Routine Methods

Malaria is considered to be immediately life threatening, and a patient with the diagnosis of P. falciparum or P. knowlesi malaria should be considered a medical emergency because the disease can be rapidly fatal. This approach to the patient is also recommended in situations where P. falciparum or P. knowlesi cannot be ruled out as a possible diagnosis. Any laboratory providing the expertise to identify malarial parasites should do so on a 24-hour basis, 7 days a week.

Examination of a single blood specimen is not sufficient to exclude the diagnosis of malaria, especially when the patient has received partial prophylaxis or therapy and has a low number of organisms in the blood. Patients with a relapse case or an early primary case may also have few organisms in the blood smear. Regardless of the presence or absence of any fever periodicity, both thick (Figure 1) and thin blood films should be prepared immediately, and at least 200 to 300 oil immersion fields should be examined on both films before a negative report is issued. If the initial specimen is negative, additional blood specimens should be examined over a 36-hour time frame. Although Giemsa stain is recommended for all parasitic blood work, the organisms can also be seen with other blood stains, such as Wright’s stain. Using any of the blood stains, the white blood cells (WBCs) serve as the built-in quality control; if the WBCs look good, any parasites present will also look good. Figure 2 compares the multinucleated stages (schizont) of Plasmodium malariae and Plasmodium vivax. Fluorescent nucleic acid stains, such as acridine orange, may also be used to identify organisms in infected RBCs. However, this may be more difficult to interpret because of the presence of white blood cell nuclei or RBC Howell Jolly bodies.

Fig1. Plasmodium vivax in thick smear. 1, Ameboid trophozoites. 2, Schizont, two divisions of chromatin. 3, Mature schizont.  4, Microgametocyte. 5, Blood platelets. 6, Nucleus of neutrophil. 7, Eosinophil. 8, Blood platelet associated with cellular remains of young  erythrocytes. (From Wilcox A: Manual for the microscopical diagnosis of malaria in man, Washington, DC, 1960, U.S. Public Health Service.)

Fig2. A, Plasmodium malariae schizont. B, Plasmodium vivax schizont. (Courtesy Dr. Henry Travers, Sioux Falls, SD.)

Blood collected using ethylenediaminetetraacetic acid (EDTA) anticoagulant is preferred; however, if the blood remains in the tube for any length of time before blood film preparation, Schüffner’s dots may not be visible after staining (P. vivax, as an example) and other morphologic changes in the parasites will be seen. Also, the proper ratio between blood and anticoagulant is required for good organism morphology, so each col lection tube should be filled to the top. Finger-stick blood is recommended, particularly when the volume of blood required is minimal (i.e., when no other hematologic procedures have been ordered). The blood should be free flowing when taken for smear preparation, and should not be contaminated with alcohol used to clean the finger before the stick. However, the use of finger-stick blood is currently much less common, and venipuncture blood is the normal specimen collected for the laboratory. Identification to the species level is highly desirable, because this information determines which drug(s) is (are) recommended. In early infections, patients with P. falciparum infections may not have the crescent-shaped gametocytes in the blood. Also, low parasitemia with the delicate ring forms may be missed; consequently, oil immersion examination at 1000× is mandatory.

Serologic Methods

Several rapid malaria tests (RMTs) are now commercially available, some of which use monoclonal antibodies against the histidine-rich protein 2 (HRP2) whereas others detect species-specific parasite lactate dehydrogenase (pLDH). These procedures are based on an antigen capture approach in dipstick or cartridge formats. The BinaxNOW rapid malaria test (Alere, Waltham, MA) is FDA approved for use within the United States. The kit is designed to detect primarily P. falciparum and P. vivax; detection of the other species is less sensitive. However, because of sensitivity limitations in patients with a low parasitemia, the gold standard is still considered the examination of thick and thin blood films. If the rapid test is negative, the thick and thin blood films must be examined on a STAT basis.

Molecular Diagnostics

Other methods include direct detection of the five species by using a specific DNA probe after PCR amplification of target DNA sequences. Some laboratories are now using PCR for detection of malaria; the high sensitivity, rapidity, and simplicity of some of the methods are becoming much more relevant for diagnosis. Detection is possible for as few as 5 or 10 parasites per microliter of blood; thus, PCR detects many more cases of low-level parasitemia than do thick blood films. This approach is also valuable when the determination of species is questionable, or in situations where mixed infections are suspected.

Automated Instruments

 Using automated flow cytometry hematology instruments, there are potential limitations related to the diagnosis of blood parasite infections. Plasmodium spp. and Babesia infections have been completely missed using instruments. Failure to detect a light parasitemia is highly likely. Unfortunately, failure to make the diagnosis in many of these patients has resulted in delayed therapy. Although the majority of these instruments are not designed to detect intracellular blood parasites, the inability of the automated systems to discriminate between uninfected RBCs and those infected with para sites may pose serious diagnostic problems in situations where the parasitemia is 0.5% or less.

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المزيد

مواضيع ذات صلة

African Trypanosomiasis

Babesia spp.

Therapy of Plasmodium spp

Plasmodium falciparum (Malignant Tertian Malaria)

Plasmodium malariae (Quartan Malaria)

Plasmodium ovale

Plasmodium vivax (Benign Tertian Malaria)

Pathogenesis and Spectrum of Disease of Microsporidia

Sarcocystis SPP.

Isospora (Cystoisospora) belli

Cyclospora cayetanensis

.Cryptosporidium spp