Implementation of an FTIR spectral library of 486 filamentous fungi strains for rapid identification of molds.

Filamentous fungi may cause food and feed spoilage and produce harmful metabolites to human and animal health such as mycotoxins. Identification of fungi using conventional phenotypic methods is time-consuming and molecular methods are still quite expensive and require specific laboratory skills. In the last two decades, it has been shown that Fourier transform infrared (FTIR) spectroscopy was an efficient tool for microorganism identification. The aims of this study were to use a simple protocol for the identification of filamentous fungi using FTIR spectroscopy coupled with a partial least squares discriminant analysis (PLS-DA), to implement a procedure to validate the obtained results, and to assess the transferability of the method and database. FTIR spectra of 486 strains (43 genera and 140 species) were recorded. An IR spectral database built with 288 strains was used to identify 105 different strains. It was found that 99.17% and 92.3% of spectra derived from these strains were correctly assigned at the genus and species levels, respectively. The establishment of a score and a threshold permitted to validate 80.79% of the results obtained. A standardization function (SF) was also implemented and tested on FTIR data from another instrument on a different site and permitted to increase the percentage of well predicted spectra for this set from 72.15% to 89.13%. This study confirms the good performance of high throughput FTIR spectroscopy for fungal identification using a spectral library of molds of industrial relevance.

Scedosporium apiospermum Otitis Complicated by a Temporomandibular Arthritis: A Case Report and Mini-Review.

Scedosporium apiospermum is an ubiquitous fungus responsible for various infections in immunocompromised and immunocompetent patients. Ear infections are infrequent. We report an exceptional case of S. apiospermum external otitis complicated by temporomandibular joint arthritis. After 6 months of antibiotherapy, diagnosis was established by mycological analysis of external auditory canal and infratemporal fossae needle sampling. A satisfactory outcome was obtained after 2 months of voriconazole alone. We have reviewed 15 cases of S. apiospermum otitis. Seven of these patients were immunocompromised. Most common clinical presentation included a chronic external otitis lasting months or years before complication stage. Most common clinical features included recurrent unilateral otalgia (11/15) and purulent otorrhea (13/15). Diagnosis was often made at later stage (12/15) with local extension to bones and/or soft tissues (9/15) or cerebral lethal dissemination (3/15).The extremely low incidence of S. apiospermum otomycosis and its non-specific presentation results in a frequent diagnosis delay. A mycological investigation should be performed in case of persistent external otitis and/or osteolysis despite prolonged antibiotic treatment to prevent further extension of the disease.

Reactivity of (1→3)-ß-d-glucan assay in bacterial bloodstream infections.

To diagnose invasive fungal infections, the detection of (1 → 3)-ß-d-glucan in serum has shown variable specificity, depending on the targeted population. Several circumstances for false-positive results of beta-glucan tests have been identified, among which are severe bacterial infections. In this study, we measured (1 → 3)-ß-d-glucan by the Fungitell test in the serum of 62 patients (one serum sample tested per patient) for whom invasive fungal infection was not suspected: 19 control subjects and 43 patients with bacteraemia. The test was interpretable for 58 sera: all 19 control subjects had negative beta-glucan test; among the 39 bacteraemic patients, we report 16 false-positive results. For the 22 patients undergoing bacteraemia due to Gram-negative bacilli, we observed 13 false-positive results (59%). Among the 17 patients with bloodstream infection involving Gram-positive cocci, three false-positive tests were recorded, but none in the eight cases of Streptococcus pneumoniae bacteraemia. Statistical analysis showed that beta-glucan levels were significantly higher in patients with Gram-negative bacilli bloodstream infection in comparison to those with bacteraemia due to Gram-positive cocci. These results were independent from other previously described causes for false-positive beta-glucan tests. These data might help physicians to interpret positive beta-glucan detection when an invasive fungal infection is suspected, especially for patients with bacterial infections.

Investigation of nosocomial pneumocystis infections: usefulness of longitudinal screening of epidemic and post-epidemic pneumocystis genotypes.

BACKGROUND: Twenty-five patients, of whom 22 were renal transplant recipients, developed Pneumocystis jirovecii infections at the nephrology department of Reims University Hospital (France) from September 2008 to October 2009, whereas only four sporadic cases had been diagnosed in this department over the 14 previous years. AIM: This outbreak was investigated by analysing patient encounters and P. jirovecii types. METHODS: A transmission map was drawn up. P. jirovecii typing at DHPS, ITS and mtLSU rRNA sequences was performed in the patients of the cluster (18 patients with Pneumocystis pneumonia (PCP) and seven colonized patients), 10 unlinked control patients (six PCP patients and four colonized patients), as well as 23 other patients diagnosed with P. jirovecii (nine PCP patients and 14 colonized patients) in the same department over a three-year post-epidemic period. FINDINGS: Eleven encounters between patients harbouring the same types were observed. Three PCP patients and one colonized patient were considered as possible index cases. The most frequent types in the cluster group and the control group were identical. However, their frequency was significantly higher in the first than in the second group (P < 0.01). Identical types were also identified in the post-epidemic group, suggesting a second outbreak due to the same strain, contemporary to a disruption in prevention measures. CONCLUSIONS: These results provide additional data on the role of both PCP and colonized patients as infectious sources. Longitudinal screening of P. jirovecii types in infected patients, including colonized patients, is required in the investigation of the fungus's circulation within hospitals.

Early diagnosis and monitoring of mucormycosis by detection of circulating DNA in serum: retrospective analysis of 44 cases collected through the French Surveillance Network of Invasive Fungal Infections (RESSIF).

The main objective of this study was to assess the diagnostic performance of a set of three Mucorales quantitative PCR assays in a retrospective multicentre study. Mucormycosis cases were recorded thanks to the French prospective surveillance programme (RESSIF network). The day of sampling of the first histological or mycological positive specimen was defined as day 0 (D0). Detection of circulating DNA was performed on frozen serum samples collected from D-30 to D30, using quantitative PCR assays targeting Rhizomucor, Lichtheimia, Mucor/Rhizopus. Forty-four patients diagnosed with probable (n = 19) or proven (n = 25) mucormycosis were included. Thirty-six of the 44 patients (81%) had at least one PCR-positive serum. The first PCR-positive sample was observed 9 days (range 0-28 days) before diagnosis was made using mycological criteria and at least 2 days (range 0-24 days) before imaging. The identifications provided with the quantitative PCR assays were all concordant with culture and/or PCR-based identification of the causal species. Survival rate at D84 was significantly higher for patients with an initially positive PCR that became negative after treatment initiation than for patients whose PCR remained positive (48% and 4%, respectively; p <10-6). The median time for complete negativity of PCR was 7 days (range 3-19 days) after initiation of l-AmB treatment. Despite some limitations due to the retrospective design of the study, we showed that Mucorales quantitative PCR could not only confirm the mucormycosis diagnosis when other mycological arguments were present but could also anticipate this diagnosis. Quantification of DNA loads may also be a useful adjunct to treatment monitoring.

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.

Enzyme-linked immuno-filtration assay applied to accelerated immunodetection of gel transferred proteins on immobilizing matrices.

The enzyme-linked immunofiltration assay (ELIFA) has been used for the rapid detection of electrophoresed proteins transferred from gels to immobilizing matrices. By controlled filtration, ELIFA permits the saturation of nylon or nitrocellulose membranes and the immunodetection of blotted antigens to be carried out in 15 min. The method is simple, can be automated and requires no handling of membranes. It complements the well standardized steps of gel electrophoresis and semi-dry horizontal electroblotting which can themselves be carried out in less than an hour. The sensitivity is at least 1-5 ng. The same process can be extended to the accelerated characterization of glycoproteins using appropriate ligands, or to the identification of antigens in a variety of biological fluids.

Genotyping human and bovine isolates of Cryptosporidium parvum by polymerase chain reaction-restriction fragment length polymorphism analysis of a repetitive DNA sequence.

In order to define transmission routes of cryptosporidiosis and develop markers that distinguish Cryptosporidium parvum isolates, we have identified 2 polymorphic restriction enzyme sites in a C. parvum repetitive DNA sequence. The target sequence was amplified by polymerase chain reaction from 100 to 500 oocysts and the amplified product was subjected to restriction enzyme digestion. Typing of 23 isolates showed that 10/10 calf isolates had the same profile. In contrast, 2 patterns were observed among human isolates: 7/13 displayed the calf profile, and 6/13 presented another pattern. The PCR-RFLP assay described here is a sensitive tool to distinguish C. parvum isolates.

[Characterization of specific IgG, IgM, IgA and IgE isotypes in profound candidiasis]. / Caractérisation des isotypes spécifiques IgG, IgM, IgA et IgE dans les candidoses profondes.

Enzyme-linked immunofiltration assay technique (Elisa) has been applied to the characterization of G, M, A and E anti-Candida antibodies isotypes specific to cell wall mannans in 201 sera from 126 patients. These sera were studied at the same time using Co-immunoelectrodiffusion and indirect immunofluorescence. In 18 of 21 patients with systemic candidiasis, Elisa demonstrated the presence of antimannan IgG antibodies in sera contemporary of Candida positive blood culture. These IgG were associated with antimannan IgM, A and E in 15 patients. In 37 patients colonized with Candida, used as negative controls, antimannan IgG were detected in 3 cases, and in 2 were associated with specific IgMs. The sensitivity and specificity of Elisa IgM and IgA in the diagnosis of systemic Candidiasis were 85.7% and 81%, respectively. The kinetic study shows that the different isotypes appeared most of the time simultaneously. The evolution of the 4 isotypes beyond the acute episode was variable and without correlation with the clinical status. The decrease of IgG was slower than the one of IgM, IgA or IgE. The systematic research, in at risk patients, of antimannan antibodies using Elisa required simple technology. A simple method should allow to aim at other functional antigens which could be used in a quantitative manner to determine the efficacy of the medical treatment.

[Multicenter study of 435 yeast strains isolated from 116,000 hemocultures]. / Etude multicentrique de 435 souches de levures isolées de 116000 hémocultures.

During a multicentric study on the diagnosis of yeast septicemia, 5 blood culture media have been compared. 435 yeast strains have been isolated from 116,372 blood cultures received during year 1989 in the mycological laboratories of 5 university hospitals from east of France. The results show that Candida albicans is always the first septicemia agent with 52% of yeasts isolation from blood. C. parapsilosis comes next with 17%, then C. glabrata, 6.2%. The average delay of yeasts isolation is 2.3 days for Bactec NR and Sabouraud media, 3.7 days for other bacteriological media. Compared with polyvalent media, Sabouraud diphasic medium is significantly the best for yeasts isolation. It is recommended to add chloramphenicol in the medium, to culture 10 ml of blood and to keep blood cultures for at least 8 days, better 15 days.

[Comparison of ELIFA and IGM-immunocapture technics in the diagnosis of 50 cases of congenital toxoplasmosis]. / Comparaison entre les techniques ELIFA et immunocapture-IgM dans le diagnostic de cinquante cas de toxoplasmose congénitale.

The authors have compared the diagnostic value in congenital toxoplasmosis of two recently developed immunological techniques, Enzyme Linked immunofiltration Assay (ELIFA) and IgM-immunocapture (or immunosorbent agglutination assay). The study involved 50 children suffering from congenital toxoplasmosis and 300 unscathed control children, whose mothers suffered from toxoplasmosis during their pregnancies. The ELIFA technique showed a sensitivity of 74 p. cent at the end of the first month after birth (day 30) and 84 p. cent after two months (day 60), whereas the IgM-immunocapture technique gave a sensitivity of 64 p. cent at day 30 and 70 p. cent at day 60. Both methods were at least 95 p. cent specific. The two techniques seem to be complementary to each other, and their association allowed the diagnosis to be confirmed in 80 p. cent of cases at day 30 and in 90 p. cent of cases at day 60. A serum sample taken on the tenth day after birth (day 10) was important both to eliminate the rare cases (5 p. cent) of transplacental transmission of maternal IgM antibodies and to detect transient neonatal synthesis of IgG or IgM toxoplasmic antibodies.

[Comparative study of antitoxoplasmic IgG isotypes titrated by high sensitivity direct agglutination and indirect immunofluorescence: consequence of the choice of methods on the expression of results in international units]. / Etude comparée des isotypes antitoxoplasmiques IgG titrés par agglutination directe haute sensibilité et immunofluorescence indirecte: incidence du choix des méthodes sur l'expression des résultats en unités internationales.

The use of International Units per ml (IU/ml) to express antitoxoplasmic IgG antibody titers in the various diagnostic systems presently proposed, is misleading owing to discrepancies in the values found from one test to the other for a given serum. The authors compared the results of high sensitivity direct agglutination (HSDA) to those of indirect immunofluorescence (IIF) in two studies, systematic and longitudinal, dealing with 158 sera stratified for values ranging from 102,400 to 5 IU/ml. Discordances between the methods, which are greater for high values, prompt the use of low-titer sera for standardization. From the systematic study, a correlation table was established and proposed to convert the HSDA results into the theoretical corrected values close to those that would be obtained by IIF. Although this may be of interest in maintaining a coherent language, this table has its limits, particularly in acute episodes where the various antibody kinetics vary and amplify further the discrepancies. In such situations, it seems advisable for both clinicians and biologists to raise any equivocal kept going by what is termed as International Units. Consequently, if the titers obtained by one method cannot be correlated to those of the technique of reference (IIF, Dye test), on the basis of using I.U., it would be appropriate to express the results in units related to the method or kit used (e.g. U/ml/HSDA for high sensitivity direct agglutination). Finally, whatever the technique, it is still mandatory to conserve a significant threshold value of protective immunization, common and identical to those classically adopted (8-12 IU/ml).

[Immunohistochemical study of plasmocytes and T-lymphocyte subpopulations in Hashimoto's thyroiditis]. / Etude immunohistochimique des plasmocytes et des sous-populations T lymphocytaires dans la thyroïdite de Hashimoto.

In Hashimoto's thyroiditis, there is a diffuse interfollicular infiltration of lymphocytes and plasmocytes and lymphoid collections with germinal centers. Eleven cases of Hashimoto's disease were studied with immunocytochemical methods for characterization of intracytoplasmic immunoglobulins (IgA, IgG, IgM, Kappa and lambda light chains). Most of the cells are plasmocytes stained positively for intracytoplasmic IgG; IgA positive cells were less frequent and IgM positive cells are rare. Kappa positive plasma cells are more numerous than lambda positive cells in the germinal centers and the interfollicular infiltration. By an indirect immunofluorescence technique in frozen tissue, we have studied with four monoclonal antibodies (OKT3, OKT4, OKT8, B1) the T cells subsets populations in two cases.

[Extrinsic allergic alveolitis of occupational origin]. / Les alvéolites allergiques extrinsèques d'origine professionnelle.

Occupational factors encountered in farming and other agricultural activities produce a particular risk for respiratory diseases. For some, such as extrinsic allergic alveolitis, diagnosis depends upon a range of epidemiological, clinical, radiological and immunological arguments. Farmer's lung is one of the most common form of extrinsic allergic alveolitis. Bird breeder's lung is another, the list is long. The environmental allergens likely to affect alveoli and interstitial tissues have been identified, but simple detection of antibodies does not constitute a pathognomonic criterion of extrinsic allergic alveolitis. Co-immuno-electrodiffusion is a rapid and sensitive technique for the demonstration of remarkable precipitating systems of extrinsic allergic alveolitis. This investigation enables subjects who really have the disease to be distinguished from contact subjects. Diagnosis is important to prevent development of a disabling and irreversible pulmonary fibrosis.

[Should immunologic monitoring of toxoplasmosis seronegative pregnant women stop at delivery?]. / La surveillance immunologique d'une femme enceinte séronégative pour la toxoplasmose doit-elle s'arrêter à l'accouchement?

The authors report 2 cases of congenital toxoplasmosis fortuitously diagnosed in 2 newborn infants aged 12 and 35 days respectively whose mothers had no anti-Toxoplasma antibodies detectable at the time of birth. These cases prompted us to carry out, over an 18 months' period, a systematic postnatal control of all pregnant women who were still seronegative at the time of delivery. This enabled us to detect 4 cases of perinatal maternal infection with Toxoplasma contamination in 2 neonates. In view of these results, and in order not to miss any maternal infection at the very end of pregnancy, it seems advisable to complete the control of seronegative women by taking a last blood sample about 30 days after they have given birth.

Differentiation and identification of filamentous fungi by high-throughput FTIR spectroscopic analysis of mycelia.

Routine identification of fungi based on phenotypic and genotypic methods can be fastidious and time-consuming. In this context, there is a constant need for new approaches allowing the rapid identification of molds. Fourier-transform infrared (FTIR) spectroscopy appears as such an indicated method. The objective of this work was to evaluate the potential of FTIR spectroscopy for an early differentiation and identification of filamentous fungi. One hundred and thirty-one strains identified using DNA sequencing, were analyzed using FTIR spectroscopy of the mycelia obtained after a reduced culture time of 48 h compared to current conventional methods. Partial least square discriminant analysis was used as a chemometric method to analyze the spectral data and for identification of the fungal strains from the phylum to the species level. Calibration models were constructed using 106 strains pertaining to 14 different genera and 32 species and were used to identify 25 fungal strains in a blind manner. Identification levels of 98.97% and 98.77% achieved were correctly assigned to the genus and species levels respectively. FTIR spectroscopy with its high discriminating power and rapidity therefore shows strong promise for routine fungal identification. Upgrading of our database is ongoing to test the technique's robustness.

[Post-traumatic mucormycosis due to Lichtheimia corymbifera: three case reports]. / Mucormycoses post-traumatiques à Lichtheimia corymbifera: à propos de 3 cas.

We report 3 cases of post-traumatic cutaneous mucormycosis caused by Lichtheimia corymbifera, two of them occurring after a farm working accident. Management of post-traumatic mucormycoses consists of a wide excision of the infected tissue, combined with immediate antifungal therapy. Liposomal amphotericin B is the recommended first line treatment. Few studies have evaluated the efficacy of posaconazole. All 3 patients received a surgical debridement and liposomal amphotericin B, which was followed by posaconazole in 2 cases. The duration of the antifungal treatment is not yet well defined. All three patients received a treatment of five weeks with a favorable outcome.

Usefulness of matrix-assisted laser desorption ionisation-time-of-flight mass spectrometry for identifying clinical Trichosporon isolates.

Trichosporon spp. have recently emerged as significant human pathogens. Identification of these species is important, both for epidemiological purposes and for therapeutic management, but conventional identification based on biochemical traits is hindered by the lack of updates to the species databases provided by the different commercial systems. In this study, 93 strains, or isolates, belonging to 16 Trichosporon species were subjected to both molecular identification using IGS1 gene sequencing and matrix-assisted laser desorption ionisation-time-of-flight (MALDI-TOF) analysis. Our results confirmed the limits of biochemical systems for identifying Trichosporon species, because only 27 (36%) of the isolates were correctly identified using them. Different protein extraction procedures were evaluated, revealing that incubation for 30 min with 70% formic acid yields the spectra with the highest scores. Among the six different reference spectra databases that were tested, a specific one composed of 18 reference strains plus seven clinical isolates allowed the correct identification of 67 of the 68 clinical isolates (98.5%). Although until recently it has been less widely applied to the basidiomycetous fungi, MALDI-TOF appears to be a valuable tool for identifying clinical Trichosporon isolates at the species level.

Isotypic characterization of anti-Toxoplasma gondii antibodies in 18 cases of congenital toxoplasmic chorioretinitis.

A serological study was carried out by ELIFA (Enzyme Linked Immuno-Filtration Assay) for 50 children with congenital toxoplasmosis diagnosed by several parasitological and serological methods showing that the fetus had been infected by the parasite and had developed it's own specific immune response. At birth, anti-Toxoplasma gondii IgE antibodies were detected in the sera of 66% of the 18 children who had retinochoroiditis and in 32% of the 32 children without this complication. During the 4 months before or at the time of diagnosis of retinochoroiditis, specific IgE antibodies were detected in 70% of the 20 cases (2 children with 2 successive lesions); but during the 4 months following the discovery of ocular lesions, anti-Toxoplasma IgE antibodies were only detected in 30% of the 20 cases. Among all the 50 children, the prolonged detection of specific IgM + IgE association (for at least 4 months) was followed in 46% of cases by the appearance of chorioretinitis (predictive value).

[Diagnosis and prevention of candidiasis in intensive care patients]. / Diagnostic et prévention des candidoses en réanimation.

Invasive candidiasis infections remain a major complication in I.C.U. patients. Numerous attempts have been made to evaluate potential prophylactic methods. Various agents have been tested. Gastrointestinal tract constitutes one of the major reservoirs for Candida species. One major problem is the difficulty in establishing an accurate, early, diagnosis of invasive fungal infection. A prospective randomized, controlled blind study was performed to assess the ability of oral Amphotericin B to prevent candidiasis in selected I.C.U. patients. Fifty one patients with serious infection and antibiotherapy were randomized to receive either oral Amphotericin B (2 g/day) or placebo, and observed until discharge. All patients were screened weekly for sites culture positive, sero-conversion and oesophagitis. Invasive candidiasis developed in 45% of patients receiving Amphotericin B compared with 41% receiving placebo. C. Albicans persists in the surveillance cultures. However a significant reduction of the colonization by the yeasts and a significant reduction of oesophagitis was demonstrated among the Amphotericin B group. No benefit was found in the total number of hospital days. Digestive decontamination can be successfully managed by Amphotericin B in I.C.U. patients but failed to prevent invasive candidiasis.