Philippe M. Hauser

Prospective multicenter international surveillance of azole resistance in Aspergillus fumigatus.

To investigate azole resistance in clinical Aspergillus isolates, we conducted prospective multicenter international surveillance. A total of 3,788 Aspergillus isolates were screened in 22 centers from 19 countries. Azole-resistant A. fumigatus was more frequently found (3.2% prevalence) than previously acknowledged, causing resistant invasive and noninvasive aspergillosis and severely compromising clinical use of azoles.

β-Glucan Antigenemia Anticipates Diagnosis of Blood Culture–Negative Intraabdominal Candidiasis

RATIONALE Life-threatening intraabdominal candidiasis (IAC) occurs in 30 to 40% of high-risk surgical intensive care unit (ICU) patients. Although early IAC diagnosis is crucial, blood cultures are negative, and the role of Candida score/colonization indexes is not established. OBJECTIVES The aim of this prospective Fungal Infection Network of Switzerland (FUNGINOS) cohort study was to assess accuracy of 1,3-β-d-glucan (BG) antigenemia for diagnosis of IAC. METHODS Four hundred thirty-four consecutive adults with abdominal surgery or acute pancreatitis and ICU stay 72 hours or longer were screened: 89 (20.5%) at high risk for IAC were studied (68 recurrent gastrointestinal tract perforation, 21 acute necrotizing pancreatitis). Diagnostic accuracy of serum BG (Fungitell), Candida score, and colonization indexes was compared. MEASUREMENTS AND MAIN RESULTS Fifty-eight of 89 (65%) patients were colonized by Candida; 29 of 89 (33%) presented IAC (27 of 29 with negative blood cultures). Nine hundred twenty-one sera were analyzed (9/patient): median BG was 253 pg/ml (46-9,557) in IAC versus 99 pg/ml (8-440) in colonization (P < 0.01). Sensitivity and specificity of two consecutive BG measurements greater than or equal to 80 pg/ml were 65 and 78%, respectively. In recurrent gastrointestinal tract perforation it was 75 and 77% versus 90 and 38% (Candida score ≥ 3), 79 and 34% (colonization index ≥ 0.5), and 54 and 63% (corrected colonization index ≥ 0.4), respectively. BG positivity anticipated IAC diagnosis (5 d) and antifungal therapy (6 d). Severe sepsis/septic shock and death occurred in 10 of 11 (91%) and 4 of 11 (36%) patients with BG 400 pg/ml or more versus 5 of 18 (28%, P = 0.002) and 1 of 18 (6%, P = 0.05) with BG measurement less than 400 pg/ml. β-Glucan decreased in IAC responding to therapy and increased in nonresponse. CONCLUSIONS BG antigenemia is superior to Candida score and colonization indexes and anticipates diagnosis of blood culture-negative IAC. This proof-of-concept observation in strictly selected high-risk surgical ICU patients deserves investigation of BG-driven preemptive therapy.

Molecular evidence of interhuman transmission of Pneumocystis pneumonia among renal transplant recipients hospitalized with HIV-infected patients.

Molecular evidence indicates that P. jirovecii may be nosocomially transmitted to severely immunosuppressed patients.

Mutations of Pneumocystis jirovecii Dihydrofolate Reductase Associated with Failure of Prophylaxis

ABSTRACT Most drugs used for prevention and treatment of Pneumocystis jirovecii pneumonia target enzymes involved in the biosynthesis of folic acid, i.e., dihydropteroate synthase (DHPS) and dihydrofolate reductase (DHFR). Emergence of P. jirovecii drug resistance has been suggested by the association between failure of prophylaxis with sulfa drugs and mutations in DHPS. However, data on the occurrence of mutations in DHFR, the target of trimethoprim and pyrimethamine, are scarce. We examined polymorphisms in P. jirovecii DHFR from 33 patients diagnosed with P. jirovecii pneumonia who were receiving prophylaxis with a DHFR inhibitor (n = 15), prophylaxis without a DHFR inhibitor (n = 11), or no prophylaxis (n = 7). Compared to the wild-type sequence present in GenBank, 19 DHFR nucleotide substitution sites were found in 18 patients with 3 synonymous and 16 nonsynonymous mutations. Of 16 amino acid changes, 6 were located in positions conserved among distant organisms, and five of these six positions are probably involved in the putative active sites of the enzyme. Patients with failure of prophylaxis, including a DHFR inhibitor, were more likely to harbor nonsynonymous DHFR mutations than those who did not receive such prophylaxis (9 of 15 patients versus 2 of 18; P = 0.008). Analysis of the rate of nonsynonymous versus synonymous mutations was consistent with selection of amino acid substitutions in patients with failure of prophylaxis including a DHFR inhibitor. The results suggest that P. jirovecii populations may evolve under selective pressure from DHFR inhibitors, in particular pyrimethamine, and that DHFR mutations may contribute to P. jirovecii drug resistance.

Molecular Detection and Identification of Zygomycetes Species from Paraffin-Embedded Tissues in a Murine Model of Disseminated Zygomycosis: a Collaborative European Society of Clinical Microbiology and Infectious Diseases (ESCMID) Fungal Infection Study Group (EFISG) Evaluation

ABSTRACT The present study was performed to assess the interlaboratory reproducibility of the molecular detection and identification of species of Zygomycetes from formalin-fixed paraffin-embedded kidney and brain tissues obtained from experimentally infected mice. Animals were infected with one of five species (Rhizopus oryzae, Rhizopus microsporus, Lichtheimia corymbifera, Rhizomucor pusillus, and Mucor circinelloides). Samples with 1, 10, or 30 slide cuts of the tissues were prepared from each paraffin block, the sample identities were blinded for analysis, and the samples were mailed to each of seven laboratories for the assessment of sensitivity. A protocol describing the extraction method and the PCR amplification procedure was provided. The internal transcribed spacer 1 (ITS1) region was amplified by PCR with the fungal universal primers ITS1 and ITS2 and sequenced. As negative results were obtained for 93% of the tissue specimens infected by M. circinelloides, the data for this species were excluded from the analysis. Positive PCR results were obtained for 93% (52/56), 89% (50/56), and 27% (15/56) of the samples with 30, 10, and 1 slide cuts, respectively. There were minor differences, depending on the organ tissue, fungal species, and laboratory. Correct species identification was possible for 100% (30 cuts), 98% (10 cuts), and 93% (1 cut) of the cases. With the protocol used in the present study, the interlaboratory reproducibility of ITS sequencing for the identification of major Zygomycetes species from formalin-fixed paraffin-embedded tissues can reach 100%, when enough material is available.

Association between a Specific Pneumocystis jiroveci Dihydropteroate Synthase Mutation and Failure of Pyrimethamine/Sulfadoxine Prophylaxis in Human Immunodeficiency Virus–Positive and –Negative Patients

To investigate the possible association between different prophylactic sulfa drugs and the genotype of the Pneumocystis jiroveci dihydropteroate synthase (DHPS) gene, we examined DHPS polymorphisms in clinical specimens from 158 immunosuppressed patients (38 HIV-negative and 120 HIV-positive), using polymerase chain reaction-single-strand conformation polymorphism. Fifty-seven (36.1%) of 158 patients were infected with a mutant DHPS genotype. All patients who developed P. jiroveci pneumonia (PcP) while receiving pyrimethamine/sulfadoxine (PM/SD) prophylaxis (n=14) had a strain harboring DHPS with an amino acid change at position 57 (Pro-->Ser). This mutation was only present in 20 (14%) of 144 patients not receiving prophylaxis (P<.001). Hospitalization in a specific hospital was an independent risk factor for having P. jiroveci harboring the same DHPS mutation, which indirectly supports that interhuman transmission may affect the dissemination of the mutant strains.

ECIL guidelines for the diagnosis of Pneumocystis jirovecii pneumonia in patients with haematological malignancies and stem cell transplant recipients

The Fifth European Conference on Infections in Leukaemia (ECIL-5) convened a meeting to establish evidence-based recommendations for using tests to diagnose Pneumocystis jirovecii pneumonia (PCP) in adult patients with haematological malignancies. Immunofluorescence assays are recommended as the most sensitive microscopic method (recommendation A-II: ). Real-time PCR is recommended for the routine diagnosis of PCP ( A-II: ). Bronchoalveolar lavage (BAL) fluid is recommended as the best specimen as it yields good negative predictive value ( A-II: ). Non-invasive specimens can be suitable alternatives ( B-II: ), acknowledging that PCP cannot be ruled out in case of a negative PCR result ( A-II: ). Detecting β-d-glucan in serum can contribute to the diagnosis but not the follow-up of PCP ( A-II: ). A negative serum β-d-glucan result can exclude PCP in a patient at risk ( A-II: ), whereas a positive test result may indicate other fungal infections. Genotyping using multilocus sequence markers can be used to investigate suspected outbreaks ( A-II: ). The routine detection of dihydropteroate synthase mutations in cases of treatment failure is not recommended ( B-II: ) since these mutations do not affect response to high-dose co-trimoxazole. The clinical utility of these diagnostic tests for the early management of PCP should be further assessed in prospective, randomized interventional studies.

Multicenter, Prospective Clinical Evaluation of Respiratory Samples from Subjects at Risk for Pneumocystis jirovecii Infection by Use of a Commercial Real-Time PCR Assay

ABSTRACT Pneumocystis jirovecii pneumonia (PCP) is a common opportunistic infection. Microscopic diagnosis, including diagnosis using the Merifluor-Pneumocystis direct fluorescent antigen (MP-DFA) test, has limitations. Real-time PCR may assist in diagnosis, but no commercially validated real-time PCR assay has been available to date. MycAssay Pneumocystis is a commercial assay that targets the P. jirovecii mitochondrial large subunit (analytical detection limit, ≤3.5 copies/μl of sample). A multicenter trial recruited 110 subjects: 54 with transplants (40 with lung transplants), 32 with nonmalignant conditions, 13 with leukemia, and 11 with solid tumors; 9 were HIV positive. A total of 110 respiratory samples (92% of which were bronchoalveolar lavage [BAL] specimens) were analyzed by PCR. Performance was characterized relative to investigator-determined clinical diagnosis of PCP (including local diagnostic tests), and PCR results were compared with MP-DFA test results for 83 subjects. Thirteen of 14 subjects with PCP and 9/96 without PCP (including 5 undergoing BAL surveillance after lung transplantation) had positive PCR results; sensitivity, specificity, and positive and negative predictive values (PPV and NPV, respectively) were 93%, 91%, 59%, and 99%, respectively. Fourteen of 83 subjects for whom PCR and MP-DFA test results were available had PCP; PCR sensitivity, specificity, PPV, and NPV were 93%, 90%, 65%, and 98%, respectively, and MP-DFA test sensitivity, specificity, PPV, and NPV were 93%, 100%, 100%, and 98%. Of the 9 PCR-positive subjects without PCP, 1 later developed PCP. The PCR diagnostic assay compares well with clinical diagnosis using nonmolecular methods. Additional positive results compared with the MP-DFA test may reflect low-level infection or colonization.

Genetic diversity of Pneumocystis carinii in HIV-positive and -negative patients as revealed by PCR-SSCP typing.

ObjectiveTo describe the epidemiology of severe Pneumocystis carinii pneumonia (PCP) in HIV-infected and non HIV-infected patients. MethodsBronchoalveolar lavage specimens from 212 European patients with PCP were typed using PCR–single strand conformation polymorphism analysis of four genomic regions of P. carinii f. sp. hominis. Demographic and clinical information was obtained from all patients. ResultsTwenty-three per cent of the patients were presumably infected with a single P. c. hominis type. The other patients presented with two (50%) or more (27%) types. Thirty-five genetically stable and ubiquitous P. c. hominis types were found. Their frequency ranged from 0.4% to 10% of all isolates, and up to 15% of those from a given hospital. There was no significant association between the P. c. hominis type or number of co-infecting types per patient and geographical location, year of collection, sex, age, or HIV status. No more than three patients infected with the same type were observed in the same hospital within the same 6 month period, and no epidemiological link between the cases was found. ConclusionsThe broad diversity of types observed seems to indicate that multiple sources of the pathogen co-exist. There was no evidence that in our study population inter-human transmission played a significant role in the epidemiology of P. carinii.

De Novo Assembly of the Pneumocystis jirovecii Genome from a Single Bronchoalveolar Lavage Fluid Specimen from a Patient

ABSTRACT Pneumocystis jirovecii is a fungus that causes severe pneumonia in immunocompromised patients. However, its study is hindered by the lack of an in vitro culture method. We report here the genome of P. jirovecii that was obtained from a single bronchoalveolar lavage fluid specimen from a patient. The major challenge was the in silico sorting of the reads from a mixture representing the different organisms of the lung microbiome. This genome lacks virulence factors and most amino acid biosynthesis enzymes and presents reduced GC content and size. Together with epidemiological observations, these features suggest that P. jirovecii is an obligate parasite specialized in the colonization of human lungs, which causes disease only in immune-deficient individuals. This genome sequence will boost research on this deadly pathogen. IMPORTANCE Pneumocystis pneumonia is a major cause of mortality in patients with impaired immune systems. The availability of the P. jirovecii genome sequence allows new analyses to be performed which open avenues to solve critical issues for this deadly human disease. The most important ones are (i) identification of nutritional supplements for development of culture in vitro, which is still lacking 100 years after discovery of the pathogen; (ii) identification of new targets for development of new drugs, given the paucity of present treatments and emerging resistance; and (iii) identification of targets for development of vaccines. Pneumocystis pneumonia is a major cause of mortality in patients with impaired immune systems. The availability of the P. jirovecii genome sequence allows new analyses to be performed which open avenues to solve critical issues for this deadly human disease. The most important ones are (i) identification of nutritional supplements for development of culture in vitro, which is still lacking 100 years after discovery of the pathogen; (ii) identification of new targets for development of new drugs, given the paucity of present treatments and emerging resistance; and (iii) identification of targets for development of vaccines.