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Clinical Infection & Immunity

Case Report

Volume 4, Number 2, October 2019, pages 27-30

Peripheral Microscopic Localization of Plasmodium falciparum Garnham Body in an Immature Stage-III Gametocyte in Fatal Cerebral Malaria

Gametocytes of Plasmodium falciparum (P. falciparum) (stage I-IV) are strictly localized in the bone marrow and spleen for their development, that is, until their maturation as stage-V gametocytes [1, 2]. Upon maturation, the stage-V gametocytes only get released to the peripheral circulation to be eventually picked up by the vector (mosquito) for sporogony and subsequent reproductive phases. It is worthwhile to mention that the initial switch for sequestration of merozoites released from the “committed” schizonts (< 10% of total parasites) to the marrow and spleen starts as early as 24 h of merozoites leaving the hepatocytes [3, 4]. Therefore undoubtedly, the dynamics of transition between asexual differentiation and gametocytogenesis in P. falciparum has been one of the tremendous areas of research in malaria, pertaining to the exploration of transmission-blocking drugs and several vaccine design strategies for malaria eradication [5-7].

Garnham (G) bodies were first discovered in 1933 by Garnham himself [8] in the peripheral blood smear of a Kenyan child who died of severe malaria and their identifications were confirmed later in 1935 [9]. Since then, the clinical demonstration and imaging of G bodies have been extremely rare in P. falciparum malaria literature. Since the immature gametocytes (stage-III) are exclusively localized in bone marrow and spleen for development, it is indeed intriguing for the fact that their peripheral occurrence of a P. falciparum immature gametocyte (with G body) as shown in this case report from India.

A 23-year-old man hailing from a small village of Sundergarh District, Odisha, India was admitted in an unconscious state to the emergency ward. Clinical history revealed fever, chills and rigors since past 5 - 6 days and he was passing high colored urine. He was disorientated and had convulsions since past 2 days. Interestingly, he was treated at a local hospital for 2 days, the microscopic test for malaria parasite was negative and therefore he was clinically diagnosed there to be a case of hepatic encephalopathy. He got treated with ciprofloxacin injection, diphenylhydantoin sodium injection with little dosing information. As he had no further improvement on his condition, he was eventually referred to our facility for further management.

On admission to our hospital, his detailed history taken from the family members revealed the young man to be a daily-wage laborer who worked in and around his native village, an area surrounded by forested hills, small streams and undulating landscape. In the past 2 months, he hardly travelled out of his native place and had no history of malaria before. He was an intermittent alcohol consumer, no co-morbid conditions known to his family. The mosquito-vectors in his area are predominantly Anopheles fluviatilis and the geographic location is an ideal setting for malaria transmission and endemicity known before [10]. On examination, his Glasgow coma score was 9, with deep icterus and mild pallor. His initial blood pressure was recorded to be 100/70 mm Hg and pulse was 80/min. No pupillary abnormalities were noticed and funduscopy revealed no papillededama, hemorrhages or whitening of the retina. Deep tendon jerks were grossly diminished and plantars were flexors with no signs of meningeal irritation. Chest was clinically clear and per abdominal examination showed no hepatosplenomegaly. Biochemical investigations of the serum showed random blood sugar 114 g/dL, urea 331 mg/dL, creatinine 8.0 mg/dL, bilirubin 20.7 mg/dL, glutamic pyruvic transaminase 180.4 U/L, sodium 128 mmol/L, potassium 6.6 mmol/L and lactate 3.6 mmol/L. Hematological tests revealed a total leucocyte count of 21,400/mm³ (neutrophil 45%, eosinophil 4%, lymphocyte 42% and monocyte 9%) with hemoglobin 12.0 g%, hematocrit of 38% and a platelet count of 52,000/mm³. Blood, urine and cerebrospinal fluid were cultured to rule out the other co-infections or sepsis, for which the results were revealed to be negative.

The patient was diagnosed to have cerebral malaria with multi-organ failure, that is, acute kidney injury and jaundice. The patient was treated with intravenous (IV) artesunate (2.4 mg/kg of body weight) at 0 and 12 h along with other IV fluids and antibiotics ceftriaxone (1 g) at 0 at 12 h and anticonvulsant diphenylhydantoin sodium (5 mg/kg IV every 8 h) as the starting dose received at the previous hospital was unknown according to the referral documents. Unfortunately, the patient deteriorated within 2 h, became hypotensive (90/60 mm Hg) and started having respiratory distress, for which he was immediately shifted to the intensive care unit where high flow oxygen and inotropic support (adrenaline in infusion pump) was given. Since oxygen saturation remained low despite high flow oxygen, patient was put on a mechanical ventillator. Though acute kidney injury (deranged creatinine and urea) was detected with severe oliguria, hemodialysis could not be attempted due to severe hypotension. With all measures of treatment and intensive care, there was no improvement in the patient’s condition and he succumbed within 19 h, possibly due to cerebral malaria, acute kidney injury, severe malarial jaundice and severe hypotension.

Detailed microscopic examination of the peripheral blood smear taken at the time of admission revealed P. falciparum asexual stages (mostly mid and late stage of trophozoites and schizonts) and free-floating parasites including trophozoites. A species-specific semi-nested multiplex malaria PCR was performed to rule out presence of other Plasmodia species, which revealed the exclusive presence of P. falciparum DNA. The patient had hyperparasitemia of 12.7% (635,000 parasites/µL). The peripheral blood microscopy for the parasite observation and parasite calculation was then performed by two independent microscopists unaware of the patient’s clinical details. A single immature gametocyte of P. falciparum was also located containing a “G body”, which was further identified using differential interference contrast imaging in a confocal laser scanning microscope (Fig. 1a, b). A single, crescent shaped, stage-III gametocyte was seen in the peripheral blood smear which contained the G body (Fig. 1), resembling the description by Garnham [8]. It was an immature gametocyte with an integrated structure attached to the lower end of the gametocyte. The G body appeared like a spring “S”-shaped amorphous structure (arrowheads, red) and contained hemozoin pigments at the center of the cytoplasm (Fig. 1b, blue arrowhead) which can be seen clearly in the additional video file (Supplementary video, www.ciijournal.org) and illustrating the visibility of the internal organelles and hemozoin pigments inside.

Click for large image

Figure 1. Demonstration of the Plasmodium falciparum Garnham body in the peripheral blood by (a) Giemsa-stained light microscopy and (b) differential interference contrast imaging in confocal laser scanning microscope.

It is intriguing to disseminate the observation on this immature stage-III gametocyte containing the G body as it was localized in the peripheral circulation of the patient who died eventually. It presents some previously unseen features rarely published in malaria literature. Nonetheless, it is acknowledgeable that G bodies inside immature P. falciparum gametocytes have been under focus earlier, albeit in synchronized gametocyte cultures. Functions like storage of hemozoin pigments and their protective role inside the P. falciparum immature gametocytes have been demonstrated in vitro by Orjih and group in 2012 [11]. Remarkably, peripheral occurrence of immature gametocytes and their expressed proteins have been characterized as well, based on qPCR-based findings in patients from Mozambique, attributable mostly to hematological disturbances leading to deformability of gametocytic development in bone marrow [12]. We hypothesize that the bone marrow may present transient saturation and/or hematological perturbations for stage III-IV gametocytes undergoing maturation. This in turn, may result in either spontaneous “leaks” of the immature gametocytes or a genetically programmed response for release to the peripheral circulation in order to enhance their chances of survival through gamete formation. However, focal characterization of tissue-specific (biopsy-based) differential gene expression profiles of gametocytogenesis genes, backed by immuno-histochemical and microscopic evidences would be highly necessary, to prove a conclusive and mechanistic basis for such premature release of immature gametocytes [5]. It is admissible that this investigation was limited by the unavailability of bone marrow or spleen biopsy samples under practical conditions of obtaining ethical permissions and consents within a short time-span in the hospital. It may be however noted that, the timing of appearances of mature gametocytes (stage-V) in the peripheral circulation may not statistically correlate with hyperparasitemia [13] or anemia [14] in clinical malaria seen from Africa, which is unlikely in this case report which is described. Therefore, spontaneously leaking or genetically programmed release of immature stage II-IV gametocytes (irrespective of containing G bodies) from the bone marrow or spleen to the periphery, can explain in part by the evolutionary sensing of parasite to the increasing hyperparasitemia (in this case 12.5%), towards an imminent end (death of the host), and thus showing up the urgency of getting back into peripherally circulated blood to enhance the chances of transmission.

Taken together, the present case, to the best of our knowledge, is the first ever clinical report on fatal malaria from India with the microscopic demonstration a unique G body in the peripheral blood, only second to the Garnham’s original report in 1933 from Kenya. The microscopic localization of G body particularly in peripheral circulation, inside the P. falciparum immature state-III gametocyte is of high clinical significance and an uncommon phenomenon in parasite’s life cycle which presents an exciting area to develop further insights on the expression profiles of gametocyte genes and the possible “trade-offs” of immature and mature P. falciparum gametocytes [4, 5]. Interestingly, the recently characterized role of P. falciparum export proteins such as Gametocyte EXported Protein-5 can be the new biomarker to identify sexually committed ring stages of P. falciparum [15] in this context. Advances and insights from further characterization could certainly augment the stage-specific vaccine designs and improvisation of transmission-blocking drugs, besides defining G bodies as a novel prognostic indicator for severe falciparum malaria.

Acknowledgments

Authors are grateful to the patient’s family for their consent for presentation of this case report as a publication. We acknowledge Director I/c, IGH, Rourkela and Director, ILS, Bhubaneswar for their help and necessary guidance.

Financial Disclosure

None to declare.

Conflict of Interest

The authors declare there is no conflict of interest with regard to the submission of this manuscript.

Informed Consent

Written informed consent from the patient’s family members has been obtained for the publication of this case report containing the data and images used in it.

Author Contributions

AKM and SS performed the light microscopy and major identification studies. KP performed confocal microscopy and imaging analysis. SM analyzed patient’s clinical data and helped in organizing the manuscript. PKS conceived the study, analysed the experimental, imaging, clinical data, performed PCR and wrote the paper. All the authors read and approved the final manuscript.

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