Plasmodium falciparum parasites with histidine-rich protein 2 (pfhrp2) and pfhrp3 gene deletions in two endemic regions of Kenya

Abstract

Deletions of the Plasmodium falciparum hrp2 and hrp3 genes can affect the performance of HRP2-based malaria rapid diagnostic tests (RDTs). Such deletions have been reported from South America, India and Eritrea. Whether these parasites are widespread in East Africa is unknown. A total of 274 samples from asymptomatic children in Mbita, western Kenya, and 61 genomic data from Kilifi, eastern Kenya, were available for analysis. PCR-confirmed samples were investigated for the presence of pfhrp2 and pfhrp3 genes. In samples with evidence of deletion, parasite presence was confirmed by amplifying three independent genes. We failed to amplify pfhrp2 from 25 of 131 (19.1%) PCR-confirmed samples. Of these, only 8 (10%) samples were microscopic positive and were classified as pfhrp2-deleted. Eight microscopically-confirmed pfhrp2-deleted samples with intact pfhrp3 locus were positive by HRP2-based RDT. In addition, one PCR-confirmed infection showed a deletion at the pfhrp3 locus. One genomic sample lacked pfhrp2 and one lacked pfhrp3. No sample harbored parasites lacking both genes. Parasites lacking pfhrp2 are present in Kenya, but may be detectable by HRP-based RDT at higher parasitaemia, possibly due to the presence of intact pfhrp3. These findings warrant further systematic study to establish prevalence and diagnostic significance.

A Novel Multiplex qPCR Assay for Detection of Plasmodium falciparum with Histidine-rich Protein 2 and 3 (pfhrp2 and pfhrp3) Deletions in Polyclonal Infections

“…The multiplex qPCR assay was designed to provide advantages over existing detection methods (5,13,23). Firstly, the qPCR assay uses only one parasite target to confirm the presence of DNA and to assess its quality while the gel-electrophoresis based nested PCR (nPCR) methods use three target genes (5,24,25). This reduces the cost and throughput time as well as simplifies the algorithm during the interpretation of results.…”

“…This characteristic of the assay is useful because one of the criteria for confirming deletion of pfhrp2/3 is estimation of parasite density using microscopy examination to rule out low parasite density as a factor for lack of parasite target amplification (13). However, microscopic results are often absent or inaccurate from samples collected in community surveys, so quantification of parasite density using qPCR is required (5,12). The qPCR assay not only detects P. falciparum parasites with pfhrp2/3 deletions but also simultaneously estimates parasite density in the same experiments and hence reducing time and cost.…”

“…The various nPCR methods used differ in limit of detection and this can cause type I and type II errors. Further, the pfhrp2 and pfhrp3 PCR assays do not reliably detect P. falciparum DNA derived from dried bloodspots (DBS) particularly at low parasite density (5,12), and gel-electrophoresis approaches do not detect deletions masked in polyclonal infections (6).…”

“…We report the validation and application of this novel method using DNA samples derived from DBS and whole blood of field isolates and clinical samples. We also deploy the assay to estimate P. falciparum parasite density to rule out low parasite density as a factor for false RDT negative results (5,12) when microscopic data is unavailable or if it is not reliable.…”

Quantitative and rapid Plasmodium falciparum malaria diagnosis and artemisinin-resistance detection using a CMOS Lab-on-Chip platform

“…Consequently, there is a significant need to specifically detect this pathogen with high sensitivity and rapid detection for adequate therapy prescription. Most rapid diagnostic tests developed for this purpose are no longer reliable due to the loss of expression of targeted proteins, such as HRP-II (leading to high occurrence of false negatives) [17,18]. As a result, subsequent efforts have focused their attention on NAATs, primarily targeting the gene 18S rRNA.…”