Pneumocystis in free-living wild squirrels and river rats in Italy.

We used a real-time PCR (rtPCR) targeting a 150-bp amplicon of the mitochondrial small subunit of ribosomal RNA (mtSSU rRNA) to screen for Pneumocystis DNA in lungs of wild squirrels ( Callosciurus finlaysonii, n = 85) and river rats ( Myocastor coypus, n = 43) in Italy. The rtPCR revealed Pneumocystis DNA in 20 of 85 (24%) squirrels and in 35 of 43 (81%) river rats, and was more sensitive than a nested PCR that targets a portion of the mtSSU rRNA and the mitochondrial large subunit of rRNA (mtLSU rRNA). Phylogenetic analysis based on mtSSU rRNA and mtLSU rRNA sequences showed distinct Pneumocystis sequence types in these rodents. The rtPCR assay should be reliable for screening large populations for this potential pathogen, thereby allowing cost-effective monitoring of the disease in wild animals.

A Molecular Window into the Biology and Epidemiology of Pneumocystis spp.

Pneumocystis, a unique atypical fungus with an elusive lifestyle, has had an important medical history. It came to prominence as an opportunistic pathogen that not only can cause life-threatening pneumonia in patients with HIV infection and other immunodeficiencies but also can colonize the lungs of healthy individuals from a very early age. The genus Pneumocystis includes a group of closely related but heterogeneous organisms that have a worldwide distribution, have been detected in multiple mammalian species, are highly host species specific, inhabit the lungs almost exclusively, and have never convincingly been cultured in vitro, making Pneumocystis a fascinating but difficult-to-study organism. Improved molecular biologic methodologies have opened a new window into the biology and epidemiology of Pneumocystis. Advances include an improved taxonomic classification, identification of an extremely reduced genome and concomitant inability to metabolize and grow independent of the host lungs, insights into its transmission mode, recognition of its widespread colonization in both immunocompetent and immunodeficient hosts, and utilization of strain variation to study drug resistance, epidemiology, and outbreaks of infection among transplant patients. This review summarizes these advances and also identifies some major questions and challenges that need to be addressed to better understand Pneumocystis biology and its relevance to clinical care.