Community-based surveys for Plasmodium falciparum pfhrp2 and pfhrp3 gene deletions in selected regions of mainland Tanzania

Abstract

Background Histidine-rich protein 2 (HRP2)-based malaria rapid diagnostic tests (RDTs) are effective and widely used for the detection of wild-type Plasmodium falciparum infections. Although recent studies have reported false negative HRP2 RDT results due to pfhrp2 and pfhrp3 gene deletions in different countries, there is a paucity of data on the deletions of these genes in Tanzania. Methods A community-based cross-sectional survey was conducted between July and November 2017 in four regions: Geita, Kigoma, Mtwara and Ruvuma. All participants had microscopy and RDT performed in the field and provided a blood sample for laboratory multiplex antigen detection (for Plasmodium lactate dehydrogenase, aldolase, and P. falciparum HRP2). Samples showing RDT false negativity or aberrant relationship of HRP2 to pan-Plasmodium antigens were genotyped to detect the presence/absence of pfhrp2/3 genes. Results Of all samples screened by the multiplex antigen assay (n = 7543), 2417 (32.0%) were positive for any Plasmodium antigens while 5126 (68.0%) were negative for all antigens. The vast majority of the antigen positive samples contained HRP2 (2411, 99.8%), but 6 (0.2%) had only pLDH and/or aldolase without HRP2. Overall, 13 samples had an atypical relationship between a pan-Plasmodium antigen and HRP2, but were positive by PCR. An additional 16 samples with negative HRP2 RDT results but P. falciparum positive by microscopy were also chosen for pfhrp2/3 genotyping. The summation of false negative RDT results and laboratory antigen results provided 35 total samples with confirmed P. falciparum DNA for pfhrp2/3 genotyping. Of the 35 samples, 4 (11.4%) failed to consistently amplify positive control genes; pfmsp1 and pfmsp2 and were excluded from the analysis. The pfhrp2 and pfhrp3 genes were successfully amplified in the remaining 31 (88.6%) samples, confirming an absence of deletions in these genes. Conclusions This study provides evidence that P. falciparum parasites in the study area have no deletions of both pfhrp2 and pfhrp3 genes. Although single gene deletions could have been missed by the multiplex antigen assay, the findings support the continued use of HRP2-based RDTs in Tanzania for routine malaria diagnosis. There is a need for the surveillance to monitor the status of pfhrp2 and/or pfhrp3 deletions in the future.

Plasmodium falciparum pfhrp2 and pfhrp3 Gene Deletions from Persons with Symptomatic Malaria Infection in Ethiopia, Kenya, Madagascar, and Rwanda

“…The antigen screening methodology provided phenotypic rationale for categorizing the infecting P. falciparum as a high producer of HRP2/3 antigens or a HRP2/3 low-producer requiring subsequent characterization though genetic assays ( 25 , 26 ). Correlation of antigen assay signal with parasite density (as determined by microscopy during enrollment for each TES) ( Appendix Figure 1) showed that the 2 pan- Plasmodium antigens displayed a moderate correlation with microscopy-estimated P. falciparum parasite density, whereas the HRP2 antigen showed higher variability, as seen previously ( 25 ).…”

“…However, by initially employing a low-cost, high-throughput antigen screening step, fewer samples can be carefully selected for more definitive investigation into production of these RDT targets ( 25 – 27 ). This strategy of phenotypic screening and genetic confirmation is not unique for the TES sampling design and has also been used for healthcare facility ( 25 , 27 ) and community ( 26 ) surveys. Further exploration of this strategy with large datasets is needed throughout global P. falciparum populations to determine the overall accuracy of this methodology and its ability to generalize antigen levels with deletions of pfhrp2 and pfhrp3 .…”

“…Using the strategy reported previously ( 26 , 27 ), we selected samples for further genetic assays on the basis of the relationship between the 2 pan- Plasmodium antigens (aldolase and lactate dehydrogenase [LDH]) and the HRP2/3 signal. Samples were selected if they completely lacked an assay signal for HRP2/3 or if the assay signal for HRP2/3 was atypically lower compared with the level of pAldolase or pLDH antigens.…”

Plasmodium falciparum pfhrp2 and pfhrp3 gene deletions among patients in the DRC enrolled from 2017 to 2018

“…Additionally, for wild-type P. falciparum isolates, the assay signal for HRP2 is higher than either pan- Plasmodium antigen since the HRP2 protein is more highly expressed throughout the erythrocytic stage of infection and HRP2 persists in the blood for several weeks following antimalarial treatment 7 . Using a strategy described previously 23 , 24 samples were classified as HRP2 low (or HRP2 negative) based on the relationship between the two pan- Plasmodium antigens (aldolase and LDH) and the HRP2/3 signal and were selected for further characterization by genetic assays because these samples show phenotypic evidence of potential pfhrp2 and/or pfhrp3 deletions.…”

Investigation of Plasmodium falciparum pfhrp2 and pfhrp3 gene deletions and performance of a rapid diagnostic test for identifying asymptomatic malaria infection in northern Ethiopia, 2015

“…DBS samples showing an atypically low signal for HRP2 antigen (compared to pLDH or pAldolase) [ 21 , 22 ] were selected for DNA extraction (DNA Mini Kit, Qiagen, Valencia, CA) and Plasmodium species-specific photo-induced electron transfer (PET)-PCR and quantification of DNA [ 23 ]. Samples positive for P. falciparum DNA were subjected to nested PCR (nPCR) for the single-copy pfmsp1 and pfmsp2 genes as quality control for DNA quantity and integrity [ 24 ].…”