Impact of azole antifungal treatment on outcome in acute invasive fungal rhinosinusitis with orbitocranial involvement: a surgical perspective.

PURPOSE: To provide real-life data on azole treatment outcomes and the role of surgery in the current management of invasive fungal rhinosinusitis complicated by orbitocranial fungal infection (OCFI). METHODS: Data was collected retrospectively from a chart review from four participating centers and a systematic literature review. The study group included patients with OCFI treated with azole antifungals. The control cases were treated with other antifungal agents. The cranial and orbital involvement degree was staged based on the imaging. The extent of the surgical resection was also classified to allow for inter-group comparison. RESULTS: There were 125 patients in the azole-treated group and 153 in the control group. Among the patients with OCFI cranial extension, 23% were operated on in the azole-treated group and 18% in the control group. However, meninges and brain resection were performed only in the controls (11% of patients) and never in the azole antifungals group. Orbital involvement required surgery in 26% of azole-treated cases and 39% of controls. Despite a more aggressive cranial involvement, azole-treated patients' mortality was significantly lower than in controls, with an OCFI-specific mortality rate of 21% vs. 52%. A similar, though not statistically significant, trend was found for the extent of the orbital disease and surgery. CONCLUSION: Despite less aggressive surgical intervention for cranial involvement, OCFI patients treated with azoles had a higher survival rate. This finding suggests we may improve morbidity with a more conservative surgical approach in conjunction with azole treatment. The same trend is emerging for orbital involvement.

Candida auris: Diagnostic Challenges and Emerging Opportunities for the Clinical Microbiology Laboratory.

Purpose of Review: This review summarises the epidemiology of Candida auris infection and describes contemporary and emerging diagnostic methods for detection and identification of C. auris. Recent Findings: A fifth C. auris clade has been described. Diagnostic accuracy has improved with development of selective/differential media for C. auris. Advances in spectral databases of matrix-associated laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF MS) systems have reduced misidentification. Direct detection of C. auris in clinical specimens using real time PCR is increasingly used, as is whole genome sequencing (WGS) to track nosocomial spread and to study phylogenetic relationships and drug resistance. Summary: C. auris is an important transmissible, nosocomial pathogen. The microbiological laboratory diagnostic capacity has extended beyond culture-based methods to include PCR and WGS. Microbiological techniques on the horizon include the use of MALDI-TOF MS for early echinocandin antifungal susceptibility testing (AST) and expansion of the versatile and information-rich WGS methods for outbreak investigation.

Laboratory diagnosis of severe acute respiratory syndrome coronavirus 2.

The first laboratory confirmed case of Coronavirus disease 2019 (COVID-19) in Australia was in Victoria on 25 January 2020 in a man returning from Wuhan city, Hubei province, the People's Republic of China. This was followed by three cases in New South Wales the following day. The Australian Government activated the Australian Health Sector Emergency Response Plan for Novel Coronavirus on 27 February 2020 in anticipation of a pandemic. Subsequently, the World Health Organization declared COVID-19 to be a Public Health Emergency of International Concern followed by a pandemic on 30 January 2020 and 11 March 2020, respectively. Laboratory testing for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus responsible for COVID-19, is key in identifying infected persons to guide timely public health actions of contact tracing and patient isolation to limit transmission of infection. This article aims to provide a comprehensive overview of current laboratory diagnostic methods for SARS-CoV-2, including nucleic acid testing, serology, rapid antigen detection and antibody tests, virus isolation and whole genome sequencing. The relative advantages and disadvantages of the different diagnostic tests are presented, as well as their value in different clinical, infection control and public health contexts. We also describe the challenges in the provision of SARS-CoV-2 diagnostics in Australia, a country with a relatively low COVID-19 incidence in the first pandemic wave but in which prevalence could rapidly change.

Interpret with caution: An evaluation of the commercial AusDiagnostics versus in-house developed assays for the detection of SARS-CoV-2 virus.

INTRODUCTION: There is limited data on the analytical performance of commercial nucleic acid tests (NATs) for laboratory confirmation of COVID-19 infection. METHODS: Nasopharyngeal, combined nose and throat swabs, nasopharyngeal aspirates and sputum was collected from persons with suspected SARS-CoV-2 infection, serial dilutions of SARS-CoV-2 viral cultures and synthetic positive controls (gBlocks, Integrated DNA Technologies) were tested using i) AusDiagnostics assay (AusDiagnostics Pty Ltd); ii) in-house developed assays targeting the E and RdRp genes; iii) multiplex PCR assay targeting endemic respiratory viruses. Discrepant SARS-CoV-2 results were resolved by testing the N, ORF1b, ORF1ab and M genes. RESULTS: Of 52 clinical samples collected from 50 persons tested, respiratory viruses were detected in 22 samples (42 %), including SARS CoV-2 (n = 5), rhinovirus (n = 7), enterovirus (n = 5), influenza B (n = 4), hMPV (n = 5), influenza A (n = 2), PIV-2 (n = 1), RSV (n = 2), CoV-NL63 (n = 1) and CoV-229E (n = 1). SARS-CoV-2 was detected in four additional samples by the AusDiagnostics assay. Using the in-house assays as the "gold standard", the sensitivity, specificity, positive and negative predictive values of the AusDiagnostics assay was 100 %, 92.16 %, 55.56 % and 100 % respectively. The Ct values of the real-time in-house-developed PCR assay targeting the E gene was significantly lower than the corresponding RdRp gene assay when applied to clinical samples, viral culture and positive controls (mean 21.75 vs 28.1, p = 0.0031). CONCLUSIONS: The AusDiagnostics assay is not specific for the detection SARS-CoV-2. Any positive results should be confirmed using another NAT or sequencing. The case definition used to investigate persons with suspected COVID-19 infection is not specific.

Candidaemia and a risk predictive model for overall mortality: a prospective multicentre study.

BACKGROUND: Candidaemia is associated with high mortality. Variables associated with mortality have been published previously, but not developed into a risk predictive model for mortality. We sought to describe the current epidemiology of candidaemia in Australia, analyse predictors of 30-day all-cause mortality, and develop and validate a mortality risk predictive model. METHODS: Adults with candidaemia were studied prospectively over 12 months at eight institutions. Clinical and laboratory variables at time of blood culture-positivity were subject to multivariate analysis for association with 30-day all-cause mortality. A predictive score for mortality was examined by area under receiver operator characteristic curves and a historical data set was used for validation. RESULTS: The median age of 133 patients with candidaemia was 62 years; 76 (57%) were male and 57 (43%) were female. Co-morbidities included underlying haematologic malignancy (n = 20; 15%), and solid organ malignancy in (n = 25; 19%); 55 (41%) were in an intensive care unit (ICU). Non-albicans Candida spp. accounted for 61% of cases (81/133). All-cause 30-day mortality was 31%. A gastrointestinal or unknown source was associated with higher overall mortality than an intravascular or urologic source (p < 0.01). A risk predictive score based on age > 65 years, ICU admission, chronic organ dysfunction, preceding surgery within 30 days, haematological malignancy, source of candidaemia and antibiotic therapy for ≥10 days stratified patients into < 20% or ≥ 20% predicted mortality. The model retained accuracy when validated against a historical dataset (n = 741). CONCLUSIONS: Mortality in patients with candidaemia remains high. A simple mortality risk predictive score stratifying patients with candidaemia into < 20% and ≥ 20% 30-day mortality is presented. This model uses information available at time of candidaemia diagnosis is easy to incorporate into decision support systems. Further validation of this model is warranted.

The epidemiology and clinical manifestations of mucormycosis: a systematic review and meta-analysis of case reports.

BACKGROUND: The epidemiology of mucormycosis in the era of modern diagnostics is relatively under-explored. OBJECTIVES: To examine the contemporary epidemiology, clinical manifestations, diagnosis and causative pathogens of mucormycosis. DATA SOURCES: Ovid MEDLINE and Ovid EMBASE from January 2000 to January 2017. STUDY ELIGIBILITY CRITERIA: Published case reports/series of proven/probable mucormycosis. PARTICIPANTS: Patients ≥18 years old. METHODS: Patient characteristics, disease manifestations and causative pathogens were summarized descriptively. Categorical variables were assessed by chi-square test or Fischer's exact test, and continuous variables by the Wilcoxon-Mann-Whitney or Kruskal-Wallis test. Risk factors for the different clinical manifestations of mucormycosis were identified using multivariate logistic regression. RESULTS: Initial database searches identified 3619 articles of which 600 (851 individual patient cases) were included in the final analysis. Diabetes mellitus was the commonest underlying condition (340/851, 40%) and was an independent risk for rhino-orbital-cerebral mucormycosis (odds ratio (OR) 2.49; 95% CI 1.77-3.54; p < 0.001). Underlying haematological malignancy was associated with disseminated infection (OR 3.86; 95% CI 1.78-8.37; p 0.001), whereas previous solid organ transplantation was associated with pulmonary (OR 3.19; 95% CI 1.50-6.82; p 0.003), gastrointestinal (OR 4.47; 95% CI 1.69-11.80; p 0.003), or disseminated (OR 4.20; 95% CI 1.68-10.46; p 0.002) mucormycosis. Eight genera (24 species) of Mucorales organisms were identified in 447/851 (53%) cases, of which Rhizopus spp. (213/447, 48%) was the most common. Compared with other genera, Rhizopus spp. was predominantly observed in patients with rhino-orbital-cerebral mucormycosis (75/213, 35% versus 34/234, 15%; p < 0.001). Death was reported in 389/851 (46%) patients. Mortality associated with Cunninghamella infections was significantly higher than those caused by other Mucorales (23/30, 71% versus 185/417, 44%; p < 0.001). However, Cunninghamella spp. were isolated primarily in patients with pulmonary (17/30, 57%) or disseminated disease (10/30, 33%). CONCLUSIONS: Findings from the current review have helped ascertain the association between various manifestations of mucormycosis, their respective predisposing factors and causative organisms.

Molecular mechanisms of azole resistance in Candida tropicalis isolates causing invasive candidiasis in China.

OBJECTIVE: We investigated molecular mechanisms responsible for azole resistance in Candida tropicalis isolates. METHODS: We studied 507 C. tropicalis isolates causing invasive candidiasis from ten hospitals over 5 years. Antifungal susceptibility was determined by broth microdilution methods. Point mutations in the C. tropicalis ERG11 gene that may confer azole resistance were explored and verified. The expression levels of ERG11, CYTb, MDR1 and CDR1 genes were compared in 20 fluconazole-susceptible and 20 fluconazole-resistant isolates. RESULTS: Fluconazole-susceptible, -susceptible dose-dependent and -resistant strains accounted for 76.7% (389/507), 10.5% (53/507) and 12.8% (65/507) of C. tropicalis isolates, respectively. The ERG11 mutation A395T/W occurred in 10.7% (54/507) of isolates, all of which were resistant to fluconazole. The nucleotide mutation C461T/Y was the second most common (50/507 isolates, 9.9%), and all isolates carrying C461T/Y also had the mutation A395T/W. However, the presence of C461T did not contribute to the azole-resistant phenotype. Substitutions V125A, Y257H and G464S (<2% of isolates), which were reported for the first time in C. tropicalis, also conferred fluconazole non-susceptible phenotypes. Compared with fluconazole susceptible isolates, fluconazole-resistant isolates had higher ERG11 (fold expression level 1.42 versus 0.79, p < 0.01) but lower CYTb (fold expression level 1.26 versus 2.67, p < 0.01) gene expression levels. Three azole-resistant isolates carrying the wild-type ERG11 gene had higher levels of CDR1 and MDR1 expression. CONCLUSIONS: ERG11 missense mutations were the major mechanism responsible for azole resistance in C. tropicalis isolates, but overexpression of ERG11, CDR1 and MDR1, as well as reduced expression of CYTb, also contributed to resistance.

Rapid detection of Group B streptococcus directly from vaginal-rectal specimens using liquid swabs and the BD Max GBS assay.

OBJECTIVE: We adapted the BD Max GBS assay, an automated platform for the detection of Group B streptococcus (GBS) DNA in vaginal-rectal swab specimens after LIM broth enrichment, to directly detect GBS in specimens collected using cellular foam swabs in Amies liquid medium. We compared the BD Max GBS assay performance to that of enriched culture and the BD GeneOhm StrepB assay. METHODS: Seventy-two reference vaginal-rectal specimens were employed to determine the limit of GBS detection and the preferred test volume for direct detection of GBS. A total of 304 clinical specimens were then tested by the optimized BD Max GBS assay, both by direct testing and following broth enrichment. RESULTS: The limit of GBS detection was 75 CFU/mL and the preferred test volume was 100 µL. Of 304 clinical specimens tested, GBS was detected in 62 specimens by enriched culture (20.4%); 61 of these yielded GBS by the BD Max GBS assay when performed directly from the liquid swab (sensitivity 98.4%). All 242 culture-negative specimens also yielded negative results by the BD Max GBS assay (specificity 100%). When this assay was performed following broth enrichment, GBS was detected in all 62 culture-positive specimens (100% sensitivity). The sensitivity and specificity of the BD GeneOhm StrepB assay was 90.3% and 99%, respectively. CONCLUSIONS: The BD Max GBS assay is highly sensitive, requires minimal technical skill with <2 min required to set-up, and results are available in under 80 min (versus 24-48 h for culture). It is configured for 'on demand' testing and vaginal-rectal specimens can be rapidly screened for GBS without the need for enrichment. The results obtained in this study demonstrate that rapid GBS screening using the BD Max GBS assay at the time of delivery is a viable alternative to the current recommended screening at 35-37 weeks of gestation with pre-enrichment testing methods.

Laboratory Approach to the Diagnosis of Culture-Negative Infective Endocarditis.

Blood-culture negative endocarditis (BCNE) accounts for up to 35% of all cases of infective endocarditis (IE) and is a serious life-threatening condition with considerable morbidity and mortality. Rapid detection and identification of the causative pathogen is essential for timely, directed therapy. Blood-culture negative endocarditis presents a diagnostic and therapeutic challenge. Causes of BCNE are varied including: treatment with antibiotic agents prior to blood culture collection; sub-optimal specimen collection; and/or infection due to fastidious (eg. nutritionally variant streptococci), intracellular (eg. Coxiella burnetii, Bartonella species) or non-culturable or difficult to culture organisms (eg. Mycobacteria, Tropheryma whipplei and fungi); as well as non-infective aetiologies. Here, we review aetiological and diagnostic approaches to BCNE including newer molecular based techniques, with a brief summary of imaging investigation and treatment principles.

Identification of genetic markers of resistance to echinocandins, azoles and 5-fluorocytosine in Candida glabrata by next-generation sequencing: a feasibility study.

OBJECTIVES: Multi-antifungal drug resistance in Candida glabrata is increasing. We examined the feasibility of next-generation sequencing (NGS) to investigate the presence of antifungal drug resistance markers in C. glabrata. METHODS: The antifungal susceptibility of 12 clinical isolates and one ATCC strain of C. glabrata was determined using the Sensititre YeastOne® YO10 assay. These included three isolate pairs where the second isolate of each pair had developed a rise in drug MICs. Single nucleotide polymorphisms (SNPs) in genes known to be linked to echinocandin, azole and 5-fluorocytosine resistance were analysed in all isolates through NGS. RESULTS: High-quality non-synonymous SNPs in antifungal resistance genes such as FKS1, FKS2, CgCDR1, CgPDR1 and FCY2 were identified. For two of three isolate pairs, there was a >60-fold rise in MICs to all echinocandins in the second isolate from each pair; one echinocandin-resistant isolate harboured a mutation in FKS1 (S629P) and the other in FKS2 (S663P). Of the third pair, both the 5-fluorocytosine-susceptible, and resistant isolates had a mutation in FCY2 (A237T). SNPs in CgPDR1 were found in pan-azole-resistant isolates. SNPs in other genes linked to azole resistance (CgCDR1, ERG9 and CgFLR1) were present in both azole-susceptible and azole-resistant isolates. SNPs were also identified in Candida adhesin genes EPA1, EPA6, PWP2 and PWP5 but their presence was not associated with higher drug MICs. CONCLUSIONS: Genome-wide analysis of antifungal resistance markers was feasible and simultaneously revealed mutation patterns of genes implicated in resistance to different antifungal drug classes.

New developments and directions in the clinical application of the echinocandins.

The echinocandins-caspofungin, anidulafungin and micafungin-are semi-synthetic cyclic hexapeptide antimicrobial agents with modified N-linked acyl lipid side chains which anchor the compounds to the phospholipid bilayer of the fungal cell membrane, thereby inhibiting synthesis of fungal cell wall glucan. Over the last 10 years, echinocandins have become the first-line antifungal treatment of candidaemia and other forms of invasive candidiasis (IC). Echinocandins are generally well tolerated, but their use is limited by their requirement for daily intravenous dosing, lack of oral formulation and limited spectrum. In critically ill patients, it is also recognised that achievement of their pharmacokinetic/pharmacodynamic targets shows large inter-individual variability. As a drug class, they are safe to use and are associated with few adverse reactions and few drug-drug interactions of significance. Recent discovery of their ability to prevent and treat Candida biofilm formation particularly in the presence of invasive medical devices and also their ability to penetrate into mucosal surfaces such as vulvovaginal candidiasis has opened up new opportunities for research into their drug delivery. New dosing intervals are being explored to allow less frequent intravenous dosing in the ambulatory setting, and a new long-acting echinocandin, CD101, is being developed for weekly and topical administration.

Is a biomarker-based diagnostic strategy for invasive aspergillosis cost effective in high-risk haematology patients?

Empirical antifungal therapy is frequently used in hematology patients at high risk of invasive aspergillosis (IA), with substantial cost and toxicity. Biomarkers for IA aim for earlier and more accurate diagnosis and targeted treatment. However, data on the cost-effectiveness of a biomarker-based diagnostic strategy (BDS) are limited. We evaluated the cost effectiveness of BDS using results from a randomized controlled trial (RCT) and individual patient costing data. Data inputs derived from a published RCT were used to construct a decision-analytic model to compare BDS (Aspergillus galactomannan and PCR on blood) with standard diagnostic strategy (SDS) of culture and histology in terms of total costs, length of stay, IA incidence, mortality, and years of life saved. Costs were estimated for each patient using hospital costing data to day 180 and follow-up for survival was modeled to five years using a Gompertz survival model. Treatment costs were determined for 137 adults undergoing allogeneic hematopoietic stem cell transplant or receiving chemotherapy for acute leukemia in four Australian centers (2005-2009). Median total costs at 180 days were similar between groups (US$78,774 for SDS [IQR US$50,808-123,476] and US$81,279 for BDS [IQR US$59,221-123,242], P = .49). All-cause mortality was 14.7% (10/68) for SDS and 10.1% (7/69) for BDS, (P = .573). The costs per life-year saved were US$325,448, US$81,966, and US$3,670 at 180 days, one year and five years, respectively. BDS is not cost-sparing but is cost-effective if a survival benefit is maintained over several years. An individualized institutional approach to diagnostic strategies may maximize utility and cost-effectiveness.

Emerging infections caused by non-Aspergillus filamentous fungi.

There are three broad groups of non-Aspergillus moulds: the mucormycetes, the hyalohyphomycetes and the phaeohyphomycetes. Infections with these pathogens are increasingly reported, particularly in the context of increasing use of immunosuppressant agents and improved diagnostics. The epidemiology of non-Aspergillus mould infections varies with geography, climate and level of immunosuppression. Skin and soft-tissue infections are the predominant presentation in the immunocompetent host and pulmonary and other invasive infections in the immunocompromised host. The more common non-Aspergillus moulds include Rhizopus, Mucor, Fusarium and Scedosporium species; however, other emerging pathogens are Rasamsonia and Verruconis species, which are discussed in this article. Outbreaks of non-Aspergillus mould infections have been increasingly reported, with contaminated medical supplies and natural disasters as common sources. Currently culture and other conventional diagnostic methods are the cornerstone of diagnosis. Molecular methods to directly detect and identify mould pathogens in tissue and body fluids are increasingly used.

Mucormycosis in Australia: contemporary epidemiology and outcomes.

Mucormycosis is the second most common cause of invasive mould infection and causes disease in diverse hosts, including those who are immuno-competent. We conducted a multicentre retrospective study of proven and probable cases of mucormycosis diagnosed between 2004-2012 to determine the epidemiology and outcome determinants in Australia. Seventy-four cases were identified (63 proven, 11 probable). The majority (54.1%) were caused by Rhizopus spp. Patients who sustained trauma were more likely to have non-Rhizopus infections relative to patients without trauma (OR 9.0, p 0.001, 95% CI 2.1-42.8). Haematological malignancy (48.6%), chemotherapy (42.9%), corticosteroids (52.7%), diabetes mellitus (27%) and trauma (22.9%) were the most common co-morbidities or risk factors. Rheumatological/autoimmune disorders occurred in nine (12.1%) instances. Eight (10.8%) cases had no underlying co-morbidity and were more likely to have associated trauma (7/8; 87.5% versus 10/66; 15.2%; p <0.001). Disseminated infection was common (39.2%). Apophysomyces spp. and Saksenaea spp. caused infection in immuno-competent hosts, most frequently associated with trauma and affected sites other than lung and sinuses. The 180-day mortality was 56.7%. The strongest predictors of mortality were rheumatological/autoimmune disorder (OR = 24.0, p 0.038 95% CI 1.2-481.4), haematological malignancy (OR = 7.7, p 0.001, 95% CI 2.3-25.2) and admission to intensive care unit (OR = 4.2, p 0.02, 95% CI 1.3-13.8). Most deaths occurred within one month. Thereafter we observed divergence in survival between the haematological and non-haematological populations (p 0.006). The mortality of mucormycosis remains particularly high in the immuno-compromised host. Underlying rheumatological/autoimmune disorders are a previously under-appreciated risk for infection and poor outcome.

Clinical utility of panfungal polymerase chain reaction for the diagnosis of invasive fungal disease: a single center experience.

The role of panfungal polymerase chain reaction (PCR) assays for diagnosis of invasive fungal disease (IFD) is inadequately defined. We describe the use of an internal transcribed spacer 1 (ITS-1) region-directed panfungal PCR in this context at a tertiary referral transplant center. A retrospective review of patients at Alfred Health, Melbourne, Australia (2009-2014) who had clinical samples referred for panfungal PCR testing was conducted. Baseline patient characteristics, antifungal drug history, fungal culture/histopathology, and radiology results were recorded. For bronchoalveolar lavage (BAL) fluid samples, identification of a fungus other than a Candida spp. was defined as a potential pathogen.Of 138 panfungal PCR tests (108 patients), 41 (30%) were positive for a fungal product. Ninety-seven percent (134/138) of specimens were from immunocompromised hosts. Thirteen percent (19/138) of panfungal PCR positive results were for potential pathogens and potential pathogens were detected more frequently in tissue as compared with BAL (12/13 vs. 6/26; P = .0001). No positive panfungal PCR results were obtained from CSF specimens. If histopathology examination was negative, panfungal PCR identified a potential pathogen in only 12% (11/94) of specimens. For the 20 culture negative/histopathology positive specimens, diagnosis of IFD to causative species level by panfungal PCR occurred in 35% (6/20).Sterile site specimens, in particular tissue, were more frequently panfungal PCR positive for potential pathogens than BAL. The utility of panfungal PCR appears greatest in tissue specimens, as an adjunct to histopathology to improve diagnostic sensitivity and specificity. Based on the results of this study we are now only testing tissue specimens by panfungal PCR.

Microsphaeropsis arundinis skin and soft tissue infection in renal transplant recipients: three case reports and a review of the literature.

Microsphaeropsis arundinis, a dematiaceous mold, is emerging as a cause of skin and soft tissue infection in immunocompromised hosts. Diagnosis is challenging because of the difficulty in identifying Microsphaeropsis species morphologically and few data are available to guide optimal management. We report 3 renal transplant recipients with M. arundinis soft tissue infection, where the etiological agent was diagnosed using DNA sequencing, and who were successfully treated with prolonged courses of extended-spectrum triazole antifungal agents.

Pulmonary Cryptococcosis.

Inhalation of Cryptococcus into the respiratory system is the main route of acquisition of human infection, yet pulmonary cryptococcosis goes mostly unrecognized by many clinicians. This delay in diagnosis, or misdiagnosis, of lung infections is due in part to frequently subtle clinical manifestations such as a subacute or chronic cough, a broad differential of diagnostic possibilities for associated pulmonary masses (cryptococcomas) and, on occasion, negative respiratory tract cultures. Hematogenous dissemination from the lung can result in protean manifestations, the most severe of which is meningoencephalitis. There are few clinical studies of pulmonary cryptococcosis and its pathogenesis is poorly understood. The main purpose of this review is to describe the epidemiology, clinical presentation, diagnosis, and treatment of pulmonary cryptococcosis to increase clinician's awareness of this diagnostic possibility and to enhance clinical management. Useful pointers to the approach and management of pulmonary cryptococcosis and the implications of disseminated disease are included, together with recommendations for future research.

Multicenter study of epidemiological cutoff values and detection of resistance in Candida spp. to anidulafungin, caspofungin, and micafungin using the Sensititre YeastOne colorimetric method.

Neither breakpoints (BPs) nor epidemiological cutoff values (ECVs) have been established for Candida spp. with anidulafungin, caspofungin, and micafungin when using the Sensititre YeastOne (SYO) broth dilution colorimetric method. In addition, reference caspofungin MICs have so far proven to be unreliable. Candida species wild-type (WT) MIC distributions (for microorganisms in a species/drug combination with no detectable phenotypic resistance) were established for 6,007 Candida albicans, 186 C. dubliniensis, 3,188 C. glabrata complex, 119 C. guilliermondii, 493 C. krusei, 205 C. lusitaniae, 3,136 C. parapsilosis complex, and 1,016 C. tropicalis isolates. SYO MIC data gathered from 38 laboratories in Australia, Canada, Europe, Mexico, New Zealand, South Africa, and the United States were pooled to statistically define SYO ECVs. ECVs for anidulafungin, caspofungin, and micafungin encompassing ≥97.5% of the statistically modeled population were, respectively, 0.12, 0.25, and 0.06 µg/ml for C. albicans, 0.12, 0.25, and 0.03 µg/ml for C. glabrata complex, 4, 2, and 4 µg/ml for C. parapsilosis complex, 0.5, 0.25, and 0.06 µg/ml for C. tropicalis, 0.25, 1, and 0.25 µg/ml for C. krusei, 0.25, 1, and 0.12 µg/ml for C. lusitaniae, 4, 2, and 2 µg/ml for C. guilliermondii, and 0.25, 0.25, and 0.12 µg/ml for C. dubliniensis. Species-specific SYO ECVs for anidulafungin, caspofungin, and micafungin correctly classified 72 (88.9%), 74 (91.4%), 76 (93.8%), respectively, of 81 Candida isolates with identified fks mutations. SYO ECVs may aid in detecting non-WT isolates with reduced susceptibility to anidulafungin, micafungin, and especially caspofungin, since testing the susceptibilities of Candida spp. to caspofungin by reference methodologies is not recommended.

Molecular diagnostic methods for invasive fungal disease: the horizon draws nearer?

Rapid, accurate diagnostic laboratory tests are needed to improve clinical outcomes of invasive fungal disease (IFD). Traditional direct microscopy, culture and histological techniques constitute the 'gold standard' against which newer tests are judged. Molecular diagnostic methods, whether broad-range or fungal-specific, have great potential to enhance sensitivity and speed of IFD diagnosis, but have varying specificities. The use of PCR-based assays, DNA sequencing, and other molecular methods including those incorporating proteomic approaches such as matrix-assisted laser desorption ionisation-time of flight mass spectroscopy (MALDI-TOF MS) have shown promising results. These are used mainly to complement conventional methods since they require standardisation before widespread implementation can be recommended. None are incorporated into diagnostic criteria for defining IFD. Commercial assays may assist standardisation. This review provides an update of molecular-based diagnostic approaches applicable to biological specimens and fungal cultures in microbiology laboratories. We focus on the most common pathogens, Candida and Aspergillus, and the mucormycetes. The position of molecular-based approaches in the detection of azole and echinocandin antifungal resistance is also discussed.