***Reminder: Slides are copyrighted and cannot be copied for publication.
A patient is a 47 year-old male with multiple medical problems who was seen in an emergency room complaining of diarrhea, cough, and fever. He had recently returned from a trip to India. Although malaria prophylaxis was recommended, he failed to take the medication properly. 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 possible identification of the organisms seen.
Both the thick and thin blood films were stained using a rapid blood stain.
After the appropriate diagnosis was made, additional blood was drawn with the following images seen in the thick and thin blood films. Please comment on the objects seen below.
(Scroll Down for Answers and Discussion)
ANSWER AND DISCUSSION OF DIAGNOSTIC QUIZ #65
ANSWERS TO QUESTIONS:
The images presented after the first blood specimen was drawn represent Plasmodium vivax developing trophozoites in the thick blood film (left, at a lower magnification), a developing trophozoite in the thin blood film (middle), and an RBC containing two ring forms (right). Note the presence of pale stippling and the enlarged RBC (typical of P. vivax).
After the second blood draw, the image on the left represents developing rings in a thick blood film, the image in the middle represents a mature schizont in the thin blood film (again P. vivax), and the image on the right shows an early developing trophozoite (notice there is no stippling). The number of merozoites in the mature schizont is always a clue to the species identification.
Although stippling (Schüffner's dots) is present, this morphologic characteristic may not always be found in EDTA-preserved blood, particularly if the blood is stored prior to blood film preparation. Lack of stippling should not prevent the correct identification of Plasmodium vivax. Also, remember that stippling does not occur in the young rings (P. vivax), while they are seen in all stages in infections with P. ovale, including very young rings. Visualization of stippling depends on the lag time present between blood collection in EDTA and preparation of the blood films - too long a lag time will prevent stippling from being seen.
Key characteristics of P. vivax include the following:
1. 48-hour cycle
2. Tends to infect young cells
3. Enlarged RBCs, RBC may be normal size until ring fills half of cell
4. Schüffner's dots after 8-10 hours (providing storage, buffer pH criteria are met)
5. Delicate ring, but heavy chromatin dot
6. Very ameboid trophozoite
7. Mature schizont contains 12-24 merozoites, usually around 16-1
COMMENTS ON THE PATIENT:
Since this patient had not taken proper prophylaxis during his trip to India, malaria was suspected and his illness had been diagnosed as P. vivax malaria. With proper treatment, he recovered. Treatment included drugs for both the blood stages, as well as the liver stages (liver stages = hypnozoites, which could lead to a relapse at a later time without the use of the second drug).
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. If blood is going to be sent to a reference laboratory, it is important that both thick and thin blood films be prepared as quickly as possible after receipt of the blood; both thick and thin blood films, as well as the tube of blood should be sent to the reference laboratory.
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 (0.01%, particularly with P. falciparum). 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 BOTH 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 has not been excluded as a diagnosis.
Clinical Laboratory Standards Institute/National Committee for Clinical Laboratory Standards. 2000. Laboratory Diagnosis of Blood-borne Parasitic Diseases. Approved Guideline, M15-A. CLSI/NCCLS, Villanova, Pa.
Garcia, LS, 2016. Diagnostic Medical Parasitology, 6th Ed., ASM Press, Washington, DC.
Isenberg, H. D. (ed.). 2004. Clinical Microbiology Procedures Handbook, ed. 2, vol. 1, 2, 3. American Society for Microbiology, Washington, D.C.
Isenberg, H. D. (ed.). 1995. Essential Procedures for Clinical Microbiology, ASM Press, Washington, D.C.
Wilcox, A., 1960. Manual for the Microscopical Diagnosis of Malaria in Man. U.S. Department of Health, Education, and Welfare, Washington, D.C.