Community-acquired invasive aspergillosis and outdoor filamentous fungal spore load: a relationship?

The daily number of outdoor spores was counted and the cases of community-acquired invasive aspergillosis (IA) were observed over a period of 31 months. The outdoor fungal load preceding IA occurrences was significantly higher than that measured during IA-free periods, underlining the importance of preventive measures to protect high-risk patients, even at home.

Development of an innovative method for the evaluation of fungal contamination of surfaces.

The objective of this technical report is to compare the ability to capture fungal spores through samples performed with three different methods: Rodac contact plates, cotton pad and a pad prepared with a dusting cloth (DC pads) selected from those available on the market. The tests were conducted using a suspension of Aspergillus niger conidia equal to 0.5 MacFarland diluted 1/30, 1/40, 1/50, 1/100. With each of these dilutions 3 sterile tiles of stainless steel were contaminated, each divided into 16 small squares, in the center of which 0.1 ml of the dilution chosen was placed and left to dry (for a total of 12 sheets). In addition, we have used other 6 tiles to repeat the experience with dilutions 1/40 and 1/50. A total of 288 squared surfaces were contaminated: 96 of these were sampled with Rodac contact plates, 96 with cotton pads and 96 with DC and then inseminated in Petri plates. Sabouraud dextrose agar was used as culture medium for the first 12 plates, while, for the other 6 plates Sabouraud dextrose agar added with lecithin and polysorbate 80 was used. All plates were incubated at 37 degrees for 18 hours. To estimate the differences among the sampling methods and the dilutions tested, multiple linear regression was used. The analysis showed that the number of colonies harvested at dilution 1/40 is 13% higher (P = 0.09) than the number harvested at dilution 1/50 and the number of colonies harvested at dilution 1/30 is 6% higher than dilution 1/50 (P = 0.52). With regard to the comparison between the number of colonies harvested with Rodac contact plates, with cotton pads and DC pads, regression analysis shows that cotton pads harvest a number of fungal cfu 5 times higher than those detected with Rodac plates, while DC pads harvest a number of fungal ufc 6 times higher than those detected with Rodac plates (P < 0.00005). These results, although preliminary, indicate that DC pads are a sensitive and simple approach for the environmental control of fungal contamination.

Multicenter evaluation of a commercial PCR-enzyme-linked immunosorbent assay diagnostic kit (Onychodiag) for diagnosis of dermatophytic onychomycosis.

We prospectively evaluated a new PCR-enzyme-linked immunosorbent assay kit (Onychodiag; BioAdvance, France) for the diagnosis of dermatophytic onychomycosis by testing nail samples from 438 patients with suspected onychomycosis and from 108 healthy controls in three independent laboratories. In two laboratories, samples were collected by trained mycologists as close as possible to the lesions (proximal samples). In one laboratory, samples were collected by other physicians. All samples were processed by conventional mycological techniques and by Onychodiag, blindly to the mycological results. An additional distal sample, collected by clipping the nail plate, was obtained from 75 patients and tested with Onychodiag alone. In patients with culture-proven dermatophytic onychomycosis, the sensitivity of Onychodiag was 83.6% (87.9% including the gray zone) and ranged from 75 to 100% according to the laboratory and the sampling conditions. The specificity was 100% when healthy subjects were considered true negative controls. Onychodiag was positive on 68 patient samples that were sterile or yielded nondermatophyte species in culture. Based on the results of Onychodiag for mycologically proven positive samples and true-negative samples, these results were considered true positives, and the poor performance of mycology on these samples was attributed to inconvenient sampling conditions or to contaminants. When tested on distal samples, Onychodiag was positive in 49/53 (92%) cases of proven dermatophytic onychomycosis. Finally, with either proximal or distal samples, Onychodiag provided a diagnosis of dermatophytic onychomycosis within 24 to 48 h after sampling, and its sensitivity was close to that of mycological techniques applied to proximal samples.

Ten-year surveillance of fungal contamination of food within a protected haematological unit.

Since 1992, we have established a protocol of food management (restrictive diet, food distribution protocol and fungal surveillance) for allogeneic stem-cell transplant (SCT) recipients hospitalised in protected ward. This study analyses the results of 10-year surveillance of fungal contamination of this diet. Among the 456 types of foods tested filamentous fungi were isolated in 37 of them (8.1%). Aspergillus fumigatus was isolated in one type of food only, while the majority of the food was contaminated to a lower extent.

Hospital-wide prospective mandatory surveillance of invasive aspergillosis in a French teaching hospital (2000-2002).

A multidisciplinary working group devoted to epidemiological surveillance of invasive aspergillosis (IA) was created in January 2000 in Grenoble University Hospital. This article presents the results of a three-year IA surveillance. The multidisciplinary working group surveyed all hospitalized patients, and the mycology laboratory detected most suspected IA cases. Cases were reviewed monthly by the Aspergillosis Committee, and were classified according to international consensus criteria. Possible nosocomial acquisition was determined. Among the 490 alerts, 74 IA cases were observed: six proven cases (8%), 36 (49%) probable cases and 32 (43%) possible cases. The incidence was 4.4 (95% CI 3.4-5.4) IA/100 000 patient-days. Among the proven and probable IA cases, we observed 10 nosocomial cases and six cases of undetermined origin. No cases were noted in the protected rooms in the haematology unit. Only one cluster of cases (three nosocomial cases) was detected in the haematology unit. Forty-three percent of cases (N=32) were hospitalized in the haematology unit, and all other cases were hospitalized elsewhere. This three-year survey found a high rate of non-nosocomial IA cases and a high frequency of IA cases hospitalized in units other than haematology. Thus, this study shows the importance of IA surveillance in haematology units and all high-risk units.

Performance of the Now Malaria rapid diagnostic test with returned travellers: a 2-year retrospective study in a French teaching hospital.

Malaria caused by Plasmodium falciparum remains the major life-threatening parasitic infection in the world. The number of cases in non-endemic countries continues to increase, and it is important that misdiagnosis of malaria should not occur, especially in non-immune travellers, because of the high risk of a fatal outcome. In a retrospective study of 399 sera, the Now Malaria rapid test was compared with the quantitative buffy coat (QBC) test and microbiological examination of thin blood films. Compared with the QBC test and thin blood films, the Now Malaria test had sensitivity and specificity values of 96.4% and 97%, respectively, for the detection of pure P. falciparum infection. A negative predictive value of 99.4% allows this test to be included in diagnostic strategies for patients presenting with clinical suspicion of malaria. Two false-negative results were associated with low levels of parasitaemia in the specimens. Thus, use of the Now Malaria test alone to detect P. falciparum infection in non-endemic countries could lead to misdiagnosis of malaria. This rapid diagnostic test should therefore be performed in association with another prompt traditional method such as examination of thin blood films.

Syntheses, in vitro antibacterial and antifungal activities of a series of N-alkyl, 1,4-dithiines.

A series of dithiines were synthesized by cyclization of 4-(alkylamino)-4-oxobutanoic acids under the action of SOCl2. Their in vitro antibacterial and antifungal activities have been evaluated against reference strains and versus reference compounds. The so-called 'isoimides' 2a, 2b were totally inactive whereas some imides had low MICs for few bacteria and for few fungal microorganisms.

In vitro susceptibility testing of Candida and Aspergillus spp. to voriconazole and other antifungal agents using Etest: results of a French multicentre study.

Minimum inhibitory concentrations (MICs) of the antifungal agent voriconazole were determined using the Etest and compared with those of amphotericin B, itraconazole and fluconazole using 1986 clinical isolates of Candida spp. Voriconazole MICs were also compared with those of amphotericin B and itraconazole using 391 clinical isolates of Aspergillus spp. Voriconazole was found to have more potent activity and lower MIC values than amphotericin B, itraconazole and fluconazole against C. albicans, C. tropicalis, C. parapsilosis and C. kefyr. Against C. glabrata and C. krusei, voriconazole was more active than either of the other two azole antifungals but had similar activity to amphotericin B. For species of Aspergillus, MIC values of voriconazole were lower than those of amphotericin B and itraconazole against A. fumigatus and A. flavus, and were similar to those of amphotericin B against A. niger. Against A. terreus, MIC values for voriconazole and itraconazole were similar. A. terreus is known to be resistant to amphotericin B, and this was reflected in higher MIC values compared with those of voriconazole and itraconazole. Voriconazole therefore compares very favourably with other antifungal agents against a large number of clinical isolates of Candida and Aspergillus spp.

Epidemiology of candidaemia in Europe: results of 28-month European Confederation of Medical Mycology (ECMM) hospital-based surveillance study.

In order to update the epidemiological and mycological profile of candidaemia in Europe, the European Confederation of Medical Mycology conducted a prospective, sequential, hospital population-based study from September 1997 to December 1999. A total of 2,089 cases were documented by 106 institutions in seven European countries. Rates of candidaemia ranging from 0.20 to 0.38 per 1,000 admissions were reported. Candida albicans was identified in 56% of cases. Non-albicans Candida species were most frequently isolated from patients with haematological malignancies (65%). With increasing age, an increasing incidence of Candida glabrata was seen. The 30-day mortality rate was 37.9%. The survey results underline the burden of candidaemia in a wide range of patient populations, confirm the importance of non- albicans species, and provide baseline data for future surveillance studies at a European level.

In vitro susceptibility of Aspergillus spp. clinical isolates to albendazole.

The in vitro antifungal activity of albendazole, a benzimidazole widely used as an antihelmintic drug in humans, was investigated and assessed for its activity against Aspergillus spp. Forty-eight isolates, representing the most frequent species found in human pathology [Aspergillus fumigatus (n = 27), Aspergillus flavus (n = 10), Aspergillus terreus (n = 7), Aspergillus nidulans (n = 3) and Aspergillus niger (n = 1)], and one quality control strain (A. niger ATCC 9804 83435) were tested according to the NCCLS M38-P methodology for moulds. All the strains were susceptible to albendazole, with homogeneous MICs for each species; three strains were resistant to itraconazole.

Hospital-acquired Aspergillus fumigatus infection: can molecular typing methods identify an environmental source?

Aspergillus fumigatus infection in hospitalized immunocompromised patients often raises suspicion regarding the potential for hospital acquisition. Hospital staff have an important responsibility in implementing preventive measures, especially since the advent of current legislation concerning hospital-acquired infections. There have been high expectations that molecular typing methods might determine the source of Aspergillus fumigatus, a ubiquitous mould. The aim of the present epidemiological study, was therefore, to identify the origin(s) of Aspergillus infection in six well-documented patients. All the clinical strains (N=33), and those from hospital (N=14) and home environments (N=34) were isolated according to a standardized protocol and typed by sequence-specific DNA primer analysis. The results confirmed the huge biodiversity of the A. fumigatus population, and consequently the difficulty in ascertaining a hospital source of the infection, as opposed to infections due to other Aspergillus species less frequently encountered.

Effect of medium composition on static and cidal activity of amphotericin B, itraconazole, voriconazole, posaconazole and terbinafine against Aspergillus fumigatus: a multicenter study.

The effect of the medium composition on the fungistatic (MIC) and fungicidal (MLC) activity of amphotericin B, itraconazole, voriconazole, posaconazole and terbinafine against four Aspergillus fumigatus strains has been investigated by four European laboratories. MICs were determined by broth microdilution, using RPMI 1640 and Antibiotic Medium 3 (AM3), three times in three independent determinations by the four laboratories. MLCs were determined for the three independent determinations by the four laboratories, subculturing 100 microl from each well showing no visible growth after 48 hours. Except for a 2-dilution difference observed in three cases, no differences were observed between MICs determined on the two media. In contrast, a 3- to 6-dilution discrepancy between the MLCs was observed for the azoles. Endpoints on RPMI were higher than those on AM3. A 1-2 dilution difference was noted between both the endpoints of amphotericin B and of terbinafine. The highest inter- and intra-laboratory agreements were reached on AM3. The azoles showed a medium-dependent fungicidal activity.

[Emerging parasitosis ans mycosis: risk and threats for the new millenium]. / Emergence de parasitoses et mycoses: risques et menaces au seuil de troisième millénaire.

For multiple reasons, the emergent infectious risks do not stop increasing these last twenty years. The climatic modifications and the human interventions modifying the biotope as well as the rapid spreading of resistant strains to treatments, generate re-emergence or emergence, all the more dramatic as the means of fight are reduced. These emergent or re-emergent diseases are extremely worrisome as their diagnosis and their prevention are often difficult. The important infesting power of parasites and the particularly effective capacities of adaptation of these eucaryotes contributed to the public health problems. Anthropozoonoses and zoonoses constitute a permanent risk the control of which is imaginary. The new pathogenic agents, the unusual clinical demonstrations in the context of deficiency of the host immune functions imply attentiveness and a permanent up to date of the knowledge of the biologist and of the different professionals of health. The risks with which are confronted the humanity during this century underline the necessity of determining mechanisms involved in the pathogenesis. The determination of the specific and vital biologic processes for the microorganism, could allow to define the most appropriated targets and the most effective and original means of fight.

Eight-year surveillance of environmental fungal contamination in hospital operating rooms and haematological units.

An eight-year fungal environmental surveillance was carried out in 15 operating theatres and two haematological units. Sampling was performed twice a year in each room, using contact plates for plane surfaces and sterile swabs for grids. From 1992 to 1999, individual rooms in the 17 units were sampled on 1094 occasions and 3822 samples were collected. The percentage of rooms without fungus increased regularly between 1992 and 1999 (41.1% and 74.8%, respectively). The units were classified according to the fungal contamination during the eight years: the operating theatres which required the highest protection (cardiological, thoracic, vascular, hand, orthopaedic and neurosurgery) and the adult haematological unit showed least contamination (71.8% rooms were negative). The most frequent species isolated were Penicillium spp. (28.4%), Cladosporium spp. (15.6%) and Aspergillus spp. (7.6%). Aspergillus fumigatus was rarely isolated (3.7%), and was mainly isolated at the beginning of the study. This study demonstrates that environmental control programmes are effective in reducing environmental mould contamination and could be useful in establishing exposure guidelines, especially by defining an acceptable level of biocontamination in zones at risk.

[Role of local immunity in invasive pulmonary aspergillosis]. / Le rôle de l'immunité locale dans l'aspergillose pulmonaire invasive.

A LEADING CAUSE OF MORTALITY: Inhalation of fungal spores may cause infection and/or inflammation, which is dependent on the nature of the fungus as well as the individual's immune status. Aspergillus fumigatus is responsible for a dramatic pathology, invasive aspergillosis (IA). IA has become a leading cause of death, mainly among bone marrow transplantation or solid-organ recipients, but also among AIDS patients. IMMUNE RESPONSE: The diversity of immune failure suggests that many lines of immunity are implicated in the A. fumigatus elimination process. Non specific immunity plays a major role in the defence against A. fumigatus, including three major lines: anatomical barriers, humoral components, phagocytic cells. But acquired immunity plays a role with different T-cell subsets and their associated cytokines. FUNGUS-HOST RELATIONSHIPS: The relationship between the fungus and its host is complex and could be again study to improve the prevention and the treatment of IA. The aim of this review is a synthesis of the knowledge about the immunity response against Aspergillus fumigatus in IA.

A longitudinal study of lung transplant recipients infected with Aspergillus: genetic polymorphism of A fumigatus.

BACKGROUND: Aspergillus infection is a well-known complication of lung transplantation and remains associated with high mortality rates. Molecular typing methods are required to elucidate the complex epidemiology of Aspergillus disease in lung transplant recipients. METHODS: Eight lung transplant recipients from one hospital were followed for A fumigatus colonization or infection. Forty-four sequential isolates from these patients were selected and typed by three molecular methods (random amplified polymorphic DNA, sequence-specific DNA primer and multi-locus enzyme electrophoresis). RESULTS: Sixteen different types were identified of which 14 were specific to 1 patient. A factorial correspondence analysis showed that variability between sequential isolates from a single patient was as high as between isolates from the other patients. Lung transplant recipients presented many different genotypes, reflecting the environmental diversity of A fumigatus. Nevertheless, throughout their follow-up, 2 of the 8 lung transplant recipients harbored a common genotype that was not replaced by others. CONCLUSIONS: These results confirm the important genetic polymorphism of the A fumigatus population. The observed genotypes were not related to the type of Aspergillus disease or anti-fungal treatment used nor to the outcome of the patient. These data confirm that all A fumigatus molecular types present the same pathogenic risk.

Evaluation of Candida ID, a new chromogenic medium for fungal isolation and preliminary identification of some yeast species.

Candida ID, a new chromogenic medium, allows identification of Candida albicans (blue colonies) and preliminary identification into a group of four species (pink colonies). In comparison with Albicans ID2 and Sabouraud gentamicin chloramphenicol on 446 fungal strains, Candida ID allowed the isolation of more species than Albicans ID 2 (95.5% versus 91.2%).

Genetic polymorphism of Aspergillus fumigatus in clinical samples from patients with invasive aspergillosis: investigation using multiple typing methods.

The genotypes of 52 strains of Aspergillus fumigatus isolated from 12 patients with invasive aspergillosis were investigated using three typing methods (random amplified polymorphic DNA, sequence-specific DNA polymorphism, and microsatellite polymorphism) combined with multilocus enzyme electrophoresis. Isolates were from patients hospitalized in three different geographic areas (Lyon, France; Grenoble, France; and Milan, Italy). In each case, the genetic polymorphism of several colonies (two to five) within the first respiratory clinical sample was studied. For the 52 isolates tested, random amplified polymorphic DNA identified 8 different genotypes, sequence-specific DNA polymorphism identified 9 different types, and microsatellite polymorphism identified 14 types. A combination of these results with multilocus enzyme electrophoresis study identified 25 different types within the sample studied. We identified 3 patients (of the 12 studied) who carried a single genotype; 6 patients were infected by two genotypes, 1 patient had four genotypes, while the last patient had five. A combination of typing methods provided better discrimination than the use of a single method. Typing methods revealed a population structure within each geographical site, suggesting that the epidemiology of A. fumigatus should be considered separately for each of these geographic areas. This study demonstrates the usefulness of combining several typing methods in reaching an understanding of the epidemiology of A. fumigatus and clarifies whether it is sufficient to type one isolate from each specimen to determine the strain involved in invasive aspergillosis.

Hydrocortisone, prednisolone and dexamethasone act on Aspergillus fumigatus in vitro susceptibility to itraconazole.

In a previous in vitro investigation from the same laboratory a therapeutic level of hydrocortisone enhanced the itraconazole susceptibility of a single strain of Aspergillus fumigatus. In the present work, the influence of therapeutic levels of hydrocortisone (1 microM), prednisolone (0.125 microM 0.25 microM and 0.5 microM) and dexamethasone (0.25 microM and 0.5 microM) on the itraconazole susceptibility of four A. fumigatus strains, was determined. A. fumigatus conidia were germinated either in the absence or in the presence of a glucocorticoid. The germinated conidia were then spread onto plates and grown either in the presence or in the absence of a glucocorticoid, together with increasing concentrations of itraconazole. The mean colony forming units (CFU) were measured. Two factor analyses of variance showed that hydrocortisone significantly (p <0.001) potentiated the action of itraconazole. The cytotoxic effect of prednisolone on the fungal strains added significantly to the effect of itraconazole (p <0.001). Dexamethasone was also cytotoxic to the fungus but, when used in conjunction with itraconazole, it effectively increased (p <0.01) the number of CFU. This study showed a direct effect of glucocorticoids, currently in use for patient therapy, on in vitro A. fumigatus susceptibility to itraconazole.