Case 1: At the village
A 30-year-old male farmer with no past history of malaria presented to a malaria post close to the Myanmar–Thai border in November 2015, with 3 days of fever and headache. He showed no signs of severity and was found to be positive for P. falciparum with an HRP-2-based rapid diagnostic test. He was treated with oral artemether–lumefantrine by the village malaria post (first dose supervised). The next morning, he returned complaining of anuria, when the village malaria worker referred him to a clinic in Thailand.
At the clinic
On arrival, the patient appeared slightly jaundiced, was apyrexial and fully conscious. The blood smear revealed a high P. falciparum asexual parasitaemia of 20% of infected red blood cells (iRBC) or 757,368 µL. The presence of schizonts (352 µL) and gametocytes (1216 µL) was noted, as well as malaria pigment in the neutrophils. Capillary blood haematocrit (Hct) was 30%, blood glucose 82 mg%, pulse 88 beats/min, respiratory rate 32/min, SPO2 98% in air, blood pressure 110/60 mmHg. His liver was palpable, 3 cm below the costal margin and his spleen was not palpable. Physical examination was otherwise unremarkable. The patient was treated with intravenous artesunate (2.4 mg/kg) because of the oligo-anuria and high parasitaemia [6] and rehydrated with 2.5 L of normal saline over 6 h. His vital signs, blood glucose, conscious level and parasitaemia were monitored 4-hourly and these remained within normal limits.
However, the patient remained oligo-anuric and was referred to another clinic equipped with a biochemistry analyser. Blood urea nitrogen [BUN] (normal range) was 91.8 (2.5–7.1 mmol/L), creatinine 2.5 (0.67–1.17 mg/dL), potassium 4.47 (3.5–5.1 mmol/L), sodium 135.8 (136–145 mmol/L), bicarbonate 4.9 (23–29 mmol/L), blood glucose was 74 mg/dL and Hct dropped to 22%, so 350 mL whole blood was transfused. His Glasgow Coma Score (GCS) was 15/15; he was eating and walking unsupported, had a respiratory rate of 40/min, clear lungs and a temperature of 36.9 °C, blood pressure of 90/40 mmHg. His asexual parasitaemia was now 27% of iRBC or 746,064 µL (> 80% of early ring stage), gametocytaemia 1824 µL, schizontaemia 32 µL. A loading dose (20 mg/kg) of quinine 4-hourly infusion was added. Four hours after the start of the loading dose quinine infusion (quinine in 500 mL of dextrose 10%) the patient’s condition was unchanged and his asexual parasitaemia was now 26% iRBC or 718,432 µL (> 80% early ring stage), gametocytaemia 1920 µL, schizontaemia 16 µL (Fig. 1), blood glucose was 104 mg/dL. A second dose of intravenous artesunate 2.4 mg/kg was given. After 4.5 L of fluid (including fluid bolus) the patient passed 60 mL of high-coloured urine and his temperature rose to 39.7 °C. Fourteen hours after arrival, the patient became unconscious, had a generalized seizure followed by cardiac arrest. Resuscitation was unsuccessful.
Case 2
On 3 April, 2017, an 18 years old male was admitted with cerebral malaria: P. falciparum asexual parasitaemia 38% iRBC or 1,050,016 µL (late stage trophozoite 40%). HIV rapid test was positive, which was confirmed subsequently with a baseline CD4 count of 39 cells/mL. The patient’s GCS was 8/15, tympanic temperature 39.0 °C, blood glucose 51 mg/dL, and blood pressure 120/80 mmHg. His liver was palpable, 3 cm below the costal margin and his spleen was not palpable, although confirmed to be present on abdominal ultrasonography. Intravenous artesunate (2.4 mg/kg) was given on admission, after 12 h and again after 24 h as per protocol [1], i.e., three doses within the first 24 h. However, 24 h after the start of treatment, the parasitaemia was still 34% of iRBC or 1,069,107 µL (> 90% late trophozoites) and a loading dose of quinine (20 mg/kg) was added. At hour 24, he received a blood transfusion of 350 mL whole blood.
After 72 h, the patient recovered consciousness and started eating. However, the parasitaemia had increased again (Fig. 1). Atovaquone–proguanil combination was given in addition to the intravenous artesunate and quinine. By hour-90, the asexual parasitaemia was 7% iRBC or 200,221 µL and he received a second transfusion. Intravenous artesunate and quinine infusion were given for 7 days in total. By day 10 his malaria slide was negative. The patient was discharged on day 12 of hospital admission.
Anti-malarial drug quality
The anti-malarials used were artemether-lumefantrine (Macleods Pharmaceuticals: EAB 5412B) and artesunate injection (Atlantic pharma: LA170333) provided by the Global Fund malaria programme. Quinine injection (A.N.B laboratories Co. Ltd: 555009) and atovaquone–proguanil (Malarone GSK: GS 0009 Dec 2018) were purchased from the supplier. All the drugs were in date.
Laboratory tests
All blood slides were double-checked by an expert microscopist of Shoklo Malaria Research Unit.
Parasite genotyping
Leftover blood from the patients was sent for parasite Kelch-13 genotyping with patient or relative written consent. Both had kelch mutant parasites: patient 1 with the P441L mutation and patient 2 the C580Y mutation. Microsatellite genotyping of flanking regions of C580Y haplotype of the Kelch-13 gene revealed a different C580Y lineage from those described recently in Western Cambodia/Northeastern Thailand/Southern Laos/Myanmar, suggesting a different parental origin [7].
Plasma drug concentrations
In Case 1, artesunate or DHA were not detected which was not unexpected since the blood sampling time was ~ 24 h after the second (last) dose of artemether-lumefantrine. However, lumefantrine was also undetectable, which could be due to poor absorption of drug since the tablets were taken with water [8] or non-adherence since the second dose was not supervised.
In Case 2, the plasma concentration of artesunate after ~ 2 h of intravenous injection was 105 ng/mL and DHA was 1530 ng/mL, which were well above the plasma levels providing maximum parasiticidal effect [9].
In vitro parasite culture
Parasite isolates taken from patient 2, after two doses of intravenous artesunate injection (~ 2 h of second dose) grew normally in drug free plate (Additional file 1).
In vitro sensitivity assay
The in vitro Ring‐stage Survival Assay (RSA0–3h) was performed on the parasites obtained from these two patients (Fig. 2). In both cases in vitro assay confirmed that these isolates were resistant to artemisinin (see Methods in Additional file 2).
