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Informational Tables

- 1.1 Parasite Classification | - 1.2 Body Site, Specimens, Procedures, Parasites, Comments | - 1.3 STAT Testing in Parasitology | - 1.4 Test Issues and Reports: Computer Report Comments| - 1.5 Rapid Diagnostic Testing
- 2.1 Stool Testing Order Recommendations | - 2.2 Fecal specimens for parasites: options for collection and processinga2 | - 2.3 Preservatives used for Stool Specimens
- 3.1 Body Sites and Specimen Collection | - 3.2 Body sites and the most common parasites recovered | - 3.3 Body Site, Specimens and Recommended Stain | - 3.4 Examination of tissues and body fluids | - 3.5 Parasitic Infections: Clinical Findings Healthy/Compromised Hosts | - 3.6 Microscope Calibration | - 3.7 Serologic, Antigen, and Probe Tests for Parasite Diagnosis
- 4.1 Protozoa: Intestinal Tract, Urogenital System: Key Characteristics | - 4.2 Tissue Protozoa: Characteristics | - 4.3 Tips on Performance of Fecal Immunoassays for Intestinal Protozoa
5.1 Helminths: Key Characteristics | 5.2 Helminth Parasites Associated with Eosinophilia
6.1 Reference Laboratory for Parasite Blood Testing | 6.2 Parasites Found in Blood: Characteristics
7.1 Malaria (5 Species) (2 P. ovale subspecies) | 7.2 Malaria (5 Species, Images) | 7.3 Rapid Malaria Testing (BinaxNOW Malaria Test) | 7.4 Malaria Parasitemia Method | 7.5 Malaria Parasitemia Interpretation

- HELMINTH PARASITES ASSOCIATED WITH EOSINOPHILIA | - Histology: Staining Characteristics - Table 1 | - Histological Identification of Parasites - Table 2 | - Microscope Calibration | - Figures for Histology Identification Table 2

Procedure/Table 7.4 Determination of Malaria Parasitemia



It is important to report the level of parasitemia when blood films are examined and found to be positive for malaria parasites. Because of the potential for drug resistance in some of the Plasmodium species, particularly Plasmodium falciparum, it is important that every positive smear be assessed and the parasitemia reported exactly the same way for follow-up specimens as for the initial specimen (1–7). This allows the parasitemia to be monitored after therapy has been initiated. In cases where the patient is hospitalized, monitoring should be performed at 24, 48, and 72 h after initiating therapy. Generally, the parasitemia will drop very quickly within the first 2 h; however, in cases of drug resistance, the level may not decrease but increase over time. Although there may be shortcut methods for estimating the percent parasitemia, the protocol seen here will provide the most accurate results. Molecular tests, including PCR-based assays, for malaria detection and species identification exist and have been shown to be more sensitive in low-parasitemia infections (8). However, this testing exists in relatively few laboratories and microscopy remains the gold standard for malaria detection and parasitemia monitoring.


Observe standard precautions.

The specimen consists of stained thick or thin blood films that have been examined a minimum of 300 oil immersion fields per blood film to determine that the film is positive for malaria or Babesia parasites.


  • A. Reagents>

  • None

  • B. Supplies

  • None

  • C. Equipment

  • Microscope, binocular with mechanical stage; low-power (10×), high dry power (40×), and oil immersion (100×) objectives; 10× oculars; calibrated ocular micrometer; light source equivalent to a 20-W halogen or 100-W tungsten bulb; blue and white ground-glass diffuser filters



  • A. Prepare and stain films from “normal” blood, and microscopically evaluate the staining reactions of RBCs, platelets, and WBCs (normally done during staining procedure using Giemsa or Wright’s stain). Other blood stains, including the rapid stains, are also acceptable.

  • B. The microscope should be calibrated, and the objectives and oculars used for the calibration procedure should be used for all measurements on the microscope. Post the calibration factors for all objectives on the microscope for easy access (multiplication factors can be pasted right on the body of the microscope) (see procedures 9.1 and 9.3.2). Although there is not universal agreement, the microscope should probably be recalibrated once each year. This recommendation should be considered with heavy use or if the microscope has been bumped or moved multiple times. If the microscope does not receive heavy use, then recalibration is not required on a yearly basis.


  • A. Thin blood film: counting > several hundred to > thousands of RBCs, report the percentage of infected RBCs per 100 RBCs counted (0.5%, 1.0%, etc.). The exact number of RBCs (infected and uninfected) should be counted and never estimated. Estimating numbers of RBCs in a microscope field of view may result in inaccurate parasitemia calculations. The number of RBCs counted will vary depending on the overall number of parasites present. If very few parasites are seen, more RBCs will need to be counted to arrive at an accurate parasitemia percentage.
    Note: Plasmodium spp. gametocytes are not counted, as these are dead-end stage parasites in humans. Also, because some anti-malaria medications are not anti-gametocidal, counting gametocytes in parasitemia quantification will not accurately reflect a patient’s response to therapy.

  • B. Thick or thin blood film: counting 100 WBCs (or more), report the number of parasites per 100 WBCs on the smear.

    • 1. This figure can be converted to the number of parasites per microliter of blood; divide the number of parasites per 100 WBCs by 100, and multiply that figure by the number of WBCs per microliter of blood.

    • 2. Depending on the parasitemia, 200 or more WBCs may have to be counted, so the denominator may vary (it may be 200 or even more).

    • 3. Using this method, blood for both the peripheral smears and cell counts must be collected at the same time.

    • 4. This method may be inaccurate in patients with immunodeficiency and/or leukopenic patients.


VI. REPORTING RESULTS (see table below)

  • A. Using the thin blood film method, report the percentage of parasite-infected RBCs per 100 RBCs counted.tions of RBCs, platelets, and WBCs (normally done during staining procedure using Giemsa or Wright’s stain). Other blood stains, including the rapid stains, are also acceptable.
    Example:Plasmodium falciparum, parasitemia = 0.5%

  • B. Using the thick-thin blood film method, report the number of parasites per microliter of blood.
    Example:Plasmodium falciparum, parasitemia = 10,000 per μl of blood

Table. Parasitemia determined from conventional light microscopy: clinical correlationa

% Parasitemia

No. of parasites/μl

Clinical correlationb



Number of organisms that are required for a positive thick film (sensitivity)

Note: Examination of 100 TBF fields (0.25 μl) may miss infections up to 20% (sensitivity of 80–90%); at least 300 TBF fields should be examined before reporting a negative result.

Both TBF and THBF should be examined for every specimen submitted for a suspected malaria case. One set (TBF + THBF) of negative blood films does not rule out a malaria infection.



Patients may be symptomatic below this level, particularly if they are immunologically naïve (no prior exposure to malaria [travelers])

Level above which immune patients will exhibit symptoms. Level can be seen in travelers (immunologically naïve); however, results may also be much lower in this type of patient (traveler).



(>5,000 = 93.5% sensitivity for P. vivax). Below this level of parasitemia, the test sensitivity declines. A negative test does not rule out malaria.



Usual maximum parasitemia of P. vivax and Plasmodium ovale (infect young RBCs only)

a Adapted from references 3 and 7.

b TBF, thick blood film; THBF, thin blood film. The BinaxNOW malaria test (Abbott Laboratories, Abbott Park, IL) is FDA approved and detects antigen from both viable and nonviable malaria organisms, including gametocytes and sequestered P. falciparum parasites. Test performance depends on antigen load in the specimen and may not directly correlate with microscopy performed on the same specimen. Samples with positive rheumatoid factor titers may produce false-positive results. Analytical reactivity testing demonstrates that the pan-malarial test line on the BinaxNOW test is capable of detecting all four malaria species. However, during clinical trials, insufficient data were generated to support clinical performance claims for the detection of P. malariae or P. ovale. Although this test was developed without considering the fifth human malaria organism, Plasmodium knowlesi, cases have been documented to be positive using the BinaxNOW. However, use of this test for the detection of P. knowlesi should not be considered acceptable. Clinical performance claims for this test are made for P. falciparum and P. vivax detection only. The test is not intended for use in screening asymptomatic populations. If this test is negative, STAT thick and thin blood films must be examined immediately (BinaxNOW malaria test package insert; Inverness Medical, Scarborough, ME). Delayed examination of blood films may be detrimental to patient care and outcomes. The BinaxNOW Positive Malaria Control, as well as the BinaxNOW malaria test, is available commercially. Delayed testing.

c World Health Organization criteria for severe malaria are parasitemia of >10,000/μl and severe anemia (hemoglobin <5 g/liter). Prognosis is poor if >20% of parasites are pigment-containing trophozoites and schizonts and/or if >5% of neutrophils contain visible pigment.


  • A. It is critical that the same reporting method be used consistently for every subsequent set of blood films so that the parasitemia can be tracked for decrease or possible increase, indicating resistance. For consistency, it is also recommended that the same individual who calculated the original result perform any additional calculations on subsequent blood films.

  • B. Remember that drug resistance may not become evident for several days; the parasitemia may even appear to be dropping before it begins to increase again.

  • C. It is very important that any patient with P. falciparum infection be monitored; drug tolerance and resistance have also been reported for Plasmodium vivax infections.

  • D. It is critical to remember that mixed malarial infections occur, many of which include P. falciparum.


  • A. A light infection may be missed in a thin film, whereas the increased volume of blood present on a thick film may allow the detection of the infection, even with a low parasitemia.

  • B. If the smears are prepared from anticoagulated blood that is more than 1 h old, the morphology of both parasites and infected RBCs may not be typical. Also, if the blood has been standing in anticoagulant for 4 to 6 h prior to blood film preparation, some of the parasites may become very distorted and will not identified as parasites, thus leading to a false low or negative parasitemia (9).

  • C. It is important that good-quality blood films be examined and counted according to directions; poorly prepared and/or stained blood films will lead to incorrect assessments of the parasitemia (both too low and too high).

  • D. Follow-up counts are critical in monitoring the patient’s response to treatment. Drug resistance in malaria cases may manifest in various ways, such as decreased parasitemia followed by recrudescence or prolonged low-level parasitemia. (see Table below)

  • E. Technologist training is important to ensure that personnel are reporting similar counts from the same slides for standardized results.

Table._Malaria resistancea

Resistance definition



From initiation of therapy, asexual parasites are cleared by day 6; no evidence of recrudescence up to day 28

Peripheral blood films appear to go from positive to negative very quickly (can be a change from one draw to the second draw 6 h later).

Resistance type I

From initiation of therapy, asexual parasites have cleared for at least two consecutive days (the latest day being day 6); recrudescence follows within 28 days.

Parasite count initially drops and blood films appear to be negative; patient should be monitored for a period of days, particularly if drug-resistant P. falciparum is suspected.

Resistance type II

Within 48 h of initiation of therapy, marked reduction of asexual parasitemia to <25% of pretreatment count; however, no subsequent disappearance of parasitemia (smear positive on day 6)

Patient appears to be improving; parasite count drops, but blood films always appear positive.

Resistance type III

Modest reduction in parasitemia may be seen; less than 75% reduction in parasitemia seen during first 48 h after treatment; no clearing of asexual parasites

In some cases, the parasite count continues to increase with no visible decrease at any time; blood films show overall parasite increase.

a Adapted from references 3 and 4


Smear-positive asymptomatic malaria infections detectable using microscopy are an important reservoir because these infections can persist for months and harbor gametocytes, the parasite stage infectious to mosquitoes. However, many asymptomatic infections are submicroscopic; detection is not possible using routine microscopy, but requires molecular methods (10). Additional research is required to clarify to what extent these cases of submicroscopic malaria contribute to the overall infectious reservoir. Diagnostic detection methods for infection confirmation are also necessary to define in order to effectively interrupt the normal transmission cycle. In light of these problems, there has been a shift from the emphasis on symptomatic patients to those who are asymptomatic, but continue to serve as potential reservoirs for infection. Diagnostic methods such as PCR-based molecular tests may not be routinely available to confirm these submicroscopic cases.

A number of questions remain to be clarified; a few of these are listed below.

  1. What percent of submicroscopic infections persist asymptomatically?

  2. How many of these infections will become symptomatic and identifiable through microscopy?

  3. Do treated patients develop subsequent submicroscopic parasitemia?

  4. Do these submicroscopic infections develop and maintain gametocytes? If so, at what level of parasitemia?

  5. What human and/or mosquito factors influence transmission?

  6. What level of sensitivity is required to confirm infections in this particular reservoir?

  7. We need to define the ethics of therapy considering the risk-benefit ratios of the options in these patients who are asymptomatic and have submicroscopic parasitemia.


  1. Garcia LS. 2016. Diagnostic Medical Parasitology, 6th ed. ASM Press, Washington, DC.

  2. Garcia LS, Johnston SP, Linscott AJ, Shimizu RY. 2008. Cumitech 46, Laboratory Procedures for Diagnosis of Blood-Borne Parasitic Diseases. Coordinating ed, Garcia LS. ASM Press, Washington, DC.

  3. Hansheid T. 1999. Diagnosis of malaria: a review of alternatives to conventional microscopy. Clin Lab Haematol 21:235–245.

  4. Homel M, Gilles HM. 1998. Malaria, p 361–409. In Cox FEG, Krier JP, Wakelin D (ed), Topley & Wilson’s Microbiology and Microbial Infections, 9th ed. Arnold, London, United Kingdom.

  5. Milhouse WK, Kyle DE. 1998. Introduction to the modes of action of and mechanisms of resistance to antimalarials, p 303–316. In Cox FEG, Krier JP, Wakelin D (ed), Topley & Wilson’s Microbiology and Microbial Infections, 9th ed. Arnold, London, United Kingdom.

  6. NCCLS. 2000. Laboratory Diagnosis of Blood-Borne Parasitic Diseases. Approved guideline M15-A. NCCLS, Wayne, PA.

  7. Wilkinson RJ, Brown JL, Pasvol G, Chiodini PL, Davidson RN. 1994. Severe falciparum malaria: predicting the effect of exchange transfusion. Q J Med 87:553–557.

  8. Mathison BA, Pritt BS.2017. Update on Malaria Diagnostics and Test Utilization. J Clin Microbiol 55:2009–2017.

  9. Antwi-Baffour S, Quao E, Kyeremeh R, Mahmood SA. 2014. Prolong storage of blood in EDTA has an effect on the morphology and osmotic fragility of erythrocytes. Intl J Biomedical Sci and Eng. DOI:10.11648/j.ijbse.20130102.11

  10. Lin JT, Saunders DL, Meshnick SR.2014. The role of submicroscopic parasitemia in malaria transmission: what is the evidence?Trends Parasitol.30:183-190