Semiquantitative Real-Time PCR to Distinguish Pneumocystis Pneumonia from Colonization in a Heterogeneous Population of HIV-Negative Immunocompromised Patients.

Pneumocystis jirovecii is a threat to iatrogenically immunosuppressed individuals, a heterogeneous population at rapid growth. We assessed the ability of an in-house semiquantitative real-time PCR assay to discriminate Pneumocystis pneumonia (PCP) from colonization and identified risk factors for infection in these patients. Retrospectively, 242 PCR-positive patients were compared according to PCP status, including strata by immunosuppressive conditions, human immunodeficiency virus (HIV) infection excluded. Associations between host characteristics and cycle threshold (CT) values, semiquantitative real-time PCR correlates of fungal loads in lower respiratory tract specimens, were investigated. CT values differed significantly according to PCP status. Overall, a CT value of 36 allowed differentiation between PCP and colonization with sensitivity and specificity of 71.3% and 77.1%, respectively. A CT value of less than 31 confirmed PCP, whereas no CT value permitted exclusion. A considerable diversity was uncovered; solid organ transplant (SOT) recipients had significantly higher fungal loads than patients with hematological malignancies. In SOT recipients, a CT cutoff value of 36 resulted in sensitivity and specificity of 95.0% and 83.3%, respectively. In patients with hematological malignancies, a higher CT cutoff value of 37 improved sensitivity to 88.5% but reduced specificity to 66.7%. For other conditions, assay validity appeared inferior. Corticosteroid usage was an independent predictor of PCP in a multivariable analysis and was associated with higher fungal loads at PCP expression. Semiquantitative real-time PCR improves differentiation between PCP and colonization in immunocompromised HIV-negative individuals with acute respiratory syndromes. However, heterogeneity in disease evolution requires separate cutoff values across intrinsic and iatrogenic predisposition for predicting non-HIV PCP. IMPORTANCE Pneumocystis jirovecii is potentially life threatening to an increasing number of individuals with compromised immune systems. This microorganism can cause severe pneumonia in susceptible hosts, including patients with cancer and autoimmune diseases and people undergoing solid organ transplantation. Together, these patients constitute an ever-diverse population. In this paper, we demonstrate that the heterogeneity herein has important implications for how we diagnose and assess the risk of Pneumocystis pneumonia (PCP). Specifically, low loads of microorganisms are sufficient to cause infection in patients with blood cancer compared to those in solid organ recipients. With this new insight into host versus P. jirovecii biology, clinicians can manage patients at risk of PCP more accurately. As a result, we take a significant step toward offering precision medicine to a vulnerable patient population. One the one hand, these patients have propensity for adverse effects from antimicrobial treatment. On the other hand, this population is susceptible to life-threatening infections, including PCP.

Genetic diversity of Pneumocystis jirovecii isolates among Turkish population based on mitochondrial large subunit ribosomal RNA and dihydropteroate synthase gene typing.

Pneumocystis jirovecii (P. jirovecii) is an atypical fungus that can cause severe interstitial pneumonia in immunocompromised patients. In this study, mitochondrial large subunit ribosomal RNA (mtLSU-rRNA) and dihydropteroate synthase (DHPS) gene polymorphism in P. jirovecii isolates were investigated in Western Turkey's Izmir province and its surroundings. For this purpose, a total of 157 P. jirovecii isolates obtained from bronchoalveolar lavage samples of hospitalized cases and lung tissue samples of autopsy cases who died outside hospital were examined. Genotypes were identified by direct sequencing of mtLSU-rRNA restriction fragment length polymorphism analysis of the DHPS gene amplicons. The mtLSU-rRNA analysis revealed that genotype 2 was the most common genotype with 58%. The following genotypes were genotype 3 (13%), genotype 1 (11.6%) and genotype 4 (5.1%), while genotype 5 (0.7%) was detected in only one autopsy case. In addition, 16 (11.6%) cases had dual or triple different genotypes (mixed infection). It was observed that the genotype distribution was not affected by characteristics such as age, gender and immune status. However, the predominance of genotype 2 in solid organ tumors and the predominance of mixed infection in patients with chronic pulmonary disease were statistically significant. On the other hand, DHPS gene amplification was positive in 137 (87.3%) of 157 samples. While no mutation was observed in 135 samples, the association of wild-type and 57th codon mutation was detected in two hospitalized cases (1.5%). In this study, important epidemiological data on the distribution of mtLSU-rRNA genotypes were obtained. Also the existence of DHPS gene mutations associated with potential drug resistance in our community was shown for the first time. Further studies are needed to evaluate the possible effects of genotypes on the prognosis of the disease to help with the clinician's treatment decisions. LAY ABSTRACT: Pneumocystis jirovecii (P. jirovecii) is an atypical fungus that can cause life-threatening pneumonia in immunocompromised patients. In this study, we investigated the mtLSU-rRNA and DHPS gene polymorphisms in P. jirovecii isolates from both hospital and autopsy cases.

Genomic insights into the host specific adaptation of the Pneumocystis genus.

Pneumocystis jirovecii, the fungal agent of human Pneumocystis pneumonia, is closely related to macaque Pneumocystis. Little is known about other Pneumocystis species in distantly related mammals, none of which are capable of establishing infection in humans. The molecular basis of host specificity in Pneumocystis remains unknown as experiments are limited due to an inability to culture any species in vitro. To explore Pneumocystis evolutionary adaptations, we have sequenced the genomes of species infecting macaques, rabbits, dogs and rats and compared them to available genomes of species infecting humans, mice and rats. Complete whole genome sequence data enables analysis and robust phylogeny, identification of important genetic features of the host adaptation, and estimation of speciation timing relative to the rise of their mammalian hosts. Our data reveals insights into the evolution of P. jirovecii, the sole member of the genus able to infect humans.

The prevalence of laboratory-confirmed Pneumocystis jirovecii in HIV-infected adults in Africa: A systematic review and meta-analysis.

BACKGROUND: The epidemiology of Pneumocystis jirovecii, known to colonize the respiratory tract and cause a life-threatening HIV-associated pneumonia (PCP), is poorly described in Africa. We conducted a systematic review to evaluate P. jirovecii prevalence in African HIV-positive adults with or without respiratory symptoms. METHODS: We searched Medline, Embase, Cochrane library, Africa-Wide, and Web of Science for studies employing PCR and/or microscopy for P. jirovecii detection in respiratory samples from HIV-positive adults in Africa between 1995 and 2020. Prevalence with respiratory symptoms was pooled using random-effect meta-analysis, and stratified by laboratory method, sample tested, study setting, CD4 count, and trimethoprim/sulfamethoxazole prophylaxis. Colonization prevalence in asymptomatic adults and in adults with non-PCP respiratory disease was described, and quantitative PCR (qPCR) thresholds to distinguish colonization from microscopy-confirmed PCP reviewed. RESULTS: Thirty-two studies were included, with 27 studies (87%) at high risk of selection bias. P. jirovecii was detected in 19% [95% confidence interval (CI): 12-27%] of 3583 symptomatic and in 9% [95% CI: 0-45%] of 140 asymptomatic adults. Among symptomatic adults, prevalence was 22% [95% CI: 12-35%] by PCR and 15% [95% CI: 9-23%] by microscopy. Seven percent of 435 symptomatic adults had PCR-detected Pneumocystis colonization without evidence of PCP [95% CI: 5-10%, four studies]. One study established a qPCR cutoff of 78 copies/5µl of DNA in 305 induced sputum samples to distinguish Pneumocystis colonization from microscopy-confirmed PCP. CONCLUSION: Despite widened access to HIV services, P. jirovecii remains common in Africa. Prevalence estimates and qPCR-based definitions of colonization are limited, and overall quality of studies is low.

The shift from pulmonary colonization to Pneumocystis pneumonia.

Pulmonary specimen pairs from five patients who presented with pulmonary colonization and later developed Pneumocystis Pneumonia (PcP) were retrospectively examined for P. jirovecii genotyping. A match of genotypes in pulmonary specimen pairs of three patients was observed, whereas a partial match and a mismatch were observed in the fourth and fifth patients, respectively. The genotyping results suggest that the colonization state can differ from PcP but can also represent the incubation period of PcP. Clinicians should not systematically rule out the treatment of putative colonized patients and should at least discuss the initiation of prophylaxis on a case-by-case basis.

The results suggest that clinicians should not systematically rule out the treatment of putative patients colonized by Pneumocystis jirovecii and should at least discuss prophylaxis initiation on a case-by-case basis.

Ability of quantitative PCR to discriminate Pneumocystis jirovecii pneumonia from colonization.

Introduction. Pneumocystis jirovecii pneumonia (PCP) is a severe disease affecting immunocompromised patients. Diagnosis is difficult due to the low sensitivity of direct examination and inability to grow the pathogen in culture. Quantitative PCR in bronchoalveolar lavage fluid (BAL) has high sensitivity, but limited specificity for distinguishing PCP from colonization.Aim. To assess the performance of an in-house quantitative PCR to discriminate between PCP and colonization.Methodology. This was a single-centre retrospective study including all patients with a positive PCR result for P. jirovecii in BAL between 2009 and 2017. Irrespective of PCR results, PCP was defined as the presence of host factors and clinical/radiological criteria consistent with PCP and (i) the presence of asci at direct examination of respiratory sample or (ii) anti-PCP treatment initiated with clinical response and absence of alternative diagnosis. Colonization was considered for cases who did not receive anti-PCP therapy with a favourable outcome or an alternative diagnosis. Cases who did not meet the above mentioned criteria were classified as 'undetermined'.Results. Seventy-one patients with positive P. jirovecii PCR were included (90 % non-HIV patients). Cases were classified as follows: 37 PCP, 22 colonization and 12 undetermined. Quantitative PCR values in BAL were significantly higher in patients with PCP versus colonization or undetermined (P<0.0001). The cut-off of 5×103 copies/ml was able to discriminate PCP cases from colonization with 97 % sensitivity, 82 % specificity, 90 % positive predictive value and 95 % negative predictive value.Conclusions. Our quantitative PCR for P. jirovecii in BAL was reliable to distinguish PCP cases from colonization in this predominantly non-HIV population.

Long-read sequencing based clinical metagenomics for the detection and confirmation of Pneumocystis jirovecii directly from clinical specimens: A paradigm shift in mycological diagnostics.

The advent of next generation sequencing technologies has enabled the characterization of the genetic content of entire communities of organisms, including those in clinical specimens, without prior culturing. The MinION from Oxford Nanopore Technologies offers real-time, direct sequencing of long DNA fragments directly from clinical samples. The aim of this study was to assess the ability of unbiased, genome-wide, long-read, shotgun sequencing using MinION to identify Pneumocystis jirovecii directly from respiratory tract specimens and to characterize the associated mycobiome. Pneumocystis pneumonia (PCP) is a life-threatening fungal disease caused by P. jirovecii. Currently, the diagnosis of PCP relies on direct microscopic or real-time quantitative polymerase chain reaction (PCR) examination of respiratory tract specimens, as P. jirovecii cannot be cultured readily in vitro. P. jirovecii DNA was detected in bronchoalveolar lavage (BAL) and induced sputum (IS) samples from three patients with confirmed PCP. Other fungi present in the associated mycobiome included known human pathogens (Aspergillus, Cryptococcus, Pichia) as well as commensal species (Candida, Malassezia, Bipolaris). We have established optimized sample preparation conditions for the generation of high-quality data, curated databases, and data analysis tools, which are key to the application of long-read MinION sequencing leading to a fundamental new approach in fungal diagnostics.

Beta-d-Glucan for Diagnosing Pneumocystis Pneumonia: a Direct Comparison between the Wako ß-Glucan Assay and the Fungitell Assay.

Measuring serum beta-d-glucan (BDG) is a useful tool for supporting a quantitative PCR (qPCR)-based diagnosis of suspected Pneumocystis pneumonia (PCP) with bronchoalveolar lavage (BAL) fluid. Since the 2000s, the Fungitell assay was the only BDG assay which was FDA cleared and Conformité Européenne (CE) marked. However, the Wako ß-glucan test was also recently CE marked and commercialized. We analyzed archived sera from 116 PCP cases (who were considered to have PCP based on compatible clinical and radiological findings plus a BAL fluid qPCR threshold cycle value of ≤28) and 114 controls (those with a BAL fluid qPCR threshold cycle value of >45 and no invasive fungal infection) using the Fungitell and Wako assays in parallel and assessed their diagnostic performance using the manufacturer's proposed cutoffs of 80 pg/ml and 11 pg/ml, respectively. We found the Wako assay to be more specific (0.98 versus 0.87, P < 0.001) and the Fungitell assay to be more sensitive (0.78 versus 0.85, P = 0.039) at the proposed cutoffs. Overall performance, as determined by the area under the receiver operating characteristic curve, was similar for both assays. We determined a new Wako assay cutoff (3.616 pg/ml) to match the sensitivity of the Fungitell assay (0.88 at a cutoff of ≥60 pg/ml). Using this newly proposed cutoff, the specificity of the Wako assay was significantly better than that of the Fungitell assay (0.89 versus 0.82, P = 0.011). In conclusion, the Wako assay performed excellently compared to the Fungitell assay for the diagnosis of presumed PCP based on qPCR. In addition, contrary to the Fungitell assay, the Wako assay allows for single-sample testing with lower inter- and intrarun variability. Finally, we propose an optimized cutoff for the Wako assay to reliably exclude PCP.

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.

Prevalence and genotyping of Pneumocystis jirovecii in renal transplant recipients-preliminary report.

Pneumocystis jirovecii is an opportunistic fungus occurring in human lungs. The group at highest risk consists of HIV-infected and non-HIV-infected immunosuppressed individuals. In these patients, P. jirovecii infection may lead to Pneumocystis pneumonia; it may, however, persist also in an asymptomatic form. This study aimed to determine the prevalence of P. jirovecii and potential risk factors for infection in a group of renal transplant recipients and to characterize the genetic diversity of this fungus in the studied population. Sputum specimens from 72 patients were tested for presence of P. jirovecii using immunofluorescence microscopy, as well as nested PCR targeting the mtLSU rRNA gene. Genotyping involving analysis of four loci-mtLSU rRNA, CYB, DHPS, and SOD-was used to characterize the diversity of the detected organisms. Pneumocystis DNA was detected in eight (11.11%) patients. It has been shown that low eosinophil count and dual immunosuppressive treatment combining prednisone and calcineurin inhibitors are potential risk factors for colonization. Analysis of genotype distribution showed an association of the wild-type genotype of mtLSU rRNA with lower average age of patients and shorter time after kidney transplantation. Furthermore, CYB 2 genotype was detected only in patients with the ongoing prophylaxis regimen. In conclusion, renal transplant recipients are at risk of Pneumocystis colonization even a long time after transplantation. The present preliminary study identifies specific polymorphisms that appear to be correlated with certain patient characteristics and highlights the need for deeper investigation of these associations in renal transplant recipients.

Genotyping of Pneumocystis jirovecii in colonized patients with various pulmonary diseases.

Pneumocystis jirovecii is an opportunistic fungus causing Pneumocystis pneumonia primarily in immunosuppressed patients. However, immunocompetent individuals may become colonized and, as asymptomatic carriers, serve as reservoirs of the pathogen. Moreover, these asymptomatic carriers are at higher risk of developing pneumonia if favorable conditions occur. This study aimed to determine the prevalence of P. jirovecii in patients with various pulmonary diseases and to characterize the genetic diversity of organisms circulating in the studied population. Bronchial washing specimens from 105 patients were tested for presence of P. jirovecii using nested polymerase chain reaction (PCR) targeting the mtLSU rRNA gene, as well as immunofluorescence microscopy. Multilocus sequence typing involving analysis of three loci-mtLSU rRNA, CYB, and SOD-was used for genotyping analysis. P. jirovecii DNA was detected in 17 (16.2%) patients. Amplification of the SOD locus was successful only in five cases (29.4% of the positive patients), while mtLSU rRNA and CYB were genotyped in all positive samples. Therefore, combined genotypes were identified based only on mtLSU rRNA and CYB loci. Eight different genotypes were identified, with Pj 1 and Pj 2 being the most prevalent (29.4% of patients each). There was no statistical correlation between these genotypes and demographic or clinical data; however, we found that infection with mutant CYB strains occurred only in patients diagnosed with lung cancer. Of the potential predictors examined, only immunosuppressive treatment was significantly associated with colonization. In conclusion, patients with various respiratory diseases, especially when immunosuppressed, are at risk of Pneumocystis colonization.

Investigation of nosocomial pneumocystis infections: usefulness of longitudinal screening of epidemic and post-epidemic pneumocystis genotypes.

BACKGROUND: Twenty-five patients, of whom 22 were renal transplant recipients, developed Pneumocystis jirovecii infections at the nephrology department of Reims University Hospital (France) from September 2008 to October 2009, whereas only four sporadic cases had been diagnosed in this department over the 14 previous years. AIM: This outbreak was investigated by analysing patient encounters and P. jirovecii types. METHODS: A transmission map was drawn up. P. jirovecii typing at DHPS, ITS and mtLSU rRNA sequences was performed in the patients of the cluster (18 patients with Pneumocystis pneumonia (PCP) and seven colonized patients), 10 unlinked control patients (six PCP patients and four colonized patients), as well as 23 other patients diagnosed with P. jirovecii (nine PCP patients and 14 colonized patients) in the same department over a three-year post-epidemic period. FINDINGS: Eleven encounters between patients harbouring the same types were observed. Three PCP patients and one colonized patient were considered as possible index cases. The most frequent types in the cluster group and the control group were identical. However, their frequency was significantly higher in the first than in the second group (P < 0.01). Identical types were also identified in the post-epidemic group, suggesting a second outbreak due to the same strain, contemporary to a disruption in prevention measures. CONCLUSIONS: These results provide additional data on the role of both PCP and colonized patients as infectious sources. Longitudinal screening of P. jirovecii types in infected patients, including colonized patients, is required in the investigation of the fungus's circulation within hospitals.

Colonización por Pneumocystis jirovecii en la enfermedad pulmonar obstructiva crónica / Colonization by Pneumocystis jirovecii in Chronic Obstructive Pulmonary Disease

La enfermedad pulmonar obstructiva crónica (EPOC) es una enfermedad prevalente en la población colombiana, que característicamente tiene un deterioro progresivo y altera la calidad de vida de los pacientes. Solo del 15% al 20% de los pacientes con antecedente de tabaquismo desarrollan la enfermedad, por lo que factores ambientales y genéticos adicionales influyen en su progresión. De las causas infecciosas que han tomado importancia, el Pneumocystis jiroveái, un hongo ubicuo que entra en contacto con la vía aérea de los humanos desde la infancia, es causa de neumonía en pacientes inmunosuprimidos. Se han descrito tasas elevadas de colonización en pacientes con EPOC, que aumentan con la severidad de la enfermedad. EPOC e infección por P jiroveái parecen compartir una respuesta inmunológica similar; lo cual podría explicar el papel de la colonización por el hongo en su progresión y gravedad.

Chronic obstructive pulmonary disease (COPD) is a prevalent disease in our population, which characteristically has a Progressive deterioration and alters the quality of Iife of patients. Only 15-20% of patients with a history of smoking develop the disease, so there are additional environmental and genetic factors that influence the progression of the disease. Of the infectious causes that have taken importance is Pneumocystis jiroveái, this is a ubiquitous fungus that comes into contact with the airway of humans since childhood and is a cause of pneumonía in immunosuppressed patients. In addition, high rates of colonization have been reported in patients with COPD, which increase with the severity of the disease. COPD and P jiroveái infection appear to share a similar immune response, which may explain the role of fungal colonization in the progression and severity of the disease.

Pneumocystis jirovecii genotyping: experience in a tertiary-care hospital in Northern Italy.

Respiratory samples from Pneumocystis jirovecii pneumonia (PJP) cases collected at a tertiary-care university hospital in Modena were analyzed for the presence of specific polymorphisms in the mitochondrial large subunit ribosomal RNA (mtLSU-rRNA). Retrospectively, 57 cases were selected in a six-year period and 34 out of the 57 processed BAL samples returned PCR positive results, thus allowing further molecular analysis. The following P.jirovecii genotype distribution was observed: genotype 3 (50%), genotype 2 (23%), genotype 1 (18%), genotypes 1 or 4 (9%). These data add novel insights on P.jirovecii epidemiology, investigating a previously unstudied area of Northern Italy. A peculiar local distribution is highlighted with respect to other areas within the national panorama, thus encouraging further in-depth studies in an attempt to better understand the overall situation concerning P.jirovecii genotype circulation.

Added Value of Next-Generation Sequencing for Multilocus Sequence Typing Analysis of a Pneumocystis jirovecii Pneumonia Outbreak1.

Pneumocystis jirovecii is a major threat for immunocompromised patients, and clusters of pneumocystis pneumonia (PCP) have been increasingly described in transplant units during the past decade. Exploring an outbreak transmission network requires complementary spatiotemporal and strain-typing approaches. We analyzed a PCP outbreak and demonstrated the added value of next-generation sequencing (NGS) for the multilocus sequence typing (MLST) study of P. jirovecii strains. Thirty-two PCP patients were included. Among the 12 solid organ transplant patients, 5 shared a major and unique genotype that was also found as a minor strain in a sixth patient. A transmission map analysis strengthened the suspicion of nosocomial acquisition of this strain for the 6 patients. NGS-MLST enables accurate determination of subpopulation, which allowed excluding other patients from the transmission network. NGS-MLST genotyping approach was essential to deciphering this outbreak. This innovative approach brings new insights for future epidemiologic studies on this uncultivable opportunistic fungus.

Diversity of Pneumocystis jirovecii Across Europe: A Multicentre Observational Study.

Pneumocystis jirovecii is an airborne human-specific ascomycetous fungus responsible for Pneumocystis pneumonia (PCP) in immunocompromised patients, affecting >500,000 patients per year (www.gaffi.org). The understanding of its epidemiology is limited by the lack of standardised culture. Recent genotyping data suggests a limited genetic diversity of P. jirovecii. The objective of the study was to assess the diversity of P. jirovecii across European hospitals and analyse P. jirovecii diversity in respect to clinical data obtained from the patients. Genotyping was performed using six already validated short tandem repeat (STR) markers on 249 samples (median: 17 per centre interquartile range [11-20]) from PCP patients of 16 European centres. Mixtures of STR markers (i.e., ≥2 alleles for ≥1 locus) were detected in 67.6% (interquartile range [61.4; 76.5]) of the samples. Mixture was significantly associated with the underlying disease of the patient, with an increased proportion in HIV patients (78.3%) and a decreased proportion in renal transplant recipients (33.3%) (p<0.001). The distribution of the alleles was significantly different (p<0.001) according to the centres in three out of six markers. In analysable samples, 201 combinations were observed corresponding to 137 genotypes: 116 genotypes were country-specific; 12 in two; six in three; and two in four and one in five countries. Nine genotypes were recorded more than once in a given country. Genotype 123 (Gt123) was significantly associated with France (14/15, p<0.001) and Gt16 with Belgium (5/5, p<0.001). More specifically, Gt123 was observed mainly in France (14/15/16 patients) and in renal transplant patient (13/15). Our study showed the wide population diversity across Europe, with evidence of local clusters of patients harbouring a given genotype. These data suggest a specific association between genotype and underlying disease, with evidence of a different natural history of PCP in HIV patients and renal transplant recipients.

Infectious disease ward admission positively influences P. jiroveci pneumonia (PjP) outcome: A retrospective analysis of 116 HIV-positive and HIV-negative immunocompromised patients.

P. jiroveci (Pj) causes a potentially fatal pneumonia in immunocompromised patients and the factors associated with a bad outcome are poorly understood. A retrospective analysis on Pj pneumonia (PjP) cases occurring in Tor Vergata University Hospital, Italy, during the period 2011-2015. The patients' demographic, clinical and radiological characteristics and the Pj genotypes were considered. The study population included 116 patients, 37.9% of whom had haematological malignancy or underwent haematological stem cell transplantation (HSCT), 22.4% had HIV infection, 16.4% had chronic lung diseases (CLD), 7.8% had a solid cancer, and 3.4% underwent a solid organ transplant (SOT). The remaining 12.1% had a miscellaneous other condition. At univariate analysis, being older than 60 years was significantly correlated with a severe PjP (OR [95%CI] 2.52 [0.10-5.76]; p = 0.031) and death (OR [95%CI] 2.44 [1.05-5.70]; p = 0.036), while a previous trimethoprim/sulfamethoxazole (TMP/SMX) prophylaxis were significantly associated with a less severe pneumonia (OR[95%CI] 0.35 [0.15-0.84], p = 0.023); moreover, death due to PjP was significantly more frequent in patients with CLD (OR[95%CI] 3.26 [1.17-9.05]; p = 0.019) while, admission to the Infectious Diseases Unit was significantly associated with fewer deaths (OR[95%CI] 0.10 [0.03-0.36], p = 0.002). At multivariate analysis, a better PjP outcome was observed in patients taking TMP/SMX prophylaxis and that were admitted to the Infectious Diseases Unit (OR[95%CI] 0.27 [0.07-1.03], p = 0.055, OR[95%CI] 0.16 [0.05-0.55]; p = 0.004, respectively). In conclusion, in our study population, TMP/SMX prophylaxis and infectious disease specialist approach were variables correlated with a better PjP outcome.

Circulating genotypes of Pneumocystis jirovecii and its clinical correlation in patients from a single tertiary center in India.

The present study was carried out with the objectives of genotyping Pneumocystis jirovecii at three distinct loci, to identify the single nucleotide polymorphisms (SNPs), and to study its clinical implications in patients with Pneumocystis pneumonia (PCP). Analysis of genetic diversity in P. jirovecii from immunocompromised patients was carried out by genotyping at three distinct loci encoding mitochondrial large subunit rRNA (mtLSU rRNA), cytochrome b (CYB), and superoxide dismutase (SOD) using polymerase chain reaction (PCR) assays followed by direct DNA sequencing. Of the 300 patients enrolled in the present study, 31 (10.33%) were positive for PCP by a specific mtLSU rRNA nested PCR assay, whereas only 15 P. jirovecii could be amplified at the other two loci (SOD and CYB). These positives were further subjected to sequence typing. Important genotypic combinations between four SNPs (mt85, SOD110, SOD215, and CYB838) and clinical outcomes could be observed in the present study, and mt85A, mt85T, and SOD110C/SOD215T were frequently associated with "negative follow-up". These SNPs were also noted to be relatively more prevalent amongst circulating genotypes in our study population. The present study is the first of its kind from the Indian subcontinent and demonstrated that potential SNPs of P. jirovecii may possibly be attributed to the clinical outcome of PCP episodes in terms of severity or fatality in different susceptible populations likely to develop PCP during their course of illness.

Molecular diagnosis of Pneumocystis pneumonia in dogs.

Pneumocystis pneumonia (PCP) is a life-threatening fungal disease that can occur in dogs. The aim of this study was to provide a preliminary genetic characterisation of Pneumocystis carinii f.sp.'canis' (P. canis) in dogs and thereby develop a reliable molecular protocol to definitively diagnose canine PCP. We investigated P. canis in a variety of lung specimens from dogs with confirmed or strongly suspected PCP (Group 1, n = 16), dogs with non-PCP lower respiratory tract problems (Group 2, n = 65) and dogs not suspected of having PCP or other lower respiratory diseases (Group 3, n = 11). Presence of Pneumocystis DNA was determined by nested PCR of the large and small mitochondrial subunit rRNA loci and by a real-time quantitative polymerase chain reaction (qPCR) assay developed using a new set of primers. Molecular results were correlated with the presence of Pneumocystis morphotypes detected in cytological/histological preparations. Pneumocystis DNA was amplified from 13/16 PCP-suspected dogs (Group 1) and from 4/76 dogs of control Groups 2 and 3 (combined). The latter four dogs were thought to have been colonized by P. canis. Comparison of CT values in 'infected' versus 'colonized' dogs was consistent with this notion, with a distinct difference in molecular burden between groups (CT ≤ 26 versus CT range (26