Evaluation of Plasmodium falciparum histidine rich protein 2 and 3 genes deletion prevalence in Kenya and it implication to RDTs use

Abstract

Globally, Rapid Diagnostic Tests (RDTs) diagnoses about 75% of all suspected malaria cases. The accuracy of the most frequently used Plasmodium falciparum histidine rich protein 2 (Pfhrp2) based RDTs can be compromised by deletion in Pfhrp2 gene or cross-reaction with Pfhrp3 antigens. The criteria of World Health Organisation (WHO) necessitate accuracy above 95 % as the threshold for selection or withdrawal of RDTs advocating for vigorous Pfhrp2 gene deletions mapping. As part of continuing study on epidemiology of malaria drug resistance, the objective of this study was to determine occurrence and trends of Pfhrp2 and/or Pfhrp3 genes deletion as well as their polymorphisms in three of five malaria transmission zones in Kenya. This study used samples from the epidemiology of malaria drug resistance in Kenya study that started in 1992. This specific study used 350 samples comprising 255 samples collected between 2013 and 2017 (Post-RDTs) in Kombewa, Kericho, and Malindi and 95 samples collected between 2003 and 2005 (Pre-RDTs) in Kericho, Malindi, Alupe and Kisumu. The samples were diagnosed for malaria by microscopy, Pfhrp2/pLDH RDTs and real time PCR using primers targeting 18S ribosomal RNA. RDTs were not conducted on these samples, as only DNA samples were available. To identify genes deletions and amino acid repeat types, primers targeting Pfhrp2 and Pfhrp3 genes were used to amplify and sequence all P. falciparum PCR positive samples. Out of 350 samples, 327 (93.4%) were P. falciparum positive by PCR, comprising 93 (28.4%) pre-RDTs and 234 (71.6%) post RDTs. The prevalence of single Pfhrp2 gene deletion was 0.3% while that of Pfhrp3 was 2.1% of the 327 samples genotyped. No sample had double deletion of both Pfhrp2 and Pfhrp3 genes. All pre RDTs samples had intact Pfhrp2 and Pfhrp3 genes. For both Pfhrp2 and Pfhrp3, deletion of the gene was not associated with deletion of any of the flanking genes. Twenty one (10.8%) of the 195 samples positive by P. falciparum PCR (with 30 parasites per microliter) and pLDH RDTs were negative by Pfhrp2 based RDTs. This appeared to be false negative Pfhrp2 based RDTs results, but genotype analysis showed that all the 21 samples had Pfhrp2 and only one sample (4.8%) had deletion of Pfhrp3 gene. Out of 327 samples sequenced, most had poor coverage at the intron and at the start of exon 2. However, 69 clean Pfhrp3 sequences were obtained. Thirty-one of the 69 sequences had no deletion while the other 38 sequences had partial deletion. Amino acid position 94 to 100 had the highest prevalence of deletion occurring in 27 (71.1%) of all 38 sequences. Amino acid repeat type 4 and type 16 were the most frequent, occurring 4 to 14 times in all 69 sequences. This study findings shows that Pfhrp2 and Pfhrp3 genes deletion are indeed present in Kenya among the symptomatic individuals enrolled in this study. However, these genes deletion could not be associated to false negative RDTs results. This finding heralds the need for investigating additional mechanisms of false-RDTs negativity. Further work needs to be done using concurrent cohort and include asymptomatic cases as the method used for screening in the positive could present a bias. In addition, Whole genome sequencing approach may be used rather than Sanger sequencing since it is more informative. Research on new targets of RDTs need to continue in order to increase chances of having better and more reliable RDTs.