Clinical Parasitology Laboratory Procedures - Specimen Collection & Handling - PDF

CLINICAL PARASITOLOGY  Barium LABORATORY 001 – PRELIMS  Bismuth SPECIMEN COLLECTION & HANDLING  Mineral Oil -...

CLINICAL PARASITOLOGY  Barium LABORATORY 001 – PRELIMS  Bismuth SPECIMEN COLLECTION & HANDLING  Mineral Oil - Samples from patients whose therapy includes those Introduction interfering elements must be collected 5-7 days - Parasitic disease continues to be a significant threat before or after taking the drug throughout the world and they appeared to be - Samples from patients who are taking antibiotic or prevalent in underdeveloped tropical and anti-malarial drug must be collected 2 weeks subtropical countries. following the therapy - The disease is usually brought about the climate conditions desirable for parasitic survival as well as Stool Collection poor sanitation and personal hygiene practices of the - Stool specimen must be collected in a clean, water inhabitants. tight container with a tight-fitting lid - It is critical for clinicians to obtain knowledge of the - Acceptable amount of stool for analysis is 2.5 grams clinical manifestation of parasitic disease and (walnut or pea size) understand the laboratory test order - Stool must not be contaminated by urine for it is known to destroy parasite 3 Phases in Laboratory Analysis - Stool must not be retrieved from the toilet bowl Successful laboratory identification of parasites requires because presence of free-living protozoa and the knowledge and practice of laboratory testing in the 3 nematodes may be confused with human parasite phases of analysis: - Water may destroy selected parasites such as 1. PRE-ANALYTIC PHASE schistosome eggs and amoebic trophozoites - Includes the specimen collection, timing, labeling, - The specimen container must be labelled with the handling, and transport following information 2. ANALYTIC PHASE  Patient’s name - Includes the specimen processing, analysis,  ID number identification, and verification  Physicians name 3. POST ANALYTIC PHASE  Date and Time of collection - Involves the evaluation, interpretation, and release - Time frame is essential to demonstrate the motility of the results in timely manner of a certain parasite. - A freshly collected specimen is required to Ova and Parasite Examination demonstrate the motility of protozoan trophozoites. - The most common procedure performed in the area  Watery stool – trophozoites; within 30 mins of parasitology is the examination of a stool  Semi-formed stools - may contain both specimen for ova and parasite (O&P) trophozoite or cyst form; within 1 hour 2 general components of routine parasitology  Formed stools – cysts; within 24 hours; with procedure: preservative 1. Macroscopic examination 2. Microscopic examination Specimen Preservation - Freshly collected stool sample must be immediately Specimen Collection and Transport processed and analyzed in the laboratory. If not - Morphologic forms and protozoa and helminths may possible, sample must be preserved to maintain its be detected from a properly collected and prepared integrity. stool specimen - Fixatives are substances that preserve the - Parasites are shed intermittently on stool thus; they morphology of protozoa and prevent further may not appear in a stool specimen on a daily basis; development of certain helminth eggs and larvae. multiple specimens are recommended for adequate - Ratio of fixative stool is important for the successful detection recovery of parasite. - Certain medications and substances may interfere - The recommended ratio is 3:1 fixative to stool with the detection of parasites. Patients who - The specimen must be fixed in the preservative for at undergo therapy with medicines containing the least 30 minutes before processing. following: Stool Preservatives ADVANTAGES 1. FORMALIN 1. Easy to prepare - Used as an all-purpose fixative for recovery of 2. Long shelf life protozoa and helminths 3. Can be used of preparing smear for staining of  5% - preserves protozoan and cyst modified acid-fast stain to detect coccidian  10% - helminth eggs and larvae; eggs oocysts - Used in direct examination and concentration 4. Compatible with immunoassay procedures but not for permanent stains - Compatible with immunoassay kits and UV DISADVANTAGE fluorescence 1. Poor adhesion; need to use albumin 2. Protozoa morphology preserved is not as clear ADVANTAGES in permanent stains as when mercury- 1. Easy to prepare containing preservatives are used 2. Preserves specimen for several years 3. Has long shelf life 4. Modified PVA - Alternative to mercury-based PVA DISADVANTAGES - Contains copper sulfate or zing sulfate in substitute 1. Does not preserve parasite morphology to mercuric chloride adequately for permanent smear - Used in concentration methods and permanent 2. Trophozoites can’t be recovered and stained smears morphologic details of cysts and egg may fade - Zinc sulfate fixatives provide better result than with time copper sulfate reagents 2. POLYVINYL ALCOHOL (PVA) DISADVANTAGE - Comprised of plastic powder that acts as an adhesive 1. Not same quality of preservation compared to for the stool specimen when preparing slides for mercury chloride fixative staining - Mostly combined with Schaundinn solution, which 5. Schaundinn’s Fixative contains zinc, sulfate, copper sulfate, and mercuric - Preserves morphology of protozoan trophozoite and chloride as a base cysts - Easy to prepare ADVANTAGES 1. Detects trophozoites, cyst of protozoa, and DISADVANTAGE most helminth eggs 1. Less suitable for concentration 2. Can be used for preparation of permanent 2. Has mercuric chloride stained smear 3. Inadequate of morphology of helminth eggs and 3. PVA-preserved specimen has a long shelf life larvae, coccidian and microsporidia when stored at RT 4. Poor adhesion of liquid or mucoid specimens to slides DISADVANTAGES 1. PVA that contains Schaundinn solution contains 6. Alternative Single-Vial System mercuric chloride. Exposure may lead to - Single vials system is free of formalin and mercury potential health problems and can be used for concentration techniques and 2. Difficult to dispose permanent stained smears - Some of these products can be used for performing 3. SODIUM ACETATE FORMALIN (SAF) fecal immunoassay - Used in concentration techniques and permanent - Similar with PVA, these vials do not provide the same stained smears quality of preservation as mercury-based fixative - Viable alternative to the use of PVA and Schaundinn fixative - Mercury free CLINICAL PARASITOLOGY Consistency Gross Gross Appearance LABORATORY 002 – PRELIMS Terms Abnormalities Terms SPECIMEN PROCESSING in Stool Hard Adult worms Conspicuously MACROSCOPIC ANALYSIS fibrous - Color and consistency of the stool submitted Soft Proglottids Fiber scanty to must be initially determined; followed by screening moderate and examination for any gross abnormalities Mushy Pus Colloidal - Stool consistency or degree of moisture in a (homogenous) specimen may serve as an indication of potential Loose Mucus Scanty mucus parasite present Diarrheic Much mucus o Soft or liquid stool may suggest presence (watery or of protozoan trophozoite liquid) o In formed stools, protozoan cyst are likely to be Watery liquid Mucus with scanty recovered blood - The color of stool is important because it may Formed indicate a patient’s condition or status (or hard) Semi-formed (deformed) MICROSCOPIC ANALYSIS - Done to detect presence of parasites in stool specimen - Microscopic examination of stool for presence of ova and parasites involves three distinct procedures: 1. Direct wet preparation - can’t be performed for examination of O&P if the specimen is received in a fixative (preserved) 2. Concentration technique 3. Permanent staining Stool Color Possible Cause A. Direct Wet Preparation (Direct Wet Mount) Stool Color Possible Cause - Performed with a slide mixing a small portion of unfixed stool with saline or iodine and subsequent examination of the mixture (stool and saline/iodine) under the microscope Black Upper GI Bleeding - Motility can be only demonstrated using Iron Therapy this method (saline prep) on fresh specimens Charcoal - Organisms that are detected with direct Bismuth (antacid) wet preparation Red Lower GI bleeding  Protozoan and flagellate trophozoite Beets and food coloring  Protozoan cysts Rifampin (for TB)  Oocyst Pale Yellow Bile duct obstruction  Helminth eggs  Larvae White, Gray Barium sulfate 2 Types of Direct Wet Preparation Green Biliverdin/Oral antibiotics 1. Direct saline wet preparation Green vegetables 2. Direct iodine wet preparation 1. Direct Saline Wet Preparation debris is usually lighter and rises to the upper layers - For trophozoites of the tube - Made by placing a drop of 0.85% saline on glass slide - Advantage of this technique is that it provides good and mixing a with a small portion of unfixed stool recovery of most parasites and is easy to perform using a wooden applicator stick or another mixing - Disadvantage is that the preparation contains more tool fecal debris than a floatation technique and is more - Mixture of stool and saline must be thin enough for challenging to the microscopist newspaper print to be read through the smear - 22-mm square coverslip is placed on the slide and 2. Zinc Sulfate Flotation Technique the preparation is examined under the microscope - Also based on differences in specific in a systematic fashion - But in contrast to the Formalin-Ethyl Acetate, - Entire coverslip is scanned using LPO (10x) sample debris sink on the bottom and the potential parasite float on the top 2. Direct Iodine Preparation - Advantage is that more fecal debris is removed and - Uses a single drop of Lugol’s Iodine or D’Antoni’s it yields a cleaner preparation, making it easier for formula instead of saline microscopic examination - It enhances the detail of protozoan cysts (glycogen - Disadvantage is that some of the helminth eggs are and nuclei) very dense and will not float - Iodine kills trophozoite present C. Permanent Stains B. Concentration Method - Microscope slide that contains fixed sample that has - Provide the ability to detect small numbers been allowed to dry and subsequently stained of parasite that might not be detected using - Designed to confirm the presence of protozoa cyst direct preparations or trophozoites and it allows to observe detailed - Purpose of concentration is to aggregate features of protozoa by staining intracellular the parasites present into a small volume of organelles sample and to remove as much as debris possible - Not the method of choice for identification of that may hinder to see parasites clearly helminth eggs and larvae because these parasites - Concentration techniques can be performed often stain too dark or appear distorted on fresh or preserved specimens - Sample of choice for staining is thinly prepared slide - Concentration techniques enables of see through thickness made from PVA preserved  Protozoan cyst sample  Oocyst - Specimen fixed with SAF may be used but staining is  Helminth eggs limited only to iron-hematoxylin  Larvae - Slides can also be prepared from fresh - The two techniques use differences in stool specimen but it should be placed immediately specific gravity and centrifugation to separate into a fixative such as Schaundinn’s fixative parasites from fecal debris and increase their - Once staining is complete, the slides can be sealed recovery with a permanent mounting sealant and can be kept 1. In sediment techniques, parasites for years are concentrated in the sediment of the tube - Slides are viewed under OIO (100x) ; 300 fields are following centrifugation and the sediment is reviewed before the slide can be examined microscopically. considered negative 2. In flotation techniques, the parasites are less dense than the solutions used and, 2 COMMON STAINS FOR ROUTINE O&P TESTING during centrifugation, they float to the surface. 1. Trichrome stain (Wheatley Trichrome) - Most widely used permanent stain 1. Formalin-Ethyl Acetate Sedimentation Procedure - Long shelf life and easy procedure - Most widely used sedimentation 2. Iron hematoxylin - Principle of technique is based on specific gravity - Considered to be time-consuming - Wherein parasites are heavier than the solution and - Reveals excellent morphology of the intestinal settle in the sediment of the tube, whereas fecal protozoa SPECIALIZED STAINS STOOL SCREENING METHODS 1. Acid fast stain - Alternative tests for detection of parasitic Detection of the oocysts of Cryptosporidium, as infection have been developed and are referred to well as those of Isospora and Cyclospora as ‘rapid test’ or stool screening methods 2. Modified iron hematoxylin - These methods can be obtained as a kit that contain Detection of acid-fast parasites in addition to a monoclonal antibody other protozoa normally recovered in - This commercial antibody is used to detect antigen iron hematoxylin stain in patient’s specimen 3. Modified trichrome stain - Assays include: Detection of spores of microsporidia 1. Enzyme Immunoassay 2. Direct fluorescent antibody 3. Membrane flow cartridge antibody - Rapid test or stool screening methods are highly sensitive and specific - Antigen detection methods are available for specific intestinal protozoa such as Entamoeba histolytica, etc OTHER SPECIMENS AND LABORATORY TECHNIQUES - There are parasitic infections wherein examination of stool specimens may not be detecting the infectious agent. There are additional procedures that can be performed to reveal presence of specific parasites - Other intestinal specimens: 1. Duodenal Material - Parasites that reside in the small intestine may be difficult to recover in a stool specimen thus duodenal material may yield success - Specimen is collected by nasogastric intubation or enteric capsule test (entero test) - Parasites that may be recovered includes:  Giardia intestinalis trophozoites  Cryptosporidum spp  Isospera belli  Strongyloides stercoralis  Ova of Fasciola hepatica or Clonorchi sinensis - Duodenal material recovered can be examined as a wet preparation - If volume is >2mL, it must be centrifuged and sediment is examined - Material can be fixed with PVA and can be stained using trichrome, iron hematoxylin, or modified acid- fast chain 2. Sigmoidoscopy Material - Colon material is often helpful in detecting E. histolytica. - Coccidian parasites and microsporidia may also be recovered from examining sigmoid material - Material from ulcers obtained by aspiration or longer period leads to distortion and possible loss of scraping must be examined by direct wet mount malarial parasite prep and permanent stain - Timing for collecting blood sample varies with the parasite suspected 3. Cellophane Tape Preparation - Typical blood sample processing - The specimen of choice for recovering Enterobius incudes preparation of thick and thin vermicularis (pinworms) eggs. Adult female smears, staining with permanent stain pinworm may also be seen then examined microscopically - Eggs are released on the perianal region - Other blood sample processing - Specimen must be collected in the morning before o Knott technique the patient washes or defecates o Buffy coat slides - To rule out negative infection of E. vermicularis. 5 o Culture specimens will be collected daily and must yield a negative result on all samples collected  Thick Smear - Other specimen for Parasitic recovery - Frequently satisfactory for screening purposes, particularly when malaria is suspected Other Specimens for Parasitic Recovery - Used when parasites are few in number or thin 1. Blood smear is negative 2. CSF and other - Its advantage is an increased ability to detect 3. Tissue and biopsy the malarial parasite 4. Sputum - Disadvantage is that the red blood cells have 5. Urine and genital secretions been lysed and it is not possible to assess the 6. Eye specimens morphology of parasites that are detected 7. Mouth scrapings and nasal discharge 8. Skin snips  Thin Smear 9. Culture methods - Provide the best view of the malarial parasites 10. Animal inoculation and xenodiagnoses in RBC and are recommended for species identification Blood - Systemic or blood-borne parasitic infections are  Permanent Stain diagnosed by demonstrating the diagnostic stage(s) - Wright Stain – Contains fixative and stain in of the responsible parasite in a blood specimen one solution; Yields satisfactory result - Parasites that may be recovered: - Giemsa Stain – Fixative and stain are separate; o Leishmania donovani Considered the preferred stain because it allows o Tryoanosoma spp. for detection parasite detail necessary for - Blood smears can be prepared from fresh whole species identification blood without anticoagulant (fingertip or earlobe) or from venipuncture collection without anticoagulant - Specimens for parasite study must be collected by aseptic technique - Blood from fingertip or earlobe yields the best morphology of parasites, but improper collection can lead to unsatisfactory results - Capillary blood must be free flowing and not contaminated with alcohol used to cleanse the puncture site - Anticoagulant causes distortion to the staining process and parasite morphology - Tube containing EDTA is the anticoagulant used in blood collection - If malaria is suspected, it is best to prepare smear within 1 hour of collection. Storage of blood for Blood Sample Techniques  Hookworms 1. Knott Technique: Designed to concentrate  Ascaris lumbricoides blood specimens suspected of containing - Specimen of choice is early morning specimen low numbers of microfilariae collected in a wide mouthed container with a screw 2. Buffy Coat Slides: Used in cancer patients, capped lip. stained with GEMSA - Samples are examined via wet mount prep of 3. Cultures: Blood and other specimen such as concentrating using N-acetylcysteine bone marrow and tissues may be performed - Novy-MAcNeal Nicolle (NNN) medium: used Urine and Genital Secretions for the recovery of Leishmania spp. - Urine is the specimen of choice for detection of and Trypanosoma cruzi Schistosoma haematobium eggs. Trichomonas - NNN slant is inoculated by addition of single vaginalis trophozoite can also be recovered from drop of collected blood or ground tissue this specimen - In heavy filarial infection, microfilariae can be Sterile Fluids Other Than CSF sometimes found on urine specimens 1. Fluid in cysts - Vaginal, urethral specimens and prostatic secretions 2. Aspirates are usually collected to examine for the presence of 3. Peritoneal fluids Trichomonas vaginalis trophozoites 4. Pleural fluids - Wet preparations are the method of choice for 5. Bronchial washing demonstrating motility Cerebrospinal Fluid and Other Sterile Fluids Eye Specimens - CSF must be examined promptly to detect the - Acanthamoeba keratitis is best diagnosed by the motility of these parasites collection and examination of corneal scrapings. - Special stains can also be performed on CSF These scrapings should be placed into an airtight including Giemsa, Trichrome, and modified container. It is important that small tissue samples trichrome stains be kept moist with sterile saline - Sterile fluids other than CSF include several specimen - Other specimens that may be tested include a types, such as fluid present in cysts, aspirates, contact lens or contact lens solution. peritoneal fluid, pleural fluid, and bronchial - The samples may be processed in several ways. First, washings it may be cultured on an agar plate seeded with - The parasites that may be detected, as well as the gram-negative bacteria. Examining the culture plate specific processing techniques necessary to identify under low dry magnification every day for 1 week them, vary by specimen type should reveal the trophozoites (usually in less than 4 - All these samples may be examined using wet preps days) and the cysts (in 4 to 5 days). Second, the and/or permanent stains scrapings may be transferred to glass slides and stained using the calcofluor white stain, followed Tissue and Biopsy Specimen by microscopic examination using - Recommended for recovery of several parasites fluorescent microscopy  Leishmania spp - Other eye pathogens  T. gondii  Toxoplasma gondii - Surgical removal of the specimen followed by the  Microsporidia preparation of histologic tissue sections and  Loa loa impression smears is the preferred method for handling these specimens Mouth Scraping and Nasal Discharges - Mouth scrapings are the sample of choice for the Sputum detection of E. gingivalis and Trichomonas tenax - Sputum is typically collected and tested from (Chapter 4), whereas nasal discharge specimens are suspected patient infected with Paragonium helpful for the recovery of parasites such as N. westermani (lung fluke) fowleri. - Other parasite that might be seen on sputum - Material obtained via mouth scrapings and nasal  Motile larvae discharge should be placed in a clean airtight  E. histolytica collection container, such as on a swab or in a cup.  The material may then be extracted from the swab the specific parasite(s) that is (are) tested for in the or transferred from the cup for examination assay. purposes. - In general, for most parasites, quantitation is - Wet preps are typically made from mouth scrapings not indicated. Situations in which quantitation is and nasal discharge samples. Permanent stains may important are as follows: Blastocystis also be used if appropriate. hominis, helminth eggs, including Trichuris trichiura, Clonorchis sinensis and Schistosoma spp., Skin Nips and Plasmodium and Babesia spp. Charcot- - Useful in the detection of Onchocerca volvulus Leyden are also reported when found and can (Chapter 9), skin snips may be made using one of two be quantitated. collection techniques. - One of the methods involves making a firm (scleral) punch into skin with a specially designed tool. The other technique uses a razor blade with which a small cut into the skin is made. - The resulting material obtained by both techniques may then be placed in approximately 0.2 mL of saline. After a 30- minute incubation period, the sample may be microscopically examined. - The jerky movement of the microfilariae should be visible, if present, in the saline because they tend to migrate into the liquid from the skin snip itself. Culture methods - Not a common means of detecting parasites. There are a few techniques available but they are not usually performed in the routine laboratory. - Specialized laboratories and research facilities may offer these services. - Parasites that can be isolated with culture include E. histolytica, T. vaginalis, Leishmania spp., T. cruzi, and T. gondii. IMMUNOLOGIC TESTING - Immunologic tests include methods for antigen and antibody detection - Antigen detection are more reliable and a positive test result is indicative of current infection - Test that detects antibody are more complex and must be interpreted cautiously - Immunologic testing are not usually offered by routine lab and specimens must be sent out to reference lab that perform them REPORTING OF RESULTS - When reporting a positive specimen, the report should state the scientific name (genus and species), along with the stage that is present (e.g., cyst, trophozoite, larvae, eggs, adults). Example: “Entamoeba histolytica cyst seen.” - Presence of blood cells should be reported semi quantitatively—rare, few, moderate, many - The results of fecal immunoassays should indicate