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Lecture - 8 (1st DDS Program)


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GENETICS of HUMAN PARASITES

Parasitic life style has important effects on genetic structure as well as on the pattern of genetic variation within and between populations of parasites. Parasite populations are relatively small and isolated from each other in different hosts. It leads to gradual genetic differentiation. Heterogeneity of parasites will produce parasite populations that could be medically and epidemiologically important.
Genetic variation in medically important parasites and the nature of the reproductive strategies which predispose to such a variation are currently the subject of much interest and controversy. The epidemiological significance of such variation and knowledge of the selection processes have important implications for the control of parasites. For these reasons molecular epidemiology increases and the principles of epidemiological analysis are the same as those used in traditional epidemiology.

Molecular bases of parasite species identification and molecular epidemiology

Giardia sp. and Trichinella sp.

Giardia is one of the most commonly identified intestinal protozoan parasites in the world. More than 50 Giardia species have been described, primarily on the basis of host occurrence, but the lack of distinguishing morphological features and increasing evidence that Giardia is not host specific testify to the fact, that many of the species are not valid. Filice recognised only three morphological groups (species) in this genus: G. agilis, G. muris and G. duodenalis. However, five species of Giardia are currently recognised as valid: G. agilis, G. muris, G. duodenalis, G. psittaci, G. ardeae. Unfortunately, this apparently simple scheme fails to reflect the considerable phenotypic, and genetic heterogeneity that exists within the species of G. duodenalis. This species (or morphological group) is the most widespread and is known to occur in at least 100 species of vertebrates, including humans. The occurrence of morphological indistinguishable G. duodenaiis populations in human and animals complicates our understanding of the epidemiology of giardiosis and hampers the control of this parasite. Determination of species in organisms that reproduce entirely asexually is very difficult.
The parasite exhibits considerable genetic diversity, with eight assemblages (A–H) having been reported. These assemblages differ in host specificity: only G. duodenalis assemblages A (except sub-assemblage AIII) and B cause human giardiosis. Because Giardia dispersive stages are resistant to environmental factors and remain infective for a long time, they may cause singular infections as well waterborne outbreaks seriously threatening public health. A considerable degree of genetic diversity has been found in Giardia isolated from humans and other animals from many geographic regions. Using modern molecular techniques (e.g. Whole Genome Sequencing; WGS) in epidemiological studies it has been shown that Giardia found in beavers were the source of a waterborne outbreak of giardiosis in humans in Canada whereas in Norway the source was water reservoir supplying city with drinking water. Though a necessity of using methods of molecular biology exists in epidemiological studies, some care must be applied to how we use the modern molecular tools. The molecular data must be interpreted rigorously and correctly which will require molecular geneticists, parasitologists and epidemiologists to work together so as to avoid creating a confusing picture of genetic diversity which is of no practical or biological relevance.

Trichinella is nematode parasite of cosmopolitan distribution. It is the etiological agent of trichinellosis in humans. Human infections with Trichinella have been closely associated with the ingestion of raw or undercooked pork, but more recently infection sources have included meat of other animal species (e,g. bear, walrus, bush pig).†
Changing views on taxonomic status of Trichinella genus perfectly mirror the development of the research techniques. Until 1970 year the Trichinella genus was considered as a monospecific and therefore it was accepted that all human cases of trichinellosis werecaused by one species - T. spiralis. At the beginning of 1970 three new species of Trichinella were described on the basis of differences in geographic distributionand biological properties (e.g. infectivity, virulence, capacity to reproduce and so on). In the last years, both identification and taxonomic classification of the different populations in Trichinella genus have been studied. Several methods have been used (proteins and nucleic acids analyses). The results showed the existence of eight genetic clusters (species) within Trichinella genus: T. spiralis sensu stricto, T. nativa. T. britovi, T. pseudospiralis, T. nelsoni, Trichinella papuae, T. zimbwawensis and T. papuae.

Genetic markers of drug resistance

Schistosoma sp. and Leischmania sp.

Variable sensitivity of different species of parasites to drugs has been demonstrated. It has also been found that inter- and intrapopulation heterogeneity exist in sensitivity to antiparasitic drugs. Moreover, in various parasite genus or species there develop different molecular mechanisms of drug resistance. In some cases, drug resistance comes from the acquisition of a new activity in organisms (e. g. the ability to degrade or inactive the drug), while in another organisms resistance comes from the loss of a pre-existing activity (e. g. the loss of drug-activating mechanisms).
Drug resistance - a genetically transmitted loss of sensitivity in a parasite populations that was primarily susceptible to a drug.
Tolerance (natural resistance) - the lack of sensitivity to a drug in a previously unexposed population.
The crosses of
Schistosoma mansoni individuals showed that drug resistance was controlled by a single, autosomal, recessive gene. It means that the resistance is due to the loss of a single activity. Indeed, a drug requires some trematode activity in order to exert its schistosomicidal effects. Sensitive schistosomes have activating (estrifying) enzyme, while resistant/tolerant schistosomes lack it.
Leishmania infections that are refractory to chemotherapy have been recognised since the early 1940s and are increasing in the endemic regions. Some modes of action of drugs (pentavalent antimonials such as Pentostam) are still poorly understood. Sodium arsenite, an oxyanion related to antimony, is a potent inducer of gene amplification in Leishmania. The first amplified region characterised was the H region (part of extrachromosomal genome of Leishmania). The region H is a fragment of DNA that is often amplified as a part of extrachromosomal circles after selection with drugs. The first gene described in the H region was P-glycoprotein gene ltpgpA. P-glycoproteins are large membrane proteins that can function as ATP-dependent extrusion pumps, conferring resistance to cytotoxic drugs by an active drug efflux system. It is also possible that other drug-resistance genes are present in this region. The region R is another part of extrachromosomal circles that is amplified after drug selection.
Leishmania spp. are refractory to the action of cytotoxic drugs by amplifying either:
target genes (dhfr-ts, impdh, odc, nagt),†
genes encoding for putative pumps (Idmrdt 1, ltpgpA) or
genes involved in alternative metabolic pathway (Itdh).

Genetic aspects of virulence in the parasites and in human

Plasmodium falciparum

Pathogenicity - ability to induce the pathological changes or disease without specifying the conditions;parasites are pathogenic or are not.
Virulence - level of pathogenecity; various degrees of virulence may be found depending on parasite strain or on conditions.
Malaria is one of the most prevalent infections that afflict humans. According to World Health Organization estimates there are 300-500 million clinical cases of malaria per year, 90% of them in Africa. There are also 1.5-3 million deaths due to malaria each year, one million of them being children under the age of five living in sub-Saharan Africa. Among several Plasmodium species occurring in humans, P. faiciparum causes the most severe disease. However, genetic polymorphism exists within the host and parasite populations which have major influences on the manifestations and outcome of malaria infections. The recognition of host and parasite genetic traits in malaria is a fundamental problem.
Human genetic traits that determine the manifestations of malarial infection are more amenable to study than are the virulence-determining genes of the parasites. The number of such traits have been described in the past. These include the thalassaemias and other abnormalities of red blood cells, among the most notable of which are the protection against severe P. falciparum malaria conferred by the HbS (sickle-cell) gene and the total refractoriness for the Duffy negative gene.
The recognition of the mechanisms of pathogenesis of malaria due to Plasmodium falciparum concerns two types of the phenomenon:†
the adherence of parasitized erythrocytes to host cells, either endothelial or circulating blood cells, and†
the induction of host cytokines by parasites (TNF- tumor necrosis factor) and their products and their effects on brain tissues through secondary mediators (e.g. NO and free oxygen radicals).
In vitro studies showed that bloodstage parasites from different isolates of P. falciparum have variable potency in inducing TNF. Moreover, an association was found between the severity of P. falciparum infection in African children and the ability of the parasites derived from those infections to induce TNF. This cytokine is responsible for the expression of specific receptors on endothelial cells and sequestration of parasitized cells in the brain microvasculature.
A group of proteins expressed on the surface of parasitized erythrocytes was found. These membrane proteins (PfEMP-1 - P. falciparum-infected erythrocyte membrane protein 1) cause cytoadherence to endothelial cells. Different populations of parasite exhibit substantial variation in PfEMIJ-1expression. Genes encoding PfEMP-1, which are represented by a gene family whose members are distributed through at least several chromosomes of P. falciparum have been identified.
Distinct from the phenomenon of cytoadherence to endothelial cells is that of rosetting. Rosetting is the binding of uninfected erythrocytes to circulating erythrocytes infected with parasites. This phenomenon occurs only in P. falciparum infection. On the surface of infected erythrocytes proteins which were called ‘rosettin' molecules have been identified. The rosettins have lectin-like properties and therefore bind to carbohydrate domains on uninfected erythrocytes. It has also been found that some blood-group antigens (A and B blood-groups) on uninfected erythroctes may be the receptors of rosettins.
Therefore, in patients with A and B blood-group severe form of malaria is observed as a rule. There are no evidences of the existence of a linkage between the parasite phenotype for TIFF induction and that for resetting. The suggestion that TNF-inducing and resetting phenotypes are stable genetic traits of the parasites and that they may represent virulence factors, is an appealing idea. However, further evidences should be determined because it is very well known that parasites undergo phenotypic switching during propagation. Until now. the only category of parasite phenotype that is clearly genetically inherited and which represents genotypes for parasite virulence is a drug-resistant phenotype of parasite.

Malaria vaccines

In general, the induction of immunity is a key strategy for control of every infectious disease. Nevertheless, the task of producing vaccines against any of the clinically important parasitic infections has proven to be a formidable challenge. Developing vaccines against parasites is further complicated by the lack of systematic knowledge of the nature of protective host response and heterogeneity of the parasites.†
The first and second generation vaccines were primarily based on antigenic material. Antigenic materials in the first generation vaccines are live attenuated or killed forms of whole organisms, whereas in the second generation vaccines there are defined native or recombinant protein components of the organism obtained either by biochemical purification or by genetic engineering.
Live attenuated vaccines stimulate protective cytotoxic T-cell (CTL) responses as well as T helper (Th) cell and humoral immunity. Live attenuated vaccines have the intrinsic risk, however rarely of reversion to a pathogenic form.†
Killed pathogens do not carry this risk. Such vaccines can generate Th-cell and humoral responses, they are unable to generate specific CTL responses against pathogens. Similarly, vaccination with defined protein components induces Th-cell and humoral immune responses but not CTL responses.
Mosquirix is a recombinant protein-based malaria vaccine. Pilot project for vaccination has been launched on 2019 in Malawi, Ghana and in Kenya. The results are planned to be used by the World Health Organization to advise about a possible future deployment of the vaccine. Sanaria PfSPZ vaccine uses radiation attenuated (weakened) SPZ (sporozoites which are non-dividing stages of the Plasmodium that cannot cause disease) to induce protective immune responses.

 Author: Piotr Nowosad date: 2020-04-29  print    back  
 
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