Current Development in the Diagnosis of Parasitic Infection 19 04 2013

7/28/2019 Current Development in the Diagnosis of Parasitic Infection 19 04 2013

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CURRENT DEVELOPMENT IN THE

DIAGNOSIS OF PARASITIC INFECTION

INCLUDING PCR METHODS


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DIAGNOSTIC METHODS IN

PARASITOLOGY


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CONVENTIONAL METHODS

Need trained staffs, equipments,slow

throughput BUT gold standard

Rapid molecular tests being developed


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MOLECULAR DIAGNOSTIC

Methods to study primary structure (Sequence ofDNA

Every organism contains some unique, speciesspecific DNA sequences

Molecular diagnostic make the species specificDNA visible

Needed if traditional methods provide poor results

1. Microscopy gives false positive/ negative results2. Low sensitivity

3. Cultivation methods : slow growth


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MOLECULAR METHODS

Advantages

High sensitivity &specificity

Detect pathogens , notimmune response

Quick results

High gransport

tolerations Easy to perform

Cost effective

Disadvantages

Expensive : PCR

PCR can fail :contamination and false

positive

Do not distinguish deadand living parasites


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MOLECULAR DIAGNOSIS METHODS1. MOLECULAR HYBRIDISATION

(a) RNA-RNA hybrids

(b)RNA-DNA hybrids

(c)In situ Hybridization(analyze prepared cells / histologic

sections)

2. POLYMERASE CHAIN REACTION TECHNIQUES

(a) Conventional PCR

(b) Nested PCR

(c) Multiplex PCR

(d) RT-PCR(e) Real time PCR

3. COMBINED TECHNIQUES

(a) Serological and molecular techniques

(b) Immunocapture PCR (IC PCR)


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By heating and cooling

- Target DNA is denatured to provide singlestranded template DNA

- Temperature lower , primers will anneal to

flanking regions

- Bases are added to 3 end of annealed primers

by DNA polymerase

The cycles are repeated, amount of DNAincrease exponentially


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Toxoplasma gondii Five targets are routinely used in PCR

1. 35-fold repititive gene B1

2. Single copy P30 gene, coding for the major surface

protein antigen P30, nested PCR is favored

3. 110-fold repititive ssu rRNA

4. Single copy tubulin gene

5. Single copy tubulin gene


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A: Agarose gel (2%) analysis of a

PCR diagnostic test for detection

ofCryptosporidium parvum DNA.

PCR was performed usingstandard ABI protocol.Lane S:

Molecular base pair standard

(100-bp ladder). Black arrows

show the size of standard bands.

Lane 1: C. parvum positive fecal

specimen. The red arrow shows

the diagnostic band for

Cryptosporidium parvum zoonoticgenotype (size: 435 bp).


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NESTED PCR ASSAY for detection

Cryptosporidium parvum Oocysts

These sophisticated

new "nested PCR"

systems are

significantly moresensitive than other

currently available PCR

technologies.

A schematic of nested

PCR is shown in the

figure

l l l i


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Placental Malaria

Placental infection with Plasmodim falciparum

contribute to maternal morbidity, low birthweight and pre term delivery

Post partum microscopy of placental blood films

has been used as a more accurate indicator

Sensitivity detecting Plasmodium falciparum in peripheral blood

Microscopy Immunoassay PCR42% 80% 99.9%


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Visceral leishmaniasis (kala-azar)

Routine diagnosis is by direct microscopy orculture

Microscopic detection of amastigote in Geimsa

stained spleen, bone marrow/lymph node

aspirates is simple and cheap

Disadvantages :

(a) Spleen aspiration is danger under field conditions

(b) Bone marrow and lymph node aspirates are of

limited sensitivity. And inconvenient to patient

(c) Isolation of parasites by culture is time consuming,

expensive and difficult


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PCR > sensitive than microscopy

Blood may be used for initial PCR screening

If PCR on blood is negative, PCR on lymph

node / bone marrow should be performed

PCR is cheaper test compared to microscopy

detection

Visceral leishmaniasis (kala-azar)


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PCR LABORATORY


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PCR LABORATORY

Sample Handling

DNA preparations

Laboratory

Mixing SiteThermocycler

Amplification

Detection

Documentation

R & D


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Documents

Current Development in the Diagnosis of Parasitic Infection 19 04 2013