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Chapter 31 : Procedures for Detecting Blood Parasites

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Abstract:

Depending on the life cycle, a number of parasites may be recovered in a blood specimen, either whole blood, buffy coat preparations, or various types of concentrations. These blood parasites include , , and species, , and microfilariae. Blood films can be prepared from fresh, whole blood collected with no anticoagulants, anticoagulated blood, or sediment from the various concentration procedures. The recommended stain of choice is Giemsa stain; however, the parasites can also be seen on blood films stained with Wright’s stain, a Wright-Giemsa combination stain, or one of the more rapid stains such as Diff-Quik. Delafield’s hematoxylin stain is often used to stain the microfilarial sheath; in some cases, Giemsa stain does not provide sufficient stain quality to allow differentiation of the microfilariae. It is important to report the level of parasitemia when blood films are reviewed and found to be positive for malaria parasites. Due to the potential for drug resistance in some of the Plasmodium species, particularly and , it is important that every positive smear be assessed and the parasitemia reported exactly the same way on follow-up specimens as on the initial specimen. Rapid diagnostic tests (RDTs) offer great potential to improve the diagnosis of malaria, particularly in remote areas. There are a number of new approaches to the diagnosis of malaria, including the use of fluorescent stains (QBC), dipstick antigen detection of histidine-rich protein 2 (HRP2) and parasite lactate dehydrogenase (pLDH) PCR, and automated blood cell analyzers.

Citation: Garcia L. 2007. Procedures for Detecting Blood Parasites, p 881-909. In Diagnostic Medical Parasitology, Fifth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816018.ch31

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Trypanosoma brucei gambiense
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Chagas' Disease
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Relapsing Fever
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Figures

Image of Figure 31.1
Figure 31.1

Method for preparation of thin blood film. (A) Position of spreader slide; (B) well-prepared thin film. Arrows indicate the area of the slide (feather edge) used to observe accurate cell morphology. (Illustration by Nobuko Kitamura.)

Citation: Garcia L. 2007. Procedures for Detecting Blood Parasites, p 881-909. In Diagnostic Medical Parasitology, Fifth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816018.ch31
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Image of Figure 31.2
Figure 31.2

Method of thick-thin combination blood film preparation. (a) Position of the drop of EDTA-containing blood. (b) Position of the applicator stick in contact with blood and glass slide. (c) Rotation of the applicator stick. (d) Completed thick-thin combination blood film prior to staining. (Illustration by Sharon Belkin. Reprinted from L. S. Garcia, , ASM Press, Washington, D.C., 1999.)

Citation: Garcia L. 2007. Procedures for Detecting Blood Parasites, p 881-909. In Diagnostic Medical Parasitology, Fifth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816018.ch31
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Image of Figure 31.3
Figure 31.3

(Left) Photograph of exflagellation of the malarial microgametocyte. This may occur when anticoagulated blood is left standing at room temperature for some time prior to smear preparation. The life cycle of the parasite continues in the tube of blood as it would if the parasite had been ingested by the mosquito during a blood meal. (Right) in blood. Note that the microgametes can resemble these organisms, particularly if they appear free in the background of the smear.

Citation: Garcia L. 2007. Procedures for Detecting Blood Parasites, p 881-909. In Diagnostic Medical Parasitology, Fifth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816018.ch31
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Image of Figure 31.4
Figure 31.4

spp. seen in stained thin blood films. (Left column from top to bottom) ( ) Developing ring (note the enlarged RBC, Schüffner’s dots, and ameboid ring); ( ) developing trophozoite (note the enlarged RBC and Schüffner’s dots); ( ) large trophozoite prior to the development of schizonts (note the enlarged RBC and Schüffner’s dots); ( ) mature schizont with approximately 16 merozoites. (Center column from top to bottom) ( ) Young ring form (note the nonameboid ring and Schüffner’s dots that appear earlier than in rings of ); ( ) developing trophozoites (note Schüffner’s dots and fimbriated edges of the infected RBCs); ( ) maturing schizont containing merozoites and Schüffner’s dots; ( ) gametocyte. (Right column from top to bottom) ( ) Young ring (note the normal-sized RBC and no stippling); ( ) developing trophozoite (note the normal-sized RBC and “band form” configuration of the trophozoite); ( ) developing schizont (note the size of the RBC); ( ) more mature schizont containing developing merozoites.

Citation: Garcia L. 2007. Procedures for Detecting Blood Parasites, p 881-909. In Diagnostic Medical Parasitology, Fifth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816018.ch31
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Image of Figure 31.5
Figure 31.5

spp. seen in stained thick films. (Top row from left to right) ( ) Numerous ring forms (note differences: nuclei and cytoplasm); ( ) low parasitemia with a single ring form in the field; ( ) crescent-shaped gametocyte (note that the RBC around the gametocyte is not visible). (Row two from left to right) ( ) Ring forms and developing trophozoites; ( ) some larger rings with developing trophozoite; ( ) developing schizonts. (Row three from left to right) ( ) Mature schizonts containing about eight merozoites (photographed at higher magnification); ( ) developing schizonts and two mature schizonts; ( ) single ring form and several mature schizonts. (Bottom row from left to right) ( ) Developing trophozoites (note that the organisms do not appear to be ameboid like those normally seen in ; ( ) developing trophozoite; ( ) developing trophozoite (note that panels 2 and 3 are photographed at a higher magnification).

Citation: Garcia L. 2007. Procedures for Detecting Blood Parasites, p 881-909. In Diagnostic Medical Parasitology, Fifth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816018.ch31
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Image of Figure 31.6
Figure 31.6

Trypomastigotes. (Left) (Right) (note the undulating membrane on both trypomastigotes and the larger kinetoplast in ).

Citation: Garcia L. 2007. Procedures for Detecting Blood Parasites, p 881-909. In Diagnostic Medical Parasitology, Fifth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816018.ch31
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Image of Figure 31.7
Figure 31.7

in an impression smear. Note the numerous small amastigotes containing a nucleus and bar-shaped primitive flagellum.

Citation: Garcia L. 2007. Procedures for Detecting Blood Parasites, p 881-909. In Diagnostic Medical Parasitology, Fifth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816018.ch31
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Image of Figure 31.8
Figure 31.8

sp. in a thin blood film. Note the “Maltese cross” formation that is diagnostic but not always seen in blood films. The ring forms are very pleomorphic, much more so than ring forms; there tend to be numerous rings per RBC.

Citation: Garcia L. 2007. Procedures for Detecting Blood Parasites, p 881-909. In Diagnostic Medical Parasitology, Fifth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816018.ch31
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Image of Figure 31.9
Figure 31.9

(Upper row) Note the ring forms (multiple rings per RBC and presence of the “headphone” ring configuration). The photograph at the right is a good mimic of organisms, but the rings are not quite as pleomorphic as in spp. (Lower row) Three examples of gametocytes, two of which appear to be outside the RBC and one of which is inside.

Citation: Garcia L. 2007. Procedures for Detecting Blood Parasites, p 881-909. In Diagnostic Medical Parasitology, Fifth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816018.ch31
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Image of Figure 31.10
Figure 31.10

The QBC Malaria Test components include the ParaLens UV microscope adapter (blue-violet light module providing fiber-optic illumination with AC outlet or bulb illumination) with rechargeable battery for mobile use (attachable to any conventional microscope), the ParaFuge battery-powered centrifuge, and the ParaViewer microscope tube holder, a specially designed QBC tube viewing block that accepts standard microscope oil.

Citation: Garcia L. 2007. Procedures for Detecting Blood Parasites, p 881-909. In Diagnostic Medical Parasitology, Fifth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816018.ch31
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Image of Figure 31.11
Figure 31.11

Diagram of the ParaSight F test format. (Adapted from reference with permission.)

Citation: Garcia L. 2007. Procedures for Detecting Blood Parasites, p 881-909. In Diagnostic Medical Parasitology, Fifth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816018.ch31
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Image of Figure 31.12
Figure 31.12

ParaSight F test showing (from left to right) the positive test strip with a reagent control mark above the positive test result and a negative test strip with the reagent control mark.

Citation: Garcia L. 2007. Procedures for Detecting Blood Parasites, p 881-909. In Diagnostic Medical Parasitology, Fifth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816018.ch31
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Image of Figure 31.13
Figure 31.13

Diagram of the NOW malaria test format. (Adapted from package insert, Binax, Portland, Maine.)

Citation: Garcia L. 2007. Procedures for Detecting Blood Parasites, p 881-909. In Diagnostic Medical Parasitology, Fifth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816018.ch31
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Image of Figure 31.14
Figure 31.14

Flow anti-pLDH monoclonal antibodies rapid malaria test. (Left) Diagram of blood flow on the membrane through the timed test; note the positive control line and positive test line. (Right) Diagram of negative control, a positive result, and a positive result. Note that the line for is a panspecific antibody against all four species; however, the panspecific antibody has been shown to detect only with any degree of consistency. (Photographs adapted from Flow’s website, with permission.)

Citation: Garcia L. 2007. Procedures for Detecting Blood Parasites, p 881-909. In Diagnostic Medical Parasitology, Fifth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816018.ch31
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Image of Figure 31.15
Figure 31.15

microfilaria at low power from a Knott concentration stained with Giemsa stain. Note that the sheath is not visible.

Citation: Garcia L. 2007. Procedures for Detecting Blood Parasites, p 881-909. In Diagnostic Medical Parasitology, Fifth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816018.ch31
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Image of Figure 31.16
Figure 31.16

microfilaria on a thick film stained with Delafield’s hematoxylin. Note the presence of the sheath.

Citation: Garcia L. 2007. Procedures for Detecting Blood Parasites, p 881-909. In Diagnostic Medical Parasitology, Fifth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816018.ch31
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Image of Figure 31.17
Figure 31.17

microfilaria. (Upper) Microfilaria stained with Giemsa stain. Note that the sheath is not visible. (Lower) Microfilaria stained with Delafield’s hematoxylin stain. Note that the sheath is visible.

Citation: Garcia L. 2007. Procedures for Detecting Blood Parasites, p 881-909. In Diagnostic Medical Parasitology, Fifth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816018.ch31
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Tables

Generic image for table
Table 31.1

Advantages of the thin and thick blood films

Citation: Garcia L. 2007. Procedures for Detecting Blood Parasites, p 881-909. In Diagnostic Medical Parasitology, Fifth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816018.ch31
Generic image for table
Table 31.2

Potential problems of the use of EDTA anticoagulant for the preparation of thin and thick blood films

Citation: Garcia L. 2007. Procedures for Detecting Blood Parasites, p 881-909. In Diagnostic Medical Parasitology, Fifth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816018.ch31
Generic image for table
Table 31.3

Potential problems with thick blood film preparation and staining

Citation: Garcia L. 2007. Procedures for Detecting Blood Parasites, p 881-909. In Diagnostic Medical Parasitology, Fifth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816018.ch31
Generic image for table
Table 31.4

Potential problems with thin blood film preparation and staining

Citation: Garcia L. 2007. Procedures for Detecting Blood Parasites, p 881-909. In Diagnostic Medical Parasitology, Fifth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816018.ch31
Generic image for table
Table 31.5

Advantages and disadvantages of the buffy coat films

Citation: Garcia L. 2007. Procedures for Detecting Blood Parasites, p 881-909. In Diagnostic Medical Parasitology, Fifth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816018.ch31
Generic image for table
Table 31.6

Phosphate buffer solutions

Citation: Garcia L. 2007. Procedures for Detecting Blood Parasites, p 881-909. In Diagnostic Medical Parasitology, Fifth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816018.ch31
Generic image for table
Table 31.7

Parasitemia determined from conventional light microscopy: clinical correlation

Citation: Garcia L. 2007. Procedures for Detecting Blood Parasites, p 881-909. In Diagnostic Medical Parasitology, Fifth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816018.ch31
Generic image for table
Table 31.8

Summary of commercially available kits for immunodetection of blood parasites, antigens, or antibodies

Citation: Garcia L. 2007. Procedures for Detecting Blood Parasites, p 881-909. In Diagnostic Medical Parasitology, Fifth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816018.ch31
Generic image for table
Table 31.9

Field trials of antigen-detection tests for malaria

Citation: Garcia L. 2007. Procedures for Detecting Blood Parasites, p 881-909. In Diagnostic Medical Parasitology, Fifth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816018.ch31

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