Sexually transmitted diseases. Manson's tropical diseases, 22nd ed. These report comments are extremely helpful in the clarification of reports for physicians in terms of pathogenicity and clinical relevance (5). For decolorizer, the options range from 1 to 10% H2SO4 with or without alcohol to the use of 1 to 3% HCl in alcohol. It should also be noted that although the WHO has recommended that intestinal E. histolytica infection be diagnosed with a specific test, such as E. histolytica-specific immunoassays (180), these methods have been validated only for fecal specimens and, as such, should not be used without additional validation for mucosal specimens collected after a sigmoidoscopy. The Gram-positive nature of microsporidium spores suggests that the chitin wall structure is perhaps analogous to the dense cross-linked peptidoglycan layers of Gram-positive bacteria. Since Panmede (a liver digest and a component of the TPS-1 medium) was no longer available and liver digest from other sources did not support good growth of Giardia, efforts were made to come up with a medium that supported the growth of not only Giardia but other protists, including E. histolytica. Inappropriate orders have several adverse consequences. The document is based on a comprehensive literature review and expert consensus on relevant diagnostic methods. The same types of criteria are used for protozoans depending on their stage. (70) was also referred to as a modified acid-fast stain. Foremost, the usefulness of this type of technology to fully characterize the microbiomes of individuals with a particular disease, such as gastroenteritis, provides the opportunity to fully characterize all the microorganisms present in an attempt to determine the presence and type of pathogens, regardless of their taxonomic position. Helminthiases. ), the opportunity for intermolecular interactions increases significantly with each additional primer and probe set added to the mixture. Thank you for sharing this Clinical Microbiology Reviews article. She has given over 400 presentations (international, national, and local) and published over 175 manuscripts, book chapters, and articles and is the author of Diagnostic Medical Parasitology (5) and Practical Guide to Diagnostic Parasitology (50), published by ASM Press, Washington, DC. Strongyloides stercoralis, Mini-FLOTAC and Kato-Katz: helminth eggs watching on the shore of Lake Victoria, Mini-FLOTAC, an innovative direct diagnostic technique for intestinal parasitic infections: experience from the field, Mini-FLOTAC, Kato-Katz and McMaster: three methods, one goal; highlights from north Argentina, Comparing diagnostic accuracy of Kato-Katz, Koga plate, ether-concentration, and FLOTAC for Schistosoma mansoni and soil-transmitted helminths, Comparison of the Kato-Katz and FLOTAC techniques for the diagnosis of soil-transmitted helminth infections, Diagnostic accuracy of Kato-Katz and FLOTAC for assessing anthelmintic drug efficacy, The unreliability of the Kato-Katz technique limits its usefulness for evaluating, A comparison of the sensitivity and fecal egg counts of the McMaster egg counting and Kato-Katz thick smear methods for soil-transmitted helminths, Field evaluation of an improved Kato-Katz thick smear technique for quantitative determination of helminth eggs in faeces, Independent evaluation of the Nigrosin-Eosin modification of the Kato-Katz technique, Basic laboratory methods in medical parasitology, p 25–28, A new miracidia hatching device for diagnosing schistosomiasis, District laboratory practice in tropical countries, part 1, p 238–239, The measurement of schistosome populations, International Academy of Pathology, Special Monograph, Euparal as a permanent mounting medium for helminth eggs and proglottids. This process is twice repeated with the fecal sediment obtained. Practical guide to diagnostic parasitology, 2nd ed. Laboratory-Developed Tests for Gastrointestinal Parasites, MonoplexWhether a molecular assay for a particular parasite is clinically useful (i.e., it represents a practical assay for implementation in a clinical laboratory and may improve turnaround time) is a question that will ultimately be determined in laboratories across the country. They reported an 83% sensitivity and 100% specificity for this approach and recommend it as a possible screen for enteric parasites. The first report of human illness came in 1976 with the identification of Cryptosporidium in rectal biopsy specimens of a 3-year-old child (68). can also be grown in Diamond's TP-S-1 or Balamuth's medium, although growth is not as good as that in the egg slant medium. E. cuniculi, E. hellem, E. intestinalis, and Anncaliia algerae have been cultured from urine, sputum, and corneal smears and cryopreserved indefinitely (220). compared this assay to conventional methods to study the etiologic agents of severe diarrhea in renal transplant recipients (270). For example, a Giardia-specific PCR would be appropriately negative for a patient whose stool O&P disclosed Cystoisospora as the causative agent of infection. John DT, Petri WA. Further impetus for study of Cryptosporidium came from the veterinary world with the description of Cryptosporidium meleagridis in turkey poults in Scotland in 1955 (65). The latter are usually acid fast, so Ziehl-Neelsen staining can assist identification; other schistosome eggs, such as those of S. mansoni and Schistosoma mekongi, also exhibit an acid-fast shell outline (173). Although numerous papers have disclosed excellent sensitivity and overall performance for many of these assays, there has been limited adoption in clinical microbiology laboratories for a number of reasons. It is also important to remember that some state public health laboratories require that stools positive for certain organisms (Cryptosporidium) be submitted for confirmatory testing and typing. In addition to all the challenges encountered with monoplex assays (e.g., inhibitors, primer and probe design, etc. Generally, these methods are nonautomated and require extensive bench experience for accurate performance and interpretation. Parasitic These recent figures illustrate that infection rates in developing countries have been significantly underestimated. Life cycles of the various parasites are available on the www.CDC.gov/dpdx website complete with detailed descriptions to help understand the complexity of the individual life cycles. Microscope for general use.Good-quality microscopes and light sources are required for the examination of specimens for parasites. Airflow is generally controlled by a movable sash and should be in the range of 80 to 120 ft/min (1 ft = 30.48 cm). There are specific criteria that are used to identify the trophozoite and cyst forms of amoebae. (203) cultured Giardia in TPS-1 medium by directly inoculating the parasites from the human intestines. The proglottids of tapeworms are the specimens required for identification purposes, as the eggs of all three species are indistinguishable without special procedures. These infections usually manifest with some degree of abdominal pain, bloating, and diarrhea. It is also mandatory that the lens of any oil immersion objective be cleaned with lens paper after each use. Rondanelli EG, Scaglia M. 1993. Between 1983 and 2009, approximately 64% of MLS programs were closed (42, 46). 1). recommend caution when interpreting positive signals in high-prevalence settings, suggesting that these may be secondary to asymptomatic carriage or residual DNA from previous infections (258). Although these methods have been useful because of exquisite sensitivity and the ability to genotype the parasites, Frickmann et al. Colonoscopy and CT angiogram of abdomen … Contemporaneously within our clinical service, the histology laboratory tasked with staining intestinal biopsy specimens found this stain superior to modified Gram stains for the detection of Enterocytozoon bieneusi in enterocytes. Techniques for the recovery and identification of Cryptosporidium oocysts from stool specimens. Cysticercosis of the human nervous system. The classic example is that of pernicious anemia caused by vitamin B12 deficiency that results from the absorption of this nutrient by the large fish tapeworm, Diphyllobothrium latum (18). An important advantage of in vitro culture, especially for axenic cultures, is its provision of a continuous supply of pure organisms without any interfering bacterial, fungal, or host tissue contamination. Roberts LS, Janovy J, Nadler S. 2012. Microscopy detected the presence of eight specimens positive for helminths, whereas the multiplex assay detected 46 (P < 0.001). Only one EIA kit and one rapid test are specific for Entamoeba histolytica; additional kits are due to be released in the near future. Perhaps the most concerning challenge is the likely associated loss of microscopic morphological expertise when the majority of testing is converted from traditional to molecular assays. The Williams & Wilkins Co, Baltimore, MD. Contrary to the finding of van Gool et al. The examination of sigmoid colon biopsy specimens is also considered a sensitive test for the rapid diagnosis of S. mansoni and S. japonicum infections, particularly if fecal testing is negative yet clinical suspicion remains high (181). Eldridge BF, Edman JD (ed). Monoplex Cryptosporidium species PCR.As with Giardia, a variety of molecular assays have been described for a number of applications. Parasites in human tissues. Once again the spores were described as having clear zones or a belt-like stripe girding the spores diagonally or equatorially. Strongyloides larvae in a wet preparation move and may demonstrate thrashing motility under a 10× objective magnification. Cook GC, Zumla A (ed). The stain is easy to perform and allows detection of protozoan trophozoites and cysts, white blood cells, red blood cells, Charcot-Leyden crystals, yeasts, and fecal debris. Blastocystis: pathogen or passenger? A commonly used culture medium for Blastocystis spp. ASM Press, Washington, DC. 1976. The sensitivities and specificities of the morphological assessment for the four pathogens were, respectively, as follows: 50% and 100% for Giardia, 38% and 99% for D. fragilis, 47% and 97% for E. histolytica, and 56% and 100% for Cryptosporidium. Safranin staining.An additional method used for identifying Cryptosporidium is the Safranin stain, described by Baxby et al. Also, the OSHA regulations require that employers provide hepatitis B vaccination and postexposure evaluation and follow-up, communicate the hazards to employees, and maintain appropriate records (40). Ash LR, Orihel TC. This 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. Apart from cost, a limitation of this test is the requirement that the fecal smear must be processed or cleared in some way to ensure that fluorescence can easily be detected. A wet preparation is made from a drop of the fluid, and the complete coverslip area is examined for parasites. Since the O&P is a labor-intensive, multistep test that requires a skilled microscopist, other test options have been developed. He discovered Balamuthia mandrillaris as the agent of meningoencephalitis in humans and other animals, discovered Naegleria fowleri as the agent of primary amoebic meningoencephalitis in animals, and identified Sappinia as the agent of a human brain infection. Commercial vendors have responded to the needs of the clinical laboratory through the development of syndromic test panels. and internal features (hooklets, fibrils, yolk cells, miracidia, or other larvae) (5, 47, 48). As the world continues to “shrink” in terms of exposure to infectious diseases, it becomes much more likely that patients with gastrointestinal parasitic infections will be seen in areas where parasites are not endemic and will continue to increase in number in areas where they are endemic. He did residency training in Anatomic and Clinical Pathology training at Duke University Medical Center and a Clinical Microbiology Fellowship at the Mayo Clinic. How the parasite leaves its definitive host to return to the environment. and Monocercomonas spp. In addition to a variety of PCR-based methods, other methods of nucleic acid amplification, such as loop-mediated isothermal amplification (LAMP), have been used to detect E. histolytica (245). Current fecal immunoassays for the Entamoeba histolytica/Entamoeba dispar group and Entamoeba histolytica are limited to fresh or frozen fecal specimens or Cary-Blair transport medium. Rapid trichrome stains.After the initial reports of the validity of trichrome staining for Microsporidia, further modifications to these approaches have been developed. Compared to the direct smear or FEA concentrate technique, the Baermann method increases the sensitivity of detection of S. stercoralis by 3.6 to 4 times (153). are very similar, it is important that an accurate distinction is made, as the sequelae of T. saginata and T. asiatica infection are of lesser significance. Formalin, sodium acetate-acetic acid-formalin (SAF), mercuric chloride polyvinyl alcohol (PVA), modified (nonmercury) PVA, and nonformalin, nonmercury, non-PVA preservatives are commercially available (5, 57–59) (Table 4). The saline suspension is then decanted, replaced with chlorine-free water, and added to a 500-ml sidearm flask so that the water rises 20 to 30 mm vertically in the side arm (the eggs will not hatch in saline). When choosing which method is best for your laboratory, parameters such as test performance characteristics, skill level of technologists, availability of equipment, volume of requests, specimen collection requirements, and kit cost should be considered. 2004. Concentration of Cystoisospora and Cyclospora.Commentary about the recovery of Cystoisospora and Cyclospora is more limited than for Cryptosporidium due largely to the lower frequency of detection. Clin Microbiol Rev. Nematodes, intestinal: Helminthic parasites; roundworms. The fluorescence associated with oocysts was much stronger than the dull, background staining of bacteria within these smears. It exploits the fact that most Microsporidia spores stain Gram-positive to various degrees. CRC Press Inc, Boca Raton, FL. The waterborne outbreak in Milwaukee, WI, that affected 400,000 people has been attributed to human feces (C. hominis from the sewage treatment plant) entering the drinking water system after a failure of the water treatment plant filtration system (30, 75). The developed assays commonly target the small-subunit rRNA, which contains both conserved regions for broad-range primer hybridization and taxonomically unique regions that may be used for species-level differentiation. Schistosome eggs will appear as refractile bodies, often in clusters. Incidental human infections with Gongylonema are rare, and species-level identifications are difficult and seldom confirmed. The specimen must also be accompanied by a request form indicating which laboratory procedures should be performed. The immense significance of Tyzzer's discovery was not appreciated for many decades. Therefore, when investigating the cause of a likely infectious diarrheal syndrome in an immunocompromised host, both common and less common agents should be considered (5, 36). Stensvold et al. An often-cited example is that of the drinking water sources in Milwaukee, WI, in 1993 that were contaminated with Cryptosporidium hominis from human sewage effluent that impacted the drinking water facility intake, rather than water runoff from bovine feces (30). Molecular methods have been used to study and clarify the epidemiology of cryptosporidiosis. The advantage of this stain lies in the rapidity of the test and its applicability to examination of tissues where there is perhaps less likelihood of contaminating flora. Fortunately, advances in specimen preparation (i.e., extraction methods), more-robust PCR (i.e., with DNA polymerases that are less susceptible to inhibition), and advances in PCR design software have afforded the creation of clinically useful multiplex PCR assays for enteric pathogens. The trophozoite stage is normally found in cases of diarrhea. Place the proglottid onto absorbent paper, and with a tuberculin-style syringe fitted with a 25- or 26-gauge needle, carefully inject India ink through the genital pore (which is a discrete protuberance situated about half way along one side of the segment) so that it ramifies throughout the uterine arms but not so forcefully that it ruptures into the body of the proglottid. Using 10% formalin and centrifugation at 500 × g for 2 min, the number of oocysts recovered by FEA represented a loss of 51.2% of expected numbers for liquid stool and 93.2% for the formed stool. Unless it has floated off, do not attempt to reposition it, as it will be fragile and likely to tear. Helminth egg counting is an inexact science, with many factors contributing to variations and errors in eggs per gram (EPG). This medium consisted of buffered salt solution, casein digest peptone, yeast extract, gastric mucin, heat-inactivated bovine serum, and rice starch in screw-cap tubes. Sherman IW. ASM Press, Washington, DC. Tropical medicine and parasitology classic investigations. In 1983, first reports describing Cryptosporidium in intestinal biopsy specimens of AIDS patients were seemingly in agreement with this theory. Parasites that are covered in this chapter include helminths and protozoans. Laboratory diagnosis of parasites from the gastrointestinal tract. Training Clinicians Regarding the Diagnosis of Gastrointestinal Tract Parasitic InfectionsAs travel has become more accessible, the rate of tropical infections across the world is expected to increase; more health care professionals both at home and abroad are going to encounter these diseases more often. Semiquantitation scoring of helminth eggs and Blastocystis spp.a. The numbers in the fatty plug exceeded those in the deposit in 25.9% of these. 2011. Fresh or Preserved SpecimensFresh stool specimens are mandatory for the recovery of motile protozoan trophozoites (amoebae, flagellates, ciliates). E. histolytica may cause hepatic amoebic abscesses and, rarely, pleuropulmonary disease, in addition to ulceration in the colon (227). Birkhauser Verlag AG, Basel, Germany. Test Selection and Patient PreparationThe test that is to be requested depends on which infectious agent is suspected. The formalin vial is used for the concentration and fecal immunoassays, while the PVA vial is used for the permanent-stain smear. Use of optical brighteners to detect Cryptosporidium.Harrington (103) made a brief mention of the possible fluorescence of Cryptosporidium in two reports about application of fluorescent brighteners for detection of Microsporidia. Trypan blue staining is another reliable way of determining viability (173). She has extensive international teaching experience in medical laboratory sciences, particularly parasitology. Strongyloides larvae are actively motile in the wet preparation (10× objective), but care must be taken to distinguish embryonating Strongyloides eggs from those of hookworm. Immunodetection of antigens in stool specimens using the monoclonal-antibody-based DFA, EIA, and LFA formats are available through various manufacturers. Four to five drops of bile-stained mucus can be “milked” with thumb and forefinger off the terminal end of the string and expressed into a small tube, or the whole string can be placed into a petri dish, sealed with tape, and sent immediately to a laboratory. Additional stains for Microsporidia and Cyclospora/Cystoisospora/Cryptosporidium may need to be ordered separately, and immunoassays for selected organisms may also not be included in the O&P. This 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. There are no recognized drug susceptibility differences between cryptosporidial isolates or strains (so far), and general treatment (supportive therapy) is universal for all cryptosporidial diarrhea. (125) reported the successful application of calcofluor white, also with Evans blue counterstain, and noted that the method was easy to perform and that spores could even be detected in thicker areas of smears. Formalin fixative.Formalin has been used for many years as an all-purpose fixative that is appropriate for helminth eggs and larvae and for protozoan cysts, oocysts, and spores. Although the enhanced sensitivity of the molecular approach is evident, the importance of a broadly inclusive panel was also evident, as three instances of T. trichiura infections were detected by microscopy, but this organism was not present in the helminth multiplex PCR, so it was missed by that method. To rationalize the approach to screening for opportunistic parasites in AIDS patients, Ignatius et al. There is enormous variation in the choices of recipe used for modified acid-fast stains, ranging from ZN-strength carbol-fuchsin to the stronger Kinyoun reagent, applied with or without heating. Review articles have stressed that any form of infection, even asymptomatic or postsymptomatic, can result in significantly reduced growth rates by the age of 6 to 9 years (78, 79). Despite results being comparable statistically, there was limited overlap of low-positivity results; approximately 20% of positive results were detected by a single method only (84). 1991. Once complete, the proglottid will appear parchment-like on one slide only. A total of 812 additional agents (344 of which as single pathogen) was detected by the FA-GP and not included in the clinical suspicion. Importance of Computer and Test Result CommentsComputer and test result comments can serve as excellent teaching aids for clinicians, many of whom may have little-to-no training in medical parasitology. Laboratory Methods for Diagnosis of Parasitic Infections Overview . Although the Gram-positive property of spores can be exploited in this way, conversely the possibility of misinterpretation of Gram stains from tissue specimens should not be overlooked. (209) reviewed the previous work on the culture media used and compared different culture media for their ability to support the growth of several isolates of D. fragilis, their optimum temperatures for growth, the optimum method for the cryopreservation of the isolates, and important bacterial associates in D. fragilis cultures. If viability is still undetermined, urine (preferably that collected between 10:00 a.m. and 2:00 p.m., when there is a daily peak excretion of eggs) is diluted approximately 1 in 10 with chlorine-free tap water in a large flask, in which miracidial hatching will occur in 10 to 15 min. Other fecal fixatives appropriate for permanent staining are also acceptable. The pancreatitis ranges from mild to severe. Laboratory diagnosis: Direct evidences: Laboratory confirmation of diagnosis of T. sagi­nata infection is made by demonstration of gravid segments and eggs passed in faeces.