15: Diagnostic Medical Parasitology

CHAPTER 15
Diagnostic Medical Parasitology

Lynne S. Garcia¹ and Gary W. Procop²

¹ LSG and Associates, Santa Monica, CA, USA

² Cleveland Clinic, Cleveland, OH, USA

With the increase in world travel and access to many different populations and geographic areas, it is very likely that we will see more tropical diseases and infections due to the rapidity with which people and organisms can be transmitted from one place to another. The transportation of infectious agents, as well as human travelers, has been clearly demonstrated during the past few years, particularly via air travel. Travel has become available and more affordable for many people throughout the world, including those who are in some way compromised in terms of their overall health status. With the increase in the number of patients whose immune systems are compromised, through underlying illness, chemotherapy, transplantation, AIDS, or age, we are much more likely to see increasing numbers of opportunistic infections, including those caused by parasites. Also, we continue to discover and document organisms that were thought to be nonpathogenic that, when found in the compromised host, can cause serious disease [8,20,21].

Control of parasitic infections depends on a number of different factors, including geographic location, public health infrastructure, political stability, available funding, social and behavioral customs and beliefs, trained laboratory personnel, healthcare support teams, environmental constraints, poor understanding of organism life cycles, and opportunities for control and overall commitment. Vectors and other carriers of infectious agents do not recognize political or control boundaries, both of which become meaningless. When newer infectious agents and/or diseases are recognized, there is often very little information available regarding the organism life cycle, potential reservoir hosts, and environmental requirements for survival. Priorities may change, and in areas of the world where disease epidemiology was considered important in the past, these important issues may have been moved lower on the priority list. Unfortunately, funding often plays a major role in decisions that impact disease control measures.

As new etiologic agents are discovered and the need for new therapeutics increases, more sensitive and specific diagnostic methods will become mandatory in order to assess the efficacy of newer drugs and alternative therapies.

15.1 Diagnostic parasitology testing

15.1.1 General comments

Diagnostic procedures in the field of medical parasitology require a great deal of judgment and interpretation and are generally classified by the Clinical Laboratory Improvement Amendments of 1988 (CLlA ’88) as high-complexity procedures.

Very few procedures can be automated, and organism identification relies on morphologic characteristics that can be very difficult to differentiate. Although morphology can be learned at the microscope, the amount of training to reach proficiency, and eventually mastery, is extensive. Knowledge about the life cycle, epidemiology, infectivity, geographic range, clinical symptoms, range of illness, disease presentation depending on immune status, and recommended therapy are critical to the operation of any laboratory providing diagnostic services in medical parasitology. As laboratories continue to downsize and reduce staff, cross-training will become more common and critical to financial success. Maintaining expertise in fields such as diagnostic parasitology will become more difficult, particularly when standard manual methods are used. Also, the lower the positive rate for parasitic infections (i.e., the prevalence of disease), the more likely we are to generate both false-positive and false-negative laboratory reports. It is difficult to maintain proficiency when positive specimens are rarely encountered. It is important for members of the healthcare team to thoroughly recognize those areas of the clinical laboratory that require experienced personnel and why various procedures are recommended above others. Healthcare delivery settings where physicians provide parasitology diagnostic testing may occasionally provide simple test results (CLlA ’88 waived tests) based on wet mount examinations. However, in spite of the CLIA classification of these diagnostic methods, wet mount examinations are often very difficult to perform and are not as thorough as a full examination, which includes a permanent stained smear. The key to performance of diagnostic medical parasitology procedures is formal training and experience. As the laboratory setting changes during the early part of the 21st century, it is important to recognize that these changes will continue to require a thorough understanding of the skills required to perform diagnostic parasitology procedures and the pros and cons of available diagnostic methods. Laboratories will continue to have a number of diagnostic options: whatever approach is selected by an individual laboratory, it is important that the clinical relevance of the approach be thoroughly understood and conveyed to the client user of the laboratory services.

The majority of diagnostic parasitology procedures can be performed either within the hospital setting or in an offsite location. There are very few procedures within this discipline that must be performed and reported on a short turnaround time (STAT) basis. Two procedures fall into the STAT category: request for examination of blood films for the diagnosis of malaria and examination of cerebrospinal fluid for the presence of free-living amebae, primarily Naegleria fowleri. Any laboratory providing diagnostic parasitology procedures must be prepared to examine these specimens on a STAT basis 7 days a week, 24 h a day [20,28,45]. Unfortunately, these two procedures can be very difficult to perform and interpret; cross-trained individuals with little microbiology training or experience will find this work difficult and subject to error. It has been well documented that automated hematology instrumentation lacks the sensitivity to detect malaria infections, particularly since most patients seen in an emergency room have a very low parasitemia [28]. However, even a low parasitemia can be life threatening in an infection with Plasmodium falciparum or Plasmodium knowlesi.

Diagnostic laboratories outside of the hospital setting are also appropriate settings for this type of diagnostic testing; the test requests, for the most part, are routine and are batch tested, rather than tested singly. With very few exceptions, STAT requests are not relevant and/or sent to such laboratory locations and do not require immediate testing and reporting. Point-of-care testing within the hospital (ward laboratories, intensive care units, emergency rooms, and bedside) is usually not considered appropriate sites for diagnostic parasitology testing; one exception might be the emergency room, where patients with malaria may first present with fever and general malaise. Alternative testing sites (outpatient clinics, shopping malls, senior citizen groups, and others) are generally not considered appropriate settings for diagnostic parasitology testing, although relevance might be dictated by geographic location, particularly outside of the United States, and by the development of newer, less subjective methods. The majority of physician office laboratories are not involved in diagnostic parasitology testing; however, as more molecular (nonmicroscopic) methods are developed, they may become more widely used in this setting.

15.1.2 Specific options

15.1.2.1 Diagnostic tests

The selection and use of diagnostic procedures depend on a number of factors, including geographic area, population served, overall positivity rate, client preference, number of test orders, staffing, personnel experience, turn-around time requirements, epidemiology considerations, clinical relevance of test results, and cost. Diagnostic tests generally have a wide range in sensitivity and specificity. As an example, the ova and parasite (O&P) examination (direct wet mount, concentration, and permanent stained smear) could be considered a screening method for the detection of a number of different intestinal protozoa and helminth infections; this procedure is moderately sensitive and highly specific when performed by expert microscopists. Molecular test methods tend to be very specific (generally for a single organism such as Giardia lamblia) and often more sensitive than the routine O&P examination. However, the test results are limited in scope; either the organism is present or it is not, and none of the other possible etiologic agents can be ruled in or out! In certain situations a laboratory may offer tests on request; an example would be testing for the presence of Cryptosporidium spp. If a potential waterborne outbreak were suspected, the laboratory might change its approach and begin testing all stool specimens submitted for an O&P examination rather than testing only those specimens accompanied by a specific test request. These decisions require close communication with other laboratories, water companies, public health personnel, pharmacies (e.g., communication of an increase in the purchase of antidiarrheal over-the-counter medications), and physicians.

It is critical that laboratory clients be educated regarding appropriate test ordering protocols; tests are ordered based on patient history and symptoms (Table 15.1). These ordering guidelines can be extremely helpful for the physicians and the laboratory.

Table 15.1 Stool testing order recommendations (for physicians)

Patient and/or situation	Test ordered*	Follow-up test ordered
Patient with diarrhea and AIDS or other cause of immune deficiency	Cryptosporidium or Giardia/Cryptosporidium immunoassay	If immunoassays are negative and symptoms continue, special tests for microsporidia (modified trichrome stain) and other coccidia (modified acid-fast stain) and O&P exam should be performed
Potential waterborne outbreak (municipal/city water supply)	Cryptosporidium or Giardia/Cryptosporidium immunoassay
Patient with diarrhea (nursery school, day-care center, camper, backpacker)	Giardia or Giardia/Cryptosporidium immunoassay (perform testing on two stools before reporting as negative) Particularly relevant for areas of the United States where Giardia most common organism found	If immunoassays are negative and symptoms continue, special tests for microsporidia and other coccidia (see above) and O&P exam should be performed
Patient with diarrhea and potential waterborne outbreak (resort setting)
Patient with diarrhea from areas where Giardia is the most common parasite found
Patient with diarrhea and relevant travel history	O&P exam, E. histolytica/E. dispar immunoassay; immunoassay for confirmation of E. histolytica; various tests for Strongyloides may be relevant (even in the absence of eosinophilia)	If exams are negative and symptoms continue, special tests for coccidia and microsporidia should be performed
Patient with diarrhea who is a past or present resident of a developing country
Patient in an area of the United States where parasites other than Giardia are found
Patient with unexplained eosinophilia and possible diarrhea; if chronic, patient may also have history of respiratory problems (larval migration) and/or sepsis or meningitis (hyperinfection)	Although the O&P exam is a possibility, the agar plate culture for Strongyloides stercoralis (more sensitive than the O&P exam) is recommended	If tests are negative and symptoms continue, additional O&P exams and special tests for microsporidia and other coccidia should be performed
Patient with diarrhea (suspected food-borne outbreak)	Test for Cyclospora cayetanensis (modified acid-fast stain, autofluorescence)	If tests are negative and symptoms continue, special procedures for microsporidia and other coccidia and O&P exam should be performed

* Depending on the particular immunoassay kit used, various single or multiple organisms may be included. Selection of a particular kit depends on many variables: clinical relevance, cost, ease of performance, training, personnel availability, number of test orders, training of physician clients, sensitivity, specificity, equipment, time to result, etc. Very few laboratories will handle this type of testing exactly the same. Many options are clinically relevant and acceptable for good patient care. It is critical that the laboratory report indicate specifically which organisms could be identified using the kit; a negative report should list the organisms relevant to that particular kit.

15.1.2.2 Routine methods

Routine methods may also be screening methods; however, this is not always the case. “Routine” can imply a widely used, well-understood laboratory test; it can also imply a low or moderately complex method, rather than a high complexity procedure. Routine diagnostic parasitology procedures could include the O&P exam, preparation and examination of blood films and pinworm tapes and/or paddles, occult blood tests, and examination of specimens from other body sites (urine, sputum, duodenal aspirates, urogenital sites, etc.).

Diagnostic parasitology includes laboratory procedures that are designed to detect organisms within clinical specimens using morphologic criteria and visual identification. Some clinical specimens, such as those from the intestinal tract, contain numerous artifacts that complicate the differentiation of parasites from surrounding debris. Final identification is usually based on light microscopic examination of stained preparations, often using high magnification techniques such as oil immersion (1000×) [20,38,39,42].

Specimen preparation often requires some type of concentration, all of which are designed to increase the chances of finding the organism(s). Microscopic examination requires review of the prepared clinical specimen using multiple magnifications and different examination times; organism identification also depends to a great degree on the skill of the microscopist.

Protozoa range from 1.5 μm (microsporidia) to approximately 80 μm (Balantidium coli [ciliate]) in size. Some parasites are intracellular, and multiple isolation and staining methods may be required for identification. Helminth infections are usually diagnosed by finding eggs, larvae, and/or adult worms in various clinical specimens, primarily those from the intestinal tract. Identification to the species level for many human parasites may require microscopic examination of the specimen. The recovery and identification of blood parasites can require concentration, culture, and microscopy. Confirmation of suspected parasitic infections depends on the proper collection, processing, and examination of clinical specimens, and multiple specimens may be required in order to confirm the presence of organisms [12,20,22,24,27,29,35,37,41,44,56,60].

15.1.2.3 Special testing

Special procedures such as parasite culture are performed in a limited number of laboratories. These procedures require the maintenance of positive control cultures used for quality control checks on all patient specimens and special expertise and time often not available in many clinical laboratories [59]. Although some standardized reagents are now commercially available, many clinical laboratories choose to send their request for serologic testing for parasitic disease to other laboratories. Often, the Centers for Disease Control and Prevention (CDC) perform serologic testing on specimens submitted to each state’s Department of Public Health. Generally, specimens for parasitic serologies are not submitted directly to the CDC, but are forwarded to CDC by the state public health laboratories. In an emergency situation, consultation with your city, county, or state public health laboratory may allow shipment of a specimen directly to the CDC.

15.1.3 Diagnostic medical parasitology

15.1.3.1 Equipment

Equipment required for diagnostic parasitology work is very minimal; however, the one expense that is totally justified would be for one or more microscopes with good optics. Each microscope should be equipped with high quality (flat-field) objectives (10×, 40×, 50× or 60× oil immersion, and l00× oil immersion objectives). The oculars should be a minimum of l0×. Depending on the range of immunoassay testing available, a fluorescence microscope or enzyme immunoassay (EIA) reader might be desirable. The availability of this equipment will vary from one laboratory to another and may be shared with other groups within the laboratory. Another option would be a fume hood, in which the staining could be performed; this is not required but is recommended, particularly if the laboratory is still using xylene for dehydration of permanent stained fecal smears. The rest of the equipment is quite common and can be shared with other areas within the laboratory. Equipment would include refrigerators, freezers, pipette systems, and other common items normally found within a clinical laboratory.

15.2 Solicitation of product information

Commercial companies that supply collection systems, stool concentration devices, reagents, test kits, and other relevant products used in the practice of diagnostic medical parasitology were contacted for inclusion in this book on laboratory medicine diagnostic products. Those companies that replied have been included and are presented below. Not all companies provided the same information, and you will see differences as you review the available products used in this field. In some cases, peer reviewed manuscripts on the products have not been published. Based on company responses, the data are current at the time of this writing.

15.3 Specimen collection systems

Various collection systems are available for clinical specimens suspected of containing parasites or parasitic elements (Table 15.2). The selection of collection options should be based on a thorough understanding of the value and limitations of each. The final laboratory results are based on parasite recovery and identification and will depend on the initial handling of the organisms. Unless the appropriate specimens are properly collected and processed, the examination may lead to false-negative results. Specimen rejection criteria have become much more important for all diagnostic parasitology procedures. Laboratory results based on improperly collected specimens may require inappropriate expenditures of time and supplies and may also mislead the physician.

Table 15.2 Body sites and specimen collection*

Source: adapted from references 19 and 20.

Site(s)	Specimen option	Collection method recommendations
Blood	Smears of whole blood	Thick and thin films: fresh or with anticoagulant
Blood	Anticoagulated blood	EDTA (first choice); heparin (second choice)
Bone marrow	Aspirate	Sterile
Central nervous system	Spinal fluid	Sterile
Central nervous system	Brain biopsy specimen	Sterile
Cutaneous ulcer	Aspirates from below surface	Sterile, plus air-dried smears
Cutaneous ulcer	Biopsy specimen	Sterile, nonsterile to histopathology (formalin acceptable)
Eye	Biopsy specimen	Sterile (in saline), nonsterile to histopathology
	Scrapings	Sterile (in saline)
	Contact lens	Sterile (in saline)
	Lens solution	Sterile; unopened commercial solutions not acceptable
Intestinal tract	Fresh stool	Half-pint waxed or plastic container
	Preserved stool	5 or 10% formalin, MIF, SAF, Schaudinn’s fluid (mercury, copper, or zinc base)(with PVA), single vial systems/Universal Fixative (no mercury, no formalin, no PVA)
	Sigmoidoscopy material	Fresh, preserved, or smears submitted in Schaudinn’s fluid
	Duodenal contents	Fresh, preserved, or smears submitted in Schaudinn’s fluid
	Anal impression smear	Entero-Test or aspirates
	Adult worm or worm segments	Cellulose tape (pinworm exam) or other collection device. Saline, 70% alcohol
Liver and/or spleen	Aspirates	Sterile, collected in four separate aliquots (liver)
Liver and/or spleen	Biopsy specimen	Sterile, nonsterile to histopathology
Lung	Sputum	True sputum (not saliva)
	Induced sputum	Sterile, air-dried smears
	Bronchoalveolar lavage	Sterile, air-dried smears
	Tracheobronchial aspirate	Sterile, air-dried smears
	Brush biopsy	Sterile, air-dried smears
	Open lung biopsy	Sterile
	Aspirate	Sterile, air-dried smears
Muscle	Biopsy specimen	Fresh, squash preparation, nonsterile to histopathology (formalin acceptable)
Skin	Scrapings	Aseptic, smear or vial
	Skin snip	No preservative
	BiopsysSpecimen	Sterile (in saline), nonsterile to histopathology
Urogenital system	Vaginal discharge	Saline swab, transport swab (no charcoal), culture medium, plastic envelope culture; air-dried smear for fluorescent antibody
	Urethral discharge	Saline swab, transport swab (no charcoal), culture medium, plastic envelope culture; air-dried smear for fluorescent antibody
	Prostatic secretions	Saline swab, transport swab (no charcoal), culture medium, plastic envelope culture; air-dried smear for fluorescent antibody
	Urine	Single unpreserved specimen (early morning), 24-h unpreserved specimen

* Most products used for specimen collection are available from any major medical supply house.

15.4 Fresh stool specimen collection

Fecal specimens should be collected in clean, wide mouth containers; often a waxed cardboard or plastic container with a tight fitting lid is selected for this purpose. These collection containers are available from most of the microbiology product distributors and are not difficult to locate; there are very minor differences, none of which are critical. Stool specimen containers should be placed in plastic biohazard bags when transported to the laboratory for testing. Specimens should be identified with the following information: patient’s name and identification number, physician’s name, the date and time the specimen was collected (if the laboratory is computerized, the date and time may reflect arrival in the laboratory, not the actual collection time), and the laboratory procedures requested. Although it may be helpful to have information concerning the presumptive diagnosis or relevant travel history, this information is rarely available.

Fresh specimens are required for the recovery of motile trophozoites (amebae, flagellates, or ciliates). The protozoan trophozoite stage is normally found in cases of diarrhea; the gastrointestinal tract contents are moving through the system too rapidly for cyst formation to occur. Once the stool specimen is passed from the body, trophozoites do not encyst but may disintegrate if not examined or preserved within a short time after passage. However, most helminth eggs and larvae, coccidian oocysts, and microsporidian spores will survive for extended periods. Liquid specimens should be examined within 30 min of passage, not 30 min from the time they reach the laboratory. If this general time recommendation of 30 min is not possible, then the specimen should be placed in one of the available fixatives. Soft (semiformed) specimens may have a mixture of protozoan trophozoites and cysts and should be examined within 1 h of passage; if this time frame is not possible, then preservatives should be used. Immediate examination of formed specimens is not as critical; if the specimen is examined any time within 24 h after passage, the protozoan cysts should still be intact (Table 15.3) [20–22,41,44].

Table 15.3 Stool collection: pros and cons of fresh and preserved specimens*

Stool type	Pros	Cons
Fresh	No requirements for stool fixatives. Ability to see motile trophozoites. Lower cost. Can perform direct wet exam, concentration and permanent stained smear (O&P), fecal immunoassays, special stains for coccidia and microsporidia. Relevant only if time from stool passage to laboratory is acceptable; in symptomatic patients, the trophozoite form of the intestinal protozoa is present and will not encyst when outside of the body: liquid or watery stool – 30 min semiformed – 1 h formed – 24 h	May have excessive lag time between stool passage and fixation or processing; trophozoites may disintegrate, thus giving a false negative result. Ova and parasite examination (direct wet exam, concentration, and permanent stained smear) may be negative due to lack of organism preservation and morphology integrity. Poor results may also result for special stains for coccidia and microsporidia.
Preserved	Organism morphology is preserved when lag time between stool passage and fixation is short, which can be accomplished by allowing the patients to collect and fix stool specimens at home. Once the specimen is mixed with the preservative, the delivery time to the laboratory is not critical. Can perform concentration and permanent stain (O&P), fecal immunoassays, special stains for coccidia and microsporidia. Fixatives now available that contain no mercury, no formalin, and no PVA (universal fixatives)	Cost of collection vials may represent a cost increase; however, may be overall less expensive because of much more accurate result (patient outcome). Disposal of vials may be a problem if the laboratory is using preservatives containing mercuric chloride

MIF, merthiolate–iodine–formalin; SAF, sodium acetate–acetic acid–formalin; PVA, polyvinyl alcohol; FA, fluorescent antibody; O&P, ova and parasite examination.

* Most products used for specimen collection are available from any major medical supply house.

15.5 Preservation of stool specimens

If there are likely to be delays in specimen delivery to the laboratory, fecal preservatives should be used (Table 15.4). To preserve protozoan morphology and to prevent the continued development of some helminth eggs and larvae, the stool specimens can be placed in preservative, either immediately after passage (by the patient using a collection kit) or once the specimen is received by the laboratory (Figure 15.1). There are several fixatives available, and these include formalin, sodium acetate–acetic acid–formalin (SAF), and Schaudinn’s fluid; some of these fixatives will contain polyvinyl alcohol (PVA), a plastic powder that is used as a glue to adhere the stool to the glass slide (Table 15.4). Regardless of the fixative selected, adequate mixing of the specimen and preservative is mandatory (Figures 15.1 to 15.9) [24,27,29,37,56,60]. When selecting one or more stool fixatives, remember that a permanent stained smear is mandatory for a complete examination for parasites. You may also want to perform methods such as fluorescence, EIA, or the rapid cartridge immunoassay devices; however, you will need to confirm that the fixative you are using is compatible with the diagnostic procedures you have selected [1–7,10,11,13,14,16,18,19,23,25,26,30–34,42,48,50,52–55,61]. It is also important to remember that disposal regulations for compounds containing mercury are becoming more restrictive; each laboratory will have to check applicable state and federal regulations to help determine fixative disposal options. Another responsibility may be the need to validate specific protocols and/or kits for uses that are not clearly defined in the package insert.

Table 15.4 Preservatives used in diagnostic parasitology (stool specimens)

Preservative	Concentration	Permanent stained smear	Immunoassays* *(Giardia lamblia, Cryptosporidium* spp.)**	Comments
5% or 10% formalin	Yes	No	Yes	EIA, FA, cartridge
5% or 10% buffered formalin	Yes	No	Yes	EIA, FA, cartridge
MIF	Yes	Polychrome IV stain	ND	ND
SAF	Yes	Iron hematoxylin (best)	Yes	EIA, FA, cartridge
Schaudinn’s fixative^† (mercury base) with or without PVA	Yes, but rarely used	Trichrome or iron hematoxylin	No	PVA interferes with immunoassays
Modified Schaudinn’s^‡ (copper base) with PVA	Yes, but rarely used	Trichrome or iron hematoxylin	No	PVA interferes with immunoassays
Modified Schaudinn’s^§ (zinc base) with PVA	Yes, but rarely used	Trichrome or iron hematoxylin	No	PVA interferes with immunoassays
Single-vial systems^¶ (with or without PVA)	Yes	Trichrome or iron hematoxylin	Some, but not all	Check with the manufacturer
Universal fixative^** (single-vial system)	Yes	Trichrome or iron hematoxylin	Yes	EIA, FA, cartridge; contain no mercury, no formalin, no PVA; does not require albumin for adhesion (no PVA, no albumin); has been validated for some molecular/PCR testing

EIA, enzyme immunoassay; FA, fluorescent antibody; MIF, merthiolate–iodine–formalin; ND, no data; PCR, polymerase chain reaction; PVA, polyvinyl alcohol; SAF, sodium acetate–acetic acid–formalin.

* Fecal immunoassays for the E. histolytica/E. dispar group or the true pathogen, E. histolytica require fresh or frozen specimens; in some cases Cary–Blair may be acceptable (check with the manufacturer).

^† These two fixatives use the mercuric chloride base in the Schaudinn’s fluid; this formulation is still considered to be the “gold standard”, against which all other fixatives are evaluated (organism morphology after permanent staining). Additional fixatives prepared with nonmercuric chloride-based compounds are used; however, the overall organism morphology is not as good.

^‡ This modification uses a copper sulfate base rather than mercuric chloride; organism morphology is marginal.

^§ This modification uses a zinc base rather than mercuric chloride and works well with both trichrome and iron-hematoxylin stains.

^¶ These modifications use a combination of ingredients (including zinc), but are prepared from proprietary formulas. The aim is to provide a fixative that can be used for the fecal concentration, permanent stained smear, and available immunoassays for Giardia lamblia, Cryptosporidium spp.; Entamoeba histolytica (or the E. histolytica/E. dispar group).

** Universal fixatives contain no mercury, no formalin, and no PVA. The complete O&P (concentration, permanent stained smear), fecal immunoassays for G. lamblia and Cryptosporidium spp., and special stains for the coccidia and microsporidia can be performed using these options. Fecal immunoassays for the E. histolytica/E. dispar group or the true pathogen, E. histolytica require fresh or frozen specimens; in some cases Cary–Blair may be acceptable (check with the manufacturer). An example would be TOTAL-FIX (Medical-Chemical Corporation, Torrance, CA), a universal fixative that has also been found to be compatible with a number of molecular test options, including PCR: it may be necessary to validate reagents for test options other than those appearing in the package inserts.