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Protozoan Pathogens: Identification
Abstract and Figures
The identification of protozoan pathogens is based upon direct detection of the respective causative agent in clinical specimens and/or upon detection of specific immune reactions of the host. A considerable part of protozoan diagnostics still mainly relies on microscopy, however, in the past years molecular methods, particularly polymerase chain reaction (PCR)‐based techniques, have gained more and more importance. One of the big advantages of molecular techniques is that they usually allow identification below the genus level, which is often impossible by light microscopy. Serological tests are of great value in all tissue parasites and generally in most extra‐intestinal infections, whereas they only have limited importance in acute infections with short incubation times and in immunocompromised patients. Rapid card tests detecting parasite antigens and host antibodies are available for several important protozoan parasites and serve particularly well in the field setting.
Key Concepts
Of the around 100 protozoan species that can infect humans, some are of prime importance for human health, including the causative agents of malaria, amebiasis, leishmaniasis and sleeping sickness .
Several protozoan infections show a more severe progression in the immunocompromised host , important examples are toxoplasmosis, pneumocystosis, cryptosporidiosis and visceral leishmaniasis.
The size of protozoan pathogens varies from ∼2 μm (amastigote Leishmania spp.) to ∼150 μm ( Balantidium coli ).
As parasite density in stool or body fluids may not be constant, the collection of repeated samples is often essential.
Proper collection, storage and transport of clinical specimens are of crucial importance for reliable laboratory diagnostics.
Accurate diagnosis includes both parasite detection and species (in some cases: genotype/serotype) identification .
Although numerous protocols for molecular detection of protozoan pathogens have been established in the past years, consent on the most reliable technique is missing for most protozoan taxa, thus demonstration of the causative agent in native material or stained smears is still the gold standard for detection.
Good microscopic expertise is essential: low parasite density and/or morphologic variability can result in false negative results, pseudo‐parasites and artefacts are common causes of false positive results.
In many protozoan taxa, morphology alone does not provide enough information for identification on or below the species level, this is today mainly based on molecular biological methods .
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... Altogether, almost 15% of all known protists show symbiotic (mutualistic or parasitic) life styles and around 100 protist species can infect humans (Walochnik and Aspöck 2012). The majority of these are not significantly harmful to their hosts, with several species even being commensals (Parfrey et al. 2014). ...
Protists include all eukaryotes except plants, fungi and animals. They are an essential, yet often forgotten, component of the soil microbiome. Method developments have now furthered our understanding of the real taxonomic and functional diversity of soil protists. They occupy key roles in microbial foodwebs as consumers of bacteria, fungi and other small eukaryotes. As parasites of plants, animals and even of larger protists, they regulate populations and shape communities. Pathogenic forms play a major role in public health issues as human parasites, or act as agricultural pests. Predatory soil protists release nutrients enhancing plant growth. Soil protists are of key importance for our understanding of eukaryotic evolution and microbial biogeography. Soil protists are also useful in applied research as bioindicators of soil quality, as models in ecotoxicology and as potential biofertilizers and biocontrol agents. In this review, we provide an overview of the enormous morphological, taxonomical and functional diversity of soil protists, and discuss current challenges and opportunities in soil protistology. Research in soil biology would clearly benefit from incorporating more protistology alongside the study of bacteria, fungi and animals.
Microscopy is the gold standard for diagnosis of intestinal parasitic diseases in many countries, including Cuba, although molecular approaches often have higher sensitivity as well as other advantages.
Fecal samples from 133 patients were analyzed by light microscopy and also real-time multiplex qPCR targeting Giardia duodenalis, Cryptosporidium spp., and Entamoeba histolytica, and, separately, Dientamoeba fragilis. Microscopy revealed G. duodenalis occurred most commonly (17 patients), followed by Blastocystis spp. (12 patients). In a few patients, Entamoeba histolytica/E. dispar, Cryptosporidium spp., and Cyclospora cayetanensis were identified.
Molecular analysis identified 4 more G. duodenalis infections and 2 more Cryptosporidium spp. infections; concordance between microscopy and PCR showed almost perfect agreement for G. duodenalis (κ = 0.88) and substantial agreement for Cryptosporidium (κ = 0.74). PCR indicated that E. dispar, rather than E. histolytica, had been identified by microscopy. Additionally, 16 D. fragilis infections were detected using molecular methods.
Although both microscopy and molecular techniques have a place in parasitology diagnostics, for parasites such as D. fragilis, where microscopy can underestimate occurrence, molecular techniques may be preferable, and also essential for distinguishing between morphologically similar microorganisms such as E. histolytica and E. dispar. Although in resource-constrained countries such as Cuba, microscopy is extremely important as a diagnostic tool for intestinal parasites, inclusion of molecular techniques could be invaluable for selected protozoa.
Parasites are organisms that depend on a host for feeding and reproduction and belong to various unrelated taxa, primarily protozoa, helminths and arthropods. Complex life cycles have often lead to extreme adaptations; nevertheless parasites may harm their hosts and even cause serious disease and death. Transmission of parasite stages can be environmental, nutritional or vector-borne. Among the most important human protozoan parasites in a global context are Leishmania, Plasmodium (both transmitted by bloodsucking arthropods) and Toxoplasma which is soil- or food-borne. Parasitic worms (helminths) relevant for human health include Echinococcus, Toxocara, hookworms (all soil-borne) and Dirofilaria (transmitted by mosquitoes). Various arthropods (ticks, insects) are involved in the transmission of pathogens due to their blood-feeding behaviour. They are especially involved in transmission cycles between animals and humans (zoonotic infections). Research on parasites and their interactions with the host requires suitable animal models. For parasites with a wide host range or those naturally infecting rodent species available as laboratory animals, established models are available. Others, like the human malaria parasites, require sophisticated and often costly genetic manipulation to allow for infection in non-natural rodent hosts. Alternatively, surrogate models of closely related helminth species in rodent models are used to study human parasites.
This article is a condensed review of the medically relevant protozoa in Central Europe and the infections and diseases caused by them. Information is given on modes and sources of infection, organs involved in the disease, prevalence, diagnostics, therapy, and prophylaxis. Moreover, travel-associated infections with protozoa are briefly outlined.
Includes detailed guidelines for practical, expedient and accessible diagnostic methodologies of infectious diseases in insects.
Techniques described provide reliable alternatives for preliminary pathogen identification which do not require special equipment
or intensive training, in a step by step protocol format, and for an easy orientation and accurate learning.
Changes in the organization of health services in developing countries have led to district levels assuming more responsibility for the planning, delivery and quality of community health care. This fully up-dated new edition has been produced to help those working in the district laboratory, and those responsible for the organization and management of community laboratory services and the training of district laboratory personnel. Replacing the previous publication Medical Laboratory Manual for Tropical Countries, this book provides an up-to-date practical bench manual, taking a modern approach to the provision of a quality medical laboratory service. Reviews: Review of District Laboratory Practice in Developing Countries Part 1: 'Clear and easily understood information is provided on the clinical biochemistry of laboratory analytes and the biology of parasites … The book is probably the most comprehensive source of information available today to those who work in or need to know about laboratory services in developing countries. It can be recommended as a basic document for laboratory technicians, technologists, and medical doctors at all levels …' Bulletin of the World Health Organization.
Diagnostic procedures in the field of medical parasitology require a great deal of judgment and interpretation and are generally classified by the Clinical Laboratory Improvement Amendments of 1988 (CLlA '88) as high complexity procedures. The majority of diagnostic parasitology procedures can be performed either within the hospital setting or in an offsite location. There are very few procedures within this discipline that must be performed and reported on a short turnaround time (STAT) basis. Two procedures fall into the STAT category: request for examination of blood films for the diagnosis of malaria and examination of cerebrospinal fluid for the presence of free-living amebae, primarily Naegleria fowleri. The specimen commonly submitted to the diagnostic parasitology laboratory is the stool specimen, and the most commonly performed procedure in parasitology is the ova and parasite (O&P) examination, which comprises three separate protocols: the direct wet mount, the concentration, and the permanent stained smear.
Microsporidia are long-known parasites of a wide variety of invertebrate and vertebrate hosts. The emergence of these obligate intracellular organisms as important opportunistic pathogens during the AIDS pandemic and the discovery of new species in humans renewed interest in this unique group of organisms. This review summarises recent advances in the field of molecular biology of microsporidia which (i) contributed to the understanding of the natural origin of human-infecting microsporidia, (ii) revealed unique genetic features of their dramatically reduced genome and (iii) resulted in the correction of their phylogenetic placement among eukaryotes from primitive protozoans to highly evolved organisms related to fungi. Microsporidia might serve as new intracellular model organisms in the future given that gene transfer systems will be developed.
Diagnosis of microsporidial infections is routinely performed by light microscopy, with unequivocal non-molecular species identification achievable only through electron microscopy. This study describes a single SYBR Green real-time PCR assay for the simultaneous detection and species identification of such infections. This assay was highly sensitive, routinely detecting infections containing 400 parasites (g stool sample)(-1), whilst species identification was achieved by differential melt curves on a Corbett Life Science Rotor-Gene 3000. A modification of the QIAamp DNA tissue extraction protocol allowed the semi-automated extraction of DNA from stools for the routine diagnosis of microsporidial infection by real-time PCR. Of 168 stool samples routinely analysed for microsporidian spores, only five were positive by microscopy. By comparison, 17 were positive for microsporidial DNA by real-time analysis, comprising 14 Enterocytozoon bieneusi, one Encephalitozoon cuniculi and two separate Pleistophora species infections.
To update the reader on the latest developments in the laboratory diagnosis of intestinal protozoa.
Correct identification of a diarrhoea causing pathogens is essential for the choice of treatment in an individual patient as well as to map the aetiology of diarrhoea in a variety of patient populations. Classical diagnosis of diarrhoea causing protozoa by microscopic examination of a stool sample lacks both sensitivity and specificity. Alternative diagnostic platforms are discussed.
Recent literature on the diagnosis of intestinal protozoa has focused mainly on nucleic acid-based assays, in particular the specific detection of parasite DNA in stool samples using real-time PCR. In addition, the trend has been moving from single pathogen detection to a multiplex approach, allowing simultaneous identification of multiple parasites. Different combinations of targets can be used within a routine diagnostic setting, depending on the patient population, such as children, immunocompromised individuals and those who have been travelling to tropical regions. Large-scale monitoring and evaluation of control strategies become feasible due to automation and high-throughput facilities. Improved technology also has become available for differentiating protozoa subspecies, which facilitates outbreak investigations and extensive research in molecular epidemiology.
The genus Sappinia with the single species Sappinia pedata was established for an amoeba with two nuclei and pedicellate "cysts" by Dangeard in 1896. In 1912, Alexeieff transferred an also double nucleated, but apparently sexually reproducing amoeba to this genus as Sappinia diploidea, that had been described as Amoeba diploidea by Hartmann and Nägler in 1908. As the original isolates were lost, Michel and colleagues established a neotype for S. diploidea in 2006 and Brown and colleagues established a neotype for S. pedata in 2007. Molecular analyses have corroborated the differentiation between S. pedata and S. diploidea, however, the genus splits into more than two well separated clusters. Altogether, the genus Sappinia is now classified as a member of the Thecamoebidae and, moreover, as potentially pathogenic. In 2001, Gelman and colleagues reported a case of severe encephalitis in a non-immunocompromised young man caused by Sappinia.