Peritonitis as a result of perforation has been reported in connection withsevere amebic colitis and, much less often, in patients with few or no symptoms.Other complications of intestinal amebiasis include colocutaneous fistula,perianal ulceration, urogenital infection, colonic stricture, intussusception,and hemorrhage. Most of these complications are uncommon and therefore may provedifficult to diagnose. The term post-amebic colitis is used for nonspecificcolitis following a bout of severe acute amebic colitis. In such cases, thecolon is free of parasites and the clinical findings resemble those of chroniculcerative colitis.
Amebas are difficult to demonstrate in aspirates from extraintestinal abscesses(Fig. 79-5) unless specialprecautions are taken. The contents of most amebic abscesses are relatively freeof the organism. Instead, the organisms concentrate adjacent to the wall of theabscess cavity. If care is taken during aspiration to separate serial aliquotsof aspirate, amebas may be found in the last syringe that empties the cavity.Cysts or trophozoites are only found in approximately one-half of all patientswith amebic liver abscess.
Baveja Parasitology Pdf Free 79
Life cycle of E. granulosus. Echinococcus spp. require two mammalian hosts for completion of their life cycle. Segments containing eggs (gravid proglotitids) or free eggs are passed in the feces of the definitive host, a carnivore. The eggs are ingested by an intermediate host, in which the metacestode stage and protoscoleces develop. The cycle is completed if the metacestode and protoscoleces are eaten by a suitable carnivore.
ELISA techniques using a variety of antigens have been applied to the immunodiagnosis of animal CE (138, 141, 266). In experimentally infected sheep, antibodies to hydatid antigens can be detected as early as 4 to 6 weeks postinfection (266) and persist for at least 4 years (138). However, as referred to above, serological cross-reactions between E. granulosus and other cestodes limit the specific diagnosis of hydatid infection by ELISA with crude parasite antigens (138, 266). Affinity purification of crude antigens with antibodies from animals immunized with homologous antigen (141) or affinity depletion of cross-reactive antigens with monoclonal antibody (138) only partly reduces the cross-reactivity. Components of ovine HCF can bind to sheep immunoglobulin nonspecifically and contribute to false-positive reactions, even with sera from cestode-free animals (138). After affinity depletion of crude antigen with both monoclonal antibody and sheep immunoglobulin from animals not infected with hydatid disease, background reactions were greatly reduced. Using this affinity-depleted antigen, it was possible to differentiate serologically between a flock of sheep with hydatid infection and uninfected sheep from the same locality; however, specific diagnosis of infection in individual sheep from another locality was low, and variation in antibody responses to different parasite strains was suggested as a possible cause of these differences (138).
Cell-free systems (CFS) have recently evolved into key platforms for synthetic biology applications. Many synthetic biology tools have traditionally relied on cell-based systems, and while their adoption has shown great progress, the constraints inherent to the use of cellular hosts have limited their reach and scope. Cell-free systems, which can be thought of as programmable liquids, have removed many of these complexities and have brought about exciting opportunities for rational design and manipulation of biological systems. Here we review how these simple and accessible enzymatic systems are poised to accelerate the rate of advancement in synthetic biology and, more broadly, biotechnology.
Importantly, CFS can be freeze-dried, enabling room temperature storage and distribution [46, 48]. Freeze-dried cell-free (FD-CF) systems can then be activated at the time of need simply by adding water [46]. This feature has been used to deploy biosafe, genetically encoded tools outside of the laboratory as diagnostics and as platforms for biomanufacturing [49, 50], as well as their deployment in altogether new contexts, such as global health and education.
Below we will discuss how CFS are enabling new technologies and accelerating the coming revolution in bioengineering, highlighting some of the most active areas of research in the cell-free community (Fig. 1).
Cell-free protein expression systems and their applications. Capitalizing on their open nature, CFS can be rationally assembled to include cell lysates, purified proteins, energy sources (e.g., ATP), amino acids, other substrates (such as modified tRNAs and membrane mimics) and RNA or DNA (circular or linear). CFS can be applied in portable diagnostic devices [46, 50] and also hold great potential for biomolecular manufacturing [49, 51]. Additionally, CFS can enable discovery of novel enzymes (e.g., through directed evolution) [52]
Molecular recognition underlies almost every biological process, including the nucleic acid base pairing that imparts specific syntax to the central dogma. Scientists and engineers have long worked to usher these processes into cell-free in vitro environments to understand and exploit their underlying molecular mechanisms for purposes such as diagnostics and detection of molecules. One of the fruits from such efforts is the polymerase chain reaction (PCR), which is now an indispensable tool utilized in most molecular biology laboratories, including those for clinical diagnostics. There is currently a growing need for de-centralized, portable diagnostics that can be rapidly deployed in the field, for instance during infectious disease outbreaks or for agricultural purposes. However, sensing technologies such as PCR and others have largely remained confined to laboratories in large urban centers due to their requirement for specialized equipment and personnel.
The biosafe and stable nature of FD-CF systems offers an alternative molecular venue to address the unmet need for distributed and low-cost sensing. Here, the transcription and translation properties of CFS can be used to host gene circuit-based sensors that can detect small molecules and nucleic acids with exquisite sensitivity and specificity. Many of the biosensors and circuits that have been developed for cell-based applications can be operated in the cell-free environment. These include, among others, many classic switches (e.g., TetO- and LacI-based systems), logic gates, negative feedback loops, transcriptional cascades [37, 41, 53,54,55,56] and ring oscillators [57]. This cross-compatibility between CFS and cell-based systems has also been exploited for rapid prototyping of regulatory elements that can be brought back to the cell-based environment.
This sensitivity challenge was addressed by placing an isothermal amplification step (e.g., NASBA) in the workflow upstream of the cell-free reaction. This improved the threshold of detection by orders of magnitude (106). Since isothermal amplification is a primer-directed process, combination with toehold-based sensing results in two sequence-specific checkpoints. An opportunity to test out the improved system presented itself in early 2016 when the outbreak of the mosquito-borne Zika virus was reported in Brazil. With the improved embodiment, FD-CF toehold sensors could detect all global strains of the Zika virus at clinically relevant concentrations (down to 2.8 femtomolar) from viremic plasma [50]. Moreover, powered by the first CRISPR-based system in an in vitro diagnostic system, viral genotypes could be distinguished with single base pair resolution (e.g., American vs African Zika strains). Most recently the Collins group extended these concepts in a tour de force effort that demonstrated quantitative detection of ten gut bacterial species from patient samples [59]. This work demonstrated detection at clinically relevant concentrations with sensing performance that mapped well with parallel measurements done with RT-qPCR. It also showcased the ability to detect a toxin-related sequence for the diagnosis of Clostridium difficile infections.
Another active area in CFS research is the biomanufacturing of therapeutics and other protein-based reagents. Natural biological systems have evolved a remarkable capacity to synthesize a variety of molecules ranging from metabolites to biopolymers. Cell-free protein expression systems allow the incorporation of such reactions into a highly controlled process that allows production of molecules as needed and in the field. Our primary focus here will be on a subset of biopolymers, namely therapeutic proteins. The ongoing work in this field rests on decades of research that have led to the productive and practical systems currently available [28, 29, 36,37,38, 40]. Recent advances in high-throughput preparation techniques [40, 45] and in the development of systems that can use more economical energy sources [64, 65] have made CFS highly accessible. Meanwhile, significant strides are being made towards resolving various protein folding issues and shortcomings in post-translational modifications [66] associated with traditional CFS. Recent advances have showcased the potential for scaling up cell-free reactions, with some having demonstrated reaction volumes reaching 100 liters [67, 68] to 1000 liters [69]. Cell-free expression has been used as a platform for the production of a wide range of potential therapeutics, some of which have been summarized in Table 1. A number of these products have been validated in animal models [49, 76].
Two primary modes of CFS have been pursued. The first, used by commercial efforts such as Sutro [94], focuses on large, centralized production. This approach leverages the advantages of synthesis outside of the cell for biomanufacturing. For these applications, CFS not only allow for rapid production, but also significantly speed up the drug development process [95]. Remarkably, Sutro has reportedly increased their cell-free production to an incredible 1000 liters [69], showcasing the scalability of centralized cell-free production. The second mode uses FD-CF systems to de-centralize biomanufacturing capacity for small-batch production of therapeutics, with applications in global health and emergency response [49, 73, 96, 97]. Using this mode of production, we have recently demonstrated the proof-of-concept capacity to manufacture over 50 therapeutics and lab reagents, including proteins (e.g., vaccines, antibodies, and antimicrobial peptides) and small molecules [49], with applications outside of the laboratory setting. 2ff7e9595c
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