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1.  Complete Genome Sequence of Listeria monocytogenes LL195, a Serotype 4b Strain from the 1983–1987 Listeriosis Epidemic in Switzerland 
Genome Announcements  2013;1(1):e00152-12.
The complete genome sequence of Listeria monocytogenes LL195, a serotype 4b clinical strain isolated during the 1983–1987 listeriosis epidemic in Switzerland, is presented.
PMCID: PMC3569335  PMID: 23405339
2.  Diagnosis of invasive candidiasis in the ICU 
Invasive candidiasis ranges from 5 to 10 cases per 1,000 ICU admissions and represents 5% to 10% of all ICU-acquired infections, with an overall mortality comparable to that of severe sepsis/septic shock. A large majority of them are due to Candida albicans, but the proportion of strains with decreased sensitivity or resistance to fluconazole is increasingly reported. A high proportion of ICU patients become colonized, but only 5% to 30% of them develop an invasive infection. Progressive colonization and major abdominal surgery are common risk factors, but invasive candidiasis is difficult to predict and early diagnosis remains a major challenge. Indeed, blood cultures are positive in a minority of cases and often late in the course of infection. New nonculture-based laboratory techniques may contribute to early diagnosis and management of invasive candidiasis. Both serologic (mannan, antimannan, and betaglucan) and molecular (Candida-specific PCR in blood and serum) have been applied as serial screening procedures in high-risk patients. However, although reasonably sensitive and specific, these techniques are largely investigational and their clinical usefulness remains to be established. Identification of patients susceptible to benefit from empirical antifungal treatment remains challenging, but it is mandatory to avoid antifungal overuse in critically ill patients. Growing evidence suggests that monitoring the dynamic of Candida colonization in surgical patients and prediction rules based on combined risk factors may be used to identify ICU patients at high risk of invasive candidiasis susceptible to benefit from prophylaxis or preemptive antifungal treatment.
PMCID: PMC3224461  PMID: 21906271
3.  Ultra-Performance Liquid Chromatography Mass Spectrometry and Sensitive Bioassay Methods for Quantification of Posaconazole Plasma Concentrations after Oral Dosing▿  
Antimicrobial Agents and Chemotherapy  2010;54(12):5074-5081.
Posaconazole (POS) is a new antifungal agent for prevention and therapy of mycoses in immunocompromised patients. Variable POS pharmacokinetics after oral dosing may influence efficacy: a trough threshold of 0.5 μg/ml has been recently proposed. Measurement of POS plasma concentrations by complex chromatographic techniques may thus contribute to optimize prevention and management of life-threatening infections. No microbiological analytical method is available. The objective of this study was to develop and validate a new simplified ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method and a sensitive bioassay for quantification of POS over the clinical plasma concentration range. The UPLC-MS/MS equipment consisted of a triple quadrupole mass spectrometer, an electrospray ionization (ESI) source, and a C18 analytical column. The Candida albicans POS-hypersusceptible mutant (MIC of 0.002 μg/ml) Δcdr1 Δcdr2 Δflu Δmdr1 Δcan constructed by targeted deletion of multidrug efflux transporters and calcineurin genes was used for the bioassay. POS was extracted from plasma by protein precipitation with acetonitrile-methanol (75%/25%, vol/vol). Reproducible standard curves were obtained over the range 0.014 to 12 (UPLC-MS/MS) and 0.028 to 12 μg/ml (bioassay). Intra- and interrun accuracy levels were 106% ± 2% and 103% ± 4% for UPLC-MS/MS and 102% ± 8% and 104% ± 1% for bioassay, respectively. The intra- and interrun coefficients of variation were 7% ± 4% and 7% ± 3% for UPLC-MS/MS and 5% ± 3% and 4% ± 2% for bioassay, respectively. An excellent correlation between POS plasma concentrations measured by UPLC-MS/MS and bioassay was found (concordance, 0.96). In 26 hemato-oncological patients receiving oral POS, 27/69 (39%) trough plasma concentrations were lower than 0.5 μg/ml. The UPLC-MS/MS method and sensitive bioassay offer alternative tools for accurate and precise quantification of the plasma concentrations in patients receiving oral posaconazole.
PMCID: PMC2981256  PMID: 20921320
4.  Multiplex Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry Method for Simultaneous Quantification in Human Plasma of Fluconazole, Itraconazole, Hydroxyitraconazole, Posaconazole, Voriconazole, Voriconazole-N-Oxide, Anidulafungin, and Caspofungin▿ †  
Antimicrobial Agents and Chemotherapy  2010;54(12):5303-5315.
Therapeutic drug monitoring (TDM) may contribute to optimizing the efficacy and safety of antifungal therapy because of the large variability in drug pharmacokinetics. Rapid, sensitive, and selective laboratory methods are needed for efficient TDM. Quantification of several antifungals in a single analytical run may best fulfill these requirements. We therefore developed a multiplex ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method requiring 100 μl of plasma for simultaneous quantification within 7 min of fluconazole, itraconazole, hydroxyitraconazole, posaconazole, voriconazole, voriconazole-N-oxide, caspofungin, and anidulafungin. Protein precipitation with acetonitrile was used in a single extraction procedure for eight analytes. After reverse-phase chromatographic separation, antifungals were quantified by electrospray ionization-triple-quadrupole mass spectrometry by selected reaction monitoring detection using the positive mode. Deuterated isotopic compounds of azole antifungals were used as internal standards. The method was validated based on FDA recommendations, including assessment of extraction yields, matrix effect variability (<9.2%), and analytical recovery (80.1 to 107%). The method is sensitive (lower limits of azole quantification, 0.01 to 0.1 μg/ml; those of echinocandin quantification, 0.06 to 0.1 μg/ml), accurate (intra- and interassay biases of −9.9 to +5% and −4.0 to +8.8%, respectively), and precise (intra- and interassay coefficients of variation of 1.2 to 11.1% and 1.2 to 8.9%, respectively) over clinical concentration ranges (upper limits of quantification, 5 to 50 μg/ml). Thus, we developed a simple, rapid, and robust multiplex UPLC-MS/MS assay for simultaneous quantification of plasma concentrations of six antifungals and two metabolites. This offers, by optimized and cost-effective lab resource utilization, an efficient tool for daily routine TDM aimed at maximizing the real-time efficacy and safety of different recommended single-drug antifungal regimens and combination salvage therapies, as well as a tool for clinical research.
PMCID: PMC2981284  PMID: 20855739
5.  False-Negative PCR Result Due to Gene Polymorphism: the Example of Neisseria meningitidis ▿  
Journal of Clinical Microbiology  2010;48(12):4590-4591.
Early treatment of meningococcal meningitis is mandatory but may negate the cerebrospinal fluid culture. Etiological diagnosis then mainly relies on PCR. Here, we report a case of false-negative results for real-time PCR for a Neisseria meningitidis serogroup B isolate with a polymorphism in the ctrA gene.
PMCID: PMC3008480  PMID: 20962143
6.  Multicenter, Prospective Clinical Evaluation of Respiratory Samples from Subjects at Risk for Pneumocystis jirovecii Infection by Use of a Commercial Real-Time PCR Assay▿† 
Journal of Clinical Microbiology  2011;49(5):1872-1878.
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.
PMCID: PMC3122670  PMID: 21367988
7.  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 ▿  
Journal of Clinical Microbiology  2010;48(6):2043-2046.
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.
PMCID: PMC2884487  PMID: 20375233
8.  Natural Variability of In Vitro Adherence to Fibrinogen and Fibronectin Does Not Correlate with In Vivo Infectivity of Staphylococcus aureus▿  
Infection and Immunity  2010;78(4):1711-1716.
Adherence to fibrinogen and fibronectin plays a crucial role in Staphylococcus aureus experimental endocarditis. Previous genetic studies have shown that infection and carriage isolates do not systematically differ in their virulence-related genes, including genes conferring adherence, such as clfA and fnbA. We set out to determine the range of adherence phenotypes in carriage isolates of S. aureus, to compare the adherence of these isolates to the adherence of infection isolates, and to determine the relationship between adherence and infectivity in a rat model of experimental endocarditis. A total of 133 healthy carriage isolates were screened for in vitro adherence to fibrinogen and fibronectin, and 30 isolates were randomly chosen for further investigation. These 30 isolates were compared to 30 infective endocarditis isolates and 30 blood culture isolates. The infectivities of the carriage isolates, which displayed either extremely low or high adherence to fibrinogen and fibronectin, were tested using a rat model of experimental endocarditis. The levels of adherence to both fibrinogen and fibronectin were very similar for isolates from healthy carriers and members of the two groups of infection isolates. All three groups of isolates showed a wide range of adherence to fibrinogen and fibronectin. Moreover, the carriage isolates that showed minimal adherence and the carriage isolates that showed strong adherence had the same infectivity in experimental endocarditis. Adherence was proven to be important for pathogenesis in experimental endocarditis, but even the least adherent carriage strains had the ability to induce infection. We discuss the roles of differential gene expression, human host factors, and gene redundancy in resolving this apparent paradox.
PMCID: PMC2849403  PMID: 20065030
9.  Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry for Direct Bacterial Identification from Positive Blood Culture Pellets ▿  
Journal of Clinical Microbiology  2010;48(4):1481-1483.
An ammonium chloride erythrocyte-lysing procedure was used to prepare a bacterial pellet from positive blood cultures for direct matrix-assisted laser desorption-ionization time of flight (MALDI-TOF) mass spectrometry analysis. Identification was obtained for 78.7% of the pellets tested. Moreover, 99% of the MALDI-TOF identifications were congruent at the species level when considering valid scores. This fast and accurate method is promising.
PMCID: PMC2849571  PMID: 20164269
10.  New Diagnostic Real-Time PCR for Specific Detection of Mycoplasma hominis DNA 
Mycoplasma hominis is a fastidious micro-organism causing genital and extragenital infections. We developed a specific real-time PCR that exhibits high sensitivity and low intrarun and interrun variabilities. When applied to clinical samples, this quantitative PCR allowed to confirm the role of M. hominis in three patients with severe extragenital infections.
PMCID: PMC2913506  PMID: 20706532
11.  Defining Responses to Therapy and Study Outcomes in Clinical Trials of Invasive Fungal Diseases: Mycoses Study Group and European Organization for Research and Treatment of Cancer Consensus Criteria 
Invasive fungal diseases (IFDs) have become major causes of morbidity and mortality among highly immunocompromised patients. Authoritative consensus criteria to diagnose IFD have been useful in establishing eligibility criteria for antifungal trials. There is an important need for generation of consensus definitions of outcomes of IFD that will form a standard for evaluating treatment success and failure in clinical trials. Therefore, an expert international panel consisting of the Mycoses Study Group and the European Organization for Research and Treatment of Cancer was convened to propose guidelines for assessing treatment responses in clinical trials of IFDs and for defining study outcomes. Major fungal diseases that are discussed include invasive disease due to Candida species, Aspergillus species and other molds, Cryptococcus neoformans, Histoplasma capsulatum, and Coccidioides immitis. We also discuss potential pitfalls in assessing outcome, such as conflicting clinical, radiological, and/or mycological data and gaps in knowledge.
PMCID: PMC2671230  PMID: 18637757
12.  Revised Definitions of Invasive Fungal Disease from the 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) Consensus Group 
Invasive fungal diseases are important causes of morbidity and mortality. Clarity and uniformity in defining these infections are important factors in improving the quality of clinical studies. A standard set of definitions strengthens the consistency and reproducibility of such studies.
After the introduction of the original 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) Consensus Group definitions, advances in diagnostic technology and the recognition of areas in need of improvement led to a revision of this document. The revision process started with a meeting of participants in 2003, to decide on the process and to draft the proposal. This was followed by several rounds of consultation until a final draft was approved in 2005. This was made available for 6 months to allow public comment, and then the manuscript was prepared and approved.
The revised definitions retain the original classifications of “proven,” “probable,” and “possible” invasive fungal disease, but the definition of “probable” has been expanded, whereas the scope of the category “possible” has been diminished. The category of proven invasive fungal disease can apply to any patient, regardless of whether the patient is immunocompromised, whereas the probable and possible categories are proposed for immunocompromised patients only.
These revised definitions of invasive fungal disease are intended to advance clinical and epidemiological research and may serve as a useful model for defining other infections in high-risk patients.
PMCID: PMC2671227  PMID: 18462102
13.  Extended-Spectrum β-Lactamases of the CTX-M Type Now in Switzerland▿  
The epidemiology of clavulanic acid-inhibited extended-spectrum β-lactamases (ESBLs) was investigated among infection-associated enterobacterial isolates at the University Hospital in Lausanne, Switzerland, from January 2004 to June 2005. Out of 57 nonrepetitive ESBL producers (prevalence rate of 0.7%), 45 produced CTX-M-like ESBLs. CTX-M enzymes were mostly from clonally nonrelated Escherichia coli isolates, from urinary infections and community-acquired infections. Pediatric patients (20 out of 57) accounted for a large number of CTX-M producers. CTX-M-15 was the most frequent CTX-M-type enzyme. The plasmid-located blaCTX-M genes were associated with either ISEcp1 or ISCR1 insertion sequences. This study is the first published report of CTX-M-type β-lactamases in Switzerland.
PMCID: PMC1932552  PMID: 17470647
15.  Variability of Voriconazole Plasma Levels Measured by New High-Performance Liquid Chromatography and Bioassay Methods▿  
Voriconazole (VRC) is a broad-spectrum antifungal triazole with nonlinear pharmacokinetics. The utility of measurement of voriconazole blood levels for optimizing therapy is a matter of debate. Available high-performance liquid chromatography (HPLC) and bioassay methods are technically complex, time-consuming, or have a narrow analytical range. Objectives of the present study were to develop new, simple analytical methods and to assess variability of voriconazole blood levels in patients with invasive mycoses. Acetonitrile precipitation, reverse-phase separation, and UV detection were used for HPLC. A voriconazole-hypersusceptible Candida albicans mutant lacking multidrug efflux transporters (cdr1Δ/cdr1Δ, cdr2Δ/cdr2Δ, flu1Δ/flu1Δ, and mdr1Δ/mdr1Δ) and calcineurin subunit A (cnaΔ/cnaΔ) was used for bioassay. Mean intra-/interrun accuracies over the VRC concentration range from 0.25 to 16 mg/liter were 93.7% ± 5.0%/96.5% ± 2.4% (HPLC) and 94.9% ± 6.1%/94.7% ± 3.3% (bioassay). Mean intra-/interrun coefficients of variation were 5.2% ± 1.5%/5.4% ± 0.9% and 6.5% ± 2.5%/4.0% ± 1.6% for HPLC and bioassay, respectively. The coefficient of concordance between HPLC and bioassay was 0.96. Sequential measurements in 10 patients with invasive mycoses showed important inter- and intraindividual variations of estimated voriconazole area under the concentration-time curve (AUC): median, 43.9 mg · h/liter (range, 12.9 to 71.1) on the first and 27.4 mg · h/liter (range, 2.9 to 93.1) on the last day of therapy. During therapy, AUC decreased in five patients, increased in three, and remained unchanged in two. A toxic encephalopathy probably related to the increase of the VRC AUC (from 71.1 to 93.1 mg · h/liter) was observed. The VRC AUC decreased (from 12.9 to 2.9 mg · h/liter) in a patient with persistent signs of invasive aspergillosis. These preliminary observations suggest that voriconazole over- or underexposure resulting from variability of blood levels might have clinical implications. Simple HPLC and bioassay methods offer new tools for monitoring voriconazole therapy.
PMCID: PMC1797701  PMID: 17088483
16.  First Case of Bacteremia and Multifocal Cellulitis Due to Helicobacter canis in an Immunocompetent Patient▿  
Journal of Clinical Microbiology  2006;44(12):4598-4600.
Bacteremia due to Helicobacter canis has been reported in a patient with X-linked hypogammaglobulinemia. Here we report on the first human case of H. canis bacteremia in an immunocompetent host. Identification of the organism was made by genetic and phylogenetic analyses of the complete 16S rRNA sequence.
PMCID: PMC1698384  PMID: 17005753
17.  Prospective Determination of Plasma Imipenem Concentrations in Critically Ill Children 
Plasma imipenem concentrations were measured in 19 critically ill children (median age, 0.8 year; range, 0.02 to 12.9 years). Wide interindividual variations (2 to 4× at peak and >10× at trough concentrations) resulted in unpredictable plasma levels in several children. To avoid subtherapeutic drug levels, we recommend treatment with at least 100 mg/kg of body weight/day of imipenem-cilastatin for critically ill children requiring such therapy.
PMCID: PMC1489785  PMID: 16801447
18.  Bloodstream and endovascular infections due to Abiotrophia defectiva and Granulicatella species 
Abiotrophia and Granulicatella species, previously referred to as nutritionally variant streptococci (NVS), are significant causative agents of endocarditis and bacteraemia. In this study, we reviewed the clinical manifestations of infections due to A. defectiva and Granulicatella species that occurred at our institution between 1998 and 2004.
The analysis included all strains of NVS that were isolated from blood cultures or vascular graft specimens. All strains were identified by 16S rRNA sequence analysis. Patients' medical charts were reviewed for each case of infection.
Eleven strains of NVS were isolated during the 6-year period. Identification of the strains by 16S rRNA showed 2 genogroups: Abiotrophia defectiva (3) and Granulicatella adiacens (6) or "para-adiacens" (2). The three A. defectiva strains were isolated from immunocompetent patients with endovascular infections, whereas 7 of 8 Granulicatella spp. strains were isolated from immunosuppressed patients, mainly febrile neutropenic patients. We report the first case of "G. para-adiacens" bacteraemia in the setting of febrile neutropenia.
We propose that Granulicatella spp. be considered as a possible agent of bacteraemia in neutropenic patients.
PMCID: PMC1360077  PMID: 16426445
19.  Cardiac involvement in a patient with clinical and serological evidence of African tick-bite fever 
Myocarditis and pericarditis are rare complications of rickettsiosis, usually associated with Rickettsia rickettsii and R. conorii. African tick-bite fever (ATBF) is generally considered as a benign disease and no cases of myocardial involvement due to Rickettsia africae, the agent of ATBF, have yet been described.
Case presentation
The patient, that travelled in an endemic area, presented typical inoculation eschars, and a seroconversion against R. africae, was admitted for chest pains and increased cardiac enzymes in the context of an acute myocarditis.
Our findings suggest that ATBF, that usually presents a benign course, may be complicated by an acute myocarditis.
PMCID: PMC1274315  PMID: 16242016
20.  Detection of Four Plasmodium Species in Blood from Humans by 18S rRNA Gene Subunit-Based and Species-Specific Real-Time PCR Assays 
Journal of Clinical Microbiology  2004;42(12):5636-5643.
There have been reports of increasing numbers of cases of malaria among migrants and travelers. Although microscopic examination of blood smears remains the “gold standard” in diagnosis, this method suffers from insufficient sensitivity and requires considerable expertise. To improve diagnosis, a multiplex real-time PCR was developed. One set of generic primers targeting a highly conserved region of the 18S rRNA gene of the genus Plasmodium was designed; the primer set was polymorphic enough internally to design four species-specific probes for P. falciparum, P. vivax, P. malarie, and P. ovale. Real-time PCR with species-specific probes detected one plasmid copy of P. falciparum, P. vivax, P. malariae, and P. ovale specifically. The same sensitivity was achieved for all species with real-time PCR with the 18S screening probe. Ninety-seven blood samples were investigated. For 66 of them (60 patients), microscopy and real-time PCR results were compared and had a crude agreement of 86% for the detection of plasmodia. Discordant results were reevaluated with clinical, molecular, and sequencing data to resolve them. All nine discordances between 18S screening PCR and microscopy were resolved in favor of the molecular method, as were eight of nine discordances at the species level for the species-specific PCR among the 31 samples positive by both methods. The other 31 blood samples were tested to monitor the antimalaria treatment in seven patients. The number of parasites measured by real-time PCR fell rapidly for six out of seven patients in parallel to parasitemia determined microscopically. This suggests a role of quantitative PCR for the monitoring of patients receiving antimalaria therapy.
PMCID: PMC535226  PMID: 15583293
21.  TAC1, Transcriptional Activator of CDR Genes, Is a New Transcription Factor Involved in the Regulation of Candida albicans ABC Transporters CDR1 and CDR2†  
Eukaryotic Cell  2004;3(6):1639-1652.
The ABC transporter genes CDR1 and CDR2 can be upregulated in Candida albicans developing resistance to azoles or can be upregulated by exposing cells transiently to drugs such as fluphenazine. The cis-acting drug-responsive element (DRE) present in the promoters of both genes and necessary for their upregulation contains 5′-CGG-3′ triplets that are often recognized by transcriptional activators with Zn(2)-Cys(6) fingers. In order to isolate regulators of CDR1 and CDR2, the C. albicans genome was searched for genes encoding proteins with Zn(2)-Cys(6) fingers. Interestingly, three of these genes were tandemly arranged near the mating locus. Their involvement in CDR1 and CDR2 upregulation was addressed because a previous study demonstrated a link between mating locus homozygosity and azole resistance. The deletion of only one of these genes (orf19.3188) was sufficient to result in a loss of transient CDR1 and CDR2 upregulation by fluphenazine and was therefore named TAC1 (transcriptional activator of CDR genes). Tac1p has a nuclear localization, and a fusion of Tac1p with glutathione S-transferase could bind the cis-acting regulatory DRE in both the CDR1 and the CDR2 promoters. TAC1 is also relevant for azole resistance, since a TAC1 allele (TAC1-2) recovered from an azole-resistant strain could trigger constitutive upregulation of CDR1 and CDR2 in an azole-susceptible laboratory strain. Transcript profiling experiments performed with a TAC1 mutant and a revertant containing TAC1-2 revealed not only CDR1 and CDR2 as targets of TAC1 regulation but also other genes (RTA3, IFU5, and HSP12) that interestingly contained a DRE-like element in their promoters. In conclusion, TAC1 appears to be the first C. albicans transcription factor involved in the control of genes mediating antifungal resistance.
PMCID: PMC539021  PMID: 15590837
22.  Mutations of Pneumocystis jirovecii Dihydrofolate Reductase Associated with Failure of Prophylaxis 
Antimicrobial Agents and Chemotherapy  2004;48(11):4301-4305.
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.
PMCID: PMC525445  PMID: 15504856
23.  Comparison of Gene Expression Profiles of Candida albicans Azole-Resistant Clinical Isolates and Laboratory Strains Exposed to Drugs Inducing Multidrug Transporters 
Azole resistance in Candida albicans can be due to upregulation of multidrug transporters belonging to ABC (ATP-binding cassette) transporters (CDR1 and CDR2) or major facilitators (CaMDR1). Upregulation of these genes can also be achieved by exposure to fluphenazine, resulting in specific upregulation of CDR1 and CDR2 and by exposure to benomyl, resulting in specific CaMDR1 upregulation. In this study, these two different states of gene upregulation were used to determine coregulated genes that often share similar functions or similar regulatory regions. The transcript profiles of a laboratory strain exposed to these drugs were therefore determined and compared with those of two matched pairs of azole-susceptible and -resistant strains expressing CDR1 and CDR2 (CDR strains) or CaMDR1 (MDR isolates). The results obtained revealed that, among 42 commonly regulated genes (8.6% of all regulated genes) between fluphenazine-exposed cells and CDR isolates, the most upregulated were CDR1 and CDR2 as expected, but also IFU5, RTA3 (which encodes putative membrane proteins), HSP12 (which encodes heat shock protein), and IPF4065 (which is potentially involved in stress response). Interestingly, all but HSP12 and IPF4065 contain a putative cis-acting drug responsive element in their promoters. Among the 57 genes (11.5% of all regulated genes) commonly regulated between benomyl-exposed cells and MDR isolates, the most upregulated were CaMDR1 as expected but also genes with oxido-reductive functions such as IFD genes, IPF5987, GRP2 (all belonging to the aldo-keto reductase family), IPF7817 [NAD(P)H oxido-reductase], and IPF17186. Taken together, these results show that in vitro drug-induced gene expression only partially mimics expression profiles observed in azole-resistant clinical strains. Upregulated genes in both drug-exposed conditions and clinical strains are drug resistance genes but also genes that could be activated under cell damage conditions.
PMCID: PMC478486  PMID: 15273122
24.  Interlaboratory Comparison of Results of Susceptibility Testing with Caspofungin against Candida and Aspergillus Species 
Journal of Clinical Microbiology  2004;42(8):3475-3482.
Seventeen laboratories participated in a study of interlaboratory reproducibility with caspofungin microdilution susceptibility testing against panels comprising 30 isolates of Candida spp. and 20 isolates of Aspergillus spp. The laboratories used materials supplied from a single source to determine the influence of growth medium (RPMI 1640 with or without glucose additions and antibiotic medium 3 [AM3]), the same incubation times (24 h and 48 h), and the same end point definition (partial or complete inhibition of growth) for the MIC of caspofungin. All tests were run in duplicate, and end points were determined both spectrophotometrically and visually. The results from almost all of the laboratories for quality control and reference Candida and Aspergillus isolates tested with fluconazole and itraconazole matched the NCCLS published values. However, considerable interlaboratory variability was seen in the results of the caspofungin tests. For Candida spp. the most consistent MIC data were generated with visual “prominent growth reduction” (MIC2) end points measured at 24 h in RPMI 1640, where 73.3% of results for the 30 isolates tested fell within a mode ± one dilution range across all 17 laboratories. MIC2 at 24 h in RPMI 1640 or AM3 also gave the best interlaboratory separation of Candida isolates of known high and low susceptibility to caspofungin. Reproducibility of MIC data was problematic for caspofungin tests with Aspergillus spp. under all conditions, but the minimal effective concentration end point, defined as the lowest caspofungin concentration yielding conspicuously aberrant hyphal growth, gave excellent reproducibility for data from 14 of the 17 participating laboratories.
PMCID: PMC497639  PMID: 15297486
25.  Candida albicans Mutations in the Ergosterol Biosynthetic Pathway and Resistance to Several Antifungal Agents 
The role of sterol mutations in the resistance of Candida albicans to antifungal agents has not been thoroughly investigated. Previous work reported that clinical C. albicans strains resistant to both azole antifungals and amphotericin B were defective in ERG3, a gene encoding sterol Δ5,6-desaturase. It is also believed that a deletion of the lanosterol 14α-demethylase gene, ERG11, is possible only under aerobic conditions when ERG3 is not functional. We tested these hypotheses by creating mutants by targeted deletion of the ERG3 and ERG11 genes and subjecting those mutants to antifungal susceptibility testing and sterol analysis. The homozygous erg3/erg3 mutant created, DSY1751, was resistant to azole derivatives, as expected. This mutant was, however, slightly more susceptible to amphotericin B than the parent wild type. It was possible to generate erg11/erg11 mutants in the DSY1751 background but also, surprisingly, in the background of a wild-type isolate with functional ERG3 alleles under aerobic conditions. This mutant (DSY1769) was obtained by exposure of an ERG11/erg11 heterozygous strain in a medium containing 10 μg of amphotericin B per ml. Amphotericin B-resistant strains were obtained only from ERG11/erg11 heterozygotes at a frequency of approximately 5 × 10−5 to 7 × 10−5, which was consistent with mitotic recombination between the first disrupted erg11 allele and the other remaining functional ERG11 allele. DSY1769 was also resistant to azole derivatives. The main sterol fraction in DSY1769 contained lanosterol and eburicol. These studies showed that erg11/erg11 mutants of a C. albicans strain harboring a defective erg11 allele can be obtained in vitro in the presence of amphotericin B. Amphotericin B-resistant strains could therefore be selected by similar mechanisms during antifungal therapy.
PMCID: PMC166068  PMID: 12878497

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