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CASE REPORT Table of Contents   
Year : 2009  |  Volume : 52  |  Issue : 2  |  Page : 252-254
Exflagellated microgametes of Plasmodium vivax in human peripheral blood: A case report and review of the literature


Department of Pathology, Tata Memorial Hospital (TMH), Mumbai, India

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   Abstract 

Peripheral blood smear examination is the most specific as well as the most common test performed for the diagnosis of malaria. Schizonts, ring forms (trophozoites) and gametocytes are the stages of malarial parasite that are commonly seen in the peripheral blood smear of a patient. Here, we report an extremely rare case of a 40-year-old male patient who presented with Plasmodium vivax infection with multiple exflagellated microgametes in the peripheral blood smear with review of the literature. Exflagellation of microgametes in malarial parasites is only seen in the definitive host, mosquito, and is very unusual to see during the developmental phases in the intermediate host, human. It is important to recognize these exflagellated microgametes in the peripheral blood smear as they may lead to diagnostic confusion with organisms such as spirochetes and trypanosomes.

Keywords: Exflagellated, microgametes, Plasmodium vivax

How to cite this article:
Tembhare P, Shirke S, Subramanian P G, Sehgal K, Gujral S. Exflagellated microgametes of Plasmodium vivax in human peripheral blood: A case report and review of the literature. Indian J Pathol Microbiol 2009;52:252-4

How to cite this URL:
Tembhare P, Shirke S, Subramanian P G, Sehgal K, Gujral S. Exflagellated microgametes of Plasmodium vivax in human peripheral blood: A case report and review of the literature. Indian J Pathol Microbiol [serial online] 2009 [cited 2020 Mar 30];52:252-4. Available from: http://www.ijpmonline.org/text.asp?2009/52/2/252/48936



   Introduction Top


Plasmodium vivax malaria constitutes about 60-65% of the total cases of malaria in India. [1] Peripheral blood smear is the first investigation performed routinely for its diagnosis. The malarial parasite undergoes two types of reproduction: sexual (gametogony) and asexual (schizogony). Male gametes (microgamatocytes) can be seen in the human peripheral blood. However, occurrence of exflagellated microgametes is a part of the sexual life cycle and is found only within the mosquito, which is extremely rare to find in the human peripheral blood. [2]


   Case Report Top


A 40-year-old man, a case of well-differentiated squamous cell carcinoma of buccal mucosa, presented with high-grade fever, chills, malaise and headache of 3-day duration. The patient's peripheral blood in an ethylene diamine tetraacetic acid (EDTA) vacutainer tube was received in the laboratory approximately 50 min after the collection of blood. A complete blood cell count was immediately generated on a CELL-DYN Sapphire (by Abbott USA - optical and impedance cell counting technology), which also detects the malarial parasite in depolarization against a size scatter plot. It revealed a total leukocyte count of 8.73 × 10 9 /L, with 52.2% neutrophils, 31.2% lymphocytes, 15.2% monocytes and 1.52% basophils. The platelet count was 35.4 × 10 9 /L. Depolarization against a size scatter plot showed a well-distinguished cluster of events representing a parasite population [Figure 1]. A peripheral blood smear was prepared from the same sample after approximately 2h of collection, then stained with Wright's stain and examined, which revealed a heavy parasitemia (approximately 2%) consisting of the ring forms, gametocytes and ameboid trophozoites of P. vivax. In addition, several filamentous microgametes were seen outside the erythrocytes. These thin, undulating structures were approximately 12-15µm in length and showed a central dark chromatined oval to rod-like nucleus [Figure 2]. A few of them also showed a small dot-like knob at one end. The finding was correlated with the literature and confirmed to be exflagellated microgametes of P. vivax. The patient received chloroquine and primaquine therapy. Three days later, a post-therapy follow-up peripheral blood examination revealed a complete response to treatment with the absence of parasite.


   Discussion Top


Only eight cases of the presence of exflagellated microgametes in human peripheral blood have been published till date to our knowledge. [3],[4],[5],[6],[7] This is the ninth case of that kind. Published literature in the standard medical textbooks mention that trophozoites, schizonts and gametocytes of the malarial parasite are typically found in human blood and are useful for diagnosing and typing malarial infection. The remaining forms, like exflagellated microgametes, ookinetes and oocysts are found in the mosquitoes and are not expected to appear in the human blood.

The gametogony (sexual reproduction) occurs in the mosquito gut. When a mosquito ingests mature gametocytes in its blood meal, the gametocytes transform into gametes. The macrogametocytes (female) transform into only one macrogamete each while microgametocytes (male) undergo exflagellation to produce several highly motile, very slender microgametes. A microgamete then penetrates a macrogamete and the resulting zygote transforms into a fusiform, motile ookinete. Schizogony occurs in humans (vertebrates). Sporozoites are injected into the circulation from the salivary glands of a carrier mosquito. After an extra-erythrocytic (hepatic) merozoite stage, the parasite infects the erythrocytes. Maturing into the trophozoite (ring form) stage, the parasite may divide still further to form more merozoites, or may undergo gametogony, to form male gametes and female gametes. [2]

In 1897, MacCallum [8] reported the presence of flagellated gametocytes while examining infected human blood under a microscope. Pavlova et al . [3] detected exflagellating male gametocytes of P. vivax in a thick drop of blood. Later, in 1981, Gerber et al. [4] reported two cases presenting with exflagellation of the microgametocyte in the human peripheral blood smear. In late nineties, Hummert [8] published a case of detection of P. vivax ookinetes in the human peripheral blood. In the last decade, an occasional case of occurrence of exflagellating malarial parasite has been published in the literature. [5],[6],[7] This is the first case to be reported of its kind from India, a country with many endemic areas for malarial infection.

Factors required for exflagellation of the microgametocyte include increase in pH, which occurs in the mosquito following dissipation of carbon dioxide from the blood taken up into its gut, and decease in temperature. [7],[9] Exflagellated forms are not identified in freshly prepared peripheral blood smears but may instead be seen after the blood has been exposed to air for several minutes. As the CO 2 level in the blood quickly falls, to equilibrate with the surrounding air, the pH of the plasma rises and exflagellation may begin. [9]

In usual clinical laboratory conditions ( in vitro ), it is not common to find the high blood pH (7.7) that is thought necessary to allow exflagellation. Sufficiently high pH levels may be obtained in the plasma of blood samples collected into EDTA-containing tubes if the samples are left unstoppered and unagitated overnight. [4] Another possibility for the appearance of exflagellated microgametes in the human blood may be a drop of Plasmodium-infected blood left exposed to air on a microscope slide. Exposure to ambient air causes a decrease in pCO 2 , with a consequent rise in pH from the normal 7.4 to at least 7.7. [8] In addition, some unidentified factors may be responsible for exflagellation in human blood. Few published suggestions include phosphodiesterase inhibitors and caffeine but the exact mechanism is not known. [4]

In case of P. falciparum gametocytes, it has now been shown that they exflagellate at room temperature at pH 7.4 as long as the human serum concentration exceeds 1.5% and forms ookinetes within 8 h in the culture. [8]

In our case, the blood was collected in an EDTA vacutainer tube and kept in a stand for approximately 2h before smear preparation. The laboratory temperature was between 20 and 22°C.

The relative rarity found in a search of "Pubmed.com" (1965 through 2008) of exflagellated microgamagtocytes in human blood might reflect the fact that diseased specimens obtained in endemic countries generally consist of finger-stick smears rather than blood collected in test tubes. Another possibility of this rarity may be that these are not recognized and often discounted as an artifact.

Symptoms and signs of both malaria and relapsing fever (Borreliosis) are similar and appearance of spirochete is similar to that of exflagellated microgametes of P. vivax. [10] However, exflagellated microgametes are filamentous, thin, undulating structures measuring approximately 12-15µm in length, seen outside the erythrocytes. They also show a dark, cigar-shaped nucleus after staining with Wright's or Geimsa stains. Spirochete are microaerophilic helical bacteria, about 5-20µm long, with 4-30 coils. [11] The nucleus is absent, which is a major differentiating point between the two. But, lack of knowledge of exflagellating microgametes may lead to misidentification of the spirochete infection as Borrelia or, rarely, trypanosomes. In conclusion, knowledge about exflagellated microgametes in addition to other forms of malarial parasite is essential to avoid errors in diagnosis.

 
   References Top

1.Sharma VP. Re-emergence of malaria in India. Indian J Med Res 1996;103:26-45.  Back to cited text no. 1  [PUBMED]  
2.Fritsche TR, Smith JW: Medical parasitology. In: Henry JB, editor. Clinical diagnosis and management by laboratory methods. 20 th ed. Philadelphia, Pennsylvania: Saunders; 2001. p. 1203-8.  Back to cited text no. 2    
3.Pavlova EA. Detection of exflagellating male gamonts of Plasmodia vivax in a thick drop of blood. Med Parazitol (Mosk) 1969;38:284-6.  Back to cited text no. 3  [PUBMED]  
4.Gerber JE, Ukena TE, Cote L, Wyllie JM, Winn WC Jr. Exflagellation of malarial parasites in human peripheral blood. J Clin Microbiol 1981;13:236-7.  Back to cited text no. 4  [PUBMED]  [FULLTEXT]
5.Ford JC, Wadsworth LD. Exflagellating Plasmodium vivax in peripheral blood. Arch Pathol Lab Med 2003;127:117-8.  Back to cited text no. 5  [PUBMED]  [FULLTEXT]
6.Enger A, Strand OA, Ranheim T, Hellum KB. Exflagellation of microgametocytes in Plasmodium vivax malaria: A diagnostic conundrum. Med Princ Pract 2004;13:298-300.  Back to cited text no. 6    
7.Karplus R, Johnston SP. Images in clinical medicine: Plasmodium vivax microgametes. N Engl J Med 2006;354:1064.  Back to cited text no. 7  [PUBMED]  [FULLTEXT]
8.Hummert BA. Plasmodium vivax ookinetes in human peripheral blood. J Clin Microbiol 1994;32:2578-80.  Back to cited text no. 8  [PUBMED]  [FULLTEXT]
9.Nijhout MM, Carter R. Gamete development in malaria parasites: bicarbonate-dependent stimulation by pH in vitro . Parasitology 1978;76:39-53.  Back to cited text no. 9  [PUBMED]  
10.Poulsen LW, Iversen G. Relapsing fever: A differential diagnosis to malaria. Scand J Infect Dis 1996;28:419-20.  Back to cited text no. 10  [PUBMED]  
11.Smith MB, Hayden RT, Persing DH, Woods GL. Medical parasitology. In: Henry JB, editor. Clinical diagnosis and management by laboratory methods. 20th ed. Philadelphia, Pennsylvania: Saunders; 2001. p. 1131-41.  Back to cited text no. 11    

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Correspondence Address:
Sumeet Gujral
Department of Pathology, Tata Memorial Hospital, Mumbai - 400 012
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0377-4929.48936

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    Figures

  [Figure 1], [Figure 2]

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