One-step PCR: A novel protocol for determination of pfhrp2 deletion status in Plasmodium falciparum

Jones, Sophie; Subramaniam, Gireesh; Pluciński, Mateusz M.; Patel, Dhruviben; Padilla, Jasmine; Aidoo, Michael; Talundzic, Eldin

doi:10.1371/journal.pone.0236369

Cited by 19 publications

(19 citation statements)

References 49 publications

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“…We used nested PCR to genotype pfmsp1, pfmsp2, and pfhrp3 as described previously ( 29 ). For pfhrp2 genotyping, we performed PCR on these samples under conditions described previously ( 30 ). Results for pfhrp2/3 genotyping were only reported if both pfmsp1 and pfmsp2 (both single-copy genes in the P. falciparum genome) were successfully amplified for a DNA sample ( 31 ).…”

Section: Methodsmentioning

confidence: 99%

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

Rogier¹,

McCaffery²,

Nace³

et al. 2022

Emerg. Infect. Dis.

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Histidine-rich protein 2 (HRP2)–based rapid diagnostic tests detect Plasmodium falciparum malaria and are used throughout sub-Saharan Africa. However, deletions in the pfhrp2 and related pfhrp3 ( pfhrp2/3 ) genes threaten use of these tests. Therapeutic efficacy studies (TESs) enroll persons with symptomatic P. falciparum infection. We screened TES samples collected during 2016–2018 in Ethiopia, Kenya, Rwanda, and Madagascar for HRP2/3, pan- Plasmodium lactate dehydrogenase, and pan- Plasmodium aldolase antigen levels and selected samples with low levels of HRP2/3 for pfhrp2/3 genotyping. We observed deletion of pfhrp3 in samples from all countries except Kenya. Single-gene deletions in pfhrp2 were observed in 1.4% (95% CI 0.2%–4.8%) of Ethiopia samples and in 0.6% (95% CI 0.2%–1.6%) of Madagascar samples, and dual pfhrp2/3 deletions were noted in 2.0% (95% CI 0.4%–5.9%) of Ethiopia samples. Although this study was not powered for precise prevalence estimates, evaluating TES samples revealed a low prevalence of pfhrp2/3 deletions in most sites.

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Section: Methodsmentioning

confidence: 99%

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

Rogier¹,

McCaffery²,

Nace³

et al. 2022

Emerg. Infect. Dis.

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“…Samples that failed to amplify by pfmsp1 or pfmsp2 were omitted from further genotyping analysis because DNA quality (or quantity) was assumed to be compromised. A one-step PCR was used to determine the presence of the pfhrp2 gene 27 , and two separate nested PCRs were used for the confirmation of the presence of the pfhrp3 gene: a pfhrp3 exon 1–2 PCR and an exon 2 PCR 22 , 26 . Deletion of the pfhrp3 gene was confirmed if the sample was unable to amplify the exon 1–2 PCR product.…”

Section: Methodsmentioning

confidence: 99%

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

McCaffery

Nace

Herman

et al. 2021

Sci Rep

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Rapid diagnostic tests (RDTs) detecting histidine-rich protein 2 (HRP2) and HRP3 are widely used throughout sub-Saharan Africa (SSA) to diagnose Plasmodium falciparum malaria. However, multiple SSA countries have reported pfhrp2 and pfhrp3 (pfhrp2/3) gene deletions. Blood samples (n = 1109) collected from patients with P. falciparum infection from six health facilities throughout the Democratic Republic of the Congo (DRC) from March 2017 to January 2018 were evaluated for pfhrp2/3 deletions. Samples were assayed for HRP2, pan-Plasmodium LDH (pLDH) and aldolase (pAldolase) antigens by bead-based multiplex antigen assay. Samples with low HRP2 concentration compared to pLDH and pAldolase antigens were selected for further pfhrp2/3 genotyping PCRs. The majority of blood samples (93.3%, 1035/1109) had high concentrations of the HRP2 antigen. Single deletions of pfhrp2 were identified in 0.27% (3/1109) of screened samples, with one sample from each of the Kapolowe, Mikalayi, and Rutshuru study sites. A pfhrp3 single deletion (0.09%, 1/1109) was found in the Kapolowe site. Dual pfhrp2 and pfhrp3 deletions were not observed. Due to, the low numbers of pfhrp2 deletions and the sporadic locations of these deletions, the use of HRP2-based RDTs appears to still be appropriate for these locations in DRC.

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“…Studies of pfhrp2/3- deleted P. falciparum are difficult due to the challenges of confirming the absence of these genes using conventional approaches 11 , 12 . These challenges are compounded by inconsistent laboratory methodologies across studies and inherent limitations of pfhrp2/3 assays that can suffer from variable performance and cross-reactivity 13 , 14 . In addition, false-negative RDT results are common throughout Africa and typically caused by factors other than pfhrp2/3 deletions, including operator error, lot-to-lot RDT variability, low-density infections below the RDT’s limit of detection, and infection by non-falciparum species 15 , 16 .…”

Section: Introductionmentioning

confidence: 99%

Analysis of false-negative rapid diagnostic tests for symptomatic malaria in the Democratic Republic of the Congo

Parr

Kieto

Phanzu

et al. 2021

Sci Rep

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The majority of Plasmodium falciparum malaria diagnoses in Africa are made using rapid diagnostic tests (RDTs) that detect histidine-rich protein 2. Increasing reports of false-negative RDT results due to parasites with deletions of the pfhrp2 and/or pfhrp3 genes (pfhrp2/3) raise concern about existing malaria diagnostic strategies. We previously identified pfhrp2-negative parasites among asymptomatic children in the Democratic Republic of the Congo (DRC), but their impact on diagnosis of symptomatic malaria is unknown. We performed a cross-sectional study of false-negative RDTs in symptomatic subjects in 2017. Parasites were characterized by microscopy; RDT; pfhrp2/3 genotyping and species-specific PCR assays; a bead-based immunoassay for Plasmodium antigens; and/or whole-genome sequencing. Among 3627 symptomatic subjects, 427 (11.8%) had RDT-/microscopy + results. Parasites from eight (0.2%) samples were initially classified as putative pfhrp2/3 deletions by PCR, but antigen testing and whole-genome sequencing confirmed the presence of intact genes. 56.8% of subjects had PCR-confirmed malaria. Non-falciparum co-infection with P. falciparum was common (13.2%). Agreement between PCR and HRP2-based RDTs was satisfactory (Cohen’s kappa = 0.66) and superior to microscopy (0.33). Symptomatic malaria due to pfhrp2/3-deleted P. falciparum was not observed. Ongoing HRP2-based RDT use is appropriate for the detection of falciparum malaria in the DRC.

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One-step PCR: A novel protocol for determination of pfhrp2 deletion status in Plasmodium falciparum

Cited by 19 publications

References 49 publications

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

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

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

Analysis of false-negative rapid diagnostic tests for symptomatic malaria in the Democratic Republic of the Congo

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