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Presentation of Quiz #63

A patient is a 44 year-old male living within the United States. He presented admitted to the clinic with 10 day history of irregular recurrent fever, shivering, nausea, and anorexia. Several weeks before, he had returned from an extended business to Thailand and India. On examination of both thick and thin blood films prepared from blood collected in EDTA tube (purple top), the following images were seen. Please comment on the identification of the organisms seen.

The thin blood films were stained using a blood stain.

After the appropriate diagnosis of Plasmodium spp. was made, additional history information was obtained.  The tentative diagnosis was the possibility of a mixed infection of Plasmodium falciparum and P. malariae.

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Answer and Discussion of Quiz #63

The images presented after the first blood specimen was drawn resemble a Plasmodium malariae band form. Note the small size of the infected RBC (typical of P. malariae). The middle image demonstrates a ring form that could be any one of the five species.  The image on the right contains an RBC containing double rings, characteristic of P. falciparum





Additional information was obtained from the following:

The patient had a 0.8% parasitemia that appeared to be a mixed infection with P. falciparum (multiple rings/RBC) and P. malariae (band form trophozoites).  Rapid diagnostic tests (RPDS) were performed (BinaxNOW test – Inverness Medical, Sevres, France) with the following results:

The kit was positive for the Pan-Plasmodium (aldolase common to the four species of human malaria) line (often interpreted as P. vivax); however, the histidine-rich protein 2 (HRP2), a specific antigen of P. falciparum, was negative.

Given the discrepancy between the thin blood films and the rapid test results, another laboratory was asked to confirm the results.  Their results with the rapid test were identical (pos Pan-Plasmodium line, neg P. falciparum line).

Although PCR was used and only the P. vivax PCR was positive, the parasite morphology on the blood films was not compatible with P. vivax.

Comments on the Patient:

Since this patient had spent time in Southeast Asia, followup sequencing was done to confirm the malarial species. A basic local alignment search tool (BLAST) search of the GeneBank database with the sequence obtained (1,000 bp), showed 100% identify with Plasmodium knowlesi sequences.


Plasmodium knowlesi (simian malaria)*

1.         24-hour cycle

2.         Tends to infect any cell regardless of age, thus very heavy infection may result

3.         All sizes of RBCs, but most tend to be normal size       

4.         No Schüffner’s dots (faint, clumpy dots later in cycle)

5.         Multiple rings/cell (may have 2-3)

6.         Delicate rings, may have two or three dots of chromatin/ring, appliqué forms

7.         Band form trophozoites commonly seen

8.         Mature schizont contains 16 merozoites, no rosettes

9.         Gametocytes round, tend to fill the cell

A relatively large focus of human infections with the simian malaria, P. knowlesi, has been reported in Malaysian Borneo; cases have also been reported from Thailand, Myanmar, China, Philippines, and Singapore. The early blood stages of P. knowlesi resemble those of P. falciparum, whereas the mature blood stages and gametocytes resemble those of P. malariae. Unfortunately, these infections are often misdiagnosed as the relatively benign P. malariae; however, infections with P. knowlesi can be fatal. P. knowlesi infection should be considered in patients with a travel history to forested areas of Southeast Asia, especially if P. malariae is diagnosed, unusual forms are seen

with microscopy, or if a mixed infection with P. falciparum/P. malariae is diagnosed. 

Because the disease is potentially fatal, proper identification to the species level is critical.  Patients exhibit chills, minor headaches, and daily low-grade fever. Patients who present with P. malariae hyperparasitemia diagnosis by microscopy should receive intensive management as appropriate for severe P. falciparum malaria, assuming the infection is actually caused by P. knowlesi.


The upper image shows the endemic areas for P. knowlesi; however, it appears that the range continues to expand.  The lower image demonstrates typical morphology for P. knowlesi (courtesy of CDC).

Comments on Diagnostic Methods:

It is very important to realize that a single set of thick and thin blood films can be negative, although the patient may be positive. In this case, both thick and thin blood films were positive. A second draw was taken to examine the thick and thin blood films for additional stages and/or evidence of a mixed infection. Venipunctures were performed for both blood draws, with the recommended EDTA anticoagulant in the lavender (purple) top tube. It is important that the slides be prepared as quickly as possible after the blood draw, in order to prevent organism distortion and possible loss that can occur if the blood is allowed to stand for a period of hours prior to slide preparation. Remember, every request for malaria blood films should always be considered a STAT request and the laboratory coverage should be 24 hours/day, 7 days/week.  


Examination of the thin blood film is relatively simple when the parasitemia is high, as in this slide. However, a returning traveler with his or her first malaria infection may experience the typical clinical symptoms of high fever, chills, myalgia, and headache with a much lower parasitemia. Also, these patients may present to the emergency room with vague symptoms that do not represent the typical textbook description; they may have malaise, a steady low-grade fever, and may even have diarrhea. These low levels of parasitemia are often impossible to detect using thin blood film examination only. For this reason, the key to successful detection of malaria parasites in the peripheral blood is the examination of thick and thin blood films from every patient suspected of having malaria (or any patient from whom blood is submitted to the laboratory for blood film examination).

Thick films allow a larger amount of blood to be examined, which increases the possibility of detecting light infections. Species identification from the thick film examination, particularly in the case of malaria, may be difficult for those with little experience examining thick blood films. The morphological characteristics of blood parasites are best seen in thin films, particularly the relationship between the size of the infected RBC and those that are uninfected. However, in cases with a low parasitemia, the identification to the species level may have to be accomplished by thick film examination.

The accurate examination of thick and thin blood films and identification of parasites depends on the use of absolutely clean, grease-free slides for preparation of all blood films. Old (unscratched) slides should be cleaned first in detergent and then with 70% ethyl alcohol; new slides should also be cleaned with alcohol before use.

Blood films are usually prepared when the patient is admitted; in instances in which malaria is a possible diagnosis, after the first set of negative smears, samples should be taken at intervals of 6 to 8 h for at least 3 successive days, particularly if P. falciparum or P. knowlesi have not been excluded as a diagnosis.


  1. Berry, A. et al. 2011.  Case Report: Imported Plasmodium knowlesi Malaria in a French Tourist Returning from Thailand.  Am J Trop Med Hyg 84:535-538.
  2. Garcia, LS, 2016. Diagnostic Medical Parasitology, 6th Ed., ASM Press, Washington, DC.
  3. Garcia, L.S. (ed.). 2010. Clinical Microbiology Procedures Handbook, 3rd Ed., vol. 1, 2, and 3. ASM Press, Washington, D.C.
  4. Isenberg, H. D. (ed.). 2004. Clinical Microbiology Procedures Handbook, 2nd Ed, vol. 1, 2 and 3. ASM Press, Washington, D.C.
  5. Isenberg, H. D. (ed.). 1995. Essential Procedures for Clinical Microbiology, ASM Press, Washington, D.C.
  6. Khim, N. et al. 2011.  Plasmodium knowlesi Infection in Humans, Cambodia, 2007-2010.  Emerg Inf Dis 17:1900-1902.
  7. National Committee for Clinical Laboratory Standards. 2000. Laboratory Diagnosis of Blood-borne Parasitic Diseases. Approved Guideline, M15-A. National Committee for Clinical Laboratory Standards, Villanova, Pa.
  8. Wilcox, A., 1960. Manual for the Microscopical Diagnosis of Malaria in Man. U.S. Department of Health, Education, and Welfare, Washington, D.C.
  9. William, T. et al. 2011.  Severe Plasmodium knowlesi Malaria in a Tertiary Care Hospital, Sabah, Malaysia.  Emerg. Inf. Dis. 17:1248-1255.