Molecular epidemiological surveillance of pfhrp2 and pfhrp3 gene deletions in plasmodium falciparum parasite populations in Uganda
Bekiita Agaba, Bosco
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Background: The emergence of Plasmodium falciparum parasites lacking the histidine-rich protein 2 and 3 (pfhrp2 and pfhrp3) threatens the usefulness of histidine-rich protein 2 rapid diagnostic tests (RDTs). However, data on their presence, magnitude, extent of spread, associated factors, parasite relatedness, transmission dynamics and contribution to false negative RDTs in Uganda are limited. Molecular epidemiological surveillance was conducted to investigate pfhrp2/3 gene deletions in P. falciparum parasite populations in Uganda. Methods: Investigation occurred in four sub-studies. Sub-study I was a systematic review that examined the status, methods and approaches that have been used for the investigation, confirmation and reporting of pfhrp2/3 gene deletion in Africa. Sub-studies II–IV analyzed 300, 359 and 147 P. falciparum isolates, respectively, collected from symptomatic individuals aged 2–10 years from 48 districts in Uganda between 2017–2019. Parasite DNA was confirmed by amplification of the 18s ribosomal DNA and the merozoite surface protein 1 and 2 genes. Gene deletion was confirmed by amplification of the exon 1 and exon 2 of pfhrp2/3 using gene specific polymerase chain reaction (PCR) and controls. Sequencing of the pfhrp2 gene and genotyping of microsatellite markers were performed for molecular characterization of parasites. Geographic information system was used to map all locations where isolates originated. Statistical testing was done using the exact binomial test, chi-square or Fisher’s exact test, Wilcoxon rank test and logistic regression to explore factors associated with deletions and false-negative RDTs. Results: In Sub-study I, the level of pfhrp2/3 gene deletion across the reviewed studies was highest in Eritrea (62% and lowest in Angola (0.4%); wide variation exists in the methods and approaches used for investigating pfhrp2 and pfhrp3 gene deletion across Africa. In Sub-study provide the first large-scale evidence reporting the presence of pfhrp2/3 gene deletions in P. falciparum isolates in Uganda, demonstrate that deletions are not confined but rather spread across regions, and explore possible factors associated with gene deletions. Overall, pfhrp2/3 gene deletions were detected in 29/300 (9.7%, 95% CI: 6.6–13.6%) parasite isolates. Thepfhrp2 gene was deleted in 10/300 (3.3%, 95% CI: 1.6–6.0%) – and pfhrp3 in 9/300 (3.0%,95% CI: 1.4–5.6%) – parasite isolates, whilst both pfhrp2 and pfhrp3 were deleted in 10/300(3.3%, 95% CI: 1.6–6.0%). The proportion of pfhrp2/3 deletions was higher in the eastern (14.7%, 95% CI: 9.7–20.0%) compared to western (3.1%, 95% CI: 0.8–7.7%) region(P=0.001). Geographic location was associated with a gene deletion adjusted odds ratio (aOR)of 6.25 (95% CI: 2.02–23.55, P=0.003). In Sub-study III, we investigated the contribution of xvi gene deletions and other parasite factors to false negative HRP2 RDTs in this population of parasites. Using PCR as reference, the overall agreement between RDTs and PCR in sample sub-sets that had earlier tested RDT+/blood smear + and RDT-/blood smear+ was 97.8% (137/140) and 10.9% (24/219), respectively. We show for the first time that gene deletions contribute to false-negative RDTs in field settings in Uganda. Other parasite-related factors that contributed to false negative HRP2 RDTs in these isolates were low density infections and no falciparum species. Factors associated with false-negative RDTs were pfhrp2/3 gene deletion and low-density infections (aOR=4.4,95% CI 1.72–13.66, P=0.004; aOR=2.65, 95% CI: 1.62–4.38, P=0.001) respectively. In Sub-study IV,i examine parasite diversity, genetic variability of the pfhrp2 gene, possible explanations on how pfhrp2 deletions emerge and spread as well as parasite relatedness, transmission dynamics and challenges for detection. Overall, 75.5%(95% CI: 61.1–85.8%) and 24.5% (95% CI:14.2–38.9%) of parasites examined were of multiclonal and single-clone infections, respectively, whilst multiplicity of infection was 1.9 (95% CI: 1.7–2.1). Sanger sequencing of pfhrp2 exon 2 revealed a high level of genetic diversity, reflected in 96.8% (60/62) unique sequence types. Most commonly observed pfhrp2 repeat sequence type was AHHAHHAAD. Genetic relatedness analysis suggests independent spontaneous emergence and clonal expansion of gene deletions. Conclusions: Investigative approaches and methods of pfhrp2/3 deletions have varied across Africa. The presence of gene deletions was confirmed in the Ugandan isolates; deletions were not confined but spread across regions. Geographical location was associated with parasite gene deletion. Deletions, non-P. falciparum species and low-density infections contributed to false-negative HRP2 RDTs. Genetic relatedness analysis suggested the independent and spontaneous emergence of gene-deleted parasites. Finally, these samples revealed a high level of genetic variability of the pfhrp2 gene. In view of these findings, the use of HRP2 RDTs for malaria surveillance and case management may need to be reconsidered including options for the introduction of combination RDTs that target alternative antigens particularly in areas where gene deletions were mapped.