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Examination of Blood, Other Body Fluids, Tissues, and Sputum
154.2
Examination of Blood, Other Body Fluids, Tissues, and Sputum Brett E Swierczewski, John H Cross†
EXAMINATION OF FRESH BLOOD Microscopic examination of fresh blood is useful for detection of trypanosomes and microfilariae. A small drop of blood is placed on a slide and covered with a coverglass. The high-dry objective with reduced illumination is suitable for trypanosomes, while low-power is used for detection of microfilariae.
THE THIN FILM Thin and thick blood films are used for species identification of malarial parasites (Chapter 96), trypanosomes (Chapters 97 and 98), and microfilariae (Chapters 110–113). To prepare a thin film, place a small drop of blood from the fingertip, earlobe, or vial of blood near one end of a microscope slide. Raise the end of the slide farthest from the drop of blood and take a second slide as a spreader, making an angle of approximately 30 degrees with the first. Allow the blood to touch the spreader slide and begin to run out toward the edges. Before the blood has a chance to reach the edges, pull the spreader slide in an even, quick motion so that the drop is drawn out into a thin film. This will form a “comet’s tail” toward the end of the slide. Thin films must be fixed in absolute methyl alcohol for 1 minute and air-dried before being placed in diluted Giemsa (1 part Giemsa to 9 parts buffered water) and stained. After 45 minutes, slides are washed in buffered water and allowed to dry. If the slide contains a thick film and a thin film, fix only the thin portion then stain both parts of the film simultaneously.
THICK BLOOD FILMS When a thick layer of blood is used, many more parasites will be present in each field; however, increased distortion of the parasites is common. To prepare a thick film, place three drops of blood close together near one end of the slide. With a corner of another slide, stir the blood, mingling the three drops over an area 2 cm in diameter. After the films are thoroughly dry, immerse the slide in buffer solution prior to staining or in Giemsa’s stain itself. When Giemsa’s stain is used for thick films, the procedure is the same as for thin films except that the fixation step is omitted. Thick and thin films can be made on the same slide.
ESTIMATING NUMBERS OF MALARIA PARASITES IN BLOOD Determine the patient’s white blood cell count. On a thick blood smear, count the number of parasites seen per 100 white blood cells; the total number per cubic millimeter of blood can then be determined. Parasites can also be quantified as a percent of the number of red blood cells infected, for example 2%. †
Deceased
BLOOD CONCENTRATION PROCEDURES Buffy coat films serve to concentrate white cells (in which Leishmania may be found) and are useful for detection of trypanosomes and microfilariae. A centrifugation technique is used to check for trypanosomes when they are too sparse to be seen in thick blood films. Small numbers of microfilariae in the blood can be detected by a membrane filtration technique.
QUANTITATIVE BUFFY COAT The QBC Malaria Test is a rapid diagnostic method used for malaria parasites.
RAPID DIAGNOSTIC TESTS FOR MALARIA Histidine-rich protein (PF HRP-2) is released by Plasmodium falciparum and can be detected on a nitrocellulose and glass fiber dipstick pretreated with a monoclonal antibody against HRP-2. The dipstick is placed in hemolyzed blood followed by a drop of reagent and read visually. There are several other malaria rapid diagnostic tests (RDTs) on the market and the World Health Organization has reviewed these (www.wpro.who.int/sites/rdt/home.htm).
EXAMINATION OF CEREBROSPINAL FLUID Trophozoites of Naegleria (Chapter 102) and trypanosomes (Chapter 97) can be found in the cerebrospinal fluid (CSF). Detection of helminths in the CSF may also be possible in patients with severe infections. The CSF must be examined promptly, as trypanosomes will survive for only about 20 minutes, and Naegleria may become rounded and non-motile. The CSF is centrifuged (7000 × g for 10 minutes), the supernatant removed and the sediment examined under reduced illumination.
TISSUE IMPRESSIONS Tissue impression smears stained with Giemsa’s stain can be used for detection of Leishmania and Toxoplasma. Fresh lymph nodes, liver biopsy material, or bone marrow is lightly impressed on a slide, allowed to dry at room temperature and stained like a thin blood film. When dealing with lymph nodes or other tissue, it is best to prepare the smear from a freshly cut surface.
BIOPSY AND ASPIRATION Spleen, liver, and bone marrow biopsies are used in the diagnosis of visceral leishmaniasis. Aspiration of enlarged posterior cervical or other involved lymph nodes will at times reveal African trypanosomes when the blood is apparently free of them. The lymph nodes are less often involved in Rhodesian (East African) sleeping sickness than in
G e n e ra l Pr inciples
the Gambian (West African) form, or in Chagas’ disease (American trypanosomiasis). Aspiration of fluid from a hydatid cyst (done under ultrasonographic guidance) may reveal hydatid sand, but it must be remembered that certain hydatid cysts are sterile so the absence of scoleces or hooklets from the sediment is not necessarily evidence against the parasitic nature of the cyst. Aspiration of an amebic abscess will yield few parasites as they tend to occur in the tissue surrounding the abscess cavity. Eggs of Schistosoma mansoni and Schistosoma japonicum can be found in tissue from the rectal mucosa when they cannot be recovered from the stool. Mucosa from the bladder wall, taken at cystoscopy or a rectal snip biopsy, may likewise reveal eggs of Schistosoma haematobium (Chapter 122). Larvae of Trichinella spiralis may be abundant in muscle. Microfilariae of Onchocerca volvulus, Mansonella ozzardi, and Mansonella streptocerca can be demonstrated in skin snips.
EXAMINATION OF SPUTUM Examination of the sputum is indicated when there is a question of pulmonary paragonimiasis; eggs can also found in the feces. Entamoeba histolytica can be detected in the sputum of patients with pulmonary abscesses. Pneumocystis jirovecii (now designated a fungus) can be found in sputum, but is more readily seen in bronchial aspirates or in impression smears of lung biopsy material (Chapter 86). Cryptosporidium has occasionally been found in sputum and lung biopsy. Ruptured hydatid cysts can be recognized by the presence of hooklets in the sputum. Strongyloides larvae may also be found in sputum in disseminated infections. Sputum specimens should be induced, if possible. Specimens should be examined by wet mount while fresh or preserved in polyvinyl alcohol (PVA) fixative for protozoa.
RAPID METHENAMINE SILVER STAIN FOR PNEUMOCYSTIS JIROVECII The best results for detection of Pneumocystis are obtained with a methenamine silver impregnation technique [1]. Pneumocystis jirovecii will have a delicately stained wall, usually brownish or grayish, and rather transparent. Mucin will be taupe to dark gray.
CULTURE METHODS Acanthamoeba and Naegleria, the leishmanias (Chapter 99), Trypanosoma cruzi, and Toxoplasma (Chapter 101) can be cultured with specific media.
OTHER CULTURE METHODS Acanthamoebaa, Naegleria, and Balamuthia can be established in axenic cultures, as well as in cell culture. Novy–Mac Neal–Nicolle
(NNN) medium is used for Leishmania and Trypanosoma cruzi, and Culbertson’s medium for Acanthamoeba. Culture of Toxoplasma has been described [2] and is applicable to blood, CSF, placental and other tissues.
ANIMAL INOCULATION Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense infections can be established in a number of laboratory animals. White rats, white mice, and guinea pigs are most useful for diagnosis and the maintenance of laboratory strains. Young animals are most easily infected. Rats infected with T. brucei gambiense will survive for several months with a low-grade parasitemia; if infected with T. brucei rhodesiense, they die within a short time with an overwhelming parasitemia. Trypanosoma rangeli multiplies in common laboratory animals but does not cause apparent disease. Young white rats and white mice can be infected with T. cruzi; the white mouse is best for diagnostic inoculation. Intraperitoneal or subcutaneous inoculation should be used; amounts of up to 2 ml of blood are injected, depending on the size of the animal. It is important to check rats for the presence of their common parasite, Trypanosoma lewisi, before inoculation. For isolation of Leishmania, the hamster is most satisfactory. Following intraperitoneal or intratesticular inoculation, hamsters will develop a generalized infection with any form of Leishmania, and the organisms can be demonstrated in spleen impression smears or in testicular aspirates. This infection develops slowly, and culture methods are generally regarded as being superior for diagnostic use. Toxoplasma gondii shows little host specificity and will infect all common laboratory animals. White rats and mice are generally used; rats develop a chronic infection and are good for maintenance of the strain, whereas intraperitoneal infection of mice results in proliferation of the organisms in the ascitic fluid and death of the mice within a few days. Mouse peritoneal fluid, rich in organisms, is used as a source of Toxoplasma for the Sabin-Feldman dye test and other diagnostic procedures. Xenodiagnosis can be considered a special case of animal inoculation; the term was originally applied to the diagnosis of Chagas disease by feeding uninfected reduviid bugs on a patient suspected of having the disease. Subsequent examination of the bugs will reveal developmental stages of the parasites if the test result is positive.
REFERENCES 1. Yu PKW, Uhl JR, Anhalt JP. Rapid methenamine silver stain. Arch Pathol Lab Med 1989;113:111. 2. Shepp DH, Mackman RC, Conley FK, et al. Toxoplasma gondii reactivation identified by detection of parasitemia in tissue culture. Ann Intern Med 1985;103:218–21.
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Examination of Blood, Other Body Fluids, Tissues, and Sputum Brett E Swierczewski, John H Cross†
EXAMINATION OF FRESH BLOOD Microscopic examination of fresh blood is useful for detection of trypanosomes and microfilariae. A small drop of blood is placed on a slide and covered with a coverglass. The high-dry objective with reduced illumination is suitable for trypanosomes, while low-power is used for detection of microfilariae.
THE THIN FILM Thin and thick blood films are used for species identification of malarial parasites (Chapter 96), trypanosomes (Chapters 97 and 98), and microfilariae (Chapters 110–113). To prepare a thin film, place a small drop of blood from the fingertip, earlobe, or vial of blood near one end of a microscope slide. Raise the end of the slide farthest from the drop of blood and take a second slide as a spreader, making an angle of approximately 30 degrees with the first. Allow the blood to touch the spreader slide and begin to run out toward the edges. Before the blood has a chance to reach the edges, pull the spreader slide in an even, quick motion so that the drop is drawn out into a thin film. This will form a “comet’s tail” toward the end of the slide. Thin films must be fixed in absolute methyl alcohol for 1 minute and air-dried before being placed in diluted Giemsa (1 part Giemsa to 9 parts buffered water) and stained. After 45 minutes, slides are washed in buffered water and allowed to dry. If the slide contains a thick film and a thin film, fix only the thin portion then stain both parts of the film simultaneously.
THICK BLOOD FILMS When a thick layer of blood is used, many more parasites will be present in each field; however, increased distortion of the parasites is common. To prepare a thick film, place three drops of blood close together near one end of the slide. With a corner of another slide, stir the blood, mingling the three drops over an area 2 cm in diameter. After the films are thoroughly dry, immerse the slide in buffer solution prior to staining or in Giemsa’s stain itself. When Giemsa’s stain is used for thick films, the procedure is the same as for thin films except that the fixation step is omitted. Thick and thin films can be made on the same slide.
ESTIMATING NUMBERS OF MALARIA PARASITES IN BLOOD Determine the patient’s white blood cell count. On a thick blood smear, count the number of parasites seen per 100 white blood cells; the total number per cubic millimeter of blood can then be determined. Parasites can also be quantified as a percent of the number of red blood cells infected, for example 2%. †
Deceased
BLOOD CONCENTRATION PROCEDURES Buffy coat films serve to concentrate white cells (in which Leishmania may be found) and are useful for detection of trypanosomes and microfilariae. A centrifugation technique is used to check for trypanosomes when they are too sparse to be seen in thick blood films. Small numbers of microfilariae in the blood can be detected by a membrane filtration technique.
QUANTITATIVE BUFFY COAT The QBC Malaria Test is a rapid diagnostic method used for malaria parasites.
RAPID DIAGNOSTIC TESTS FOR MALARIA Histidine-rich protein (PF HRP-2) is released by Plasmodium falciparum and can be detected on a nitrocellulose and glass fiber dipstick pretreated with a monoclonal antibody against HRP-2. The dipstick is placed in hemolyzed blood followed by a drop of reagent and read visually. There are several other malaria rapid diagnostic tests (RDTs) on the market and the World Health Organization has reviewed these (www.wpro.who.int/sites/rdt/home.htm).
EXAMINATION OF CEREBROSPINAL FLUID Trophozoites of Naegleria (Chapter 102) and trypanosomes (Chapter 97) can be found in the cerebrospinal fluid (CSF). Detection of helminths in the CSF may also be possible in patients with severe infections. The CSF must be examined promptly, as trypanosomes will survive for only about 20 minutes, and Naegleria may become rounded and non-motile. The CSF is centrifuged (7000 × g for 10 minutes), the supernatant removed and the sediment examined under reduced illumination.
TISSUE IMPRESSIONS Tissue impression smears stained with Giemsa’s stain can be used for detection of Leishmania and Toxoplasma. Fresh lymph nodes, liver biopsy material, or bone marrow is lightly impressed on a slide, allowed to dry at room temperature and stained like a thin blood film. When dealing with lymph nodes or other tissue, it is best to prepare the smear from a freshly cut surface.
BIOPSY AND ASPIRATION Spleen, liver, and bone marrow biopsies are used in the diagnosis of visceral leishmaniasis. Aspiration of enlarged posterior cervical or other involved lymph nodes will at times reveal African trypanosomes when the blood is apparently free of them. The lymph nodes are less often involved in Rhodesian (East African) sleeping sickness than in
G e n e ra l Pr inciples
the Gambian (West African) form, or in Chagas’ disease (American trypanosomiasis). Aspiration of fluid from a hydatid cyst (done under ultrasonographic guidance) may reveal hydatid sand, but it must be remembered that certain hydatid cysts are sterile so the absence of scoleces or hooklets from the sediment is not necessarily evidence against the parasitic nature of the cyst. Aspiration of an amebic abscess will yield few parasites as they tend to occur in the tissue surrounding the abscess cavity. Eggs of Schistosoma mansoni and Schistosoma japonicum can be found in tissue from the rectal mucosa when they cannot be recovered from the stool. Mucosa from the bladder wall, taken at cystoscopy or a rectal snip biopsy, may likewise reveal eggs of Schistosoma haematobium (Chapter 122). Larvae of Trichinella spiralis may be abundant in muscle. Microfilariae of Onchocerca volvulus, Mansonella ozzardi, and Mansonella streptocerca can be demonstrated in skin snips.
EXAMINATION OF SPUTUM Examination of the sputum is indicated when there is a question of pulmonary paragonimiasis; eggs can also found in the feces. Entamoeba histolytica can be detected in the sputum of patients with pulmonary abscesses. Pneumocystis jirovecii (now designated a fungus) can be found in sputum, but is more readily seen in bronchial aspirates or in impression smears of lung biopsy material (Chapter 86). Cryptosporidium has occasionally been found in sputum and lung biopsy. Ruptured hydatid cysts can be recognized by the presence of hooklets in the sputum. Strongyloides larvae may also be found in sputum in disseminated infections. Sputum specimens should be induced, if possible. Specimens should be examined by wet mount while fresh or preserved in polyvinyl alcohol (PVA) fixative for protozoa.
RAPID METHENAMINE SILVER STAIN FOR PNEUMOCYSTIS JIROVECII The best results for detection of Pneumocystis are obtained with a methenamine silver impregnation technique [1]. Pneumocystis jirovecii will have a delicately stained wall, usually brownish or grayish, and rather transparent. Mucin will be taupe to dark gray.
CULTURE METHODS Acanthamoeba and Naegleria, the leishmanias (Chapter 99), Trypanosoma cruzi, and Toxoplasma (Chapter 101) can be cultured with specific media.
OTHER CULTURE METHODS Acanthamoebaa, Naegleria, and Balamuthia can be established in axenic cultures, as well as in cell culture. Novy–Mac Neal–Nicolle
(NNN) medium is used for Leishmania and Trypanosoma cruzi, and Culbertson’s medium for Acanthamoeba. Culture of Toxoplasma has been described [2] and is applicable to blood, CSF, placental and other tissues.
ANIMAL INOCULATION Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense infections can be established in a number of laboratory animals. White rats, white mice, and guinea pigs are most useful for diagnosis and the maintenance of laboratory strains. Young animals are most easily infected. Rats infected with T. brucei gambiense will survive for several months with a low-grade parasitemia; if infected with T. brucei rhodesiense, they die within a short time with an overwhelming parasitemia. Trypanosoma rangeli multiplies in common laboratory animals but does not cause apparent disease. Young white rats and white mice can be infected with T. cruzi; the white mouse is best for diagnostic inoculation. Intraperitoneal or subcutaneous inoculation should be used; amounts of up to 2 ml of blood are injected, depending on the size of the animal. It is important to check rats for the presence of their common parasite, Trypanosoma lewisi, before inoculation. For isolation of Leishmania, the hamster is most satisfactory. Following intraperitoneal or intratesticular inoculation, hamsters will develop a generalized infection with any form of Leishmania, and the organisms can be demonstrated in spleen impression smears or in testicular aspirates. This infection develops slowly, and culture methods are generally regarded as being superior for diagnostic use. Toxoplasma gondii shows little host specificity and will infect all common laboratory animals. White rats and mice are generally used; rats develop a chronic infection and are good for maintenance of the strain, whereas intraperitoneal infection of mice results in proliferation of the organisms in the ascitic fluid and death of the mice within a few days. Mouse peritoneal fluid, rich in organisms, is used as a source of Toxoplasma for the Sabin-Feldman dye test and other diagnostic procedures. Xenodiagnosis can be considered a special case of animal inoculation; the term was originally applied to the diagnosis of Chagas disease by feeding uninfected reduviid bugs on a patient suspected of having the disease. Subsequent examination of the bugs will reveal developmental stages of the parasites if the test result is positive.
REFERENCES 1. Yu PKW, Uhl JR, Anhalt JP. Rapid methenamine silver stain. Arch Pathol Lab Med 1989;113:111. 2. Shepp DH, Mackman RC, Conley FK, et al. Toxoplasma gondii reactivation identified by detection of parasitemia in tissue culture. Ann Intern Med 1985;103:218–21.
1083