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Chapter 7 : Procedures for Detecting Blood Parasites

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Abstract:

  • Preparation of thick and thin blood films
    • Thick blood films
    • Thin blood films
    • Combination thick and thin blood films (on the same slide)
    • Combination thick and thin blood films (can be stained as either)
    • Buffy coat blood films
  • Staining blood films
    • Giemsa stain
    • Wright's stain
    • General notes on staining procedures
  • Proper examination of thin and thick blood films
    • Thin blood films
    • Thick blood films
    • Determination of parasitemia
  • Diagnosis of malaria: review of alternatives to conventional microscopy
    • QBC microhematocrit centrifugation method
    • ParaSight F test
    • NOW malaria test
    • Flow anti-pLDH monoclonal antibodies
    • Molecular testing
    • Automated blood cell analyzers
  • Diagnosis of leishmaniasis: review of alternatives to conventional microscopy
    • ICT for detection of anti-rK-39 antibodies
  • Concentration procedures
    • Cytocentrifugation technique
    • Knott concentration procedure
    • Membrane filtration technique
    • Gradient centrifugation technique
    • Triple-centrifugation method for trypanosomes
  • Special stain for microfilarial sheath
    • Delafield's hematoxylin

Citation: Garcia L. 2016. Procedures for Detecting Blood Parasites, p 129-155. In Diagnostic Medical Parasitology, Sixth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555819002.ch7
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Figures

Image of Figure 7.1
Figure 7.1

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 Sharon Belkin.) doi:10.1128/9781555819002.ch7.f1

Citation: Garcia L. 2016. Procedures for Detecting Blood Parasites, p 129-155. In Diagnostic Medical Parasitology, Sixth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555819002.ch7
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Image of Figure 7.2
Figure 7.2

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 . doi:10.1128/9781555819002.ch7.f2

Citation: Garcia L. 2016. Procedures for Detecting Blood Parasites, p 129-155. In Diagnostic Medical Parasitology, Sixth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555819002.ch7
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Image of Figure 7.3
Figure 7.3

(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. doi:10.1128/9781555819002.ch7.f3

Citation: Garcia L. 2016. Procedures for Detecting Blood Parasites, p 129-155. In Diagnostic Medical Parasitology, Sixth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555819002.ch7
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Image of Figure 7.4
Figure 7.4

spp. seen in stained thin blood films. (Left column from top to bottom) : ( ) developing ring (note the enlarged RBC, lack of Schüffner's dots [EDTA blood], and ameboid rings); ( ) developing trophozoite (note the enlarged RBC and Schüffner's dots); ( ) mature schizont with approximately 16 merozoites; ( ) thick blood film showing developing ring forms; ( ) normal WBCs (including polymorphonuclear leukocytes [PMNs], eosinophil, basophil, monocyte, and lymphocyte). (Second 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 malarial pigment; ( ) thick blood film showing developing schizont; ( ) normal PMNs. (Third 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); ( ) mature schizont (note the size of the RBC) containing approximately eight merozoites and brownish malarial pigment; ( ) thick blood film with mature schizonts containing developing merozoites; ( ) normal WBCs (including PMN, eosinophil, basophil, monocyte and lymphocyte). (Last column from top to bottom) : ( ) typical ring forms (note appliqué form at the side of the RBC and multiple rings/cell); ( ) typical ring forms (note the ring appearing to be partially outside the RBC—typical for ); ( ) crescent-shaped gametocyte; ( ) thick blood film showing many ring forms; ( ) typical PMNs. doi:10.1128/9781555819002.ch7.f4

Citation: Garcia L. 2016. Procedures for Detecting Blood Parasites, p 129-155. In Diagnostic Medical Parasitology, Sixth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555819002.ch7
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Image of Figure 7.5
Figure 7.5

Trypomastigotes. (Left) . (Right) (note the undulating membrane on both trypomastigotes and the larger kinetoplast in ). doi:10.1128/9781555819002.ch7.f5

Citation: Garcia L. 2016. Procedures for Detecting Blood Parasites, p 129-155. In Diagnostic Medical Parasitology, Sixth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555819002.ch7
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Image of Figure 7.6
Figure 7.6

sp. (Top) Note some of the ring forms are outside the RBCs (box, rare to see in infections). (Bottom) Note the numerous ring forms per RBC; also note the Maltese cross ring formation in the lowermost RBC (arrow). doi:10.1128/9781555819002.ch7.f6

Citation: Garcia L. 2016. Procedures for Detecting Blood Parasites, p 129-155. In Diagnostic Medical Parasitology, Sixth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555819002.ch7
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Image of Figure 7.7
Figure 7.7

in an impression smear. Note the numerous small amastigotes containing a nucleus and bar-shaped primitive flagellum. doi:10.1128/9781555819002.ch7.f7

Citation: Garcia L. 2016. Procedures for Detecting Blood Parasites, p 129-155. In Diagnostic Medical Parasitology, Sixth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555819002.ch7
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Image of Figure 7.8
Figure 7.8

. (Upper row) Note the ring forms (multiple rings per RBC and presence of the “headphone” ring configuration). The photograph at the far right is a good mimic of organisms, but the rings are not quite as pleomorphic as in spp. (Lower row) Two examples of gametocytes, two of which appear to be outside the RBC. However, although the RBC membrane is not visible, the gametocyte is intracellular. The image on the far right is an ookinete from the mosquito cycle; this can occur within the EDTA blood specimen if the blood cools and is left standing with the cap removed. In the cooled, aerated blood, the parasites begin the cycle that normally occurs within the mosquito. This artifact can easily be confused with the crescent-shaped gametocyte. doi:10.1128/9781555819002.ch7.f8

Citation: Garcia L. 2016. Procedures for Detecting Blood Parasites, p 129-155. In Diagnostic Medical Parasitology, Sixth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555819002.ch7
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Image of Figure 7.9
Figure 7.9

(Upper) microfilaria on a blood film stained with Giemsa stain. Note the presence of the sheath (stains pink with Giemsa stain). (Lower) microfilaria on a thick blood film stained with Giemsa stain (note some of the individual nuclei are visible). (Images courtesy of the CDC Public Health Image Library; lower, photograph by Dr. Mae Melvin.) doi:10.1128/9781555819002.ch7.f9

Citation: Garcia L. 2016. Procedures for Detecting Blood Parasites, p 129-155. In Diagnostic Medical Parasitology, Sixth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555819002.ch7
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Image of Figure 7.10
Figure 7.10

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. doi:10.1128/9781555819002.ch7.f10

Citation: Garcia L. 2016. Procedures for Detecting Blood Parasites, p 129-155. In Diagnostic Medical Parasitology, Sixth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555819002.ch7
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Image of Figure 7.11
Figure 7.11

Diagram of the ParaSight F test format. (Adapted from with permission.) doi:10.1128/9781555819002.ch7.f11

Citation: Garcia L. 2016. Procedures for Detecting Blood Parasites, p 129-155. In Diagnostic Medical Parasitology, Sixth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555819002.ch7
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Image of Figure 7.12
Figure 7.12

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. doi:10.1128/9781555819002.ch7.f12

Citation: Garcia L. 2016. Procedures for Detecting Blood Parasites, p 129-155. In Diagnostic Medical Parasitology, Sixth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555819002.ch7
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Image of Figure 7.13
Figure 7.13

Diagram of the NOW malaria test format. (Adapted from package insert, Binax, Portland, ME.) doi:10.1128/9781555819002.ch7.f13

Citation: Garcia L. 2016. Procedures for Detecting Blood Parasites, p 129-155. In Diagnostic Medical Parasitology, Sixth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555819002.ch7
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Image of Figure 7.14
Figure 7.14

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.) doi:10.1128/9781555819002.ch7.f14

Citation: Garcia L. 2016. Procedures for Detecting Blood Parasites, p 129-155. In Diagnostic Medical Parasitology, Sixth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555819002.ch7
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Image of Figure 7.15
Figure 7.15

Microfilariae at low power from a Knott concentration, unstained. Note that the sheath is not visible. doi:10.1128/9781555819002.ch7.f15

Citation: Garcia L. 2016. Procedures for Detecting Blood Parasites, p 129-155. In Diagnostic Medical Parasitology, Sixth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555819002.ch7
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Image of Figure 7.16
Figure 7.16

African trypanosomes obtained using the triple centrifugation method. doi:10.1128/9781555819002.ch7.f16

Citation: Garcia L. 2016. Procedures for Detecting Blood Parasites, p 129-155. In Diagnostic Medical Parasitology, Sixth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555819002.ch7
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Image of Figure 7.17
Figure 7.17

microfilaria on a thick film stained with Delafield's hematoxylin. Note the presence of the sheath. (Image courtesy of the CDC Public Health Image Library.) doi:10.1128/9781555819002.ch7.f17

Citation: Garcia L. 2016. Procedures for Detecting Blood Parasites, p 129-155. In Diagnostic Medical Parasitology, Sixth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555819002.ch7
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Image of Figure 7.18
Figure 7.18

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. doi:10.1128/9781555819002.ch7.f18

Citation: Garcia L. 2016. Procedures for Detecting Blood Parasites, p 129-155. In Diagnostic Medical Parasitology, Sixth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555819002.ch7
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References

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Tables

Generic image for table
TABLE 7.1

Advantages of the thin and thick blood films

Citation: Garcia L. 2016. Procedures for Detecting Blood Parasites, p 129-155. In Diagnostic Medical Parasitology, Sixth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555819002.ch7
Generic image for table
TABLE 7.2

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

Citation: Garcia L. 2016. Procedures for Detecting Blood Parasites, p 129-155. In Diagnostic Medical Parasitology, Sixth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555819002.ch7
Generic image for table
TABLE 7.3

Potential problems with thick blood film preparation and staining

Citation: Garcia L. 2016. Procedures for Detecting Blood Parasites, p 129-155. In Diagnostic Medical Parasitology, Sixth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555819002.ch7
Generic image for table
TABLE 7.4

Potential problems with thin blood film preparation and staining

Citation: Garcia L. 2016. Procedures for Detecting Blood Parasites, p 129-155. In Diagnostic Medical Parasitology, Sixth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555819002.ch7
Generic image for table
TABLE 7.5

Advantages and disadvantages of the buffy coat films

Citation: Garcia L. 2016. Procedures for Detecting Blood Parasites, p 129-155. In Diagnostic Medical Parasitology, Sixth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555819002.ch7
Generic image for table
TABLE 7.6

Phosphate buffer solutions

Citation: Garcia L. 2016. Procedures for Detecting Blood Parasites, p 129-155. In Diagnostic Medical Parasitology, Sixth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555819002.ch7
Generic image for table
TABLE 7.7

Parasitemia determined from conventional light microscopy: clinical correlation

Citation: Garcia L. 2016. Procedures for Detecting Blood Parasites, p 129-155. In Diagnostic Medical Parasitology, Sixth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555819002.ch7
Generic image for table
TABLE 7.8

Rapid diagnostic test kits for detection of antigen of species that are currently available for sale

Citation: Garcia L. 2016. Procedures for Detecting Blood Parasites, p 129-155. In Diagnostic Medical Parasitology, Sixth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555819002.ch7
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TABLE 7.9

Summary of commercially available kits for immunodetection of blood parasite antigens or antibodies (other than sp.)

Citation: Garcia L. 2016. Procedures for Detecting Blood Parasites, p 129-155. In Diagnostic Medical Parasitology, Sixth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555819002.ch7

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