María D. Moragues

A Monoclonal Antibody Directed against a Candida albicans Cell Wall Mannoprotein Exerts Three Anti-C. albicans Activities

ABSTRACT Antibodies are believed to play a role in the protection against Candida albicans infections by a number of mechanisms, including the inhibition of adhesion or germ tube formation, opsonization, neutralization of virulence-related enzymes, and direct candidacidal activity. Although some of these biological activities have been demonstrated individually in monoclonal antibodies (MAbs), it is not clear if all these anti-C. albicans activities can be displayed by a single antibody. In this report, we characterized a monoclonal antibody raised against the main target of salivary secretory immunoglobulin A in the cell wall of C. albicans, which exerts three anti-C. albicans activities: (i) inhibition of adherence to HEp-2 cells, (ii) inhibition of germination, and (iii) direct candidacidal activity. MAb C7 reacted with a proteinic epitope from a mannoprotein with a molecular mass of >200 kDa predominantly expressed on the C. albicans germ tube cell wall surface as well as with a number of antigens from Candida lusitaniae, Cryptococcus neoformans, Aspergillus fumigatus, and Scedosporium prolificans. MAb C7 caused a 31.1% inhibition in the adhesion of C. albicans to HEp-2 monolayers and a 55.3% inhibition in the adhesion of C. albicans to buccal epithelial cells, produced a 38.5% decrease in the filamentation of C. albicans, and exhibited a potent fungicidal effect against C. albicans, C. lusitaniae, Cryptococcus neoformans, A. fumigatus, and S. prolificans, showing reductions in fungal growth ranging from 34.2 to 88.7%. The fungicidal activity showed by MAb C7 seems to be related to that reported by antibodies mimicking the activity of a killer toxin produced by the yeast Pichia anomala, since one of these MAbs also reacted with the C. albicans mannoprotein with a molecular mass of >200 kDa. Results presented in this study support the concept of a family of microbicidal antibodies that could be useful in the treatment of a wide range of microbial infections when used alone or in combination with current antimicrobial agents.

Evaluation of the New Chromogenic Medium Candida ID 2 for Isolation and Identification of Candida albicans and Other Medically Important Candida Species

ABSTRACT The usefulness of Candida ID 2 (CAID2) reformulated medium (bioMérieux, France) has been compared with that of the former Candida ID (CAID; bioMérieux), Albicans ID 2 (ALB2; bioMérieux), and CHROMagar Candida (CAC; Chromagar, France) chromogenic media for the isolation and presumptive identification of clinically relevant yeasts. Three hundred forty-five stock strains from culture collections, and 103 fresh isolates from different clinical specimens were evaluated. CAID2 permitted differentiation based on colony color between Candida albicans (cobalt blue; sensitivity, 91.7%; specificity, 97.2%) and Candida dubliniensis (turquoise blue; sensitivity, 97.9%; specificity, 96.6%). Candida tropicalis gave distinguishable pink-bluish colonies in 97.4% of the strains in CAID2 (sensitivity, 97.4%; specificity, 100%); the same proportion was reached in CAC, where colonies were blue-gray (sensitivity, 97.4%; specificity, 98.7%). CAC and CAID2 showed 100% sensitivity values for the identification of Candida krusei. However, with CAID2, experience is required to differentiate the downy aspect of the white colonies of C. krusei from other white-colony-forming species. The new CAID2 medium is a good candidate to replace CAID and ALB2, and it compares well to CAC for culture and presumptive identification of clinically relevant Candida species. CAID2 showed better results than CAC in some aspects, such as quicker growth and color development of colonies from clinical specimens, detection of mixed cultures, and presumptive differentiation between C. albicans and C. dubliniensis.

Point prevalence, microbiology and antifungal susceptibility patterns of oral Candida isolates colonizing or infecting Mexican HIV/AIDS patients and healthy persons.

We have conducted a longitudinal study over a 3-year period to address the point prevalence, microbiological characteristics and antifungal susceptibility patterns of yeast isolates colonizing or infecting the oral cavities of 111 HIV-infected (51 adults, 60 children) and 201 non HIV-infected (109 adults, 92 children) Mexican persons. Regarding the epidemiology of oral candidiasis, Candida albicans was the most frequent species isolated. Seventy-one out of 85 isolates from colonized persons were C. albicans (83.5%), 27 isolates of them were from HIV-infected children and 44 from non HIV-infected patients. Sixty-two isolates belonged to serotype A which was the most prevalent serotype of C. albicans. Non-albicans species (Candida glabrata, Candida tropicalis and Candida parapsilosis, and Saccharomyces cerevisiae) were isolated from 16.5% of colonized patients and from 38.5% patients with candidiasis or Candida-related lesions. There were nine episodes of infection or colonization by at least 2 different yeast species. In the case of HIV/AIDS patients, it was determined that yeast carriage was not associated with the number of CD4+ cells or the viral load, but HAART reduced the prevalence of oral candidiasis. Overall, most patients harbored strains in vitro susceptible to fluconazole, however 10.8% of the yeasts were resistant to one or more azole antifungal agents and 29% were intermediate susceptible to them. On the contrary, 5-fluorocytosine was very active against all isolates tested, and amphotericin B was active against 97.9% of them.

Oral Candida Isolates Colonizing or Infecting Human Immunodeficiency Virus-Infected and Healthy Persons in Mexico

ABSTRACT Oral yeast carriage was studied in 312 Mexican subjects. Candida albicans was the most frequent species, but other Candida spp. were isolated from 16.5 to 38.5% of patients. Colonization did not correlate with CD4+ number or viral load, but highly active antiretroviral therapy reduced the frequency of candidiasis. Most isolates were susceptible to fluconazole, but 10.8% were resistant to one or more azoles.

Casein Agar: a Useful Medium for Differentiating Candida dubliniensis from Candida albicans

ABSTRACT Production of chlamydospores on casein agar at 24°C for 48 h provides a simple means for differentiating Candida dubliniensis from Candida albicans based on chlamydospore production. Of 109 C. dubliniensis isolates tested on this medium, 106 (97.2%) produced abundant chlamydospores and three produced few chlamydospores. In contrast, of the 120 C. albicans isolates tested, 111 (92.5%) failed to produce any chlamydospores, whereas the remaining nine isolates produced few chlamydospores. These findings indicate that abundant chlamydospore production on casein agar is a useful test for discriminating between C. dubliniensis and C. albicans.

EvaluaciÓn de una nueva técnica comercializada (Candida albicans IFA IgG) para el diagnÓstico de la candidiasis invasiva

Introduccion Se comparan dos pruebas para detectar anticuerpos antimicelio en pacientes con candidiasis invasiva: una nueva tecnica comercializada (Candida albicans IFA IgG) y la tecnica de inmunofluorescencia indirecta tradicionalmente empleada para la deteccion de anticuerpos contra la fase micelial de C. albicans (anticuerpos antimicelio) Metodos Mediante dos tecnicas de inmunofluorescencia indirecta se estudiaron retrospectivamente un total de 172 sueros de 51 pacientes clasificados en dos grupos: el grupo I incluia 123 sueros de 32 pacientes con candidiasis invasiva y el grupo II, utilizado como control, comprendia 49 sueros de 19 pacientes sin evidencia de infeccion por Candida Resultados El 84,4% de los pacientes con candidiasis invasiva (grupo I) presentaron titulos de anticuerpos antimicelio ≥ 1:160 con la prueba Candida albicans IFA IgG, mientras que el 78,1% lo hicieron con la tecnica tradicional. Ambas tecnicas presentaron una alta correlacion al ser comparadas entre si (R2= 0,9512 por pacientes; R2= 0,8986 por sueros). Utilizando un titulo de corte de anticuerpos antimicelio ≥ 1:160 para el diagnostico de la candidiasis invasiva, la prueba Candida albicans IFA IgG presento una sensibilidad del 84,4% y una especificidad del 94,7%, mientras que la tecnica tradicional presento una sensibilidad del 78,1% y una especificidad del 100%. Conclusion. La prueba comercializada Candida albicans IFA IgG es muy similar a la prueba tradicionalmente empleada para la deteccion de anticuerpos antimicelio, permitiendo un diagnostico mas rapido y sencillo de la candidiasis invasiva en los laboratorios de microbiologia hospitalarios

Antibody complementarity-determining regions (CDRs) can display differential antimicrobial, antiviral and antitumor activities.

Background Complementarity-determining regions (CDRs) are immunoglobulin (Ig) hypervariable domains that determine specific antibody (Ab) binding. We have shown that synthetic CDR-related peptides and many decapeptides spanning the variable region of a recombinant yeast killer toxin-like antiidiotypic Ab are candidacidal in vitro. An alanine-substituted decapeptide from the variable region of this Ab displayed increased cytotoxicity in vitro and/or therapeutic effects in vivo against various bacteria, fungi, protozoa and viruses. The possibility that isolated CDRs, represented by short synthetic peptides, may display antimicrobial, antiviral and antitumor activities irrespective of Ab specificity for a given antigen is addressed here. Methodology/Principal Findings CDR-based synthetic peptides of murine and human monoclonal Abs directed to: a) a protein epitope of Candida albicans cell wall stress mannoprotein; b) a synthetic peptide containing well-characterized B-cell and T-cell epitopes; c) a carbohydrate blood group A substance, showed differential inhibitory activities in vitro, ex vivo and/or in vivo against C. albicans, HIV-1 and B16F10-Nex2 melanoma cells, conceivably involving different mechanisms of action. Antitumor activities involved peptide-induced caspase-dependent apoptosis. Engineered peptides, obtained by alanine substitution of Ig CDR sequences, and used as surrogates of natural point mutations, showed further differential increased/unaltered/decreased antimicrobial, antiviral and/or antitumor activities. The inhibitory effects observed were largely independent of the specificity of the native Ab and involved chiefly germline encoded CDR1 and CDR2 of light and heavy chains. Conclusions/Significance The high frequency of bioactive peptides based on CDRs suggests that Ig molecules are sources of an unlimited number of sequences potentially active against infectious agents and tumor cells. The easy production and low cost of small sized synthetic peptides representing Ig CDRs and the possibility of peptide engineering and chemical optimization associated to new delivery mechanisms are expected to give rise to a new generation of therapeutic agents.

Detection of (1 → 3)-β-D-glucan as an adjunct to diagnosis in a mixed population with uncommon proven invasive fungal diseases or with an unusual clinical presentation.

ABSTRACT This single-center observational prospective study evaluated the performance of (1→3)-β-d-glucan as an adjunct diagnostic tool in 12 patients with proven invasive fungal disease with different risk factors. The infections were due to either uncommon fungal pathogens such as dematiaceous molds (Scedosporium apiospermum, Alternaria infectoria, and Cladosporium macrocarpum) and hyaline septate molds (Fusarium solani and Blastoschizomyces capitatus) or Aspergillus spp. with unusual clinical presentations.

Effect of salivary secretory IgA on the adhesion of Candida albicans to polystyrene.

Attachment of Candida albicans to plastic materials of dental prostheses or to salivary macromolecules adsorbed on their surface is believed to be a critical event in the development of denture stomatitis. In an earlier study, it was shown that adhesion of C. albicans to polystyrene, a model system to study the adhesion of C. albicans to plastic materials, can be partially inhibited with an mAb directed against cell wall polysaccharides of C. albicans. In the present study, the role of whole saliva in the adhesion of C. albicans to polystyrene has been investigated, and three mAbs directed against epitopes of cell wall mannoproteins have been used to mimic the inhibitory effect observed with salivary secretory IgA (sIgA) on the adhesion of C. albicans to polystyrene. In the absence of whole saliva, adherence of C. albicans 3153 increased with germination. However, the presence of whole saliva enhanced the adhesion to polystyrene of C. albicans 3153 yeast cells but decreased the adhesion of germinated cells. The enhancement of adhesion of yeast cells to polystyrene mediated by saliva was confirmed with an agerminative mutant of C. albicans 3153. The inhibition of the adhesion of C. albicans 3153 germ tubes to polystyrene was due to the salivary sIgA since sIgA-depleted saliva enhanced the adhesion of C. albicans 3153 to polystyrene. The inhibitory effect mediated by sIgA was not related to the inhibition of germination but to the blockage of adhesins expressed on the cell wall surface of the germ tubes. The three mAbs studied reduced the adhesion of C. albicans 3153 to polystyrene at levels equivalent to those for purified sIgA. The highest reduction in the adhesion was obtained with the IgA mAb N3B. The best results were obtained when the three mAbs were combined. The results suggest that whole saliva plays a different role in the adhesion of C. albicans to polystyrene depending on the morphological phase of C. albicans. These results may give new insights into the conflicting role of saliva in the adhesion of C. albicans to plastic materials of dental prostheses.

Supplementation of CHROMagar Candida Medium with Pal's Medium for Rapid Identification of Candida dubliniensis

ABSTRACT CHROMagar Candida medium is used for the isolation and identification of Candida species, but it does not differentiate Candida albicans from Candida dubliniensis. This differentiation can be achieved by using Pals agar, which cannot be used in primary isolation. We have combined both media to obtain a new medium that can be used for the isolation and identification of C. dubliniensis in primary cultures.