Failure of rapid diagnostic tests in Plasmodium falciparum malaria cases among travelers to the UK and Ireland: Identification and characterisation of the parasites

Abstract

Objectives: Our objective was to systematically investigate false-negative histidine-rich protein 2 rapid diagnostic tests (HRP2-RDT) in imported Plasmodium falciparum malaria cases from travelers to the UK and the Republic of Ireland (RoI).

Methods: Five imported malaria cases in travellers returning to the UK and RoI from East Africa were reported to the PHE Malaria Reference Laboratory as negative according to histidine-rich protein (HRP2)-RDT. The cases were systematically investigated using microscopic, RDT, molecular, genomic, and in in vitro approaches.

Results: In each case, HRP2-RDT was negative, whereas microscopy confirmed the presence of P. falciparum. Further analysis revealed that the genes encoding HRP2 and HRP3 were deleted in three of the five cases. Whole-genome sequencing in one of these isolates confirmed deletions in P. falciparum chromosomes 8 and 13. Our study produced evidence that the fourth case, which had high parasitemia at clinical presentation, was a rare example of antigen saturation ('prozone-like effect'), leading to a false negative in the HRP2-RDT, while the fifth case was due to low parasitemia.

Conclusions: False-negative HRP2-RDT results with P. falciparum are concerning. Our findings emphasise the necessity of supporting the interpretation of RDT results with microscopy, in conjunction with clinical observations, and sets out a systematic approach to identifying parasites carrying pfhrp2 and pfhrp3 deletions.

Keywords: Deletion; Malaria; Plasmodium; RDT; pfhrp2; pfhrp3.

Figure 1

Amplification curves for four different genes in five samples. Three samples (S105, S194, and S204) showed no amplification curves for pfhrp2 and pfhrp3 genes, while two samples (S160 and S227) showed amplification curves for both pfhrp2 and pfhrp3 genes. All samples showed good amplification curves for both parasite (pfldh) and human (humTuBB) internal controls. As expected, all the controls (Dd2, INT, 3BD5, and HB3) showed amplification curves or no amplification curves according to their pfhrp2/3 status.

Figure 2

Carestart™ Malaria Rapydtest® detection of LDH but not HRP antigens in six independent culture wells 4 weeks after commencing in vitro propagation of P. falciparum isolate S204. The upper panel shows four representative Giemsa-stained microscopy fields after magnetic purification, which selectively enriches for late-stage asexual parasites and gametocytes. The lower panel shows readouts from neat culture supernatants from six different culture wells. Following successful cryopreservation, this line was designated HL2004. C, control line; red arrow, expected position of PfLDH antigen detection line; blue arrow, expected position of PfHRP2/3 antigen detection line.

Figure 3

Genomic coverage of pfhrp2, pfhrp3, and flanking regions for two clinical samples. Short-read sequence coverage is missing around the pfhrp2 and pfhrp3 in sample S105 (top panel) but not in sample S160 (bottom panel). (A) pfhrp2 and pfhrp3 (dark orange) in clinical sample S105 (top panel) are deleted together with flanking regions that include PHIST, STEVOR, EBL1, RIFIN, PHISTb, and HSP70x (purple) but not PfEMP1 and CLAG8 (green). (B) Both pfhrp2 and pfhrp3, as well as the adjacent genes (colored), show good genomic coverage in clinical sample S160 (bottom panel).