CONCENTRATIONS TECHNIQUES IN PARASITOLOGY PRESENTATION.pptxShreyayadav91
INTRODUCTION
Concentration procedure separate parasites from fecal debris and increase the chances of detecting parasitic organisms when these are in small numbers.
If number of organisms in stool specimen is low, examination of a direct wet mount may not detect parasites.
Thus, whenever possible, the stool should be concentrated.
Advantages
Maximizes the numbers of organisms detected which may be too scanty to be seen by direct microscopy alone. Worm eggs, larvae, and protozoan cysts may be recovered.
Disadvantages
Destroys trophozoite stages. Most concentration methods destroy trophozoites stages.
Concentration techniques can be classified as the floatation or sedimentation methods.
Floatation technique
Here solutions with higher specific gravity than the organisms to be floated so that the organisms rise to the top and debris sink to the bottom.
Principle
This technique involves suspending the specimen in a medium of greater density than that of the helminthic eggs and protozoan cysts.
Eggs and cysts float to the top and are collected by placing a glass slides on the surface of the meniscus at the top of the tube.
Floatation Methods includes:
Saturated salt solution technique
Zinc sulfate centrifugal floatation
Sugar floatation technique
Saturated salt solution technique
Procedure:
About half tea spoon (about 4 gm) of fresh stool or preserved stool in a flat bottomed container with 20 ml capacity.
Now, few drops of saturated salt solution (specific gravity 1.20) is added and stirred to make a fine emulsion.
More salt solution is added with stirring throughout to fill the container up to the brim, until a convex meniscus is formed.
A glass slide (3”*2”) is carefully laid on the top of the container so that the center is in contact with the fluid.
This preparation is allowed to stand for 20 minutes after which the glass slide is quickly lifted and examined under microscope after putting coverslip.
Zinc sulfate centrifugal floatation
Procedure
Make a fine suspension of about 1 g of feces in 10 m L of water and strain through gauze to remove coarse particles.
Collect the liquid in a small test tube and centrifuge for 1 minute at 2,500 revolutions per minute. Pour off the supernatant, add water, resuspend, and centrifuge in the same manner, repeating the process, till the supernatant is clear.
Pour off the clear supernatant, add a small quantity of zinc sulfate solution (specific gravity 1.18- 1.2) and resuspend the sediment well.
Add zinc sulfate solution to a little below the brim and centrifuge at 2,500 revolution per minute for 1 minute.
Take samples care fully from the surface, using a wire loop, transfer to slide and examine under the microscope. A drop of dilute iodine helps to bring out the protozoan cysts in a better way.
This technique is useful for protozoan cysts and eggs of nematodes and small tapeworms, but it does not detect unfertilized roundworm eggs, nematode larvae, and eggs of most trematodes and large tapeworms.
CONCENTRATIONS TECHNIQUES IN PARASITOLOGY PRESENTATION.pptxShreyayadav91
INTRODUCTION
Concentration procedure separate parasites from fecal debris and increase the chances of detecting parasitic organisms when these are in small numbers.
If number of organisms in stool specimen is low, examination of a direct wet mount may not detect parasites.
Thus, whenever possible, the stool should be concentrated.
Advantages
Maximizes the numbers of organisms detected which may be too scanty to be seen by direct microscopy alone. Worm eggs, larvae, and protozoan cysts may be recovered.
Disadvantages
Destroys trophozoite stages. Most concentration methods destroy trophozoites stages.
Concentration techniques can be classified as the floatation or sedimentation methods.
Floatation technique
Here solutions with higher specific gravity than the organisms to be floated so that the organisms rise to the top and debris sink to the bottom.
Principle
This technique involves suspending the specimen in a medium of greater density than that of the helminthic eggs and protozoan cysts.
Eggs and cysts float to the top and are collected by placing a glass slides on the surface of the meniscus at the top of the tube.
Floatation Methods includes:
Saturated salt solution technique
Zinc sulfate centrifugal floatation
Sugar floatation technique
Saturated salt solution technique
Procedure:
About half tea spoon (about 4 gm) of fresh stool or preserved stool in a flat bottomed container with 20 ml capacity.
Now, few drops of saturated salt solution (specific gravity 1.20) is added and stirred to make a fine emulsion.
More salt solution is added with stirring throughout to fill the container up to the brim, until a convex meniscus is formed.
A glass slide (3”*2”) is carefully laid on the top of the container so that the center is in contact with the fluid.
This preparation is allowed to stand for 20 minutes after which the glass slide is quickly lifted and examined under microscope after putting coverslip.
Zinc sulfate centrifugal floatation
Procedure
Make a fine suspension of about 1 g of feces in 10 m L of water and strain through gauze to remove coarse particles.
Collect the liquid in a small test tube and centrifuge for 1 minute at 2,500 revolutions per minute. Pour off the supernatant, add water, resuspend, and centrifuge in the same manner, repeating the process, till the supernatant is clear.
Pour off the clear supernatant, add a small quantity of zinc sulfate solution (specific gravity 1.18- 1.2) and resuspend the sediment well.
Add zinc sulfate solution to a little below the brim and centrifuge at 2,500 revolution per minute for 1 minute.
Take samples care fully from the surface, using a wire loop, transfer to slide and examine under the microscope. A drop of dilute iodine helps to bring out the protozoan cysts in a better way.
This technique is useful for protozoan cysts and eggs of nematodes and small tapeworms, but it does not detect unfertilized roundworm eggs, nematode larvae, and eggs of most trematodes and large tapeworms.
Infection characterized by severe local inflammation, usually with pus formation, generally caused by one of the pyogenic bacteria.
Sepsis:The term sepsis covers numerous and diverse pyogenic infections which includes superficial skin infections,wound infections,infection of burns,infection of eyes,peritonitis and abscesses.
Pus is an exudate typically white yellow or yellow formed at the site of inflammation during infection.
Abscesses are localized collection of pus composed of living and dead WBC, components of tissue break down.
70% of tissue infection is mainly caused by
Staphylococcus aureus.
Etymology:
Greek word, pyon meaning pus, genein, meaning to produce
Pus is a fluid composed of : dead & dying WBC, dead & dying bacteria (in bacterial cause of pus),tissue debris, edema, fibrin, lipid and nucleic acid.
Pus cells : it is degranulated wbc, neutrophils.
The body responds to invasion by a wide variety of bacteria by an increased blood supply to the area and by an outpouring of serous fluid and white blood cells.
This is the typical inflammatory response.
The white cells which pass from the blood into the infected tissues attempt to ingest the bacteria (phagocytosis), many cells die and the resultant material consisting of both living and dead white cells (leucocytes or pus cells) and bacteria, together with damaged local tissues and blood proteins, constitutes PUS.
Infections in which pus is produced are known as pyogenic, i.e. pus-producing infections.
Pus may be present as a localised collection deep in the tissues—an ABSCESS, it may be produced on a surface, e.g. the mucosa of the pharynx, the mucosa of the bladder, the méninges, indeed any body surface, it is then known as a PURULENT EXUDATE.
Alternatively infection may spread evenly through the tissues causing a diffuse inflammation :CELLULITIS.
The type of pus production will depend on the organism causing the infection, on the tissue in which the infective process is taking place, and also on the body resistance to the infection.
Although the pyogenic infections have very similar appearances whatever the causative organism, different sites of the body have a tendency to be infected with particular species of bacteria.
Always submit two swabs so that Gram stain can be performed.
Limit swab sampling to wounds that are clinically infected or those that are chronic and are not healing.
To minimize contamination, it is important to cleanse the wound to remove superficial debris by thorough irrigation and cleansing with non-bacteriostatic sterile saline.
If the wound is relatively dry, collect the specimen with two cotton-tipped swabs moistened with sterile non-bacteriostatic saline. Gently roll the swab over the surface of the wound approximately five times, focusing on an area where there is evidence of pus or inflamed tissue.
pseudomonas aeruginosa is one of the leading cause of hospital-associated infection. mainly Pseudomonas is a multi drug resistant bacteria.
they are oxidase positive, non fermenters, strictly aerobic bacteria.
they are pigment producing, pigment can be appreciated on nutrient agar.
Infection characterized by severe local inflammation, usually with pus formation, generally caused by one of the pyogenic bacteria.
Sepsis:The term sepsis covers numerous and diverse pyogenic infections which includes superficial skin infections,wound infections,infection of burns,infection of eyes,peritonitis and abscesses.
Pus is an exudate typically white yellow or yellow formed at the site of inflammation during infection.
Abscesses are localized collection of pus composed of living and dead WBC, components of tissue break down.
70% of tissue infection is mainly caused by
Staphylococcus aureus.
Etymology:
Greek word, pyon meaning pus, genein, meaning to produce
Pus is a fluid composed of : dead & dying WBC, dead & dying bacteria (in bacterial cause of pus),tissue debris, edema, fibrin, lipid and nucleic acid.
Pus cells : it is degranulated wbc, neutrophils.
The body responds to invasion by a wide variety of bacteria by an increased blood supply to the area and by an outpouring of serous fluid and white blood cells.
This is the typical inflammatory response.
The white cells which pass from the blood into the infected tissues attempt to ingest the bacteria (phagocytosis), many cells die and the resultant material consisting of both living and dead white cells (leucocytes or pus cells) and bacteria, together with damaged local tissues and blood proteins, constitutes PUS.
Infections in which pus is produced are known as pyogenic, i.e. pus-producing infections.
Pus may be present as a localised collection deep in the tissues—an ABSCESS, it may be produced on a surface, e.g. the mucosa of the pharynx, the mucosa of the bladder, the méninges, indeed any body surface, it is then known as a PURULENT EXUDATE.
Alternatively infection may spread evenly through the tissues causing a diffuse inflammation :CELLULITIS.
The type of pus production will depend on the organism causing the infection, on the tissue in which the infective process is taking place, and also on the body resistance to the infection.
Although the pyogenic infections have very similar appearances whatever the causative organism, different sites of the body have a tendency to be infected with particular species of bacteria.
Always submit two swabs so that Gram stain can be performed.
Limit swab sampling to wounds that are clinically infected or those that are chronic and are not healing.
To minimize contamination, it is important to cleanse the wound to remove superficial debris by thorough irrigation and cleansing with non-bacteriostatic sterile saline.
If the wound is relatively dry, collect the specimen with two cotton-tipped swabs moistened with sterile non-bacteriostatic saline. Gently roll the swab over the surface of the wound approximately five times, focusing on an area where there is evidence of pus or inflamed tissue.
pseudomonas aeruginosa is one of the leading cause of hospital-associated infection. mainly Pseudomonas is a multi drug resistant bacteria.
they are oxidase positive, non fermenters, strictly aerobic bacteria.
they are pigment producing, pigment can be appreciated on nutrient agar.
Amebiasis is an intestinal (bowel) illness caused by a microscopic (tiny) parasite called Entamoeba histolytica, which is spread through human feces (poop). Often there are no symptoms, but, sometimes it causes diarrhea (loose stool/poop), nausea (a feeling of sickness in the stomach), and weight loss.
Common Terms in Parasitology 22-02-2017 (1).pptxssuser12303b
Common Terms in Parasitology 22-02-2017 (1).pptxCommon Terms in Parasitology 22-02-2017 (1).pptxCommon Terms in Parasitology 22-02-2017 (1).pptxCommon Terms in Parasitology 22-02-2017 (1).pptx
Similar to Diagnostic methods in intestinal parasites (20)
The Gram stain is a fundamental technique in microbiology used to classify bacteria based on their cell wall structure. It provides a quick and simple method to distinguish between Gram-positive and Gram-negative bacteria, which have different susceptibilities to antibiotics
Local Advanced Lung Cancer: Artificial Intelligence, Synergetics, Complex Sys...Oleg Kshivets
Overall life span (LS) was 1671.7±1721.6 days and cumulative 5YS reached 62.4%, 10 years – 50.4%, 20 years – 44.6%. 94 LCP lived more than 5 years without cancer (LS=2958.6±1723.6 days), 22 – more than 10 years (LS=5571±1841.8 days). 67 LCP died because of LC (LS=471.9±344 days). AT significantly improved 5YS (68% vs. 53.7%) (P=0.028 by log-rank test). Cox modeling displayed that 5YS of LCP significantly depended on: N0-N12, T3-4, blood cell circuit, cell ratio factors (ratio between cancer cells-CC and blood cells subpopulations), LC cell dynamics, recalcification time, heparin tolerance, prothrombin index, protein, AT, procedure type (P=0.000-0.031). Neural networks, genetic algorithm selection and bootstrap simulation revealed relationships between 5YS and N0-12 (rank=1), thrombocytes/CC (rank=2), segmented neutrophils/CC (3), eosinophils/CC (4), erythrocytes/CC (5), healthy cells/CC (6), lymphocytes/CC (7), stick neutrophils/CC (8), leucocytes/CC (9), monocytes/CC (10). Correct prediction of 5YS was 100% by neural networks computing (error=0.000; area under ROC curve=1.0).
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APPLICATION OF PHARMACOKINETICS : TARGETED DRUG DELIVERY SYSTEMS By - AKANKSHA ASHTANKAR
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The Central Drugs Standard Control Organization (CDSCO) is India's national regulatory body for pharmaceuticals and medical devices. Operating under the Directorate General of Health Services, Ministry of Health & Family Welfare, Government of India, the CDSCO is responsible for approving new drugs, conducting clinical trials, setting standards for drugs, controlling the quality of imported drugs, and coordinating the activities of State Drug Control Organizations by providing expert advice.
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In India, pharmacovigilance activities are monitored by the Pharmacovigilance Programme of India (PvPI), which works closely with CDSCO to collect, analyze, and act upon data regarding adverse drug reactions (ADRs). Together, they play a critical role in ensuring that the benefits of drugs outweigh their risks, maintaining high standards of patient safety, and promoting the rational use of medicines.
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Muktapishti is a traditional Ayurvedic preparation made from Shoditha Mukta (Purified Pearl), is believed to help regulate thyroid function and reduce symptoms of hyperthyroidism due to its cooling and balancing properties. Clinical evidence on its efficacy remains limited, necessitating further research to validate its therapeutic benefits.
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2. Lab diagnosis in parasitic infections
• Detection of parasite or its stage (trophozoite,
cyst, egg, larva, adult etc.) in different
specimens
• Culture and egg counting
• Serological techniques and immunodiagnosis
• Molecular methods – DNA probes, PCR
• Imaging techniques
2
4. Stool examination
• Collection – wide mouthed, clean, leak-proof
container without contamination of urine, water
or disinfectants
• Time of collection –
- Close to the onset of symptoms
- Before anti-parasitic therapy
- First morning sample
- Free from oil, barium, bismuth
- i/c/o barium enema – after 1 week
4
5. • Three specimens for eggs/larvae, six for
amoebiasis or giardiasis
• Avoid purgatives with oil base
• Liquid stools – examine within 30 minutes
for trophozoites
• Semi-formed stools – in 60 min
• Formed stools – only cysts, examine up to
24 hrs
5
6. Macroscopy:
1. Mucoid bloody stool – Amoebic dysentery,
Intestinal schistosomiasis and invasive
balantidiasis
2. Colour: Dark red= Upper GIT bleeding and
bright red= Lower GIT bleeding
3. Frothy pale offensive stool(Steatorrhoea or fat in
stools)- Giardiasis
6
7. Preservation of stool
• To maintain morphology of protozoa, eggs
• Analysis in reference lab
• Prevent further development i/c/o
helminthic eggs, larva
• Trophozoite motility – lost
7
8. Methods of preservation
1. Formalin
- 10% formalin saline solution
- 3 parts formalin + 1 part stool
- Long shelf life
- Maintains eggs, larvae, cysts
- Not adequate for trophozoites
2. Polyvinyl alcohol (PVA)
- Preserves trophozoites well
- 3 parts PVA + 1 part stool
- Not adequate for helminth eggs, larvae, coccidia, microsporidia
8
9. 3. Merthiolate-iodine-formalin (MIF) solution
- Stains and fixes parasitic cysts, eggs, larvae
- Not suitable for trophozoites
- Preserves specimen for > 1 year
4. Schaudinn’s solution
- Mercuric chloride, ethyl alcohol, glacial acetic acid,
glycerol, D/W
- Trophozoites, cysts – well preserved
- Used for fixing stool smears before permanent staining
(e.g. Trichrome stain)
- Not adequate for helminth eggs, larvae, coccidia,
microsporidia
9
10. Examination of stool
• Macroscopic examination
- Consistency, colour, odour
- Presence of blood, mucus
- Adult helminths – Ascaris, Enterobius, segments of tapeworms
- Trophozoites (liquid stool), cysts (semiformed or formed)
- Coccidian oocysts (Cryptosporidium parvum) – any type of stool
- Helminthic eggs (any stool)
- Blood & mucus – amoebic dysentery, schistosomiasis, invasive
Balantidium coli infection, heavy infection with Trichuris trichiura
- Fat coloured, frothy stool – giardiasis
10
11. • Microscopic exam –
1. Saline wet mounts
2. Iodine wet mounts
3. Smear after concentration
4. Permanent stained smears
i. Iron hematoxylin stain
ii. Trichrome stain
iii. Modified acid fast stain
11
12. Saline wet mount
• Avoid air bubbles
• Thick enough so that
newspaper print can be read
through it
• 1st low power, then high power
• Detects trophozoites, cysts,
eggs, larvae
• Motile trophozoites –
Entamoeba, Giardia, B. coli
Larva of Strongyloides
sterocralis
12
13. Iodine wet mount
• Five times diluted soln of Lugol’s iodine is used.
• Lugol’s iodine contains –
1. Potassium iodide 10 gm
2. Iodine crystals 5 gm
3. Distilled water 100 ml
• Potassium iodide renders elemental iodine soluble in water by
forming triiodide.
• Useful to see nuclear character of cysts, trophozoites
• Glycogen mass – stained brown, chromatin bars – not stained
• Trophozoite motility – not seen
13
21. Eggs that float in saturated salt solution
• Fertilized egg of Ascaris
• Ankylostoma duodenale
• Trichuris trichiura
• Enterobius vermicularis
• Hymenolepis nana
F A T E H
21
23. 23
A B
C
D
E
F
D
G
H
C
G
A– Fertilized egg of Ascaris lumbricoides
B– Giardia lamblia
C- Taenia saginata/solium
D- Unfertilized egg of Ascaris lumbricoides
E—Egg of H. nana
F--- Larva of Strongyloides sterocrolis
G—Egg of Trichuris trichuria
H – Egg of Ancylostoma
Egg of
Enterobius
vermicularis
24. 24
Microscopic Character Amoebic Dysentery Bacillary Dysentery
RBCs In clumps Discrete or in Rouleaux
Pus cells Few Numerous
Macrophages Few Numerous, many have
RBCs and may mimic EH
Eosinophils Present Sacrce
Charcot-Leyden crystals Present Absent
Pyknotic bodies Present Absent
Ghost cells Absent Present
Parasites Trophozoites of EH Absent
Bacteria Many motile bacteria Few or absent
Difference b/w amoebic and bacillary dysentery
25. Quantification of worm burden
• Direct smear egg counting (2 mg) – wet
mount preparation
• Stoll’s method
– 4 gm feces + 56 ml of N/10 NaOH
– Mix to make uniform suspension
– 0.075 ml is removed with pipette to make wet
mount
– Count the eggs and multiply by 200
25
26. How to detect eggs of
Enterobius vermicularis ?
1. Scotch cellulose
adhesive tape method
2. NIH swab
26
27. • Examination of urine
– Trichomonas vaginalis – in centrifuged urine sediment
– Eggs of S. hematobium, Dioctophyma renale
– Microfilaria of W. bancrofti in c/o chyluria
• Examination of sputum
– Eggs of P. westermani, Capillaria aerophila
– Trophozoites of E. histolytica in c/o pleuro-pulmonary
amoebiasis
– Fragments of laminated membrane and free scolices of E.
granulosus in c/o pulmonary hydatid cyst
– A. lumbricoides, A. duodenale, N. americanus, S.
stercoralis – during migratory phase
27
28. • Examination of aspirates
– Liver aspirates – amoebic liver abscess, hydatid cyst
– Proctoscopic aspirates and scrapings – amoebic ulcers
– Duodenal aspirates – G. lamblia, larvae of S. stercoralis
– Aspirates from L.N., spleen, liver, bone marrow, CSF –
African trypanosomiasis (T. brucei), leishmaniasis, Chagas
disease (T. cruzi), toxoplasmosis
• Examination of CSF
– N. fowleri, Acanthmoeba spp., Balamuthia spp.
– T. brucei gambiense and T. brucei rhodeiense –
sleeping sickness
28
29. Biopsy examination
• Skin biopsy – Leishmania, Onchocerca volvulus,
Mansonella, encysted larva of Spirometra
• L.N. biopsy – Trypanosoma brucei, T. cruzi,
Toxoplasma, filarial nematodes
• Muscle biopsy – T. solium, larvae of Trichinella
spiralis, Ancylostoma, Toxocara, sarcocysts of
Sarcocystis lindemanni
• Gastric biopsy – Anisakis simplex
29
30. • Colon and rectum biopsy – eggs of S. japonicum, S.
mansoni, trophozoites of E. histolytica and B. coli
• Brain biopsy – trophozoites of E. histolytica, N. fowleri,
Acanthamoeba spp., Balamuthia spp., cysticercus
cellulose of T. solium, hydatid cyst of E. granulosus
• Corneal scrapings – Acanthamoeba keratitis
(trophozoites)
• Liver biopsy – E. histolytica, L. donovani, E. granulosus
• Lung biopsy – E. histolytica, E. granulosus, P.
westermani, Capillaria aerophila
30
34. 34
Molecular diagnosis
Disease/Parasite Test format
Amoebiasis Nested PCR , Real time PCR,
Biofire film assay
Giardiasis PCR, Biofire film assay
Cryptosporiodiosis PCR, Biofire film assay
Intestinal Taeniasis PCR
Hydatid cyst PCR, PCR-RFLP
Hook worm PCR and Real time PCR
assays
Strongyloides stercoralis Real time PCR
Ascaris lumbricoides Multiplex PCR