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.

Strain Improvement of Native Saccharomyces cerevisiae LN ITCC 8246 Strain Through Protoplast Fusion To Enhance Its Xylose Uptake.

Co-utilization of xylose and glucose and subsequent fermentation using Saccharomyces cerevisiae could enhance ethanol productivity. Directed engineering approaches have met with limited success due to interconnectivity of xylose metabolism with other intrinsic, hidden pathways. Therefore, random approaches like protoplast fusion were used to reprogram unidentified mechanisms. Saccharomyces cerevisiae LN, the best hexose fermenter, was fused with xylose fermenting Pichia stipitis NCIM 3498. Protoplasts prepared using glucanex were fused under electric impulse and fusants were selected using 10% ethanol and cycloheximide (50 ppm) markers. Two fusants, 1a.23 and 1a.30 showing fast growth on xylose and tolerance to 10% ethanol, were selected. Higher extracellular protein expression observed in fusants as compared to parents was corroborated by higher number of bands resolved by two-dimensional analysis. Overexpression of XYL1, XYL2, XKS, and XUT4 in fusants as compared to S. cerevisiae LN as observed by RT-PCR analysis was substantiated by higher specific activities of XR, XDH, and XKS enzymes in fusants. During lignocellulosic hydrolysate fermentation, fusants could utilize glucose faster than the parent P. stipitis NCIM 3498 and xylose consumption in fusants was higher than S. cerevisiae LN.

Serological Proteome Analysis for the Characterization of Secreted Fungal Protein Antigens.

Defining the humoral immune response to infectious agents is important for gaining insights into infectious diseases and the response of the immune system. It can further aid development of serodiagnostic tests, discovery of vaccine antigen candidates, and immuno-epidemiological research. During the last three decades, serological proteome analyses (SERPAs) have played a significant role in characterizing the antibody response of humans or animals to fungal pathogens. SERPA combines 2D-gel electrophoresis with Western blotting. The introduction of multiplexing approaches by means of fluorescent dyes has greatly improved the reliability of the 2D technique and has boosted also the qualitative capabilities of the SERPA approach. In this chapter, we detail a SERPA protocol using fungal extracellular proteins from a fungal culture, here as an example the mold Aspergillus fumigatus.

[Preparation and Biological Characterization of Limulus Factor G-activating Substance of Aspergillus spp.]

Aspergillus is a medically important fungal genus that causes a life-threatening infection known as aspergillosis in immunocompromised patients. ß-1,3-Glucan is detected in the plasma of patients with aspergillosis and appears to be useful for the diagnosis of aspergillosis. In this study, we cultured Aspergillus spp. in a chemically defined liquid medium and prepared an Aspergillus water-soluble fraction (ASWS) from the culture supernatants. ASWS was found to be primarily composed of polysaccharides and proteins. Nuclear magnetic resonance analysis suggested that ASWS is a complex carbohydrate, consisting of α-1,3-glucan, ß-1,3-glucan, galactomannan, and protein. The ASWS from Aspergillus fumigatus showed limulus factor G activity, whereas zymolyase-treated ASWS did not. ASWS was eliminated from the blood more rapidly than Aspergillus solubilized cell wall ß-glucan. We analyzed the reactivity of human immunoglobulin towards ASWS by an enzyme-linked immunosorbent assay. Anti-ASWS antibodies were detected in human sera, with titers differing among individuals. This study demonstrated that the ASWS corresponds to the limulus factor G-activating substance found in the blood of patients with aspergillosis.

Recombinant 60-kDa heat shock protein from Paracoccidioides brasiliensis: is it a good antigen for serological diagnosis of paracoccidioidomycosis?

Paracoccidioides brasiliensis and P. lutzii are fungi that cause paracoccidioidomycosis (PCM), the most prevalent systemic mycosis in South America. For serological diagnosis, although 43-kDa glycoprotein (gp43) is regarded as highly specific for PCM, the occurrence of false negative reactions in sera from patients infected with P. lutzii suggests that preparation with only one antigen is not recommended. Heat shock proteins are feasible alternatives as a second antigen because they are often highly immunogenic. In this study, we evaluated the usefulness of recombinant 60-kDa heat shock protein from P. brasiliensis (rPbHsp60) for the serological diagnosis of PCM. Using western blotting assay, we observed that 77.3% of the sera from PCM patients were positive to rPbHsp60, with 90.9% positivity to recombinant gp43 (rgp43). More importantly, sera from healthy subjects had 27% positivity to rPbHsp60 and none to rgp43. When rPbHsp60 was used in ELISA, we did not observe significant differences between the reactions with sera from PCM patients and healthy subjects, while the difference was clearly evident when the antigen was rgp43. Furthermore, rPbHsp60 was recognized by sera from patients with histoplasmosis, aspergillosis, sporotrichosis or tuberculosis in an ELISA test. These results show that rPbHsp60 is not a good antigen for PCM diagnosis.

Recombinant 60-kDa heat shock protein from Paracoccidioides brasiliensis: is it a good antigen for serological diagnosis of paracoccidioidomycosis?

Paracoccidioides brasiliensis and P. lutzii are fungi that cause paracoccidioidomycosis (PCM), the most prevalent systemic mycosis in South America. For serological diagnosis, although 43-kDa glycoprotein (gp43) is regarded as highly specific for PCM, the occurrence of false negative reactions in sera from patients infected with P. lutzii suggests that preparation with only one antigen is not recommended. Heat shock proteins are feasible alternatives as a second antigen because they are often highly immunogenic. In this study, we evaluated the usefulness of recombinant 60-kDa heat shock protein from P. brasiliensis (rPbHsp60) for the serological diagnosis of PCM. Using western blotting assay, we observed that 77.3% of the sera from PCM patients were positive to rPbHsp60, with 90.9% positivity to recombinant gp43 (rgp43). More importantly, sera from healthy subjects had 27% positivity to rPbHsp60 and none to rgp43. When rPbHsp60 was used in ELISA, we did not observe significant differences between the reactions with sera from PCM patients and healthy subjects, while the difference was clearly evident when the antigen was rgp43. Furthermore, rPbHsp60 was recognized by sera from patients with histoplasmosis, aspergillosis, sporotrichosis or tuberculosis in an ELISA test. These results show that rPbHsp60 is not a good antigen for PCM diagnosis.

Development and evaluation of a real-time polymerase chain reaction assay for the rapid detection of Talaromyces marneffei MP1 gene in human plasma.

Penicilliosis caused by Talaromyces marneffei is a common AIDS-defining illness in South and Southeast Asia. Diagnosis is based on culture which can take up to 14 days for identification, leading to treatment delay and increased mortality. We developed a TaqMan real-time PCR assay targeting the MP1 gene encoding an abundant cell wall protein specific to T. marneffei. The assay's performance was evaluated in MP1-containing plasmids, clinical isolates, and plasma from HIV-infected patients with and without penicilliosis. The assay consistently detected 10 copies of MP1-containing plasmids per reaction and 100 T. marneffei yeast cells per millilitre plasma. There were no amplification with seven other Penicillium species and six other HIV-associated fungal pathogens tested. The assay was evaluated in 70 patients with AIDS: 50 patients with culture-confirmed penicilliosis and 20 patients with opportunistic infections other than penicilliosis. The diagnostic sensitivity was 70.4% (19/27, 95% CI: 51.5-84.1%) and 52.2% (12/23, 95% CI: 33.0-70.8%) in plasma samples collected prior to and within 48 h of antifungal therapy respectively. The diagnostic specificity was 100% (20/20, 95% CI: 83.9-100%). This assay provides a useful tool for the rapid diagnosis of T. marneffei infection and has the potential to improve the management of patients with penicilliosis.

[Use of MALDI-TOF in the rapid diagnosis of sepsis]. / Utilización de MALDI-TOF en el diagnóstico rápido de la sepsis.

The introduction of mass spectrometry through MALDI-TOF (matrix-assisted laser desorption ionization time-of-flight) in the diagnosis of bacteraemia and fungaemia has represented a revolution due to the rapidity and reliability of the results that it can offer to microbiology services and laboratories through analysis of the mass spectrum of the bacterial protein directly from positive blood culture bottles. These data are more useful if they are used in conjunction with other techniques able to identify the antibiotic resistance pattern of the microorganism. There is a need for a process of standardising sample processing protocols and for perfecting the identification of the agents causing bacteraemia, especially in some species of Gram-positive cocci and in polymicrobial processes. The introduction of this methodology provides rapid information that is highly important for the clinical management of bacteraemia. The availability of a multidisciplinary working group that applies all this information quickly and correctly in hospitals will improve the quality of care, reduce antibiotic expenditure and hospital stay and help to control the serious problem of antibiotic resistance.

A Murine, Bispecific Monoclonal Antibody Simultaneously Recognizing ß-Glucan and MP65 Determinants in Candida Species.

There is a real medical need of new diagnostic tools for the early recognition of invasive Candida infections. We exploited a rather simple and rapid redox methodology to construct a bispecific monoclonal antibody (bsmAb) that combines a monoclonal antibody (mAb) directed against 1,3-ß-D-glucan, a well-known, pan-fungal diagnostic biomarker, with a mAb recognizing MP65, a major immunogenic mannoprotein secreted by C.albicans and other Candida species. The bsmAb (MP65/bglu mAb) was successfully produced and purified at high yields and proved to bind and reveal simultaneously, with high sensitivity, the ß-glucan and MP65 antigens in both purified and native forms. The MP65/bglu mAb is the first bispecific antibody generated against a fungal microorganism and may prove useful for the concurrent detection of different and clinically significant Candida biomarkers in patient sera.

Seroprofiling at the Candida albicans protein species level unveils an accurate molecular discriminator for candidemia.

Serum antibodies to specific Candida proteins have been reported as potential diagnostic biomarkers for candidemia. However, their diagnostic usefulness at the protein species level has hardly been examined. Using serological proteome analysis, we explored the IgG-antibody responses to Candida albicans protein species in candidemia and control patients. We found that 87 discrete protein species derived from 34 unique proteins were IgG-targets, although only 43 of them were differentially recognized by candidemia and control sera. An increase in the speciation of the immunome, connectivity and modularity of antigenic species co-recognition networks, and heterogeneity of antigenic species recognition patterns was associated with candidemia. IgG antibodies to certain discrete protein species were better predictors of candidemia than those to their corresponding proteins. A molecular discriminator delineated from the combined fingerprints of IgG antibodies to two distinct species of phosphoglycerate kinase and enolase accurately classified candidemia and control patients. These results provide new insight into the anti-Candida IgG-antibody response development in candidemia, and demonstrate that an immunoproteomic signature at the molecular level may be useful for its diagnosis. Our study further highlights the importance of defining pathogen-specific antigens at the chemical and molecular level for their potential application as immunodiagnostic reagents or even vaccine candidates.

Immunodiagnosis of paracoccidioidomycosis due to Paracoccidioides brasiliensis using a latex test: detection of specific antibody anti-gp43 and specific antigen gp43.

BACKGROUND: Paracoccidioidomycosis (PCM) is a life-threatening systemic disease and is a neglected public health problem in many endemic regions of Latin America. Though several diagnostic methods are available, almost all of them present with some limitations. METHOD/PRINCIPLE FINDINGS: A latex immunoassay using sensitized latex particles (SLPs) with gp43 antigen, the immunodominant antigen of Paracoccidioides brasiliensis, or the monoclonal antibody mAb17c (anti-gp43) was evaluated for antibody or antigen detection in sera, cerebrospinal fluid (CSF), and bronchoalveolar lavage (BAL) from patients with PCM due to P. brasiliensis. The gp43-SLPs performed optimally to detect specific antibodies with high levels of sensitivity (98.46%, 95% CI 91.7-100.0), specificity (93.94%, 95% CI 87.3-97.7), and positive (91.4%) and negative (98.9%) predictive values. In addition, we propose the use of mAb17c-SLPs to detect circulating gp43, which would be particularly important in patients with immune deficiencies who fail to produce normal levels of immunoglobulins, achieving good levels of sensitivity (96.92%, 95% CI 89.3-99.6), specificity (88.89%, 95% CI 81.0-94.3), and positive (85.1%) and negative (97.8%) predictive values. Very good agreement between latex tests and double immune diffusion was observed for gp43-SLPs (k = 0.924) and mAb17c-SLPs (k = 0.850), which reinforces the usefulness of our tests for the rapid diagnosis of PCM in less than 10 minutes. Minor cross-reactivity occurred with sera from patients with other fungal infections. We successfully detected antigens and antibodies from CSF and BAL samples. In addition, the latex test was useful for monitoring PCM patients receiving therapy. CONCLUSIONS/SIGNIFICANCE: The high diagnostic accuracy, low cost, reduced assay time, and simplicity of this new latex test offer the potential to be commercialized and makes it an attractive diagnostic assay for use not only in clinics and medical mycology laboratories, but mainly in remote locations with limited laboratory infrastructure and/or minimally trained community health workers.

Detection of Candida albicans Sap2 in cancer patient serum samples by an indirect competitive enzyme-linked immunosorbent assay for the diagnosis of candidiasis.

BACKGROUND: The secreted aspartyl proteinases 2 (Sap2) of Candida albicans (C. albicans) is a potential marker of candididasis. It is a virulence factor associated with adherence and tissue invasion. AIM: In order to detect Sap2 in clinical sera, we developed an indirect competitive enzyme-linked immunosorbent assay (ELISA). MATERIALS AND METHODS: Polyclonal antibodies were produced for Sap2 by injecting Sap2 into a New Zealand White inbred rabbit. They could be used at a dilution exceeding 1:1200 in an indirect ELISA, and detected Sap2 concentration up to 1 ng/mL. RESULTS: Of the 286 cancer serum samples tested, 16.8% were found as candidiasis. The test was simple and economical to perform and had a level of sensitivity for detection of low-titer positive sera; thus, it may be proven to be of value in epidemiological studies on candidiasis.

Identification of aero-allergens from Rhizopus oryzae: an immunoproteomic approach.

Airborne fungal spores bearing allergens are the causative agent for inducing immediate hypersensitive reaction in sensitive individuals. In this study the potential aeroallergens have been reported for the first time from Rhizopus oryzae a common airborne mold. Clinical data based on SPT was further confirmed by ELISA. IgE reactive bands were revealed by one-dimensional immunoblotting. A 44 kDa major reactive band was found in all immunoblots. For precise identification of allergens, an immuno-proteomic approach was taken with a combination of 2-Dimensional gel electrophoresis and Mass-spectrometry. 2D map of spore-mycelial protein was confronted with pooled sera and several IgE reactive spots were detected, most of which were glycoproteins and except for one, which has no antigenic determinacy after metaperiodate modification. Each of those spots was identified by MALDI-TOF-TOF. Some bioinformatic approaches were taken to predict the signal peptide and subcellular localization of each protein. Major 44 kDa allergen was identified as Aspartyl endopeptidase. Sequence information was extracted from MS/MS spectra of two tryptic peptides generated from the 44 kDa endopeptidase. Multiple alignments with other reported aspartyl protease allergens showed significant homology. Allergenicity assessment of this protein was performed in silico and identified as a potential putative allergen.

DIGE enables the detection of a putative serum biomarker of fungal origin in a mouse model of invasive aspergillosis.

Invasive aspergillosis (IA) is a major threat for immunocompromised patients. Diagnostic difficulties often delay specific treatment initiation, which increases mortality. Finding new biomarkers to improve and speed accurate diagnosis is thus vital. To investigate the ability of proteomic methods for discovering new biomarkers of IA, we used a DIGE approach to perform a proteomic analysis on both bronchoalveolar lavages (BAL) and sera at different time-points of infection in a mouse model of invasive pulmonary aspergillosis. Progression of the infection was monitored using a bioluminescent strain of Aspergillus fumigatus. Sera proteins were enriched using the ProteoMiner kit (Biorad). This method allowed us to identify a fungal protein, the A. fumigatus major allergen Asp f 2, in sera of mice one day after the infection. However, this fungal protein was not detected three days after the infection. Importantly, in BAL, this work provides evidence of an in vivo complement evasion mechanism through the cleavage of C3b into three fragments during aspergillosis. Finally, our results underlining the inflammatory host response to IA in both lung and blood compartments at different times of infection may provide new insights into the pathophysiology of this disease.

Serologic responses to recombinant Pneumocystis jirovecii major surface glycoprotein among Ugandan patients with respiratory symptoms.

BACKGROUND: Little is known about the serologic responses to Pneumocystis jirovecii major surface glycoprotein (Msg) antigen in African cohorts, or the IgM responses to Msg in HIV-positive and HIV-negative persons with respiratory symptoms. METHODS: We conducted a prospective study of 550 patients, both HIV-positive (n = 467) and HIV-negative (n = 83), hospitalized with cough ≥2 weeks in Kampala, Uganda, to evaluate the association between HIV status, CD4 cell count, and other clinical predictors and antibody responses to P. jirovecii. We utilized ELISA to measure the IgM and IgG serologic responses to three overlapping recombinant fragments that span the P. jirovecii major surface glycoprotein: MsgA (amino terminus), MsgB (middle portion) and MsgC1 (carboxyl terminus), and to three variations of MsgC1 (MsgC3, MsgC8 and MsgC9). RESULTS: HIV-positive patients demonstrated significantly lower IgM antibody responses to MsgC1, MsgC3, MsgC8 and MsgC9 compared to HIV-negative patients. We found the same pattern of low IgM antibody responses to MsgC1, MsgC3, MsgC8 and MsgC9 among HIV-positive patients with a CD4 cell count <200 cells/µl compared to those with a CD4 cell count ≥200 cells/µl. HIV-positive patients on PCP prophylaxis had significantly lower IgM responses to MsgC3 and MsgC9, and lower IgG responses to MsgA, MsgC1, MsgC3, and MsgC8. In contrast, cigarette smoking was associated with increased IgM antibody responses to MsgC1 and MsgC3 but was not associated with IgG responses. We evaluated IgM and IgG as predictors of mortality. Lower IgM responses to MsgC3 and MsgC8 were both associated with increased in-hospital mortality. CONCLUSIONS: HIV infection and degree of immunosuppression are associated with reduced IgM responses to Msg. In addition, low IgM responses to MsgC3 and MsgC8 are associated with increased mortality.

Can chitotriosidase be a surrogate marker for invasive fungal disease?

BACKGROUND: Chitotriosidase (CHT) enzyme has been known to be secreted from the activated macrophages. We infer with these data that CHT activity is an indicator for the defence. METHODS: In this study, we evaluated CHT levels in both neutropenic and non neutropenic patients. CHT enzyme activity was measured and compared to each other groups. RESULTS: Chitotriosidase levels were found to be significantly higher in neutropenic patients with candidemia. CONCLUSION: In the comparison between neutropenic and non neutropenic patients, there was a significant difference for CHT levels. The use of this enzyme as a surrogate marker for candidemias were evaluated in neutropenic and non neutropenic patients.

Cyclosporin A treatment and decreased fungal load/antigenemia in experimental murine paracoccidioidomycosis.

Paracoccidioidomycosis (PCM) is a systemic mycosis caused by the fungus Paracoccidioides brasiliensis (Pb). The cyclosporin A (CsA) is an immunosuppressant drug that inhibits calcineurin and has been described as a potential antifungal drug. The present study investigated the effect of CsA on the immune response, fungal load/antigenemia in experimental murine PCM. It was used four groups of BALB/c mice: (a) infected with 1 x 105 Pb18 yeast cells (Pb), (b) infected and treated with CsA every other day 10 mg/kg of CsA (s.c.) during 30 days (Pb/CsA), (c) treated with CsA (CsA) and (d) no infected/treated (PBS). The immune response was evaluated by lymphocyte proliferation, DTH assays to exoAgs, ELISA for IgG anti-gp43 (specific immune responses) and cytokine serum levels (IFN-γ, TNF-α, IL-4 and IL-10). Fungal load was determined by lung colony-forming units (CFU) counts, lung and liver histopathology analysis and antigenemia determined by inhibition-ELISA. As expected, CsA was able to inhibit the specific cellular and humoral immune response (P < 0.05), with decrease in serum IFN-γ, TNF-α and IL-4 levels (P < 0.05). Cyclosporin A treatment also resulted in significantly decreased lung Pb CFU (P < 0.05) as well as a lower number of yeasts in the lung and liver (P < 0.05) by histopathology. In concordance, the decreased antigenemia was observed in Pb/CsA group (P < 0.05). In conclusion, even with immunosuppressive action, treatment with CsA results in decreased lung fungal load/antigenemia in experimental PCM in BALB/c mice. Further study is required to determine whether this represents less severe disease or protection by CsA.

Spore-forming microsporidian encephalitozoon: current understanding of infection and prevention in Japan.

Microsporidia are spore-forming obligate intracellular parasites. They are known to cause opportunistic infections in humans through zoonotic, waterborne and foodborne transmission routes. This article reviews the present knowledge regarding microsporidian Encephalitozoon cuniculi infections in animals living in the human environment in Japan and discusses the basic measures required for the effective disinfection of Encephalitozoon. The article also discusses seroepidemiologic data from healthy people in order to shed light on the mechanisms of protective immunity and to identify potential strategies for preventive medicine.

Serology of paracoccidioidomycosis.

This review provides the background for understanding the role of a battery of diagnostic methods in paracoccidioidomycosis (PCM). This systemic mycosis is a disease endemic in many regions of Latin America, with sporadic cases also occurring throughout the world (mycosis of importation). Although excellent laboratory methods for diagnosis are available, there are deficiencies that must be met by continued research. Understanding the uses and limitations of a battery of laboratory methods is essential to diagnose PCM. Clinicians and laboratory directors must be familiar with the uses and limitations of a battery of serologic and mycological tests to accurately diagnose of PCM. Antibody and antigen detections are valuable adjuncts to histopathology and culture. More recently, the gp43 and gp70 antigen detection assay have improved the methodology of diagnosis of this mycosis, which improves reproducibility and facilitates monitoring antigen clearance during antifungal treatment. Furthermore, detection of antigen in cerebrospinal fluid and in bronchoalveolar lavage fluid increases the sensitivity for diagnosis of PCM in central nervous system and in pulmonary infections, respectively.

Use of sera from humans and dolphins with lacaziosis and sera from experimentally infected mice for Western Blot analyses of Lacazia loboi antigens.

Antibodies in the sera of patients with lacaziosis recognized an approximately 193-kDa antigen and other Lacazia loboi antigens. Paracoccidioides brasiliensis gp43 antigen was detected by all evaluated sera, but they failed to detect a protein with the same molecular mass in L. loboi extracts. This study is the first to examine the humoral response to L. loboi antigens by using multiple host sera.