Lab Discussion-1.pdf

Diagnostic Parasitology MedBio4: Medical Parasitology 1st Semester, A.Y. 24-25  Laboratory Diagnosis A parasitology laboratory should be able to: confirm a clinical impression that the condition has a parasitic nature; rule out differential diagnoses; aid a clinic...

Diagnostic Parasitology MedBio4: Medical Parasitology 1st Semester, A.Y. 24-25  Laboratory Diagnosis A parasitology laboratory should be able to: confirm a clinical impression that the condition has a parasitic nature; rule out differential diagnoses; aid a clinician in the choice of ??? proper medication; and help in monitoring the effect of a treatment regimen.  Laboratory Diagnosis Example: Dysentery is a disease that is characterized by diarrhea and contains both blood and mucus (macroscopic examination). Diagnosis through clinical signs and Stool stated symptoms, but confirmed by microscopic examination.  Laboratory Diagnosis Example: Dysentery is a disease that is characterized by diarrhea and Bacillary dysentery Amoebic dysentery contains both blood and mucus (macroscopic examination). Feces: Feces: no forms of trophozoites bacteria are present Diagnosis through clinical signs identified as and stated symptoms, but trophozoites confirmed by microscopic examination.  Laboratory Diagnosis factors that generate reliable results: proper collection, handling, and processing of specimens prior to examination the skill of the laboratory analyst (examiner) the quality of equipment used in the examination.  Laboratory Diagnosis There are two ways of diagnosing parasitic infections: Taenia saginata 1. Demonstration of parasite or parasite components - definitive diagnosis egg scolex mature proglottid 2. Detection of host immune response (humoral) to the parasites - presumptive evidence of infection  Laboratory Diagnosis factors that generate reliable results: proper collection, handling, and processing of specimens prior to examination the skill of the laboratory analyst (examiner) the quality of equipment used in the examination.  Laboratory Protocols in Specimen Collection MedBio4: Medical Parasitology 1st Semester, A.Y. 24-25  Specimen Collection Protocols for parasitologic study depend on type of sample and examination When this part is compromised, the results can be affected dramatically Proper specimen selection and processing are crucial to parasite recovery Collection and Processing: Environmental Samples a. Water Samples Cryptosporidium sp. and Giardia sp. Procedures: EPA Method 1623 Modified Method 1623 Elution – the process of extracting one material from another by washing with a solvent b. Soil Samples Protozoan and helminths hookworms (filariform larvae) Procedure: Collect using auger/spade - 20 cm deep Put in sterile plastic bags Sent to lab for immediate processing Amphizoic - organism that can exist either as a parasite or as free-living organism Collection and Processing: Human Biological Samples a. Urine Trichomonas vaginalis (trophozoites) and Schistosoma haematobium (eggs) Terminal urine specimen (last 10- 20mL) - 24-hrs Excretion of eggs - highest around midday In case of delay: 0.5 mL of 10% formalin to prevent eggs from hatching Trichomonas vaginalis a. Urine Microfilariae can sometimes be found in the urine of patients with a heavy filarial infection. Schistosoma haematobium b. Stool Most common (intestinal in origin) 3-5 grams in sterile containers Prevent contamination w/ urine Immediate processing In case of delay: Preservatives b. Stool - Macroscopic Examination Consistency: Watery/Loose - diarrheic stool (trophozoites) Formed/Hard or Semi-formed/Soft - cysts, helminth eggs, and larvae b. Stool - Macroscopic Examination Presence of: Tapeworm proglottids, and adult worms Blood, mucus, and pus Fresh (bright red) - acute lower GI bleeding or irritation Bloody w/ mucus - amebic ulceration in the colon or large intestine b. Stool - Microscopic Examination 1. Ocular Micrometer Calibration 2. Processing of Stool Samples 3. Examining the mounted slides microscopically Egg of Trichuris sp. measured with an ocular micrometer. b. Stool - Microscopic Examination 1. Ocular Micrometer Calibration size difference (microns) 2. Processing of Stool Samples DFS Kato-thick Concentration Techniques 3. Examining the mounted slides microscopically Egg of Trichuris sp. measured with an ocular micrometer. c. Perianal swabs Enterobius vermicularis scotch tape tongue depressor glass slide PPE (gloves and mask) - Highly infective Collect early in the day, before taking a bath Collection of E. vermicularis eggs using perianal swab (scotch tape method) c. Perianal swabs Enterobius vermicularis scotch tape tongue depressor glass slide PPE (gloves and mask) - Highly infective Collect early in the day, before taking a bath Collection of E. vermicularis eggs using perianal swab (scotch tape method) d. Sputum Paragonimus westermani Lung migration (larval stages of ASH): Ascaris lumbricoides Strongyloides stercolaris Hookworms E. histolytica (trophozoite) Echinococcus granulosus (Scolex) Collect early morning and put deep sputum Procedure for deep sputum: rinse mouth with water and expel the sputum by breathing and in sterile containers coughing at 2-minutes interval Why deep sputum? High sens/concentrated d. Sputum Direct wet mount using saline or iodine N-acetyl-L-cysteine (mucolytic) Centrifugation Paragonimus westermani egg e. Aspirates Body fluids smeared and stained on glass slides Duodenal - Giardia lamblia, Strongyloides stercoralis Nasogastric intubation Entero Test or Nasogastric intubation Liver and Lung - E. histolytica Entero test or String test f. Blood Leishmania donovani and Trypanosoma spp. Plasmodium and Babesia spp. Trypanosoma cruzi Thick and thin blood smear using Giemsa stain Serum for serologic tests to detect antibodies in a patient g. CSF Naegleria fowleri Acanthamoeba spp. (also obtained in corneal scrapings) Cultured on non-nutrient agar seeded Naegleria fowleri with Escherichia coli. Other pathogen recovered form central nervous system Trypanosoma spp. (trypomastigote) Toxoplasma gondii Taeniasolium solium cysticercus larvae Acanthamoeba spp. g. CSF Lumbar Puncture Naegleria fowleri Centrifuged Examined in wet mount for trophozoites or permanent stained smears Acanthamoeba spp. Laboratory Diagnossis of Parasitic Diseases MedBio4: Medical Parasitology 1st Semester, A.Y. 24-25 Outline 1. Parasitic (morphological) diagnosis Macroscopic Microscopic 2. Immunological diagnosis Antibody detection Antigen detection 3. Molecular diagnosis 4. Culture 5. Animal inoculation (Xenodiagnosis) Morphological Diagnosis Macroscopic and Microscopic  Morphological Diagnosis A. Macroscopic examination Ex. stool specimen examined with the naked eye for: occult blood parasite component color consistency B. Microscopic examination Ex. blood, stool, urine (stained or unstained preparation)  Morphological Diagnosis B. Microscopic examination Elements that may be found in stool specimens White blood cells Polymorphonuclears (PMNs) Eosinophils Macrophages Red blood cells Charcot-Leyden crystals Epithelial cells Eggs of arthropods Fungal spores Elements of plant origin Animal hairs  Morphological Diagnosis Special stains and corresponding parasites Temporary stain Permanent stain in histopathologic slides Giemsa/field Eosin stain Thompson’s stain Trichrome stain Iron-Hematxylin Lugol’s iodine solution stain Modified Sargeaunt’s stain Ziehl-Neelson stain Phenol-Auromine Burrow’s stain method Acridine orange  Permanent Stain PVA-preserved specimen Iron-hematoxylin Stain stains intestinal protozoa Images captured from a trichrome stained smear were submitted helminth egg and larvae may be obscured to DPDx for diagnostic assistance. The smears were made from a good stain for fresh, PVA, or SAF-preserved polyvinyl-alcohol (PVA) preserved fecal specimen, but no other fecal smears patient or specimen information was given. What is your diagnosis? Based on what criteria? What valuable information Wheatley's Trichrome Stain would have been useful if provided? one of the most commonly used stain for intestinal protozoa stain for fresh and PVA-preserved but not w/ SAF-preserved smears Acid-Fast Stain Cryptosporidium, Isospora, Cyclospora  Immunodiagnosis A. Antibody detection serum Ab is produced in response to a particular parasitic infection Ab may persist for a long period of time in the serum after an infection has ended unable to distinguish between past or present infection  Immunodiagnosis A. Antibody detection Microscopy - gold standard However, parasites can be low in numbers during pre-patent and chronic periods of infection, hence, microscopic examination may yield false negative results. Specimen preparation for microscopic examination can also be laborious and tedious when a lot of samples need to be examined during epidemiologic investigations.  Immunodiagnosis Immunodiagnostic techniques are required when: Parasites live in the tissue of internal organ and cannot be easily obtained for examination. Parasites can be found in specimens only in certain stages of infection, e.g., in the acute stage not in the chronic Trichinella spiralis stage.  Immunodiagnosis Immunodiagnosis is of particular value for: South American trypanosomiasis Trichinosis (Chronic stage) Toxoplasmosis African trypanosomiasis (when Toxocarisis parasitaemia is low) Hydatid disease Leishmaniasis Schistosomiasis Filariasis Malaria Amoebic liver abscess  Culture Methods Harada-Mori or the Test Tube Culture Method Hookworm and S. stercoralis presence of rhabditiform larvae distinguish bet. Strongyloides sp. and hookworm development of into filariform stage filariform larvae will generally move downwards Culture Methods  Culture Methods Hemoflagellates: Novy-MacNeal-Nicolle (NNN) NNN slant + 1 drop of blood or ground tissue  Animal Inoculation Xenodiagnosis Lab-cultured bug/reduviid bug/kissing bug takes a blood meal on the patient The bugs are dissected after 20-25 days to examine for T. cruzi epimastigote  Molecular Diagnosis Microscopic examination is still considered the “gold standard” for the diagnosis of parasitic diseases. The stool specimen can be analyzed using molecular techniques such as polymerase chain reaction (PCR). PCR amplified fragments can be analyzed by: using restriction fragment length polymorphisms (RFLP) or DNA sequencing if further characterization is needed. Examination of Stool Sample  Laboratory Diagnosis Ova and Parasite (O&P) Examination stool eggs, larvae, adults, trophozoites, cysts urine blood sputum cerebrospinal fluid tissue aspirates tissue biopsies orifice swabs  What is Feces? Feces (Stool) waste product or substance formed in the digestive tract and excreted out through the rectum (rear end). Why is it called feces? Feces comes from the Latin word "faex,“ It means "dregs." Dregs means the most undesirable part. Feces are also known as stool. Stool comes from the Anglo Saxon word "stol," which means "seat  Examination of Stool Purpose of examining stool: To identify intestinal parasitic infection associated Severe anemia especially in pregnant & child Series ill-health Persistent diarrhea Weight loss, malabsorbtion Impairment of development etc To identify chronic infection with serious complication if untreated To identify parasitic causes of blood and mucus To assist in surveillance &control of parasitic infection  Examination of Stool A. Macroscopic examination Ex. stool specimen examined with the naked eye for: occult blood parasite component color consistency B. Microscopic examination Ex. blood, stool, urine, mucus (stained or unstained preparation)  Examination of Stool B. Microscopic examination Elements that may be found in stool specimens White blood cells Polymorphonuclears (PMNs) Eosinophils Macrophages Red blood cells Charcot-Leyden crystals Epithelial cells Eggs of arthropods Fungal spores Elements of plant origin Animal hairs  Biosafety Potential risks with stool specimens includes: ingestion of eggs or cysts, skin penetration by infective larvae, and infection by non-parasitic agents found in stool and biologic fluids. These risks can be minimized: by adopting universal precautions as well as standard microbiological laboratory practices (Biosafety Level 2). Collection of Stool or Fecal Sample clean, wide-mouthed containers with tight fitting lid retain moisture, prevent spillage, and contamination 3 specimens within 10 days (Amebiasis - 6 in 14 days)  Collection of Stool or Fecal Sample After treatment, stools are rechecked: protozoan: 3 - 4 weeks helminth: 1 - 2 weeks Taenia sp.: 5 - 6 weeks How many stool samples should be collected when following the typical O&P collection protocol? A. 1 B. 2 C. 3 D. 4  Important factors in stool analysis A. Intake of drugs/medicinal All of these drugs have been substances found to leave crystalline 1. antacids residues. 2. anti-diarrheals 3. barium - samples should be collected a 4. bismuth week after 5. laxatives A barium enema is a type of x-ray that allows your doctor to see your colon and rectum. It is also called a colon x-ray or lower GI exam. Barium enemas can help diagnose changes to your large intestine, such as your colon and rectum.  Important factors in stool analysis B. Intake of antibiotics decreases the number of protozoans for several weeks C. Amount of stool dictated routine stool examination - by techniques thumb/walnut size (formed) or 5-6 tbsp (watery) D. Contamination w/ toilet destroy trophozoites and may water, urine, or soil contain free-living organisms  Important factors in stool analysis E. Age of stool sample Liquid specimen should be examined within 30mins and formed within 1 hour F. Delay in examination may ensure that parasites are present require preservation in identifiable stage E. Temporary storage of fecal prolonged refrigeration = samples in refrigerator (3-5℃) desiccation trophozoites are killed eggs, cysts survive Important factors in stool analysis  Gross/Macroscopic - Color Normal: Adult: brown New born infants: Black (meconium) Breast feed infants: scrambled egg Infant feed on animal milk: “curd like” CONTENT WARNING: POOP PICS AHEAD!!!  Gross/Macroscopic - Color Meconium: Baby’s first stool First stool a baby will pass thick Green, tar-like substance Lines the intestines of the fetus First bowel movement within a few hours after birth. Transitional Stool - Stage One Newborn slowly begins to pass the meconium after birth Meconium will begin to change in consistency Slightly lighter in color than meconium.  Gross/Macroscopic - Color Transitional Stool - Stage Two Stool is lighter in color and slightly less thick than meconium. Transitional Stool - Stage Three Much lighter and thinner than meconium. Occurs just before regular stooling begins  Gross/Macroscopic - Color Breastfed Stool Yellow Runny Small seed like objects in the stool Often called baby poop mustard  Gross/Macroscopic - Color Changes in the color, consistency, and frequency of bowel movements is known as a "change in bowel habits.’’ In some cases, an unusual stool color is harmless and can be attributed to a particular food or medication Changes in stool color that persist can be a serious matter and should always be investigated by a physician.  Gross/Macroscopic - Color Abnormal: Clay or white absence of bile pigment (bile obstruction) or diagnostic study using barium ´Black or tarry drug (e.g., iron), bleeding from upper gastrointestinal tract (e.g.,stomach, small intestine), diet high in red meat and dark green vegetables (e.g., spinach)  Gross/Macroscopic - Color Abnormal: Red bleeding from lower gastrointestinal tract (e.g., rectum), hemorrhoids some foods red gelatin, tomato juice or soup large amounts beets Pale malabsorption of fats, diet high in milk and milk products and low in meat  Gross/Macroscopic - Consistency 1 - resists puncture 2 - can be punctured 3 - can be cut with applicator 4 - can be reshaped 5 - shaped into container 6 - can flow 7 - can pour  Gross/Macroscopic - Consistency Normal: Formed, soft, semisolid or mushy Abnormal: Hard, dry, constipated stool Dehydration, decreased intestinal motility resulting from lack of fiber in diet, lack of exercise, emotional upset, laxative abuse Diarrhea Increased intestinal motility (e.g., irritation of the colon by bacteria) Cleary watery, loose mixed with mucus and blood  Gross/Macroscopic - Consistency Classification of the form, (appearance in a toilet) of feces into seven groups. The form of the stool depends on the time it spends in the colon. Chart breakdown Types 1 and 2 indicate constipation; Types 3 and 4 are usually the most comfortable to pass, Types 5-6 tend to be associated with urgency, while Type 7 is diarrhea.  Gross/Macroscopic - Shape Normal: cylindrical (contour of rectum) about 2.5 cm (1 inch) in diameter in adults Abnormal: narrow, pencil-shaped, or stringlike stool obstructive conditional of the rectum  Gross/Macroscopic Amount Normal: varies with diet about 100 to 400 g per day Size Normal: healthy piece of feces is about one foot long.  Gross/Macroscopic Frequency Normal: three times a day to once every three days. average person poops about once a day. Odor Normal: aromatic, affected by ingested food and person’s own bacterial flora Abnormal Pungent (infection, blood, sloughed tissue)  Gross/Macroscopic - Odor What makes feces smell so bad? The distinctive odor of feces is due to bacterial action. Specifically, the bacteria produce various compounds and gases that lead to the infamous smell of feces. Gut flora produce compounds such as indole, skatole, and thiols (sulfur containing compounds), as well as the inorganic gas hydrogen sulfide The bad smell of feces will usually be reduced by eating more natural foods that do not contain any artificial flavors or chemicals  Gross/Macroscopic - Odor What makes feces smell so bad? The distinctive odor of feces is due to bacterial action. Specifically, the bacteria produce various compounds and gases that lead to the infamous smell of feces. Gut flora produce compounds such as indole, skatole, and thiols (sulfur containing compounds), as well as the inorganic gas hydrogen sulfide The bad smell of feces will usually be reduced by eating more natural foods that do not contain any artificial flavors or chemicals  Gross/Macroscopic - Constituents Normal: water (about 75%). dead bacteria that helped us digest our food, living bacteria, undigested food residue (known as fiber), cellular linings, sloughed epithelial cells substances released from the intestines (such as mucus) and the liver fat, protein, dried constituents of digestive juices (e.g., bile pigments), inorganic matter (e.g., calcium, phosphates) Abnormal: pus: bacterial infection mucus: inflammatory condition blood: gastrointestinal bleeding large quantities of fat: malabsorption foreign objects: accidental ingestion  Techniques A. Direct Fecal Smear (DFS) B. Kato Thick Smear C. Concentration Techniques  Techniques A. Direct Fecal Smear (DFS)  Techniques B. Kato Thick Smear STH Mass epidemiologic studies  Techniques C. Concentration Techniques Sedimentation Flotation  Preservation Formalin 5% - protozoan cyst 10% - helminth eggs and larvae FECT (formalin-ether/ethyl acetate concentration technique)  Preservation Schaudinn’s solution fresh stool for staining the stool smear mercuric choride (toxic)  Preservation Polyvinyl alcohol (PVA) protozoan cysts and trophozoites for permanent staining mercuric chloride FECT  Preservation Merthiolate-iodine-formalin (MIF) intestinal protozoans, helminth eggs, and larvae merthiolate and iodine (stain) formalin (preservative) wet mount using preserved stools Sodium acetate-acetic acid formalin (SAF) liquid fixative w/ long shelf-life does not contain mercuric chloride image not as sharp as PVA or Schaudinn’s Preservation