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

iv

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.