Diagnosis and Characterization of Leishmania Species Using PCR-RFLP

1. DIAGNOSIS AND CHARACTERIZATION
OF LEISHMANIA SPECIES USING PCRRFLP
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Prepared by: Hercolanium GDeath
Supervised by: Dr. N. H.

2. INTRODUCTION
 In
Jordan there are several species of Leishmania;
Leishmania infantum, Leishmania tropica, and
Leishmania major.
 Leishmania
tropica is the major species of
Leishmania parasite in Jordan but recent
revolutions in Syria and other countries resulted in
the migration of high number of refugees and with
them came new species mainly Leishmania major
from Syria.
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3.  The
problem with that is the prediction of an L.
major outbreak that might take place several
years from now.
 Identification
of Leishmania species will be so
important to decide the best treatment.
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4. 4

5. DIAGNOSIS

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1.
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2.
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To minimize the predicament there should be a ‘gold standard’ to diagnose
patients if the need calls for it. Several methods are available:
Traditional diagnostic methods:
Microscopic Examination
Examination of intracellular amastigotes from Giemsa-stained lesion
biopsy smears (CL), or lymph node, bone marrow aspiration (VL)
It depends on skilled clinicians, has low sensitivity, and the procedures are
invasive in the case of VL.
Culture
Growth of promastigotes in days to weeks
NNN medium is the medium of choice
Sensitivity is considerably mediocre
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6. Microscopy
Culture
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7. CONT’
Serological Diagnosis
1. Indirect Fluorescence Antibody (IFA)
2. ELISA
 Both are not suitable for field conditions
3. Direct Agglutination Test (DAT)
4. rK39
5. Fast Agglutination Screening Test (FAST)
 Both DAT and rK39 dipsticks are easy to use, require
minimal technological expertise or lab. Set-up and are
ideal for field and lab. Use.

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8. 8

9. CONT’
 rK39
is an abbreviation for a Recombinant Kinesin-related protein of 39 kDa
that is a part of a kinesis-related protein of Leishmania chagasi which is
conserved within Leishmania donovani complex, and antibody against it from
cutaneous and mucocotaneous leishmaniasis cannot be detected.
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10. CONT’

PCR and Leishmaniasis:
There has been a lot or research has been done and over 400 articles
on it have been published
2. High-copy-number target genes are chosen
 rRNA genes
 ITS1 Region of rRNA genes
 Kinetoplast DNA minicircles
 Mini-exon genes
Other genes
 Gp63 gene
 Hsp70 gene
 Cysteine proteinase gene
1.
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11. CONT’



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kDNA minicircle gene:
An excellent target for a
sensitive and rapid detection
methods
Present at thousands of
copies per cell
Show the highest sensitivity
by PCR
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12. CONT’

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
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Mini-exon (spliced leader) gene repeat
Involved in the transsplicing process of nuclear mRNA
Present 100-200 tandemly repeated copies per nuclear genome
Sequence variation of non-transcribed spacer was observed between
individual species
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13. CONT’

ITS (Internal Transcribed Spacer) refers to a piece of non-functional
RNA situated between structural ribosomal RNAs (rRNA) on a
common precursor transcript.

Read from 5' to 3', this polycistronic rRNA precursor transcript
contains the 5' external transcribed sequence (5' ETS), 18S rRNA,
ITS1, 5.8S rRNA, ITS2, 28S rRNA and finally the 3'ETS.
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14. 
During rRNA maturation, ETS and ITS pieces are excised and as
non-functional maturation by-products rapidly degraded. Genes
encoding ribosomal RNA and spacers occur in tandem repeats that are
thousands of copies long, each separated by regions of nontranscribed DNA termed intergenic spacer (IGS) or non-transcribed
spacer (NTS).
Sequence comparison of the ITS region is widely used in taxonomy
and molecular phylogeny because:
a) It is easy to amplify even from small quantities of DNA (due to the
high copy number of rRNA genes).
b) It has a high degree of variation even between closely related species
(due to the relatively low evolutionary pressure acting on such nonfunctional sequences).

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16. SPECIES IDENTIFICATION
•
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•
PCR-RFLP
ITS1 of SSU
rRNA gene;
restriction enzyme
Hae III
Mini-exon gene;
restriction enzyme
Eae I
DNA sequencing
of PCR products
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17. RFLP


Stands for Restriction
Fragment Length
Polymorphism
Takes advantage of
differences in DNA between
individuals that result in
different fragments when
digested with restriction
enzymes
How many fragments will
result when each of these
alleles are digested with
DdeI?
3 fragments
2 fragments
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18. RFLP

To see RFLP, DNA is digested
with the appropriate restriction
enzymes and run on an agarose
gel.

A Southern Blot is performed to
complete the analysis.
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19. SOUTHERN BLOTTING

A method to visualize specific
segments of DNA– usually a
particular gene.

Uses radioactive probes that
bind to the specific DNA
segments
 Ex.
When testing for the
hemoglobin alleles, the probe
would bind to these regions
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21. POLYMERASE CHAIN
REACTION
PCR allows scientists
to amplify small,
specific segments of
DNA = make millions
of copies of segment
 Allows for
amplification at an
exponential rate
 DNA Replication in a
test tube

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23. FILTER PAPER PCR

A new method for obtaining of samples from fields is Filter
paper; in which the clinician impress a filet paper on the
lesion.

Filter papers were then allowed to air-dry, and 6-mm punches
were obtained and stored in 1.5-mL Eppendorf tubes
containing 700 mL 100% ethanol for qualitative PCR testing.

To avoid contamination between specimens, the single-hole
punch was used on clean filter paper 10 times, then immersed
and washed in soapy water for 10 min, allowed to air-dry, and
then cleaned again with 2 separate isopropyl alcohol wipes.
Specimen collection using the Fisher brand 7-cm filter paper lesion impression
method. A, cleansing of an ulcer suspected to be cutaneous
 Sensitivity and specificity of filter paper PCR were 92.3%
leishmaniasis with topical antiseptic; B, preparation of the filter paper for
specimen collection; C, application of the filter paper to the ulcer base; D,
evidence of tissue fluid and ulcer exudates wicked onto the filter paper
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26. REFERENCES

Hijjawi N.S., Atoum M, Kanani K, Rasheed M, Abdel-dayem M, Irhimeh M.
R. Molecular diagnosis and identification of different Leishmania species for
Jordanian cutaneous leishmaniasis patients. Unpublished

Phramongkutklao College of Medicine; Saovanee Leelayoova, Mathirut
Mungthin, Suradej Siripattanapipong, and Tawee Naaglor

Hajjaran H, Vasigheh F, Mohebali M, Rezaei S, Mamishi S, Charedar S.
Direct diagnosis of Leishmania species on serosity materials punctured from
cutaneous leishmaniasis patients using PCR-RFLP. J Clin Lab Anal.
2011;25(1):20–4. doi: 10.1002/jcla.20377

Van Thiel P-P A.M., Van Gool T., Faber W.R., Leenstra T., Kager P.A.
Variation in Clinical Presentation and Genotype of Causative Leishmania
major Strain in Cutaneous Leishmaniasis in North and South Afghanistan. Am
J Trop Med Hyg. 2011 July 1; 85(1): 60–63.

Toz SO, Nasereddin A, Ozbel Y, Ertabaklar H, Culha G, et al. Leishmaniasis
in Turkey: molecular characterization of Leishmania from human and canine
clinical samples. Trop Med Int Health. 2009;14:1401–1406

Some internet sites
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