12

Zygomycete infections can be devastating in immunocompromised hosts. Difficulties in the histopathologic differentiation of this class from other filamentous fungi (e.g., Aspergillus spp., Fusarium spp.) may lead to delays in diagnosis and initiation of appropriate treatment, thereby significantly affecting patient outcome. A real-time PCR assay was developed to detect species of the zygomycete genera Absidia, Apophysomyces, Cunninghamella, Mucor, Rhizopus, and Saksenaea in culture and tissue samples. Primers and fluorescence resonance energy transfer hybridization probes were designed to detect a 167-bp conserved region of the multicopy zygomycete cytochrome b gene. A plasmid containing target sequence from Mucor racemosus was constructed as a positive control. The analytical sensitivity of the assay is 10 targets/l, and a specificity panel consisting of other filamentous fungi, yeasts (Candida spp.), and bacteria demonstrated no cross-reactivity in the assay. The clinical sensitivity and specificity of the assay from culture isolates were 100% (39/39) and 92% (59/64), respectively. Sensitivity and specificity determined using a limited number of fresh tissue specimens were both 100% (2/2). The sensitivity seen with formalin-fixed, paraffin-embedded tissues was 56% (35/62), and the specificity was 100% (19/19). The speed, sensitivity, and specificity of the PCR assay indicate that it is useful for the rapid and accurate detection of zygomycetes.Zygomycetes are common environmental fungi capable of producing serious disease in immunocompromised hosts. The vast majority of infections occur in individuals who are neutropenic, those receiving cytotoxic therapies, or those who have underlying metabolic acidosis. Although previously considered a relatively rare cause of disease, the incidence of opportunistic infections due to zygomycetes has increased over the last decade. Studies from multiple centers have indicated infection rates from 2.5% in bone marrow transplant recipients to 5.7% in solid-organ transplant recipients (10). Depending on the patient population, underlying disease, clinical presentation, and an early, accurate diagnosis, overall mortality rates due to zygomycete infections have ranged from 20% in localized infections to 100% in cases of disseminated disease (5, 13).Members of the order Mucorales include species of the genera Rhizopus, Absidia, Mucor, Rhizomucor, Apophysomyces, Cunninghamella, and Saksenaea, and all have been implicated in human disease. Approximately 90% of all infections are caused by members of the genus Rhizopus, most commonly, R. arrhizus, followed by R. microsporus var. rhizopodiformis.Currently, a diagnosis of zygomycosis is based upon identification of broad, ribbon-like, pauciseptate hyphae by histopathology or the use of macroscopic and microscopic morphology analysis following fungal culture. Histopathology determinations suffer from subjectivity that is dependent upon the experience of the reader. In addition, tissue processing, fixation, and staining may require sever...

The performance of the new VITEK 2 Advanced Colorimetry yeast identification (YST) card for use with the VITEK 2 system (bioMérieux, Inc., Hazelwood, MO) was compared to that of the API 20C AUX (API) system (bioMérieux SA, Marcy-l'Etoile, France) in a multicenter evaluation. A total of 12 quality control, 64 challenge, and 623 clinical yeast isolates were used in the study. Comparisons of species identification, platform reliability, and substrate reproducibility were made between YST and API, with API considered the reference standard. Quality control testing to assess system and substrate reproducibility matched expected results >95% of the time. The YST card correctly identified 100% of the challenge strains, which covered the species range of the manufacturer's performance claims. Using clinical isolates, the YST card correctly identified 98.5%, with 1.0% of isolates incorrectly identified and 0.5% unidentified. Among clinical isolates, the YST card generated fewer low-discrimination results (18.9%) than did API (30.0%). The time to identification with YST was 18 h, compared to 48 to 72 h with API. The colorimetric YST card used with the VITEK 2 provides a highly automated, objective yeast identification method with excellent performance and reproducibility. We found this system useful for timely and accurate identification of significant yeast species in the clinical microbiology laboratory.Candida species have emerged as notable pathogens over the last decade, especially among hospitalized and immunosuppressed populations. Recent studies have implicated Candida spp. as one of the leading causes of nosocomial fungemia, with a crude mortality rate of approximately 40% (5). In addition, there has been a shift in the dominant causative agent from C. albicans to non-C. albicans species (i.e., C. parapsilosis, C. glabrata, and C. tropicalis) (14). Concern about antifungal resistance, particularly with agents of the azole class of antifungals and amphotericin B (3, 13, 15), necessitates the rapid and accurate identification of yeasts to the species level by the clinical microbiology laboratory. Although nonculture methods (e.g., PCR and antigen detection) (12,17,18,22) have been applied to Candida diagnostics, they exhibit variable sensitivities, can be time-and labor-intensive, and may not be applicable for routine clinical use (19). A number of commercial systems which use enzymatic reactivity or carbohydrate utilization as the basis for yeast identification have been developed (2, 20).VITEK 2 (bioMérieux, Inc. Hazelwood, MO) is a fully automated microbiology identification system that evaluates an optical signal generated by individual biochemical reactions contained within a variety of microbe identification cards. After inoculation with a standardized suspension of the unknown organism, each self-contained card is incubated and read by the instrument's internal optics. Comparison of results to known species-specific reactions in the VITEK 2 database yields organism identification.This study utilized a new co...

Background As invasive mucormycosis (IM) numbers rise, clinicians suspect prior voriconazole worsens IM incidence and severity, and believe combination anti-fungal therapy improves IM survival. Objectives To compare the cumulative incidence (CI), severity and mortality of IM in eras immediately before and after the commercial availability of voriconazole. Methods All IM cases from 1995–2011 were analyzed across four risk-groups (hematologic/oncologic malignancy (H/O), stem cell transplantation (SCT), solid organ transplantation (SOT), and other), and two eras, E1, (1995–2003), and E2, (2004–2011). Results Of 101 IM cases, (79 proven, 22 probable): 30 were in E1 (3.3/year) and 71 in E2 (8.9/year). Between eras, the proportion with H/O or SCT rose from 47% to 73%, while “other” dropped from 33% to 11% (p=0.036). Between eras, the CI of IM did not significantly increase in SCT (p=0.27) or SOT (p=0.30), and patterns of anatomic location (p=0.122) and surgical debridement (p=0.200) were similar. Significantly more patients received amphotericin-echinocandin combination therapy in E2 (31% vs. 5%, p=. 01); however, 90-day survival did not improve (54% vs. 59%, p=0.67). Conclusions Since 2003, the rise of IM reflects increasing numbers at risk, not prior use of voriconazole. Frequent combination anti-fungal therapy has not improved survival.

No abstract

BackgroundPeptide nucleic acid fluorescent in situ hybridization (PNA-FISH) is a rapid and established method for identification of Candida sp., Gram positive, and Gram negative bacteria from positive blood cultures. This study reports clinical experience in the evaluation of 103 positive blood cultures and 17 positive peritoneal fluid cultures from 120 patients using PNA-FISH. Our study provides evidence as to potential pharmaceutical cost savings based on rapid pathogen identification, in addition to the novel application of PNA-FISH to peritoneal fluid specimens.MethodsIdentification accuracy and elapsed time to identification of Gram positives, Gram negatives, and Candida sp., isolated from blood and peritoneal fluid cultures were assessed using PNA-FISH (AdvanDx), as compared to standard culture methods. Patient charts were reviewed to extrapolate potential pharmaceutical cost savings due to adjustment of antimicrobial or antifungal therapy, based on identification by PNA-FISH.ResultsIn blood cultures, time to identification by standard culture methods for bacteria and Candida sp., averaged 83.6 hours (95% CI 56.7 to 110.5). Identification by PNA-FISH averaged 11.2 hours (95% CI 4.8 to 17.6). Overall PNA-FISH identification accuracy was 98.8% (83/84, 95% CI 93.5% to 99.9%) as compared to culture. In peritoneal fluid, identification of bacteria by culture averaged 87.4 hours (95% CI −92.4 to 267.1). Identification by PNA-FISH averaged 16.4 hours (95% CI −57.3 to 90.0). Overall PNA-FISH identification accuracy was 100% (13/13, 95% CI 75.3% to 100%). For Candida sp., pharmaceutical cost savings based on PNA-FISH identification could be $377.74/day. For coagulase-negative staphylococcus (CoNS), discontinuation of vancomycin could result in savings of $20.00/day.ConclusionsIn this retrospective study, excellent accuracy of PNA-FISH in blood and peritoneal fluids with reduced time to identification was observed, as compared to conventional culture-based techniques. Species-level identification based on PNA-FISH could contribute to notable cost savings due to adjustments in empiric antimicrobial or antifungal therapy as appropriate to the pathogen identified.

No abstract

Context.— Candida auris is an emerging yeast species that was first described in 2009. This ascomycetous yeast is notable for resistance to azole antifungal agents, for environmental persistence, and for its ability to contaminate health care environments, resulting in patient colonization and nosocomial infections. Objective.— To review the state of current knowledge addressing challenges in the accurate identification of C auris in the diagnostic microbiology laboratory, including application of phenotypic, proteomic, and genomic methodologies; characteristics that may predispose the human host to acquiring C auris; transmission; clinical presentations; treatment modalities; environmental decontamination; and infection prevention in health care settings. Data Sources.— The PubMed search engine was used to access peer-reviewed literature published from 2009 to 2019. Conclusions.— The rapid emergence of C auris has presented unique challenges for the areas of laboratory diagnostics and infection prevention and in options for antifungal treatment, which are limited. The current lack of established antifungal susceptibility test breakpoints complicates therapeutic decision making. Enhanced awareness of this pathogen is essential to monitor outbreaks and to reduce the risk of spread within health care environments.