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. 2016 Feb 18:15:101.
doi: 10.1186/s12936-016-1159-z.

Plasmodium falciparum histidine rich protein-2 diversity and the implications for PfHRP 2: based malaria rapid diagnostic tests in Ghana

Linda Eva Amoah  1 , Joana Abankwa  2 , Akua Oppong  3
Affiliations

Plasmodium falciparum histidine rich protein-2 diversity and the implications for PfHRP 2: based malaria rapid diagnostic tests in Ghana

Linda Eva Amoah et al. Malar J. .

Abstract

Background: Malaria rapid diagnostic tests (RDTs) play a key role in malaria management and control. The PfHRP-2 based RDT is the most widely used RDT for malaria diagnosis in Ghana. Deletion of pfhrp2 in Plasmodium falciparum parasites affect the diagnostic accuracy of PfHRP-2 based RDT kits. Identifying the prevalence and distribution of P. falciparum parasites with deleted pfhrp2 is important for malaria control.

Aim: The purpose of this study was to identify and confirm the prevalence of pfhrp2 deletant P. falciparum parasites circulating within different regions of Ghana.

Methods: DNA was extracted from the membrane of spent CareStart™ PfHRP-2 RDT kits and dried filter paper blood blots using Chelex-100. Exon 2 of pfhrp2 and pfhrp3 genes were amplified by polymerase chain reaction (PCR), resolved by agarose gel electrophoresis and visualized under UV light.

Results: Microscopic analysis of blood smears from samples that were PfHRP-2 RDT positive revealed a parasite prevalence of 54/114 (47.4 %) and 2/26 (7.7 %) in Accra and Cape Coast, respectively. PCR analysis increased parasite prevalence in the RDT positive samples to 94/114 (82.5 %) and 6/26 (23.1 %) in Accra and Cape Coast respectively. The exon 2 of the pfhrp2 gene was deleted in 18/54 (33.3 %) of the microscopy confirmed and 36.2 % (34/94) of the PCR confirmed RDT positive samples collected in Accra. No RDT sample, confirmed to contain parasites by either PCR or microscopy was negative by pfhrp2 exon 2 PCR in Cape Coast. A survey of an additional 558 DBS revealed that 22.4 % (46/205) and 40 % (44/110) of PCR positive samples in Accra and Cape Coast, respectively, lacked the exon 2 region of pfhrp2 and possibly the entire pfhrp2 gene.

Conclusions: A high number of P. falciparum parasites, which lack pfhrp2 exon 2 gene have been identified in two communities in Ghana. Continuous nationwide monitoring of the prevalence of pfhrp2 deletant parasites would be essential to malaria control. The use of RDT kits that are effective at malaria diagnosis despite deletion of pfhrp2, such as the PfHRP-2/PfLDH combo RDT kit could enhance the diagnosis of clinical malaria in Ghana.

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Figures

Fig. 1
Fig. 1
Prevalence of Plasmodium falciparum in samples collected in 2015. a Giemsa-stained thick blood smears were read and parasite prevalence estimated by microscopy was compared with data obtained from the PCR genotyping of genomic DNA extracted from a DBS. Each Giemsa-stained blood smear analyzed had a corresponding DBS sample. b The frequencies of positivity (obtaining a positive test results) obtained by PCR, PfHRP-2 RDT and microscopy in the samples collected in April 2015 from Accra and Cape Coast
Fig. 2
Fig. 2
Contributions of pfhrp2 and pfhrp3 to PfHRP-2 RDT read out. Genomic DNA obtained from either the membrane of the PfHRP-2 RDT kit or the corresponding DBS sample was subjected to pfhrp2 and pfhrp3 exon 2 PCR amplification. The presence or absence of pfhrp2 and or pfhrp3 in negative (a) and positive (b) banded PfHRP-2 RDT kits collected from Accra and Cape Coast was identified. Each RDT kit was confirmed as P. falciparum positive by PCR and microscopy
Fig. 3
Fig. 3
Prevalence of P. falciparum parasites lacking exon 2 of pfhrp2 and or pfhrp3. Samples that were confirmed positive for P. falciparum by PCR genotyping were further analyzed for the presence of pfhrp2 and pfhrp3 by PCR amplification of exon 2. Samples were grouped according to the presence or absence of either or both pfhrp2 and pfhrp3
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