Anti-Candida Antibodies of Patients with Invasive Candidiasis Inhibit Growth, Alter Cell Wall Structure, and Kill Candida albicans In Vitro.

Invasive candidiasis (IC), caused by Candida yeasts, particularly Candida albicans, poses a significant threat with high mortality rates. Diagnosis is challenging due to Candida's common presence in human microbiota. To address this, our research group developed an immunofluorescence assay detecting Candida albicans Germ Tube Antibodies (CAGTA) in IC patients. CAGTA, indicative of invasive processes, is associated with a lower mortality rate in ICU patients. Based on this premise, this study aims to provide results regarding the lack of knowledge about the potential activity of CAGTA against invasive infections in humans caused by the fungus Candida albicans. Therefore, in order to characterize the activity of CAGTA produced by patients with IC, we used sera from 29 patients with IC caused by either C. albicans or non-albicans Candida species. Whole serum IgG antibodies were fractionated into anti-blastospores, CAGTA-enriched, and purified CAGTA and the assessments included XTT colorimetric assays for metabolic activity, CFU counts for viability, and microscopy for growth, viability, and morphological analysis. The CAGTA-enriched IgG fraction significantly reduced the metabolic activity and viability of C. albicans compared to anti-blastospores. Purified CAGTA altered germ tube cell wall surfaces, as revealed by electron microscopy, and exhibited fungicidal properties by DiBAC fluorescent staining. In conclusion, antibodies in response to invasive candidiasis have antifungal activity against Candida albicans, influencing metabolic activity, viability, and cell wall structure, leading to cell death. These findings suggest the potential utility of CAGTA as diagnostic markers and support the possibility of developing immunization protocols against Candida infections.

Low sensitivity of conventional fungal agars in fungemia by Rhodotorula mucilaginosa: description of two cases.

BACKGROUND: Although most bloodstream yeast infections are caused by Candida spp., infections by rare or less common species have increased in recent years. Diagnosis of infections caused by these species is difficult due to the lack of specific symptoms and adequate diagnostic tools. CASES PRESENTATION: We describe two cases of fungemia by Rhodotorula mucilaginosa within a few months of each other, in a secondary Spanish hospital. In both cases, diagnosis was challenging. Blood subcultures in conventional fungal media were persistently negatives and the use of non-conventional fungal media was essential for isolating the yeasts and achieving a correct diagnosis. 1-3 beta-D-glucan detection and a panfungal PCR assay were helpful techniques to confirm the diagnosis CONCLUSION: It is highly important to establish an early diagnosis for fungemia. The process is challenging because often non-specific symptoms are presents. When yeasts grow in blood cultures other genera than Candida spp. could be the cause of infection. Patient risk factors should be assessed to incorporate alternative culture media and the available rapid diagnostic test, in order to provide an early recognition of the pathogen.

Antibacterial and Antifungal Activity of the Human Endometrial Fluid during the Natural Cycle.

Purpose: Some microbiota patterns have been associated with favorable IVF prognosis and others with pathological conditions. The endometrial fluid aspirate (EFA) contains antibacterial proteins that are enriched in implantative IVF cycles, but the antimicrobial effect of EFA has not been addressed. We aimed to evaluate the antimicrobial activity of the human endometrial fluid during the natural cycle. Methods: EFA was obtained through an embryo transfer catheter in 38 women, aged 18-40 years, with regular cycles attending to a fertility clinic. The antimicrobial activity of EFAs was tested against two strains of Staphylococcus aureus; one strain each of Streptococcus agalactiae, Enterococcus faecalis, Escherichia coli, and Klebsiella pneumoniae; and three yeasts (Candida albicans, Candida glabrata, and Candida krusei). Results: All samples exhibited antibacterial activity against S. aureus. In addition, 32.4% of EFAs were active against one of the other microorganisms assayed, 16.2% against two, and 5.4% against four of them. In contrast, none exhibited antibacterial activity against E. coli or K. pneumoniae. The antimicrobial activity differs considerably between EFA samples, and we failed to observe a cycle-related pattern. Conclusions: EFA presented two antimicrobial activity patterns: (a) one common to all the samples, exhibiting activity against S. aureus and lack of activity against E. coli and K. pneumoniae, and (b) an individualized pattern, showing activity against some of the other microorganisms tested. The intensity of antibacterial activity differs between EFA samples. Our data suggest that the uterine microbiota is controlled by means of endometrial fluid components.

Sequencing of FKS Hot Spot 1 from Saprochaete capitata To Search for a Relationship to Reduced Echinocandin Susceptibility.

Saprochaete capitata, formerly known as Geotrichum capitatum, is an emerging fungal pathogen with low susceptibility to echinocandins. Here, we report the nucleotide sequence of the S. capitata hot spot 1 region of the FKS gene (FKS HS1), which codifies for the catalytic subunit of ß-1,3-d-glucan synthase, the target of echinocandins. For that purpose, we first designed degenerated oligonucleotide primers derived from conserved flanking regions of the FKS1 HS1 segment of 12 different fungal species. Interestingly, analysis of the translated FKS HS1 sequences of 12 isolates of S. capitata revealed that all of them exhibited the same F-to-L substitution in a position that is highly related to reduced echinocandin susceptibility.

Candida albicans and cancer: Can this yeast induce cancer development or progression?

There is currently increasing concern about the relation between microbial infections and cancer. More and more studies support the view that there is an association, above all, when the causal agents are bacteria or viruses. This review adds to this, summarizing evidence that the opportunistic fungus Candida albicans increases the risk of carcinogenesis and metastasis. Until recent years, Candida spp. had fundamentally been linked to cancerous processes as it is an opportunist pathogen that takes advantage of the immunosuppressed state of patients particularly due to chemotherapy. In contrast, the most recent findings demonstrate that C. albicans is capable of promoting cancer by several mechanisms, as described in the review: production of carcinogenic byproducts, triggering of inflammation, induction of Th17 response and molecular mimicry. We underline the need not only to control this type of infection during cancer treatment, especially given the major role of this yeast species in nosocomial infections, but also to find new therapeutic approaches to avoid the pro-tumor effect of this fungal species.

A novel Candida albicans Als3, Hwp1 and Met6 derived complex peptide protects mice against hematogenously induced candidiasis.

Candida albicans can cause superficial or systemic infections in humans, particularly in immunocompromised individuals. Vaccination strategies targeting specific antigens of C. albicans have shown promise in providing protection against invasive candidiasis. This study aimed to evaluate the immuno-protective capacity of a KLH conjugated complex peptide, 3P-KLH, containing epitopes from C. albicans antigens Als3, Hwp1, and Met6 in a murine model of hematogenously induced candidiasis. Mice immunized with 3P-KLH raised a specific antibody response, and protection against C. albicans infection was assessed. Immunized mice exhibited significantly lower fungal load in their kidneys compared to the control group. Moreover, 37.5 % of immunized mice survived 21 days after the infection, while all control animals died within the first nine days. These findings suggest that the 3P-KLH complex peptide, targeting C. albicans key antigens, elicits a protective immune response and reduces the severity of systemic Candida infection. In addition, the high binding affinity of the selected epitopes with MHC II alleles further supports the potential immunogenicity of this peptide in humans. This research provides insights into the development of novel immunotherapeutic approaches against invasive candidiasis.

Candida albicans cDNA library screening reveals novel potential diagnostic targets for invasive candidiasis.

The detection of patterns associated with the invasive form of Candida albicans, such as Candida albicans germ tube antibodies (CAGTA), is a useful complement to blood culture for Invasive Candidiasis (IC) diagnosis. As CAGTA are detected by a non-standardisable and non-automatable technique, a Candida albicans cDNA expression library was screened with CAGTA isolated from serum of an animal model of invasive candidiasis, and five protein targets were identified: hyphally regulated cell wall protein 1 (Hyr1), enolase 1 (Eno1), coatomer subunit gamma (Sec21), a metallo-aminopeptidase (Ape2) and cystathionine gamma-lyase (Cys3). Homology with proteins from other organisms rules out Cys3 as a good biomarker while Sec21 results suggest that it is not in the germ tubes surface but secreted to the external environment. Our analysis propose Ape2, Sec21 and a region of Hyr1 different from the one currently being studied for immunoprotection as potential biomarker candidates for the diagnosis of IC.

Molecular Identification of Fungal Species through Multiplex-qPCR to Determine Candidal Vulvovaginitis and Antifungal Susceptibility.

Vulvovaginal candidiasis (VVC) is a prevalent condition affecting women worldwide. This study aimed to develop a rapid qPCR assay for the accurate identification of VVC etiological agents and reduced azole susceptibility. One hundred and twenty nine vaginal samples from an outpatient clinic (Bilbao, Spain) were analyzed using culture-based methods and a multiplex qPCR targeting fungal species, which identified Candida albicans as the predominant species (94.2%). Antifungal susceptibility tests revealed reduced azole susceptibility in three (3.48%) isolates. Molecular analysis identified several mutations in genes associated with azole resistance as well as novel mutations in TAC1 and MRR1 genes. In conclusion, we developed a rapid multiplex qPCR assay that detects C. albicans in vulvovaginal specimens and reported new mutations in resistance-related genes that could contribute to azole resistance.

Pga13 in Candida albicans is localized in the cell wall and influences cell surface properties, morphogenesis and virulence.

The fungal cell wall is an essential organelle required for maintaining cell integrity and also plays an important role in the primary interactions between pathogenic fungi and their hosts. PGA13 encodes a GPI protein in the human pathogen Candida albicans, which is highly up-regulated during cell wall regeneration in protoplasts. The Pga13 protein contains a unique tandem repeat, which is present five times and is characterized by conserved spacing between the four cysteine residues. Furthermore, the mature protein contains 38% serine and threonine residues, and therefore probably is a highly glycosylated cell wall protein. Consistent with this, a chimeric Pga13-V5 protein could be localized to the cell wall, but only after deglycosylation was performed. Disruption of PGA13 led to increased sensitivity to Congo red, Calcofluor white, and zymolyase, and to a diminished ability of protoplasts to recover their cell wall. In addition, pga13Δ mutants exhibited delayed filamentation, a higher surface hydrophobicity, and increased adherence and flocculation (cell-cell interactions). Furthermore, transcript profiling showed that expression of four members of the ALS family (adhesin-encoding genes) is up-regulated in the pga13Δ null mutant. Altogether, these results indicate that Pga13 is a wall-localized protein that contributes to cell wall synthesis and is important for acquiring normal surface properties. The contribution of Pga13 to surface hydrophilicity may be important for cell dispersal during development of invasive infections, and possibly for morphological development. This is consistent with the observed reduced virulence of pga13Δ mutants in a mouse model of disseminated candidiasis.

Biomarkers for the diagnosis of invasive candidiasis in immunocompetent and immunocompromised patients.

Blood culture methods show low sensitivity, so reliable non-culture diagnostic tests are needed to help clinicians with the introduction, de-escalation, and discontinuation of antifungal therapy in patients with suspected invasive candidiasis (IC). We evaluated different biomarkers for the diagnosis of IC in immunocompetent and immunocompromised patients at risk for developing invasive fungal diseases. The specificity of Candida albicans germ-tube antibodies (CAGTA) detection was high (89%-100%), but sensitivity did not exceed 61% even after raising the cut-off from 1/160 to 1/80. We developed enzyme-linked immunoassays detecting antibodies against C. albicans proteins (Als3-N, Hwp1-N, or Met6) that resulted more sensitive (66%-92%) but less specific than CAGTA assay. The combination of 1,3-beta-D-glucan (BDG) detection and CAGTA results provided the highest diagnostic usefulness in immunocompetent patients. However, in immunocompromised patients, anti-Met6 antibodies was the best biomarker, both, alone or in combination with BDG.

In-depth proteomics and natural peptidomics analyses reveal antibacterial peptides in human endometrial fluid.

The composition of endometrial fluid reflects the status of the endometrium; it is a good atraumatic source of information on embryo implantation processes and possible pathological conditions. Although some attempts have been made to characterise its proteome, the catalogue of its proteins remains incomplete and little has been done to analyse the natural peptides it contains. Here, we present a comprehensive analysis of the proteins and natural peptides of the endometrial fluid. The protein content of samples from 11 individuals was analysed using the novel timsTOF Pro mass spectrometer. We identified 4694 proteins with at least one peptide with FDR < 1%, of which 2261 were found in >50% of the samples. A pooled endometrial fluid sample was used for isolation and analysis of the natural peptides. Mass spectrometry analysis identified 3899 naturally occurring peptides from 238 different proteins. Among these, there were some putative natural antibacterial peptides. Antimicrobial activity of peptides derived from elafin and Cu/Zn superoxide dismutase was confirmed using microbiological assays. Our results substantially expand the catalogue of known endometrial fluid proteins and provide extensive new information on the natural peptide content of this fluid. SIGNIFICANCE: The endometrial fluid contains many proteins whose clinical relevance is still unknown. Some might be merely markers of endometrial function, but others might play a role in embryo nutrition and/or implantation. Human endometrial fluid analysis might open the door to new developments in embryo transfer strategies in in-vitro fertilisation programmes and lead to improvements in the composition of embryo culture media. Here, we report, for the first time, antimicrobial activity of endometrial fluid peptides. Such peptides could play an important role in the balance of the recently described uterine microbiota.

Anti-Candidaalbicans germ tube antibodies reduce in vitro growth and biofilm formation of C. albicans.

BACKGROUND: Invasive candidiasis by Candida albicans is associated with high morbidity and mortality, due in part to the late implementation of an appropriate antifungal therapy hindered by the lack of an early diagnosis. AIMS: We aimed to evaluate the in vitro antifungal activity of the antibodies against C. albicans germ tubes (CAGTA) raised in a rabbit model of candidemia. METHODS: We measured the effect of CAGTA activity by colorimetric XTT and crystal violet assays, and colony forming units count, both on C. albicans planktonic cells and during the course of biofilm formation and maturation. Viability and cell morphology were assessed by optical, fluorescent or scanning electron microscopy. RESULTS: CAGTA ≥50µg/ml caused a strong inhibition of C. albicans blastospores growth, and DiBAC fluorescent staining evidenced a fungicidal activity. Moreover, electron microscopy images revealed that CAGTA induced morphological alterations of the surface of C. albicans germ tubes grown free as well as in biofilm. Interestingly, CAGTA ≥80µg/ml reduced the amount of C. albicans biofilm, and this effect started at the initial adhesion stage of the biofilm formation, during the first 90min. CONCLUSIONS: This is the first report showing that CAGTA reduce C. albicans growth, and impair its metabolic activity and ability to form biofilm in vitro. The antigens recognized by CAGTA could be the basis for the development of immunization protocols that might protect against Candida infections.

Use of recombinant antigens for the diagnosis of invasive candidiasis.

Invasive candidiasis is a frequent and often fatal complication in immunocompromised and critically ill patients. Unfortunately, the diagnosis of invasive candidiasis remains difficult due to the lack of specific clinical symptoms and a definitive diagnostic method. The detection of antibodies against different Candida antigens may help in the diagnosis. However, the methods traditionally used for the detection of antibodies have been based on crude antigenic fungal extracts, which usually show low-reproducibility and cross-reactivity problems. The development of molecular biology techniques has allowed the production of recombinant antigens which may help to solve these problems. In this review we will discuss the usefulness of recombinant antigens in the diagnosis of invasive candidiasis.

Isolation of Aspergillus lentulus in Spain from a critically ill patient with chronic obstructive pulmonary disease.

Aspergillus lentulus was first described in the year 2005, and since it cannot be phenotypically distinguished from Aspergillus fumigatus, it is conceivable that earlier descriptions (before 2005) could be attributed to this new species. Currently invasive infections caused by A. lentulus are rare and very few cases have been previously published in neutropenic patients, all of them with fatal outcome. Here we report a critically ill non neutropenic patient with chronic obstructive pulmonary disease (COPD) who was admitted to the medical intensive care unit with an exacerbation of COPD and who had been treated with long term corticosteroids. A. fumigatus was cultured from two bronchial aspirates and in a third bronchial aspirate both A. lentulus and A. fumigatus were isolated. On two consecutive days detection of galactomannan in serum was negative whilst detection of (1-3) beta-D glucan was positive (> 518 pg/ml). Minimal inhibitory concentrations (MIC) for itraconazole, voriconazole, caspofungin and amphotericin B were high for this strain of A. lentulus. Given the high MIC values of A. lentulus to available antifungals, the accurate identification of this new species is warranted. To our knowledge, this is the first report of the isolation of A. lentulus in a non-neutropenic critically ill patient, although we note that since it was isolated only once from respiratory specimens, its implication as an etiologic agent of infection for this patient remains to be established.

Diagnostic potential of (1,3)-beta-D-glucan and anti-Candida albicans germ tube antibodies for the diagnosis and therapeutic monitoring of invasive candidiasis in neutropenic adult patients.

The usefulness to diagnose and monitor invasive candidiasis (IC) using beta-glucan (BG) and antibodies against Candida albicans germ tubes (CAGT) was evaluated in a twice-weekly screening of 35 episodes in neutropenic adults at high risk. Three proven IC and three probable IC were assessed. Diagnostic levels of both markers were detected in 100% of proven IC and in 66% of probable IC. Sensitivity, specificity, positive and negative predictive values of BG and anti-CAGT antibodies detection were 83.3%, 89.6%, 62.5% and 96.3%, and 83.3%, 86.2%, 55.5%, 96.1%, respectively. False positive reactions occurred at a rate of 10.3% and 13.8% for the detection of BG and anti-CAGT antibodies, respectively. However, the patients with false positive results were different by each test. Both tests anticipated the clinical and radiological diagnosis, and the initiation of antifungal therapy in most patients. Combination of both tests improved specificity and positive predictive value to 100%.

Isolation of Issatchenkia occidentalis from the esophagus of a leukemic patient.

Issatchenkia occidentalis was isolated from an esophageal biopsy of a young leukemic male patient who underwent bone marrow transplantation. At the time the specimen was collected, the patient was also suffering from esophageal herpetic lesions. The identification of the isolate was not possible by the use of the available commercial methods. Thus, its identification was done by PCR and DNA sequencing using panfungal primers.

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.

Developing chick embryos express a protein which shares homology with the nuclear pore complex protein Nup88 present in human tumors.

Nup88 is a nuclear pore complex protein which is overexpressed in a variety of human tumors of the stomach, colon, liver, pancreas, breast, lung, ovary, uterus, prostate and kidney. A monoclonal antibody crossreacting with the yeast Candida albicans and Nup88 was used to investigate the expression of cross-reactive antigens in chick embryos, in an attempt to identify an experimental model for studying the role played by Nup88 during cell development and differentiation. All cells in the trilaminar embryo were labeled with the antibody, but as development advanced and organogenesis was completed, expression of the corresponding antigen became more restricted. Thus, some structures continued to be intensely labeled (skin epithelium, oropharyngeal endothelium, perichondral mesenchymal tissue), whereas others ( muscular tissue, vascular endothelium, respiratory endothelium, digestive tract mucosa, peripheral nerves, medullary white matter and the retinal axons) were more moderately stained. No immunoreactivity was observed in the medullary grey matter or cartilage. A specific band of 53 kDa observed by Western blotting of chick embryo extracts suggested that the chicken antigen recognized by the monoclonal antibody is the homologue of human Nup88, which is associated with the high proliferation and low differentiation of tumor cells. The present results indicate that the role of Nup88 in cell differentiation and organ development could be fruitfully investigated using the developing chick embryo as an experimental model.

Disseminated fusariosis and hematologic malignancies, a still devastating association. Report of three new cases.

BACKGROUND: Fungi of the genus Fusarium are primarily plant pathogens and saprobes that produce disseminated infections in immunologically deficient humans. After aspergillosis, disseminated fusariosis is the second most common cause of invasive infection by filamentous fungi in patients with hematologic malignancies or those undergoing transplants of hematopoietic progenitors. AIMS: Disseminated fusariosis (DF) is considered an extremely rare infection and has reached a stable incidence rate, but its high mortality rate and the lack of an optimal management protocol have raised increasing interest in this mycosis. METHODS: We present three cases of DF produced by Fusarium oxysporum species complex, Fusarium solani species complex and the highly unusual Fusarium dimerum in patients with advanced hematological malignancies diagnosed in our hospital between 2007 and 2011. The species level identification of the Fusarium isolates was established by sequencing their TEF1 gene. RESULTS: The isolates showed low susceptibility to most of the antifungal agents analyzed, except that observed for F. dimerum to amphotericin B (AmB) and terbinafine, and F. oxysporum species complex to AmB. Interestingly, the strain of F. solani species complex exhibited high MIC values for AmB and voriconazole, notwithstanding these drugs were used for treatment with good results. Other relevant aspects to be considered in the treatment of DF are surgically cleaning foci of infection, withdrawing presumably contaminated catheters and recovery from neutropenia. CONCLUSIONS: The prevention of infection in colonized patients, the maintenance of a high level of diagnostic suspicion for early diagnosis, and the combined, vigorous and prolonged use of L-AmB and voriconazole are essential to decrease the mortality rate of this devastating infection.

Humoral immunity links Candida albicans infection and celiac disease.

OBJECTIVE: The protein Hwp1, expressed on the pathogenic phase of Candida albicans, presents sequence analogy with the gluten protein gliadin and is also a substrate for transglutaminase. This had led to the suggestion that C. albicans infection (CI) may be a triggering factor for Celiac disease (CeD) onset. We investigated cross-immune reactivity between CeD and CI. METHODS: Serum IgG levels against recombinant Hwp1 and serological markers of CeD were measured in 87 CeD patients, 41 CI patients, and 98 healthy controls (HC). IgA and IgG were also measured in 20 individuals from each of these groups using microchips sensitized with 38 peptides designed from the N-terminal of Hwp1. RESULTS: CI and CeD patients had higher levels of anti-Hwp1 (p=0.0005 and p=0.004) and anti-gliadin (p=0.002 and p=0.0009) antibodies than HC but there was no significant difference between CeD and CI patients. CeD and CI patients had higher levels of anti-transglutaminase IgA than HC (p=0.0001 and p=0.0039). During CI, the increase in anti-Hwp1 paralleled the increase in anti-gliadin antibodies. Microchip analysis showed that CeD patients were more reactive against some Hwp1 peptides than CI patients, and that some deamidated peptides were more reactive than their native analogs. Binding of IgG from CeD patients to Hwp1 peptides was inhibited by γIII gliadin peptides. CONCLUSIONS: Humoral cross-reactivity between Hwp1 and gliadin was observed during CeD and CI. Increased reactivity to Hwp1 deamidated peptide suggests that transglutaminase is involved in this interplay. These results support the hypothesis that CI may trigger CeD onset in genetically-susceptible individuals.