recent diagnostic methods for intestinal parasitic...
TRANSCRIPT
![Page 1: Recent Diagnostic Methods for Intestinal Parasitic Infectionssmh.mans.edu.eg/files/pdf/conf/2012/11_Recent_Diagnostic_Methods... · Recent Diagnostic Methods for Intestinal Parasitic](https://reader031.vdocuments.mx/reader031/viewer/2022021821/5af89af87f8b9aff288c0698/html5/thumbnails/1.jpg)
![Page 2: Recent Diagnostic Methods for Intestinal Parasitic Infectionssmh.mans.edu.eg/files/pdf/conf/2012/11_Recent_Diagnostic_Methods... · Recent Diagnostic Methods for Intestinal Parasitic](https://reader031.vdocuments.mx/reader031/viewer/2022021821/5af89af87f8b9aff288c0698/html5/thumbnails/2.jpg)
Recent Diagnostic
Methods for
Intestinal Parasitic
Infections
By
Dr. Doaa Abdel Badie Salem
Lecturer of Medical
Parasitology, Mansoura
Faculty of Medicine
![Page 3: Recent Diagnostic Methods for Intestinal Parasitic Infectionssmh.mans.edu.eg/files/pdf/conf/2012/11_Recent_Diagnostic_Methods... · Recent Diagnostic Methods for Intestinal Parasitic](https://reader031.vdocuments.mx/reader031/viewer/2022021821/5af89af87f8b9aff288c0698/html5/thumbnails/3.jpg)
Agenda• Intestinal parasites.
• Traditional Diagnostic
Methods.
• Why we are looking for
new techniques for
diagnosis of intestinal
parasitic tract?
• Commercially available
techniques
• Hoping future techniques
• Summary5/26/2012 3
![Page 4: Recent Diagnostic Methods for Intestinal Parasitic Infectionssmh.mans.edu.eg/files/pdf/conf/2012/11_Recent_Diagnostic_Methods... · Recent Diagnostic Methods for Intestinal Parasitic](https://reader031.vdocuments.mx/reader031/viewer/2022021821/5af89af87f8b9aff288c0698/html5/thumbnails/4.jpg)
![Page 5: Recent Diagnostic Methods for Intestinal Parasitic Infectionssmh.mans.edu.eg/files/pdf/conf/2012/11_Recent_Diagnostic_Methods... · Recent Diagnostic Methods for Intestinal Parasitic](https://reader031.vdocuments.mx/reader031/viewer/2022021821/5af89af87f8b9aff288c0698/html5/thumbnails/5.jpg)
Most common Egyptian intestinal parasites
Protozoa Helminthes
Entamoebahistolytica Trematodes Cectodes Nematodes
Coccidea(Cryptosporidium parvum, Isosporabelli, Cyclosporacayetanensis)
Schistosomamansoni
Teania spp. ( T. saginata and T. solium )
Strongoiloidesstercoralis
Giardia duodenalis Hetrophyeshetrophyes
Hymenolipes ( H. nana and H. diminuta).
Soil transmitted
helminths STH
(Ascaris lumbricoid,
Trichuristrichiura, Hook
worm)
Enterobiusvermicularis
5/26/2012 5
![Page 6: Recent Diagnostic Methods for Intestinal Parasitic Infectionssmh.mans.edu.eg/files/pdf/conf/2012/11_Recent_Diagnostic_Methods... · Recent Diagnostic Methods for Intestinal Parasitic](https://reader031.vdocuments.mx/reader031/viewer/2022021821/5af89af87f8b9aff288c0698/html5/thumbnails/6.jpg)
5/26/2012 6
Giardia duodenalis
Hetrophyes hetrophyes,Teania spp., Hymenolipes spp.
STH, Strongoiloides stercoralis
Entamoeba histolytica
Enterobius vermicularis
![Page 7: Recent Diagnostic Methods for Intestinal Parasitic Infectionssmh.mans.edu.eg/files/pdf/conf/2012/11_Recent_Diagnostic_Methods... · Recent Diagnostic Methods for Intestinal Parasitic](https://reader031.vdocuments.mx/reader031/viewer/2022021821/5af89af87f8b9aff288c0698/html5/thumbnails/7.jpg)
Mode of transmission• IPIs are linked to lack of sanitation, lack of
access to safe water and improper hygiene.
• Protozoan mainly transmitted by feco-oral (auto
infection, person to person, food, water,….).
• Intestinal trematodes by fish eating, and skin
penetration.
• Intestinal cectodes by ingestion of meat
auto infection, insect ingestion.
• Intestinal nematodes by ingestion of contaminated food, autoinfection, skin penetration 5/26/2012 7
![Page 8: Recent Diagnostic Methods for Intestinal Parasitic Infectionssmh.mans.edu.eg/files/pdf/conf/2012/11_Recent_Diagnostic_Methods... · Recent Diagnostic Methods for Intestinal Parasitic](https://reader031.vdocuments.mx/reader031/viewer/2022021821/5af89af87f8b9aff288c0698/html5/thumbnails/8.jpg)
Diagnosis of intestinal
parasitic infections
CLINICAL PICTUR
LABORATORY
5/26/2012 8
![Page 9: Recent Diagnostic Methods for Intestinal Parasitic Infectionssmh.mans.edu.eg/files/pdf/conf/2012/11_Recent_Diagnostic_Methods... · Recent Diagnostic Methods for Intestinal Parasitic](https://reader031.vdocuments.mx/reader031/viewer/2022021821/5af89af87f8b9aff288c0698/html5/thumbnails/9.jpg)
Clinical picture
• Diarrhea
• Dysentery
• Anemia
• Allergy
• Constipation
• Acute abdomen
• Peri-anal itching
• Bloating, dyspepsia
5/26/2012 9
![Page 10: Recent Diagnostic Methods for Intestinal Parasitic Infectionssmh.mans.edu.eg/files/pdf/conf/2012/11_Recent_Diagnostic_Methods... · Recent Diagnostic Methods for Intestinal Parasitic](https://reader031.vdocuments.mx/reader031/viewer/2022021821/5af89af87f8b9aff288c0698/html5/thumbnails/10.jpg)
I-Traditional Diagnostic Methods
5/26/2012 10
![Page 11: Recent Diagnostic Methods for Intestinal Parasitic Infectionssmh.mans.edu.eg/files/pdf/conf/2012/11_Recent_Diagnostic_Methods... · Recent Diagnostic Methods for Intestinal Parasitic](https://reader031.vdocuments.mx/reader031/viewer/2022021821/5af89af87f8b9aff288c0698/html5/thumbnails/11.jpg)
1-Macroscopic stool
examinationSearching for:
• 1- Blood and mucus.
• 2-Some adult helminths as Ascarislumbricoid or segments as Tenia spp. or
Hymenolips spp.
5/26/2012 11
![Page 12: Recent Diagnostic Methods for Intestinal Parasitic Infectionssmh.mans.edu.eg/files/pdf/conf/2012/11_Recent_Diagnostic_Methods... · Recent Diagnostic Methods for Intestinal Parasitic](https://reader031.vdocuments.mx/reader031/viewer/2022021821/5af89af87f8b9aff288c0698/html5/thumbnails/12.jpg)
2-Microscopic stool examination.
Relies on the detection of
• Trophozoites
• Cysts
• Eggs
• Larvae
5/26/2012 12
![Page 13: Recent Diagnostic Methods for Intestinal Parasitic Infectionssmh.mans.edu.eg/files/pdf/conf/2012/11_Recent_Diagnostic_Methods... · Recent Diagnostic Methods for Intestinal Parasitic](https://reader031.vdocuments.mx/reader031/viewer/2022021821/5af89af87f8b9aff288c0698/html5/thumbnails/13.jpg)
• How to improve the sensitivity of
microscopic examination?
- Multiple stool specimens.
- Concentration procedures.
- Variety of staining methods, are needed
to achieve ample sensitivity for intestinal
coccidean as
- Modified ZN stain, non specific
fluorescent stain, and some have the
ability for auto fluorescence.
5/26/2012 13
![Page 14: Recent Diagnostic Methods for Intestinal Parasitic Infectionssmh.mans.edu.eg/files/pdf/conf/2012/11_Recent_Diagnostic_Methods... · Recent Diagnostic Methods for Intestinal Parasitic](https://reader031.vdocuments.mx/reader031/viewer/2022021821/5af89af87f8b9aff288c0698/html5/thumbnails/14.jpg)
5/26/2012 14
cyst
trophozoite
Entamoeba
![Page 15: Recent Diagnostic Methods for Intestinal Parasitic Infectionssmh.mans.edu.eg/files/pdf/conf/2012/11_Recent_Diagnostic_Methods... · Recent Diagnostic Methods for Intestinal Parasitic](https://reader031.vdocuments.mx/reader031/viewer/2022021821/5af89af87f8b9aff288c0698/html5/thumbnails/15.jpg)
Giardia intestinalis
5/26/2012 15
![Page 16: Recent Diagnostic Methods for Intestinal Parasitic Infectionssmh.mans.edu.eg/files/pdf/conf/2012/11_Recent_Diagnostic_Methods... · Recent Diagnostic Methods for Intestinal Parasitic](https://reader031.vdocuments.mx/reader031/viewer/2022021821/5af89af87f8b9aff288c0698/html5/thumbnails/16.jpg)
Intestinal coccidean
5/26/2012 16
![Page 17: Recent Diagnostic Methods for Intestinal Parasitic Infectionssmh.mans.edu.eg/files/pdf/conf/2012/11_Recent_Diagnostic_Methods... · Recent Diagnostic Methods for Intestinal Parasitic](https://reader031.vdocuments.mx/reader031/viewer/2022021821/5af89af87f8b9aff288c0698/html5/thumbnails/17.jpg)
5/26/2012 17
Auto fluorescence
Isospora belli
Non specific
fluorescent stain for
Cryptosporidium
![Page 18: Recent Diagnostic Methods for Intestinal Parasitic Infectionssmh.mans.edu.eg/files/pdf/conf/2012/11_Recent_Diagnostic_Methods... · Recent Diagnostic Methods for Intestinal Parasitic](https://reader031.vdocuments.mx/reader031/viewer/2022021821/5af89af87f8b9aff288c0698/html5/thumbnails/18.jpg)
Some intestinal helminths eggs
5/26/2012 18
![Page 19: Recent Diagnostic Methods for Intestinal Parasitic Infectionssmh.mans.edu.eg/files/pdf/conf/2012/11_Recent_Diagnostic_Methods... · Recent Diagnostic Methods for Intestinal Parasitic](https://reader031.vdocuments.mx/reader031/viewer/2022021821/5af89af87f8b9aff288c0698/html5/thumbnails/19.jpg)
Some cectode eggs
5/26/2012 19
![Page 20: Recent Diagnostic Methods for Intestinal Parasitic Infectionssmh.mans.edu.eg/files/pdf/conf/2012/11_Recent_Diagnostic_Methods... · Recent Diagnostic Methods for Intestinal Parasitic](https://reader031.vdocuments.mx/reader031/viewer/2022021821/5af89af87f8b9aff288c0698/html5/thumbnails/20.jpg)
Some trematodes eggs
5/26/2012 20
![Page 21: Recent Diagnostic Methods for Intestinal Parasitic Infectionssmh.mans.edu.eg/files/pdf/conf/2012/11_Recent_Diagnostic_Methods... · Recent Diagnostic Methods for Intestinal Parasitic](https://reader031.vdocuments.mx/reader031/viewer/2022021821/5af89af87f8b9aff288c0698/html5/thumbnails/21.jpg)
3- Antibody-detection assays
1- CFT
2- IHA
3-ELISA
5/26/2012 21
![Page 22: Recent Diagnostic Methods for Intestinal Parasitic Infectionssmh.mans.edu.eg/files/pdf/conf/2012/11_Recent_Diagnostic_Methods... · Recent Diagnostic Methods for Intestinal Parasitic](https://reader031.vdocuments.mx/reader031/viewer/2022021821/5af89af87f8b9aff288c0698/html5/thumbnails/22.jpg)
Why we are searching for new
methods for diagnosis of
intestinal parasitic infections?
5/26/2012 22
![Page 23: Recent Diagnostic Methods for Intestinal Parasitic Infectionssmh.mans.edu.eg/files/pdf/conf/2012/11_Recent_Diagnostic_Methods... · Recent Diagnostic Methods for Intestinal Parasitic](https://reader031.vdocuments.mx/reader031/viewer/2022021821/5af89af87f8b9aff288c0698/html5/thumbnails/23.jpg)
• Although microscopy is considered to
be the gold standard,
-It is labour-intensive
-Its diagnostic performance critically depends
on well-trained microscopists.
-It is not sensitive and may result in
misdiagnosis leading to delayed or
inadequate treatment.
-
5/26/2012 23
![Page 24: Recent Diagnostic Methods for Intestinal Parasitic Infectionssmh.mans.edu.eg/files/pdf/conf/2012/11_Recent_Diagnostic_Methods... · Recent Diagnostic Methods for Intestinal Parasitic](https://reader031.vdocuments.mx/reader031/viewer/2022021821/5af89af87f8b9aff288c0698/html5/thumbnails/24.jpg)
- Diagnosis of E. histolytica cannot be done
any longer by microscopy, since this
parasite is morphologically similar to the
non-pathogenic parasite E. dispar.
- Increase prevalence of immune
compromised sectors in our locality
(chronic liver disease, CKD, DM,
malignancy…….).
- Empirical treatment is not preferable
5/26/2012 24
![Page 25: Recent Diagnostic Methods for Intestinal Parasitic Infectionssmh.mans.edu.eg/files/pdf/conf/2012/11_Recent_Diagnostic_Methods... · Recent Diagnostic Methods for Intestinal Parasitic](https://reader031.vdocuments.mx/reader031/viewer/2022021821/5af89af87f8b9aff288c0698/html5/thumbnails/25.jpg)
Limitations for Ab detection:
1- Some intestinal parasites do not have
commercially or FDA approved antibody
detection tests for their diagnosis.
2-Experimental results have been too
variable due to the type of antigen
preparations used (e.g., crude,
recombinant purified, adult worm, egg)
and also because of the use of non
standardized test procedures.
5/26/2012 25
![Page 26: Recent Diagnostic Methods for Intestinal Parasitic Infectionssmh.mans.edu.eg/files/pdf/conf/2012/11_Recent_Diagnostic_Methods... · Recent Diagnostic Methods for Intestinal Parasitic](https://reader031.vdocuments.mx/reader031/viewer/2022021821/5af89af87f8b9aff288c0698/html5/thumbnails/26.jpg)
• 3- Cross-reaction leading to false-positives
and misdiagnosis, especially in regions
where more than one parasite is endemic.
(cross-reactivity exists between filarial and
other helminth antigens).
• 4- Inability of antibody-detection tests to
differentiate between past and currently
active infections
5/26/2012 26
![Page 27: Recent Diagnostic Methods for Intestinal Parasitic Infectionssmh.mans.edu.eg/files/pdf/conf/2012/11_Recent_Diagnostic_Methods... · Recent Diagnostic Methods for Intestinal Parasitic](https://reader031.vdocuments.mx/reader031/viewer/2022021821/5af89af87f8b9aff288c0698/html5/thumbnails/27.jpg)
Commercially available techniques
5/26/2012 27
![Page 28: Recent Diagnostic Methods for Intestinal Parasitic Infectionssmh.mans.edu.eg/files/pdf/conf/2012/11_Recent_Diagnostic_Methods... · Recent Diagnostic Methods for Intestinal Parasitic](https://reader031.vdocuments.mx/reader031/viewer/2022021821/5af89af87f8b9aff288c0698/html5/thumbnails/28.jpg)
5/26/2012 28
![Page 29: Recent Diagnostic Methods for Intestinal Parasitic Infectionssmh.mans.edu.eg/files/pdf/conf/2012/11_Recent_Diagnostic_Methods... · Recent Diagnostic Methods for Intestinal Parasitic](https://reader031.vdocuments.mx/reader031/viewer/2022021821/5af89af87f8b9aff288c0698/html5/thumbnails/29.jpg)
• Immunology-based diagnosis tools can
be divided into two categories:
1-Antigen-detection assays
2- Antibody-detection assays.
5/26/2012 29
![Page 30: Recent Diagnostic Methods for Intestinal Parasitic Infectionssmh.mans.edu.eg/files/pdf/conf/2012/11_Recent_Diagnostic_Methods... · Recent Diagnostic Methods for Intestinal Parasitic](https://reader031.vdocuments.mx/reader031/viewer/2022021821/5af89af87f8b9aff288c0698/html5/thumbnails/30.jpg)
• ELISA and direct fluorescent antibody assays
have been accepted as cost-effective alternative
diagnostic methods for the detection of G.
lamblia and Cryptosporidium in stools.
5/26/2012 30
![Page 31: Recent Diagnostic Methods for Intestinal Parasitic Infectionssmh.mans.edu.eg/files/pdf/conf/2012/11_Recent_Diagnostic_Methods... · Recent Diagnostic Methods for Intestinal Parasitic](https://reader031.vdocuments.mx/reader031/viewer/2022021821/5af89af87f8b9aff288c0698/html5/thumbnails/31.jpg)
Coproantigen ELISA:
• Principle:
The various components involved in
ELISA are a solid phase to which
specific antibody is coated, an
antibody enzyme conjugate as probe
and enzyme substrate. Solid supports
used are polystyrene or PVC
microtitre plates
5/26/2012 31
![Page 32: Recent Diagnostic Methods for Intestinal Parasitic Infectionssmh.mans.edu.eg/files/pdf/conf/2012/11_Recent_Diagnostic_Methods... · Recent Diagnostic Methods for Intestinal Parasitic](https://reader031.vdocuments.mx/reader031/viewer/2022021821/5af89af87f8b9aff288c0698/html5/thumbnails/32.jpg)
5/26/2012 32
![Page 33: Recent Diagnostic Methods for Intestinal Parasitic Infectionssmh.mans.edu.eg/files/pdf/conf/2012/11_Recent_Diagnostic_Methods... · Recent Diagnostic Methods for Intestinal Parasitic](https://reader031.vdocuments.mx/reader031/viewer/2022021821/5af89af87f8b9aff288c0698/html5/thumbnails/33.jpg)
Applications:
• For Strongyloides
• Cryptosporidium
• Giardia
• E. histolytica
5/26/2012 33
![Page 34: Recent Diagnostic Methods for Intestinal Parasitic Infectionssmh.mans.edu.eg/files/pdf/conf/2012/11_Recent_Diagnostic_Methods... · Recent Diagnostic Methods for Intestinal Parasitic](https://reader031.vdocuments.mx/reader031/viewer/2022021821/5af89af87f8b9aff288c0698/html5/thumbnails/34.jpg)
Disadvantage
• Requires multiple reagent additions
• Washing steps.
• Incubations.
Advantages(i) Numerous samples can be screened at
one time.
(ii) Tests can be read objectively on a
spectrophotometer instead of subject.
5/26/2012 34
![Page 35: Recent Diagnostic Methods for Intestinal Parasitic Infectionssmh.mans.edu.eg/files/pdf/conf/2012/11_Recent_Diagnostic_Methods... · Recent Diagnostic Methods for Intestinal Parasitic](https://reader031.vdocuments.mx/reader031/viewer/2022021821/5af89af87f8b9aff288c0698/html5/thumbnails/35.jpg)
Direct fluoresce antibodies (DFA)
Principle:
• Utilize fluorescein-labeled antibodies
directed against cell wall antigens of
Giardia cysts and Cryptosporidium oocysts
5/26/2012 35
![Page 36: Recent Diagnostic Methods for Intestinal Parasitic Infectionssmh.mans.edu.eg/files/pdf/conf/2012/11_Recent_Diagnostic_Methods... · Recent Diagnostic Methods for Intestinal Parasitic](https://reader031.vdocuments.mx/reader031/viewer/2022021821/5af89af87f8b9aff288c0698/html5/thumbnails/36.jpg)
5/26/2012 36
![Page 37: Recent Diagnostic Methods for Intestinal Parasitic Infectionssmh.mans.edu.eg/files/pdf/conf/2012/11_Recent_Diagnostic_Methods... · Recent Diagnostic Methods for Intestinal Parasitic](https://reader031.vdocuments.mx/reader031/viewer/2022021821/5af89af87f8b9aff288c0698/html5/thumbnails/37.jpg)
5/26/2012 37
![Page 38: Recent Diagnostic Methods for Intestinal Parasitic Infectionssmh.mans.edu.eg/files/pdf/conf/2012/11_Recent_Diagnostic_Methods... · Recent Diagnostic Methods for Intestinal Parasitic](https://reader031.vdocuments.mx/reader031/viewer/2022021821/5af89af87f8b9aff288c0698/html5/thumbnails/38.jpg)
Advantage of DFA:
• Allow visualization of the intact parasites,
providing a definitive diagnosis
5/26/2012 38
Disadvantage of DFA
• Requires fluorescence microscopy
![Page 39: Recent Diagnostic Methods for Intestinal Parasitic Infectionssmh.mans.edu.eg/files/pdf/conf/2012/11_Recent_Diagnostic_Methods... · Recent Diagnostic Methods for Intestinal Parasitic](https://reader031.vdocuments.mx/reader031/viewer/2022021821/5af89af87f8b9aff288c0698/html5/thumbnails/39.jpg)
Immunecromatographic assay
Principle:
It is the same as ELISA, but differs only in
that the immunological reaction carried out
on cromatographic paper by capillary action.
5/26/2012 39
![Page 40: Recent Diagnostic Methods for Intestinal Parasitic Infectionssmh.mans.edu.eg/files/pdf/conf/2012/11_Recent_Diagnostic_Methods... · Recent Diagnostic Methods for Intestinal Parasitic](https://reader031.vdocuments.mx/reader031/viewer/2022021821/5af89af87f8b9aff288c0698/html5/thumbnails/40.jpg)
• Two kinds of specific Ab :
-one of the Ab (immoblised on clear
defined line (test line)
-the 2nd labled with detection agent as
colloidal gold and infiltrated in
congugated pad
• The Ag to be detected or anti Ab
deposited on the same strip in
another line (control line)
5/26/2012 40
![Page 41: Recent Diagnostic Methods for Intestinal Parasitic Infectionssmh.mans.edu.eg/files/pdf/conf/2012/11_Recent_Diagnostic_Methods... · Recent Diagnostic Methods for Intestinal Parasitic](https://reader031.vdocuments.mx/reader031/viewer/2022021821/5af89af87f8b9aff288c0698/html5/thumbnails/41.jpg)
Cromatographic paper structure
5/26/2012 41
![Page 42: Recent Diagnostic Methods for Intestinal Parasitic Infectionssmh.mans.edu.eg/files/pdf/conf/2012/11_Recent_Diagnostic_Methods... · Recent Diagnostic Methods for Intestinal Parasitic](https://reader031.vdocuments.mx/reader031/viewer/2022021821/5af89af87f8b9aff288c0698/html5/thumbnails/42.jpg)
5/26/2012 42
![Page 43: Recent Diagnostic Methods for Intestinal Parasitic Infectionssmh.mans.edu.eg/files/pdf/conf/2012/11_Recent_Diagnostic_Methods... · Recent Diagnostic Methods for Intestinal Parasitic](https://reader031.vdocuments.mx/reader031/viewer/2022021821/5af89af87f8b9aff288c0698/html5/thumbnails/43.jpg)
Applications
• -Hymenolipes
• -Cryptosporidium and Girdia
• -E. hisolytica
• -Urine-CCA (Circulating Cathodic
Antigen) for Schistosoma mansoni.
5/26/2012 43
![Page 44: Recent Diagnostic Methods for Intestinal Parasitic Infectionssmh.mans.edu.eg/files/pdf/conf/2012/11_Recent_Diagnostic_Methods... · Recent Diagnostic Methods for Intestinal Parasitic](https://reader031.vdocuments.mx/reader031/viewer/2022021821/5af89af87f8b9aff288c0698/html5/thumbnails/44.jpg)
Forms of immunchromatography tests
5/26/2012 44
![Page 45: Recent Diagnostic Methods for Intestinal Parasitic Infectionssmh.mans.edu.eg/files/pdf/conf/2012/11_Recent_Diagnostic_Methods... · Recent Diagnostic Methods for Intestinal Parasitic](https://reader031.vdocuments.mx/reader031/viewer/2022021821/5af89af87f8b9aff288c0698/html5/thumbnails/45.jpg)
5/26/2012 45
![Page 46: Recent Diagnostic Methods for Intestinal Parasitic Infectionssmh.mans.edu.eg/files/pdf/conf/2012/11_Recent_Diagnostic_Methods... · Recent Diagnostic Methods for Intestinal Parasitic](https://reader031.vdocuments.mx/reader031/viewer/2022021821/5af89af87f8b9aff288c0698/html5/thumbnails/46.jpg)
5/26/2012 46
![Page 47: Recent Diagnostic Methods for Intestinal Parasitic Infectionssmh.mans.edu.eg/files/pdf/conf/2012/11_Recent_Diagnostic_Methods... · Recent Diagnostic Methods for Intestinal Parasitic](https://reader031.vdocuments.mx/reader031/viewer/2022021821/5af89af87f8b9aff288c0698/html5/thumbnails/47.jpg)
ImmunoCard STAT! Cryptosporidium/Giardia Rapid Assay (Meridian Bioscience, Inc.).
Garcia L S et al. J. Clin. Microbiol. 2003;41:209-212
![Page 48: Recent Diagnostic Methods for Intestinal Parasitic Infectionssmh.mans.edu.eg/files/pdf/conf/2012/11_Recent_Diagnostic_Methods... · Recent Diagnostic Methods for Intestinal Parasitic](https://reader031.vdocuments.mx/reader031/viewer/2022021821/5af89af87f8b9aff288c0698/html5/thumbnails/48.jpg)
parasite panel demonstrating positive results.
Garcia L S et al. J. Clin. Microbiol. 2000;38:3337-3340
![Page 49: Recent Diagnostic Methods for Intestinal Parasitic Infectionssmh.mans.edu.eg/files/pdf/conf/2012/11_Recent_Diagnostic_Methods... · Recent Diagnostic Methods for Intestinal Parasitic](https://reader031.vdocuments.mx/reader031/viewer/2022021821/5af89af87f8b9aff288c0698/html5/thumbnails/49.jpg)
Advantage
• Rapid
• Stable at temperatures up to 40°C,
• Easy to use,
• Cost-effective
5/26/2012 49
Disadvantage
• They are useless at detecting very low-
density infections.
![Page 50: Recent Diagnostic Methods for Intestinal Parasitic Infectionssmh.mans.edu.eg/files/pdf/conf/2012/11_Recent_Diagnostic_Methods... · Recent Diagnostic Methods for Intestinal Parasitic](https://reader031.vdocuments.mx/reader031/viewer/2022021821/5af89af87f8b9aff288c0698/html5/thumbnails/50.jpg)
Hoping future techniques
5/26/2012 50
![Page 51: Recent Diagnostic Methods for Intestinal Parasitic Infectionssmh.mans.edu.eg/files/pdf/conf/2012/11_Recent_Diagnostic_Methods... · Recent Diagnostic Methods for Intestinal Parasitic](https://reader031.vdocuments.mx/reader031/viewer/2022021821/5af89af87f8b9aff288c0698/html5/thumbnails/51.jpg)
1-Flowcytometry
5/26/2012 51
![Page 52: Recent Diagnostic Methods for Intestinal Parasitic Infectionssmh.mans.edu.eg/files/pdf/conf/2012/11_Recent_Diagnostic_Methods... · Recent Diagnostic Methods for Intestinal Parasitic](https://reader031.vdocuments.mx/reader031/viewer/2022021821/5af89af87f8b9aff288c0698/html5/thumbnails/52.jpg)
![Page 53: Recent Diagnostic Methods for Intestinal Parasitic Infectionssmh.mans.edu.eg/files/pdf/conf/2012/11_Recent_Diagnostic_Methods... · Recent Diagnostic Methods for Intestinal Parasitic](https://reader031.vdocuments.mx/reader031/viewer/2022021821/5af89af87f8b9aff288c0698/html5/thumbnails/53.jpg)
5/26/2012 53
![Page 54: Recent Diagnostic Methods for Intestinal Parasitic Infectionssmh.mans.edu.eg/files/pdf/conf/2012/11_Recent_Diagnostic_Methods... · Recent Diagnostic Methods for Intestinal Parasitic](https://reader031.vdocuments.mx/reader031/viewer/2022021821/5af89af87f8b9aff288c0698/html5/thumbnails/54.jpg)
Cryptosporidium in stoolFSC (x-axis) versus SSC (yaxis).
The scatter-plots for Cryptosporidium oocystsrepresenting FSC vs SSC (a): The scatter-plot for oocysts without staining.
(b): The scatter-plot for oocysts stained with
direct immunofluorescent antibody
![Page 55: Recent Diagnostic Methods for Intestinal Parasitic Infectionssmh.mans.edu.eg/files/pdf/conf/2012/11_Recent_Diagnostic_Methods... · Recent Diagnostic Methods for Intestinal Parasitic](https://reader031.vdocuments.mx/reader031/viewer/2022021821/5af89af87f8b9aff288c0698/html5/thumbnails/55.jpg)
2- Molecular
Based
Approaches
5/26/2012 55
![Page 56: Recent Diagnostic Methods for Intestinal Parasitic Infectionssmh.mans.edu.eg/files/pdf/conf/2012/11_Recent_Diagnostic_Methods... · Recent Diagnostic Methods for Intestinal Parasitic](https://reader031.vdocuments.mx/reader031/viewer/2022021821/5af89af87f8b9aff288c0698/html5/thumbnails/56.jpg)
A-Conventional PCR
Four basic components:
• A DNA template .
• Primers
• A heat-stable DNA-polymerase enzyme -
• Free nucleotides
5/26/2012 56
![Page 57: Recent Diagnostic Methods for Intestinal Parasitic Infectionssmh.mans.edu.eg/files/pdf/conf/2012/11_Recent_Diagnostic_Methods... · Recent Diagnostic Methods for Intestinal Parasitic](https://reader031.vdocuments.mx/reader031/viewer/2022021821/5af89af87f8b9aff288c0698/html5/thumbnails/57.jpg)
5/26/2012 57
![Page 58: Recent Diagnostic Methods for Intestinal Parasitic Infectionssmh.mans.edu.eg/files/pdf/conf/2012/11_Recent_Diagnostic_Methods... · Recent Diagnostic Methods for Intestinal Parasitic](https://reader031.vdocuments.mx/reader031/viewer/2022021821/5af89af87f8b9aff288c0698/html5/thumbnails/58.jpg)
5/26/2012 58
![Page 59: Recent Diagnostic Methods for Intestinal Parasitic Infectionssmh.mans.edu.eg/files/pdf/conf/2012/11_Recent_Diagnostic_Methods... · Recent Diagnostic Methods for Intestinal Parasitic](https://reader031.vdocuments.mx/reader031/viewer/2022021821/5af89af87f8b9aff288c0698/html5/thumbnails/59.jpg)
Disadvantage of PCR
• The introduction of these methods in daily
laboratory practice is still uncommon
especially in rural endemic regions.
• PCR-based methods also suffer by the
lack of standardization. DNA extraction,
choice of primer sets, and use of various
amplification protocols are all factors that
may cause this diversification in results
5/26/2012 59
![Page 60: Recent Diagnostic Methods for Intestinal Parasitic Infectionssmh.mans.edu.eg/files/pdf/conf/2012/11_Recent_Diagnostic_Methods... · Recent Diagnostic Methods for Intestinal Parasitic](https://reader031.vdocuments.mx/reader031/viewer/2022021821/5af89af87f8b9aff288c0698/html5/thumbnails/60.jpg)
Advantage
• DNA-based methods have shown excellent
sensitivity and specificity
5/26/2012 60
![Page 61: Recent Diagnostic Methods for Intestinal Parasitic Infectionssmh.mans.edu.eg/files/pdf/conf/2012/11_Recent_Diagnostic_Methods... · Recent Diagnostic Methods for Intestinal Parasitic](https://reader031.vdocuments.mx/reader031/viewer/2022021821/5af89af87f8b9aff288c0698/html5/thumbnails/61.jpg)
B-Real-time PCR (RT-PCR)
• Principle:
This method utilizes an additional primer,
the probe, which also binds specifically to
the target DNA sequence. Probes have a
fluorescent 'reporter' dye at one end and a
'quencher' dye, which inhibits fluorescence,
at the other. During the extension stage the
probe is broken apart by the DNA-
polymerase and begins to fluoresce more
strongly. 5/26/2012 61
![Page 62: Recent Diagnostic Methods for Intestinal Parasitic Infectionssmh.mans.edu.eg/files/pdf/conf/2012/11_Recent_Diagnostic_Methods... · Recent Diagnostic Methods for Intestinal Parasitic](https://reader031.vdocuments.mx/reader031/viewer/2022021821/5af89af87f8b9aff288c0698/html5/thumbnails/62.jpg)
• The fluorescence can be measured at
each cycle and increases in proportion to
the number of target sequence copies
produced. This means that the assay can
be made quantitative by recording the
cycle at which the fluorescence intensity
rises above the background level for
each test sample and for a set of
standards run at the same time. A
standard curve can then be drawn and
the amount of target DNA present in the
sample can be calculated from the
standard curve.5/26/2012 62
![Page 63: Recent Diagnostic Methods for Intestinal Parasitic Infectionssmh.mans.edu.eg/files/pdf/conf/2012/11_Recent_Diagnostic_Methods... · Recent Diagnostic Methods for Intestinal Parasitic](https://reader031.vdocuments.mx/reader031/viewer/2022021821/5af89af87f8b9aff288c0698/html5/thumbnails/63.jpg)
5/26/2012 63
![Page 64: Recent Diagnostic Methods for Intestinal Parasitic Infectionssmh.mans.edu.eg/files/pdf/conf/2012/11_Recent_Diagnostic_Methods... · Recent Diagnostic Methods for Intestinal Parasitic](https://reader031.vdocuments.mx/reader031/viewer/2022021821/5af89af87f8b9aff288c0698/html5/thumbnails/64.jpg)
Advantage
• Quantitation.
• This eliminates gel electrophoresis thereby
greatly reducing the risk of contamination
and the introduction of false-positives
5/26/2012 64
![Page 65: Recent Diagnostic Methods for Intestinal Parasitic Infectionssmh.mans.edu.eg/files/pdf/conf/2012/11_Recent_Diagnostic_Methods... · Recent Diagnostic Methods for Intestinal Parasitic](https://reader031.vdocuments.mx/reader031/viewer/2022021821/5af89af87f8b9aff288c0698/html5/thumbnails/65.jpg)
Disadvantage
• It requires expensive equipment.
• Specialized personnel for analysis of the
results
5/26/2012 65
![Page 66: Recent Diagnostic Methods for Intestinal Parasitic Infectionssmh.mans.edu.eg/files/pdf/conf/2012/11_Recent_Diagnostic_Methods... · Recent Diagnostic Methods for Intestinal Parasitic](https://reader031.vdocuments.mx/reader031/viewer/2022021821/5af89af87f8b9aff288c0698/html5/thumbnails/66.jpg)
C-loop-mediated isothermal
amplification (LAMP) • Principle:
- The target sequence is amplified at a
constant temperature of 60 - 65 °C using 4
sets of primers and a polymerase.
- Amplification results in visible turbidity due
to precipitation, which allows easy
visualization by the naked eye,
5/26/2012 66
![Page 67: Recent Diagnostic Methods for Intestinal Parasitic Infectionssmh.mans.edu.eg/files/pdf/conf/2012/11_Recent_Diagnostic_Methods... · Recent Diagnostic Methods for Intestinal Parasitic](https://reader031.vdocuments.mx/reader031/viewer/2022021821/5af89af87f8b9aff288c0698/html5/thumbnails/67.jpg)
Advantage
• Results in higher yields
• Eliminates the need to buy a thermal cycler
• Shortens the reaction time by eliminating
time lost during thermal changes.
5/26/2012 67
The future adoption of LAMP as
a diagnostic tool for parasite
infections in rural endemic
regions shows promise
![Page 68: Recent Diagnostic Methods for Intestinal Parasitic Infectionssmh.mans.edu.eg/files/pdf/conf/2012/11_Recent_Diagnostic_Methods... · Recent Diagnostic Methods for Intestinal Parasitic](https://reader031.vdocuments.mx/reader031/viewer/2022021821/5af89af87f8b9aff288c0698/html5/thumbnails/68.jpg)
Application :
E. histolytica
G. lamblia
Cryptosporidium
Schistosoma mansoni
5/26/2012 68
![Page 69: Recent Diagnostic Methods for Intestinal Parasitic Infectionssmh.mans.edu.eg/files/pdf/conf/2012/11_Recent_Diagnostic_Methods... · Recent Diagnostic Methods for Intestinal Parasitic](https://reader031.vdocuments.mx/reader031/viewer/2022021821/5af89af87f8b9aff288c0698/html5/thumbnails/69.jpg)
5/26/2012 69
![Page 70: Recent Diagnostic Methods for Intestinal Parasitic Infectionssmh.mans.edu.eg/files/pdf/conf/2012/11_Recent_Diagnostic_Methods... · Recent Diagnostic Methods for Intestinal Parasitic](https://reader031.vdocuments.mx/reader031/viewer/2022021821/5af89af87f8b9aff288c0698/html5/thumbnails/70.jpg)
5/26/2012 70
![Page 71: Recent Diagnostic Methods for Intestinal Parasitic Infectionssmh.mans.edu.eg/files/pdf/conf/2012/11_Recent_Diagnostic_Methods... · Recent Diagnostic Methods for Intestinal Parasitic](https://reader031.vdocuments.mx/reader031/viewer/2022021821/5af89af87f8b9aff288c0698/html5/thumbnails/71.jpg)
![Page 72: Recent Diagnostic Methods for Intestinal Parasitic Infectionssmh.mans.edu.eg/files/pdf/conf/2012/11_Recent_Diagnostic_Methods... · Recent Diagnostic Methods for Intestinal Parasitic](https://reader031.vdocuments.mx/reader031/viewer/2022021821/5af89af87f8b9aff288c0698/html5/thumbnails/72.jpg)
5/26/2012 72