EXAMINATION OF SPECIMENS FOR PARASITES

dr. RONALD TAMBUNAN, MKT

PREFACE
LAB DIAGNOSIS IS A BASIC STEP IN THE

EVALUATION OF DISEASE PROCESSES (CONFIRMING A PRESUMPTIVE CLINICAL DIAGNOSIS & PROVIDING EVIDENCE OF AN UNSUSPECTED AGENT OF DISEASE) EXAMINATION OF SPECIMENS INVOLVES 2 STEPS: 1. DETECTION OF PARASITE (SKILLS OF CARRYING OUT PROCEDURE) 2. IDENTIFICATION (SKILLS IN RECOGNIZING) ALSO NEEDS EXPERIENCES TO DISTINGUISH PARASITE FROM ARTIFACTS (FALSE POSITIVE/NEGATIVE) RELIABLE PARASITOLOGY SERVICE: NEED APPROPRIATE AND ADEQUATE FACILITIES,

PREFACE (contd.)
THE MOST BASIC ITEM:

1.

2. 3. 4.

5.
6.

A MICROSCOPE EQUIPPED WITH A SUITABLE LIGHT SOURCE & CLEAR, CLEAN LENSES AN OCULAR MICROMETER & A CALIBRATING MEANS A CENTRIFUGE AN INCUBATOR A DRYING OVEN A MEANS OF STERILIZING GLASSWARE & INSTRUMENTS

PREFACE (contd.)
SIZE IS AN IMPORTANT CHARACTERISTIC OF

ALL LIVING THINGS MICROSCOPE MAGNIFIES THE SIZE OF OBJECTS & WITH THE SEVERAL LENS COMBINATIONS, OBJECTS ARE SEEN AT SEVERAL DIFFERENT MAGNIFICATIONS FOR RECOGNIZING & DESCRIPTING PARASITES & THEIR VARIOUS STAGES, A MICROSCOPE MUST BE PROVIDED WITH A CALIBRATED MICROMETER SCALE

EXAMINATION OF FECES
A FECAL EXAMINATION INVOLVES RECOGNIZING

& RECORDING : 1. GROSS ELEMENTS 2. DISTINCTIVE PATHOLOGICAL ODORS OR COLORS 3. UNUSUAL QUANTITIES OF NORMAL OR ABNORMAL ELEMENTS (BLOOD, CELLULAR EXUDATE, TISSUE FRAGMENTS, CHARCOTLEYDEN CRYSTALS, & UNDIGESTED FOOD) 4. RESIDUES OF MEDICATIONS (MINERAL OIL, BISMUTH, IRON, MAGNESIUM, OR BARIUM) 5. DETECTING THE PRESENCE OR ABSENCE OF PARASITES

EXAMINATION OF FECES (contd.)

STOOL SPECIMENS USUALLY ARE

DESCRIBED WITH REFERENCE TO 3 QUALITIES: 1. CONSISTENCY 2. COLOR 3. ELEMENTS AN ODOR WOULD ASSIST IN INTERPRETING THE OTHER FINDINGS A TERM USED IN DESCRIBING AN ODOR IS FOUL-SMELLING (SUBJECTIVE)

STOOL CLASSIFICATION & CODE

CONSISTENCY 1. HARD, RESIST PUNCTURE 2. FORMED, CAN BE PUNCTURED 3. SOFT, CAN BE CUT WITH APPLICATOR 4. MUSHY, CAN BE RESHAPED 5. LOOSE, SHAPE TO CONTAINER 6. DIARRHEIC, FLOWS 7. WATERY, POURS 1. 2. 3. 4. 5. 6. 7. 8. COLOR BLACK DARK BROWN BROWN PALE BROWN YELLOW GREEN CLAY OTHER* ELEMENTS 1. PULP & FIBER NEARLY PURE 2. CONSPICUOUSLY FIBROUS 3. FIBER MODERATE TO SCANTY 4. PURE COLLOIDAL FECES 5. FECES WITH SCANTY MUCUS 6. FECES WITH MUCH MUCUS 7. MUCUS WITH SCANTY FECES+ 8. OTHER (BLOOD, BARIUM)

STOOL CLASSIFICATION & CODE (contd.)

*COLOR, SUCH AS BLUE, RED, OR PURPLE, CAN INDICATE SPECIAL MEDICATION +A SPECIMEN CONTAINING GROSS BLOOD & MUCUS, WITH OR WITHOUT PUS, WOULD BE RECORDED AS DYSENTRIC & WOULDNT BE CODED FOR CONSISTENCY OR COLOR

COLLECTION OF THE SPECIMEN

COLLECTED IN A CLEAN CONTAINER

WITHOUT CONTAMINATION WITH URINE, WATER, etc. AMOUNT: WHOLE STOOL, SERIES OF STOOLS OVER A SPECIFIED PERIOD, MILIGRAMS SCRAPED FROM MEDICAL GLOVE USED AFTER RECTAL EXAMINATION ROUTINE: 1. 2 5 GRAM, PROTECTED FROM DRYING 2. FREE OF OIL, BARIUM & BISMUTH 3. BETTER TAKEN FROM NORMALLY PASSED STOOL THAN PURGATION OR ENEMA

COLLECTION OF THE SPECIMEN (contd.)

A FORMED STOOL CAN BE KEPT OVERNIGHT

AT MODERATE OR LOW ROOM TEMPERATURE DIARRHEIC & DYSENTRIC STOOLS SHOULD BE EXAMINED PROMPTLY OR PRESERVED OTHERS SHOULD BE EXAMINED IN 3 4 HRS PRESERVATION METHODS: 1. REFRIGERATOR 2. CHEMICALLY (FORMALIN, POLYVINYL ALCOHOL (PVA), MERTHIOLATE-IODINEFORMALIN (MIF), SCHAUDINNS FIXATIVE)

CHOICE OF METHOD
DIRECT EXAMINATION

DIRECT SALINE SMEAR 2. KATO THICK SMEAR PERMANENT, STAINED, FRESH FECAL SMEAR 1. IRON-HEMATOXYLIN-STAINED SMEAR 2. TRICHROME-STAINED SMEAR 3. PVA SMEAR 4. SCHAUDINNS PRESERVED SPECIMEN 5. MIF PRESERVED SPECIMEN
1.

CONCENTRATION METHODS FOR FECAL SPECIMENS

GOAL: SEPARATE CYSTS OF PROTOZA &

EGGS OF HELMINTHS FROM OTHER ELEMENTS OF FECES HOW TO ACHIEVE: SEDIMENTATION, FLOTATION, OR COMBINING OF THE 2 SEDIMENTATION: SUSPENDING THE SPECIMEN IN WATER OR AN AQUEOUS SOLUTION OR BY CENTRIFUGATION FLOTATION : SUSPENDING THE SPECIMEN IN A MEDIUM OF GREATER DENSITY THAN CYSTS & EGGS

CONCENTRATION METHODS FOR FECAL SPECIMENS (contd.)

SEDIMENTATION IN WATER:

THE RESULT DEPENDS ON THE ACCURACY OF TIMING OF SEDIMENTATION 2. SKILLS 3. EFFICACY OF THE STRAINING PROCEDURES THUS IT ISNT GENERALLY USED MODIFICATION METHOD FOR CONCENTRATING: USE OF ETHER (ABSORBED BY FECAL DEBRIS AND CONSEQUENTLY BECOMES LIGHTER THAN WATER) ETHER: FLAMMABLE & EXPLOSIVE
1.

CONCENTRATION METHODS FOR FECAL SPECIMENS (contd.)

FORMALIN-ETHER SEDIMENTATION

ACID-ETHER SEDIMENTATION
FLOTATION METHODS

BRINE FLOTATION 2. ZINC SULFATE CENTRIFUGAL FLOTATION

1.

SCHISTOSOME EGGHATCHING
DETECTING EGGS IN FECES

DETECTING MIRACIDIA IN FECED, URINE,

MACERATED TISSUES, OR POST MORTEM INTESTINAL SCRAPPINGS CONCENTRATING TECHNIQUE: USING A SIDEARM FLASK OR ERLENMEYER FLASK & TEST TUBE WITH FILTER

QUANTITATIVE DIAGNOSIS
TECHNIQUES FOR BOTH QUANTITATIVE &

QUALITATIVE: DIRECT SALINE & THE CELOPHANE-COVERED THICK SMEAR OTHER TECHNIQUE: UNRELIABLE FOR EGG COUNTS BECAUSE THE YIELDS ARE VARIABLE, DUE TO DIFFERENCES IN THE CHARACTER OF SPECIMENS & THE PROFICIENCY OF TECHNICIANS QUANTITATIVE TECHNIQUE: 1. DIRECT-SMEAR EGG COUNTS 2. DILUTION EGG COUNTS 3. THICK-SMEAR EGG COUNTS 4. DILUTION-FILTRATION EGG COUNTS

FECAL CULTURE FOR DIAGNOSIS OF NEMATODE LARVAE

TEST-TUBE CULTURES (HARADA-MORI

CULTURES) CHARCOAL CULTURES FILTER PAPER SLANT CULTURE (LITTLE, 1966) WORMS MIGRATING FORM THE ANUS: PRESERVED IN ALCOHOL (70%) OR FORMALIN (5 10%) ANAL SCRAPINGS & SWABS: E. HISTOLYTICA & ENTEROBIUS RECOVERY OF WORMS FROM STOOLS: 1. STRAINING AN AQUEOUS SUSPENSION OF THE SPECIMEN THROUGH WIRE SIEVES

OTHER SPECIMENS
URINE 1. TRICHOMONAS VAGINALIS 2. SCHISTOSOMA HAEMATOBIUM 3. STRONGYLOIDES (RARE) SPUTUM & GASTRIC WASHINGS 1. TROPHOZOITES OF E. HISTOLYTICA 2. ECHINOCOCCUS GRANULOSUS 3. EGGS OF P. WESTERMANI 4. EGGS OF STRONGYLOIDES 5. ASCARIS 6. NECATOR 7. ANCYLOSTOMA

OTHER SPECIMENS (contd.)

BLOOD

1.
2. 3. 4. 5.

MALARIA BABESIOSIS AFRICAN TRYPANOSOMIASIS FILARIASIS CHAGAS DISEASE

PROTOZOA IN BLOOD
THIN FILM

THICK FILM
MICROFILARIAE

HEMATOXYLIN-STAINED BLOOD FILM 2. CONCENTRATION OF MICROFILARIAE (KNOTTS METHOD) 3. MEMBRANE FILTRATION

1.

EXAMINATION OF ASPIRATE
PROCTOSCOPIC ASPIRATE: NONMOTILE

TROPHOZOITE OR CYSTS OF E. HISTOLYTICA DUODENAL ASPIRATION: G. LAMBLIA, S. STERCORALIS, FASCIOLOPSIS BUSKI ASPIRATION FROM LIVER & LUNG LESIONS ASPIRATES FROM LYMPH NODES, SPLEEN, LIVER, BONE MARROW, & SPINAL FLUID: AFRICAN TRYPANOSOMIASIS, VISCERAL LEISHMANIASIS (KALA-AZAR), CHAGAS DISEASE & TOXOPLASMOSIS

BIOPSY MATERIAL
SKIN

AMEBIASIS CUTIS 2. CUTANEOUS LEISHMANIASIS 3. O. VOLVULUS 4. MANSONELLA STREPTOCERCA & MANSONELLA OZZARDI LYMPH NODE 1. AFRICAN TRYPANOSOMIASIS 2. KALA-AZAR 3. CHAGAS DISEASE 4. TOXOPLASMOSIS
1.

BIOPSY MATERIAL (contd.)

MUSCLE

TRICHINELLA LARVAE 2. T. SOLIUM CYSTICERCUS 3. ANCYLOSTOMA LARVAE 4. TOXOCARA LARVAE COLON & RECTUM 1. S. MANSONI 2. S. JAPONICUM 3. S. HAEMATOBIUM
1.

CULTURE METHOD

CULTIVATION OF PROTOZOA
AMEBAE & OTHER INTESTINAL PROTOZOA

1.
2. 3. 4. 5.

BALAMUTHS MONOPHASIC MEDIUM BOECK & DRBOHLAVS DIPHASIC MEDIUM AXENIC CULTIVATION OF E. HISTOLYTICA AXENIC CULTIVATION OF GIARDIA IN VITRO EXCYSTATION OF GIARDIA

CULTIVATION OF PROTOZOA (contd.)

GENITOURINARY PROTOZOA: T. VAGINALIS

TRUSSEL & JOHNSONS MEDIUM 2. SIMPLIFIED TRYPTICASE SERUM (STS) MEDIUM CEREBROSPINAL PROTOZOA: NAEGLERIA & ACANTHAMOEBA 1. CULBERTSONS MEDIUM 2. WILLAERTS MEDIUM
1.

CULTIVATION OF PROTOZOA (contd.)

BLOOD & TISSUE PROTOZOA

1.
2. 3. 4. 5. 6.

NNN (NOVY & MacNEAL) MEDIUM HOCKMEYERS MEDIUM WEINMANS MEDIUM TOBIES DIPHASIC MEDIUM CULTIVATION OF MALARIA PARASITES (BASS & JOHNS) USING RPMI 1640 MEDIUM XENODIAGNOSIS

SPECIMENS TRANSPORT
STUARTS TRANSPORT MEDIUM

AMIES TRASNPORT MEDIUM

CARY-BLAIR MEDIUM

STAINING
GRAM

ZIEHL-NEELSEN
HAEMATOXYLIN & EOSIN (H&E) PAPANICOLAOU PERIODIC ACID-SCHIFF (PAS) ROMANOWSKY SILVER SUDAN CONKLINS

STAINING (contd.)
ACRIDINE ORANGE BISMARCK BROWN CARMINE COOMASSIE BLUE CRYSTAL VIOLET

DAPI
EOSIN ETHIDIUM BROMIDE ACID FUCHSINE HAEMATOXYLIN IODINE

STAINING (contd.)
MALACHITE GREEN METHYL GREEN METHYLENE BLUE NEUTRAL RED NILE RED

OSMIUM TETRAOXIDE
RHODAMINE SAFRANIN

PHOSPHOTUNGSTIC ACID
RUTHENIUM TETROXIDE