Use of 65kDa mannoprotein gene primers in PCR methods for the identification of five medically important Candida species.

We have developed PCR and Multiplex PCR assays for the detection of medically important Candida spp. using different species and genus-specific PCR primers selected within the MP65 gene, a recently cloned gene encoding a mannoprotein adhesin. The genus-specific PCR primers were able to amplify Candida species DNA (100% positivity) whereas DNA from all other isolates tested, belonging to other fungal genera, was not amplified. The species-specific PCR primers allowed differentiation of each of five Candida species by the amplicon length produced. No amplicons were detected using species- or genus-specific primers in several bacterial or human DNA templates. The methods described in this study are reproducible, simple and specific. The total time required for each PCR method was less than 4 h from the extraction to the visualized amplicons after PCR. In conclusion, we developed PCR methods to differentiate the five most medically important Candida species using primers directed to the MP65 gene.

An outline of the role of anti-Candida antibodies within the context of passive immunization and protection from candidiasis.

The role played by antibodies (Abs) in the anticandidal defense has long been a matter of controversy, mostly due to the past inability to clearly define antigen specificity, the relationship between the type of immune response within the different settings of experimental and human candidiasis and, last but not least, a misunderstanding about the role of T helper cell in cell-mediated versus the humoral immunity. Contributory was also the lack of precise identification of virulence traits of the fungus which are the best candidates for a protective Ab response. In recent years, an impressive amount of experimental evidence, and also some clinical proof, have been generated which assign to Abs of defined specificity an important role in the anticandidal defense both at systemic and mucosal sites. Paradigmatic among them, Abs against defined virulence factors such as adhesins or aspartyl-proteinase enzymes, or against critical viability molecules such as beta-glucan, have been detected or generated which hold great promise for immunotherapeutic interventions in humans.

Construction and use of PCR primers from a 65 kDa mannoprotein gene for identification of C. albicans.

A method for detection of Candida albicans in biological samples (blood, serum, urine) was developed by the use of polymerase chain reaction (PCR) amplification of a DNA fragment of a gene coding for a 65 kDa mannoprotein of C. albicans (CaMP65). The PCR amplifies a 220 bp fragments whose specificity for C. albicans was demonstrated by Southern blot with a non-radioactive probe, leading to the differentiation from all other yeast species or human and bacterial DNA. The sensitivity of this assay was 5-10 C. albicans cells per milliliter of biological sample.

Aspartyl proteinases of Candida albicans and their role in pathogenicity.

Among the putative virulence factors of Candida albicans, secreted aspartic proteinases (Sap, encoded by a family of at least nine genes) continue to attract the attention of many investigators studying the pathogenesis of candidiasis. Several early studies documented a correlation between the levels of Sap secretion and the virulence of different strains, but much stronger support for this role has been provided by more recent data on differential SAP gene(s) expression in ex vivo and in vivo models, the outcome of infections with SAP-deleted mutants, and use of Sap immunogens. In particular, some SAP-deleted strains suffered a substantial loss of virulence, and, more interestingly, this was specifically associated with selected gene products and selected experimental pathologies. Moreover, anti-Sap antibodies have been shown to mediate a degree of protection in an experimental, mucosal candidiasis model. There is now initial evidence that distinct Saps are differentially produced in various Candida illnesses or stages of them. The exact mechanisms of each Sap involvement in any particular Candida disease, with special regard to human infections, and how the immune system deals with Sap, are critical issues for future research. An answer to these questions will possibly facilitate the generation of Sap-based anticandidal drugs or immunotherapeutics.

Recombinant Streptococcus gordonii for mucosal delivery of a scFv microbicidal antibody.

The gram-positive bacterium Streptococcus gordonii was engineered to express the microbicidal molecule H6, which is an antiidiotypic single chain antibody mimicking a yeast killer toxin. S. gordonii is a human commensal which we developed as a model system for mucosal delivery of heterologous proteins. The in vivo candidacidal activity of both H6-secreting and H6-surface-displaying streptococcal strains were assayed in a well-established rat model of vaginal candidiasis. At day 21 full clearance of Candida albicans infection was observed in 75% of animals treated with the H6-secreting strain, and in 37.5% of animals treated with the strain expressing H6 on the surface, while all animals treated with the control strain were still infected. The observed candidacidal effect was comparable with that observed with the antimycotic drug fluconazole. These data confirm the potential of H6 as a candidacidal agent and show how promising is the approach of using recombinant bacteria for mucosal delivery of biologically active molecules.

Therapy of mucosal candidiasis by expression of an anti-idiotype in human commensal bacteria.

Two recombinant strains of Streptococcus gordonii, secreting or displaying a microbicidal single-chain antibody (H6), and stably colonizing rat vagina, were used to treat an experimental vaginitis caused by Candida albicans. A post-challenge intravaginal delivery of the H6-secreting strain was as efficacious as fluconazole in rapidly abating the fungal burden. Three weeks after challenge, 75% and 37.5% of the rats treated with the H6-secreting or displaying bacteria, respectively, were cured of the infection, which persisted in 100% of the animals treated with a S. gordonii strain expressing an irrelevant single-chain antibody. Thus, a human commensal bacterium can be suitably engineered to locally release a therapeutic antibody fragment.

Local anticandidal immune responses in a rat model of vaginal infection by and protection against Candida albicans.

Humoral (antibody [Ab]) and cellular Candida-specific immune responses in the vaginas of pseudoestrus rats were investigated during three successive infections by Candida albicans. After the first, protective infection, Abs against mannan and aspartyl proteinase antigens were present in the vaginal fluid, and their titers clearly increased during the two subsequent, rapidly healing infections. In all animals, about 65 and 10% of vaginal lymphocytes (VL) were CD3(+) (T cells) and CD3(-) CD5(+) (B cells), respectively. Two-thirds of the CD3(+) T cells expressed the alpha/beta and one-third expressed the gamma/delta T-cell receptor (TCR). This proportion slightly fluctuated during the three rounds of C. albicans infection, but no significant differences between infected and noninfected rats were found. More relevant were the changes in the CD4(+)/CD8(+) T-cell ratio, particularly for cells bearing the CD25 (interleukin-2 receptor alpha) marker. In fact, a progressively increased number of both CD4(+) alpha/beta TCR and CD4(+) CD25(+) VL was observed after the second and third Candida challenges, reversing the high initial CD8(+) cell number of controls (estrogenized but uninfected rats). The CD3(-) CD5(+) cells also almost doubled from the first to the third infection. Analysis of the cytokines secreted in the vaginal fluid of Candida-infected rats showed high levels of interleukin 12 (IL-12) during the first infection, followed by progressively increasing amounts of IL-2 and gamma interferon during the subsequent infections. No IL-4 or IL-5 was ever detected. During the third infection, VL with in vitro proliferative activity in response to an immunodominant mannoprotein antigen of C. albicans were present in the vaginal tissue. No response to this antigen by mitogen-responsive blood, lymph node, and spleen cells was found. In summary, the presence of protective Ab and T helper type 1 cytokines in the vaginal fluids, the in vitro proliferation of vaginal lymphocytes in response to Candida antigenic stimulation, and the increased number of activated CD4(+) cells and some special B lymphocytes after C. albicans challenge constitute good evidence for induction of locally expressed Candida-specific Ab and cellular responses which are potentially involved in anticandidal protection at the vaginal level.

Biotyping and virulence properties of skin isolates of Candida parapsilosis.

The biotype and virulence of skin isolates of Candida parapsilosis were compared with blood isolates of the same fungus. Morphotype, resistotype, and electrophoretic karyotype determinations did not reveal any special cluster with a unique or dominant pathogenic feature among all of the isolates, regardless of their source. However, all cutaneous isolates had uniformly elevated secretory aspartyl-protease (Sap) activity, more than four times higher than the enzyme activity of the blood isolates. They were also highly vaginopathic in a rat vaginitis model, being significantly more virulent than blood isolates in this infection model. In contrast, skin isolates were nonpathogenic in systemic infection of cyclophosphamide-immunodepressed mice, while some blood isolates were, in this model, highly pathogenic (median survival time, 2 days, with internal organ invasion at autopsy). Finally, skin isolates did not differ, as a whole, from blood isolates in their adherence to plastic. This property was associated with a morphotype, as defined by a colony with continuous fringe, which was present among both skin and blood isolates. While confirming the genetic heterogenicity of C. parapsilosis, our data strongly suggest that the potential of this fungus to cause mucosal disease is associated with Sap production and is substantially distinct from that of systemic invasion.

Avirulence of Candida albicans CaHK1 mutants in a murine model of hematogenously disseminated candidiasis.

Deletion of both alleles of the Candida albicans CaHK1 gene, which causes cells to flocculate when grown at pH 7.5, a pH comparable to that of mammalian blood, abolishes the ability of the yeast to establish a successful infection in a murine model of hematogenously disseminated candidiasis. Within 72 h all mice inoculated with the parental C. albicans strain had died. The mice infected with either the heterozygote or revertant strain, either of which harbors only one functional CaHK1 allele, also succumbed to the infection, although survivors were observed for up to 16 days postinfection. However, mice inoculated with the Deltacahk1 null strain survived for the course of the infection. These results indicate that CaHK1 is required for the virulence of C. albicans in a murine model of hematogenously disseminated candidiasis. In contrast, CaHK1 is not required for the virulence of C. albicans in a rat model of vaginal candidiasis.

In vitro and in vivo anticandidal activity of human immunodeficiency virus protease inhibitors.

Highly active antiretroviral therapy that includes human immunodeficiency virus (HIV) aspartyl protease inhibitors (PIs) causes a decline in the incidence of some opportunistic infections in AIDS, and this decline is currently attributed to the restoration of specific immunity. The effect of two PIs (indinavir and ritonavir) on the enzymatic activity of a secretory aspartyl protease (Sap) of Candida albicans (a major agent of mucosal disease in HIV-infected subjects) and on growth and experimental pathogenicity of this fungus was evaluated. Both PIs strongly (>/=90%) and dose dependently (0.1-10 microM) inhibited Sap activity and production. They also significantly reduced Candida growth in a nitrogen-limited, Sap expression-dependent growth medium and exerted a therapeutic effect in an experimental model of vaginal candidiasis, with an efficacy comparable to that of fluconazole. Thus, besides the expected immunorestoration, patients receiving PI therapy may benefit from a direct anticandidal activity of these drugs.

Role of protease inhibitors in preventing recurrent oral candidosis in patients with HIV infection: a prospective case-control study.

This study was conducted to evaluate the efficacy of highly active anti-retroviral therapy (HAART) in preventing recurrence of oral candidosis (OC) associated with HIV. A prospective case-controlled observational study was performed in an inner-city university-hospital HIV/AIDS clinic. Ninety-three HIV-positive study subjects with a history of recurrent OC were divided into two groups: protease inhibitors (PI)-treated patients (group 1, n = 30) and non-PI-treated patients (group 2, n = 63). Study subjects were matched for sex, age, stage of HIV infection, and peripheral CD4+ T-cell counts. The non-PI-treated group was further subdivided into the following three subgroups: HIV-positive study subjects treated with reverse transcriptase inhibitors (RTI; groups 2a and 2c) and HIV-positive study subjects not treated with RTIs (group 2b). Group 2c met the same inclusion criteria as group 2a had but was matched 6 months after the beginning of the study. We also assessed in vitro peripheral blood mononuclear cells (PBMC) and their lymphoproliferative response, as well as cutaneous delayed-type hypersensitivity (DTH) response to Candida-associated antigens in a randomly selected sample of study subjects divided into those treated with PIs and those who were not. During a 1-year follow-up, OC was diagnosed in 2 (7%) PI-treated and 23 (36%) non-PI-treated patients (p<.001). In addition to comparing findings in group 1 with those in group 2c, OC was detected in 14 (50%) non-PI-treated patients compared with no HAART-treated study subjects (p<.001). Only 41% of PI-treated study subjects had positive lymphoproliferative response in PBMCs and none was positive in terms of DTH to Candida antigens (p = not significant versus non-PI-treated study subjects). While objectively demonstrating a beneficial effect of HAART in preventing recurrence of OC infections, our findings suggest this effect cannot be not fully accounted for by reconstitution of anti-Candida cell-mediated immunity, given that other mechanisms, even of a nonimmune nature, could have some effect.

High aspartyl proteinase production and vaginitis in human immunodeficiency virus-infected women.

Vaginal isolates of Candida albicans from human immunodeficiency virus-positive (HIV+) and HIV- women with or without candidal vaginitis were examined for secretory aspartyl proteinase (Sap) production in vitro and in vivo and for the possible correlation of Sap production with pathology and antimycotic susceptibility in vitro. HIV+ women with candidal vaginitis were infected by strains of C. albicans showing significantly higher levels of Sap, a virulence enzyme, than strains isolated from HIV+, C. albicans carrier subjects and HIV- subjects with vaginitis. The greater production of Sap in vitro was paralleled by greater amounts of Sap in the vaginal fluids of infected subjects. In an estrogen-dependent, rat vaginitis model, a strain of C. albicans producing a high level of Sap that was isolated from an HIV+ woman with vaginitis was more pathogenic than a strain of C. albicans that was isolated primarily from an HIV-, Candida carrier. In the same model, pepstatin A, a strong Sap inhibitor, exerted a strong curative effect on experimental vaginitis. No correlation was found between Sap production and antimycotic susceptibility, as most of the isolates were fully susceptible to fluconazole, itraconazole, and other antimycotics, regardless of their source (subjects infected with strains producing high or low levels of Sap, subjects with vaginitis or carrier subjects, or subjects with or without HIV). Thus, high Sap production is associated with virulence of C. albicans but not with fungal resistance to fluconazole in HIV-infected subjects, and Sap is a potentially new therapeutic target in candidal vaginitis.

Detection of Candida mannoproteinemia in patients with neutropenic enterocolitis.

To assess the role of Candida spp. in the etiology of neutropenic enterocolitis complicating aggressive cytotoxic chemotherapy, a dot immunobinding assay for an immunodominant Candida mannoprotein antigen was employed in 20 patients with hematologic malignancies. Candida antigen was detected in at least one serum sample from 12 (60%) patients. Eleven (92%) patients were cured when an antifungal agent was added to the antibacterial treatment. In eight patients a selective anticandidal therapy with fluconazole was administered on the basis of positive Candida mannoproteinemia, and treatment was successful in all cases but one. Detection of Candida mannoproteinemia seems to be a useful diagnostic tool in patients with neutropenic enterocolitis and represents an additional tool for selecting a less empiric, low toxic antifungal treatment with fluconazole.

Evidence that members of the secretory aspartyl proteinase gene family, in particular SAP2, are virulence factors for Candida vaginitis.

Virulence of Candida albicans strains with targeted disruption of secretory aspartyl proteinase genes (SAP1 to SAP6) was assessed in an estrogen-dependent rat vaginitis model. Null sap1 to sap3 but not sap4 to sap6 mutants lost most of the virulence of their parental strain SC5314. In particular, the sap2 mutant was almost avirulent in this model. Reinsertion of the SAP2 gene into this latter mutant led to the to recovery of the vaginopathic potential. The vaginal fluids of the animals infected by the wild type strain or by the sap1 or sap3 mutants expressed a pepstatin-sensitive proteinase activity in vitro. No traces of this activity were found in the vaginal fluid of rats challenged by the sap2 mutant. All strains were capable of developing true hyphae during infection. Thus, members of SAP family, in particular SAP2, play a clear pathogenic role in vaginitis and may constitute a novel target for chemoimmunotherapy of this infection.

The pH of the host niche controls gene expression in and virulence of Candida albicans.

Little is known of the biological attributes conferring pathogenicity on the opportunistic fungal pathogen Candida albicans. Infection by this pathogen, as for bacterial pathogens, may rely upon environmental signals within the host niche to regulate the expression of virulence determinants. To determine if C. albicans responds to the pH of the host niche, we tested the virulence of strains with mutations in either of two pH-regulated genes, PHR1 and PHR2. In vitro, PHR1 is expressed when the ambient pH is at 5.5 or higher and deletion of the gene results in growth and morphological defects at neutral to alkaline pHs. Conversely, PHR2 is expressed at an ambient pH below 5.5, and the growth and morphology of the null mutant is compromised below this pH. A PHR1 null mutant was avirulent in a mouse model of systemic infection but uncompromised in its ability to cause vaginal infection in rats. Since systemic pH is near neutrality and vaginal pH is around 4.5, the virulence phenotype paralleled the pH dependence of the in vitro phenotypes. The virulence phenotype of a PHR2 null mutant was the inverse. The mutant was virulent in a systemic-infection model but avirulent in a vaginal-infection model. Heterozygous mutants exhibited partial reductions in their pathogenic potential, suggesting a gene dosage effect. Unexpectedly, deletion of PHR2 did not prevent hyphal development in vaginal tissue, suggesting that it is not essential for hyphal development in this host niche. The results suggest that the pH of the infection site regulates the expression of genes essential to survival within that niche. This implies that the study of environmentally regulated genes may provide a rationale for understanding the pathobiology of C. albicans.

Expression of the complement-binding protein (MP60) of Candida albicans in experimental vaginitis.

The expression of the Candida albicans complement-binding C3d protein (MP60) was investigated both in vitro and in vivo by immunogold labelling and electron microscopy. In vivo expression was determined in a rat vaginitis model. Reactivity of in vitro-grown cells to an anti-MP60 rabbit serum was associated with both cytoplasmic and cell wall sites. Immunostaining in the cell wall of both yeast and hyphae was most concentrated in the inner, electron-lucid layer. Immunogold stained preparations of C. albicans from vaginal smears of infected animals also showed intense localization of the MP60 in the inner cell wall, plasma membrane. However, immunogold label was also intense at the cell surface in these samples, mostly in the area of close adherence with the keratinocytes of the vaginal epithelia. These observations indicate that MP60 is expressed both in vitro and in vivo, but to a different degree in the different cell wall layers.

Protective role of antimannan and anti-aspartyl proteinase antibodies in an experimental model of Candida albicans vaginitis in rats.

The role of antibodies (Abs) in the resistance to vaginal infection by Candida albicans was investigated by using a rat vaginitis model. Animals receiving antimannoprotein (anti-MP) and anti-aspartyl proteinase (Sap) Ab-containing vaginal fluids from rats clearing a primary C. albicans infection showed a highly significant level of protection against vaginitis compared to animals given Ab-free vaginal fluid from noninfected rats. Preabsorption of the Ab-containing fluids with either one or both proteins MP and Sap sequentially reduced or abolished, respectively, the level of protection. A degree of protection against vaginitis was also conferred by postinfectious administration of anti-Sap and anti-MP monoclonal antibodies (provided the latter were directed against mannan rather than protein epitopes of MP) and by intravaginal immunization with a highly purified, polysaccharide-free Sap preparation. Postinfectious administration of pepstatin A, a potent Sap inhibitor, greatly accelerated the clearance of C. albicans from rat vagina. No anti-MP or anti-Sap Abs were elicited during a C. albicans vaginal infection of congenitally athymic nude rats. Although they were as able as their euthymic counterparts to clear the primary infection, these animals did not show increased resistance to a rechallenge, demonstrating that induction of anticandidal protection in normal rats was a thymus-dependent Ab response. Overall, our data strengthen the concept that Abs against some defined Candida antigens are relevant in the mechanism of acquired anticandidal protection in vaginitis. The T-cell dependence of this protection may also provide a link between cell-mediated and humoral immunity in vaginal infection.

Assessment of detection of Candida mannoproteinemia as a method to differentiate central venous catheter-related candidemia from invasive disease.

The proper management of candidemic patients is controversial because of the difficulties of an early differentiation of central venous catheter (CVC)-related candidemia from deep-seated invasive Candida infection. In particular, more information on possible markers of invasive disease is needed. We performed a retrospective, pilot investigation to assess the diagnostic potential of a dot immunobinding assay for Candida mannoprotein antigen in serial serum samples from 31 candidemic patients in the setting of hematologic malignancy. Mannoproteinemia (antigenemia) was detected in 1 of 14 (7.1%) patients with transient or CVC-related candidemia and in 13 of 17 (76.5%) patients with non-CVC-related persistent candidemia. Of the 11 subjects of this latter group with documented tissue invasion, 10 (91%) were antigenemic. The patients belonging to the different categories did not significantly differ in the duration of candidemia, nor was there any significant difference among the different groups of subjects either in the number of serum samples examined or in their collection time during candidemia. The day of the first antigenemic sample during candidemia greatly varied among subjects with invasive infection, although on average mannoproteinemia was detectable by the first week of candidemia. In summary, our data demonstrate a correlation between mannoproteinemia and tissue invasion by Candida spp. in candidemic patients and suggest that mannoprotein detection by our method has a potential for the diagnosis of invasive candidiasis in these subjects.