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2.1B DIAGNOSTIC PARASITOLOGY

PARASITOLOGY

DIAGNOSTIC PARASITOLOGY

GENERAL PRINCIPLES

1. Consider the possibility of parasitic infection in diagnosis. “Think of it.”

2. Take a complete history of travel, includes the recent and past. “Where

have you been? “

3. Should have knowledge of the parasite biology.

4. Consider the facilities in deciding what lab tests are necessary.

5. Finally, interpret the result of the tests in the light of the clinical picture,

then treat the disease not the tests

METHODS OF DIAGNOSIS OF PARASITIC INFECTIONS

Demonstration or Identification of parasites

developmental stages

eggs, larvae and adults

cysts, oocysts and trophozoites

trophozoites, schizonts and gametocytes

Detection of host immune response to the parasites

antigen

and antibody

different immunodiagnostic tests

THINGS TO CONSIDER IN DIAGNOSTIC PARASITOLOGY

Proper collection and transport of sample

Processing of specimen prior to examination

Skills of the laboratory personnel

Quality of equipments

SPECIMENS FOR PARASITIC EXAMINATION

Stool

Blood

Sputum

Urine and Genital Secretions

Tissue aspirate and tissue biopsy material

Eye Specimens

Cerebrospinal Fluid (CSF)‏

Mouth Scrapings and Nasal Discharge

Skin Snips

FACTORS TO CONSIDER IN COLLECTION & PROCESSING OF STOOL SAMPLES

Intake of drugs/substances

Intake of anti-malarial/antibiotics

Collect in tight fitting lid container

– Contamination w/ urine, water and soil

– Amount of sample

– Label

Time frame (age of stool sample)‏

Preservation

– Preservatives

– Temporary storage

– Never freeze or keep the sample in an incubator

COLLECTION: FIXATIVES

Formalin

PVA (Mercuric oxide)‏

Sodium Acetate formalin

Modified PVA (copper and zinc sulfate)‏

Alternative Single-Vial systems (non-toxic fixatives)‏

PROCESSING OF STOOL SAMPLE

Method of examination

• Macroscopic or Physical

– Consistency

– Color

• Microscopic

– Stages of parasites

MACROSCOPIC

CONSISTENCY COLOR

• Hard • Soft • Mushy • Loose • Diarrheic • Watery, liquid • Formed • Semiformed

• Dark brown • Black • Brown • Pale brown • Clay • Yellow • Red-brown • Green, others

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2.1B DIAGNOSTIC PARASITOLOGY Parasitology

CONSISTENCY

DISTRIBUTION OF PROTOZOA IN RELATION TO CONSISTENCY

MICROSCOPIC

Different stages of the parasites

Elements (WBC,RBC, macrophages and others)‏

Normal fecal constituents like fat globules, fibres and others

MICROSCOPIC TECHNIQUES

Direct fecal smear (DFS)‏

Concentration techniques

Permanent stain

Culture method

Egg counting

Cellophane Tape preparation

DIRECT WET PREPARATION

UNSTAINED STAINED

– NSS – Use to detect motile

protozoan parasite – Dis. - low yield of

parasites

– Lugol's solution – Enhance the details of

protozoan cysts – Dis. - Iodine kills

trophozoites

CONCENTRATION TECHNIQUES

SEDIMENTATION

– Formalin Ethyl Acetate – Provides good recovery

– Easy to perform – Dis. - contains more fecal

debris

FLOTATION

– Zinc sulfate – Yields cleaner preparation – Easy to examine – Dis. - some parasites will

be missed

PERMANENT STAINS

TRICHROME IRON HEMATOXYLIN

– Widely used – Long shelf life – Easy to perform – Differentiates the color of

parasites and background

– Time consuming – Reveals excellent

morphology of the intestinal protozoans

– Nuclear details are stained clearer and sharper than trichrome

OTHER PROCEDURES

STOOL CULTURE METHOD EGG COUNTING

- Harada-Mori or Test tube culture method

- Kato Katz - Kato Thick smear

OTHER INTESTINAL SPECIMENS

Duodenal materials

Sigmoidoscopy materials

Sample from Cellophane Tape preparation

DUODENAL MATERIALS SIGMOIDOSCOPY MATERIALS

– Collected by nasogastric intubation

– Or enteric capsule test (Enterotest)‏

– Giardia intestinalis trop, I. Belli, Cryptosporidium spp, S. stercoralis, and eggs of F. hepatica or C. sinensis

– Helpful for detecting E.histolytica

– Obtained by aspiration or scraping fr ulcers

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2.1B DIAGNOSTIC PARASITOLOGY Parasitology

CELLOPHANE TAPE

BLOOD

COLLECTION

– Aseptic technique

– whole blood fr fingertip or earlobe

– or venipuncture specimen w/ anticoagulant (EDTA)‏

– Time of collection

– L. donovani, Trypanosoma spp., Plasmodium and

Babesia specie

PROCESSING

– Thick and thin smear

– Permanent stains

– Knott method

– Buffy coat slides

– Culture using NNN medium

CSF AND OTHER STERILE FLUID

Collect aseptically

Examined promptly

Wet preparation and/or permanent stains

N. fowleri, Acanthamoeba spp, Trypanosoma spp,T. Gondii, T.

solium cysticercus larvae and Echinococcus spp

Culture on non-nutrient agar if Naeglaria and Acanthamoeba

are the suspected pathogens

TISSUE & BIOPSY SPECIMENS

– Recommended for the recovery of many parasites

includes intracellular organisms

– Surgical removal of the specimens for Leishmania

spp and T. gondii

– Liver abscess for E. histolytica

– Trypanosoma spp and T. spiralis

SPUTUM

– Collected and tested from patients suspected of

being infected by the fluke. P. westermani

– S. stercolaris hyperinfection

– E. histolytica and E. gingivalis

– A. lumbrecoides and hookworm

– Examine directly using wet preps and permanent

stain

URINE & GENITAL SECRETIONS

– S. haematobium eggs

– Microfilariae fr patients w/ heavy infection

– Sample should be centrifuged and examine the

sediment

– T. vaginalis troph (urine and genital secretions)‏

– Genital secretions are collected in a swab or in a

cup, examined by wet preps to demonstrate motile

trophozoite

EYE SPECIMENS

CORNEAL SCRAPING FOR A. KERATITIS

• Culture

• Stained with calcofluor using fluorescent

microscope

• Histologic method

• Loa loa and T. gondii

MOUTH SCRAPINGS AND NASAL DISCHARGE

– Mouth scrapings for detection of E. gingivalis and T.

tenax

– Recovery of N. fowleri fr nasal samples

– Both are collected on a swab or in a cup

– Wet preps

SKIN SNIPS

ONCHOCERCA VOLVOLUS

• Two techniques of collection:

– Scleral punch into skin

– Using razor blade with w/c small cut into skin is

made

– Put sample into saline solution and incubate for

30min

– Examine sample and look for the jerky movement of

the microfilariae

OTHER LAB METHODS

CULTURE ANIMAL INOCULATION

– E. histolytica – T. vaginalis

– Leishamnia spp. – T. cruzi – T. gondii

– Specimens fr patients suspected of suffering fr Leismania, Trypanosoma and Toxoplasma

– Collected aseptically – Mice, guinea pigs and

hamsters

XENODIAGNOSIS

– Chaga's disease – T. cruzi

RECENT ADVANCES IN DIAGNOSTIC PARASITOLOGY

MICROSCOPY

IMMUNODIAGNOSIS

– Rapid diagnostic test

MOLECULAR DIAGNOSIS

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2.1B DIAGNOSTIC PARASITOLOGY Parasitology

MICROSCOPY

FLUORESCENT

Trophozoites of P.

falciparum

stained with AO in the

QBC

UV fluorescence method.

Trophozoites of P.

falciparum stained with

BCP in the fluorescence

method.

IMMUNODIAGNOSIS

• Immunofluorescent Assay

• Enzyme Linked Immunosorbent Assay

• Indirect Hemagglutination Assay

• Radioimmunoassay

• Dot blot

RAPID DIAGNOSTIC TESTS

RDT’S FOR MALARIA RDT’S FOR MALARIA

HRP based assay

pLDH based assay

RDT’S FOR OTHER PARASITES

FAST ( fast agglutinating screening test) for visceral leishmania

LAT (latex agglutination test) for Taenia solium

Immunochromatographic test for filaria

MOLECULAR DIAGNOSIS

DNA-BASED METHOD

• Detection of parasite using DNA-probe

– Hybridization assays

• Detection of specific nucleic acid

sequences

– PCR- based assays

– Amebiasis, used to differentiate

E. histolytica and E.dispar

• Malaria, filariasis, leishmaniasis,

trypanosomiasis, and onchocerciasis for

both method

• Toxoplasmosis, taeniasis and

trichomoniasis for PCR only

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