Biomarkers for Diagnosis of Candidal Infections

Candidemia and invasive candidiasis are associated with significant mortality, in part because of inadequate diagnostic tests. The current gold standard is blood culture, which is negative in 50% of cases. As such, development of novel diagnostics is a top priority. In Europe, studies of combined mannan antigen and anti-mannan antibody detection assays have demonstrated sensitivity of 71–100% and specificity of 86–93%. In the United States, there is more experience with (1 → 3)-β-D-glucan detection as a broad-spectrum assay for fungal pathogens. Sensitivity and specificity for diagnosing invasive candidiasis have ranged widely in studies (47–95% and 76–100%, respectively). Nucleic acid detection assays such as polymerase chain reaction (PCR) are potentially powerful tools that are now approaching the clinic. Overall, optimized PCR assays have sensitivities and specificities for candidemia and proven or probable invasive candidiasis that exceed 90%. Studies to determine the precise clinical roles of non–culture-based diagnostics are in progress.     

Pathogen Inactivating Properties and Increased Sensitivity in Molecular Diagnostics by PAXgene,a Novel Non-Crosslinking Tissue Fixative

Background

Requirements on tissue fixatives are getting more demanding as molecular analysis becomes increasingly relevant for routine diagnostics. Buffered formaldehyde in pathology laboratories for tissue fixation is known to cause chemical modifications of biomolecules which affect molecular testing. A novel non-crosslinking tissue preservation technology, PAXgene Tissue (PAXgene), was developed to preserve the integrity of nucleic acids in a comparable way to cryopreservation and also to preserve morphological features comparable to those of formalin fixed samples.

Methods

Because of the excellent preservation of biomolecules by PAXgene we investigated its pathogen inactivation ability and biosafety in comparison to formalin by in-vitro testing of bacteria, human relevant fungi and human cytomegalovirus (CMV). Guidelines for testing disinfectants served as reference for inactivation assays. Furthermore, we tested the properties of PAXgene for detection of pathogens by PCR based assays.

Results

All microorganisms tested were similarly inactivated by PAXgene and formalin except Clostridium sporogenes, which remained viable in seven out of ten assays after PAXgene treatment and in three out of ten assays after formalin fixation. The findings suggest that similar biosafety measures can be applied for PAXgene and formalin fixed samples. Detection of pathogens in PCR-based diagnostics using two CMV assays resulted in a reduction of four to ten quantification cycles of PAXgene treated samples which is a remarkable increase of sensitivity.

Conclusion

PAXgene fixation might be superior to formalin fixation when molecular diagnostics and highly sensitive detection of pathogens is required in parallel to morphology assessment.     

Challenges to developing proteomic-based breast cancer diagnostics

Over the past decade, multiple genetic and histological approaches have accelerated development of new breast cancer diagnostics and treatment paradigms. Multiple distinct genetic subtypes of breast cancers have been defined, and this has progressively led toward more personalized medicine in regard to treatment options. There still remains a deficiency in the development of molecular diagnostic assays that can be used for breast cancer detection and pretherapy clinical decisions. In particular, the type of cancer-specific biomarker typified by a serum or tissue-derived protein. Progress in this regard has been minimal, especially in comparison to the rapid advancements in genetic and histological assays for breast cancers. In this review, some potential reasons for this large gap in developing protein biomarkers will be discussed, as well as new strategies for improving these approaches. Improvements in the study design of protein biomarker discovery strategies in relation to the genetic subtypes and histology of breast cancers is also emphasized. The current successes in use of genetic and histological assays for breast cancer diagnostics are summarized, and in that context, the current limitations of the types of breast cancer-related clinical samples available for protein biomarker assay development are discussed. Based on these limitations, research strategies emphasizing identification of glycoprotein biomarkers in blood and MALDI mass spectrometry imaging of tissues are described.     

Detection of mutations in human DNA

Efficient methods for the detection of mutations are of fundamental importance in research and in diagnostics. By detection of a DNA sequence alteration that cosegregates with a clinical phenotype in an affected family, the gene at fault may be identified and assigned a function. Mutation detection methods are also a rate-limiting factor for the clinical application of DNA diagnostics. Currently a large number of techniques are in use to scan for new mutations and to distinguish among previously established sequence variants. Here, some of the problems connected with mutation detection are discussed together with principles on which current and future mutation detection assays can be based.     

Polymerase Chain Reaction (PCR)–Based Diagnostic Assays for Invasive Candidiasis

Candidemia and other types of invasive candidiasis are associated with mortality rates as high as 40% despite antifungal therapy. In part, the poor outcomes stem from the inadequacies of diagnostic tests for invasive candidiasis. The current gold standard is blood culture, which is negative in 50% of autopsy-proven cases. As such, development of novel diagnostics is a top priority. Assays for invasive candidiasis based on polymerase chain reaction (PCR) are potentially attractive because of their high sensitivity, rapid turnaround time, capacity for speciation, and ability to quantitate the candidal burden. Research has refined PCR methods and performance parameters to the point that diagnostic assays for invasive candidiasis are approaching the clinic. Overall, optimized PCR assays have sensitivities and specificities for candidemia and proven or probable invasive candidiasis that exceed 90%. Studies are now needed to determine the precise clinical roles of PCR diagnostic assays and their impact on patient outcomes.     

Bioconjugated nanoparticle detection of respiratory syncytial virus infection

The integration of nanotechnology with biology has produced major advances in molecular diagnostics, therapeutics, and bioengineering. Recent advances have led to the development of functionalized nanoparticles (NPs) that are covalently linked to biological molecules such as antibodies, peptides, proteins, and nucleic acids. These functionalized NPs allow for development of novel diagnostic tools and methods, particularly for pathogens, as rapid and sensitive diagnostics are essential for defining the emergence of infection, determining the period that preventive measures should be applied, for evaluating drug and vaccine efficacy, and for controlling epidemics. In this study, we show that functionalized NPs conjugated to monoclonal antibodies can be used to rapidly and specifically detect respiratory syncytial virus in vitro and in vivo. These results suggest that functionalized NPs can provide direct, rapid, and sensitive detection of viruses and thereby bridge the gap between current cumbersome virus detection assays and the burgeoning need for more rapid and sensitive detection of viral agents.     

Assays for aptamer-based platforms

Aptamers are single stranded DNA or RNA oligonucleotides that have high affinity and specificity towards a wide range of target molecules. Aptamers have low molecular weight, amenable to chemical modifications and exhibit stability undeterred by repetitive denaturation and renaturation. Owing to these indispensable advantages, aptamers have been implemented as molecular recognition element as alternative to antibodies in various assays for diagnostics. By amalgamating with a number of methods that can provide information on the aptamer-target complex formation, aptamers have become the elemental tool for numerous biosensor developments. In this review, administration of aptamers in applications involving assays of fluorescence, electrochemistry, nano-label and nano-constructs are discussed. Although detection strategies are different for various aptamer-based assays, the core of the design strategies is similar towards reporting the presence of specific target binding to the corresponding aptamers. It is prognosticated that aptamers will find even broader applications with the development of new methods of transducing aptamer target binding.     

Quantum dots and prion proteins

A diagnostics of infectious diseases can be done by the immunologic methods or by the amplification of nucleic acid specific to contagious agent using polymerase chain reaction. However, in transmissible spongiform encephalopathies, the infectious agent, prion protein (PrP^Sc), has the same sequence of nucleic acids as a naturally occurring protein. The other issue with the diagnosing based on the PrP^Sc detection is that the pathological form of prion protein is abundant only at late stages of the disease in a brain. Therefore, the diagnostics of prion protein caused diseases represent a sort of challenges as that hosts can incubate infectious prion proteins for many months or even years. Therefore, new in vivo assays for detection of prion proteins and for diagnosis of their relation to neurodegenerative diseases are summarized. Their applicability and future prospects in this field are discussed with particular aim at using quantum dots as fluorescent labels.     

High-throughput antibody production

Proteome-wide sets of antibodies would be an invaluable research resource for use in highly parallel assays such as microarrays. Such assays could provide deeper insights into biology and a wealth of information for clinical diagnostics. However, the rate of discovery of new proteins far exceeds the antibody supply currently produced from traditional animal-based systems. To address this problem, a variety of improvements in antibody production have been developed, including improved animal-based technologies, new antibody structures with superior performances, faster and more discriminating screening techniques, and rapid validation methods. Many of these technologies are amenable to automation, allowing antibody production throughput to significantly increase.     

Rapid diagnostics for methicillin-resistant Staphylococcus aureus: current status

Methicillin-resistant Staphylococcus aureus (MRSA) is a significant cause of healthcare- and community-associated infections, and its prevalence continues to increase. These infections are associated with morbidity and excessive mortality compared with infections caused by methicillin-susceptible S. aureus (MSSA). Numerous studies have cited the increased healthcare costs associated with MRSA infections. Infection control guidelines that combine active surveillance with aggressive patient management, such as patient isolation, decontamination, and other strategies, have been shown to reduce transmission and subsequent infections. The availability of rapid molecular diagnostics has strengthened infection control programs by providing results in hours rather than days, as the time required for culture-based methods. This review summarizes the current status of rapid diagnostic methods available for MRSA detection from nasal surveillance specimens, and assays available for rapid identification of MRSA from positive blood cultures containing Gram-positive cocci in clusters. Both amplification- and probe-based assays are highlighted and discussed in detail. Future technological advances are likely to see real-time assays that combine multiple gene targets for assessment of microbial identification, virulence detection, and mechanisms of resistance beyond mecA.     

Using aptamers as capture reagents in bead-based assay systems for diagnostics and hit identification

Most applications of xMAP (Luminex) bead-based assay technology in diagnostics and drug discovery use immobilized antigens or antibodies. Here the authors describe the development of novel assay systems in which synthetic oligonucleotides that specifically bind and inhibit other biomolecules--so-called aptamers--are directly immobilized on beads. The robustness, specificity, and sensitivity of aptamer-based assays were demonstrated in a test system that detected human alpha-thrombin in serum samples. xMAP technology was also adapted to competitive screening formats where an aptamer/protein complex was disrupted by a functionally analogous competitor. The results indicate that such assays are excellently suited for diagnostic applications or drug screening, where aptamers serve as competitive binding probes for the identification of small-molecule hits. These methods should be transferable to a large number of applications because specific aptamers can be rapidly generated for almost any protein target.     

Multiplexed microsphere diagnostic tools in gene expression applications: factors and futures

Microarrays have received significant attention in recent years as scientists have firstly identified factors that can produce reduced confidence in gene expression data obtained on these platforms, and secondly sought to establish laboratory practices and a set of standards by which data are reported with integrity. Microsphere-based assays represent a new generation of diagnostics in this field capable of providing substantial quantitative and qualitative information from gene expression profiling. However, for gene expression profiling, this type of platform is still in the demonstration phase, with issues arising from comparative studies in the literature not yet identified. It is desirable to identify potential parameters that are established as important in controlling the information derived from microsphere-based hybridizations to quantify gene expression. As these evolve, a standard set of parameters will be established that are required to be provided when data are submitted for publication. Here we initiate this process by identifying a number of parameters we have found to be important in microsphere-based assays designed for the quantification of low abundant genes which are variable between studies.     

Current and emerging polymyxin resistance diagnostics: A systematic review of established and novel detection methods

The emergence of polymyxin resistance, due to transferable mcr genes, threatens public and animal health as there are limited therapeutic options. As polymyxin is one of the last-line antibiotics, there is a need to contain the spread of its resistance to conserve its efficacy. Herein, we describe current and emerging polymyxin resistance diagnostics to inform faster clinical diagnostic choices. A literature search in diverse databases for studies published between 2016 and 2020 was performed. English articles evaluating colistin resistance methods/diagnostics were included. Screening resulted in the inclusion of 93 journal articles. Current colistin resistance diagnostics are either phenotypic or molecular. Broth microdilution is currently the only gold standard for determining colistin MICs (minimum inhibitory concentration). Phenotypic methods comprise of agar-based methods such as CHROMagar™ Col-APSE, SuperPolymyxin, ChromID^® Colistin R, LBJMR and LB medium; manual MIC-determiners viz., UMIC, MICRONAUT MIC-Strip and ComASP Colistin; automated antimicrobial susceptibility testing systems such as BD Phoenix, MICRONAUT-S, MicroScan, Sensititre and Vitek 2; MCR-detectors such as lateral flow immunoassay (LFI) and chelator-based assays including EDTA- and DPA-based tests, that is, combined disk test, modified colistin broth-disk elution (CBDE), Colispot, and Colistin MAC test as well as biochemical colorimetric tests, that is, Rapid Polymyxin NP test and Rapid ResaPolymyxin NP test. Molecular methods only characterize mobile colistin resistance; they include PCR, LAMP and whole-genome sequencing. Due to the faster turnaround time (≤3 h), improved sensitivity (84%–100%) and specificity (93.3%–100%) of the Rapid ResaPolymyxin NP test and Fastinov^®, we recommend this test for initial screening of colistin-resistant isolates. This can be followed by CBDE with EDTA or the LFI as they both have 100% sensitivity and a specificity of ≥94.3% for the rapid screening of mcr genes. However, molecular assays such as LAMP and PCR may be considered in well-equipped clinical laboratories.     

Detection of antibodies in neuropathy patients by synthetic GM1 mimics

Antibodies to the ganglioside GM1 are associated with various forms of acute and chronic immune-mediated neuropathy, including Guillain-Barré syndrome (GBS) and multifocal motor neuropathy. In diagnostics and research, these antibodies are usually detected by GM1 preparations derived from bovine brain tissue, which are non-covalently attached to solid carriers such as enzyme-linked immunosorbent assay (ELISA) plates. Such brain-derived GM1 preparations are potentially contaminated with other glycolipids. In the current study, uncontaminated mono- and divalent synthetic analogs of the ganglioside GM1 were successfully attached via covalent bonds onto the surface of ELISA plates. The resulting modified diagnostic tool showed strong affinities and good specificities for binding of monoclonal mouse and human anti-GM1 antibodies and cholera toxin, as well as for the anti-GM1 antibodies in serum samples from neuropathy patients. While these proof-of-principle experiments reveal the potential of synthetic ganglioside mimics in diagnostics, they show the necessity of further studies to overcome certain limitations, specifically the non-specific interactions in the negative control assays with synthetic GM1.     

Sensitive detection of EGFR mutations using a competitive probe to suppress background in the SMart Amplification Process.

In a previous study, a single nucleotide polymorphism (SNP) diagnostic system named the SMart Amplification Process version 2 (SMAP 2) was reported, which enabled rapid gene diagnostics from crude samples such as whole blood. The asymmetric primer design and use of Taq MutS were reported as innovative background suppression technologies employed by SMAP 2, but Taq MutS is known to display differential affinities for various mismatch combinations, and hence may not be entirely effective for all possible applications. To address this issue we developed another approach using a competitive probe (CP) to enhance background suppression technology instead of Taq MutS. CP is a 3'-end aminated oligonucleotide that competes with 3'-end of a discrimination primer or the self-priming elongation site on intermediate product 2 (IM2) for non-target sequences, such as the alternative allele. The preferred hybridization kinetics for the full-match CP on the non-target sequence results in effective background suppression in SMAP 2 assays. By using a CP, we demonstrated the sensitive detection of EGFR gene mutations in purified genomic DNA from mixed cell populations. The CP approach is another tool enhancing the effectiveness and versatility of SMAP 2 assays, expanding its potential applications, and reinforcing its position as a highly effective technology for molecular diagnostics.     

Identifying and Quantifying Heterogeneity in High Content Analysis: Application of Heterogeneity Indices to Drug Discovery

One of the greatest challenges in biomedical research, drug discovery and diagnostics is understanding how seemingly identical cells can respond differently to perturbagens including drugs for disease treatment. Although heterogeneity has become an accepted characteristic of a population of cells, in drug discovery it is not routinely evaluated or reported. The standard practice for cell-based, high content assays has been to assume a normal distribution and to report a well-to-well average value with a standard deviation. To address this important issue we sought to define a method that could be readily implemented to identify, quantify and characterize heterogeneity in cellular and small organism assays to guide decisions during drug discovery and experimental cell/tissue profiling. Our study revealed that heterogeneity can be effectively identified and quantified with three indices that indicate diversity, non-normality and percent outliers. The indices were evaluated using the induction and inhibition of STAT3 activation in five cell lines where the systems response including sample preparation and instrument performance were well characterized and controlled. These heterogeneity indices provide a standardized method that can easily be integrated into small and large scale screening or profiling projects to guide interpretation of the biology, as well as the development of therapeutics and diagnostics. Understanding the heterogeneity in the response to perturbagens will become a critical factor in designing strategies for the development of therapeutics including targeted polypharmacology.