Introduction to Parasitology Required for reliable diagnosis of infection: Knowledge of patient - travel history, day care attendance, refugee? Appropriate specimen - number, frequency, time of collection. Use of appropriate procedures - flotation, sedimentation, staining, etc. Adequate training of technologist - college courses, workshops, continuing education. North Americans Do Not Suffer From a Multitude of Parasites High standards of education - better housing, higher standard of living. General good health - poor health = more susceptible to disease. Nutrition - adequate diet. Sanitation - sewers and septic systems keeps raw sewage out of streams. Temperate climate - parasites do better in the warmth of the tropics. Absence of certain vectors - intermediate hosts such as the tsetse fly, certain snails, etc. Reasons We Have Parasitic Infections in This Country Increased travel - in some areas of the world parasitic diseases are very common. Low understanding about parasitic infections results in an increased likelihood of transmission. Definitions Parasite - one animal deriving its sustenance from another without making compensation. The uncompensated animal is the host. Parasitology - the science or study of host-parasite relationships. Medical parasitology - study of parasites which infect humans. Host - the partner providing food and/or protection. Some parasites require more than one host to complete their life cycle; Or may not require a host during some stage(s). Definitions, Continued. Types of Hosts Definitive host - the host in which sexual maturity and reproduction takes place. Intermediate host - the host in which the parasite undergoes essential development. Reservoir (carrier) host - the host harboring a parasite in nature, serving as a source of infection for other susceptible hosts. Reservoir hosts show no sign or symptom of disease. Paratenic host - an accidental host serving as a holding place for a parasite. Definitions, Continued. Vector - “carrier” of a parasite from one host to another. Often an insect. Symbiosis - “living together,” a close association between two organisms. Mutualism - both organisms are benefited (bacteria in bowel). Commensalism - “eating at the same table;” One organism is benefited, the other is unaffected. Parasitism - one organism is benefited at the expense of another (the host). A Successful Parasite A parasite is successful - when it is in delicate balance with the host. If the balance is upset, the host may destroy or expel the parasite; If the host is overly damaged, it may die - as will the parasite. Parasitology is important - because this balance is not always maintained. Parasitic Damage to Host: Trauma - damage to tissues, intestine, liver, eye. Lytic action - activity of enzymes elaborated by organism. Tissue response - localized inflammation, eosinophilia. Blood loss - heavy infection with hookworm may cause anemia. Secondary infections - weakened host susceptible to bacterial infection, etc. Modes of Infection Filth-borne or contaminative - where personal hygiene and community sanitation lacking. Infectious stages remain viable for long periods in contaminated soil. Soil or water-borne - water or dirt which can contain eggs, etc.; Larvae can penetrate skin of bare feet or enter skin in infested water. Food-borne - inadequately cooked beef, pork, fish, shell fish. Arthropod-borne - the most difficult of all to control. Mosquitoes transmitting malaria, etc. Collection, Processing, & Examination of Specimens Microscope - objectives must be calibrated in order to insure accurate measurement of organisms. Centrifuges - swinging bucket type is required. Collection, Processing, & Examination of Specimens, continued. Types of specimens which can be examined for diagnosis of parasites: Natural secretions - feces, sputum, and urine are used to detect lumen dwelling parasites of GI, pulmonary and genitourinary tracts. Blood - usual specimen for detection of blood and tissue parasites, along with tissue biopsies, aspirates, etc. Collection, Processing, & Examination of Specimens, continued. Intestinal dwelling parasites - fecal specimens. Patient preparation - must avoid substances which can interfere with stool examination. No antimicrobial medications should be taken during the 10 days prior to collection of specimens. Medications containing antimicrobial agents – Bismuth, Barium, Mineral oil, Kaolin, Anti-diarrheal preparations, Laxatives. Contaminant free specimens - no urine, water or dirt - these may destroy organisms, or could contain confusing free-living organisms. Types of Stool Specimens. Liquid specimens - trophozoite stages are more likely to be present. Procedures - direct wet mounts for detecting motility; permanent stains exhibit the best morphology. Must be examined within 30 minutes of passage or placed into an appropriate preservative. Freshly passed specimens are necessary in order to recover motile trophozoites. Cyst formation will not occur once the organism is outside the body; trophozoites disintegrate rapidly after passage. Types of Stool Specimens, Continued. Semi-formed specimens (soft specimens) - all stages may be present. Procedures - one should perform all procedures (direct examinations, concentration, acid fast, & permanent stain). Types of Stool Specimens, Continued. Formed specimens - cysts of protozoa and eggs/larvae of helminths may be present. Procedures - direct wet mounts will serve to detect those organisms which do not concentrate well. Concentration is necessary to detect light infections, but permanent stains are equivocal. If blood or mucous is present on the specimen, it should be removed and stained with a permanent staining procedure. Collection Methods Submit fresh specimens directly to lab - these must be examined within 30 minutes to one hour. Store these at room temperature if examined within 30 minutes of collection. Refrigerate them otherwise, but never incubate these specimens. Commercially prepared preservation kits - use a kit if not able to examine within 30 minute frame. Preservation will insure the integrity of the specimen. Fixatives and Preservatives Introduction. An ideal preservative would preserve all diagnostic stages, and not interfere with concentration & staining techniques. If a specimen cannot be processed immediately, at least prepare a slide for permanent staining on the day it is received. Then it can be stained during the next work day, avoiding delaying results. Commonly Used Preservatives MIF (merthiolate - iodine - formalin) - for wet smear & concentration only. Cannot permanent stain. SAF (sodium acetate - acetic acid) - OK for concentration, can permanent stain with iron hemotoxylin only, trichrome stain will not produce satisfactory results. Commonly Used Preservatives, Continued PVA (polyvinyl alcohol)/Formalin kit - a popular method using two vials. Vial #1: 5 -10% formalin - preserves helminth eggs and larvae, and protozoan cysts. Specimen can be used for direct wet mounts and in a concentration procedure. Vial #2: polyvinyl alcohol fixative - preserves protozoan trophozoites (and cysts) for permanent staining. There are a variety of “fixing agents” in PVA including mercury, zinc, and copper. Zinc is the best alternative to mercury, which remains the “gold standard.” Commonly Used Preservatives, Continued Schaudinn's Fixative - used for staining fresh specimens, contains mercury. 10% buffered formalin - for concentration only. Stool Collection Kits Ideally, a kit should have three (3) containers: Formalin (5% or 10%) - for concentration. PVA-Fixative - for permanent staining. Clean vial - for unpreserved portion culture and assessment. Considerations in the Selection of a Kit Vial size - should be of adequate size to allow for a representative sample. Child proof - children should not be able to open the vials. Stirrers, scoops, etc. - should be provided to assist in getting a specimen into the vial and mixed with the preservative. Labels - that clearly indicate presence of poison; should be present in several languages. Patient label information - name, date, & time of collection. Considerations in the Selection of a Kit, Continued. Collection instructions - in several languages representing local dialects. Mailers - must meet postal regulations (triple barrier). Cost - must be affordable. Specimen Collection Collect in a clean container - without urine or water (these may be damaging to trophozoites). Minimum number of specimens - due to irregular shedding patterns of parasites, a series of three normally passed specimens is preferred. Frequency of collection - collect on alternate days. Never on same day. No laxatives permitted - these can mask infections or damage organisms. Date and time of collection - important, should be required information. Techniques of Stool Examination Gross examination - grade consistency of the specimen; decide on best methods of examination to allow for detecting most likely stages/parasites. Look for worms, segments of worms. Remove these for separate identification. If present, blood and/or mucous should be examined with wet mounts & permanent stains. Techniques of Stool Examination, Continued. Direct wet mounts: Used primarily to detect motility - fresh, unpreserved specimens are examined for motility; preserved specimens are examined for organisms which do not concentrate well. Procedure - use large slide & coverslip. For motility, mix small amount of specimen with physiological saline and add coverslip. For a stained preparation, in place of saline, use iodine stain to reveal nuclear morphology. Density - should be able to be read newsprint through the smear. Techniques of Stool Examination, Continued. Direct wet mounts, Continued: Advantages - will reveal helminth eggs and larvae; may reveal motile trophozoite and nonmotile cysts; will reveal other cells indicative of an inflammatory process, (macrophages, leucocytes). Disadvantages - does not lend itself to oil immersion examination; may not reveal adequate morphology causing misinterpretation; if preparation is too thick, organisms will be missed; cannot be used on PVA-preserved specimens (iodine stain coagulates the PVA). Techniques of Stool Examination, Continued. Stained Wet Mounts: Liquid specimens - more likely to contain trophozoites, buffered methylene blue may be used. Iodine is too harsh for trophozoites - often damages morphology of nucleus. Formed specimens - more likely to contain only cysts, popular stains include Dobell, Lugol, or D'Antoni iodine stains. Do not make smears too thick; examine them systematically. Techniques of Stool Examination, Continued. Concentration Techniques - Purpose: Reduce background fecal debris Increase relative number of parasites Preserve morphology of parasites Techniques of Stool Examination, Continued. Types of concentration procedures: Flotation Procedures - Floats parasites free of fecal debris by using a solution having a specific gravity greater than the parasites, and less than background fecal matter. Can lose operculated eggs, and other larger eggs since they are too heavy to float. Techniques of Stool Examination, Continued. Flotation Procedures, Continued: Advantages - can provide clean concentrate; reagents have long shelf life, & are available commercially; morphology is adequate. Disadvantages - specific gravity must be checked frequently; the larger helminth eggs will not float; must be examined immediately or organisms will begin to settle. Techniques of Stool Examination, Continued. Types of concentration procedures, Continued: Sedimentation Procedures - Concentrate diagnostic stages in sediment. Use of ethyl acetate cleans the specimen by dissolving and floating fat. Techniques of Stool Examination, Continued. Sedimentation Procedures, Continued: Formalin - Ethyl Acetate Procedure - Most commonly used sedimentation procedure (good for either fresh or preserved specimens). Reagents Used: 5% or 10% Formalin - kills organisms & preserves morphology. Ethyl Acetate - dissolves fat & floats artifacts. Techniques of Stool Examination, Continued. Formalin - Ethyl Acetate Procedure, Continued. Advantages - allows the recovery of all helminth eggs, larvae, and protozoan cysts; easy to perform; can be read anytime following concentration; several stopping places exist in the procedure. Disadvantages - must exercise caution since ethyl acetate is flammable; preparations not as clean as with floatation; amount of specimen used in relation to reagents must be carefully monitored (too much specimen can interfere with efficiency of cleansing & concentration). Techniques of Stool Examination, Continued. Permanently Stained Smears - aimed at trophozoite stages, and are most useful in the identification of organisms. Factors Affecting Permanent Staining: Age of specimen - organisms deteriorate with time. Consistency - specimens with significant mucus difficult to stick to the slide. Fixation - must thoroughly mix specimen with preservatives. Smear preparation - must not be too thick or thin. Reagents - must be replaced every 40 slides or weekly. Techniques of Stool Examination, Continued. Permanent Staining Procedures: Iron Hematoxylin Procedure - provides excellent morphology; time consuming and difficult to perform. Wheatley’s Trichrome Stain Procedure - rapid procedure and technically easier to perform; stable reagents; more reproducible results. Special Stains Pneumocystis carnii - methenamine-silver; Giemsa; Periodic acid Schiff. Cryptosporidium parvum - modified acid fast stain examined with fluorescent microscopy. Immunoserologic Detection (Parasitic Serology Tests) Test kits detect presence of antigen (organisms) or antibody - Those detecting antigen are satisfactory but do not concentrate the amount of antigen present. Other procedures may demonstrate the antigen as well, and less expensively. Tests that measure antibody include - Enzyme Immunoassay (EIA), Complement Fixation (CF), Latex Agglutination (LA), Direct & Indirect Immunofluorescence (DIF & IIF), Indirect hemagglutination (IHA), Bentonite flocculation (BF), Immunoblot (IB). Procedures & testing could improve - Standardization of antigens, reference reagents, and procedures would improve interpretation of results. Identification by Molecular Methods Nucleic acid probes and molecular techniques to aid in detection of malaria, toxoplasmosis, amebiasis and leishmaniasis are available for diagnostic and epidemiologic purposes. Quantitative Worm Egg Count This test is best used to estimate worm burden. This must be performed on unpreserved specimens. Preservatives dilute the specimen, eliminating the ability to calculate “eggs per gram” of feces. In Vitro Cultivation of Parasites Primarily used for blood & tissue protozoa. Can culture for intestinal protozoa, but not generally done due to time and sensitivity of test issues. Animal Inoculation Not routinely done - expensive, time consuming, lacks sensitivity. Use - primarily used for isolation of blood and tissue parasites (trypanosomes, etc.). Xenodiagnosis - may be considered a “special case” of animal inoculation. The term was originally applied to the diagnosis of Chagas' disease. After placing uninfected reduviid bugs on a patient suspected of having the disease, and allowing them to feed, the bugs are examined for developmental stages of the parasite Trypanosoma cruzi. Use of Other Specimens Anal Swabs / Scotch Tape Preparation for Enterobius vermicularis - must be collected in the morning prior to bathing or bowel movement; used for diagnosis of pinworm infections but other helminth eggs can be seen also, especially Taenia spp. eggs. Procedure - make impressions with sticky paddle or clear cellophane tape around the anus of the patient; examine for eggs under the microscope. Use of Other Specimens, Continued. Urogenital Specimens: Trichomonas vaginalis - vaginal, urethral, prostatic exudates are examined via wet mounts, looking for motile organisms. Urine Specimens: T. vaginalis - often seen in urine of infected individuals. Schistosoma hematobium - inhabits blood vessels around the urinary bladder, eggs “pop” into bladder as result of expansion and contraction of the bladder along with the aid of a terminal spine on the egg. Use of Other Specimens, Continued. Sputum Specimens: Paragonimus westermani (the lung fluke) - the worm lives in lung tissue; eggs are shed into alveoli, and are present in the sputum. Ascaris lumbricoides, Strongyloides stercoralis, and Hookworm – larvae can be present in sputum as a result of lung migration. Entamoeba histolytica – trophozoites may be present as a result of pulmonary amebic abscesses. Pneumocystis carinii - organisms may be seen in the sputum of AIDS patients. Use of Other Specimens, Continued. Aspirates and Biopsies: Aspiration of duodenal contents - Can be examined for Giardia lamblia and Strongyloides stercoralis. Entero-Test - a capsule containing a free-wheeling piece of yarn used to sample duodenal contents. Sigmoidoscopy/tissue biopsies - material from mucosa may be examined for parasites. Abscess aspirates - usually for extra-intestinal amoebiasis (wall of abscess is best area to examine). Use of Other Specimens, Continued. Biopsies: Muscle - Direct microscopic examination for presence of Trichinella spiralis larvae. Intestinal or bladder mucosa - Direct microscopic examination for Schistosoma spp. eggs. Procedures for Detecting Blood Parasites Collection of Blood Samples: Finger, heel or earlobe sticks - preferred for thick and thin blood smears. EDTA samples - best if smear made within one hour. Sodium citrate specimens - used for larger amounts of blood to be used in concentration or cultivation. Clot tubes - for serological procedures; lets clot retract. Procedures for Detecting Blood Parasites, Continued. Examination of Blood Samples: Wet Mounts - screening for motile organisms (trypanosomes & filariae). Permanent Stained Smears: Stains - are methylene blue-eosin based. Wright's stain - alcohol based, can not adjust pH. Giemsa - water based, can adjust pH; preferred stain for malaria examination. Procedures for Detecting Blood Parasites, Continued. Thick Blood Films: Will detect - malaria parasites, trypanosomes, and microfilariae. Preparation - 3 or 4 drops of blood stirred together to size of a dime; must dehemoglobinize in buffered water prior to staining with Wright’s stain (not necessary if using Giemsa stain); newsprint just legible through smear. Advantage - concentrates blood, picks up light infections. Disadvantage - infected red blood cells are lost; more experience is needed to recognize organisms. Must dry overnight before staining. Procedures for Detecting Blood Parasites, Continued. Thin Blood Films: Will detect - malaria parasites, trypanosomes, and microfilariae. Preparation - same as for CBC differential. Advantages - allows for observation of infected red blood cell. Morphology of organisms is better. Disadvantages - must examine for 30 minutes or 100 fields. Light infections may be missed.