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Bronchoalveolar lavage fluid (BALF) is a standard respiratory sample for diagnosing invasive fungal diseases like Pneumocystis pneumonia (PCP) and invasive pulmonary aspergillosis (IPA). However, procedural variations exist across medical centers and wards. This study aimed to compare the diagnostic potential of BALF and bronchial aspirate (BA) obtained during bronchoscopy in 173 patients suspected of fungal infections. A prospective observational study was conducted from April 2020 to November 2021. BALF and BA were collected during bronchoscopy and subjected to direct examination, fungal culture, Aspergillus fumigatus qPCR (AfqPCR), and Pneumocystis jirovecii qPCR (PjqPCR). Galactomannan detection was performed on BALF. Patients were classified based on established European Organization for Research and Treatment of Cancer (EORTC) criteria. Out of 173 patients, 75 tested positive for at least one test in BA or BALF. For Aspergillus, proportion of positive AfqPCR (14.5% vs. 9.2%; P < 0.0001) and fungal loads (Cq of 31.3 vs. 32.8; P = 0.0018) were significantly higher in BA compared to BALF. For Pneumocystis, fungal loads by PjqPCR was also higher in BA compared to BALF (Cq of 34.2 vs. 35.7; P = 0.003). BA only detected A. fumigatus and P. jirovecii in 12 (42.9%) and 8 (19.5%) patients, respectively. BA obtained during a BAL procedure can be a suitable sample type for increased detection of P. jirovecii and A. fumigatus by qPCR. The use of BA in diagnostic algorithms requires further investigation in prospective studies., (© The Author(s) 2023. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology.)

Pneumocystis jirovecii is a transmissible fungus responsible for severe pneumonia (Pneumocystis pneumonia [PCP]) in immunocompromised patients. Missense mutations due to atovaquone selective pressure have been identified on cytochrome b (CYB) gene of P. jirovecii. It was recently shown that atovaquone prophylaxis can lead to the selection of specific P. jirovecii CYB mutants potentially resistant to atovaquone among organ transplant recipients. In this context, our objectives were to provide data on P. jirovecii CYB mutants and the putative selective pressure exerted by atovaquone on P. jirovecii organisms in France. A total of 123 patients (124 P. jirovecii specimens) from four metropolitan hospitals and two overseas hospitals were retrospectively enrolled. Fourteen patients had prior exposure to atovaquone, whereas 109 patients did not at the time of P. jirovecii detection. A 638 base-pair fragment of the CYB gene of P. jirovecii was amplified and sequenced. A total of 10 single nucleotide polymorphisms (SNPs) were identified. Both missense mutations C431T (Ala144Val) and C823T (Leu275Phe), located at the Qo active site of the enzyme, were significantly associated with prior atovaquone exposure, these mutations being conversely incidental in the absence of prior atovaquone exposure (P < 0.001). Considering that the aforementioned hospitals may be representative of the national territory, these findings suggest that the overall presence of P. jirovecii CYB mutants remains low in France., (© The Author(s) 2023. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology.)

Pneumocystis pneumonia (PCP) is the most frequent fungal opportunistic infection defining AIDS in HIV-infected patients, and is of growing importance in HIV-negative patients. In this latter category of patients, the diagnosis mainly relies on real-time polymerase chain reaction (qPCR) detection of Pneumocystis jirovecii (Pj) on respiratory samples. The PneumoGenius® kit (PathoNostics) allows the simultaneous detection of Pj mitochondrial large subunit (mtLSU) and dihydropteroate synthase (DHPS) polymorphisms, which could be of interest to anticipate therapeutic failure. This study aimed at evaluating its clinical performance on 251 respiratory specimens (239 patients), (i) for P. jirovecii detection in clinical samples, and (ii) for DHPS polymorphisms detection in circulating strains. Patients were classified according to modified European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and the National Institute of Allergy and Infectious Diseases Mycoses Study Group (EORTC/MSG) criteria, as having proven PCP (n = 62), probable PCP (n = 87), Pneumocystis colonization (n = 37), and no PCP (n = 53). Compared with in-house qPCR, the sensitivity of PneumoGenius® assay for P. jirovecii detection reached 91.9% (182/198), the specificity was excellent (100%, 53/53) and the global concordance was 93.6% (235/253). A total of four diagnoses of proven/probable PCP were missed by the PneumoGenius® assay, reaching a 97.5% sensitivity (157/161) in this sub-group. The 12 other 'false-negative' results were obtained in patients diagnosed as colonized using the in-house PCR. DHPS genotyping was successful for 147/182 samples with PneumoGenius® and revealed dhps mutation in 8 samples, which were all confirmed by sequencing. In conclusion, PneumoGenius® assay missed the detection of low-burden PCP. This lower sensitivity for PCP diagnosis can be balanced by a higher specificity (P. jirovecii colonization less frequently detected) and the efficient detection of DHPS hot spot mutations., (© The Author(s) 2023. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology.)

Pneumocystis jirovecii (Pj) is a fungal pathogen that can cause severe and potential fatal pneumonia (Pneumocystis pneumonia, PCP) in immunocompromised patients. Microbiological diagnosis is necessary to confirm PCP, for which mainly real-time PCR assays are used by detecting Pj from bronchoalveolar lavage (BAL) specimens. In this study, we evaluate the performance of the CE-IVD PneumoGenius® assay and CE-IVD RealStar® Pneumocystis jirovecii PCR assay in comparison to the lab developed test (LDT) that is used in routine diagnostics. Comparison was done by including 100 BAL specimens: 25 retrospective specimens, selected based on results obtained with LDT (15 positive/10 negative), and 75 prospectively collected specimens. LDT (targeting MSG) was performed according to local procedures and the PneumoGenius® (targeting mtLSU and DHPS fas) and RealStar® assays (targeting mtLSU) according to the manufacturer's instructions. Combining results of retrospective and prospective analysis, sensitivity was 69.7, 100 and 100% for the LDT, PneumoGenius® and RealStar®, respectively. Specificity was 100% for LDT and Pneumogenius®, whereas RealStar® showed a specificity of 97%. Correlation of fungal loads found with the PneumoGenius® and RealStar® assays was high (R2: 0.98). The PneumoGenius® and RealStar® assays performed comparable, and both showed high sensitivity in comparison to the LDT. For optimal diagnosis of PCP, the LDT has to be replaced by another, more sensitive assay., Lay Summary: In this study, we evaluated the performance of two commercially available CE-IVD cleared real-time PCR assays to detect Pneumocystis jirovecii in comparison to the lab-developed test as used in routine diagnostics. Performance of the CE-IVD real-time PCR assay was superior to the lab-developed test., (© The Author(s) 2022. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology.)

Pneumocystis jirovecii is associated with non-noxious colonization or severe pneumonia in immunocompromised hosts. Epidemiological investigations have been hampered by the lack of a standardized typing scheme. Thus, only partial molecular data on Spanish P. jirovecii cases are available. Recently, a new ISHAM consensus multilocus sequence typing scheme (MLST) targeting β-TUB, mt26S, CYB, and SOD with a publicly accessible database has been launched to overcome this problem. The molecular epidemiology of P. jirovecii from immunocompromised patients either colonized (n = 50) or having pneumonia (n = 36) seen between 2014 and 2018 at a single center in Barcelona, Spain, was studied. The new ISHAM consensus MSLT scheme was used to investigate the local epidemiology and identify possible unnoticed outbreaks. Mutations in the DHPS gene, not included in the scheme but giving information about potential sulfa treatment failure, were also studied. The study assigned 32 sequence types (ST) to 72.2% pneumonia and 56% colonization cases. The most frequent STs were ST21 (18.5%), ST22 (14.8%), and ST37(14.8%). For non-unique STs, ST3, ST30 and ST31 were found only in pneumonia cases, whereas ST27 was associated exclusively to colonizations. Despite 38 patients sharing similar STs, only two were involved in a potential cross transmission event. No DHPS mutations were identified. The new consensus typing scheme was useful to ascertain the molecular epidemiology of P. jirovecii in our center revealing a high genetic diversity and the potential association of specific STs to colonization and pneumonia cases., Lay Summary: A newly described MLST scheme aims at providing a standardized tool to study and compare Pneumocystis jirovecii epidemiology. A high diversity among P. jirovecii isolates from patients in Barcelona, Spain, and a potential association between specific STs and infection/colonization were identified., (© The Author(s) 2021. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology.)

We conducted a pilot study of patients with cystic fibrosis (CF) to assess intra-family transmission of P. jirovecii and compare it with data on other prevalent pathogens such as P. aeruginosa and S. pneumoniae, in which respiratory transmission has already been documented. Oral swab samples from 10 patients with CF and 15 household members were collected at baseline and 2 weeks later. P. aeruginosa and S. pneumoniae were assessed using standardized culture methods and PCR, and P. jirovecii was assessed using real and nested PCR, genotyping the positive samples by direct sequencing. P. aeruginosa cultures were positive for 7/10 (70%) of patients with CF at baseline and was identified by PCR in 8/10 (80%) of cases at baseline and 2 weeks later. S. pneumoniae cultures were negative for all patients, but the microorganism was identified by PCR in two cases. P. jirovecii was detected by real time and nested PCR in 5/10 (50%) of the patients at the two time points. In the household members, P. aeruginosa and P. jirovecii were identified in 7/15 (46.7%), and S. pneumoniae was identified in 8/15 (53,3%). The concordance of positive or negative pairs of patients with CF and their household members was 33.3% (5/15) for P. aeruginosa, 46.7% (7/15) for S. pneumonia and 93.3% (14/15) for P. jirovecii. The concordance for P. jirovecii genotypes among five pairs with available genotype was 100%. This study suggests for the first time the possible transmission of Pneumocystis in the home of patients with CF, indicating that patients and their household members are reservoirs and possible sources of infection., Lay Summary: This study suggests for the first time the possible transmission of Pneumocystis in the family environment of patients with cystic fibrosis, indicating that patients and their household members are reservoirs and possible sources of this infection., (© The Author(s) 2021. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology.)

Optimal sensitivity to detect low Pneumocystis loads is of importance to take individual and collective measures to avoid evolution towards Pneumocystis pneumonia and outbreaks in immunocompromised patients. This study compares two qPCR procedures, a new automated RTqPCR using the GeneLEAD VIII extractor/thermocycler (GLVIII; ∼2.2 h workflow) and a previously validated in-house qPCR assays (IH; ∼5 h workflow) both targeting mtSSU and mtLSU for detecting P. jirovecii in 213 respiratory samples. GLVIII was found to be more sensitive than IH, detecting eight more specimens. Bland-Altman analysis between the two procedures showed a Cq bias of 1.17 ± 0.07 in favor of GLVIII., Lay Summary: The fungus Pneumocystis needs to be detected early in respiratory samples to prevent pneumonia in immunocompromised hosts. We evaluated a new commercial RTqPCR on 213 respiratory samples to detect Pneumocystis and found it more sensitive and faster than our routine sensitive in-house qPCR assay., (© The Author(s) 2021. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology.)

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., (© The Author(s) 2021. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology.)

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 Author(s) 2020. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology.)

Pneumocystis jirovecii and microsporidia species are recognized as opportunistic infectious pathogens in AIDS patients. Coinfection of both in one patient has been rarely reported. The aim of the present study was to investigate the coinfection of P. jirovecii and microsporidia in different tissues from AIDS deceased patients. Post mortem histological finding of P. jirovecii and microsporidia was demonstrated by means of the Grocott's methenamine silver and Brown Brenn staining, respectively. Molecular technique was used for identification and characterization of both fungi. Out of the 514 autopsied cases P. jirovecii and microsporidia species were identified in 53 (10.3%) and 62 (12.1%) cases respectively. A total of five cases (0.97%) coinfected with Pneumocystis and microsporidia were recovered from all analyzed autopsies. Coinfection of Pneumocystis and microsporidia is very challenging and raises interesting issues about host-parasite relationship. The early diagnosis of both pathogens must be crucial to establish correct and early treatments, improve the patient's evolution, reducing the risk of death., (© The Author(s) 2020. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology.)

Quantitative real-time PCR (qPCR) is increasingly used to detect Pneumocystis jirovecii for the diagnosis of Pneumocystis pneumonia (PCP), but there are differences in the nucleic acids targeted, DNA only versus whole nucleic acid (WNA), and also the target genes for amplification. Through the Fungal PCR Initiative, a working group of the International Society for Human and Animal Mycology, a multicenter and monocenter evaluation of PCP qPCR assays was performed. For the multicenter study, 16 reference laboratories from eight different countries, performing 20 assays analyzed a panel consisting of two negative and three PCP positive samples. Aliquots were prepared by pooling residual material from 20 negative or positive- P. jirovecii bronchoalveolar lavage fluids (BALFs). The positive pool was diluted to obtain three concentrations (pure 1:1; 1:100; and 1:1000 to mimic high, medium, and low fungal loads, respectively). The monocenter study compared five in-house and five commercial qPCR assays testing 19 individual BALFs on the same amplification platform. Across both evaluations and for all fungal loads, targeting WNA and the mitochondrial small sub-unit (mtSSU) provided the earliest Cq values, compared to only targeting DNA and the mitochondrial large subunit, the major surface glycoprotein or the beta-tubulin genes. Thus, reverse transcriptase-qPCR targeting the mtSSU gene could serve as a basis for standardizing the P. jirovecii load, which is essential if qPCR is to be incorporated into clinical care pathways as the reference method, accepting that additional parameters such as amplification platforms still need evaluation., (© The Author(s) 2019. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology.)

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., (© The Author(s) 2019. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology.)

Data on features of Pneumocystis primary infection in infancy are still fragmented. To study Pneumocystis primary infection, 192 infants who were monitored for acute pulmonary disease or fever over a 40-month period were retrospectively investigated. P. jirovecii detection on archival nasopharyngeal aspirates was performed using a qPCR assay. Factors associated with P. jirovecii were assessed using univariate and multivariate analyses. P. jirovecii genotypes in infants and a control group of adults contemporaneously diagnosed with Pneumocystis pneumonia were identified using unilocus, bilocus, and multilocus sequence typing (MLST). P. jirovecii was detected in 35 infants (18.2%). The univariate analysis pointed out four factors: viral infection (P = .035, OR [IC 95], 2.2 [1.1-4.7]), lower respiratory tract infection (P = .032, OR [IC 95], 2.5 [1.1-5.9]), absence of hospital discharge after birth (P = .003, OR (IC 95), 0.1 (0.02-0.5]), and the 63-189-day group (P < .001, OR [IC 95], 42.2 [5.4-332]). The multivariate analysis confirmed these two latter factors (P = .02, OR [IC 95], 0.1 [0.02-0.72]; P = .005, OR [IC 95], 11.5 [2.1-63.5]). Thus, P. jirovecii acquisition mostly takes place in the community. A comparison of these data with those of previously published studies showed that median and interquartile range of positive-infant ages were close to those observed in Chile, Denmark, and Peru, highlighting similar characteristics. Common unilocus or bilocus genotypes were identified in infants and adults, whereas no MLST genotypes were shared. Therefore, a common reservoir made up of infected infants and adults is still hypothetical. Finally, primary infection is a worldwide phenomenon occurring at the same time in childhood regardless of geographical location, rather than an incidental event., (© The Author(s) 2019. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology.)

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.

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

In a prospective bicentric study, Pneumocystis jirovecii excretion and diffusion was explored in air samples collected in the rooms occupied by 17 Pneumocystis-colonized patients. P. jirovecii DNA was detected by real-time PCR in the air collected from 3 patients' rooms (17.6%), with identical genotypes in corresponding clinical and air samples. Pneumocystis DNA was detected for 2/3 patients with autoimmune disease treated with corticosteroids versus 1/6 patients with hematologic disease and 0/5 kidney transplant recipients. These data confirm the possible excretion of the fungus by Pneumocystis-colonized patients and thus bring additional arguments for the prevention of airborne transmission in hospital wards., (© The Author 2016. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

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., (© The Author 2016. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Pneumocystis is mostly found in the alveolar spaces, but circulation of viable organisms also occurs and suggests that the detection of DNA in blood could be used as a noninvasive procedure to improve the diagnosis of Pneumocystis pneumonia (PcP). In order to determine the optimal compartment for Pneumocystis DNA detection, we used a rat model of PcP and tested the presence of Pneumocystis with a quantitative mtLSU targeting real-time PCR in four blood compartments: whole blood, clot, serum and Platelet-Rich-Plasma (PRP). All samples from 4 Pneumocystis-free control rats were negative. Pneumocystis was detected in 79, 64, 57, and 57% of samples from 14 PcP rats, respectively, but DNA release was not related to pulmonary loads. These data confirm the potential usefulness of Pneumocystis DNA detection in the blood for PcP diagnosis and suggest that whole blood could be the most appropriate compartment for Pneumocystis detection., (© The Author 2015. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The dihydropteroate sulfate (DHPS) gene is associated with resistance to sulfa/sulfone drugs in Pneumocystis jirovecii. We investigated the DHPS mutation rate in three groups of Iranian HIV-positive and HIV-negative patients by polymerase chain reaction-restricted fragment length polymorphism analysis. Furthermore, an association between P. jirovecii DHPS mutations and strain typing was investigated based on direct sequencing of internal transcribed spacer region 1 (ITS1) and ITS2. The overall P. jirovecii DHPS mutation rate was (5/34; 14.7%), the lowest rate identified was in HIV-positive patients (1/16; 6.25%) and the highest rate was in malignancies patients (3/11; 27.3%). A moderate rate of mutation was detected in chronic obstructive pulmonary disease (COPD) patients (1/7; 14.3%). Most of the isolates were wild type (29/34; 85.3%). Double mutations in DHPS were detected in patients with malignancies, whereas single mutations at codons 55 and 57 were identified in the HIV-positive and COPD patients, respectively. In this study, two new and rare haplotypes were identified with DHPS mutations. Additionally, a positive relationship between P. jirovecii strain genotypes and DHPS mutations was identified. In contrast, no DHPS mutations were detected in the predominant (Eg) haplotype. This should be regarded as a warning of an increasing incidence of drug-resistant P. jirovecii strains., (© The Author 2015. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Detection of viral and bacterial DNA in nasopharyngeal aspirates (NPAs) is now a routine practice in emergency cases of febrile pneumonia. We investigated whether Pneumocystis jirovecii DNA could also be detected in these cases by conducting retrospective screening of 324 consecutive NPAs from 324 adult patients (198 or 61% were immunocompromised) admitted with suspected pulmonary infections during the 2012 influenza epidemic season, using a real-time quantitative polymerase chain reaction (PCR) assay (PjqPCR), which targets the P. jirovecii mitochondrial large subunit ribosomal RNA gene. These NPAs had already been tested for 22 respiratory pathogens (18 viruses and 4 bacteria), but we found that 16 NPAs (4.9%) were PjqPCR-positive, making P. jirovecii the fourth most prevalent of the 23 microorganisms in the screen. Eleven of the 16 PjqPCR-positive patients were immunocompromised, and five had underlying pulmonary conditions. Nine NPAs were also positive for another respiratory pathogen. Six had PjqPCR-positive induced sputa less than 3 days after the NPA procedure, and five were diagnosed with pneumocystis pneumonia (four with chronic lymphoproliferative disorders and one AIDS patient). In all six available pairs quantification of P. jirovecii DNA showed fewer copies in NPA than in induced sputum and three PjqPCR-negative NPAs corresponded to PjqPCR-positive bronchoalveolar lavage fluids, underscoring the fact that a negative PjqPCR screen does not exclude a diagnosis of pneumocystosis. Including P. jirovecii DNA detection to the panel of microorganisms included in screening tests used for febrile pneumonia may encourage additional investigations or support use of anti-pneumocystis pneumonia prophylaxis in immunocompromised patients., (© The Author 2014. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

A high prevalence of Pneumocystis jirovecii colonization was observed in patients positive for the human immunodeficiency virus (HIV) admitted to a tertiary hospital in southern Brazil between August 2012 and December 2012. Amplification of the mitochondrial large subunit ribosomal RNA gene in oropharyngeal samples through nested polymerase chain reaction identified P. jirovecii colonization in 26 of 58 (44.8%) HIV-positive patients admitted for causes other than Pneumocystis pneumonia. Colonization was more frequent among patients with an absolute CD4 count ≤200 cells/μl. These findings suggest that the HIV-infected population is a major reservoir and source of P. jirovecii infection and that identification of such individuals may contribute to future strategies for improving management of HIV-infected patients., (© The Author 2014. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Pneumocystis jirovecii pneumonia (PJP) is increasingly seen in association with the use of new and potent immunosuppressive therapies in populations not infected with human immunodeficiency virus. Today, molecular methods are widely used to improve diagnostic yield; however, the relationship between clinical findings and quantitative polymerase chain reaction (qPCR) results is undefined. Our objective was to describe characteristics of PJP in patients with malignancies and determine if qPCR results were correlated with clinical findings. From 2007 to 2012, all patients at the Peter MacCallum Cancer Centre with positive Pneumocystis PCR were identified from a microbiology database. Clinical, radiological, and microbiological records were reviewed. PJP was defined as the presence of positive PCR for Pneumocystis on a respiratory specimen, radiological abnormalities consistent with a pneumonic process, and receipt of targeted PJP treatment. qPCR was performed on all diagnostic specimens, and values were reported according to clinical findings. Forty-five patients fulfilled inclusion criteria: 44.4% had underlying solid organ tumors and 55.6% had hematological malignancies. Nonsmall cell lung carcinoma and lymphoma were the most frequent predispositions. Shortness of breath, cough, and fever were reported in 64.4%, 48.9%, and 42.2% of the patients, respectively. Admission to the intensive care unit and mortality rates were lower than in previous reports. Overall, a relationship between other clinical features and qPCR results was not identified. In the era of routine molecular diagnostics, patients with malignancy and PJP have improved outcomes. However, there was no demonstrable relationship between qPCR results and clinical features or PCR data and outcomes.

Pneumocystis jirovecii, a transmissible fungus, is the causative agent of pulmonary infections. Its genomic diversity has appeared in reports from around the world but data on P. jirovecii genotypes in France are still limited. This study describes the typing of P. jirovecii isolates from 81 HIV-negative patients monitored at Brest University Hospital, Brittany, France, 40 of whom developed Pneumocystis pneumonia (PcP), and remaining 41 patients were colonized by the fungus. The isolates were assayed at the internal transcribed spacer (ITS)1 and ITS2 under improved amplification conditions to avoid in vitro ITS recombination. P. jirovecii ITS haplotypes were identified in 56/81 patients (31 PcP patients and 25 patients who were colonized) which revealed a high diversity in that 27 different haplotypes were identified. Eg was the most frequent haplotype (31/56, 55.3%), followed by Ec and Ai (5/56, 8.9% each). In contrast, Ne, usually the second most frequent haplotype in Europe and the USA, was observed in only 2/56 patients (3.6%). Mixed infections were detected in 18/56 patients (32.1%; 12 PcP patients and six who were colonized). No significant differences were observed in haplotype diversity, frequency of peculiar haplotypes, and mixed infection occurrence, between the two patient populations. The study, conducted with the largest HIV-negative patient population investigated so far, shows that ITS typing remains an efficient method for characterizing P. jirovecii among human populations, whatever their clinical presentation of Pneumocystis infections.

Since there have been no published molecular studies of Pneumocystis jirovecii isolates from Iranian patients, we investigated the genotypes of such isolates recovered from HIV-infected patients, those undergoing cancer chemotherapy and patients with chronic obstructive pulmonary disease (COPD). P. jirovecii typing, based on ITS1 and ITS2 sequence analysis, was performed on 34 isolates from Iranian immunosuppressed patients. In total, 44 genotypes were detected of which relative to ITS1, eight known genotypes (A, B, C, E, G, H, N and O) and one novel sequence were noted. Eight known genotypes (b, c, e, g, h, i, j and n) were also found with ITS2. The most frequent ITS1 and ITS2 genotypes were E (21/44, 47.7%) and g (22/44, 50%), respectively. From determined haplotypes, the four most frequent ones were Eg (11/44, 25%), Gg (5/44, 11.3%), Gi (4/44, 9.1%), Ei (3/44, 6.8%), and Hg (3/44, 6.8%). Two novel haplotypes (Hb and Hi) were also identified, along with mixed infections as seven (20.5%) patients were found to have more than one haplotype. It is suggested that novel haplotypes in Iranian patients may be generated through sexual recombination within the host.

Pneumocystis jirovecii is an important opportunistic pathogen that causes severe pneumonia in immunocompromised patients. The aim of the present study was to investigate the genetic diversity of P. jirovecii strains by direct sequencing and analysis of the Upstream Conserved Sequence (UCS) region, mitochondrial large-subunit (mtLSU) rRNA and dihydrofolate reductase (DHFR) genes. We identified the polymorphisms in P. jirovecii strains of 15 immunocompromised patients, as well as detecting a new tandem repeat of 5 nucleotides in UCS region. The following three different types of repeat unit were found: type a GCCCA; type b GCCCT; and type c GCCTT. In addition, we identified the repeat unit which consisted of 10 nucleotides and three different patterns of UCS repeats with 3 and 4 repeats, i.e., 1, 2, 3 (86.7%), 1, 2, 3, 3 (6.6%) and a new genotype 2, 2, 3, 3 (6.6%). The polymorphism in the mtLSUrRNA gene was seen primarily at position 85 where we detected three different genotypes. Genotype 3 and genotype 2 were the most abundant with frequencies of 53.3% and 40%, respectively. With regard to the DHFR gene, only two (20%) patients had nucleotide substitution in position 312. In conclusion, the multilocus analysis facilitated the typing of P. jirovecii strains and proved the important genetic biodiversity of this fungus.

Pneumocystis jirovecii dihydropteroate synthase (DHPS) gene mutations are well-reported. Although sulfa prophylaxis generally is associated with DHPS mutant infection, whether mutant infection is associated with poorer clinical outcomes is less clear. The differing definitions of sulfa prophylaxis and the different mortality endpoints used in these studies may be one explanation for the conflicting study results. Applying different definitions of prophylaxis, mortality endpoints and DHPS mutant to 301 HIV-infected patients with Pneumocystis pneumonia, we demonstrate that prophylaxis, irrespective of definition, increased the risk of infection with pure mutant (any prophylaxis: AOR 4.00, 95% CI: 1.83-8.76, P < 0.001) but not mixed genotypes (any prophylaxis: AOR 0.78, 95% CI: 0.26-2.36, P = 0.65). However, infection with mutant DHPS, irrespective of definition, was not associated with increased mortality (all-cause or PCP death) at the three time-intervals examined (all P > 0.05). Future studies should standardize key variables associated with DHPS mutant infection as well as examine DHPS mutant subtypes (pure mutant vs. mixed infections) - perhaps even individual DHPS mutant genotypes - so that data can be pooled to better address this issue.

Genetic diversity of Pneumocystis jirovecii isolates based on internal transcribed spacer (ITS) of the nuclear rRNA locus has previously been reported. The information about ITS genotype and epidemiology of this organism in Chinese human immunodeficiency virus-infected patients has not been available. In this study, 12 bronchoalveolar lavage fluid specimens obtained from HIV-infected patients were analyzed by PCR followed by cloning, sequencing and typing. Three ITS1 genotypes (E, B and 'H') and four ITS2 genotypes (b, g, i and r) as previously reported were identified, the most common of which were E, b and i. Five ITS haplotypes (Eg, Eb, Bi, Er and 'H'r) and 19 new combination types were also identified with the most common types being Eg (four of 12 patients, 10 of 60 clones), Eb (three of 12 patients, 11 of 60 clones) and Bi (three of 12 patients, 10 of 60 clones). Nine patients were found to be co-infected with more than one ITS genotype of P. jirovecii. The prevalence of ITS genotypes in HIV patients from one Chinese hospital did not seem to be significantly different when compared to reports from other countries.

Pneumocystis has been identified in various mammalian species, including domestic, wild and zoo animals. This study's main objectives were: (1) to estimate the prevalence of the Pneumocystis carinii f. sp. suis infection in slaughtered pigs in Portugal, (2) assess the prevalence differences within distinct age groups of animals, (3) determine the possible associations between pulmonary lesions and the infection, and (4) genetically characterize the P. carinii f. sp. suis isolates recovered from infected animals using PCR with DNA sequencing. An epidemiological cross-sectional study was conducted using 215 pig lung tissue samples which demonstrated a global prevalence of 7% (14 positive samples). This value was later validated by statistical analysis as being representative of the national population prevalence. Regarding the assessment of relations between the different variables investigated during the study (age, gender, geographical region, type of farming, weight and pulmonary lesion) and the P. carinii f. sp. suis infection, no significant statistical differences were found, and apparently, no predisposing factors could be defined. Nevertheless, infection by Pneumocystis in pigs is ubiquitous and it can be detected in healthy animals. Thus, the colonization of P. carinii f. sp. suis among healthy individuals suggests that asymptomatic carriers can be an effective reservoir for susceptible animals and participate in the transmission of infection. The present data confirmed that porcine Pneumocystis is genetically distinct from Pneumocystis DNA detected in other mammalian hosts.

A high rate of Pneumocystis jirovecii colonization was observed in Brazilian cystic fibrosis (CF) patients (13 out of 34; 38.2%) who underwent bronchoscopy between March 2006 and August 2009 at the Hospital de Clinicas de Porto Alegre, Brazil. Bronchoalveolar lavage samples were collected from these patients and studied by nested PCR amplification of the mitochondrial gene coding for the large subunit ribosomal RNA (mtLSUrDNA). The observed rate of colonization was higher than that reported in European populations. Genotypic characterization of the mtLSUrDNA locus revealed a predominance of the polymorphisms 85C/248C (genotype 1) and 85T/248C (genotype 3), with all samples possessing the wild-type genotype of dihydropteroate synthase. These findings suggest that cystic fibrosis patients could be an important reservoir and source of P. jirovecii infection. Further studies are required to elucidate the role of this common fungal colonization in the evolution of CF patients.

Genotypes of two different loci of the Pneumocystis jirovecii mitochondrial gene were studied in specimens from a total of 75 Pneumocystis pneumonia patients in Spain, France and Cuba. A new genotype of the mitochondrial small subunit rRNA gene of P. jirovecii (160A/196T) was identified, which was revealed to be the most common in these three countries, especially in Cuba where its proportion reached 93.8%. Our data imply that the new genotype might be circulating worldwide and also suggests that the distribution of P. jirovecii genotypes could be narrower in islands such as Cuba.

Pneumocystis jirovecii remains an important cause of pneumonia in the immunocompromised host, with the largest group of patients at risk for P. jirovecii pneumonia (PCP) in Sweden being those with haematological diseases. Widespread prophylaxis and treatment for P. jirovecii with sulfa-containing drugs have effectively decreased the incidence of PCP, but concerns have been raised about the possible emergence of P. jirovecii isolates that are resistant to these drugs. Two point mutations in the gene coding for the dihydropteroate synthase enzyme (DHPS) in P. jirovecii have been shown to be associated with prior exposure to sulfa drugs. We retrospectively studied the occurrence of P. jirovecii DHPS mutations in isolates recovered from 103 Swedish patients. The DHPS gene, including the polymorphic positions 165 and 171, were amplified and sequenced by pyrosequencing technology. All the clinical specimens showed a wild-type pattern indicating that the occurrence of P. jirovecii DHPS mutations in Sweden is very low or absent.

The evaluation of quantitative polymerase chain reaction (PCR) characteristics can increase the accuracy of the laboratory diagnosis of Pneumocystis pneumonia (PCP). Between July 2008 and September 2009, 66 non-sequential prospective bronchoalveolar lavage (BAL) samples, obtained from five HIV-infected and 49 non HIV-infected patients were investigated, using a quantitative-touch-down-PCR to determine the number of copies of major surface glycoprotein (MSG) genes of Pneumocystis jirovecii (q-TD-MSG-PCR). PCP was confirmed by microscopic observation of Pneumocystis, radio-clinical and therapeutic data in 18/54 patients. For PCP, the cut-off was 54.3 MSG copies per ml of BAL fluid. The PCR was positive in these same 18 cases and it was the only positive assay in two cases and the earliest diagnosis test in one case of PCP relapse. The likelihood positive ratio, sensitivity and specificity of the q-TD-MSG-PCR were 44, 100% and 97.7%, respectively. The Predictive Negative Value was 100% and the Predictive Positive Value of 95.5%, the intra- and inter-assay variability values were 2.7% (at more than 30 MSG copies) and 11.7% (at 10,000 MSG copies), respectively. Quantitative PCR can help diagnose PCP even in cases of low Pneumocystis load and might decrease morbidity in association with very early specific treatments.

Studies on Pneumocystis jirovecii dihydropteroate synthase (DHPS) genotypes among non-HIV immunocompromised patients from developing countries are rare. In the present prospective investigation, 24 (11.8%) cases were found to be positive for Pneumocystis jirovecii out of 203 non-HIV patients with a clinical suspicion of Pneumocystis pneumonia (PCP). Dihydropteroate synthase (DHPS) genotype 1 (Thr55+Pro57) was noted in 95.8% P. jirovecii isolates in the present study in contrast to only 4.1% of patients with DHPS genotype 4 (Thr55Ala + Pro57Ser).

Pneumocystis jirovecii is an atypical opportunistic fungus with lung tropism and worldwide distribution that causes pneumonia in immunosuppressed individuals. The development of sensitive molecular techniques has led to the recognition of a colonization or carrier state of P. jirovecii, in which low levels of the organism are detected in persons who do not have pneumonia. Pneumocystis colonization has been described in individuals with various lung diseases, and accumulating evidence suggests that it may be a relevant issue with potential clinical impact. Only a few published studies carried out in Europe have evaluated the prevalence of Pneumocystis colonization in patients with cystic fibrosis, reporting ranges from 1.3-21.6%. The evolution of P. jirovecii colonization in cystic fibrosis patients is largely unknown. In a longitudinal study, none of the colonized patients developed pneumonia during a 1-year follow-up. Since patients with cystic fibrosis could act as major reservoirs and sources of infection for susceptible individuals further research is thus warranted to assess the true scope of the problem and to design rational preventive strategies if necessary. Moreover, it's necessary to elucidate the role of P. jirovecii infection in the natural history of cystic fibrosis in order to improve the clinical management of this disease.

The combination of trimethoprim-sulfamethoxazole is widely used for the prophylaxis and treatment of Pneumocystis pneumonia (PCP) caused by Pneumocystis jirovecii. Many studies have shown that mutations in the drug target, the dihydropteroate synthase (DHPS) gene, are presumably involved with the failure of prophylaxis and treatment. We have analyzed dihydropteroate synthase (DHPS) and dihydrofolate reductase (DHFR) mutations in P. jirovecii isolates recovered from Thai patients. Out of 17 samples, 11.7% (2) of the DHPS gene contained a double mutation at codon 55 and codon 57, whereas out of 18 samples, 61.1% (11) of the DHFR genes contained the silent mutation at codon 104. In comparison to previous reports, we have found a higher number of DHFR mutations but a lower prevalence of DHPS mutations.

This paper describes the development of a rapid probe-based real-time polymerase chain reaction assay (PCR) for the diagnosis of Pneumocystis pneumonia. To develop the PCR, primers and fluorescent resonance energy transfer probes were designed after sequencing products obtained using previously published primers. This gave results that were concordant with conventional cytological staining techniques, but were available within 2 h instead of greater than 24 h.