Impact of  Plasmodium falciparum  gene deletions on malaria rapid diagnostic test performance

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Abstract

Background

Malaria rapid diagnostic tests (RDTs) have greatly improved access to diagnosis in endemic countries. Most RDTs detect Plasmodium falciparum histidine-rich protein 2 (HRP2), but their sensitivity is seriously threatened by the emergence of pfhrp2-deleted parasites. RDTs detecting P. falciparum or pan-lactate dehydrogenase (Pf- or pan-LDH) provide alternatives. The objective of this study was to systematically assess the performance of malaria RDTs against well-characterized pfhrp2-deleted P. falciparum parasites.

Methods

Thirty-two RDTs were tested against 100 wild-type clinical isolates (200 parasites/µL), and 40 samples from 10 culture-adapted and clinical isolates of pfhrp2-deleted parasites. Wild-type and pfhrp2-deleted parasites had comparable Pf-LDH concentrations. Pf-LDH-detecting RDTs were also tested against 18 clinical isolates at higher density (2,000 parasites/µL) lacking both pfhrp2 and pfhrp3.

Results

RDT positivity against pfhrp2-deleted parasites was highest (> 94%) for the two pan-LDH-only RDTs. The positivity rate for the nine Pf-LDH-detecting RDTs varied widely, with similar median positivity between double-deleted ( pfhrp2/3 negative; 63.9%) and single-deleted ( pfhrp2-negative/ pfhrp3-positive; 59.1%) parasites, both lower than against wild-type P. falciparum (93.8%). Median positivity for HRP2-detecting RDTs against 22 single-deleted parasites was 69.9 and 35.2% for HRP2-only and HRP2-combination RDTs, respectively, compared to 96.0 and 92.5% for wild-type parasites. Eight of nine Pf-LDH RDTs detected all clinical, double-deleted samples at 2,000 parasites/µL.

Conclusions

The pan-LDH-only RDTs evaluated performed well. Performance of Pf-LDH-detecting RDTs against wild-type P. falciparum does not necessarily predict performance against pfhrp2-deleted parasites. Furthermore, many, but not all HRP2-based RDTs, detect pfhrp2-negative/ pfhrp3-positive samples, with implications for the HRP2-based RDT screening approach for detection and surveillance of HRP2-negative parasites.

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Most cited references 20

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Human malaria parasites in continuous culture

W Trager, J. J. Jensen (1976)

Plasmodium falciparum can now be maintained in continuous culture in human erythrocytes incubated at 38 degrees C in RPMI 1640 medium with human serum under an atmosphere with 7 percent carbon dioxide and low oxygen (1 or 5 percent). The original parasite material, derived from an infected Aotus trivirgatus monkey, was diluted more than 100 million times by the addition of human erythrocytes at 3- or 4-day intervals. The parasites continued to reproduce in their normal asexual cycle of approximately 48 hours but were no longer highly synchronous. The have remained infective to Aotus.

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False-negative malaria rapid diagnostic tests in Rwanda: impact of Plasmodium falciparum isolates lacking hrp2 and declining malaria transmission

Christina Kozycki, Noella Umulisa, Stephen Rulisa … (2017)

Background Rapid diagnostic tests (RDTs) for histidine rich protein 2 (HRP2) are often used to determine whether persons with fever should be treated with anti-malarials. However, Plasmodium falciparum parasites with a deletion of the hrp2 gene yield false-negative RDTs and there are concerns the sensitivity of HRP2-based RDTs may fall when the intensity of transmission decreases. Methods This observational study enrolled 9226 patients at three health centres in Rwanda from April 2014 to April 2015. It then compared the sensitivity of RDTs based on HRP2 and the Plasmodium lactate dehydrogenase (pLDH) to microscopy (thick smears) for the diagnosis of malaria. PCR was used to determine whether deletions of the histidine-rich central repeat region of the hrp2 gene (exon 2) were associated with false-negative HRP2-based RDTs. Results In comparison to microscopy, the sensitivity and specificity of HRP2- and pLDH-based RDTs were 89.5 and 86.2% and 80.2 and 94.3%, respectively. When the results for both RDTs were combined, sensitivity rose to 91.8% and specificity was 85.7%. Additionally, when smear positivity fell from 46 to 3%, the sensitivity of the HRP2-based RDT fell from 88 to 67%. Of 370 samples with false-negative HRP2 RDT results for which PCR was performed, 140 (38%) were identified as P. falciparum by PCR. Of the isolates identified as P. falciparum by PCR, 32 (23%) were negative for the hrp2 gene based on PCR. Of the 32 P. falciparum isolates negative for hrp2 by PCR, 17 (53%) were positive based on the pLDH RDT. Conclusion This prospective study of RDT performance coincided with a decline in the intensity of malaria transmission in Kibirizi (fall in slide positivity from 46 to 3%). This decline was associated with a decrease in HRP2 RDT sensitivity (from 88 to 67%). While P. falciparum isolates without the hrp2 gene were an important cause of false-negative HRP2-based RDTs, most were identified by the pLDH-based RDT. Although WHO does not recommend the use of combined HRP2/pLDH testing in sub-Saharan Africa, these results suggest that combination HRP2/pLDH-based RDTs could reduce the impact of false-negative HRP2-based RDTs for detection of symptomatic P. falciparum malaria. Electronic supplementary material The online version of this article (doi:10.1186/s12936-017-1768-1) contains supplementary material, which is available to authorized users.

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

Khalid Beshir, Nuno Sepúlveda, Jameel Bharmal … (2017)

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.

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Author and article information

Contributors

Michelle L. Gatton:

ORCID: http://orcid.org/0000-0003-1188-609X

m.gatton@qut.edu.au

Journal

Journal ID (nlm-ta): Malar J

Journal ID (iso-abbrev): Malar J

Title: Malaria Journal

Publisher: BioMed Central (London )

ISSN (Electronic): 1475-2875

Publication date (Electronic): 4 November 2020

Publication date PMC-release: 4 November 2020

Publication date Collection: 2020

Volume: 19

Electronic Location Identifier: 392

Affiliations

[1 ]GRID grid.1024.7, ISNI 0000000089150953, Queensland University of Technology, ; Brisbane, QLD Australia

[2 ]GRID grid.474959.2, ISNI 0000 0004 0528 628X, The CDC Foundation, ; Atlanta, GA USA

[3 ]GRID grid.416738.f, ISNI 0000 0001 2163 0069, Centers for Disease Control and Prevention, ; Atlanta, USA

[4 ]GRID grid.439634.f, ISNI 0000 0004 0612 2527, Hospital for Tropical Diseases, ; London, UK

[5 ]GRID grid.8991.9, ISNI 0000 0004 0425 469X, London School of Hygiene and Tropical Medicine, ; London, UK

[6 ]GRID grid.452485.a, ISNI 0000 0001 1507 3147, Foundation for Innovative New Diagnostics (FIND), ; Geneva, Switzerland

[7 ]GRID grid.237081.f, Australian Defence Force Malaria and Infectious Diseases Institute (FORMERLY Australian Army Malaria Institute), ; Brisbane, QLD Australia

[8 ]GRID grid.3575.4, ISNI 0000000121633745, World Health Organization, ; Geneva, Switzerland

Author information

Michelle L. Gatton http://orcid.org/0000-0003-1188-609X

Article

Publisher ID: 3460

DOI: 10.1186/s12936-020-03460-w

PMC ID: 7640408

PubMed ID: 33148265

SO-VID: 0698d928-b099-489b-b66c-d398711b8c22

License:

Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

History

Date received : 27 May 2020

Date accepted : 23 October 2020

Funding

Funded by: UNITAID

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ScienceOpen disciplines: Infectious disease & Microbiology

Keywords: rapid diagnostic tests,hisidine rich protein 2,hrp2,gene deletion

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ScienceOpen disciplines: Infectious disease & Microbiology

Keywords: rapid diagnostic tests, hisidine rich protein 2, hrp2, gene deletion

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Impact of Plasmodium falciparum gene deletions on malaria rapid diagnostic test performance

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Human malaria parasites in continuous culture

False-negative malaria rapid diagnostic tests in Rwanda: impact of Plasmodium falciparum isolates lacking hrp2 and declining malaria transmission

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

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