PREVALENCE OF PFHRP2 AND/OR PFHRP3 GENE DELETIONS IN PLASMODIUM FALCIPARUM ISOLATES AND THE PERFORMANCE OF HRP2 BASED MALARIA RAPID DIAGNOSTIC TESTS

ABSTRACT

BACKGROUND: Malaria rapid diagnostic tests (MRDTs) are important for malaria disease management. However, performance of the RDTs is affected when the targeted antigens in the parasite have a variation or are altogether absent. The most common parasite target antigen in RDTs, Plasmodium falciparum histidine-rich protein 2 (HRP2), has been reported to be absent in some P. falciparum parasites. Loss of the pfhrp2 in P. falciparum parasites affects the accuracy of PfHRP2 based RDT kits when they are used in malaria diagnosis. Thus, to control malaria, determining where and how often P. falciparum parasites not having pfhrp2 occur, is very important.

AIM: The aim was to investigate the prevalence of pfhrp2 and/or pfhrp3 gene deletions in P. falciparum isolates from southern Ghana and the performance of the currently used PfHRP2 based MRDTs.

METHODS: Samples were collected from sites in the southern part of Ghana: three Cocoa Clinics (Accra, Tafo, and Kumasi) and from three other health facilities, Ussher and Mamprobi polyclinics and 37 Military Hospital, all in Accra. Patients with febrile illness, referred by a clinician to the laboratories of these health facilities for a malaria test, were recruited. Blood samples for thick and thin blood smears, for malaria microscopy, and filter paper blots were obtained. All blood samples were tested using a HRP2-based malaria RDT. DNA was extracted from the dried filter paper blood blots using the TNES (Tris HCl, EDTA, NaCl, and SDS) buffer protocol. Plasmodium falciparum infection was confirmed by polymerase chain reaction (PCR). The presence of pfhrp2 and pfhrp3 genes was investigated by PCR.

RESULTS: A total of 371 patient samples, from Accra (58.5%), Kumasi (21.3%) and from Tafo (20.2%), were used in the study. PCR provided the highest number, 14.8% (55/371), of positive detections for falciparum infections. Microscopy detected parasites in 20/261 (7.7%) samples and the minimum parasite density by microscopy was 430 parasites/μL. Out of the 371 samples, 27 (7.3%) were positive by RDT. The highest RDT positivity rate, 13.3% (10/75), was observed at Tafo. False negative RDT results were obtained in 43/55 (78.2%) of the negative branded RDT kits. Only two microscopy positive sample were RDT positive. Using 18SrDNA PCR, 55 (14.8%) samples were positive for P. falciparum. In Accra, 79.2 % (19/24) of the PCR positive samples had P. falciparum parasites that lacked exon 2 of pfhrp2. In Tafo, on the other hand, only 7.4% (2/27) of the PCR positive samples had P. falciparum parasites that lacked exon 2 of pfhrp2. None of PCR positive samples had P. falciparum parasites that lacked exon 2 of pfhrp2 in Kumasi. Only 33.3% (8/24) samples, all from Accra, lacked exon 2 of pfhrp3. In total, 38.1% (8/21) of the samples contained parasites that lacked exon 2 of both pfhrp2 and pfhrp3. Fourteen negative- branded PfHRP2 RDT isolates, consisting of 13 (92.9%) samples from Accra and 1 (7.1%) from Tafo, were negative for the pfhrp2 gene (pfhrp2-). Two samples, both negative- branded PfHRP2 RDT, were lacking the pfhrp3 gene (pfhrp3-). Both samples were from Accra.

CONCLUSION: An overall RDT positivity rate of 7.3% (27/371) and false negative rate of 78.2% (43/55) was observed for the study sites. Plasmodium falciparum parasite populations with deletions of the pfhrp2 and pfhrp3 genes are present in the country. There is an urgent need for investigation of the prevalence and geographic distribution of these parasites.