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Chapter 31 : Procedures for Detecting Blood Parasites
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 Plasmodium, Babesia, and Trypanosoma species, Leishmania donovani, 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 P. falciparum and P. vivax, 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.
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.)
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, Practical Guide to Diagnostic Parasitology, ASM Press, Washington, D.C., 1999.)
(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.
Plasmodium spp. seen in stained thin blood films. (Left column from top to bottom) Plasmodium vivax. ( 1 ) Developing ring (note the enlarged RBC, Schüffner’s dots, and ameboid ring); ( 2 ) developing trophozoite (note the enlarged RBC and Schüffner’s dots); ( 3 ) large trophozoite prior to the development of schizonts (note the enlarged RBC and Schüffner’s dots); ( 4 ) mature schizont with approximately 16 merozoites. (Center 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 Schüffner’s dots; ( 4 ) gametocyte. (Right 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 ) developing schizont (note the size of the RBC); ( 4 ) more mature schizont containing developing merozoites.
Plasmodium spp. seen in stained thick films. (Top row from left to right) Plasmodium falciparum. ( 1 ) Numerous ring forms (note differences: nuclei and cytoplasm); ( 2 ) low parasitemia with a single ring form in the field; ( 3 ) crescent-shaped gametocyte (note that the RBC around the gametocyte is not visible). (Row two from left to right) Plasmodium vivax. ( 1 ) Ring forms and developing trophozoites; ( 2 ) some larger rings with developing trophozoite; ( 3 ) developing schizonts. (Row three from left to right) Plasmodium malariae. ( 1 ) Mature schizonts containing about eight merozoites (photographed at higher magnification); ( 2 ) developing schizonts and two mature schizonts; ( 3 ) single ring form and several mature schizonts. (Bottom row from left to right) Plasmodium ovale. ( 1 ) Developing trophozoites (note that the organisms do not appear to be ameboid like those normally seen in P. vivax; ( 2 ) developing trophozoite; ( 3 ) developing trophozoite (note that panels 2 and 3 are photographed at a higher magnification).
Trypomastigotes. (Left) Trypanosoma brucei gambiense. (Right) Trypanosoma cruzi (note the undulating membrane on both trypomastigotes and the larger kinetoplast in T. cruzi).
Leishmania donovani in an impression smear. Note the numerous small amastigotes containing a nucleus and bar-shaped primitive flagellum.
Babesia 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 P. falciparum ring forms; there tend to be numerous rings per RBC.
Plasmodium falciparum. (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 Babesia organisms, but the rings are not quite as pleomorphic as in Babesia spp. (Lower row) Three examples of P. falciparum gametocytes, two of which appear to be outside the RBC and one of which is inside.
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.
Diagram of the ParaSight F test format. (Adapted from reference 30 with permission.)
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.
Diagram of the NOW malaria test format. (Adapted from package insert, Binax, Portland, Maine.)
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 Plasmodium 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.)
Loa loa microfilaria at low power from a Knott concentration stained with Giemsa stain. Note that the sheath is not visible.
Brugia malayi microfilaria on a thick film stained with Delafield’s hematoxylin. Note the presence of the sheath.
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.
Advantages of the thin and thick blood films
Potential problems of the use of EDTA anticoagulant for the preparation of thin and thick blood films
Potential problems with thick blood film preparation and staining
Potential problems with thin blood film preparation and staining
Advantages and disadvantages of the buffy coat films
Phosphate buffer solutions
Parasitemia determined from conventional light microscopy: clinical correlation a
Summary of commercially available kits for immunodetection of blood parasites, antigens, or antibodies
Field trials of antigen-detection tests for malaria