Molecular surveillance reveals the presence of  pfhrp2  and  pfhrp3  gene deletions in  Plasmodium falciparum  parasite populations in Uganda, 2017–2019

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Abstract

Background

Histidine-rich protein-2 (HRP2)-based rapid diagnostic tests (RDTs) are the only RDTs recommended for malaria diagnosis in Uganda. However, the emergence of Plasmodium falciparum histidine rich protein 2 and 3 ( pfhrp2 and pfhrp3) gene deletions threatens their usefulness as malaria diagnostic and surveillance tools. The pfhrp2 and pfhrp3 gene deletions surveillance was conducted in P. falciparum parasite populations in Uganda.

Methods

Three-hundred (n = 300) P. falciparum isolates collected from cross-sectional malaria surveys in symptomatic individuals in 48 districts of eastern and western Uganda were analysed for the presence of pfhrp2 and pfhrp3 genes. Presence of parasite DNA was confirmed by PCR amplification of the 18s rRNA gene , msp1 and msp2 single copy genes. Presence or absence of deletions was confirmed by amplification of exon1 and exon2 of pfhrp2 and pfhrp3 using gene specific PCR.

Results

Overall, pfhrp2 and pfhrp3 gene deletions were detected in 29/300 (9.7%, 95% CI 6.6–13.6%) parasite isolates. The pfhrp2 gene was deleted in 10/300 (3.3%, 95% CI 1.6–6.0%) isolates, pfhrp3 in 9/300 (3.0%, 95% CI 1.4–5.6%) while both pfhrp2 and pfhrp3 were deleted in 10/300 (3.3%, 95% CI 1.6–6.0%) parasite isolates. Proportion of pfhrp2/3 deletions was higher in the eastern 14.7% (95% CI 9.7–20.0%) compared to the western region 3.1% (95% CI 0.8–7.7%), p = 0.001. Geographical location was associated with gene deletions aOR 6.25 (2.02–23.55), p = 0.003.

Conclusions

This is the first large-scale survey reporting the presence of pfhrp2/3 gene deletions in P. falciparum isolates in Uganda. Roll out of RDTs for malaria diagnosis should take into consideration the existence of pfhrp2/3 gene deletions particularly in areas where they were detected. Periodic pfhrp2/3 surveys are recommended to inform future decisions for deployment of alternative RDTs.

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Most cited references 14

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Use of a rapid, single-round, multiplex PCR to detect malarial parasites and identify the species present.

Ian J. Saldanha, P Chiodini, Sally Moody … (2003)

A new, rapid assay, based on a single-round, multiplex PCR, can be used to detect Plasmodium falciparum, P. vivax, P. malariae or P. ovale in human blood. The PCR, which targets the conserved 18S small-subunit RNA genes of the parasites, not only permits a malarial infection to be detected but also allows each Plasmodium species present to be identified, even in cases of mixed infection.

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Semi-nested, multiplex polymerase chain reaction for detection of human malaria parasites and evidence of Plasmodium vivax infection in Equatorial Guinea.

J. Rubio, A Benito, J. Roche … (1999)

A semi-nested, multiplex polymerase chain reaction (PCR) based on the amplification of the sequences of the 18S small subunit ribosomal RNA (ssrRNA) gene was tested in a field trial in Equatorial Guinea (a hyperendemic focus of malaria in west central Africa). The method uses a primary PCR amplification reaction with a universal reverse primer and two forward primers specific for the genus Plasmodium and to mammals (the mammalian-specific primer was included as a positive control to distinguish uninfected cases from inhibition of the PCR). The second amplification is carried out with the same Plasmodium genus-specific forward primer and four specific reverse primers for each human Plasmodium species. The PCR amplified products are differentiated by fragment size after electrophoresis on a 2% agarose gel. Four villages from three regions of the island of Bioko (Equatorial Guinea) and two suspected Plasmodium vivax-P. ovale infections from the hospital of Malabo were tested by microscopy and PCR. The PCR method showed greater sensitivity and specificity than microscopic examination and confirmed the existence of a focus of P. vivax infections in Equatorial Guinea suspected by microscopic examination. It also provided evidence of several mixed infections, mainly P. falciparum and P. malariae, the two predominant species causing malaria in Equatorial Guinea.

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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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Author and article information

Contributors

Agaba B. Bosco:

ORCID: http://orcid.org/0000-0003-4601-5171

bagaba2011@gmail.com

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): 26 August 2020

Publication date PMC-release: 26 August 2020

Publication date Collection: 2020

Volume: 19

Electronic Location Identifier: 300

Affiliations

[1 ]GRID grid.11194.3c, ISNI 0000 0004 0620 0548, College of Health Sciences, , Makerere University, ; Kampala, Uganda

[2 ]National Malaria Control Division, Kampala, Uganda

[3 ]GRID grid.1049.c, ISNI 0000 0001 2294 1395, QIMR Berghofer Medical Research Institute, ; Brisbane, QLD Australia

[4 ]Australian Defence Force Malaria and Infectious Disease Institute, Enoggera, Australia

[5 ]GRID grid.463352.5, Infectious Diseases Research Collaboration, ; Kampala, Uganda

[6 ]World Health Organization Country Office, Kampala, Uganda

[7 ]GRID grid.415705.2, National Health Information Division, , Ministry of Health, ; Kampala, Uganda

[8 ]GRID grid.31501.36, ISNI 0000 0004 0470 5905, Department of Public Health Sciences, , Seoul National University, ; Seoul, South Korea

[9 ]GRID grid.222754.4, ISNI 0000 0001 0840 2678, Department of Laboratory Medicine, College of Health Sciences, , Korea University, ; Seoul, South Korea

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

Author information

Agaba B. Bosco http://orcid.org/0000-0003-4601-5171

Article

Publisher ID: 3362

DOI: 10.1186/s12936-020-03362-x

PMC ID: 7449024

PubMed ID: 32843041

SO-VID: dd964ef7-9e77-4f23-b0c0-eb48a5cc6118

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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 : 21 April 2020

Date accepted : 5 August 2020

Funding

Funded by: FundRef http://dx.doi.org/10.13039/100000061, Fogarty International Center;

Award ID: D43TW010526

Funded by: FundRef http://dx.doi.org/10.13039/100014035, Armed Forces Health Surveillance Branch;

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ScienceOpen disciplines: Infectious disease & Microbiology

Keywords: malaria rapid diagnostic tests,plasmodium falciparum,histidine rich protein 2,histidine rich protein 3,gene deletion,deoxyribonucleic acid,microscopy

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ScienceOpen disciplines: Infectious disease & Microbiology

Keywords: malaria rapid diagnostic tests, plasmodium falciparum, histidine rich protein 2, histidine rich protein 3, gene deletion, deoxyribonucleic acid, microscopy

Molecular surveillance reveals the presence of pfhrp2 and pfhrp3 gene deletions in Plasmodium falciparum parasite populations in Uganda, 2017–2019

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Malaria vaccine development

Most cited references 14

Use of a rapid, single-round, multiplex PCR to detect malarial parasites and identify the species present.

Semi-nested, multiplex polymerase chain reaction for detection of human malaria parasites and evidence of Plasmodium vivax infection in Equatorial Guinea.

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

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