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.

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.

Perinuclear Anti-Neutrophil Cytoplasmic Antibodies (pANCA) Impair Neutrophil Candidacidal Activity and Are Increased in the Cellular Fraction of Vaginal Samples from Women with Vulvovaginal Candidiasis.

Vulvovaginal candidiasis (VVC) is primarily caused by Candida albicans and affects 75% of childbearing age women. Although C. albicans can colonize asymptomatically, disease is associated with an increased Candida burden, a loss of epithelial tolerance and a breakdown in vaginal microbiota homeostasis. VVC symptoms have been ascribed to a powerful inflammatory response associated with the infiltration of non-protective neutrophils (PMN). Here, we compared the immunological characteristics of vaginal fluids and cellular protein extracts obtained from 28 VVC women and from 23 healthy women colonized by Candida spp. We measured the levels of antibodies against fungal antigens and human autoantigens (anti-Saccharomyces cerevisiae antibodies (ASCA), C. albicans germ tube antibodies (CAGTAs) and perinuclear anti-neutrophil cytoplasmic antibodies (pANCA)), in addition to other immunological markers. Our results show that the pANCA levels detected in the cellular protein extracts from the vaginal fluids of symptomatic women were significantly higher than those obtained from healthy colonized women. Consistent with a potential physiologically relevant role for this pANCA, we found that specific anti-myeloperoxidase antibodies could completely neutralize the ex vivo killing capacity of polymorphonuclear cells. Collectively, this preliminary study suggests for the first time that pANCA are found in the pathogenic vaginal environment and can promptly impair neutrophil function against Candida, potentially preventing a protective response.

Biomarkers of fungal infection: Expert opinion on the current situation.

The introduction of non-culture-based diagnostic techniques is revolutionizing the world of microbiological diagnosis and infection assessment. Fungi are no exception, and the introduction of biomarkers has opened up enormous expectations for better management of these entities. Biomarkers are diverse, their targets are also diverse and their evaluation has been done preferably in an individualized use and with deficient designs. Less is known about the value of the combined use of biomarkers and the impact of the negativity of two or more biomarkers on antifungal treatment decisions has been poorly studied. Given the paucity of prospective, randomized and definitive studies, we have convened experts from different fields, with an interest in invasive fungal infections, to answer some questions about the current relevant use of fungal biomarkers. This document summarizes the answers of these experts to the different questions.

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.

Diagnostic accuracy of Candida albicans germ tube antibody for invasive candidiasis: systematic review and meta-analysis.

BACKGROUND: Candida albicans germ tube antibody (CAGTA) may be helpful as a marker for the diagnosis of invasive candidiasis (IC). However, the performance has been variable. We conducted a meta-analysis to assess the diagnostic accuracy of this assay for diagnosing IC. METHOD: We searched MEDLINE, EMBASE, Cochrane Collaboration databases, reference lists of retrieved studies, and review articles. The sensitivity, specificity, positive likelihood ratio, negative likelihood ratio, diagnostic odds ratio, and a summary receiver-operating characteristic curve of CAGTA for diagnosing IC were pooled using meta-analysis. RESULTS: A total of 976 patients (262 with proven or probable IC), included in 7 studies, were analyzed. The pooled sensitivity, specificity, positive and negative likelihood ratios, and diagnostic odds ratios and area under the curve were 66% (95% confidence interval [95% CI], 59% to 73%), 76% (95% CI, 58% to 88%), 2.8 (95% CI, 1.5 to 5.8), 0.44 (95% CI, 0.34 to 0.57), 6 (95% CI, 3 to 5), and 0.68 (95% CI, 0.64 to 0.72), respectively. Heterogeneity of specificity was significant. CONCLUSION: The diagnostic accuracy of the CAGTA assay is moderate for IC. Since the CAGTA assay is not absolutely sensitive and specific for IC, the CAGTA results should be interpreted in parallel with other biomarkers and clinical findings.

Diagnosis of invasive fungal disease in hospitalized patients with chronic obstructive pulmonary disease.

BACKGROUND: The role of culture-independent techniques (galactomannan, (1-3)-ß-d-glucan) in the early diagnosis of invasive fungal diseases (IFD) is well assessed in hematological patients, but there are no clear conclusions in patients with chronic obstructive pulmonary disease (COPD). AIMS: To study the usefulness of nonculture-based techniques in the diagnosis of IFD in COPD-patients at risk for IFD. METHODS: A prospective observational study based on monitoring COPD patients at risk for IFD during 2007-2010 was carried out. The presence of galactomannan, (1-3)-ß-d-glucan and an indirect immunofluorescence of Candida albicans germ tube specific antibodies (CAGTA) were performed. RESULTS: Among 43 COPD patients, 16 (37.2%) were diagnosed with IFD: seven cases were proven IFD (five invasive candidemia - IC, one invasive aspergillosis - IA and a rhinocerebral zygomycosis) and nine probable IFD (seven IA and two IC). In the diagnosis of IC and IA, the negative predictive value (NPV) of (1-3)-ß-d-glucan was 100%. Regarding CAGTA in IC, NPV was 96.2%. Finally, NPV of galactomannan in IA was 91.2%. The area under the ROC curve for (1-3)-ß-d-glucan in IC and for the rest of the IFD cases was 0.86 (95% CI, 0.79-0.93) and 0.60 (95% CI, 0.43-0.77), for CAGTA in IC was 0.83 (95% CI, 0.74-0.91) and for galactomannan in IA was 0.71 (95% CI, 0.56-0.85). Positive (1-3)-ß-d-glucan preceded the growth of Candida (average of 1.7 days) in blood culture. CONCLUSIONS: In COPD patients at risk for IFD the assayed techniques are especially useful to rule out the presence of IFD.

Candida albicans Germ-Tube Antibody: Evaluation of a New Automatic Assay for Diagnosing Invasive Candidiasis in ICU Patients.

Testing for Candida albicans germ-tube antibody IFA IgG assay (CAGTA) is used to detect invasive candidiasis infection. However, most suitable assays lack automation and rapid single-sample testing. The CAGTA assay was adapted in an automatic monotest system (invasive candidiasis [CAGTA] VirClia® IgG monotest (VirClia®), a chemiluminescence assay with ready-to-use reagents that provides a rapid objective result. CAGTA assay was compared with the monotest automatic VirClia® assay in order to establish the diagnostic reliability, accuracy, and usefulness of this method. A prospective study with 361 samples from 179 non-neutropenic critically ill adults patients was conducted, including 21 patients with candidemia, 18 with intra-abdominal candidiasis, 84 with Candida spp. colonization, and 56 with culture-negative samples, as well as samples from ten healthy subjects. Overall agreement between the two assays (CAGTA and VirCLIA) was 85.3%. These assays were compared with the gold-standard method to determine the sensitivity, specificity as well as positive and negative predictive values. In patients with candidemia, values for CAGTA and VirCLIA assays were 76.2 versus 85.7%, 80.3 versus 75.8%, 55.2 versus 52.9%, and 91.4 versus 94.3%, respectively. The corresponding values in patients with intra-abdominal candidiasis were 61.1 versus 66.7%, 80.3 versus 75.8%, 45.8 versus 42.9%, and 88.3 versus 89.3%, respectively. No differences were found according to the species of Candida isolated in culture, except for Candida albicans and C. parapsilosis, for which VirClia® was better than CAGTA. According to these results, the automated VirClia® assay was a reliable, rapid, and very easy to perform technique as tool for the diagnosis invasive candidiasis.

Clinical validation of a multiplex real-time PCR assay for detection of invasive candidiasis in intensive care unit patients.

BACKGROUND: New techniques, such as those based on multiplex quantitative real-time PCR (MRT-PCR), can improve the detection of invasive candidiasis (IC). METHODS: We prospectively studied 63 intensive care unit patients with suspected IC and 40 healthy controls. Blood cultures and MRT-PCR were performed at day 0 and +2, +7, +14 and +21 days in all patients. In addition, ß-d-glucan (BDG) and Candida albicans germ tube antibody (CAGTA) were quantified. RESULTS: IC was confirmed in 27 patients. Colonization was significantly higher in patients with IC (96% versus 64%, P = 0.002). The sensitivity, specificity, positive predictive value and negative predictive value of MRT-PCR for the diagnosis of IC were 96.3%, 97.3%, 92.8% and 98.7%, respectively. The positive predictive value and specificity were significantly higher for MRT-PCR than for BDG and CATGA. MRT-PCR performed very well, especially in deep-seated IC (sensitivity 90.9% versus 45.4% for blood culture; P = 0.06). As regards the most appropriate clinical sample for DNA amplification, in this study whole blood and serum presented similar results. CONCLUSIONS: MRT-PCR appears to be a useful test for confirming a diagnosis of IC in critically ill patients, especially in those with deep-seated disease. Its high sensitivity and positive predictive value make it a much more efficient tool for the management of IC than other diagnostic procedures and clinical scores.

Potential role of Candida albicans germ tube antibody in the diagnosis of deep-seated candidemia.

Patients with candidemia may have transient or catheter-related infections without involvement of deep tissues or deep-seated candidiasis. Clinical differentiation of these entities may not be evident with conventional microbiological and imaging methods. Our aim was to determine if the detection of Candida albicans germ tube-specific antibody (CAGTA) in patients with candidemia was related to the extent of the disease. This study was conducted from 2003 to 2012 with 50 patients diagnosed as having candidemia, that is, 29 with deep-seated candidiasis and 21 with non-deep-seated candidiasis. The most common species recovered from samples obtained from these patients were C. albicans, 40%; C. tropicalis, 20%; C. parapsilosis, 18%; and C. glabrata, 12%. Serum samples were processed according to the manufacturer's recommendations (Vircell Microbiologist S.L., Granada, Spain). The CAGTA tests were positive in 1/21 non-deep-seated candidemias (DSCs; 4.76%) and 20/29 DSCs (68.96%; P < 0.01). Accordingly, the values for specificity and positive predictive values of CAGTA for identifying DSC were 95%. We concluded that the presence of a positive CAGTA test in a sample from a patient with candidemia suggests deep-seated candidiasis. Extension screening studies should be considered and origins other than catheters should be searched. Prospective studies are needed to determine the clinical implications of this finding and its potential use in defining the optimal duration of therapy.

Clinical practice guidelines for the management of invasive Candida infections in adults in the Middle East region: Expert panel recommendations.

Invasive Candida infections contribute to significant morbidity and mortality in patients with healthcare-associated infections. They represent a major burden on the public health system, and are challenging to diagnose and treat. A multidisciplinary expert panel critically reviewed available evidence to provide consensus recommendations for the management of invasive Candida infections in the Middle East. Based on diagnosis, recommendations were provided for the management of Candida infections in non-neutropenic and neutropenic patients. Polyenes (amphotericin B-deoxycholate [AmB-d] and lipid formulations amphotericin B [LFAmB]), triazoles (fluconazole, itraconazole and voriconazole), echinocandins (caspofungin, anidulafungin, and micafungin) and flucytosine are the recommended categories of antifungal agents for treatment of Candida infections. Echinocandins are preferred for treatment of proven and suspected Candida infections, especially in critically ill patients or those with previous exposure to azoles. Recommendations were also provided for infections caused by specific Candida species as well as management of different disease conditions. The experts highlighted that the guidelines should be used along with clinical judgment. Given the paucity of published data from the region, research in the form of randomized clinical trials should be given priority.