1. GENOMIC MARKERS FOR
PARASITIC INFECTIONS
Dr.R.Jayaprada
1

2. 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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3. 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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4. Introduction
Problems 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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5. 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
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dissimilar to each other.

6. 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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7. 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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8. 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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9. Introduction
Human Parasites Complete sequence: Timeline
2001: 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 suum
Other parasites: Toxoplasma gondii, Leishmania
braziliensis, L. infantum, L.mexicana,(Partial/ Draft).
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10. 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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11. 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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12. 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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13. Real Time PCR
The 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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14. Real Time PCR
Various Fluorescent chemistries are being used
1. 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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15. Real Time PCR
2. Minor-groove binder(MGB) Eclipse probes
3. Molecular beacons
4. Fluorescence- (or Forster) resonance-energy-
transfer (FRET)-based assays
5. 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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16. 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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17. 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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18. 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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19. 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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20. 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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21. 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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22. 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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23. 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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24. 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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25. 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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26. Microarrays
The 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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27. 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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28. (Monis et al 2005.
Trends in
Parasitology 21:
28 340-46)

29. 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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30. 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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31. 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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32. 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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33. 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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34. 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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35. 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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36. 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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37. 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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38. 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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39. 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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40. 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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41. 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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42. 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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43. 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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44. Functional Genomic Study
Two Categories
1. 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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45. 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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46. 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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47. 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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48. 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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49. 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.
49

50. 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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51. 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.
51

52. 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
52

53. 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.
53

54. 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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55. 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.
55

56. 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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57. 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.
57

58. 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 .
58

59. 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.
59

60. 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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61. 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].
61

62. 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 .
62

63. 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.
63

64. 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.
64

65. 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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