Two Hyperparasitaemic Plasmodium Falciparum Cases Successfully Treated with Artemisinin-based Combination Therapy

Background Methods


Background
In Plasmodium falciparum infection, > 10% parasitized red blood cells (RBCs) generally known as hyperparasitaemia, one of the features leading to severe malaria (SM) [1]. Cerebral malaria (CM), severe anemia, multiple organ dysfunctions, and respiratory failure are the common complications with severe P. falciparum infection, suggestive of sequestered parasites in the vital host organs, and a threat for recrudescent infections [2,3]. Hyperparasitaemia has also been associated with high gametocyte carriage, [4] and de novo anti-malarial drug resistance, [5] which have implications on malaria elimination programme. Generally, hyperparasitaemia is found in non-immune, [6] and semi-immune individuals [7].
In Thai population, hyperparasitaemia was associated with poor prognosis in adults suffering from CM [8] whereas, in Nigerian children, it increased the risk of CM [9]. In Indian patients, hyperparasitaemia has been correlated with ampli ed risk of mortality with multi-organ dysfunction (MOD) [10], and patient's death within a short period of time [11]. Here, we report two Indian adult patients, who had P. falciparum hyperparasitaemia with no severe or adverse eventsone of them presented hypergametocytaemia stages.

Study design
The two prospective adult males infected with hyperparasitaemia of P. falciparum visited the malaria clinic at the Wenlock District Government Hospital, Mangalore, Karnataka, India for diagnosis and treatment. Clinical, biochemical, hematological, parasitological outcomes as well as treatment details of the two patients are presented in this brief report. Indian reference value system for these parameters was considered [12].

Malaria diagnosis and quanti cation of parasites
Both thick and thin blood smears on glass micro slides were prepared from spirit-swabbed nger pricking [13]. These smears were stained with 10% Giemsa and examined under 100X immersion oil objective lens using Carl Zeiss Primo Star light microscope (Germany) [14]. Images were capture on Zeiss AxioPhot microscope (Germany). Two trained expert microscopists examined both thick and thin smears for diagnosis and determination of parasite countsthe senior expert (SKG) recon rmed the nal results.
The mean values of two counts were taken for estimating nal parasitaemia. The percent parasite density in the thin lms was determined by counting the number of infected RBCs examined against 5000 RBCs in 20 microscopic elds considering the actual number of RBCs/µL for each patient i.e. 3.87 and 3.69 million/mm 3 , respectively. Similarly, parasitaemia per µl of blood in the thick smears was calculated enumerating the number of parasites counting 200 white blood cells (WBCs) in relation to the actual number of WBCs/µl i.e. 6900 and 6000/mm 3 , respectively.

Laboratory procedures
On admission, 4 ml venous blood (pre-treatment) from the two patients was collected in EDTA vacutainers (BD Vacutainer®; Cat. No. 367861) for haematological tests. Another 4 ml blood was collected in Clot Activator Vacutainer (BD Vacutainer® 367812) for biochemical analyses for liver and kidney function tests. DxH 800 Hematology (Beckman Coulter) and Cobas® 6000 (Roche) analyzers were used for hematological and biochemical tests, respectively. All the tests were performed at the National Accreditation Board for Testing and Calibration Laboratory (NABL)-accredited laboratory services at Kasturba Medical College, Mangalore, India.

Treatment and follow-up
Both the inpatients were treated orally with artemisinin-based combination therapy (ACT)artesunate 200 mg and 1500 mg sulfadoxine plus 75 mg pyrimethamine on day 0; artesunate 200 mg on day 1 and day 2, respectively, followed by a single dose of 45 mg primaquine on day 2 as per the guideline of national programme [15]. All required safety measures and prognosis of oral ACT therapy were monitored in the hospital as per the protocol.
Both the patients were discharged from the hospital on day 7 when blood smears were found negative or other parameters showed normal. Subsequently, both the patients are followed weekly up to day 63. Blood smear examination and temperature were recorded for each visit.

Clinical manifestations
The two adult patients aged 35 and 36 years, respectively presented almost similar clinical manifestations having high-graded fever of 39 0 C and 38.5 0 C, respectively. On admission both the patients had higher heart rates and abnormal breathing issues. Clinical assessment revealed no neurological and lymphatic abnormalities, but had palpable splenomegaly on admission. Detailed information on physical and clinical manifestations is presented in Table 1. On the day of admission i.e. day 0 both the patients presented abnormal haematological and biochemical parameters compared to the reference range (Table 2). However, total leukocyte count, total protein, albumin and globulin and their ratios were within the reference ranges. Patient 2 had severe anemia and acute kidney injury. Both patients presented hypoglycemia and clinical jaundice. Further details on haematological and biochemical test results are shown in Table 2. The microscopic examination of peripheral blood smears con rmed only asexual stages of P. falciparum parasites based on typical morphological characteristics. Parasitaemia was 1668824/ml of blood based on thick smear examination and 42% on thin smear. Fig. 1 A and B show thick smear images, and Fig. 1 C and D are thin smear images. A large number of typical chromatin dots of ring form are seen on thick smears ( Fig. 1 A and B). The parasitological assessment of thin smear for species identi cation was the cytoplasm, which makes the complete ring formation in young trophozoites followed by; thickening and invariably contains several vacuoles to develop the trophozoites. The chromatin (parasite nucleus) was characteristically contained as a single bead, double bead forms on thin smear examination (Fig. 1 C).
The most important feature of the rings was found on the margin or edge of the red blood cells, called accolé/appliqué' forms (Fig 1. C and D). Accolé forms were seen in early stage of P. falciparum parasites and these are three distinct types -common form, rim form and displaced form (16%). The large mass of golden brown pigment (haemozoin) was seen in the pre-schizont and schizont stage (20%). A low number of pigmented monocytes (0.19%), neutrophils (0.29%), and eosinophils (0.09%) was detected ( Fig. 1 A). The unusual of marked multi parasitism (74.02%, 1 parasite/RBC; 14.95%, 2 parasites/RBC; 7.7%, 3 parasites/RBC; 3.14%, 4 or more parasites/RBC; 0.19%) was observed ( Fig. 1 C and D). No sexual forms i.e. gametocytes were detected in this patient.

Patient 2
Patient 2 had mixed stages of parasites. Total parasitaemia on thick smear was 304000/ml of blood, and 9.5% on thin smears ( Fig. 2 A to D). Infected RBCs in thin smear were normal in size and contained young rings, and in some mature stages, showing occasionally thin accolé/appliqué forms (Fig. 2 C). Asexual stages constituted about 46% of 9.5% parasitaemia. Occasional pigments were evident in mature trophozoites and schizonts. Phagocytosed monocytes, macrophages and polymorphonuclear neutrophils were also detected. Different developmental stages of gametocytesthe sexual stage of the parasite, were seen in both thick and thin peripheral blood smears (Fig. 2 A to D) constituted about 54% of 9.5% parasitaemia. The female gametocyte or macrogametocyte is more slender and longer than the male, cytoplasm is deep blue in colour and nucleus is small, compact, staining dark red in colour, but the male gametocyte or microgametocyte is border than female, sausage shaped, cytoplasm is either pale blue or tinted pink, nucleus is staining dark pink in colour was seen in peripheral thin blood smear. Pigments and nucleus were dispersed (Fig. 2 A to D). Some fragmented gametocytes were also detected (Fig. 2 C). In this patient, 54% of 9.5% parasitaemia were gametocytes comprising 51% males and 49% females (1:1). Pigmented neutrophils (1.2) and monocytes (0.4%) were present (Fig. 2 A and B). The unusual of marked multi parasitism asexual (14.5%), sexual (4.5%) comprising male gametocyte (2.3%) and female gametocyte (2.2%), 1 parasite/RBC (4.6%), 2 parasites/RBC (0.4%), 3 or more parasites/RBC (0.2%), schizonts (0.25%) and accolé/appliqué forms 1.5% were found.
Weekly blood examination of both the patients did not show any parasite breakthrough. Only drugaffected fragmented gametocytes were seen in the second patient. Temperature for each visit were recorded below 37.5 o C. This indicated that both the patients successfully responded to ACT treatment.

Discussion
Asexual hyperparasitaemia in P. falciparum has been observed both in children and adults by many investigators. But high gametocytaemia in P. falciparum is not commonly encountered. In the present study, we found 54% gametocytaemiato our knowledge this is the rst report. Occasionally, a disastrous mistake takes place when stains and microscopes are of poor quality, microscopists misread hyperparasitaemic slides as negative [1]. Both the patients presented uncomplicated hyperparasitaemia and were treated as per national malaria drug policy and hospital protocol. Axillary temperature became normal on day 1 for both the patients. Based on their clinical assessment and prognosis, oral therapy of ACT was administered, and patients responded successfully and hyperparasitaemia started to decline. The basic de nition of hyperparasitaemia needs to be updated based on transmission intensity [16]. WHO has de ned hyperparasitaemia as > 5% or 250,000/µL in high transmission stable malaria areas or > 2% or 100,000/µL in low transmission areas [17]. However, in low transmission areas parasitaemia of 0.5% was considered a cutoff point for discrimination between severity levels of falciparum malaria patients [18]. Now WHO has clearly indicated that > 10% parasitaemia is considered to be severe malaria irrespective of transmission settings and patient conditions [19]. Here we calculated the parasitaemia taking the actual WBC and RBC counts, instead of WHO recommendations considering 8000 WBC/µl and 5 million/mm 3 for counting and estimating parasitaemia [14]. Taking actual number gives accurate estimation especially in clinical drug trials or therapeutic e cacy studies.
The accolé/appliqué forms were seen only in hyperparasitaemia of severe P. falciparum malaria cases and indicate the presence of CM symptoms [20]. In the present cases, 16% and 5% such forms were recorded, but no sign of CM was found, suggesting both the patients were semi-immune. The presence of > 20% mature parasite stages in peripheral blood has been associated with a poor prognosis in severe falciparum malaria [3,20]. However, rst patient in our study who had ≥ 20% mature parasite stages still the prognosis was found normal, suggesting early diagnosis and prompt treatment are critical for a positive outcome in SM. This observation aligns with prior results where a 4-year girl child survived 70% falciparum parasitaemia in Odisha, India [21]. This patient also presented no gametocytes despite of 42% parasitaemia, which contradicts with the pervious nding, where hyperparasitaemia was associated with enhanced gametocyte carriage [4] suggesting this patient may have gametocytes neutralizing antibodies.
Mangalore city in the southwestern coast of India is endemic for malaria and contributes 70 to 80% of total malaria cases in Karnataka [22] and Anopheles stephensi is the main urban malaria vector [23]. Recent change in the malaria control operations in Mangalore [24] and national malaria control programme [23] have led to a signi cant reduction in malaria transmission in India. Hypergametocytaemia (54%) with 1:1 of male and female gametocytes ratio in second patient further con rms the hypothesis that in low-transmission areas, parasites invest more in transmission to new hosts via reproduction and less in within-host replication than parasites found in high-transmission areas [25].

Conclusion
In summary, we show that two male adults with P. falciparum hyperparasitaemia were successfully recovered with the ACT and a single dose of primaquine. However, the presence of hypergametocytaemia may hinder the elimination efforts with increased infectivity, if not treated immediately. This report also signi es the need of accurately estimating the parasitaemia in malaria patients and their timely management to prevent complications and mortality. Images of asexual form of P. falciparum parasites X 1000 (Patient 1) Panels A and B show thick smear images with huge ring form of P. falciparum parasites. Panels C and D on thin smears. Accolé form is seen in both C and D. A double-chromatin ring is seen in panel C (arrow in the middle). X 1000