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

ABSTRACT: Histidine-rich protein 2 (HRP2) detecting rapid diagnostic tests (RDTs) have played an important role in enabling prompt malaria diagnosis in remote locations. However, emergence of pfhrp2 deleted parasites is threatening the efficacy of RDTs, and the World Health Organization (WHO) has highlighted surveillance of these deletions as a priority. Nested PCR is used to confirm pfhrp2 deletion but is costly and laborious. Due to spurious amplification of paralogue pfhrp3, the identity of nested exon 1 PCR product must be confirmed by sequencing. Here we describe a new one-step PCR method for detection of pfhrp2. To determine sensitivity and specificity, all PCRs were performed in triplicate. Using photo-induced electron transfer (PET) PCR detecting 18srRNA as true positive, one-step had comparable sensitivity of 95.0% (88.7-98.4%) to nested exon 1, 99.0% (94.6-99.9%) and nested exon 2, 98.0% (93.0-99.8%), and comparable specificity 93.8% (69.8-99.8%) to nested exon 1 100.0% (79.4-100.0%) and nested exon 2, 100.0% (74.4-100.0%). Sequencing revealed that one step PCR does not amplify pfhrp3. Logistic regression models applied to measure the 95% level of detection of the one-step PCR in clinical isolates provided estimates of 133p/?L (95% confidence interval (CI): 3-793p/?L) for whole blood (WB) samples and 385p/?L (95% CI: 31-2133 p/?L) for dried blood spots (DBSs). When considering protocol attributes, the one-step PCR is less expensive, faster and more suitable for high throughput. In summary, we have developed a more accurate PCR method that may be ideal for the application of the WHO protocol for investigating pfhrp2 deletions in symptomatic individuals presenting to health care facilities.

SUBMITTER: Jones S

PROVIDER: S-EPMC7377462 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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

Jones Sophie S Subramaniam Gireesh G Plucinski Mateusz M MM Patel Dhruviben D Padilla Jasmine J Aidoo Michael M Talundzic Eldin E

PloS one 20200723 7

Histidine-rich protein 2 (HRP2) detecting rapid diagnostic tests (RDTs) have played an important role in enabling prompt malaria diagnosis in remote locations. However, emergence of pfhrp2 deleted parasites is threatening the efficacy of RDTs, and the World Health Organization (WHO) has highlighted surveillance of these deletions as a priority. Nested PCR is used to confirm pfhrp2 deletion but is costly and laborious. Due to spurious amplification of paralogue pfhrp3, the identity of nested exon ...[more]

PMID: 32702040

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Project description:BackgroundWith the documentation of cases of falciparum malaria negative by rapid diagnostic tests (RDT), though at low frequency from natural isolates in a small pocket of Odisha, it became absolutely necessary to investigate the status of HRP-2 based RDT throughout the state and in different seasons of the year.MethodsSuspected individuals were screened for malaria infection by microscopy and RDT in 25/30 districts of Odisha, India. Discrepancies in results were confirmed by PCR. False negative RDT samples for Plasmodium falciparum mono-infection were evaluated for detection of HRP2 antigen in ELISA and genotyped for pfhrp2, pfhrp3 and their flanking genes. Multiplicity of infection was ascertained based on msp1 and msp2 genotyping and parasitaemia level was determined by microscopy.ResultsOf the total 1058 patients suspected for malaria, 384 were microscopically confirmed for P. falciparum mono-infection and RDT failure was observed in 58 samples at varying proportion in different regions of the state. The failure in detection was due to undetectable level of HRP-2. Although most of these samples were screened during rainy season (45/345), significantly high proportion (9/17) of RDT negative samples were obtained during the summer compared to rainy season (P = 0.0002; OR = 7.5). PCR genotyping of pfhrp2 and pfhrp3 in RDT negative samples showed 38/58 (65.5) samples to be pfhrp2 negative and 24/58 (41.4) to be pfhrp3 negative including dual negative in 17/58 (29.3). Most of the RDT negative samples (39/58) were with single genotype infection and high proportions of pfhrp2 deletion (7/9) was observed in summer. No difference in parasitaemia level was observed between RDT positive and RDT negative patients.ConclusionHigh prevalence of parasites with pfhrp2 deletion including dual deletions (pfhrp2 and pfhrp3) is a serious cause of concern, as these patients could not be given a correct diagnosis and treatment. Therefore, HRP2-based RDT for diagnosing P. falciparum infection in Odisha is non-reliable and must be performed in addition to or replaced by other appropriate diagnostic tools for clinical management of the disease.

Dataset Information

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

Publications

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

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