Chapter 7 : Procedures for Detecting Blood Parasites

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

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 reference 9 . doi:10.1128/9781555819002.ch7.f2

(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) Borrelia in blood. Note that the Plasmodium microgametes can resemble these organisms, particularly if they appear free in the background of the smear. doi:10.1128/9781555819002.ch7.f3

Plasmodium spp. seen in stained thin blood films. (Left column from top to bottom) Plasmodium vivax: ( 1 ) developing ring (note the enlarged RBC, lack of Schüffner's dots [EDTA blood], and ameboid rings); ( 2 ) developing trophozoite (note the enlarged RBC and Schüffner's dots); ( 3 ) mature schizont with approximately 16 merozoites; ( 4 ) thick blood film showing developing ring forms; ( 5 ) normal WBCs (including polymorphonuclear leukocytes [PMNs], eosinophil, basophil, monocyte, and lymphocyte). (Second column from top to bottom) Plasmodium ovale: ( 1 ) young ring form (note the nonameboid ring and Schüffner's dots that appear earlier than in rings of P. vivax); ( 2 ) developing trophozoites (note Schüffner's dots and fimbriated edges of the infected RBCs); ( 3 ) maturing schizont containing merozoites and malarial pigment; ( 4 ) thick blood film showing developing schizont; ( 5 ) normal PMNs. (Third column from top to bottom) Plasmodium malariae: ( 1 ) young ring (note the normal-sized RBC and no stippling); ( 2 ) developing trophozoite (note the normal-sized RBC and “band form” configuration of the trophozoite); ( 3 ) mature schizont (note the size of the RBC) containing approximately eight merozoites and brownish malarial pigment; ( 4 ) thick blood film with mature schizonts containing developing merozoites; ( 5 ) normal WBCs (including PMN, eosinophil, basophil, monocyte and lymphocyte). (Last column from top to bottom) Plasmodium falciparum: ( 1 ) typical ring forms (note appliqué form at the side of the RBC and multiple rings/cell); ( 2 ) typical ring forms (note the ring appearing to be partially outside the RBC—typical for P. falciparum); ( 3 ) crescent-shaped gametocyte; ( 4 ) thick blood film showing many ring forms; ( 5 ) typical PMNs. doi:10.1128/9781555819002.ch7.f4

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

Babesia sp. (Top) Note some of the ring forms are outside the RBCs (box, rare to see in Plasmodium 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

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

Plasmodium falciparum. (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 Babesia organisms, but the rings are not quite as pleomorphic as in Babesia spp. (Lower row) Two examples of P. falciparum 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

(Upper) Brugia malayi microfilaria on a blood film stained with Giemsa stain. Note the presence of the sheath (stains pink with Giemsa stain). (Lower) Brugia malayi 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

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

Diagram of the ParaSight F test format. (Adapted from reference 24 with permission.) doi:10.1128/9781555819002.ch7.f11

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

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

Flow anti-pLDH Plasmodium 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 P. vivax result, and a positive P. falciparum result. Note that the line for P. vivax is a panspecific antibody against all four species; however, the panspecific antibody has been shown to detect only P. vivax with any degree of consistency. (Photographs adapted from Flow's website, with permission.) doi:10.1128/9781555819002.ch7.f14

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

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

Wuchereria bancrofti 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

Wuchereria bancrofti 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