CLS 511 Medical Microbiology. Unit 25 Lab Diagnosis of Parasitic Diseases 1. Detection Methods Stool. Laboratory Diagnosis of Parasitic Infections

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1 Laboratory Diagnosis of Parasitic Infections Cryptosporidium sp. Enterobious vermicularis (pinworm) Karen Honeycutt, M.Ed., MT(ASCP)SM Macroscopic: Look for parasite evidence/structures (worm segments, entire worms) in stool specimen (usually performed on fresh, not preserved stool specimen) Identify parasitic structure passed by patient Example: Ascaris round worm (picture from ASCP) 1 Microscopic: Wheatley s trichome stain on stool prior to concentrtaion Used to detect intestinal protozoa (amoeba, Giardia lamblia) Stool specimen processed to concentrate parasitic structures Concentrated material microscopically scanned for protozoan cysts, eggs, larvae Wheatley s trichrome stain may be performed on concentrated material to detect intestinal protozoa Microscopic examination is not sensitive and labor intensive Overall, most common detection method except for and Cryptosporidium sp. as these are commonly recovered intestinal parasites in the US Antigen detection methods are available more sensitive than microscopic methods Antigen Detection Enzyme immunoassay: known antibody captures parasitic antigen, color visualizes antigen presence More sensitive than microscopic methods Most common method to detect: Cryptosporidium sp. Nucleic acid detection (NAT) Detects specific organism nucleic acid sequence in specimen Very few assays available Used when organism detection/identification very difficult by other methods, examples Entamoeba histolytica (pathogenic intestinal amoeba) Malaria organisms Unit 25 Lab Diagnosis of Parasitic Diseases 1

2 Detection Methods Serum Serologic Detects patient antibody response to a specific parasitic organism Normally used when parasite is hard to recover from patient due to the type of tissue infected Example: Cysticercosis: caused by larval forms of tapeworm that encyst in the brain causing epilepsy Toxoplasma gondii: intracellular organism found in lung, heart, lymphoid tissue Culture and Susceptibility Testing Culture is rarely used, usually performed via research/reference laboratories Trichomonas vaginalis (STD) can easily be cultured, but is most easily detected by microscopic detection in urine (i.e., usually no need to culture) Susceptibility testing is not performed on parasitic organisms Specimen Collection Stool Collect specimen in a clean bed pan or use plastic wrap placed between the toilet seat and the bowl. Stool contaminated with urine, toilet water, or barium is NOT acceptable (interferes with testing) Transfer specimen into a clean, dry container. Transport unpreserved stool at ambient temperature ASAP (no longer than 2 hours) If transport delay > 2 hours is unavoidable, place the specimen in an appropriate preservative such as Proto fix at ambient temperature. Do not fill commercial transport vials above indicator line. Overfilling of transport vial results in improper specimen preservation (walnut size stool sample or 10 ml liquid stool) Specimen Collection Stool Proto fix kills living organism in stool, but preserves morphology Stool submitted in Proto fix is NOT able to be used for culture If culture requested, the appropriate stool culture specimen transport vial must be used Specimen Collection Stool Specimens rejected: Dry, unperserved specimen Swabs Leaking container Specimencontaminated contaminated with oil, barium, toilet water or urine Multiple specimens in a 24 hour period Most parasites are shed intermittently, a single specimen per day is appropriate Fresh specimen delayed transport (>2 hours) (protozoan) Most common parasitic organism recovered in the US population Organism located throughout the world Several animals are reservoirs and serve as source of infection: beavers, muskrats Single celled organism w/two lifecycle forms: Trophozoite: active, feeding form of organism Cyst: survival state of organism when in harsh environment, this is infective form for man Unit 25 Lab Diagnosis of Parasitic Diseases 2

3 disease state Asymptomatic can serve as carriers and a source of infection for others Symptomatic: incubation period 1 to 4 weeks (10 days average) after ingestion Diarrhea which can lead to malabsorption syndrome Abdominal cramps, nausea, flatulence, watery diarrhea, weight loss Steatorrhea (increased fats in stool) can cause stool to float Humans infected Drinking contaminated water (streams) Eating contaminated, uncooked food Person to person spread (fecal oral route) Two lifecycle stages Trophozoite: active, feeding form Cyst: allows survival in harsh environment (human stomach ph), infective form for humans Prevention Cysts are resistant to normal chlorination levels Water should be filtered, boiled or treated with iodine Boil water for at least 1 minute Use a filter that t has an absolute pore size of 1 micron or smaller Use iodine chemicals may be less effective than boiling or filtering because the amount of chemical required to make the water safe is highly dependent on the temperature, ph, and cloudiness of the water. Laboratory diagnosis Laboratory Diagnosis: may be difficult Microscopic identification (may require up to 6 stools specimens/alternate days) Immunodiagnosis (most sensitive) Many labs only offer immunodiagnosis (i.e., enzyme immunoassay) Preferred method Single specimen usually sufficient Giardiasis Treatment Tinidazole Metronidazole Nitazoxanide: first liquid for giardiasis Unit 25 Lab Diagnosis of Parasitic Diseases 3

4 Cryptosporidium Protozoan Infection by: fecal/oral, ingest oocysts (lifecycle protected by outer shell that allows for survival outside the body for long periods of time) Location: Worldwide, in US in 65 97% of surface water Wide range of animal carriers Very resistant to chemical water treatment Need to filter/boil water Contaminated water, is one of the most frequent causes of waterborne disease (drinking water and recreational water) among humans in US Cryptosporidiosis Can be asymptomatic Symptoms begin 2 to 10 days (average 7 days) after ingestion Watery diarrhea, stomach cramps or pain, dehydration, nausea, vomiting, fever, weight loss Immunocompetent: symptoms usually last 1 to 2 weeks, recurrence can occur after a brief period of recovery (symptoms can come and go for up to 30 days) People with weakened immune systems (AIDS) may develop serious, chronic, and sometimes fatal illness Cryptosporidium Laboratory Diagnosis Immunodiagnosis most sensitive, single specimen Microscopic identification detect oocyst with modified acid fast stain, may require multiple specimens Cryptosporidiosis Treatment Nitazoxanide HIV no established treatment Nitazoxanide Paromomycin Intestinal Parasites Screen Test Please NOTE: Many labs offer an immunodiagnosis antigen screen for both : Cryptosporidium sp. This test is performed before other parasites are looked for, unless there is something in the patient s history (i.e., travel) that would indicate other parasites. Enterobius vermicularis Pinworm Helminth (worm): nematode (roundworm) Infected by: fecal/oral by ingestion of eggs Location: worldwide Most common helminth (worm) infection in US (40 million/yr) Adult worms live in intestine and rectum Female migrates to anus, exits, deposits her eggs (approximatey 10,000), then usually dies or returns to intestine/rectum Unit 25 Lab Diagnosis of Parasitic Diseases 4

5 Enterobius vermicularis Pinworm Pinworm eggs become infective within a few hours after being deposited on the skin around the anus Can survive for 2 to 3 weeks on clothing, bedding, or other objects Infection by ingesting i infective i pinworm eggs that are on fingers, under fingernails, or on clothing, bedding, and other contaminated objects and surfaces Because of their small size, pinworm eggs become airborne and ingested while breathing Enterobius vermicularis Disease Asymptomatic or Perianal pruritis (itching) caused by the female pinworm laying her eggs Secondary bacterial infections due to itching Irritability (inability to sleep) Females can experience vaginal itching Enterobius vermicularis Laboratory Diagnosis Macroscopic id of female worm (collected at night or first thing in morning before going to bathroom/bathing) White, opaque, about size of a staple, visible with naked eye Enterobius vermicularis Laboratory Diagnosis Microscopic id of eggs (collected at night or first thing in morning before going to bathroom/bathing) Pinworm collection device A vial with a sticky paddle Enterobius vermicularis Laboratory Diagnosis Firmly apply the sticky paddle to perianal folds Paddle will pick up pinworm eggs (and possibly adult female worm) Paddle is microscopically examined for characteristic eggs Stool is NOT an appropriate specimen low power (10x) Higher power (40x), egg with flattened side Enterobius vermicularis Treatment Albendazole, Mebendazole, or Pyrantel pamoate Treat again 2 weeks after the first dose. All household contacts/caretakers usually treatedat once Reinfection occurs easily, strict observance of good hand hygiene is essential (e.g. proper hand washing, maintaining clean short fingernails, avoiding nail biting, avoiding scratching the perianal area). Unit 25 Lab Diagnosis of Parasitic Diseases 5

6 Enterobius vermicularis Treatment Careful handling, frequent changing of underclothing, night clothes, towels, and bedding can help reduce infection These items should be laundered in hot water Overview Giardia and Cryptosporidium stool antigen detection assay is usually performed before a full ova and parasite examination is ordered (unless patient has travel history) Et Enterobius vermicularis i (pinworm) is detected dt td via microscopic identification of eggs or adult worms from perianal area (stool not acceptable specimen) Unit 25 Lab Diagnosis of Parasitic Diseases 6