Relative Sensitivity of Conventional and Real-Time PCR Assays for Detection of SFG Rickettsia in Blood and Tissue Samples from Laboratory Animals

Studies on the natural transmission cycles of zoonotic pathogens and the reservoir competence of vertebrate hosts require methods for reliable diagnosis of infection in wild and laboratory animals. Several PCR-based applications have been developed for detection of infections caused by Spotted Fever group Rickettsia spp. in a variety of animal tissues. These assays are being widely used by researchers, but they differ in their sensitivity and reliability. We compared the sensitivity of five previously published conventional PCR assays and one SYBR green-based real-time PCR assay for the detection of rickettsial DNA in blood and tissue samples from Rickettsia- infected laboratory animals (n = 87). The real-time PCR, which detected rickettsial DNA in 37.9% of samples, was the most sensitive. The next best were the semi-nested ompA assay and rpoB conventional PCR, which detected as positive 18.4% and 14.9% samples respectively. Conventional assays targeting ompB, gltA and hrtA genes have been the least sensitive. Therefore, we recommend the SYBR green-based real-time PCR as a tool for the detection of rickettsial DNA in animal samples due to its higher sensitivity when compared to more traditional assays.     

Critical review of Identification Techniques for Armillaria spp. and Heterobasidion annosum Root and Butt Rot Diseases

The need for a rapid and definite diagnosis of the Armillaria spp. and Heterohasidion annosum root and butt rot diseases as well as general requirements expected of a test to detect the pathogens are reviewed. As a spin-off from more fundamental molecular biological research there has been a remarkable increase in new methods for diagnosis (i.e. detection and identification) in recent years. These new methods all have in comtnon that they use strtjctural elements of the target organisms such as proteins, polysaccharides, glycoproteins, nucleic acids, etc. for the construction of antibodies, probes and primers for detection, or to produce electrophoretic patterns to be used for classification and identification. This paper presents a short review of principles, advantages, disadvantages and some perspectives of the new methods developed for Armillaria spp. and H. annosum diagnosis compared with conventional ones, such as interfertility testing or visual inspection of the pathogens in situ and in vitro in pure culture. It will be noted that the specificity and reproducibility of the new techniques, which are closely linked with potential experimental error, lead to principal pitfalls which should be kept in mind when conclusions are drawn on data generated by them.
Even though PCR-based methods become increasingly more widespread it is likely that most diagnostic methods will continue to be used or co-exist in some form in the future.     

Molecular techniques in wastewater: Understanding microbial communities, detecting pathogens, and real-time process control

Traditionally, the detection of pathogens in water, wastewater, and other environmental samples is restricted by the ability to culture such organisms from complex environmental samples. During the last decade the use of molecular methods have supplied the means for examining microbial diversity and detecting specific organisms without the need for cultivation. The application of molecular techniques to the study of natural and engineered environmental systems has increased our insight into the vast diversity and interaction of microorganisms present in complex environments. In this paper, we will review the current and emerging molecular approaches for characterizing microbial community composition and structure in wastewater processes. Recent studies show that advances in microarray assays are increasing our capability of detecting hundreds and even thousands of DNA sequences simultaneously and rapidly. With the current progress in microfluidics and optoelectronics, the ability to automate a detection/identification system is now being realized. The status of such a system for wastewater monitoring is discussed.     

Impact of molecular biology on the detection of foodborne pathogens

Molecular biological methods that use antibodies and nucleic acids to detect specific foodborne bacterial pathogens were scarcely known a decade and a half ago. Few scientists could have predicted that these tools of basic research would come to dominate the field of food diagnostics. Today, a large number of cleverly designed assay formats using these technologies are available commercially for the detection in foods of practically all major established pathogens and toxins, as well as of many emerging pathogens. These tests range from very simple antibody-bound latex agglutination assays to very sophisticated DNA amplification methods. Although molecular biological assays are more specific, sensitive, and faster than conventional (often cultural) microbiological methods, the complexities of food matrices continue to offer unique challenges that may preclude the direct application of these molecular biological methods. Consequently, a short cultural enrichment period is still required for food samples prior to analysis with these assays. The greater detection sensitivity of molecular biological methods may also affect existing microbiological specifications for foods; this undoubtedly will have repercussions on the regulatory agencies, food manufacturers, and also consumers. The US government has the right to retain a nonexclusive royalty-free license in and to any copyright covering this article. Use of trade names is for identification only and does not imply an endorsement by the US FDA.     

Advances in molecular detection of Aspergillus: an update

Filamentous cosmopolitan fungi of the genus Aspergillus can be harmful in two ways, directly they can be opportunistic pathogens causing aspergillosis and indirectly due to aflatoxin production on food products which can lead to aflatoxicosis. Therefore, a number of methods have been proposed so far for detection of the fungi with lowest possible concentration at the earliest. Molecular methods such as PCR and/or in combination with certain techniques have been found to be useful for Aspergillus detection. We discuss here various technologies that have emerged in recent years and can possibly be used for the molecular detection of Aspergillus in an efficient way. These methods like RSIC, C-probe, and inversion probe with pyrosequencing or direct ss/dsDNA detection have been used for the identification of fungal or bacterial pathogens and thus formulate a ‘gold standard’ for Aspergillus detection.     

Innovative tools for detection of plant pathogenic viruses and bacteria

Detection of harmful viruses and bacteria in plant material, vectors or natural reservoirs is essential to ensure safe and sustainable agriculture. The techniques available have evolved significantly in the last few years to achieve rapid and reliable detection of pathogens, extraction of the target from the sample being important for optimising detection. For viruses, sample preparation has been simplified by imprinting or squashing plant material or insect vectors onto membranes. To improve the sensitivity of techniques for bacterial detection, a prior enrichment step in liquid or solid medium is advised. Serological and molecular techniques are currently the most appropriate when high numbers of samples need to be analysed. Specific monoclonal and/or recombinant antibodies are available for many plant pathogens and have contributed to the specificity of serological detection. Molecular detection can be optimised through the automatic purification of nucleic acids from pathogens by columns or robotics. New variants of PCR, such as simple or multiplex nested PCR in a single closed tube, co-operative-PCR and real-time monitoring of amplicons or quantitative PCR, allow high sensitivity in the detection of one or several pathogens in a single assay. The latest development in the analysis of nucleic acids is micro-array technology, but it requires generic DNA/RNA extraction and pre-amplification methods to increase detection sensitivity. The advances in research that will result from the sequencing of many plant pathogen genomes, especially now in the era of proteomics, represent a new source of information for the future development of sensitive and specific detection techniques for these microorganisms.     

The polymerase chain reaction and hepatitis C virus diagnosis

Abstract: In the absence of tissue culture, electron microscopy or assays for viral antigen, the direct detection of hepatitis C virus (HCV) is by necessity dependent upon nucleic acid hybridisation methods. Of the available methods, amplification of HCV cDNA by polymerase chain reaction (PCR) commends itself by virtue of its extreme sensitivity and its consequent ability to detect the very low levels of HCV-RNA that are present in many clinical samples. In this review the development and evolution of PCR techniques for HCV detection are described and a number of clinical applications are considered in detail. The application include diagnosis of acute infection during the seronegative window period prior to the appearance of HCV antibodies, and diagnosis of HCV infection in the immunosuppressed. PCR also enables identification of chronic viraemic carrier state and it permits accurate monitoring of the antiviral effects of drugs such as interferon. Confirmation of the specificity HCV antibody assays and detection of HCV contamination of blood donations and blood products are other important areas in which PCR techniques have proved invaluable. In addition, PCR-based techniques underlie an increasing number of molecular epidemiological and genotyping studies and they are providing insights into the details of HCV cellular tropism and replication. A number of logistic problems and operational difficulties are also discussed. Despite these limitations it is concluded that PCR will continue to make significant contributions to both clinical practice and to our understanding of the basic biology of HCV infection.     

Tissue Diagnosis of Invasive Fungal Infections: Current Limitations and the Emerging Use of Molecular Techniques

Invasive fungal diseases (IFD) due to opportunistic fungi are commonly treated using empirical antifungal therapy. Therefore, a comprehensive study of organisms associated with IFD is essential to define successful empiric therapies in each setting. Current diagnostic tests, such as culture, histology and serology are suboptimal, leading to delays in the initiation of antifungal therapies and resulting in high mortality rates despite the availability of several new antifungal agents. Using molecular methods to identify fungal pathogens directly from formalin-fixed, paraffin-embedded tissues is emerging as a diagnostic approach. The goal of this molecular approach is to complement conventional diagnostic tests through the reliable detection and identification of fungal nucleic acids or antigens in tissues so as to direct antiinfective therapies and improve patient outcomes. Here we review challenges and recent advances in the identification of fungal pathogens from tissue samples by conventional and molecular methods.     

Molecular characterization of ticks and tick-borne pathogens

Ticks and their vertebrate hosts often carry several pathogens simultaneously, which either belong to different or to the same genera. Conventional methods (such as blood smear examination or tick salivary gland staining) are often unable to discriminate between pathogens. Therefore, molecular methods for the detection and differentiation of tick-borne pathogens are increasingly used. Technical problems still remain to identify pathogens within tick tissues, or within host animals when infection rates are very low. Recently we developed an integrated approach to identify several pathogens with only one molecular test. This approach, the reverse line blot hybridization (RLB) reduces costs of analysis, gives quicker results. and allows standardized inter-laboratory comparisons. Finally, this paper also focuses on the molecular diagnostic techniques currently used in the laboratories of the Mediterranean countries.     

Microchip technology applications for blood group analysis

Blood group analysis techniques are some of the most in demand immunological applications in clinical transfusion praxis and organ transplantation. In order to aid the advance towards higher throughput and increased sensitivity, analytical solutions dealing with a minimal amount of blood samples and the miniaturization of diagnostic equipment using microchip technologies have been evolving into an optimal solution. Here we review fabrication technologies for various types of microstructure on microchips, related operating procedures, and characterization approaches. Our focus is on examples of microchip technology and instrumentation used for blood group analysis ranging from classical serological methods of glycoprotein detection and solid phase assays, to nucleic acid amplification techniques. Molecular typing using microchip-based techniques is emerging as a supplement to standard serological methods. Microchip technology will play its key role to support blood group analysis at the molecular scale by using microliters of blood samples for extremely sensitive, quantitative, and high throughput analyses.     

Quantitative mRNA expression analysis in kidney glomeruli using microdissection techniques

The introduction of new tools for molecular analysis, such as RT-qPCR and microarrays, has provided researchers with powerful applications to study renal disease and development. However, the high cellular heterogeneity of the renal tissue complicates the molecular analysis of specific cells and cell groups such as glomerular or tubular cells. In the past, glomerular sieving and manual dissection were used for the isolation of glomeruli. However, these techniques cannot be used for the isolation of specific glomeruli or for the co-isolation of additional tissue fractions. In recent decades, new microdissection techniques such as laser-assisted microdissection have been developed. These applications allow the isolation of small cell groups from heterogeneous tissue for molecular analysis, including microarray and RT-qPCR. Although very promising, some drawbacks are associated with these techniques. The isolated sample material is generally small and requires sensitive assays. In addition, the long sample processing time may result in a considerable loss of RNA integrity. Careful optimization and rigorous quality analysis should overcome these drawbacks. In the present paper, the recent literature on the application of microdissection techniques in kidney research is reviewed, together with a discussion of the critical issues that are essential for the application of quantitative mRNA expression analysis with RT-qPCR on microdissected samples.     

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.     

Subcellular localization of mycobacteria in tissues and detection of lipid antigens in organelles using cryo-techniques for light and electron microscopy

The survival of intracellular pathogens within a host is determined by microbial evasion, which can be partially attributed to their subcellular trafficking strategies. Microscopic techniques have become increasingly important in understanding the cell biology of microbial infections. These recently developed techniques can be used for the subcellular localization of antigens not only in cultured cells but also within tissues such as Mycobacterium tuberculosis in lung and Mycobacterium leprae in skin. High-resolution immunofluorescence microscopy can be used in combination with cryo-immunogold electron microscopy using consecutive cryo-sections on the same tissue block forming a direct connection between the two microscopy techniques. The detection of mycobacterial lipid antigens in situ at an ultrastructural level is currently a challenge, but new modifications can be used to address this. These methods might be of interest to microbiologists and cell biologists who study host-pathogen interactions.     

A multiplexed protein microarray for the simultaneous serodiagnosis of human immunodeficiency virus/hepatitis C virus infection and typing of whole blood

All donor blood samples must be tested pretransfusion to determine the donor blood type. Standard testing protocols require that assays be performed for important bloodborne pathogens such as hepatitis C, syphilis, hepatitis B, and human immunodeficiency virus. We have demonstrated proof of the concept that a protein microarray can type whole blood and detect antibody to significant pathogens simultaneously from the same donor blood sample. The data collected demonstrate the ability of the array to accurately type blood samples while also detecting the presence of antibodies against both human immunodeficiency virus and hepatitis C virus. In conclusion, we have successfully developed a platform capable of typing human whole blood samples, while at the same time testing for the presence of antibodies specific for human immunodeficiency virus/hepatitis C virus. The major benefits of this system are its amenability to expansion with additional assays, for example, rhesus typing and syphilis and/or hepatitis B virus detection, and also the adaptability of the assay to higher-throughput analysis, currently 16 individual samples per slide, but readily expandable to a 96-well format.     

Clinical applications of molecular biology for infectious diseases

Molecular biological methods for the detection and characterisation of microorganisms have revolutionised diagnostic microbiology and are now part of routine specimen processing. Polymerase chain reaction (PCR) techniques have led the way into this new era by allowing rapid detection of microorganisms that were previously difficult or impossible to detect by traditional microbiological methods. In addition to detection of fastidious microorganisms, more rapid detection by molecular methods is now possible for pathogens of public health importance. Molecular methods have now progressed beyond identification to detect antimicrobial resistance genes and provide public health information such as strain characterisation by genotyping. Treatment of certain microorganisms has been improved by viral resistance detection and viral load testing for the monitoring of responses to antiviral therapies. With the advent of multiplex PCR, real-time PCR and improvements in efficiency through automation, the costs of molecular methods are decreasing such that the role of molecular methods will further increase. This review will focus on the clinical utility of molecular methods performed in the clinical microbiology laboratory, illustrated with the many examples of how they have changed laboratory diagnosis and therefore the management of infectious diseases.     

Human astrovirus diagnosis and typing: current and future prospects

Human astroviruses (HAstV) are important human pathogens causing gastroenteritis worldwide. The increased recognition of astroviruses as the cause of sporadic outbreaks of disease is due to the recent availability of improved diagnostic methods. During the last decade, most epidemiological surveys have chosen astrovirus-specific RT-PCR as screening methods. In addition to serotyping by molecular techniques, new typing methods are being developed that may also identify other viral properties related to virulence. The information provided by different typing assays is required for a better understanding of both the antigenic diversity and the molecular mechanisms of pathogenicity.     

Evaluation of non-radioactive labelling and detection of deoxyribonucleic acids: Part one: chemiluminescent methods

The growth of analytical methods for the detection of nucleic acid from various biological samples reflects recent advances in biotechnology development especially in the areas of genetic, infections and cancer diagnosis. The target DNA is detected by hybridization techniques derived from Southern's blotting. However such assays, based on the use of ³²P labelled DNA probes, bring with them the associated problems of handling radioactive materials. In order to overcome these difficulties, a number of chemiluminescent detection methods have recently been developed.These new, alternative probe labelling procedures and chemiluminescent detection methods are easy to use in routine assays performed in research laboratories as well as for medical applications, and can reach the level of sensitivity found in classical radiolabelling techniques.The techniques investigated include peroxydase, biotin 16-dUTP or digoxigenin 11-dUTP probe labelling. The target DNAs are transferred onto nitrocellulose or nylon membranes and further fixed by heat or UV crosslinking. Specific hybridization on the target DNA is finally revealed by the use of chemiluminescent substrates. For all these techniques the detection limit is 10 aM (attomol) of a 561 bp target DNA. However for the probes labelled with peroxydase and with digoxigenin the detection limit drops to 1.0 aM of the target DNA. In the present paper we shall compare several of these DNA labelling and detection procedures and show that the detection threshold can vary by as much as a factor of 20 from method to method. This is the first time that various chemiluminescent methods for label and detection of DNA are compared and evaluated in order to determine the best protocol.