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SUMMARYThis Practical Guidance for Clinical Microbiology document on the laboratory diagnosis of parasites from the gastrointestinal tract provides practical information for the recovery and identification of relevant human parasites. The document is based on a comprehensive literature review and expert consensus on relevant diagnostic methods. However, it does not include didactic information on human parasite life cycles, organism morphology, clinical disease, pathogenesis, treatment, or epidemiology and prevention. As greater emphasis is placed on neglected tropical diseases, it becomes highly probable that patients with gastrointestinal parasitic infections will become more widely recognized in areas where parasites are endemic and not endemic. Generally, these methods are nonautomated and require extensive bench experience for accurate performance and interpretation.

Dientamoeba fragilis is a single-celled protozoan, closely related to the trichomonads. Reported worldwide as causing human gastrointestinal symptoms, D. fragilis is very common and is second only to Blastocystis spp. Dientamoebiasis equals or exceeds the incidence of giardiasis. This minireview includes diagnostic options, clinical relevance, therapy, an animal model, the confirmed cyst stage, and sequencing data. The development of a rodent model, fulfilling Koch's postulates, and the confirmation of a cyst stage have clarified transmission routes, including fecal-oral transmission. The prevalence of D. fragilis varies between 0% to over 82%; results depend on the geographic location, group studied, and diagnostic methods used.

This chapter discusses various approaches and diagnostic methods currently in use for the diagnosis of parasitic infections. Examination of prepared wet mounts, concentrated specimens, permanent stained smears, blood films, and various culture materials can provide critical information leading to organism identification and confirmation of the suspected cause of clinical disease. The purpose of the concentration method is to separate parasites from fecal debris and to concentrate any parasites present through either sedimentation or flotation. Wet mounts prepared from concentrated stool are examined in the same manner as that used for the direct wet mount method. Although the routine ova and parasite examination consisting of the direct wet mount, the concentration, and the permanent stained smear is an excellent procedure recommended for the detection of most intestinal parasites, several other diagnostic techniques are available for the recovery and identification of specific parasitic organisms. The only human parasites for which it is reasonably possible to correlate egg production with adult worm burdens are Ascaris lumbricoides, Trichuris trichiura, and the hookworms (Necator americanus and Ancylostoma duodenale). Several parasites may be recovered and identified from urogenital specimens. Although, the most common pathogens are probably Trichomonas vaginalis and Schistosoma haematobium, other organisms such as the microsporidia are becoming much more important. Although other tests such as immunoassay diagnostic kits continue to become available commercially, the majority of medical parasitology diagnostic work depends on the knowledge and microscopy skills of the microbiologist.

Plasmodium vivax and Plasmodium falciparum are responsible for 95% of malaria infections. P. vivax has the widest distribution and is thought to be responsible for 80% of the infections throughout the tropics, subtropics, and temperate zones. As early as 2004, a fifth malaria species had been implicated in human disease. Plasmodium knowlesi , a malaria parasite of long-tailed macaque monkeys, has been confirmed in a number of human cases from Malaysian Borneo, Thailand, Myanmar, and the Philippines. It is now well established that P. knowlesi is emerging as an important zoonotic human pathogen. Malaria is considered to be immediately life-threatening, and a patient with the diagnosis of P. falciparum or P. knowlesi malaria should be considered a medical emergency because the disease can be rapidly fatal. While rapid malaria tests have been used in many parts of the world, currently one test is now FDA approved for use within the United States. The pros and cons of this rapid procedure compared with the "gold standard" thick and thin blood films are discussed for all five species of human malaria.

This chapter deals with various specimen collection, transport, and processing methods for detection of parasites. Various specimen collection methods are available for specimens suspected of containing parasites or parasitic elements. The most common specimen submitted to the diagnostic laboratory is the stool specimen, and the most commonly performed procedure in parasitology is the ova and parasite (O&P) examination, which is composed of three separate protocols: the direct wet mount, the concentration, and the permanent stained smear. The examination of aspirated material for the diagnosis of parasitic infections may be extremely valuable, particularly when routine testing methods have failed to demonstrate the organisms. These specimens should be transported to the laboratory immediately after collection. Detection of parasites in tissue depends in part on specimen collection and on having sufficient material to perform the recommended diagnostic procedures. The nucleic acid-based diagnostic tests have been developed for almost all species of parasites. The main reason for the minor role of diagnostic PCR in parasitology is the fact that many parasite stages can be adequately diagnosed using established, more traditional techniques that are generally less expensive than PCR and technically less demanding. Diagnostic PCR may become more widespread when simple, fully standardized test kits are available and costs are reduced through the implementation of pre- and post-PCR automated techniques. Furthermore, the possibilities to not only detect and identify but also quantify organisms and determine their genotypes by analyzing the diagnostic PCR product extend the diagnostic power of PCR.

Parasitic infections are felt by most individuals to have little impact on the health and well being of most inhabitants of Canada, Western Europe, and the United States. As the authors show in this article, parasites are always "emerging" somewhere and have a significant impact on those areas of the world. Moreover, as we are becoming an ever-smaller global village, catastrophes and instability in the Third World affect control of parasitic diseases endemic to those areas, ensuring greater chances of transmission to visitors there. The foundation of successful limitation of parasitic diseases in both developing and developed regions is still accurate and rapid diagnosis.

The microsporidia belong to the phylum Microspora within the taxonomic group Protozoa; there are more than 140 genera and 1,200 species that are parasitic in all major animal groups. Already, seven genera (Enterocytozoon, Encephalitozoon, Nosema, Pleistophora, Vittaforma, Trachipleistophora, and Brachiola) and some that are unclassified have been confirmed to cause human infection. Septata intestinalis, an organism that was identified in 1994 as belonging to a new genus, i.e., Septata (Encephalitozoon-like), has been reclassified as Encephalitozoon intestinalis, although there is not universal agreement on this generic change. The genus Microsporidium is essentially a catch-all genus for organisms that have not yet been classified (or may never be classified due to a lack of specimen). Depending on acceptance of these generic designations, there may be seven or nine microsporidial genera. The microsporidia are obligate intracellular, spore-forming protists with no active metabolic stages outside of the host cell. The life cycle involves a proliferative merogonic stage followed by sporogony, which results in spores containing a tubular extrusion apparatus (polar tubule) for injecting infective spore contents into the host cell (Fig. ​(Fig.11). FIG. 1. Life cycle of microsporidia. Reprinted from reference 9. Because microsporidia have a membrane-bound nucleus, an intracytoplasmic membrane system, and chromosome separation on mitotic spindles, they are considered to be true eukaryotes. However, they are somewhat unusual in that they have 70S ribosomes, have no mitrochondria or peroxisomes, and have simple Golgi membranes. Also, the microsporidial genome is smaller and less complex than those of other eukaryotes. Several characteristics, including the presence of chitin in the spore wall, suggest a potential link to the fungi. Microsporidiosis, a disease resulting from infection with these important emerging opportunistic organisms, has been noted in human immunodeficiency virus (HIV)-infected patients, as well as in other immunocompromised patients, including transplant recipients. The first human case was reported in 1959; since then, cases of microsporidiosis in both those who are immunocompromised and those who have no underlying immune problems and are considered to be immunocompetent have been reported (Table ​(Table1)1) (2, 7, 21). TABLE 1. Microsporidia causing human infection

Microecol. and Ther. 25:63-71. 26. DeZoysa, A. et al. 1995. Further studies on the molecular epidem_iology of toxigenic and non-toxigenic Corynebacterium diphtheriae from UK and Europe using ribotyping and pulsed-field gel electrophoresis. 2rid Int. Meet. European Lab. Working Group on Diphtheria, Helsinki, Finland, June 18-20. 27. Centers for Disease Control and Prevention. 1995. Diphtheria acquired by U.S. citizens in the Russian Federation and Ukraine1994. Morbid. Mortal. Weekly Rep. 44:237, 243-244. 28. Anonymous. 1997. Diphtheria acquired during a cruise in the Baltic Sea. Communicable Dis. Rep.: CDR Weekly 7:207. 29. Anonymous. 1997. Diphtheria acquired during a cruise in the Baltic Sea: update. Communicable Dis. Rep: CDR Weekly 7:217. 30. Nakao, H. et al. 1997. Analysis of heterogeneity of Corynebacterium diphtheriae toxin gene, tox, and its regulatory element, dtxR, by direct sequencing. Res. Microbiol. 148:45-54. 31. Popovic, T. et al. 2000. Molecular epidemiology of diphtheria. J. Infect. Dis. 181(Suppl. 1):S168-S177.

Summary Based on a number of variables discussed above, microbiologists performing diagnostic parasitology testing on stool specimens must decide on the appropriate test methodologies that are relevant for their own laboratories. It is neither realistic nor clinically relevant to assume or state that one approach is applicable to every diagnostic laboratory. Confirm that you understand the pros and cons of the various options and that you have conveyed this information clearly to your clients once your approach has been selected for implementation. Careful selection of testing algorithms for specific laboratory and client requirements will help ensure quality patient care and excellent client communication regarding test results.

The Triage parasite panel (BIOSITE Diagnostics, San Diego, Calif.) is a new qualitative enzyme immunoassay (EIA) panel for the detection of Giardia lamblia, Entamoeba histolytica/E. dispar, and Cryptosporidium parvum in fresh or fresh, frozen, unfixed human fecal specimens. By using specific antibodies, antigens specific for these organisms are captured and immobilized on a membrane. Panel performance was evaluated with known positive and negative stool specimens (a total of 444 specimens) that were tested by the standard ova and parasite (O&P) examination as the “gold standard,” including staining with both trichrome and modified acid-fast stains. Specimens with discrepant results between the reference and Triage methods were retested by a different method, either EIA or immunofluorescence. A number of samples with discrepant results with the Triage device were confirmed to be true positives. After resolution of discrepant results, the number of positive specimens and the sensitivity and specificity results were as follows: for G. lamblia, 170, 95.9%, and 97.4%, respectively; for E. histolytica/E. dispar, 99, 96.0%, and 99.1%, respectively; and for C. parvum, 60, 98.3%, and 99.7%, respectively. There was no cross-reactivity with other parasites found in stool specimens, including eight different protozoa (128 challenges) and three different helminths (83 challenges). The ability to perform the complete O&P examination should remain an option for those patients with negative parasite panel results but who are still symptomatic.

This article includes information on two human parasites, one protozoan flagellate, Giardia lamblia, and one ciliate, Balantidum coli. Both are transmitted through ingestion of food and water contaminated with fecal material. G. lamblia may be the most common intestinal protozoan found in humans throughout the world and causes a wide range of symptoms, all of which can be confused with other infectious and noninfectious causes. Although B. coli tends to be more restricted and associated with pigs as potential reservoir hosts, this organism can also cause mild to severe symptoms and can be found throughout the world.

It is well known that Giardia lamblia and Cryptosporidium parvum can cause severe symptoms in humans, particularly those who are immunologically compromised. Immunoassay procedures offer both increased sensitivity and specificity compared to conventional staining methods. These reagents are also helpful when screening large numbers of patients, particularly in an outbreak situation or when screening patients with minimal symptoms. The data obtained by using 9 diagnostic kits were compared: direct fluorescent-antibody assay (DFA) kits (TechLab Giardia/Crypto IF kit, TechLab Crypto IF kit, and Meridian Merifluor Cryptosporidium/Giardia) and enzyme immunoassay (EIA) kits (Alexon ProSpecT Giardia EZ Microplate Assay, Alexon ProSpecT Cryptosporidium Microplate Assay, Cambridge Giardia lamblia Antigen Microwell ELISA, Meridian Premier Giardia lamblia, Meridian Premier Cryptosporidium, TechLab Giardia CELISA, Trend Giardia lamblia EIA). The test with the Meridian Merifluor Cryptosporidium/Giardia kit was used as the reference method. In various combinations, 60 specimens positive for Giardia, 60 specimens positive for Cryptosporidium, 40 specimens positive for a Giardia-Cryptosporidium mix, and 50 negative fecal specimens were tested. Different species (nine protozoa, three coccidia, one microsporidium, five nematodes, three cestodes, and one trematode) were included in the negative specimens. The sensitivity of EIA for Giardia ranged from 94% (Alexon) to 99% (Trend and Cambridge); the specificity was 100% with all EIA kits tested. The sensitivity of EIA for Cryptosporidium ranged from 98% (Alexon) to 99% (Meridian Premier); specificities were 100%. All DFA results were in agreement, with 100% sensitivity and specificity; however, the TechLab reagents resulted in fluorescence intensity that was generally one level below that seen with the reagents used in the reference method. In addition to sensitivity and specificity, factors such as cost, simplicity, ease of interpretation of results (color, intensity of fluorescence), equipment, available personnel, and number of tests ordered are also important considerations prior to kit selection.

This chapter includes comprehensive information on intestinal protozoan pathogens not covered in other chapters, including the ciliate, Balantidium coli; the flagellate, Dientamoeba fragilis; the ameba, Entamoeba polecki, and a member of the Stramenopila, Blastocystis spp. These parasites are most often transmitted through food, water, or other materials that are contaminated with fecal material. Most produce non-specific intestinal symptoms, some of which can be as severe as those seen with giardiasis. Diagnosis requires microscopic identification of the trophozoites, cysts, or other stages present, which are usually identified on a permanent stained fecal smear, one of the key components of the ova and parasite examination. Fecal immunoassays and molecular methods are becoming available for these organisms; however, molecular options remain somewhat limited. Treatment varies by organism. Prevention rests on limiting fecal–oral transmission.