Advancements in molecular technologies have provided new platforms that are being increasingly adopted for use in the clinical microbiology laboratory. Among these, microarray methods are particularly well suited for diagnostics as they allow multiplexing, or the ability to test for multiple targets simultaneously from the same specimen. Microarray technologies commonly used for the detection and identification of microbial targets include solid-state microarrays, electronic microarrays and bead suspension microarrays. Microarray methods have been applied to microbial detection, genotyping and antimicrobial resistance gene detection. Microarrays can offer a panel approach to diagnose specific patient presentations, such as respiratory or gastrointestinal infections, and can discriminate isolates by genotype for tracking epidemiology and outbreak investigations. And, as more information has become available on specific genes and pathways involved in antimicrobial resistance, we are beginning to be able to predict susceptibility patterns based on sequence detection for particular organisms. With further advances in automated microarray processing methods and genotype-phenotype prediction algorithms, these tests will become even more useful as an adjunct or replacement for conventional antimicrobial susceptibility testing, allowing for more rapid selection of targeted therapy for infectious diseases.

Real-time PCR assays have revolutionised diagnostic microbiology over the past 15 years or more. Adaptations and improvements over that time frame have led to the development of multiplex assays. However, limitations in terms of available fluorophores has meant the number of assays which can be combined has remained in single figures. This latter limitation has led to the focus tending to be on individual pathogens and their detection. This chapter describes the development of TaqMan® Array Cards (TACs), technology which allows the detection of multiple pathogens (up to 48 targets) from a single nucleic acid extract, utilising small volumes and real-time PCR. This in turn lends itself to a syndromic approach to infectious disease diagnosis. Using the examples of TACs we have developed in our own laboratory, as well as others, we explain the design, optimisation and use of TACs for respiratory, gastrointestinal and liver infections. Refinement of individual assays is discussed as well as the incorporation of appropriate internal and process controls onto the array cards. Finally, specific examples are given of instances where the assays have had a direct, positive impact on patient care.

The current chapter summaries the world of Microbiology and boom of Nanotechnology and how both the exciting fields come together to help men kind with various new applications in water, food, medical biology and immunology. Furthermore synthesis of nano materials utilising the potential of microorganisms also opens a newer avenue for 'green' synthesis.

The availability of multiple versions of vaccines designed to help prevent COVID-19 has offered an opportunity to at least control the current pandemic, and possibly to quickly eradicate this disease fully, along with the implementation of other preventive measures. In order to accomplish this feat more effectively, as many people as possible need to be vaccinated, especially for high-risk groups having co-morbid conditions such as diabetes, obesity and old age, and possibly those with various forms of immunodeficiencies, such as HIV/AIDS. This chapter focuses primarily on some of the basic biomedical aspects on vaccine design and use, and any possible concerns that need to be considered in getting people in the high-risk category vaccinated and monitored thereafter for their continuous health and well-being.

This chapter discusses the management of immunocompromised and infected animals. The microbiological quality of laboratory animals is a direct result of colony management practices and monitoring provides an after-the-fact assessment of the adequacy of those practices. Monitoring is, therefore, of greatest value in connection with the maintenance of animals in isolation systems where vigorous microbiological control is applied. In addition to constructive measures, an appropriate management system is necessary for the prevention of infections, as well as for their detection and control. It is a major task for the management of an animal facility to understand the way micro-organisms might be introduced or spread under the specific conditions given. The management of all animal facilities in an institution is best centralized. This warrants that all information dealing with the purchase of animals, the use of experimental materials and equipment and the performance of animal experiments flows through one office. This reduces the opportunity for the failures of communication. Centralized management can best establish comprehensive monitoring programs to evaluate important risk factors, such as animals and biological materials, before they are introduced into a facility.

The outbreak of the COVID-19 pandemic in 2019 has been one of the greatest challenges modern medicine and science has ever faced. It has affected millions of people around the world and altered human life and activities as we once knew. The high prevalence as well as an extended period of incubations which usually does not present with symptoms have played a formidable role in the transmission and infection of millions. A lot of research has been carried out on developing suitable treatment and effective preventive measures for the control of the pandemic. Preventive strategies which include social distancing, use of masks, washing of hands, and contact tracing have been effective in slowing the spread of the virus; however, the infectious nature of the SARS-COV-2 has made these strategies unable to eradicate its spread. In addition, the continuous increase in the number of cases and death, as well as the appearance of several variants of the virus, has necessitated the development of effective and safe vaccines in a bid to ensure that human activities can return to normalcy. Nanotechnology has been of great benefit in the design of vaccines as nano-sized materials have been known to aid the safe and effective delivery of antigens as well as serve as suitable adjuvants to potentiate responses to vaccines. There are only four vaccine candidates currently approved for use in humans while many other candidates are at various levels of development. This review seeks to provide updated information on the current nano-technological strategies employed in the development of COVID-19 vaccines.

Seasonal behaviour is an attribute of many viral diseases. Like other 'winter' RNA viruses, infections caused by the causative agent of COVID-19, SARS-CoV-2, appear to exhibit significant seasonal changes. Here we discuss the seasonal behaviour of COVID-19, emerging viral phenotypes, viral evolution, and how the mutational landscape of the virus affects the seasonal attributes of the disease. We propose that the multiple seasonal drivers behind infectious disease spread (and the spread of COVID-19 specifically) are in 'trade-off' relationships and can be better described within a framework of a 'triangle of viral persistence' modulated by the environment, physiology, and behaviour. This 'trade-off' exists as one trait cannot increase without a decrease in another. We also propose that molecular components of the virus can act as sensors of environment and physiology, and could represent molecular culprits of seasonality. We searched for flexible protein structures capable of being modulated by the environment and identified a galectin-like fold within the N-terminal domain of the spike protein of SARS-CoV-2 as a potential candidate. Tracking the prevalence of mutations in this structure resulted in the identification of a hemisphere-dependent seasonal pattern driven by mutational bursts. We propose that the galectin-like structure is a frequent target of mutations because it helps the virus evade or modulate the physiological responses of the host to further its spread and survival. The flexible regions of the N-terminal domain should now become a focus for mitigation through vaccines and therapeutics and for prediction and informed public health decision making.

The hesitancy and resistance to get vaccinated against COVID-19, by a relatively small but significant part of the general population, has become a serious worldwide problem, and particularly in the United States, despite a vigorous and highly organized governmental advertising campaign promoting vaccination. The unwillingness to get vaccinated has its roots in mostly the spreading of non-scientific, unproven or misleading information. This chapter explains many of the reasons, including an historical connection, behind this anti-vaccine movement, and proposes several possible and feasible remedies to counter this sentiment.

The occurrence of the COVID-19 pandemic caused by the SARS-CoV-2 virus since the end of 2019 has significantly affected the entire world. Now SARS-CoV-2 diagnostic tests are not only required for screening of suspected infected people for their medical treatment, but have also become a routine diagnosis for all people at a place where new cases have emerged in order to control spread of the disease from that region. For these reasons, sensitive methods for detection of SARS-CoV-2 are highly needed in order to avoid undetected infections. In addition, sample pooling that uses pooled specimens has been routinely employed as a time- and cost-effective strategy for community monitoring of SARS-CoV-2. In this regard, the content of each viral RNA sample of an individual will be further diluted in detection; therefore, higher detection sensitivity would be rather preferred. Among nucleic acid-based detection methods, isothermal nucleic acid amplifications are considered quite promising because they typically take less time to complete the test (even less than 20 min) without the need of thermal cycles. Hence, it does not necessitate the use of highly costly real-time PCR machines. According to recently published isothermal nucleic acid amplification methods, the reverse transcription recombinase polymerase amplification (RT-RPA) approach shows outstanding sensitivity with up to single-copy sensitivity in a test reaction. This chapter will mainly focus on how to employ RT-RPA technology to sensitively detect SARS-CoV-2 RNA. Besides, recently published RT-RPA based detection methods will be summarized and compared regarding their detection parameters and the primers and probes being used. In addition, we will also highlight the key considerations on how to design an ultrasensitive RT-RPA assay and the precautions needed to conduct the assay. Moreover, based on our recent report, we will also detail the methods we developed to detect SARS-CoV-2 RNA using modified RT-RPA, or RT-ERA, with single-copy sensitivity and the possible extensions beyond this method.

The investigation of the immune response after SARS-CoV-2 infection has been the goal of many researchers worldwide. The study of humoral immune responses and T cell production after infection requires the obtaining of individualized blood samples to test the presence of antibodies or activated T cells specific for the virus. T cell studies are especially troublesome due to the need for more specialized resources often outside the daily routine of clinical laboratories. For this reason the development of a simple and objective method to achieve these T cell studies is needed. In this manuscript we reviewed the hypersensitivity reactions, the theoretical basis and the historical background of delayed type hypersensitivity (DTH) which uses the principles of use of this test in the clinical setting for the past century. In the second part of the review, we focus on COVID adaptive immune responses, to understand the differences and challenges offered by this new application of DTH to investigate immune responses elicited after infection. In the last part of the review a vision provided for the use of this test to investigate the immunogenicity elicited by the vaccines. In our opinion, the clinical guidelines of immune assessment of SARS-CoV-2-infected or vaccinated individuals should include this simple and low-cost test to measure T-cell immunity. Rationale and improved vaccination schemes could be obtained after its implementation in the routine assessment of immunity in this pandemic situation.

Since the invention of the polymerase chain reaction (PCR) and discovery of polymerase, PCR has become a staple in both research and clinical molecular laboratories. As clinical and diagnostic needs have evolved over the last few decades, demanding greater levels of sensitivity and accuracy, so too has PCR performance. Through optimisation, the present-day uses of real-time PCR and quantitative real-time PCR are enumerable. The technique, combined with adoption of automated processes and reduced sample volume requirements, makes it an ideal method in a broad range of clinical applications, especially in virology. Complementing serologic testing by detecting infections within the pre-seroconversion window period and infections with immunovariant viruses, real-time PCR provides a highly valuable tool for screening, diagnosing, or monitoring diseases, as well as evaluating medical and therapeutic decision points that allows for more timely predictions of therapeutic failures than traditional methods and, lastly, assessing cure rates following targeted therapies. All of these serve vital roles in the continuum of care to enhance patient management. Beyond this, quantitative real-time PCR facilitates advancements in the quality of diagnostics by driving consensus management guidelines following standardisation to improve patient outcomes, pushing for disease eradication with assays offering progressively lower limits of detection, and rapidly meeting medical needs in cases of emerging epidemic crises involving new pathogens that may result in significant health threats.

Since the SARS-CoV-2 virus triggered the beginning of the COVID-19 pandemic, scientists, government officials, and healthcare professionals around the world recognized the need for accessible, affordable, and accurate testing to predict and contain the spread of COVID-19. In the months that followed, research teams designed, tested, and rolled out hundreds of diagnostic assays, each with different sampling methods, diagnostic technologies, and sensitivity levels. However, the contagious virus continued to spread; SARS-CoV-2 travelled through airborne particles and spread rapidly, despite the widening use of diagnostic assays. As the pandemic continued, hundreds of millions of people contracted COVID-19 and millions died worldwide. With so many infections, SARS-CoV-2 received many opportunities to replicate and mutate, and from these mutations emerged more contagious, deadly, and difficult-to-diagnose viral mutants. Each change to the viral genome presented potential added challenges to containing the virus, and as such, researchers have continued developing and improving testing methods to keep up with COVID-19. In this chapter, we examine several SARS-CoV-2 variants that have emerged during the pandemic. Additionally, we discuss a few major COVID-19 diagnostic technique categories, including those involving real-time PCR, serology, CRISPR, and electronic biosensors. Finally, we address SARS-CoV-2 variants and diagnostic assays in the age of COVID-19 vaccines.

This chapter describes how real-time polymerase chain reaction (PCR) performs and how it may be used to detect microbial pathogens and the relationship they form with their host. Research and diagnostic microbiology laboratories contain a mix of traditional and leading-edge, in-house and commercial assays for the detection of microbes and the effects they impart upon target tissues, organs, and systems. The PCR has undergone significant change over the last decade, to the extent that only a small proportion of scientists have been able or willing to keep abreast of the latest offerings. The chapter reviews these changes. It discusses the second-generation of PCR technology-kinetic or real-time PCR, a tool gaining widespread acceptance in many scientific disciplines but especially in the microbiology laboratory.

SARS-CoV-2 continues to evolve, even after implementation of public-wide vaccination, as can be observed by an increasing number of mutations over time. Compared to responses by the United States and European countries, the disease mitigation strategies employed by the Australian government have been swift and effective. This provides a unique opportunity to study the emergence of variants of concern (VOCs) at many latitude levels in a country that has been able to control infection for the majority of the pandemic. In the present study, we explored the occurrence and accumulation of major mutations typical of VOCs in different regions of Australia and the effects that latitude has on the establishment of VOC-induced disease. We also studied the constellation of mutations characteristic of VOCs to determine if the mutation sets acted as haplotypes. Our goal was to explore processes behind the emergence of VOCs as the viral disease progresses towards becoming endemic. Most reported COVID-19 cases were in largest cities located within a -30°S to - 50°S latitude corridor previously identified to be associated with seasonal behavior. Accumulation plots of individual amino acid variants of major VOCs showed that the first major haplotypes reported worldwide were also present in Australia. A classification of accumulation plots revealed the existence of 18 additional haplotypes associated with VOCs alpha, delta and omicron. Core mutant constellations for these VOCs and curve overlaps for variants in each set of haplotypes demonstrated significant decoupling patterns, suggesting processes of emergence. Finally, construction of a "haplotype network" that describes the viral population landscape of Australia throughout the COVID-19 pandemic revealed significant and unanticipated seasonal patterns of emergence and diversification. These results provide a unique window into our evolutionary understanding of a human pathogen of great significance. They may guide future research into mitigation and prediction strategies for future VOCs.

This chapter discusses the management of immunocompromized and infected animals. The microbiological quality of laboratory animals is a direct result of colony management practices, and monitoring provides an after-the-fact assessment of the adequacy of those practices. In the case of immunocompromised animals or in infection experiments, however, monitoring for a comprehensive list of micro-organisms is reasonable. The testing of animals usually starts with necropsy and blood sampling for serology, followed by microscopic examination for parasites and sampling of organs for bacteriology, pathology, and, in rare cases, virological examinations. Biological materials represent a high risk, if they originate from or have been propagated in animals. In particular, tumors, viruses, or parasites that are serially passaged in animals often pick up pathogens, and therefore a high percentage of these are contaminated. It has been shown in mice and rats that all preimplantational stages can be revitalized successfully upon freezethaw procedures. For long-term storage, eight-cell stages have been recommended in the chapter, while two-cell stages were considered to be less suitable. One embryo batch (inbred strain) derived from a single pedigree donor pair may be regarded as a prospective breeding nucleus, if one fertile breeding pair is obtained upon revitalization. Assuming an average revitalization rate of 20% (fertile breeders), one embryo batch should contain a minimum number of 10 embryos to obtain at least one breeding pair with a 50% chance of revitalization.

Since the beginning of the COVID-19 pandemic, many diagnostic approaches (RT-qPCR, RAPID, LFA) have been adopted, with RT-qPCR being the most popular/gold standard. But, one of the major problems of COVID-19 diagnostics is the presentation of a wide range of symptoms which varies among different patients and needs early diagnosis for better management. Even though RT-qPCR is a precise molecular technique false negative results may be obtained. On the other hand, CRISPR-based SARS-CoV-2 detection approaches are cost and time efficient, highly sensitive and specific, and do not require sophisticated instruments. Moreover, they also show promise for increased scalability and diagnostic tests can be carried out at the point-of-care (POC). The CRISPR can be customized to the target of any genomic region of interest within the desired genome possessing a broad range of other applications and has been efficiently implemented for diagnosis of SARS-CoV-2. The CRISPR/Cas systems provide the specific gene targeting with immense potential to develop new generation diagnostics and therapeutics. Moreover, with the CRISPR/Cas based therapeutics, multiplexing is possible, where different sgRNAs or crRNAs can be guided to more than one target within the same gene thus decreasing the possibility of viral escape mutants. As an exceptionally efficient tool CRISPR/Cas13 and CARVER (Cas13-assisted restriction of viral expression and readout) systems can be implemented to target a broad range of ssRNA viruses that can be used for both, diagnosis and treatment for a variety of viral diseases including SARS-CoV-2. However, the efficacy and safety of the CRISPR-based therapeutics needs to be assessed in pre-clinical and clinical settings. Although the CRISPR biotechnologies are not very helpful to control the present pandemic of COVID-19 it is hopeful that the limitations of the CRISPR/Cas system can be overcome in the near future. The CRISPR based strategies may lead to a new era in the field of disease diagnosis and therapeutic development that would make us better prepared for future viral threats.

Newly developed methods for the detection of bacteria and viruses have provided microbiologists with the means to rapidly identify and monitor specific microorganisms in food and water. Traditional methods of testing involve culture techniques to increase the numbers of the organism to a detectable level, followed by isolation and biochemical identification. This chapter focuses on the methodologies to detect pathogens and indicator organisms; however, the methods described are applicable to most bacteria. As detection and isolation methods have improved, a growing number of pathogens have been identified as important food- and waterborne pathogens. This chapter describes the use of nucleic acid and antibody probes that have the potential to circumvent the need to culture the organism prior to identification. Nucleic acid probes have become a valuable diagnostic reagent in the identification of human and animal pathogens and have made possible the identification of viruses and bacteria that are difficult, if not impossible, to cultivate. DNA probes have also proved to be a useful tool for identifying and monitoring the organisms in food and the environment.