Approach Considerations
The presentation of malaria is non-specific (ie, headache, fever, chills, myalgia, nausea, vomiting, diarrhea, fatigue, abdominal pain, altered mentation), and no combination of signs or symptoms can accurately discriminate malaria from other causes of fever in an endemic area. However, malaria should be suspected in any patient presenting with fevers greater than 99.5 degrees Fahrenheit in an endemic area without other obvious cause, as a delay in diagnosis is associated with increased mortality. [18] Malaria should be suspected in children if they present with anemia and hemoglobin of less than 8g/dL. As described in the differential diagnosis section, incubation period should be accounted for if the patient of interest is a traveler to an endemic area. The 2 common methods used for parasitological diagnosis include light microscopy and immunochromatographic rapid diagnostic tests that detect parasite-specific proteins, which are discussed in depth below.
Imaging studies
Chest radiography may be helpful if respiratory symptoms are present. If CNS symptoms are present, a computed tomography (CT) scan of the head may be obtained to evaluate evidence of cerebral edema or hemorrhage.
Microhematocrit centrifugation
Using this method with the CBC tube is a more sensitive method for detection of malaria infection. However, microhematocrit centrifugation does not allow the identification of the species of Plasmodium. To determine species, a peripheral blood smear must be examined.
Fluorescent dyes/ultraviolet indicator tests
Several different dyes allow laboratory results to be obtained more quickly. These methods require the use of a fluorescent microscope. Fluorescent /ultraviolet tests may not yield speciation information.
Polymerase chain reaction assay
PCR assay testing is a very specific and sensitive means of determining if species of Plasmodium are present in the blood of an infected individual. PCR assay tests are not available in most clinical situations. However, they are very effective at detecting the Plasmodium species in patients with parasitemias as low as 10 parasites/mL of blood.
Lumbar puncture
If the patient exhibits mental-status changes, and even if the peripheral smear demonstrates P falciparum, a lumbar puncture should be performed to rule out bacterial meningitis.
Blood Smears
A diagnosis of malaria should be supported by the identification of the parasites on a thin or thick blood smear. The thick smear allows examination of a larger volume of blood and should be used for the detection of malaria parasites (typically able to detect 10-90 parasites/uL of blood depending on expertise - the thin film should be used for species identification and calculation of parasitemia, which influence treatment decisions in the case of P falciparum and P knowlesi infection.
Film Preparation
See the CDC's Malaria DPDx for blood film preparation and staining guides and videos.
Films should be prepared less than 4 hours after the blood specimen has been drawn, as parasite morphology changes the longer that it is exposed to K2EDTA (ie, anticoagulated blood). [36]
Thick smears
- Thick films should be made with 2-3 small drops of blood spread into a circle of 1cm diameter to give 4-6 RBC thickness (ie, it should be possible to read print through the blood film before staining)
- Allow the slide to dry horizontally at 98.6F for at least 15 minutes or at room temperature for 60 minutes
- Expose to acetone for 10 minutes in a Coplin staining jar and allow to dry (may help to inactivate enveloped viruses to decrease infection risk to the microscopist)
- Place the slide in staining jar with 2.5% working Giemsa for 45-60 minutes (also may use 10% Giemsa for 10 minutes - lesser quality)
- Place the slide in working Giemsa 7.2 pH buffer for 5 minutes
- Dry the slide upright in a rack
Thin smears
- Thin films should be made with a wide tail by pushing 1 drop of blood across the slide with the end of another clean slide
- Allow the slide to dry horizontally at 98.6F for at least 15 minutes or at room temperature for 60 minutes
- Expose the film to acetone for 10 minutes in a Coplin staining jar and allow to dry (may help to inactivate enveloped viruses to decrease infection risk to the microscopist)
- Fix the thin film in methanol for 30-60 seconds prior to staining with Giemsa
- Place the slide in staining jar with 2.5% working Giemsa for 45-60 minutes (also may use 10% Giemsa for 10 minutes - lesser quality)
- Dip the slide 3-4 times in Giemsa 7.2 pH buffer to rinse
- Dry the slide upright in a rack
Three thick and thin smears 12-24 hours apart should be obtained. When reading a smear, a minimum of 200 high-power fields should be examined by 2 trained observers (more if the patient recently has taken prophylactic medication, because this temporarily may decrease parasitemia). One negative smear does not exclude malaria as a diagnosis; several more smears should be examined over a 36-hour period. The highest yield of peripheral parasites occurs during or soon after a fever spike; however, smears should not be delayed while awaiting fever spikes.
Thick smears are more than 10 times more sensitive than thin smears, but species identification is more difficult. Thin smears examined toward the tail end of the blood film where red cells are not overlapping allow for species level identification and quantification of parasitemia. If there is a parasitemia greater than 5%, P falciparum (or infrequently P knowlesi) is the most likely pathogen.
Alternatives to Blood Smear Testing
Alternative diagnostic methods typically are used if the laboratory does not have sufficient expertise in detecting parasites in blood smears.
Rapid diagnostic tests (RDT)
Immunochromatographic tests based on antibody to histidine-rich protein-2 (PfHRP2), parasite LDH (pLDH), or Plasmodium aldolase appear to be very sensitive and specific. [37, 38] Only 1 RDT (BinaxNOW) has been approved for the diagnosis of malaria in the United States. [39] However, per WHO guidance, HRP2-detecting RDTs should not be used exclusively in areas where false-negative RDT rates due to non-expression of HRP2 are common (>5%). [40] The HRP2 knockout phenomenon originally was identified in Peru in 2007, then subsequently in Columbia and Brazil, but it has been found in many other malaria-endemic areas, with a major concern for high prevalence (>80% in P falciparum) in countries in the Horn of Africa (Eritrea, Djibouti, Ethiopia). [1, 2]
In other studies, RDTs have performed better than microscopy under routine conditions. RDTs performed by the health facility staff were 91.7% sensitive and 96.7% specific; microscopy was 52.5% sensitive and 77% specific. [41] A study using loop-mediated amplification technique (LAMP) also suggests that RDTs have accuracy similar to that of nested PCR, with a greatly reduced time to result, and was superior to expert microscopy. [42]
In a study from Tanzania, d'Acremont et al reported that antimalarials could be safely withheld from febrile children (< 5 y) who had negative results from an RDT based on PfHRP2. [43]
RDTs are less effective when parasite levels are below 100 parasites/mL of blood, and, in rare instances, an RDT test is negative in patients with high parasitemias. For these reasons, confirm RDT test results with a second type of screening test when possible. A false-positive result from an RDT may occur up to 2 weeks or more after treatment due to persistence of circulating antigens.
In summary, RDTs should supplement microscopy and can be helpful; however, a negative result does not rule out malaria.
Other tests
In addition to the RDT listed above, new molecular techniques, such as PCR assay testing and nucleic acid sequence-based amplification (NASBA), are available for diagnosis and species identification. They are more sensitive than thick smears but are expensive and unavailable in most developing countries. [44]
Malaria is a reportable disease. Identification of parasites by any of the above techniques should prompt notification to the local or state health department.
Histologic Findings
The table below compares histologic findings for P falciparum, P vivax, P ovale, and P malariae.
Table 1. Histologic Variations Among Plasmodium Species (Open Table in a new window)
Findings |
P falciparum |
P vivax |
P ovale |
P malariae |
Only early forms present in peripheral blood |
Yes |
No |
No |
No |
Poly-infected RBCs |
Often |
Occasionally |
Rare |
Rare |
Age of infected RBCs |
RBCs of all ages |
Young RBCs |
Young RBCs |
Old RBCs |
Schüffner dots |
No |
Yes |
Yes |
No |
Other features |
Cells have thin cytoplasm, 1 or 2 chromatin dots, and applique forms. |
Late trophozoites develop pleomorphic cytoplasm. |
Infected RBCs become oval, with tufted edges. |
Bandlike trophozoites are distinctive. |
*Slide images below were retrieved from the CDC DPDx database and are available at no cost - https://www.cdc.gov/dpdx/malaria/index.html
Plasmodium falciparum
Thin blood smear showing the ring forms of P falciparum that look like headphones with double chromatin dots. Note how P falciparum is seen infecting erythrocytes of all ages – a trait that can be utilized by the microscopist by noting the similar size of infected erythrocytes to other surrounding uninfected erythrocytes. Courtesy of the Centers for Disease Control and Prevention (CDC) [https://www.cdc.gov/dpdx/malaria/index.html].
Thick blood smear depicting the banana shaped gametocyte of P falciparum. Multiple ring-form trophozoite precursors are also visible in the background. Courtesy of the Centers for Disease Control and Prevention (CDC) [https://www.cdc.gov/dpdx/malaria/index.html].
Plasmodium vivax
Thin blood smear of the ring forms of P vivax. Note that P vivax typically has a single chromatin dot vs the two chromatin dots in P falciparum. Courtesy of the Centers for Disease Control and Prevention (CDC) [https://www.cdc.gov/dpdx/malaria/index.html].
The diagnostic form of P vivax is the amoeboid trophozoite form where the cytoplasm has finger-like projections (pseudopods) without a typical round/oval structure. These pseudopods are unique to P vivax. Numerous small pink-red dots are also seen in both P vivax and P ovale; these are known as caveola-vesicle complexes (CVCs or Schüffner’s dots) and are composed of numerous flask-like indentations on infected reticulocytes membrane skeleton associated with tube-like vesicles. CVCs are thought to play a role in nutrient uptake or release of metabolites from parasite-infected erythrocytes. Courtesy of the Centers for Disease Control and Prevention (CDC) [https://www.cdc.gov/dpdx/malaria/index.html].
Plasmodium ovale
Thin smear of P ovale in ring stage. Note that typically there is a single chromatin dot, larger cells are infected indicative of reticulocytes, and multiple ring forms may be present intracellularly. Courtesy of the Centers for Disease Control and Prevention (CDC) [https://www.cdc.gov/dpdx/malaria/index.html].
Thin smear of P ovale trophozoite. Note that this species is difficult to differentiate from P vivax as it contains CVCs (Schüffner’s dots) and infects reticulocytes; a notable unique characteristic of P ovale is the presence of fimbriae on the reticulocyte membrane, which are even more likely to be seen in gametocyte infected red blood cells. Courtesy of the Centers for Disease Control and Prevention (CDC) [https://www.cdc.gov/dpdx/malaria/index.html].
Plasmodium malariae
Thin blood smear of “band form” trophozoite of P malariae. Note that the infected erythrocyte is smaller than surrounding cells, indicating that P malariae infects older erythrocytes. As the trophozoite matures, the cytoplasm elongates and dark pigment granules of hemozoin are visualized toward the periphery. Courtesy of the Centers for Disease Control and Prevention (CDC) [https://www.cdc.gov/dpdx/malaria/index.html].
Plasmodium knowlesi
Thin blood smear of P knowlesi trophozoites. An immature ring form is seen on the right next to the mature band form trophozoite on the left. Note the small size of the infected red blood cells and how the band form is similar in appearance to P malariae. Courtesy of the Centers for Disease Control and Prevention (CDC) [https://www.cdc.gov/dpdx/malaria/index.html].
-
Malarial merozoites in the peripheral blood. Note that several of the merozoites have penetrated the erythrocyte membrane and entered the cell.
-
This micrograph illustrates the trophozoite form, or immature-ring form, of the malarial parasite within peripheral erythrocytes. Red blood cells infected with trophozoites do not produce sequestrins and, therefore, are able to pass through the spleen.
-
An erythrocyte filled with merozoites, which soon will rupture the cell and attempt to infect other red blood cells. Notice the darkened central portion of the cell; this is hemozoin, or malaria pigment, which is a paracrystalline precipitate formed when heme polymerase reacts with the potentially toxic heme stored within the erythrocyte. When treated with chloroquine, the enzyme heme polymerase is inhibited, leading to the heme-induced demise of non–chloroquine-resistant merozoites.
-
A mature schizont within an erythrocyte. These red blood cells (RBCs) are sequestered in the spleen when malaria proteins, called sequestrins, on the RBC surface bind to endothelial cells within that organ. Sequestrins are only on the surfaces of erythrocytes that contain the schizont form of the parasite.
-
Malaria life cycle. Courtesy of the Centers for Disease Control and Prevention (CDC) [https://www.cdc.gov/dpdx/malaria/index.html].
-
Proportion of 2021 Global Malaria Burden. Gray area accounts for the remaining estimated 4.4% of worldwide malaria burden. Map created using data adapted from WHO 2022 World Malaria Report [https://www.who.int/teams/global-malaria-programme/reports/world-malaria-report-2022].
-
Confirmed P falciparum or P vivax Cases Per Country 2021. The map accounts for the total of the cases per country where either species were confirmed as the primary infection. The map does not include confirmed “mixed infections.” Gray indicates that there were either no data available or there were zero endemic cases. Map created using data adapted from WHO 2022 World Malaria Report [https://www.who.int/teams/global-malaria-programme/reports/world-malaria-report-2022].
-
North American Presumed and Confirmed Malaria Cases 2021. Gray indicates that there were either no data available or there were zero endemic cases. Map created using data adapted from WHO 2022 World Malaria Report [https://www.who.int/teams/global-malaria-programme/reports/world-malaria-report-2022].
-
South American Presumed and Confirmed Malaria Cases 2021. Gray indicates that there were either no data available or there were zero endemic cases. Map created using data adapted from WHO 2022 World Malaria Report [https://www.who.int/teams/global-malaria-programme/reports/world-malaria-report-2022].
-
African Presumed and Confirmed Malaria Cases 2021. Gray indicates that there were either no data available or there were zero endemic cases. Map created using data adapted from WHO 2022 World Malaria Report [https://www.who.int/teams/global-malaria-programme/reports/world-malaria-report-2022].
-
Asian and Oceanic Presumed and Confirmed Malaria Cases 2021. Gray indicates that there were either no data available or there were zero endemic cases. Map created using data adapted from WHO 2022 World Malaria Report [https://www.who.int/teams/global-malaria-programme/reports/world-malaria-report-2022].
-
South Pacific Presumed and Confirmed Malaria Cases 2021. Gray indicates that there were either no data available or there were zero endemic cases. Map created using data adapted from WHO 2022 World Malaria Report [https://www.who.int/teams/global-malaria-programme/reports/world-malaria-report-2022].
-
Global P falciparum to P vivax Case Ratios 2021. Gray indicates that there were either no data available or there were zero endemic cases. Red indicates higher proportion of P vivax cases, whereas blue indicates higher proportion of P falciparum cases. Map created using data adapted from WHO 2022 World Malaria Report [https://www.who.int/teams/global-malaria-programme/reports/world-malaria-report-2022].
-
Thin blood smear showing the ring forms of P falciparum that look like headphones with double chromatin dots. Note how P falciparum is seen infecting erythrocytes of all ages – a trait that can be utilized by the microscopist by noting the similar size of infected erythrocytes to other surrounding uninfected erythrocytes. Courtesy of the Centers for Disease Control and Prevention (CDC) [https://www.cdc.gov/dpdx/malaria/index.html].
-
Thick blood smear depicting the banana shaped gametocyte of P falciparum. Multiple ring-form trophozoite precursors are also visible in the background. Courtesy of the Centers for Disease Control and Prevention (CDC) [https://www.cdc.gov/dpdx/malaria/index.html].
-
Thin blood smear of the ring forms of P vivax. Note that P vivax typically has a single chromatin dot vs the two chromatin dots in P falciparum. Courtesy of the Centers for Disease Control and Prevention (CDC) [https://www.cdc.gov/dpdx/malaria/index.html].
-
The diagnostic form of P vivax is the amoeboid trophozoite form where the cytoplasm has finger-like projections (pseudopods) without a typical round/oval structure. These pseudopods are unique to P vivax. Numerous small pink-red dots are also seen in both P vivax and P ovale; these are known as caveola-vesicle complexes (CVCs or Schüffner’s dots) and are composed of numerous flask-like indentations on infected reticulocytes membrane skeleton associated with tube-like vesicles. CVCs are thought to play a role in nutrient uptake or release of metabolites from parasite-infected erythrocytes. Courtesy of the Centers for Disease Control and Prevention (CDC) [https://www.cdc.gov/dpdx/malaria/index.html].
-
Thin smear of P ovale in ring stage. Note that typically there is a single chromatin dot, larger cells are infected indicative of reticulocytes, and multiple ring forms may be present intracellularly. Courtesy of the Centers for Disease Control and Prevention (CDC) [https://www.cdc.gov/dpdx/malaria/index.html].
-
Thin smear of P ovale trophozoite. Note that this species is difficult to differentiate from P vivax as it contains CVCs (Schüffner’s dots) and infects reticulocytes; a notable unique characteristic of P ovale is the presence of fimbriae on the reticulocyte membrane, which are even more likely to be seen in gametocyte infected red blood cells. Courtesy of the Centers for Disease Control and Prevention (CDC) [https://www.cdc.gov/dpdx/malaria/index.html].
-
Thin blood smear of “band form” trophozoite of P malariae. Note that the infected erythrocyte is smaller than surrounding cells, indicating that P malariae infects older erythrocytes. As the trophozoite matures, the cytoplasm elongates and dark pigment granules of hemozoin are visualized toward the periphery. Courtesy of the Centers for Disease Control and Prevention (CDC) [https://www.cdc.gov/dpdx/malaria/index.html].
-
Thin blood smear of P knowlesi trophozoites. An immature ring form is seen on the right next to the mature band form trophozoite on the left. Note the small size of the infected red blood cells and how the band form is similar in appearance to P malariae. Courtesy of the Centers for Disease Control and Prevention (CDC) [https://www.cdc.gov/dpdx/malaria/index.html].
Tables
Findings |
P falciparum |
P vivax |
P ovale |
P malariae |
Only early forms present in peripheral blood |
Yes |
No |
No |
No |
Poly-infected RBCs |
Often |
Occasionally |
Rare |
Rare |
Age of infected RBCs |
RBCs of all ages |
Young RBCs |
Young RBCs |
Old RBCs |
Schüffner dots |
No |
Yes |
Yes |
No |
Other features |
Cells have thin cytoplasm, 1 or 2 chromatin dots, and applique forms. |
Late trophozoites develop pleomorphic cytoplasm. |
Infected RBCs become oval, with tufted edges. |
Bandlike trophozoites are distinctive. |
