A large aggregate collection of clinical isolates of aspergilli (n ‫؍‬ 218) from transplant patients with proven or probable invasive aspergillosis was available from the Transplant-Associated Infection Surveillance Network, a 6-year prospective surveillance study. To determine the Aspergillus species distribution in this collection, isolates were subjected to comparative sequence analyses by use of the internal transcribed spacer and ␤-tubulin regions. Aspergillus fumigatus was the predominant species recovered, followed by A. flavus and A. niger. Several newly described species were identified, including A. lentulus and A. calidoustus; both species had high in vitro MICs to multiple antifungal drugs. Aspergillus tubingensis, a member of the A. niger species complex, is described from clinical specimens; all A. tubingensis isolates had low in vitro MICs to antifungal drugs.

Patients who recover from pneumonia subsequently have elevated rates of death after hospital discharge as a result of secondary organ damage, the causes of which are unknown. We used the bacterium , a common cause of hospital-acquired pneumonia, as a model for investigating this phenomenon. We show that infection of pulmonary endothelial cells by induces production and release of a cytotoxic amyloid molecule with prion characteristics, including resistance to various nucleases and proteases. This cytotoxin was self-propagating, was neutralized by anti-amyloid Abs, and induced death of endothelial cells and neurons. Moreover, the cytotoxin induced edema in isolated lungs. Endothelial cells and isolated lungs were protected from cytotoxin-induced death by stimulation of signal transduction pathways that are linked to prion protein. Analysis of bronchoalveolar lavage fluid collected from human patients with pneumonia demonstrated cytotoxic activity, and lavage fluid contained amyloid molecules, including oligomeric τ and Aβ. Demonstration of long-lived cytotoxic agents after infection may establish a molecular link to the high rates of death as a result of end-organ damage in the months after recovery from pneumonia, and modulation of signal transduction pathways that have been linked to prion protein may provide a mechanism for intervention.-Balczon, R., Morrow, K. A., Zhou, C., Edmonds, B., Alexeyev, M., Pittet, J.-F., Wagener, B. M., Moser, S. A., Leavesley, S., Zha, X., Frank, D. W., Stevens, T. infection liberates transmissible, cytotoxic prion amyloids.

DNA extraction from formalin-fixed paraffin-embedded (FFPE) tissues is difficult and requires special protocols in order to extract small amounts of DNA suitable for amplification. Most described methods report an amplification success rate between 60 and 80%; therefore, there is a need to improve molecular detection and identification of fungi in FFPE tissue. Eighty-one archived FFPE tissues with a positive Gomori methenamine silver (GMS) stain were evaluated using five different commercial DNA extraction kits with some modifications. Three different panfungal PCR assays were used to detect fungal DNA, and two housekeeping genes were used to assess the presence of amplifiable DNA and to detect PCR inhibitors. The sensitivities of the five extraction protocols were compared, and the quality of DNA detection (calculated for each kit as the number of housekeeping gene PCRpositive samples divided by the total number of samples) was 60 to 91% among the five protocols. The efficiencies of the three different panfungals used (calculated as the number of panfungal-PCR-positive samples divided by the number of housekeeping gene PCR-positive samples) were 58 to 93%. The panfungal PCR using internal transcribed spacer 3 (ITS3) and ITS4 primers yielded a product in most FFPE tissues. Two of the five DNA extraction kits (from TaKaRa and Qiagen) showed similar and promising results. However, one method (TaKaRa) could extract fungal DNA from 69 of the 74 FFPE tissues from which a housekeeping gene could be amplified and was also cost-effective, with a nonlaborious protocol. Factors such as sensitivity, cost, and labor will help guide the selection of the most appropriate method for the needs of each laboratory.Given the rise in the incidence of invasive fungal infections (IFIs) and the expanding spectrum of fungal pathogens, early and accurate identification of the causative microorganisms in formalin-fixed paraffin-embedded (FFPE) tissue is essential (20). Tissue samples collected and processed for pathological diagnosis represent a unique source of archived and morphologically defined disease-specific biological material (24). Histopathologic examination remains one of the major diagnostic tools in mycology because it permits rapid, presumptive identification of fungal infections. In recent years, however, there have been cases with discrepant histologic and culture results at final diagnosis; such discrepancies could lead to unnecessary pharmaceutical exposure and/or inappropriate treatment (17,24).Recent efforts to improve the sensitivity and specificity of diagnostic tests have focused on culture-independent methods, in particular, nucleic acid-based methods, such as PCR assays. PCR-based detection of fungal DNA sequences can be rapid, sensitive, and specific and can be applied to fresh and FFPE tissues (16). The majority of fungal assays target multicopy loci, in particular, the ribosomal DNA (rDNA) genes (18S, 28S, and 5.8S) and the intervening internal transcribed spacer (ITS) regions (ITS1 and ITS2) in order to maxim...

We analyzed antifungal susceptibilities of 274 clinical Aspergillus isolates from transplant recipients with proven or probable invasive aspergillosis collected as part of the Transplant-Associated Infection Surveillance Network (TRANSNET) and examined the relationship between MIC and mortality at 6 or 12 weeks. Antifungal susceptibility testing was performed by the Clinical and Laboratory Standards Institute (CLSI) M38-A2 broth dilution method for amphotericin B (AMB), itraconazole (ITR), voriconazole (VOR), posaconazole (POS), and ravuconazole (RAV). The isolate collection included 181Aspergillus fumigatus, 28 Aspergillus niger, 27 Aspergillus flavus, 22 Aspergillus terreus, seven Aspergillus versicolor, five Aspergillus calidoustus, and two Aspergillus nidulans isolates and two isolates identified as Aspergillus spp. Triazole susceptibilities were <4 g/ml for most isolates (POS, 97.6%; ITR, 96.3%; VOR, 95.9%; RAV, 93.5%). The triazoles were not active against the five A. calidoustus isolates, for which MICs were >4 g/ml. AMB inhibited 93.3% of isolates at an MIC of <1 g/ml. The exception was A. terreus, for which 15 (68%) of 22 isolates had MICs of >1 g/ml. One of 181 isolates of A. fumigatus showed resistance (MIC > 4 g/ml) to two of three azoles tested. Although there appeared to be a correlation of higher VOR MICs with increased mortality at 6 weeks, the relationship was not statistically significant (R 2 ‫؍‬ 0.61; P ‫؍‬ 0.065). Significant relationships of in vitro MIC to all-cause mortality at 6 and 12 weeks for VOR or AMB were not found.

Invasive fungal infections due to Aspergillus species have become a major cause of morbidity and mortality among immunocompromised patients. Aspergillus terreus, a less common pathogen, appears to be an emerging cause of infection at our institution, the University of Alabama hospital in Birmingham. We therefore investigated the epidemiology of A. terreus over the past 6 years by using culture data; antifungal susceptibility testing with amphotericin B, voriconazole, and itraconazole; and molecular typing with random amplification of polymorphic DNA-PCR (RAPD-PCR). During the study period, the percentage of A. terreus isolates relative to those of other Aspergillus species significantly increased, and A. terreus isolates frequently were resistant to amphotericin B. Molecular typing with the RAPD technique was useful in discriminating between patient isolates, which showed much strain diversity. Further surveillance of A. terreus may better define epidemiology and determine whether this organism is becoming more frequent in relation to other Aspergillus species.Invasive fungal infections due to Aspergillus species have become a major cause of morbidity and mortality among immunocompromised patients. Aspergillus fumigatus is most frequently isolated from clinical specimens, but other important species include A. flavus, A. niger, and A. terreus. A. terreus appears to be emerging as a cause of opportunistic infections (8,9,20) and is of concern because of in vitro resistance to amphotericin B (18). At our institution, the University of Alabama hospital in Birmingham, an increase in the frequency of A. terreus isolates and invasive infections due to A. terreus has been noticed (4). Although several recent studies have discussed clinical cases of invasive A. terreus disease and strain typing of A. terreus environmental and clinical isolates (7,9,11,19,20), questions about the epidemiology of A. terreus remain unanswered. We therefore were interested in studying the epidemiology of A. terreus at our tertiary care university hospital with the use of clinical data, antifungal susceptibility testing, and molecular genotyping using the random amplification of polymorphic DNA-PCR (RAPD-PCR) method.(This work was presented in part at the 41st Interscience Conference on Antimicrobial Agents and Chemotherapy, Chicago, Ill., 16 to 19 December 2001.) MATERIALS AND METHODSWe identified cultures positive for A. terreus and other Aspergillus species at the laboratory of the University of Alabama hospital over a 6-year period (1996 to 2001) to investigate the frequency of isolation of A. terreus from clinical samples. The Laboratory Information System was utilized to identify the number of Aspergillus isolates. A subgroup of 41 patients with cultures positive for A. terreus was selected for a more focused epidemiologic study that included medical record review. In addition, 23 of 41 patients had isolates stored and available for molecular typing and susceptibility testing. Data collected from medical records included demographics,...