In February 2021, a 29-year old pregnant woman visited the Akpet Central Cottage General Hospital in Biase local government area of Cross River State Nigeria. This hospital serves several rural communities in Nigeria’s Niger Delta region with high and perennial malaria transmission. On presentation, she complained of waist pain, chills, weakness, nausea, vomiting and high fever. Her estimated gestational age based on last menstrual period was 11–12 weeks, she has had 5 previous pregnancies with 4 live births and 1 abortion. Fever had started about 2 weeks before visiting the hospital for which she had only taken paracetamol before visiting the hospital.
She vomited twice on admission in addition to several vomits earlier that day. On examination, she was pale and moderately dehydrated with temperature of 38.7 °C, pulse of 105 beats/min, respiration rate of 32 breaths/min, and blood pressure of 80/40 mm Hg. Her lower abdomen (suprapubic region) was tender, vaginal examination revealed discharge of clear fluid and bulging membrane from the cervix. Obstetric ultrasound scan revealed the presence of a singleton active fetus with cephalic presentation and 105 beat per minutes. Initial provisional diagnosis was sepsis, secondary cervical incompetence and severe malaria, all of which could lead to irreversible miscarriage. Although she had a cerclage done for probable cervical incompetence in a previous pregnancy, the procedure did not prevent the loss of that earlier pregnancy which occurred at about the same gestational age as the current episode. Blood sample was drawn from her on arrival (and on the subsequent 2 days that she stayed in the hospital). Malaria rapid diagnostic test (mRDT) conducted using the P. falciparum histidine rich protein II test kit (SD Bioline, Abbot US) was positive for P. falciparum while light microscopy showed parasitaemia of 21,960 parasites/µl, 14,000 parasites/µl and 10,000 parasites/µl on day 1, 2 and 3, respectively. Thick blood films prepared with 10% Giemsa was used to estimate parasite density per 200 leukocytes assuming a white blood cell count of 8000 parasites/µl as detailed in a previous study [9]. She was treated with intravenous quinine 600 mg in 500 ml of 5% dextrose /0.9% saline 8-hourly for 24 h. She also received 10 mg intravenous diazepam, 1 L of 5% dextrose saline (DS) IV infusion, 1 g ceftriaxone IV daily for 48 h, 500 mg metronidazole IV 8-hourly for 48 h.
The next day, the patient complained of weakness and persistent high-grade fever (> 38.5 °C). Her packed cell volume (PCV) was evaluated to be 18%, which is lower than usual (33–38%) and consistent with the diagnosis of severe malaria. Later, in her second day of hospitalization, she complained of vaginal bleeding and closer examination revealed a bulging amnion from a widened cervix into the vaginal space. 600 µg of misoprostol was inserted into the posterior fornix of her vagina and she was taken into the delivery room for eventual expulsion which occurred around 03:00 h GMT the third day of her admission. Following expulsion, she was administered 1 ml of oxytocin and 1amp of IV vitamin K. She was given full therapeutic regimen of artemether-lumefantrine 80/480 mg tablets to be taken three days; and parenteral quinine was discontinued. She was also given fesolate (ferrous sulfate 65 mg elemental iron) tablets (twice daily) and folic acid (5 mg once daily). Her peripheral blood sample remained positive for malaria for the three days that she was on admission before absconding from the hospital.
Blood samples collected on the three consecutive days were preserved on filter papers as dried blood spots (DBS). DNA was subsequently extracted from this DBS and, P. falciparum confirmed using a real-time PCR targeting the varATS gene and an in-house conventional PCR that focused on the apical membrane protein 1 and circumsporozoite protein of P. falciparum (Oboh et al., unpublished).
Furthermore, high resolution melt (HRM) analysis for drug assay as described previously [10] using the Type IT master mix was employed to probe for molecular markers associated with resistance to the quinoline family of drug (Pfmdr1), sulfadoxine-pyrimethamine (Pfdhfr and Pfdhps) and chloroquine (Pfcrt).
Resistance markers specifically associated with the quinoline family of drugs such as quinine (Pfmdr N86Y: day 1 and 3; Pfmdr Y184F all three isolates) were observed in the collected isolates (Fig. 1). Moreover, nucleotide substitution at codons associated with resistance to pyrimethamine (Pfdhfr S108N day 1 isolate) and sulfadoxine (Pfdhps A613S—all three isolates) were noted.