genomic markers for parasitic infections
TRANSCRIPT
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GENOMIC MARKERS FOR PARASITIC INFECTIONS
Dr.R.Jayaprada
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AGENDA Introduction Tools for Gene detection and analysis . Tools For Molecular Epidemiology of Parasites Functional Genomic Study Application of Genomic Studies in
Parasitology Conclusion.
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Introduction Parasites have kept many secrets from the
researchers who have sought to eradicate them over past decades.
Little is known about them: Evade drugs, Escape the immune system, Regulate switching between genes involved in
immune evasion, and Orchestrate development.
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IntroductionProblems with Study of Parasites: Lack of reliable culture system (Difficult to
keep and breed) Shortage of material for biochemical studies; Lack of traditional genetic methods to study
gene functions. They parasitize hosts that are not ideal
experimental subjects.
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Introduction Human helminths are much more complex
organisms (e.g., they are diploid, have reproductive and other organs, nervous systems, etc.) compared to Protozoans
There are many more species of them They belong to two completely unrelated phyla
(Platyhelminthes and Nematoda) No cell lines are available The developmental cycles cannot be completed in
vitro Their developmental cycles are not only dissimilar
to those of Protozoans, but they are also generally dissimilar to each other.
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Introduction The availability of parasite genome sequences
and related genome-based tools have provided substantial opportunities to overcome these problems to a large extent.
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Introduction A genome is all of a living thing's genetic
material The genome is divided into chromosomes,
chromosomes contain genes, and genes are made of DNA.
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Introduction Gene and chromosome. Coined in 1920 by
Hans Winkler. The complete set of genes in an organism : GENOME. Frederick Sanger in 1977 introduced the “dideoxy” chain termination method to sequence DNA molecules.
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IntroductionHuman Parasites Complete sequence: Timeline2001: Encephalitozoon cuniculi 2002: P.falciparum 2004: Cryptosporidium parvum and hominis 2005: E. histolytica, Trypanosoma brucei, cruzi; L.major 2007: Brugia malayi, Giardia lamblia, Trichomonas
vaginalis 2008: P.vivax, P. knowelsi 2009: Schistosoma mansoni, and S. japonicum 2011: Ascaris suumOther parasites: Toxoplasma gondii, Leishmania
braziliensis, L. infantum, L.mexicana,(Partial/ Draft).
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Introduction The study of Genomic Markers is expected to
lead to major technological advances: New Diagnostics New therapeutics, and Vaccine development, Understanding of disease mechanisms, Elucidation of Host–parasite interactions, Insight into transmission biology.
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Tools For Gene Detection and Analysis
I. Amplification Techniques: PCR Real Time PCR NASBA LAMP LCR
2. Hybridization Techniques: FISH Microarrays
3. Biosensors Liposome Based Lateral Flow Biosensor Matrix-assisted laser desorption–ionization time-of-flight mass
spectrometry (MALDI TOF)
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Tools For Gene Analysis
Used for MOLECULAR EPIDEMIOLOGY OF PARASITES
Multi Locus Enzyme Elecrophoresis (MLEE) Amplified Fragment Length Polymorphism (AFLP) Random Amplified Polymorphic DNA (RAPD) PCR-Restriction Fragment Length Polymorphism (RFLP) Kinetoplastid minicircle DNA (kDNA) RFLP for
Leishmania Gene sequencing and Sequencing of ribosomal loci Multi locus Microsatellite Typing (MLMT) Genome wide Single Nucleotide Polymorphism (SNP) Single Strand Conformation Polymorphism (SSCP).
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Real Time PCRThe primary advantages of real-time PCR over
conventional PCR : It provides high-throughput analysis in a
closed-tube format (no post-PCR handling is required)
It can be used for quantitation over a broad dynamic range
It can be used to differentiate DNA fragments by analyzing the melting curve of DNA.
Simple, fast, closed, and automated amplification system responsible for decreasing the risk of cross-contamination.
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Real Time PCR Various Fluorescent chemistries are being used1. TaqMan Probe: One of the most widely used chemistries Assay design is simple and assays are robust. However, Taqman probes do not confirm that
the correct fragment has been amplified (other than by running a gel).
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Real Time PCR2. Minor-groove binder(MGB) Eclipse probes3. Molecular beacons4. Fluorescence- (or Forster) resonance-energy-
transfer (FRET)-based assays5. Intercalating Dyes: SYBR Green, BEBO, LC
Green: SYBR Green is the most cost-efficient chemistry and, thus, are the most popular option among researchers.
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Applications of RT-PCR Detection of Cryptosporidium, Leishmania,
Trypanosoma, Giardia, Toxoplasma Diagnosis, animal models, drug efficacy and
vectorial capacity for Leishmania Monitoring antimalarial therapy Species discrimination and SNP analysis
(FRET assay) Discrimination between species and
genotypes of Cryptosporidium
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Loop Mediated Isothermal Amplification (LAMP) LAMP is a method of nucleic acid amplification
with extremely high sensitivity and specificity to discriminate single nucleotide differences
Four primers specially designed to recognize six different sequences on the target gene . Amplification occurs only when all primers bind, thus forming a product.
DNA amplification can be achieved using simple incubators (water bath or block heater) because of isothermal conditions
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LAMP Can amplify a few copies of genetic material to
109 in less than an hour Large amounts of white magnesium
pyrophosphate are precipitated from DNA amplification . The turbidity caused by this reaction is proportional to the amount of DNA synthesized. As a consequence, it is possible to evaluate the reaction in real time by measuring the turbidity or, more importantly, by visualization by the naked eye.
The simple requirements make LAMP easily available for small laboratories, especially in rural endemic areas
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LAMP Has been used to detect several parasitic
diseases, including the human parasites Cryptosporidium, Entamoeba histolytica, Plasmodium, Trypanosoma, Taenia, Schistosoma, Fasciola hepatica and Fasciola gigantica, and Toxoplasma gondii,
Could detect the miracidium after the first day of exposure in snails, the intermediate hosts of Schistosoma
Mosquitoes carrying the parasites Plasmodium and Dirofilaria immitis have been identified
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Ligase Chain Reaction (LCR) DNA ligases are highly specific and do not
tolerate base mismatches (unlike DNA polymerases).
This makes LCR superior to PCR for the detection of SNPs, which has been its chief application
Products of the LCR are detected in real-time by
using either FRET probes as LCR primers or primers that are designed to form molecular beacons once ligated.
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LUMINEX Luminex is a bead-based xMAP technology
(multianalyte profiling), a system that combines flow cytometry, fluorescent microspheres (beads), lasers and digital signal processing
Capable of simultaneously measuring up to 100 different analytes in a single sample
Luminex was able to distinguish the species C. hominis and C. parvum in 143 DNA extractions, using oligonucleotide-specific probes for the ML-2 regions (microsatellite region-2 )of each species, without the need for DNA sequencing.
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Hybridization Techniques: 1. FISH Uses fluorescently-labeled probes [either DNA
or peptide nucleic acid (PNA)] that hybridize to complementary nucleic acid targets in whole cells to enable the direct detection of organisms in complex communities
PNA probes are pseudopeptides that hybridize to complementary nucleic acid targets (DNA and RNA) with better specificity and stability than DNA probes
PNA probes are expensive to construct.
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FISH Applying FISH to whole cells gives information
about microbial identity, cell morphology, abundance and spatial distribution of individual target species
To study the partitioning and chromosome composition of nuclei in Giardia and to detect arthropozoonotic species and genotypes of
Cryptosporidium and Giardia PNA probes have been used for the direct
FISH detection of African trypanosomes
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2. MICROARRAYS DNA microarrays provide a powerful tool for
the parallel analysis of multiple genes and gene transcript
Microarrays are arrays of either cDNAs or oligonucleotides that are either spotted onto a glass microscope slide or synthesized on a silicone chip.
DNA or mRNA extracted from cells or tissues is labeled with specific fluorescent molecules and hybridized to the microarray DNA.
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Microarrays To improve sensitivity, samples are often pre-
amplified by PCR The resulting image of fluorescent spots is
visualized in a confocal scanner and digitized for quantitative analysis
Has been used to detect and discriminate between a range of parasites, including different species and genotypes of Entamoeba, Giardia and Cryptosporidium in a single assay.
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MicroarraysThe main application of microarrays has been to
study gene expression in hosts To study differential gene expression in
different lifecycle stages of T. brucei Gender-associated gene expression in
Schistosoma japonicum Gene expression during asexual development
of T. gondii Gene expression for Plasmodium in
mosquitoes.
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BIOSENSORS A biosensor is an analytical device which
converts a biological response into an electrical signal
Sandwich hybridization is used to capture target nucleic acid and the reporter liposome, which contains a dye.
Following capture and washing, the liposomes are lyzed using detergent, which produces a signal.
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(Monis et al 2005. Trends in Parasitology 21: 340-46)
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MALDI-TOF Matrix-assisted laser desorption–ionization
time-of-flight mass spectrometry Based on the detection of diagnostic proteins;
but a database that contains the mass information of the diagnostic biomarkers is needed to use MALDI -TOF as an identification tool
Require expensive, specialized equipment and/or the establishment of appropriate sample databases.
Has been used for the study of Cryptosporidium.
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TOOLS FOR MOLECULAR EPIDEMIOLOGY: 1. MLEE Non DNA Based Based on phenotypic polymorphism
dependent on protein structures which in turn is based on structural gene sequences.
It can detect polymorphism only within the coding region of the gene, not the mutations in the promoter region or introns.
A set of 10-20 genes is analysed to generate meaningful data.
Extensively used for Leishmania, Trypanosoma, P. falciparum
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2. Amplified Fragment Length Polymorphism (AFLP) Allows detection of a DNA polymorphism
without prior information on the sequence. Employs PCR to selectively amplify the groups
of restriction fragments of totally digested DNA.
Polymorphisms are identified based on the presence or absence of DNA fragments by polyacrylamide gel analysis
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AFLP The advantages of this technique are The ability to search an entire genome for
polymorphisms, The reproducibility of the method, and The possibility of being used against parasites
about which there is no prior genetic information
Highly efficient because of the possibility of analyzing a large number of bands simultaneously, with extensive coverage of the genome
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AFLP: Uses To differentiate isolates of C. parvum into two
distinct genotypes Has revealed high genetic variability among
the genome species of Leishmania major, L. tropica and L. donovani, which was sufficient to distinguish between Cutaneous and Visceral Leishmaniasis.
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3. Random Amplified Polymorphic DNA (RAPD ) Extensively used for description of strains in
epidemiological studies RAPD is a very simple, fast, and inexpensive
technique which makes survey of parasite genome easier.
Does not require either prior knowledge of the DNA sequence or DNA hybridization
Has been used to delineate strains of microorganisms.
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RAPD Highly efficient in the differentiation of
amplification profiles, as well as its ability to distinguish polymorphisms between helminthic parasites.
Used to map genes for the characterization of species, to stimulate the genetic variability and determine the genetic structure of populations of different microorganisms.
It also reveals polymorphisms in the noncoding regions of the genome.
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RAPD: Uses Species differentiation of Leishmania, To study polymorphisms of parasites such as
Plasmodium and Trypanosoma Differentiation of endemic strains of
Wuchereria bancrofti.
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4. RFLP This reaction is based on the digestion of the
PCR products by restriction enzymes or endonucleases. These enzymes cleave DNA into fragments of certain sizes, whose analysis on agarose or polyacrylamide gel results in different patterns of fragment sizes, enabling the identification .
Diversity of Cryptosporidium spp Diagnosis of species and genotypes of
Toxoplasma gondii.
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5. Multi locus Microsatellite Typing (MLMT) Microsatellites are short DNA sequences
(about 300 base pairs) composed of tandem repeats of one to six nucleotides, with approximately one hundred repeats.
Microsatellites are abundant in eukaryotic genomes and can mutate rapidly by loss or gain of repeat units.
They show frequent polymorphism, co-dominant inheritance, high reproducibility and high resolution, require simple typing methods, and can be detected by PCR .
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MLMT Microsatellite markers have been developed
only for some parasitic nematodes such as species of Trichostrongyloid .
Strain Typing of L.donovani The low popularity of these genetic markers is
due to the high number of microsatellites, which cause technical difficulties in isolating parasites by PCR.
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Genome wide Single Nucleotide Polymorphism (SNP) SNP is a change in a single nucleotide in one
sequence relative to another caused by nucleotide mutation, gene transfer, or intragenic recombination.
Variation in a single base in the genetic code between different individuals of the same species can be detected.
SNP variation occurs when a single nucleotide, such as an A, is replaced by one of the other three nucleotide—C, G, or T. e.g. AAGGTTA to ATGGTTA
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Single Strand Conformation Polymorhism (SSCP) It is electrophoretic separation of single-
stranded nucleic acids based on subtle differences in sequence (often a single base pair) which results in a different secondary structure and a measurable difference in mobility through a gel.
Like RFLP, SSCPs are allelic variants of inherited, genetic traits that can be used as genetic markers
Unlike RFLP analysis, however, SSCP analysis can detect DNA polymorphisms and mutations at multiple places in DNA fragments.
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Functional Genomic Study Some of the available genomes are
incomplete, poorly annotated, or not annotated at all.
There are almost no tools available with which gene function can be tested directly.
Without annotation and functional genomic tools, the sequence data are not truly useful.
Annotation: a note added by way of explanation or commentary mentioning what the gene does (its function) to make sense.
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Functional Genomic Study Essential to resolve uncertainties in the
molecular physiology of parasites; and To illuminate mechanisms of pathogenesis
that may lead to development of new interventions to control and eliminate these parasites or the diseases.
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Functional Genomic StudyTwo Categories1. Bioinformatic tools for sequence mining to
generate hypotheses concerning likely biological function, and
2. Experimental tools with which gene expression can be manipulated in the target organism (or, in the case of parasites, also the host) and the consequences of that manipulation for the biology of the parasite and its relationship with the host can be observed and measured
a. RNA Interference b. Transgenesis
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Functional Genomic Study: Bioinformatic Tools The first bioinformatic tools that are applied
are generally genome-wide homologue searches, usually using variants of basic local alignment search tool (BLAST) to generate automatic annotations based on sequence homology.
Very rough guide and at worst downright misleading
In parasites the limited utility of a homology based approach is undermined further by the poor performance of gene-finding software in parasite genomic sequences.
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Functional Genomic Study: Experimental Tools: 1. RNA Interfernce
Most important functional genomic tool Gene expression is knocked down by exposing
the parasites to gene-specific dsRNA or small interfering (si)RNA (gene silencing)
Double-stranded RNA [dsRNA] triggers degradation of homologous mRNA transcripts
Efficiency of RNAi in parasites varies between species
Problems often arise with the efficiency, specificity and reproducibility of some methodologies, especially with nematode species.
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RNA Interfernce To find out the parasite biology and the possibility
of identifying and validating novel anthelmintic drug targets in B. malayi.
In contrast to the situation in nematodes, RNAi in schistosomes seems to be more robust and reproducible and is currently being used by a number of groups to elucidate the function of some key proteins and pathways in these parasites: Haemoglobin digestion, Tegument formation, and the biological role of tegumental proteins
These approaches have helped to identify vaccine/drug candidates, some of which are in various stages of clinical development.
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2. Transgenesis The converse of knock-down of expression is
manipulation of expression by gene knock-in. It is a process by which gene function is
inferred by studying the effects of transferring native or altered copies of genes into subject organisms.
Either transient or heritable transgenesis of at least three species of human parasitic nematodes and trematodes (B.malayi, S.stercoralis, S. mansoni) and in at least one cestode, E. multilocularis has been attempted; apart from malaria parasites.
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Functional Genomic Study Help in rational design of agents directed at
defined molecular targets. Important in developing new drugs and
identifying potential vaccine candidates for parasitic helminths and protozoans.
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Genomic Studies: Applications and Uses.
A. Parasite Systematics: Highly conserved coding regions e.g. Small
Subunit rDNA and certain mitochondrial genes can help in discrimination above species level.
Below the species level, relationships between genes sampled from different individuals are not hierarchical because homologous genes from the two parents combine in their offspring .
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Genomic Studies: Applications and Uses Above the species level, different taxa are
hierarchical because they are a consequence of speciation followed by long periods of reproductive isolation.
Representation of phylogenetic relationships above the species level as networks rather than as strictly bifurcating trees is gaining interest.
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Genomic Studies: Applications and Uses
B. Parasite Diagnostics and Epidemiology Discrimination between species by molecular
techniques like multiplex PCR, LAMP etc can be achieved by detecting the moderately conserved regions e.g. coding mitochondrial genes, internal transcribed spacer, rDNA, and other loci
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Genomic Studies: Applications and Uses Molecular identification is particularly
important when discriminating different parasites with morphologically identical life cycle stages, such as eggs or cysts, from faecal samples, or when attempting to match different life cycle stages of the same parasite from intermediate and definitive hosts
In some endemic areas humans may be infected with more than one species of hookworm or of taenias , the eggs of which are identical.
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Genomic Studies: Applications and Uses
C. Intraspecies variation study: Finds extensive application in population
genetics; breeding systems (e.g. cross vs. self fertilization); host specificity analysis; molecular epidemiology; conservation (e.g. predicting susceptibility to pathogens); and biosecurity (exotic and emerging pathogens).
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Genomic Studies: Applications and Uses Genes are analyzed using tools like MLEE
( allozymes), RAPD, AFLP, PFGE, PCR-RFLP, pyrosequencing, mPCR, LAMP, and qPCR.
The extent to which we recognize intraspecific groups of parasites will be a function of the extent to which intraspecific variation is structured among different hosts or among different geographic areas.
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Genomic Studies: Applications and Uses In T. brucei, for example, three subspecies have
historically been defined on the basis of geographic and host distribution, and the clinical course of disease.
T. b. gambiense : Human parasite distributed through western and central Africa, causing chronic disease.
T. b. rhodesiense : Human parasite distributed through eastern and southern Africa causing acute disease and
T. b. brucei infects domestic and game animals, but not humans and is widely distributed throughout sub- Saharan Africa.
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Genomic Studies: Applications and Uses
D. Discrimination between individual isolates:
The techniques of “fingerprinting helps in tracking transmission of subgenotypes ; to find out sources of infection and risk factors and also the course of infection
Done by using Mini/microsatellites, SSCP, and qPCR.
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Genomic Studies: Applications and Uses
E. Identifying phenotypic traits of clinical and epidemiological significance:
Linkage studies between phenotype and genotype of a particular parasite are useful to determine the virulence, infectivity, and drug sensitivity
Done by using genetic map; sequencing and/or RT-PCR of genes thought to be linked to phenotypic trait.
Drug resistance in P. falciparum , virulence in T. gondii and resistance to malaria parasites in Anopheles gambiae have been studied .
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Genomic Studies: Applications and Uses Useful to explore genetic changes involved in
resistance to anti-parasitic drugs and understanding the potential mechanisms of drug resistance in human parasites, and can be expected to facilitate development of genetic markers to monitor and manage any future appearance and spread of drug resistance.
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Application and Uses To understand how these parasite products
act on immune responses Comparisons of parasite molecules with
orthologues/paralogues from free-living relatives will strengthen efforts to decipher the strategies adopted by parasites to evade and subvert their host immune responses.
This information can be exploited for development of drugs and vaccines against the parasites.
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Application and Uses Treatment for helminthic infections, responsible for
hundreds of thousands of deaths each year, depends almost exclusively on just two or three drugs: praziquantel, the benzimidazoles, and ivermectin
Pharmacogenomics with the new helminth genomes represents a practicable route forward toward new drugs. For example, chemogenomics screening of the genome sequence of S. mansoni identified 20 parasite proteins for which potential drugs are available approved for other human ailments [schistosome thioredoxin glutathione reductase, auranofin (an anti-arthritis medication) was shown recently to exhibit potent anti-schistosomal activity].
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Conclusions Genomic studies of parasites should be done
keeping the following points in focus: Highly conserved regions can reveal information
about taxonomic relationships between species Moderately conserved regions can differentiate
strains and closely related species; Moderately variable regions can reveal
population genetic structure Highly variable regions enables tracking of
particular isolates in a population; and ‘mapping’of genetic markers can be used to find markers that correlate genotype to phenotype .
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Conclusions Sequencing detects the highest level of
variation down to changes in individual base pairs
Techniques based on restriction-fragment-length polymorphisms only detect changes that affect restriction sites, but can be used to sample across the entire genome.
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Conclusions The recent research landscape for parasites has
been dominated by rapid progress in genome sequencing of several parasites of significance to human disease.
Future genome-wide analyses will support efforts to elucidate the basic biology of parasites, including immune mediated and other host–parasite interactions that are relevant to parasitic diseases of humans.
They will help to develop novel intervention strategies such as drugs and therapeutic or prophylactic vaccines, as well as to identify parasite biomarkers and devise improved diagnostics.
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Conclusions As many of the parasitic infections are
transmitted by arthropod vectors or involve intermediate hosts, a greater understanding of the interaction between vector/intermediate hosts and parasites is also important.
The future may belong to Systems Biology approach to link high throughput molecular sciences such as genomics, proteomics and transcriptomics ; where individual streams of information can be processed as a whole, rather than remaining as an isolated descriptor of the action of individual components.
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