Hacia el diagnóstico temprano de la candidiasis invasora en el paciente crítico

The management of invasive fungal infections in critically ill patients, from diagnosis to selection of the ther- apeutic protocol, is often a challenge. Early diagnosis and treatment are associated with a better prognosis, but apart from cases with positive cultures from blood or fluid/tissue biopsy, diagnosis is neither sensitive nor specific, and there is a need for specific markers in these diseases. Serodiagnostic assays such as mannan an-tigen, mannan antibodies, Candida albicans germ-tube antibodies or (1→3)-β-D-glucan detection, and mo-lecular techniques for the detection of fungal-specific DNA have been developed with promising results in critical care settings. One of the main features in diagnosis is the evaluation of risk factors for infection, which will identify patients in need of preemptive or empirical treatment. Clinical scores were built from those risk factors. The combination of prediction rules and non-culture microbiological tools could be currently be the key to improving the diagnosis and prognosis of invasive fungal infections in critically ill patients.     

New prospects for the diagnosis of viral infections

The diagnosis of viral infections is important for the accurate management of patients with infectious diseases and for the monitoring of the course of epidemics in susceptible populations. The utility of traditional viral diagnostic assays is limited by the time, expense, and expertise required for the performance of tissue culture techniques. Similarly, the application of immunoassay techniques has been inhibited by the limited degrees of sensitivity and specificity which can be attained by most immunoassay methods. Recently, techniques for the identification of DNA and RNA have been applied to the detection of viral nucleic acids in clinical samples. Such assays have a number of potential advantages over corresponding immunoassays directed at the detection of viral antigens. In order to be generally applicable to clinical diagnosis, however, formats for the detection of viral nucleic acids have to be devised which allow for the reproducible quantitation of target DNA or RNA in human body fluids. Furthermore, formats need to be devised which allow enhanced assay sensitivity while maintaining high degrees of specificity and reproducibility. The use of non-isotopic labeling, liquid-phase hybridization, and target amplification techniques offers partial solutions to these problems. The development of practical assays for the detection of viral nucleic acids under a broad range of clinical and laboratory conditions would represent a major advance in the ability of physicians to care for patients with suspected infections.     

In vitro comparison of antifungal activity of oxiconazole and econazole against yeast.

Monoclonal antibodies (MoAbs) have had a major impact on many areas of biomedical research and almost since their advent have been used in the characterisation and identification of diagnostically important antigens of fungal pathogens. Their main significance lies in three, often inter-related areas: a) the definition and characterisation of antigens for use in detection of antibody responses, b) their direct use in the detection of diagnostically useful antigen in body fluids c) their application in immunohistochemical diagnosis. The degree to which MoAbs have been applied varies between fungal pathogens, and they have now been used, for example, in the serodiagnosis of Aspergillus spp., Cryptococcus neoformans, Histoplasma capsulatum and Paracoccidioides brasiliensis. Their use in producing diagnostic tests for other fungi such as Sporothrix schenckii and Penicillium marneffei has been more restricted but considerable potential exists for further development.     

Enzyme immunoassays for the detection of microbial antigens and prospects for improved assays

The rapid diagnosis of viral infections is an important tool in the management of patients with infectious diseases. Solid-phase enzyme immunoassays have proved to be useful tools for the direct detection of the antigens of some viruses directly in clinical specimens. Such assays have been particularly useful in the diagnosis of viral infections in the gastrointestinal and respiratory tracts. However, standard solid-phase enzyme immunoassays often do not display sufficient sensitivity for the diagnosis of all cases of viral infections. Techniques which might be utilized to increase the sensitivity of solid-phase immunoassays include the use of monoclonal antibodies to maximize the efficiency of the antigen-antibody interactions and the use of high-turnover enzymes to increase the amount of signal generated by the ensuing enzyme-substrate reactions. In addition, techniques making use of nucleic acid hybridization have a great deal of potential for the accurate detection of viral nucleic acids in human body fluids. The successful application of these techniques to the diagnosis of viral infections could lead to a marked improvement in the care of patients with suspected infectious diseases as well as to a decrease in the transmission of viral infections to high-risk individuals.     

Heterogeneous enzyme immunoassay.

During the past 10 to 15 years immunoassays have gained acceptance as the methods of choice in the diagnosis of a number of disease states. At present the immunodiagnostic techniques employed range from radioimmunoassay for haptens through immunofluorescence for autoimmune diseases to complement fixation for viral infections. All of these assays have their own individual limitations such as: safety, short shelf life and sensitivity. The development of enzyme immunoassays, in particular enzyme linked immunosorbent assay (ELISA), has led to a substantial literature which offers the view that enzyme immunoassays provide a safe, sensitive and specific alternative to standard methods for the detection of antibodies or antigens. The application of heterogeneous enzyme linked immunosorbent assays for the quantitation of haptens, macromolecular antigens and antibodies is reviewed.     

Genomic screening for Chlamydophila pneumoniae-specific antigens using serum samples from patients with primary infection

Chlamydophila pneumoniae, an obligate intracellular human pathogen, causes respiratory tract infections. The most common techniques used for the serological diagnosis of C.?pneumoniae infections are microimmunofluorescence tests and commercial serological ELISA tests; these are based on the detection of antibodies against whole chlamydial elementary bodies and lipopolysaccharide/outer membrane protein, respectively. Identification of more specific and highly immunodominant antigens is essential for the development of new serodiagnostic assays. To identify novel specific antigens from C.?pneumoniae, we screened 455 genes with unknown function in the genome of C.?pneumoniae J138. Extracts of Saccharomyces cerevisiae cells expressing GFP-tagged C.?pneumoniae proteins were subjected to Western blot analysis using serum samples from C.?pneumoniae-infected patients as the primary antibodies. From this comprehensive analysis, 58 clones expressing C.?pneumoniae open reading frames, including hypothetical proteins, were identified as antigens. These results have provided useful information for the development of new serological tools for the diagnosis for C.?pneumoniae infections and for the development of vaccines in future.     

真菌感染的实验室诊断研究进展

随着易感人群逐渐增多,对临床真菌感染标本的快速检测及真菌培养的分离鉴定日益重要。所幸目前有新的检测方法用来辅助早期诊断及指导经验性的抗真菌治疗。主要的进展集中在对标本的真菌抗原直接检测方面(如半乳甘露聚糖和β-葡聚糖);假丝酵母产色培养基等快速培养鉴定法;微生物生化自动分析系统(VITEK2)和显微扫描(MicroScan)等生化自动检测平板;多肽核苷酸原位杂交,特异性的大范围聚合酶链反应(PCR)检测以及针对临床标本或培养阳性标本直接DNA测序技术。     

Lateral Flow Assays for the Diagnosis of Invasive Aspergillosis: Current Status

Purpose of Review

Diagnosis during early stages of invasive aspergillosis (IA) and targeted antifungal treatment has the potential to improve survival significantly. Despite advances in the diagnostic arsenal, invasive mold infections remain difficult to diagnose—especially at early stages before typical radiological signs develop. Varying availability and time-to-results are important limitations of current approved biomarkers and molecular assays for diagnosis of IA. Here, we will give an update on the Aspergillus-specific lateral-flow device (LFD) test. We further review promising findings on feasibility of point-of-care (POC) detection of urinary excreted fungal galactomannan-like antigens.

Recent Findings

POC LFD assays for detection of Aspergillus antigens are currently in development. The Aspergillus-specific LFD test, which is based on the JF5 antibody (Ab), detects an extracellular glycoprotein antigen secreted during active growth of Aspergillus spp. The test has shown promising results in various studies. In addition, a monoclonal Ab476-based LFD for POC detection of urinary excreted fungal galactomannan-like antigens has been developed but needs further validation.

Summary

Important advances have been made in the development of LFD assays for IA. Most promising is the Aspergillus-specific LFD test; commercial availability is still pending, however. The search for reliable POC tests for other molds, including mucorales, continues.

     

Dengue and Zika virus multi-epitope antigen expression in insect cells

Dengue virus and Zika virus are arthropod-borne flaviviruses that cause millions of infections worldwide. The co-circulation of both viruses makes serological diagnosis difficult as they share high amino acid similarities in viral proteins. Antigens are one of the key reagents in the differential diagnosis of these viruses through the detection of IgG antibodies in serological assays during the convalescent-phase of infections. Here, we report the expression of Dengue virus (DENV) and Zika virus (ZIKV) antigens containing non-conserved and immunodominant amino acid sequences using the baculovirus expression vector system in insect cells. We designed DENV and ZIKV antigens based on the domain III of the E protein (EDIII) after analyzing previously reported epitopes and by multiple alignment of the most important flaviviruses. The ZIKV and DENV multi-epitope genes were designed as tandem repeats or impaired repeats separated by tetra- or hexa-glycine linkers. The biochemical analyses revealed adequate expression of the antigens. Then, the obtained multi-epitope antigens were semi-purified in a sucrose gradient and tested using patients’ sera collected during the convalescent-phase that were previously diagnosed positive for anti-DENV and -ZIKV IgG antibodies. The optimal serum dilution was 1:200, and the mean absorbance values in the preliminary tests show that multi-epitope antigens have been recognized by human sera. The production of both antigens using the multi-epitope strategy in the eukaryotic system and based on the EDIII regions provide a proof of concept for the use of antigens in the differentiation between DENV and ZIKV.

     

Non–Culture-Based Methods for the Diagnosis of Invasive Candidiasis

Given the limitations of current fungal diagnostics, the use of non–culture-based methods for the diagnosis of invasive candidiasis (IC) is highly warranted. The implementation of molecular diagnostic strategies could permit the timely onset of appropriate therapy and may be expected to pave the way for improved clinical outcome of IC. Polymerase chain reaction (PCR) may have higher sensitivity for the diagnosis of IC than conventional blood cultures. The detection of fungal antigens generally requires a large fungal burden, and the presence of fungus-specific antibodies may not correlate with the underlying diseases. Therefore, the combined mannan and anti-mannan antibody testing is recommended. No single test has been shown convincingly to compensate for all the limitations of culture. Real-time PCR coupled with fungal culture and/or antigen detection will likely be required to significantly ameliorate the diagnostic problems in IC.     

金抗体替代ELISA中的天然抗体用于溶菌酶检测

目的 酶联免疫吸附测定(ELISA)被广泛用于抗体或抗原的检测,并被视为临床实践中的金标准,可提供相对可靠、灵敏和特异的检测结果.ELISA的本质是抗原与相应抗体之间的特异性相互作用.然而,天然抗体固有的不稳定性是ELISA的一个难以克服的弱点,并可能导致检测结果的重现性差甚至错误的诊断结果.本课题组先前应用构象工程方...     

Biomarkers for Diagnosis and Follow-Up of Invasive Candidiasis: A Brief Review of the ECIL Recommendations

The main biomarkers for rapid and non-invasive diagnosis of invasive candidiasis include 1,3-beta-D-glucan (BG), mannan antigen (Mn), anti-mannan antibodies (A-Mn) and various molecular methods. BG, Mn and A-Mn assays are standardized, and data on BG are the most extensive. For haematology and oncology population, its performance is characterised by suboptimal sensitivity but excellent specificity. For these populations, the experts from the third European Conference on Infections in Leukemia (ECIL-3) recommended BG for detection of invasive fungal infections with grading BII, and combined Mn/A-Mn detection for the diagnosis of candidemia and hepatosplenic candidiasis, with grading CII and BIII, respectively. While very promising, PCR lacks standardization and thus could not be formally recommended. There is limited and contradictory data on the performance of BG for the monitoring of outcome in patients with invasive candidiasis, although a trend of decline or increase of BG levels was found to be associated with, respectively, a successful response or failure.     

Sero-diagnosis of invasive aspergillosis: Attempts to determine antigen and antibody relevance to infection

Attempt was made to define antigens and antisera which might prove useful in diagnosis of invasive aspergillosis in man. A convalescent antiserum (serum from rabbits after live infection withAspergillus fumigatus conidia) which might be more representative of immunological reaction to fungal growthin vivo, did not react in enzyme-linked immunosorbent assay with commercial antigens which are used at present in attempts to detect antibody response in systemic infections in man. However, this convalescent antiserum reacted with antigens from a range of fungal extracts. Antigens from young culture filtrates, in particular the 24h culture filtrate are advocated as the standard antigens for antibody detection using conventional immunoprecipitation techniques. For the detection of circulating antigens, the use of convalescent antiserum in enzyme-linked immunosorbent assay might be promising in the early diagnosis of invasive aspergillosis.     

Antibody response to Candida albicans cell wall antigens

The cell wall of Candida albicans is not only the structure where many essential biological functions reside but is also a significant source of candidal antigens. The major cell wall components that elicit a response from the host immune system are proteins and glycoproteins, the latter being predominantly mannoproteins. Both carbohydrate and protein moieties are able to trigger immune responses. Proteins and glycoproteins exposed at the most external layers of the wall structure are involved in several types of interactions of fungal cells with the exocellular environment. Thus, coating of fungal cells with host antibodies has the potential to profoundly influence the host-parasite interaction by affecting antibody-mediated functions such as opsonin-enhanced phagocytosis and blocking the binding activity of fungal adhesins to host ligands. In this review we examine various members of the protein and glycoprotein fraction of the C. albicans cell wall that elicit an antibody response in vivo. Some of the studies demonstrate that certain cell wall antigens and anti-cell wall antibodies may be the basis for developing specific and sensitive serologic tests for the diagnosis of candidiasis, particularly the disseminated form. In addition, recent studies have focused on the potential of antibodies against the cell wall protein determinants in protecting the host against infection. Hence, a better understanding of the humoral response triggered by the cell wall antigens of C. albicans may provide the basis for the development of (i) effective procedures for the serodiagnosis of disseminated candidiasis, and (ii) novel prophylactic (vaccination) and therapeutic strategies to control this type of infections.     

Recent trends in platelet antigen/antibody detection

The detection of platelet antigens and platelet antibodies has always been difficult. Recent technical achievements are due to the availability of more specific and sensitive reagents (i.e. F(ab)2 fragments; higher specific activity of labels; monoclonal antibodies directed against platelet membrane constituents etc.) and the application of advanced immunological assays to platelet immunology (i.e. blotting assays, "capture"-ELISA's and radioimmunoprecipitation in conjunction with SDS polyacrylamide gel electrophoresis). These techniques have permitted the definition and immuno-chemical characterization of new platelet allo- and autoantigens, have assisted in clinical diagnosis and promoted our understanding of pathogenic mechanisms. Illustrative examples are presented and discussed.     

Advancing the Field: Evidence for New Management Strategies in Invasive Fungal Infections

Invasive fungal infections (IFI) are a significant cause of morbidity and mortality in the immunocompromised. The traditional diagnostic methods of culture and histological examination lack sensitivity and often only make a diagnosis late when the fungal burden is high, reducing the chances of cure even with the availability of new more potent and less toxic antifungal agents. New non-culture-based serological and PCR assays have been developed. These appear more sensitive and are able to make an earlier diagnosis as compared with traditional diagnostic methods. Early diagnosis is central to reducing IFI-related morbidity and mortality. This review describes the diagnostic potential of the new serological and PCR assays and outlines how these assays have been incorporated into algorithms to improve the management of IFI.     

Fungal fragments as indoor air biocontaminants

The aerosolization process of fungal propagules of three species (Aspergillus versicolor, Penicillium melinii, and Cladosporium cladosporioides) was studied by using a newly designed and constructed aerosolization chamber. We discovered that fungal fragments are aerosolized simultaneously with spores from contaminated agar and ceiling tile surfaces. Concentration measurements with an optical particle counter showed that the fragments are released in higher numbers (up to 320 times) than the spores. The release of fungal propagules varied depending on the fungal species, the air velocity above the contaminated surface, and the texture and vibration of the contaminated material. In contrast to spores, the release of fragments from smooth surfaces was not affected by air velocity, indicating a different release mechanism. Correlation analysis showed that the number of released fragments cannot be predicted on the basis of the number of spores. Enzyme-linked immunosorbent assays with monoclonal antibodies produced against Aspergillus and Penicillium fungal species showed that fragments and spores share common antigens, which not only confirmed the fungal origin of the fragments but also established their potential biological relevance. The considerable immunological reactivity, the high number, and the small particle size of the fungal fragments may contribute to human health effects that have been detected in buildings with mold problems but had no scientific explanation until now. This study suggests that future fungal spore investigations in buildings with mold problems should include the quantitation of fungal fragments.     

Immunogens of interest for the diagnosis of Campylobacter jejuni infections

In order to identify the C. jejuni immunogens of interest for the diagnosis of Campylobacter infections, we analyzed the humoral response of 153 patients by using complement fixation (CF) and western blot assays. A first group of 79 sera was from C. jejuni infected patients suffering from enteritis (n=16), Guillain-Barré syndrome (GBS) (n=40) and arthritis (n=23). A second group of 49 sera was from healthy blood donors and a third group consisted of 25 sera from children under 4 years old. Using the CF test, 88.6% of the C. jejuni infected patients were seropositive versus 28.5% of the healthy blood donors and none of the children. The Western blot assay allowed detection of antibodies directed against seven selected antigens ranging from 14 to 67 kDa. Three of these antigens with a molecular size of 29, 37 and 43 kDa were detected by 86.0%, 84.8% and 91.1% of the C. jejuni infected patients, respectively. These three antigens seem to be good candidates for the development of assays suitable for direct and indirect diagnosis of Campylobacter infections.     

研究报告：金抗体替代ELISA中的天然抗体用于溶菌酶检测

目的酶联免疫吸附测定(ELISA)被广泛用于抗体或抗原的检测,并被视为临床实践中的金标准,可提供相对可靠、灵敏和特异的检测结果。ELISA的本质是抗原与相应抗体之间的特异性相互作用。然而,天然抗体固有的不稳定性是ELISA的一个难以克服的弱点,并可能导致检测结果的重现性差甚至错误的诊断结果。本课题组先前应用构象工程方法开发了一种基于金纳米粒子的人工抗体(简称金抗体)。金抗体可以像天然抗体一样特异性地与抗原相互作用,并且具备远优于天然抗体的稳定性。出色的稳定性使金抗体可能成为天然抗体更好的替代物,用于ELISA中。方法经过必要的优化并与辣根过氧化物酶(HRP)耦联后,制得酶标金抗体10HRP-(Au-400P1),然后用酶标金抗体代替天然酶标抗体用于ELISA检测中。结果通过一系列的实验证明,抗溶菌酶金抗体可用于ELISA特异性检测1～16 mg/L范围内的鸡蛋清溶菌酶(HEWL)样品。结论金抗体可以替代天然抗体用于ELISA检测,并具有优于传统ELISA法的检测准确性和一致性。     

Diagnosis of Midwestern Endemic Mycoses

Histoplasmosis and blastomycosis are the two most common midwestern endemic mycoses. A history of exposure to the geographic areas in which these organisms occur is central to raising suspicion for an endemic fungal infection. For infection with these organisms, the diagnosis is definitively established by recovery of the organism in tissue or body fluids, which may take weeks. A rapid diagnosis can be made by finding the distinctive yeasts in tissues or body fluids. Antigen testing allows a presumptive diagnosis of these endemic fungal infections while awaiting culture results, but cross-reactivity between assays for Histoplasma and Blastomyces is routinely seen. Antibody tests provide supportive evidence for infection with either of these endemic mycoses but are less useful in immunosuppressed patients.