The impact of anti-mould prophylaxis on Aspergillus PCR blood testing for the diagnosis of invasive aspergillosis.

BACKGROUND: The performance of the galactomannan enzyme immunoassay (GM-EIA) is impaired in patients receiving mould-active antifungal therapy. The impact of mould-active antifungal therapy on Aspergillus PCR testing needs to be determined. OBJECTIVES: To determine the influence of anti-mould prophylaxis (AMP) on the performance of PCR blood testing to aid the diagnosis of proven/probable invasive aspergillosis (IA). METHODS: As part of the systematic review and meta-analysis of 22 cohort studies investigating Aspergillus PCR blood testing in 2912 patients at risk of IA, subgroup analysis was performed to determine the impact of AMP on the accuracy of Aspergillus PCR. The incidence of IA was calculated in patients receiving and not receiving AMP. The impact of two different positivity thresholds (requiring either a single PCR positive test result or ≥2 consecutive PCR positive test results) on accuracy was evaluated. Meta-analytical pooling of sensitivity and specificity was performed by logistic mixed-model regression. RESULTS: In total, 1661 (57%) patients received prophylaxis. The incidence of IA was 14.2%, significantly lower in the prophylaxis group (11%-12%) compared with the non-prophylaxis group (18%-19%) (P < 0.001). The use of AMP did not affect sensitivity, but significantly decreased specificity [single PCR positive result threshold: 26% reduction (P = 0.005); ≥2 consecutive PCR positive results threshold: 12% reduction (P = 0.019)]. CONCLUSIONS: Contrary to its influence on GM-EIA, AMP significantly decreases Aspergillus PCR specificity, without affecting sensitivity, possibly as a consequence of AMP limiting the clinical progression of IA and/or leading to false-negative GM-EIA results, preventing the classification of probable IA using the EORTC/MSGERC definitions.

European Confederation of Medical Mycology (ECMM) epidemiological survey on invasive infections due to Fusarium species in Europe.

In order to better understand the epidemiology of fusariosis in Europe, a survey collecting information on the clinical characteristics of the patients infected by Fusarium as well as on the infecting isolates was launched. A total of 76 cases of invasive fusariosis occurring from January 2007 to June 2012 were collected and Fusarium isolates were identified by sequencing the translation elongation factor 1α (TEF) gene. Also, antifungal susceptibility was tested by broth microdilution according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) and the Etest. Disseminated disease was considered proven in 46 cases and probable in 17 cases. Localised infection was seen in 13 cases. Gibberella fujikuroi species complex (SC), including Fusarium verticillioides and F. proliferatum, and F. solani SC were the most frequent aetiology of disseminated and localised infections, respectively. The crude mortality rate was 46 %, the highest associated with F. solani SC (67 %) and F. proliferatum (62.5 %). A wide range of antifungal susceptibilities was observed. Amphotericin B was the most potent antifungal in vitro, and itraconazole the least effective. The azoles exhibited lower minimum inhibitory concentrations (MICs) against F. verticillioides strains, with posaconazole having a slightly better performance, while F. solani SC isolates were resistant to all three azoles tested. The essential agreement between the Etest and the EUCAST method was 100 % for itraconazole and voriconazole, and 96 % for amphotericin B and posaconazole. In conclusion, we confirm that fusariosis is a rare but severe event in Europe, that G. fujikuroi SC is the predominant cause of deep infections and that different species have different antifungal in vitro susceptibility patterns.

Environmental and bioclimatic factors influencing yeasts and molds distribution along European shores.

The present study employed data collected during the Mycosands survey to investigate the environmental factors influencing yeasts and molds distribution along European shores applying a species distribution modelling approach. Occurrence data were compared to climatic datasets (temperature, precipitation, and solar radiation), soil datasets (chemical and physical properties), and water datasets (temperature, salinity, and chlorophyll-a concentration) downloaded from web databases. Analyses were performed by MaxEnt software. Results suggested a different probability of distribution of yeasts and molds along European shores. Yeasts seem to tolerate low temperatures better during winter than molds and this reflects a higher suitability for the Northern European coasts. This difference is more evident considering suitability in waters. Both distributions of molds and yeasts are influenced by basic soil pH, probably because acidic soils are more favorable to bacterial growth. Soils with high nitrogen concentrations are not suitable for fungal growth, which, in contrast, are optimal for plant growth, favored by this environment. Finally, molds show affinity with soil rich in nickel and yeasts with soils rich in cadmium resulting in a distribution mainly at the mouths of European rivers or lagoons, where these metals accumulate in river sediments.

Comparison of the two blood culture systems, Bactec 9240 and BacT/Alert 3D, in the detection of Candida spp. and bacteria with polymicrobial sepsis.

The purpose of this investigation was to evaluate the performance of the Bactec 9240 and BacT/Alert 3D blood culture systems in the detection of Candida spp. and bacteria in simulated polymicrobial sepsis models. A total of 28 clinical isolates of Escherichia coli, Staphylococcus aureus, Candida albicans, and Candida glabrata were studied. Five polymicrobial models of C. albicans + S. aureus, C. albicans + E. coli, C. glabrata + S. aureus, C. glabrata + E. coli, and C. albicans + C. glabrata were prepared. Each combination was inoculated in five different blood culture vials. The two systems were compared for culture positivity and time to detection (TTD). Twenty-four mixed cultures with a yeast and a bacteria were tested. Bactec Mycosis vials could detect yeasts in all 24 cultures. The aerobic vials from both Bactec and BacT/Alert could detect both yeasts and bacteria in 22/24 (91.66 %) cultures. Bactec Plus Anaerobic/F and BacT/Alert FN vials could detect both microorganisms in 19/24 (79.16 %) and 4/24 (16.67 %) vials, respectively. Seven polymicrobial sepsis models with C. albicans + C. glabrata were also tested. Mycosis vials could detect both yeasts in 7/7 mixed cultures. The aerobic vials from Bactec and BacT/Alert could detect both yeasts in 3/7 and 2/7 mixed cultures, respectively. Bactec Plus Aerobic/F had a shorter TTD compared to BacT/Alert FA and Bactec Plus Anaerobic/F vials (p < 0.0001 and p < 0.01, respectively). The present study shows that the Bactec and BacT/Alert systems have different characteristics in the detection of yeasts and bacteria with polymicrobial sepsis.

Increased mortality in young candidemia patients associated with presence of a Candida albicans general-purpose genotype.

The yeast Candida albicans causes life-threatening candidemia. A general-purpose genotype (GPG; corresponds to clade 1) causes more infections than other C. albicans genotypes. To investigate if GPG strains also cause higher mortality, we developed a duplex PCR assay which was 98% accurate in identifying GPG strains in an international collection of strains typed with probe Ca3. We applied the assay to 635 European C. albicans candidemia isolates. Of these, 18% conformed to the GPG genotype, 4% were of a borderline genotype, and 78% were of a non-GPG genotype, broadly consistent with genotype distributions in earlier studies. The prevalence of GPG strains was increased in females and in younger patients, exceeding 40% in infants aged ≤1 year. Logistic regression confirmed sex and age as significant determinants of GPG prevalence. Across the entire patient cohort, there was no difference in mortality for patients infected with GPG strains or other strains (36% versus 37%). However, mortality in patients aged ≤48 years was 33% for infection with GPG strains but only 15% for infection with other strains (z test; P < 0.01). Mortality rates associated with GPG and non-GPG strains were comparable in older patients (39% versus 46%). A logistic regression analysis confirmed the age-dependent impact of genotype on mortality. Thus, GPG strains may be more virulent than other strains in younger patients. Because candidemia is usually caused by endogenous strains, our PCR assay could potentially be used as a risk assessment tool for identifying younger patients most at risk of death from candidemia.

Mycosands: Fungal diversity and abundance in beach sand and recreational waters - Relevance to human health.

The goal of most studies published on sand contaminants is to gather and discuss knowledge to avoid faecal contamination of water by run-offs and tide-retractions. Other life forms in the sand, however, are seldom studied but always pointed out as relevant. The Mycosands initiative was created to generate data on fungi in beach sands and waters, of both coastal and freshwater inland bathing sites. A team of medical mycologists and water quality specialists explored the sand culturable mycobiota of 91 bathing sites, and water of 67 of these, spanning from the Atlantic to the Eastern Mediterranean coasts, including the Italian lakes and the Adriatic, Baltic, and Black Seas. Sydney (Australia) was also included in the study. Thirteen countries took part in the initiative. The present study considered several fungal parameters (all fungi, several species of the genus Aspergillus and Candida and the genera themselves, plus other yeasts, allergenic fungi, dematiaceous fungi and dermatophytes). The study considered four variables that the team expected would influence the results of the analytical parameters, such as coast or inland location, urban and non-urban sites, period of the year, geographical proximity and type of sediment. The genera most frequently found were Aspergillus spp., Candida spp., Fusarium spp. and Cryptococcus spp. both in sand and in water. A site-blind median was found to be 89 Colony-Forming Units (CFU) of fungi per gram of sand in coastal and inland freshwaters, with variability between 0 and 6400 CFU/g. For freshwater sites, that number was 201.7 CFU/g (0, 6400 CFU/g (p = 0.01)) and for coastal sites was 76.7 CFU/g (0, 3497.5 CFU/g). For coastal waters and all waters, the median was 0 CFU/ml (0, 1592 CFU/ml) and for freshwaters 6.7 (0, 310.0) CFU/ml (p < 0.001). The results advocate that beaches should be monitored for fungi for safer use and better management.

A prospective international Aspergillus terreus survey: an EFISG, ISHAM and ECMM joint study.

OBJECTIVES: A prospective international multicentre surveillance study was conducted to investigate the prevalence and amphotericin B susceptibility of Aspergillus terreus species complex infections. METHODS: A total of 370 cases from 21 countries were evaluated. RESULTS: The overall prevalence of A. terreus species complex among the investigated patients with mould-positive cultures was 5.2% (370/7116). Amphotericin B MICs ranged from 0.125 to 32 mg/L, (median 8 mg/L). CONCLUSIONS: Aspergillus terreus species complex infections cause a wide spectrum of aspergillosis and the majority of cryptic species display high amphotericin B MICs.

Molecular detection and identification of Candida and Aspergillus spp. from clinical samples using real-time PCR.

This report describes the development of a real-time LightCycler assay for the detection and identification of Candida and Aspergillus spp., using the MagNa Pure LC Instrument for automated extraction of fungal DNA. The assay takes 5-6 h to perform. The oligonucleotide primers and probes used for species identification were derived from the DNA sequences of the 18S rRNA genes of various fungal pathogens. All samples were screened for Aspergillus and Candida to the genus level in the real-time PCR assay. If a sample was Candida-positive, typing to species level was performed using five species-specific probes. The assay detected and identified most of the clinically relevant Aspergillus and Candida spp. with a sensitivity of 2 CFU/mL blood. Amplification was 100% specific for all Aspergillus and Candida spp. tested. To assess clinical applicability, 1,650 consecutive samples (1,330 blood samples, 295 samples from other body fluids and 25 biopsy samples) from patients with suspected invasive fungal infections were analysed. In total, 114 (6.9%) samples were PCR-positive, 5.3% for Candida and 1.7% for Aspergillus spp. In patients with positive PCR results for Candida and Aspergillus, verification with conventional methods was possible in 83% and 50% of cases, respectively. In conclusion, the real-time PCR assay allows sensitive and specific detection and identification of fungal pathogens in vitro and in vivo.

Invasive Candida infections in surgical patients in intensive care units: a prospective, multicentre survey initiated by the European Confederation of Medical Mycology (ECMM) (2006-2008).

A prospective, observational, multicentre study of invasive candidosis (IC) in surgical patients in intensive care units (ICUs) was conducted from 2006 to 2008 in 72 ICUs in 14 European countries. A total of 779 patients (62.5% males, median age 63 years) with IC were included. The median rate of candidaemia was 9 per 1000 admissions. In 10.8% the infection was already present at the time of admission to ICU. Candida albicans accounted for 54% of the isolates, followed by Candida parapsilosis 18.5%, Candida glabrata 13.8%, Candida tropicalis 6%, Candida krusei 2.5%, and other species 5.3%. Infections due to C. krusei (57.9%) and C. glabrata (43.6%) had the highest crude mortality rate. The most common preceding surgery was abdominal (51.5%), followed by thoracic (20%) and neurosurgery (8.2%). Candida glabrata was more often isolated after abdominal surgery in patients ≥60 years, and C. parapsilosis was more often isolated in neurosurgery and multiple trauma patients as well as children ≤1 year of age. The most common first-line treatment was fluconazole (60%), followed by caspofungin (18.7%), liposomal amphotericin B (13%), voriconazole (4.8%) and other drugs (3.5%). Mortality in surgical patients with IC in ICU was 38.8%. Multivariate analysis showed that factors independently associated with mortality were: patient age ≥60 years (hazard ratio (HR) 1.9, p 0.001), central venous catheter (HR 1.8, p 0.05), corticosteroids (HR 1.5, p 0.03), not receiving systemic antifungal treatment for IC (HR 2.8, p <0.0001), and not removing intravascular lines (HR 1.6, p 0.02).

Deep Candida infection in children receiving allogeneic bone marrow transplants: incidence, risk factors and diagnosis.

Fifty-eight children, who received 60 allogeneic bone marrow transplants (BMT), were studied with regard to incidence, risk factors and diagnosis of deep Candida infection (DCI). Serum samples were analysed for the presence of Candida IgA, IgM and IgG antibodies and free C. albicans glucoprotein antigen (Ag). Five children (8.7%) had a confirmed DCI and died before engraftment of the new bone marrow. When four patients with suspected deep Candida infection (SDCI) were included, the incidence was 15.6%. Four of the five children (80%) with DCI had pathological Candida IgM antibody (Ab) titers and/or free C. albicans glucoprotein Ag, 2-50 days before DCI was verified by culture, direct microscopy and/or autopsy. Risk factors, using Fisher's exact test for DCI, included not receiving bone marrow from an HLA-identical sibling donor, having a seropositive Herpes simplex virus (HSV) donor and pathological IgA and/or IgM Ab titers against Candida before BMT. In conclusion, a child with the above-mentioned risk factors, runs a risk of acquiring fatal DCI before engraftment. The institution of systemic antifungal prophylactic treatment may prevent death from DCI. After BMT, serological examinations may be of value in the early detection of DCI.

Identification of rare Candida species and other yeasts by polymerase chain reaction and slot blot hybridization.

Invasive candidiasis has become a major cause of morbidity and mortality in immunocompromised hosts. Here we describe a fast and reliable DNA extraction and PCR amplification method in combination with a slot blot hybridization assay. A genus-specific probe was designed that allowed to detect DNA from a broad range of Candida species and 3 other yeasts. In addition, species-specific oligonucleotides for emerging Candida and other yeast species allowed to identify DNA extracted from Candida lusitaniae, Candida humicola, Candida kefyr, Candida inconspicua, Candida solani, Malassezia furfur and Trichosporon cutaneum. A sensitivity of at least 10(1) CFU, corresponding to 100 fg of fungal DNA, was documented for all species-specific probes and the common Candida probe. In addition, the 18S rRNA genes of 7 yeast species (C. humicola, C. kefyr, C. solani, C. inconspicua, C. norvegensis, C. utilis and M. furfur) were completely sequenced. The sequencing primers described bind to highly conserved primer binding sites. Therefore, these primers would allow rapid cycle sequence of additional ribosomal genes throughout the whole kingdom of fungi.

Liposomal amphotericin B (AmBisome) for fungal infections in immunocompromised adults and children.

Invasive fungal infections are rare but life-threatening infections, most often occurring in immunocompromised patients. For a long time, Amphotericin B has been the best choice for treatment, because it is fungicidal with a broad antifungal spectrum and minimal risk of resistance development. The therapeutic use of amphotericin B has, however, been limited by its toxicity-both acute as well as chronic. To counter this, amphotericin B has been encapsulated in liposomes, which reduces its toxicity and allows higher doses to be given. Ambisome is a true, spherical, small unilamellar liposome with a median size of 80 nm. The pharmacokinetic profile was changed, and the maximum concentration and AUC of amphotericin B after AmBisome treatment were greater than those found with the conventional drug. The highest tissue concentrations of AmBisome were found in the liver and spleen, and less than 1% of the administered dose was recovered in other organs. At Huddinge University Hospital, we were the first to use and report on the experience of AmBisome. We now have more than 12 years' experience in transplant recipients, with a good safety profile, improved rate of curing mycological proven infections and reduced mortality in fungal infections. In two placebo-controlled prophylactic trials, we found that AmBisome was effective for preventing fungal colonization and invasive fungal infections, respectively, in allogeneic stem cell and liver transplantation. In uncontrolled and, more recently, in randomized controlled studies at other centers, AmBisome has revealed less toxicity and an efficacy equal or superior to that of the conventional drug in treating neutropenia-associated fever and proven invasive fungal infections in both adults as well as in children. Although investigators tend to increase the dose used, the optimal dose for probable or proven infection is still under debate. Based on our own experience in using AmBisome and the experience at other centers, we can conclude that AmBisome represents a major breakthrough in the treatment of invasive fungal infections, especially in immunocompromised patients.

Candidaemia in Sweden: a nationwide prospective observational survey.

A prospective observational nationwide investigation was performed from September 2005 to August 2006 to study the epidemiology of candidaemia in Sweden. From 385 patients, 403 isolates were recovered, yielding an incidence of 4.2 cases per 100 000 inhabitants. Candida albicans was the most common species (61%), followed by Candida glabrata (20%) and Candida parapsilosis (9%). The rates of resistance to fluconazole were ≤ 1% in C. albicans and 6-29% in non-albicans species other than C. glabrata and Candida krusei. Resistance to voriconazole was rare, except for C. glabrata and C. krusei. Only three isolates had reduced susceptibility to amphotericin B, and one had reduced susceptibility to caspofungin.

Randomized PCR-based therapy and risk factors for invasive fungal infection following reduced-intensity conditioning and hematopoietic SCT.

Invasive fungal infections (IFIs) are major complications after allogeneic hematopoietic SCT (HSCT). PCR-based assays able to detect fungal DNA have been reported to precede clinical diagnosis of IFI. We performed a prospective study to evaluate a PCR-based pre-emptive approach. Ninety-nine patients undergoing reduced-intensity conditioning (RIC) HSCT were followed with fungal PCR during the first 100 days post transplantation. Patients who tested positive were randomized to receive liposomal amphotericin B, or to no intervention. After day 100, PCR tests were performed only on clinical suspicion of IFI. A single positive PCR test was not associated with IFI, irrespective of treatment. After day 100, PCR tests for Aspergillus did not contribute to diagnosis of invasive aspergillosis (IA). The cumulative incidence rates of proven or probable IA during the first year after transplantation were 9%. GVHD grades II-IV (P=0.0014), CMV-seronegative recipient with CMV-seropositive donor (P0.001), and conditioning with alemtuzumab (P=0.014) were significant risk factors for developing IA in a multivariate model. In this study, PCR on peripheral blood was a poor indicator of IFI early after RIC HSCT. Aspergillus PCR tests performed on clinical suspicion after day 100 were insufficiently sensitive to be diagnostically useful.

A prospective randomized controlled trial comparing PCR-based and empirical treatment with liposomal amphotericin B in patients after allo-SCT.

We compared the efficacy and safety of empirical plus PCR-based vs empirical liposomal amphotericin B treatment after Allo-SCT. Allo-SCT recipients were randomized to receive either PCR-based preemptive therapy (group A; n=198) or empirical antifungal therapy (group B; n=211) with liposomal amphotericin B. In group A, therapy was started after one positive PCR result or after 120 h of febrile neutropenia refractory to broad-spectrum antibacterial therapy. In group B, liposomal amphotericin B was started after 120 h of refractory febrile neutropenia. Demographic and clinical characteristics were well balanced. A total of 112 (57.1%) patients in group A and 76 (36.7%) patients in group B received antifungal therapy (P<0.0001). Twelve patients in group A and 16 patients in group B developed proven invasive fungal infection (IFI). Survival curves showed better survival until day 30 when close PCR monitoring was performed (mortality 1.5 vs 6.3%; P=0.015), but there was no difference at day 100. At day 100, no difference was observed in the incidence of IFI (primary end point) and survival between the two arms. Further studies are required to assess the benefit of using PCR in patients after SCT.

Four cases of Candida albicans infections with isolates developing pink colonies on CHROMagar Candida plates.

Candida albicans, the most commonly isolated yeast species, is typically identified by its green colony-colour on CHROMagar Candida plates. We here report four cases of Candida albicans infections, in which the initial identification was non-albicans isolates due to a clear pink colour of the colonies on CHROMagar Candida plates. However, classical phenotypic criteria, biochemical assimilation pattern and molecular characterisation identified all four isolates as C. albicans isolates.

Deep Candida infection in child liver transplant recipients: serological diagnosis and incidence.

Nineteen children who received 22 orthotopic liver grafts on 20 occasions were studied with regard to Candida infection. Serum samples were analysed to determine Candida, IgA, IgM and IgG antibodies and detect free C. albicans glucoprotein antigen. Five children (25%) had a confirmed deep C. albicans infection (DCI) during the first 2 weeks after transplantation. In all children with DCI, serology was positive, a median of 6 days (range 2-9 days) before Candida infection was verified by fungal culture, direct microscopy and/or autopsy. The positive predictive values for Candida IgG, IgM and IgA antibodies in children with DCI were 100%, 78% and 100%, respectively, and for free C. albicans antigen, 45%. Pathological titres of IgM and IgA antibodies against Candida before liver transplantation were present in three of four children who later developed a DCI and in no child without infection. In conclusion, regular screening by Candida serology is recommended both before and after liver transplantation.

Deep Candida infection in children with leukaemia: clinical presentations, diagnosis and outcome.

OBJECTIVES: To analyse the clinical features associated with deep Candida infection (DCI) and the outcome in children with leukaemia, and to evaluate various diagnostic methods. MATERIALS AND METHODS: Serum samples were analysed to determine Candida IgA, IgM and IgG antibodies and defect free C. albicans glucoprotein antigen and C. enolase antigen in eight children who had nine episodes of DCI and six with suspected DCI. RESULTS: DCI occurred shortly after the leukaemia diagnosis (median 40 days) or after the leukaemia relapse (median 30 days). Children with DCI had fever (100%), skin lesions/exanthema (45%), oral thrush (45%), oesophagitis (22%) and laryngo-tracheitis (22%). Candida endocarditis, arthritis and hepatic candidosis were diagnosed in one patient each. Two children with disseminated candidosis died in leukaemia relapse. In patients with C. albicans infections serology had a sensitivity of 83%. However, in patients with C. parapsilosis infection antibody detection was negative. As the patients were cured of their Candida infection, the IgG antibodies disappeared and the IgM and IgA antibodies fell within the normal range for age. CONCLUSION: DCI in children occurs shortly after the leukaemia diagnosis or shortly after relapse of leukaemia. The clinical features are many. Candida serology may help to diagnose or confirm DCI. The dynamics of antibody titres may help to establish the efficacy of antifungal treatment.

Chronic non-specific diarrhea of infancy successfully treated with trimethoprim-sulfamethoxazole.

Fifteen children who fulfilled the criteria of chronic non-specific diarrhea of infancy were evaluated for intestinal bacterial overgrowth. In 10 of 11 successfully investigated children we found bacterial overgrowth of the small intestine by upper respiratory tract microflora. In 9 of 10 children (group I) treated with trimethoprim-sulfamethoxazole the diarrhea ceased immediately, whereas in all children in group II (n = 5; 3 children excluded because of growth of Yersinia enterocolitica) treated with low-lactose diet the diarrhea persisted (p = 0.004). The results indicate that bacterial overgrowth of the small intestine by upper respiratory tract microflora may be a cause of chronic non-specific diarrhea and that this diarrhea may be successfully treated with trimethoprim-sulfamethoxazole.

Antibody response to Candida and its use in clinical practice.

Reference values for specific IgM, IgA and IgG antibodies against three defined Candida antigens were determined in 280 healthy Swedish children aged 1 month to 15 years. The antibody response in 10 children with Candida infections was also determined. Precipitating IgG antibodies to a mannan-free Candida protein antigen were detected only in children with Candida infection. The haemagglutinating IgM antibody response in healthy children to a polysaccharide Candida antigen was weak. Adult levels of < or = 320 (95% confidence interval, CI) were not reached even in the oldest children (< or = 160). All children with Candida infections had elevated IgM titres. Adult levels of IgA antibodies to Candida mannan were reached at 10-12 years of age; at 13-15 years the titres were higher (< or = 1600) than in healthy adults (< or = 1000, 95% CI). All children with an acute Candida infection had elevated IgA titres. Our study shows that antibody testing may be used to diagnose systemic Candida infection in children and to follow the progression and resolution of systemic Candida infection with the rise and fall of antibody titres.