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.

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.

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.

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.

Aspergillus and lung transplant recipients: a mycologic and molecular epidemiologic study.

BACKGROUND: After lung transplantation, filamentous fungi and more particularly Aspergillus fumigatus are commonly isolated, although the origin of contamination is unclear. METHODS: To investigate the fungal flora in bronchoscopic fluids, we retrospectively reviewed 20 cases of lung transplant recipients. Using sequence-specific DNA primers analysis, we typed the clinical strains of A. fumigatus isolated from 6 lung transplant recipients. For 4 of them, the strains of this species were isolated from their environment. RESULTS: At least once 90% of patients had filamentous fungi, and A. fumigatus was the most frequently isolated. Bronchial colonization was detected in 14 patients, invasive bronchial mycosis was diagnosed in 4 others, and no case of invasive pulmonary fungal infection was detected. Genome typing of the 47 clinical strains revealed that a given patient could be affected by several different strains. A very extensive polymorphism existed among the 38 environmental strains. Origin of contamination at home was possible in 1 case and in the hospital in 3 cases. CONCLUSIONS: Bronchial colonization is frequent after lung transplantation. Although the clinical strains show a polymorphism, it is less widespread than the polymorphism of environmental strains. The origin of acquisition may be in the patient's community.

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.

Diagnosis of human hydatidosis: comparison between imagery and six serologic techniques.

Echography and/or chest radiograph in association with six serologic (immunologic) methods were tested for their ability to diagnose human hydatidosis. The immunologic techniques used were latex agglutination, counter immunoelectrophoresis, enzyme-linked immunosorbent assay (ELISA) with whole hydatid fluid and with antigen 5, a thermobabile lipoprotein that elicits the arc 5 precipitin line in immunoelectrophoresis. In this report, the results of examinations of 273 patients are presented. Of these, 243 cases were surgically proven to have hydatidosis, whereas 30 of the 273 that were strongly suspected of having hydatid cysts by radiology were shown to have other pathologies. The ELISA was more sensitive than the other methods, with 204 of 243 sera shown to be positive. The remaining 39 sera that gave false-negative results were tested using two recently developed methods, enzyme-linked immunoelectrodiffusion assay (ELIEDA) and immunoblotting (electrophoretic migration of hydatid fluid antigens under denaturing but nonreducing conditions, blotting, and immunoenzymatic assay). Immunoblotting, using our experimental conditions, had a greater sensitivity than the ELIEDA with this technique. Four of 37 sera showed one to three bands of 65, 12, and 8 kD. The sensitivity of the ELISA did not increase with either the size or type of cyst. Comparison of radiology with serology confirmed a good correlation between the two methods. Nevertheless, serology is more specific but less sensitive than imagery.

Virulence of Aspergillus fumigatus strains investigated by random amplified polymorphic DNA analysis.

A possible relationship between the ability of Aspergillus fumigatus strains to invade tissues and genetic polymorphism was studied by random amplified polymorphic DNA (RAPD) analysis. One hundred randomly designed oligonucleotide decamers were examined with DNA of three reference strains, eight environmental isolates and 21 isolates from two distinct clinical situations: non-invasive aspergillosis (predominantly aspergilloma) and invasive aspergillosis. One primer (OPQ 6) was found to generate a reproducible amplification product that enabled distinction between the two groups according to the presence or absence of a 0.95-kb fragment that correlated with the nature of the infection (non-invasive or invasive) and immune status of the patient. The results indicated that the pathogenicity of A. fumigatus was related not only to the host's immune status but also to the virulence of the strain of A. fumigatus.

Variation in virulence of Aspergillus fumigatus strains in a murine model of invasive pulmonary aspergillosis.

The diversity in virulence of different Aspergillus fumigatus strains was studied in an experimental murine model of invasive pulmonary aspergillosis (IPA) and the results were correlated with possession of a putative molecular marker of virulence. Seven strains from different patients with non-invasive or invasive aspergillosis and four environmental strains were typed by PCR with specific primers and scored as positive or negative, according to whether or not a 0.95-kb DNA fragment was amplified. Immunosuppressed mice were inoculated intranasally with A. fumigatus conidia from these different strains. The mortality curves revealed differences in virulence between the strains. The environmental strains produced a weaker infection than the strains from patients and the 0.95-kb-positive patient strains caused significantly higher mortality rates in mice than the 0.95-kb-negative patient strains. These findings support the hypothesis that certain isolates of A. fumigatus are more virulent than others and that their virulence appears to be associated with the 0.95-kb molecular marker.

Combination of three typing methods for the molecular epidemiology of Aspergillus fumigatus infections. European Research Group on Biotype and Genotype of Aspergillus.

This study investigated the source of infection and strain relatedness of Aspergillus fumigatus isolates from bronchial colonisation and invasive aspergillosis (IA) in four transplant patients. Environmental isolates from the patient's home and from the hospital and infecting isolates were obtained for patient A who developed IA. Clinic environmental and colonising isolates were obtained for patient B. Sequential isolates were obtained from various organs from patient C who developed IA and also from patient D who had a bronchitic aspergillosis that developed into IA. Ninety-one A. fumigatus isolates were analysed by three typing methods: multi-locus enzyme electrophoresis (MLEE), random amplified polymorphic DNA (RAPD) and sequence-specific DNA primers (SSDP). The three combined typing methods demonstrated a greater differentiation of isolates than the typing methods used separately or in pairs. This demonstrated the genotypic variability of A. fumigatus and facilitated better epidemiological analysis. Large polymorphisms were demonstrated for each patient isolate between and colonies within various samples. The relatedness of the isolates suggested nosocomially acquired aspergillosis for patient B, but the source of infection for patient A remained unclear. The results suggested at least three multiple infections among the four patients. This study enabled the identification of the source of infection and strain relatedness, which in turn facilitates the development of preventive measures for patient management in the future.

Molecular typing of Aspergillus fumigatus strains by sequence-specific DNA primer (SSDP) analysis.

A PCR typing method has been developed and tested to investigate the polymorphism of clinical strains of Aspergillus fumigatus. Firstly, the DNA fragments from random amplified polymorphic DNA (RAPD) patterns of nine epidemiologically and geographically non-related monosporal strains of A. fumigatus were cloned and sequenced. The pairs of five sequence-specific DNA primers (SSDP), characteristic of the 5' and 3' extremities of the RAPD products, were then used in high stringency PCR to type 43 clinical strains of A. fumigatus from 13 patients, according to the presence or absence of a single amplified band. This original approach, which uses the advantages of PCR, has made it possible to overcome the difficulties resulting from the low stringency amplification. The SSDP analysis of 51 A. fumigatus strains (9 unrelated monosporal strains and 43 clinical strains from 13 patients) can be classed into 22 different types with a high reproducibility and a high level of discrimination (D = 0.96). The results suggest that seven lung transplant patients with necrotizing aspergillosis, bronchitis aspergillosis and bronchial colonization were infected by multiple strain genotypes, whereas three patients with invasive aspergillosis seem to have been infected by a single strain.

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.

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.

[Epidemic of autochthonous hepatic and intestinal amebiasis in a place near Grenoble]. / Epidémie d'amibiase autochtone hépatique et intestinale dans une localité proche de Grenoble

We had the opportunity of studying an epidemic of autochthonous amoebiasis occurring in the autumn of 1974 in a small town of 4000 inhabitants 30 km from Grenoble. Attention was originally attracted by the occurrence in this town of two cases of hepatic amoebiasis and one of intestinal amoebiasis identified by rectoscopy. Systematic investigations (coproctic examinations and serological tests for amoebiasis by indirect antibody fluorescence) were then carried out on everyone in the locality with digestive disorders which were possibly referable to amoebiasis, and on the other members of their families. A total number of 148 coproctic examinations were made and in two cases revealed the presence of vegetative forms of Entamoeba histolytica. In both cases the infestation provoked few symptoms (asthenia, vague abdominal discomfort, intermittent and apparently banal diarrhoea). On the other hand 20 out of 94 serological tests revealed positive results, 14 of which were equal to or greater than a titre of 1/100, a level at which all risks of non-specificity are virtually ruled out under our experimental conditions. Material reasons made it impossible to subject these cases to repeated faecal checks, but in two of them at least the rectoscopic appearances were very suggestive of subacute intestinal amoebiasis. Moreover, amoebic disease appears to be well confirmed by the results obtained among the patients as a whole by treatment with Metroinidazole. A variety of hypotheses on the origin of this epidemic have been put forward and then abandoned (market garden produce, receipt by certain families of exotic frut from overseas). In actual fact water seems to be the point of departure, for, although specimens of water taken at 7 different levels in the water supply system failed to reveal the presence of a single amoeba, bacteriological analyses during autumn 1974 showed signs of faecal contamination. The locality, which is situated at the foot of the Chartreuse massif, receives its water solely from springs but there is a holiday camp for the staff of an international airline situated above the main water catchment.

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.

[Free-living amoebae in the Grenoble area swimming pool water. Influence of th "winter-summer" use and of the sterilising procedure (author's transl)]. / Les amibes libres dans les eaux de piscines de la région grenobloise. Influence du fonctionnement "été-hiver" et du procédé de stérilisation.

An epidemiological study concerning the search for and identification of free-living amoebae has been carried out on swimming pool water in the Grenoble area. Eleven establishments were surveyed over a period of one year, by taking samples of water every month. The results obtained show that the positive nature of the samples and the nature of the isolated species are influenced by the two factors being studied: The rhythm of the "Summer-Winter" use of swimming pools and the nature of the sterilising procedure, chlorine, bromine and ozone. The current sterilising conditions of the swimming pool water are discussed.

[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.

[Antifungigram: interpretation and choice of a technic]. / Antifongigramme: interprétation et choix d'une technique.

Identification and in vitro susceptibility testing of fungi is necessary mainly in immunocompromised patients. So, after a survey of the epidemiological results, the authors specified the indications of susceptibility tests; their advantages and difficulties and the lots of variations related to the fungus, drug, medium and technic. The authors compared the more recent methods from their experience: susceptibility to 6 antifungals (amphotericin B, flucytosine and 4 imidazoles) was estimated in 122 yeast strains isolated from deep specimens by 5 marketed technics. They got a good correlation between Mycototal and Antifongigramme Pasteur, but the two other technics (Mycodisk and Neosensitabs) frequently showed unreliable results.