Comparison of ß-1-3-D-Glucan and Candida Mannan Biomarker Assays with Serological Tests for the Diagnosis of Candidemia.

Invasive candidiasis, including bloodstream infection (candidemia), encompasses the most severe forms of Candida infection. Several species-specific and non-specific serological assays are commercially available to aid in diagnosis. This study compared the performance of five such biomarker assays. Serum samples from 14 patients with proven or probable invasive candidiasis, and from 10 control patients, were included in the analysis. A total of 50 serum samples were tested using C. albicans germ tube antibody (CAGTA) assay (Vircell), C. albicans IgM, C. albicans IgG and Candida mannan assays (Dynamiker Biotechnology). Among these samples, the ß-1-3-D-glucan (BDG) assay (Fungitell), a laboratory standard for the diagnosis of invasive candidiasis, was positive in 20 (40%), intermediate in five (10%) and negative in 25 (50%). In cases of proven or probable candidemia, the sensitivity and specificity of the BDG assay was 86% and 80%, respectively; the Candida mannan assay, 14% and 86%; the CAGTA test, 57% and 60%; the C. albicans IgM assay, 71% and 60%; and C. albicans IgG assay 29% and 90%. In 4/8 (50%) cases with multiple serum samples, C. albicans IgM was positive sooner than BDG. Thus, when used as a rule-out test for invasive candidiasis, our data suggest that the C. albicans IgM assay may assist antifungal stewardship (over serum BDG).

Improving awareness, diagnosis and management of invasive fungal infections in Ghana: establishment of the Ghana Medical Mycology Society.

Invasive fungal infections (IFIs) and medical mycology receive little attention in Ghana. However, the present evolution of biomarker assays for IFIs, offers an opportunity for an increased access to fungal laboratory testing in resource-limited settings, and probably make a case for availability of essential antifungal agents. Using surveys and personal communications, the state of medical mycology and IFI in Ghana were highlighted. Inadequate awareness and insufficient access to fungal diagnostics and therapeutics were identified as the key challenges, the establishment of the Ghana Medical Mycology Society was discussed, and recommendations were made to improve the status quo.

Invasive fungal infections (IFIs) receive little attention in Ghana, despite its growing relevance globally. Using surveys and personal communications, the main challenges were identified, and the formation of the Ghana Medical Mycology Society was discussed as a tool to improve the status quo.

Meteorological Factors Influence the Presence of Fungi in the Air; A 14-Month Surveillance Study at an Adult Cystic Fibrosis Center.

Background: Cystic fibrosis is an inherited disease that predisposes to progressive lung damage. Cystic fibrosis patients are particularly prone to developing pulmonary infections. Fungal species are commonly isolated in lower airway samples from patients with cystic fibrosis. Fungal spores are prevalent in the air. Methods: We performed environmental air sampling surveillance at the Manchester Adult Cystic Fibrosis Centre, UK (MACFC) over a 14-month period to assess fungal growth inside and outside the CF center. Results: Airborne counts of fungal spores peaked from May to October, both in outdoor and indoor samples. Collection of meteorological data allowed us to correlate fungal presence in the air with elevated temperatures and low wind speeds. Additionally, we demonstrated patient rooms containing windows had elevated fungal counts compared to rooms not directly connected to the outdoors. Conclusions: This study suggests that airborne Aspergillus fumigatus spores were more abundant during the summer months of the survey period, which appeared to be driven by increased temperatures and lower wind speeds. Indoor counts directly correlated to outdoor A. fumigatus levels and were elevated in patient rooms that were directly connected to the outdoor environment via an openable window designed for ventilation purposes. Further studies are required to determine the clinical implications of these findings for cystic fibrosis patients who are predisposed to Aspergillus related diseases, and in particular whether there is seasonal influence on incidence of Aspergillus related conditions and if screening for such complications such be increased during summer months and precautions intensified for those with a known history of Aspergillus related disease.

Differential Proinflammatory Responses to Aspergillus fumigatus by Airway Epithelial Cells In Vitro Are Protease Dependent.

Aspergillus fumigatus is an important human respiratory mould pathogen. In addition to a barrier function, airway epithelium elicits a robust defence against inhaled A. fumigatus by initiating an immune response. The manner by which A. fumigatus initiates this response and the reasons for the immunological heterogeneity with different isolates are unclear. Both direct fungal cell wall-epithelial cell interaction and secretion of soluble proteases have been proposed as possible mechanisms. Our aim was to determine the contribution of fungal proteases to the induction of epithelial IL-6 and IL-8 in response to different A. fumigatus isolates. Airway epithelial cells were exposed to conidia from a low or high protease-producing strain of A. fumigatus, and IL-6 and IL-8 gene expression and protein production were quantified. The role of proteases in cytokine production was further determined using specific protease inhibitors. The proinflammatory cytokine response correlated with conidia germination and hyphal extension. IL-8 induction was significantly reduced in the presence of matrix metalloprotease or cysteine protease inhibitors. With a high protease-producing strain of A. fumigatus, IL-6 release was metalloprotease dependent. Dectin-1 antagonism also inhibited the production of both cytokines. In conclusion, A. fumigatus-secreted proteases mediate a proinflammatory response by airway epithelial cells in a strain-dependent manner.

Molecular Epidemiology of Aspergillus fumigatus in Chronic Pulmonary Aspergillosis Patients.

Molecular fungal genotyping techniques developed and employed for epidemiological studies have understandably concentrated on establishing the genetic diversity of Aspergillus fumigatus in invasive aspergillosis due to its severity, the urgency for treatment, and the need to demonstrate possible sources. Some early studies suggested that these strains were phenotypically, if not genotypically, different from others. However, with improved discrimination and evaluations, incorporating environmental as well as clinical isolates from other Aspergillus conditions (e.g., chronic pulmonary aspergillosis and cystic fibrosis), this premise is no longer upheld. Moreover, with the onset of increased global triazole resistance, there has been a concerted effort to incorporate resistance profiling into genotyping studies and the realisation that the wider population of non-immunocompromised aspergillosis patients are at risk. This review summarises the developments in molecular genotyping studies that incorporate resistance profiling with attention to chronic pulmonary aspergillosis and an example of our UK experience.

Effect of patient immunodeficiencies on the diagnostic performance of serological assays to detect Aspergillus-specific antibodies in chronic pulmonary aspergillosis.

BACKGROUND: Prevalence of chronic pulmonary aspergillosis (CPA) is ~3 million patients worldwide, and detection of Aspergillus-specific antibody is a critical diagnostic component. Some patients with CPA have subtle immune deficits possibly contributing to poor Aspergillus antibody production and false negative results. MATERIALS/METHODS: We analyzed patient data from 167 cases of clinically confirmed CPA previously evaluated by ImmunoCAP Aspergillus-specific IgG EIA, Bordier ELISA and LDBio Aspergillus IgG/IgM ICT lateral flow assay, to identify deficiencies in: mannose binding lectin (MBL), IgG, IgA, IgM, IFN gamma, IL12 or IL17 production, and/or low cell marker counts (CD4, CD19, CD56). We defined patients as 'sero-negative' if ImmunoCAP Aspergillus IgG was consistently and repeatedly negative (<40 mg A/L). 'Sero-positive' was defined as all other CPA cases. RESULTS: We found the rate of false negatives by ImmunoCAP Aspergillus IgG EIA (n = 23) to be more prevalent in patients with immunodeficiency markers, especially multiple defects. MBL deficiency combined with low CD19 cells (p < 0.001), pneumococcal antibody levels (p = 0.043), IgM (p = 0.047) or three combined (p = 0.001-0.018) or all four together (p = 0.018) were significant. The performance LDBio Aspergillus IgG/IgM ICT appears to be relatively unaffected by immunodeficiency (92.7% of ImmunoCap sero-negatives were positive). The Bordier assay performed significantly better than the ImmunoCAP assay (P = 0.0016) for sero-negative CPA cases. CONCLUSIONS: In select cases of CPA, ImmunoCAP EIA yields a false negative result, making serological diagnosis difficult. ImmunoCAP false negatives are more prevalent in patients with multiple immunological defects, who may still be positive with the LDBio Aspergillus ICT or Bordier EIA.

Talaromycosis in a renal transplant recipient returning from South China.

Talaromycosis is a fungal infection endemic in Southeast Asia. We report a case of a renal transplant recipient who developed infection after a trip to South China. She presented with constitutional symptoms and was found to have an FDG-avid lung mass. Histopathology demonstrated small yeast cells and culture grew Talaromyces marneffei. The patient was treated with 2 weeks of liposomal amphotericin B followed by itraconazole. The dose of tacrolimus was significantly reduced because of the interaction with itraconazole. Mycophenolate mofetil was discontinued. After 12 months of treatment, the mass had completely resolved. Talaromycosis has mainly been reported in patients with AIDS and is uncommon among solid organ transplant recipients. The immune response against T. marneffei infection is mediated predominantly by T cells and macrophages. The diagnosis may not be suspected outside of endemic areas. We propose a therapeutic approach in transplant patients by extrapolating the evidence from the HIV literature and following practices applied to other endemic mycoses.

Isavuconazole Therapeutic Drug Monitoring during Long-Term Treatment for Chronic Pulmonary Aspergillosis.

Isavuconazole is the newest triazole antifungal, and it displays a favorable pharmacokinetic and safety profile. Less is known about its long-term use in immunocompetent hosts. We performed a retrospective service evaluation of isavuconazole therapeutic drug monitoring in patients with chronic pulmonary aspergillosis. Adverse events (AEs) and dose adjustments made during routine clinical practice were recorded, and AEs were classified based on Common Terminology Criteria for Adverse Events v5.0. Forty-five patients (mean age, 64 years) had 285 isavuconazole blood drug levels measured (mean level, 4.1 mg/liter). A total of 117 measurements (41%) were performed on patients on a 100-mg daily dose instead of 200 mg, and all had blood levels of >1 mg/liter. Age (P = 0.012) and a daily dose of 200 mg versus 100 mg (P = 0.02) were independent predictors of levels of >6 mg/liter. AEs were recorded for 25 patients (56%). The mean drug level at the first measurement was 5.5 ± 2 mg/liter for patients reporting AEs, compared with 4.2 ± 1.7 mg/liter for those not reporting AEs (P = 0.032). The cutoff threshold best predictive of an AE was 4.6 mg/liter (area under the concentration-time curve, 0.710). Sixteen patients (36%) discontinued isavuconazole therapy due to AEs. Twenty-six patients (58%) continued on isavuconazole beyond 6 months. Asthma (P = 0.022) and a daily dose of 200 mg versus 100 mg (P = 0.048) were associated with AEs of grade 2 or higher. A reduced daily dose (100 mg versus 200 mg) of isavuconazole resulted in satisfactory drug levels in a substantial number of patients; it was better tolerated and enabled continuation of therapy for prolonged periods.

Absence of Azole Antifungal Resistance in Aspergillus fumigatus Isolated from Root Vegetables Harvested from UK Arable and Horticultural Soils.

The emergence of azole-resistant Aspergillus fumigatus (ARAf) complicates the treatment of aspergillosis and can nearly double the mortality from invasive aspergillosis (IA). ARAf has been isolated from many different environmental sites and indoor environments and thus presents a significant risk for susceptible patients. Local surveillance of environmental ARAf can guide antifungal prescribing and improve patient outcomes. In this study, seventy-four soils samples collected from the surface of a variety of root vegetables from farm shops and private gardens covering a wide geographical area of the UK, were cultured to assess the presence of A. fumigatus, and the prevalence and nature of any resistance mechanisms. A high-throughput in-house antifungal susceptibility screening method was developed and validated using the EUCAST MIC reference method, E.DEF 9.3.1. A total of 146 isolates were recovered and analysed. Even though the study premise was that soil-covered root vegetables and other fresh produce could represent a conduit for ARAf exposure in vulnerable patients, no ARAf were found in the soil samples despite 55% of samples harbouring A. fumigatus. The sample type and screening method used could be suitable for more extensive monitoring of the soil to detect trends in the prevalence of ARAf.

Evaluation of the LDBio Aspergillus ICT lateral flow assay for serodiagnosis of allergic bronchopulmonary aspergillosis.

BACKGROUND: Early recognition and diagnosis of allergic bronchopulmonary aspergillosis (ABPA) is critical to improve patient symptoms, and antifungal therapy may prevent or delay progression of bronchiectasis and development of chronic pulmonary aspergillosis. OBJECTIVE: A recently commercialized lateral flow assay (Aspergillus ICT) (LDBio Diagnostics, Lyons, France) detects Aspergillus-specific antibodies in <30 minutes, requiring minimal laboratory equipment. We evaluated this assay for diagnosis of ABPA compared to diseased (asthma and/or bronchiectasis) controls. METHODS: ABPA and control sera collected at the National Aspergillosis Centre (Manchester, UK) and/or from the Manchester Allergy, Respiratory and Thoracic Surgery research biobank were evaluated using the Aspergillus ICT assay. Results were read both visually and digitally (using a lateral flow reader). Serological Aspergillus-specific IgG and IgE, and total IgE titres were measured by ImmunoCAP. RESULTS: For 106 cases of ABPA versus all diseased controls, sensitivity and specificity for the Aspergillus ICT were 90.6% and 87.2%, respectively. Sensitivity for 'proven' ABPA alone (n = 96) was 89.8%, and 94.4% for 'presumed' ABPA (n = 18). 'Asthma only' controls (no bronchiectasis) and 'bronchiectasis controls' exhibited 91.4% and 81.7% specificity, respectively. Comparison of Aspergillus ICT result with Aspergillus-specific IgG and IgE titres showed no evident immunoglobulin isotype bias. Digital measurements displayed no correlation between ImmunoCAP Aspergillus-specific IgE level and ICT test line intensity. CONCLUSIONS: The Aspergillus ICT assay exhibits good sensitivity for ABPA serological screening. It is easy to perform and interpret, using minimal equipment and resources; and provides a valuable simple screening resource to rapidly distinguish more serious respiratory conditions from Aspergillus sensitization alone.

Deciphering Aspergillus fumigatus cyp51A-mediated triazole resistance by pyrosequencing of respiratory specimens.

BACKGROUND: Infections caused by triazole drug-resistant Aspergillus fumigatus are an increasing problem. The sensitivity of standard culture is poor, abrogating susceptibility testing. Early detection of resistance can improve patient outcomes, yet tools for this purpose are limited. OBJECTIVES: To develop and validate a pyrosequencing technique to detect resistance-conferring cyp51A polymorphisms from clinical respiratory specimens and A. fumigatus isolates. METHODS: Method validation was performed by Sanger sequencing and pyrosequencing of 50 A. fumigatus isolates with a spectrum of triazole susceptibility patterns. Then, 326 Aspergillus quantitative PCR (qPCR)-positive respiratory samples collected over a 27 month period (January 2017-March 2019) from 160 patients at the UK National Aspergillosis Centre were assessed by cyp51A pyrosequencing. The Sanger sequencing and pyrosequencing results were compared with those from high-volume culture and standard susceptibility testing. RESULTS: The cyp51A genotypes of the 50 isolates analysed by pyrosequencing and Sanger sequencing matched. Of the 326 Aspergillus qPCR-positive respiratory specimens, 71.2% were reported with no A. fumigatus growth. Of these, 56.9% (132/232) demonstrated a WT cyp51A genotype and 31.5% (73/232) a resistant genotype by pyrosequencing. Pyrosequencing identified the environmental TR34/L98H mutation in 18.7% (61/326) of the samples in contrast to 6.4% (21/326) pan-azole resistance detected by culture. Importantly, pyrosequencing detected resistance earlier than culture in 23.3% of specimens. CONCLUSIONS: The pyrosequencing assay described could detect a wide range of cyp51A polymorphisms associated with triazole resistance, including those not identified by commercial assays. This method allowed prompt recognition of resistance and the selection of appropriate antifungal treatment when culture was negative.

Optimising the cut-off of the Bordier Aspergillus IgG ELISA for the diagnosis of chronic pulmonary aspergillosis.

Aspergillus IgG detection is an essential tool in the diagnosis and treatment of chronic pulmonary aspergillosis (CPA), and is often positive in allergic bronchopulmonary aspergillosis and Aspergillus bronchitis. The Bordier ELISA had an 83.3% sensitivity (identical to ImmunoCap at a cut-off of 40mgA/L) and 97.3% specificity using a cut-off of 0.9 and a diagnostic accuracy of 90.9%.

Pulmonary Aspergillosis in Patients with Suspected Ventilator-associated Pneumonia in UK ICUs.

Rationale:Aspergillus infection in patients with suspected ventilator-associated pneumonia remains uncharacterized because of the absence of a disease definition and limited access to sensitive diagnostic tests.Objectives: To estimate the prevalence and outcomes of Aspergillus infection in adults with suspected ventilator-associated pneumonia.Methods: Two prospective UK studies recruited 360 critically ill adults with new or worsening alveolar shadowing on chest X-ray and clinical/hematological parameters supporting suspected ventilator-associated pneumonia. Stored serum and BAL fluid were available from 194 nonneutropenic patients and underwent mycological testing. Patients were categorized as having probable Aspergillus infection using a definition comprising clinical, radiological, and mycological criteria. Mycological criteria included positive histology or microscopy, positive BAL fluid culture, galactomannan optical index of 1 or more in BAL fluid or 0.5 or more in serum.Measurements and Main Results: Of 194 patients evaluated, 24 met the definition of probable Aspergillus infection, giving an estimated prevalence of 12.4% (95% confidence interval, 8.1-17.8). All 24 patients had positive galactomannan in serum (n = 4), BAL fluid (n = 16), or both (n = 4); three patients cultured Aspergillus sp. in BAL fluid. Patients with probable Aspergillus infection had a significantly longer median duration of critical care stay (25.5 vs. 15.5 d, P = 0.02). ICU mortality was numerically higher in this group, although this was not statistically significant (33.3% vs. 22.8%; P = 0.23).Conclusions: The estimated prevalence for probable Aspergillus infection in this geographically dispersed multicenter UK cohort indicates that this condition should be considered when investigating patients with suspected ventilator-associated pneumonia, including patient groups not previously recognized to be at high risk of aspergillosis.

The antiseptic Miramistin: a review of its comparative in vitro and clinical activity.

Miramistin is a topical antiseptic with broad antimicrobial action, including activity against biofilms and a clinical profile showing good tolerability. Miramistin was developed within a framework of the Soviet Union Cold War Space Program. It is available for clinical use in several prior Soviet bloc countries, but barely known outside of these countries and there is almost no mention of miramistin in the English literature. However, considering emerging antimicrobial resistance, the significant potential of miramistin justifies its re-evaluation for use in other geographical areas and conditions. The review consists of two parts: (i) a review of the existing literature on miramistin in English, Russian and Ukrainian languages; (ii) a summary of most commonly used antiseptics as comparators of miramistin. The oral LD50 was 1200 mg/kg, 1000 mg/kg and 100 g/L in rats, mice and fish, respectively. Based on the results of the review, we suggest possible applications of miramistin and potential benefits over currently used agents. Miramistin offers a novel, low toxicity antiseptic with many potential clinical uses that need better study which could address some of the negative impact of antimicrobial, antiseptic and disinfectant resistance.

Positive Aspergillus PCR as a marker of azole resistance or sub-therapeutic antifungal therapy in patients with chronic pulmonary aspergillosis.

BACKGROUND: Chronic pulmonary aspergillosis (CPA) is a progressive respiratory disease, caused most commonly by A fumigatus, with significant morbidity and mortality. Azole resistance in A fumigatus is a growing concern worldwide, with resistance to itraconazole reported in up to 50% of patients. AIM: The aim of this study was to determine whether a positive Aspergillus PCR (polymerase chain reaction) is a marker of resistance in CPA patients on azole therapy. METHODS: Patients were selected via a consecutive database search for the first 50 CPA patients with a positive Aspergillus PCR from January to September 2016. Data were collected regarding concurrent and subsequent culture results, current therapy and serum antifungal levels. PCR-positive patients not on therapy were included as the control group. RESULTS: Twenty-three patients were on therapy (15 itraconazole, 4 voriconazole and 4 posaconazole). Cycle threshold (Ct) values ranged from 20.8 to 37.9; no significant difference was found between each treatment and the control group (P = .47). In treated patients, concurrent azole-resistant A fumigatus was found in 75% of A fumigatus-positive cultures (6/8). All of the resistant isolates in the itraconazole group showed therapy resistance. Twenty per cent of all itraconazole levels were sub-therapeutic. No significant difference was found in serum itraconazole levels for patients on itraconazole with a positive PCR versus negative PCR (P = .44). CONCLUSION: Positive sputum, Aspergillus-specific PCR can be associated with azole resistance in CPA patients on therapy.

Biotic Environments Supporting the Persistence of Clinically Relevant Mucormycetes.

Clinically relevant members of the Mucorales group can grow and are found in diverse ecological spaces such as soil, dust, water, decomposing vegetation, on and in food, and in hospital environments but are poorly represented in mycobiome studies of outdoor and indoor air. Occasionally, Mucorales are found in water-damaged buildings. This mini review examines a number of specialised biotic environments, including those revealed by natural disasters and theatres of war, that support the growth and persistence of these fungi. However, we are no further forward in understanding exposure pathways or the chronicity of exposure that results in the spectrum of clinical presentations of mucormycosis.

Auxarthron alboluteum related to non-dermatophytic toenail infection in Kurdistan region, Iraq: A case report.

We report a rare case of non-dermatophytic onychomycosis of the big toenail caused by Auxarthron alboluteum in a 63-years old Iraqi female with a history of diabetes. The big toenail showed distal subungual onychomycosis with extensive yellow-white discoloration. Identification of the causative agent was confirmed by morphological and microscopical characteristics in culture and analysis of ITS-rDNA region. To the best of our knowledge, the isolated Auxarthron alboluteum reported here is a new etiologic species of nail infection in Iraq and this is the first case of its kind to be reported in the world.

Trichosporon japonicum Fungemia and Ventricular Assist Device Infection in an Immunocompetent Patient.

Trichosporon species are emerging opportunistic yeasts that cause life-threatening disseminated disease in severely immunocompromised patients. Trichosporon japonicum is a very rare cause of invasive trichosporonosis. We describe a case of Trichosporon japonicum fungemia in an immunocompetent patient with a transcutaneous biventricular assist device.

An introduction to current standards of CARE in invasive fungal disease.

Invasive fungal infections are life-threatening conditions that require rapid diagnosis and optimal management to alleviate their high morbidity and mortality. They are also associated with a high economic burden, owing to prolonged hospitalization, the need for intensive supportive care, and the consumption of costly new antifungal agents. Many standards of care and guidelines have been published by national and international medical societies and organizations over the past 20 years that have embraced new diagnostic technologies and strategies, and new antifungal drugs. Recognizing the ongoing need and debate on the topic of standards for optimal diagnostics and patient care, the Scientific Committee of the Continuing Antifungal Research and Education (CARE) programme devised the scientific agenda for the 10th CARE (CARE X) meeting to review current practice and recommendations. Specialists in haematology, infectious diseases, medical microbiology and medical mycology met in Barcelona in November 2017. The meeting was organized and funded by Gilead Sciences Europe Ltd.