Candida albicans biofilms on different materials for manufacturing implant abutments and prostheses.

BACKGROUND: Morphological, physical and chemical properties of both implants and prostheses can determine the biofilm formation on their surface and increase the risk of biological complications. The aim of this study was to evaluate the capacity of biofilm formation of Candida albicans on different materials used to manufacture abutments and prostheses. MATERIAL AND METHODS: Biofilm formation was analyzed on cp grade II titanium, cobalt-chromium alloy and zirconia, silicone, acrylic resin (polymethylmethacrylate) and nano-hybrid composite. Some samples were partially covered with lithium disilicate glass ceramic to study specifically the junction areas.C. albicans was incubated in a biofilm reactor at 37 °C with agitation. The biofilm formation was evaluated at 24 and 48 hours. In addition, the morphology of the biofilm was evaluated by scanning electron microscopy. RESULTS: C. albicans developed biofilms on the surface of all materials tested. Cobalt-chromium alloy showed the lowest density of adhered biofilm, followed by zirconia and titanium. Silicone and resin showed up to 20 times higher density of biofilm. A higher biofilm formation was observed when junctions of materials presented micropores or imperfections. CONCLUSIONS: The biofilm formed in the three materials used in the manufacture of abutments and prostheses showed no major differences, being far less dense than in the resins. Two clinical recommendations can be made: to avoid the presence of resins in the subgingival area of implant prostheses and to design prostheses placing cobalt-chromium alloy/ceramic or titanium/ceramic junctions as far as possible from implants.

Therapeutic tools for oral candidiasis: Current and new antifungal drugs.

BACKGROUND: Candidiasis is one of the most common opportunistic oral infections that presents different acute and chronic clinical presentations with diverse diagnostic and therapeutic approaches. The present study carries out a bibliographic review on the therapeutic tools available against oral candidiasis and their usefulness in each clinical situation. MATERIAL AND METHODS: Recent studies on treatment of oral candidiasis were retrieved from PubMed and Cochrane Library. RESULTS: Nystatin and miconazole are the most commonly used topical antifungal drugs. Both antifungal drugs are very effective but need a long time of use to eradicate the infection. The pharmacological presentations of miconazole are more comfortable for patients but this drug may interact with other drugs and this fact should be assessed before use. Other topical alternatives for oral candidiasis, such as amphotericin B or clotrimazole, are not available in many countries. Oral fluconazole is effective in treating oral candidiasis that does not respond to topical treatment. Other systemic treatment alternatives, oral or intravenous, less used are itraconazole, voriconazole or posaconazole. Available novelties include echinocandins (anidulafungin, caspofungin) and isavuconazole. Echinocandins can only be used intravenously. Isavuconazole is available for oral and intravenous use. Other hopeful alternatives are new drugs, such as ibrexafungerp, or the use of antibodies, cytokines and antimicrobial peptides. CONCLUSIONS: Nystatin, miconazole, and fluconazole are very effective for treating oral candidiasis. There are systemic alternatives for treating recalcitrant infections, such as the new triazoles, echinocandins, or lipidic presentations of amphotericin B.

Postantifungal effect of anidulafungin against Candida albicans, Candida dubliniensis, Candida africana, Candida parapsilosis, Candida metapsilosis and Candida orthopsilosis.

OBJECTIVE: Candida albicans remains the most common aetiology of invasive candidiasis, leading to high morbidity and mortality. Nevertheless, the incidence of candidiasis due to non-C. albicans species, such as Candida parapsilosis, is increasing. Postantifungal effect (PAFE) is relevant for establishing dosage schedules in antifungal therapy, as the frequency of antifungal administration could change depending on PAFE. The aim of this study was to evaluate the PAFE of anidulafungin against C. albicans, Candida dubliniensis, Candida africana, C. parapsilosis, Candida metapsilosis and Candida orthopsilosis. METHODS: Twenty-one Candida strains were evaluated. Cells were exposed to anidulafungin for 1 h at concentrations ranging from 0.12 to 8 mg/L for PAFE studies. Time-kill experiments (TK) were conducted at the same concentrations. The experiments were performed using an inoculum of 1-5 x 105 cells/mL and 48 h incubation. Readings of PAFE and TK were done at 0, 2, 4, 6, 24 and 48 h. RESULTS: Anidulafungin was fungicidal against 2 out of 14 (14%) strains of C. albicans related species in PAFE experiments. Moreover, 2 mg/L of anidulafungin exerted a prolonged PAFE (≥ 33.6 h) against 13 out of 14 (93%) strains. Similarly, fungicidal endpoint was achieved against 1 out of 7 (14%) strains of C. parapsilosis complex, being PAFE prolonged (≥ 42 h) against 6 out of 7 (86%) strains. CONCLUSIONS: Anidulafungin induced a significant and prolonged PAFE against C. albicans and C. parapsilosis and their related species.

The immunodiagnosis of Echinococcus multilocularis infection.

Alveolar echinococcosis (AE) is a severe zoonotic disease caused by the metacestode stage of Echinococcus multilocularis. The infection can have fatal consequences in humans if treatment is not provided, so early diagnosis is fundamental for initiating treatment and reducing morbidity and mortality. In addition, detection of the parasite in the definitive host plays a central role in epidemiological studies and surveillance programmes for control of AE. This review presents an overview of the present situation regarding the immunodiagnosis of E. multilocularis infection. Special attention is given to the description of the native, partially purified and recombinant antigens available currently for immunodiagnostic purposes. Recent advances in the primary serodiagnosis and follow-up of AE patients are highlighted, including the detection of specific cytokine profiles. Progress in the immunodiagnosis of intestinal E. multilocularis infection in definitive hosts, particularly the detection of excretory-secretory and integument products of the worm in faeces (copro-antigens) by ELISA, is also discussed.

Comparison of tablet and disk diffusion methods for fluconazole and voriconazole in vitro activity testing against clinical yeast isolates.

We have compared a commercially available tablet diffusion method for the in vitro antifungal susceptibility testing of fluconazole (FCZ) and voriconazole (VCZ) with the disk diffusion method M44 (CLSI) with 282 clinical yeast isolates. The superior stability of antifungal agents in tablets can explain the differences for each category of susceptibility by both methods.Neo-Sensitabs tablets antifungal susceptibility testing showed an excellent correlation (0.98 for FCZ and 0.98 for VCZ at 24h and 0.96 for FCZ and 0.94 for VCZ at 48 h ), a reduced percentage of disagreements (4.6% and 8.2% for FCZ at 24h and 48 h respectively; 1.1% and 2.1% for VCZ at 24h and 48 h respectively) and the absence of statistically significant difference in comparison with the reference protocol for performing antifungal susceptibility testing with the agar diffusion method.

Allergenic cross-reactivity between Blomia tropicalis and Blomia kulagini (Acari: Echymiopodidae) extracts from optimized mite cultures.

BACKGROUND: At present, data about the cross-reactivity of Blomia spp. comes from studies made among different genera of mites, and no results have been published involving different species of the genus Blomia. OBJECTIVE: The aim of this study was to find out the level of cross-reactivity between the two main species of Blomia causing allergy, and its implication in the diagnosis of Blomia sensitization. METHODS: Using extracts from optimal growth phases of Blomia kulagini (Zakhvatkin, 1936) and Blomia tropicalis (van Bronswijk, Cock and Oshima, 1973) as allergenic material, the allergenic cross-reactivity between both house dust mites was evaluated by means of cutaneous tests, specific IgE values, ImmunoCAP-inhibition and SDS-PAGE-IgE-immunoblotting-inhibition. RESULTS: The results demonstrated that IgE-binding components belonging to both species are very similar from the immunological point of view, showing high correlations between both species when using cutaneous tests (R2=0.915) or specific IgE (R2=0.980). ImmunoCAP-inhibition and SDS-PAGE-IgE-Immunoblotting-inhibition probed with human sera, showed a total inhibition of specific IgE reactions by the heterologous antigens. CONCLUSION: The results obtained strongly suggest the great resemblance between the allergenic composition of both species.

Cross-reactions between Dermatophagoides pteronyssinus and Dermatophagoides farinae (Acari: Pyroglyphidae) related to the different growth phases of cultures.

The allergenic cross-reactivity of both inter- and intraspecies of house dust mites, Dermatophagoides pteronyssinus (Trouessart, 1897) and Dermatophagoides farinae Hughes, 1961, taking into account the allergenic differences that exist throughout the growth curves, was evaluated by means of RAST-inhibition, using sera from patients allergic to these mites. The results demonstrate that extracts obtained from mite cultures during the maximum exponential growth phase are the best source of reagents to better discriminate cross-reactivity studies. The analyses obtained from this work, together with those obtained in previous reports, help to define the ideal conditions related to the allergenic diversity, avidity, and cross-reactivity of specific antibodies for the elaboration of allergenic extracts as a tool for use in diagnosis and specific treatment of IgE-mediated hypersensitivity caused by house dust mites.

Enzymatic analyses of house dust mite extracts from Dermatophagoides pteronyssinus and Dermatophagoides farinae (Acari: Pyroglyphidae) during different phases of culture growth.

The majority of clinically important allergens of Dermatophagoides pteronyssinus (Trouessart, 1897) and Dermatophagoides farinae Hughes, 1961 present enzymatic activity. The allergenic enzymes described include cysteine proteases in group 1 allergens, trypsins in group 3, amylases in group 4, and chymotrypsins in group 6. Apart from these, other possibly allergenic enzymes also have been identified. Therefore, enzymatic profiles were studied during the 3 growth periods of the mite population--latency phase, exponential growth phase, and death phase. The activity of 19 different enzymes was analyzed by means of the Api Zym system, a method that has been used to study both mite extracts and other allergenic materials. Our study has demonstrated that the extracts contain a large variety of enzymes. It has been observed that enzymatic activity is caused exclusively by mites because the control carried out on the culture medium was negative for all the enzymes studied. Generally, the levels of diverse enzymatic activity increased with the growth of the culture, and decreased later, in both species. However, proteases are the exception; they maintain a high level of activity during the death phase of the cultured mites. The ratio between trypsin and chymotrypsin activity can be used as an excellent tool for quality control parameters during obtention of allergenic mite extracts.

Analysis of the allergen expression of Blomia tropicalis and Blomia kulagini (Astigmata: Glycyphagidae) cultures.

Laboratory cultures of the mites Blomia tropicalis (van Bronswijk, Cock & Oshima) and Blomia kulagini (Zakhvatkin) were used to study the population dynamics of the mites and the kinetics of released allergens during the growth cycle. The analysis of extracts obtained after different incubation periods, by means of immunoblotting, and quantification of the major allergen Blo t 5, allowed definition of three different growth phases, demonstrating that mite cultures during the maximum growth (end of exponential growth curve-beginning maximum growth plateau) contain the largest amount of allergenic components as well as the highest Blo t 5 concentration.

Kinetics of allergen expression in cultures of house dust mites, Dermatophagoides pteronyssinus and D. farinae (Acari: Pyroglyphidae).

Laboratory cultures of house dust mites Dermatophagoides pteronyssinus (Trouessart, 1897) and Dermatophagoides farinae Hughes, 1961 were used to study the population dynamics of the mites and the kinetics of antigen appearance. The analysis of extracts obtained after different incubation periods, carried out by SDS-PAGE, immunoblotting, and enzyme-linked immunosorbent assay, allows for the definition of 3 different growth phases: the latency phase (F1); the exponential growth phase (F2) during which the allergenic proteins, including the Der 1 and Der 2 major allergens, were expressed more intensely and in larger quantities; and a final phase (F3), death, in which the lowest rates of allergenic components with a clearly different pattern were seen. The data obtained from this work demonstrates that mite cultures during the maximum growth phase (F2) contain the largest amount of allergenic components as well as the highest major allergen concentrations.

Application of random amplified polymorphic DNA (RAPD) assay to the study of mites related to allergic diseases.

In this study, the conditions for the successful application of the random amplified polymorphic DNA (RAPD) assay to differentiate mite populations based on genetic variation were defined. Five species of mites related to allergic diseases were studied: Dermatophagoides pteronyssinus, D. farinae (2 strains), Blomia tropical is, Glycyphagus domesticus and Tyrophagus putrescentiae. The mites were isolated from pure cultures and processed according to the method described in this paper. The banding patterns obtained were different for all the species studied. When the DNA from two different strains of D. farinae were studied, the "fingerprint" banding patterns obtained showed differences between them. The random amplified polymorphic DNA assay may be a useful tool to aid the taxonomic study of mite populations.

Total and specific IgE levels in human hydatid disease determined by enzyme immunoassay: serological follow-up after surgery.

The evolution of both specific and nonspecific IgE in a long-term follow-up after surgery in patients with human hydatid disease was studied. Enzyme immunoassays using cyanogen bromide-activated cellulose discs as solid phase were employed. One hundred and nine postoperative serum samples from 26 patients undergoing surgery for hydatid disease were studied. Imaging studies were also carried out during the follow-up. In 8 of 26 patients, remaining cysts were detected during the follow-up. One year after surgery, total IgE levels decreased to normal values in 84.6% of the total number of patients, in 94.5% of the cases in which no remaining cysts were detected, and in 62.5% of the patients with remaining hydatid cysts. These data highlight the poor value of an isolated postoperative IgE determination as a diagnostic marker for remaining hydatidosis. On the contrary, 1 year after surgery, the levels of anti-Echinococcus IgE decreased in 55% of the patients without residual cysts and in 50% of the total number of patients. In six patients without remaining hydatid cysts, the levels of specific IgE increased 1 year after the surgery. In the group of patients with remaining cysts only in three patients did the values of specific IgE decrease, although they remained significant. Thus, 1 year after surgery, anti-Echinococcus IgE levels were still evident in all patients, although in those without remaining cysts there was a predominance of decreasing values.(ABSTRACT TRUNCATED AT 250 WORDS)

Influence of mite growth culture phases on the biological standardization of allergenic extracts.

House dust mites are a well known cause of asthma and other respiratory allergies. In order to improve the standardization of allergenic extracts for diagnosis and immunotherapy, it is important to determine the frequency and concentration of the components, both the major and the minor allergens during the growth period of the mite population. In a previous paper we demonstrated that the laboratory cultures of Dermatophagoides pteronyssinus and Dermatophogoides farinae exhibited three well differentiated growth phases: latency, exponential growth, and death of the culture. Biological standardization of extracts from the two mite species were carried out by skin prick tests in a group of 20 patients, using different concentrations of the extracts at the three growth phases. The patient sera were also studied by means of the RAST technique to determine the levels of specific IgE for each phase. The extracts produced from the exponential growth phase of the cultures revealed six times more relative allergenic activity in in vivo studies, and average RAST values were approximately three times higher than those extracts from latency and death phases. The reproducibility of the extract production method was assessed by comparing different batches obtained in similar conditions. The results showed batch-to-batch homogeneity allergenic activity. In conclusion, it was demonstrated that extracts obtained from cultures with the highest concentration of live mites (maximum growth phase) render the best diagnostic results in vivo and in vitro.

Extracts from various mite species contain cross-reactive and noncross-reactive IgE epitopes. A RAST inhibition study.

Previous studies have demonstrated the high incidence of Dermatophagoides, Euroglyphus, Blomia, Lepidoglyphus and Chortoglyphus spp. sensitizations in a mite-allergic population. The aim of this study was to evaluate immunological cross-reactivity among the above mentioned groups, using sera from a nonrural population allergic to mites, from a subtropical area (Canary Islands). RAST inhibition studies demonstrated significant cross-reactivity among Dermatophagoides and Euroglyphus species (> or = 65% of maximum theoretical inhibition), as also noted by other authors. Blomia kulagini demonstrated scarce cross-reactivity with Dermatophagoides, Lepidoglyphus and Chortoglyphus species (< or = 30% of maximum theoretical inhibition) and medium level with Euroglyphus maynei (45% of maximum theoretical inhibition). Chortoglyphus arquatus demonstrated high cross-reactivity levels with the other species studied. The results obtained in this study demonstrated the scarce immunological cross-reactivity between Pyroglyphidae and non-Pyroglyphidae mites, thus suggesting the polysensitization of the studied population to different mite species.

Antifungal activity of posaconazole against Candida spp. and non-Candida clinical yeasts isolates.

The in vitro antifungal activity of posaconazole was tested against 315 yeast clinical isolates and 11 ATCC reference strains by means an agar diffusion method (Neosensitabs, Rosco,Denmark) based in CLSI M44-A2 document. Posaconazole activity was excellent against Cryptococcus and Rhodotorula species studied and showed very good activity against most species of Candida tested. A total of 13 clinical isolates (4.1%) were resistant: Candida albicans (n=5), Candida glabrata (n=5), Candida tropicalis (n=1), Geotrichum australiensis (n=1) and Geotrichum capitatum (n=1). Our results suggest posaconazole is an effective antifungal agent against the most clinically important yeasts species (92.7% of susceptibility). Agar diffusion method provides good conditions for the posaconazole susceptibility study in the routine laboratory.

Enzymatic analysis of Blomia tropicalis and Blomia kulagini (Acari: Echimyopodidae) allergenic extracts obtained from different phases of culture growth.

The majority of important allergenic extracts from arthropods present enzymatic activity. This activity has been studied particularly in Dermatophagoides house dust mites because of its implication in the stability and immunogenicity of extracts used as tools for the diagnosis and specific treatment of allergic diseases. Extracts from cultures of Blomia tropicalis [van Bronswijk (1973a, b). Acarologia 15:477-489, 490-505] and Blomia kulagini (Zakhvatkin 1936) were used to study enzymatic profiles during three growth periods of the mite population: latency phase, maximum mite concentration during exponential growth, and drop stage. The activities of 19 enzymes were analyzed using the Api Zym system. The results show a large variety of enzymes. Some enzymatic activity was found to be (almost) exclusively attributable to mites. The activity levels of proteases, glycosidases and lipases overlapped with the growth curve. Only phosphatase activity showed no significant change during mite growth when compared with the culture medium. We suggest that the glycosidases (beta-galactosidase, beta-glucuronidase, beta-N-acetylglucosaminidase, alpha-mannosidase and alpha-fucosidase) and proteases (leucine aminopeptidase and trypsin) may constitute suitable parameters for inclusion in the quality control process for the production of allergenic mite extracts, and may help define a new index for conducting environmental controls.

Quality parameters for the production of mite extracts.

Mites present in house dust are of great etiological importance in type I hypersensitivity, with those belonging to the Dermatophagoides genus (D. pteronyssinus and D. farinae), of the Pyroglyphidae family, being the most frequent and principal source of allergens. For the production of allergenic extracts destined for specific diagnostic and treatment purposes of allergic diseases, the culture of such mites is absolutely necessary. In accordance with studies carried out in our laboratories to obtain adequate extracts, one must bear in mind the culture mite phase. Three growth phases have been distinguished for both species: latency phase (F1), growth phase (F2) in which the allergenic proteins are expressed with greater intensity, and death phase of the culture (F3). In the same study, the biological standardization of the extracts demonstrated that those produced from the maximum growth phase gave both in vitro and in vivo results, at least three times more sensitive than those from the other phases. We checked the reproducibility of the production method, obtaining different batches in similar conditions with a high homogeneity regarding allergenic activity. The sensitivity and specificity of the allergenic extracts depends just as much upon the production method as the standardization method. During the biological cycle of Dermatophagoides in culture, it is only from the maximum growth phase (F2), that allergenic extracts with an excellent diagnostic value, high sensitivity and specificity, can be obtained.