High-throughput Plasmodium falciparum hrp2 and hrp3 gene deletion typing by digital PCR to monitor malaria rapid diagnostic test efficacy

Vera-Arias, Claudia A.; Holzschuh, Aurel; Oduma, Colins O.; Badu, Kingsley; Mutala, Abdul-Hakim; Yukich, Joshua; Hetzel, Manuel W; Fakih, Bakar S.; Ali, Abdullah; Ferreira, Marcelo U.; Ladeia‐Andrade, Simone; Saénz, Fabián E.; Afrane, Yaw A.; Zemene, Endalew; Yewhalaw, Delenasaw; Kazura, James W.; Yan, Guiyun; Koepfli, Cristian

doi:10.7554/elife.72083

Cited by 36 publications

(30 citation statements)

References 50 publications

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“…Such cross-reactivity has been previously observed in conventional RDTs [ 31 ]. As the majority of hrp2 -negative infections had fewer than 10 pRBC/µL, we also speculate that samples with undetected hrp2 by endpoint PCR may be positive for hrp2 if evaluated by a more sensitive laboratory detection method that can detect HRP2 antigen in very low density infections [ 32 ]. These results suggest that hrp2 deletions are rare and do not contribute to effectiveness of the us-RDT in this region.…”

Section: Discussionmentioning

confidence: 99%

Evaluation of an ultrasensitive HRP2–based rapid diagnostic test for detection of asymptomatic Plasmodium falciparum parasitaemia among children in western Kenya

Turnbull

Ayodo

Knight

et al. 2022

Malar J

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Background Accurate detection of asymptomatic malaria parasitaemia in children living in high transmission areas is important for malaria control and reduction programmes that employ screen-and-treat surveillance strategies. Relative to microscopy and conventional rapid diagnostic tests (RDTs), ultrasensitive RDTs (us-RDTs) have demonstrated reduced limits of detection with increased sensitivity to detect parasitaemia in symptomatic individuals. In this study, the performance of the NxTek™ Eliminate Malaria P.f test was compared with traditional microscopy and quantitative polymerase chain reaction (qPCR) testing methods of detection for P. falciparum parasitaemia among asymptomatic children aged 7–14 years living in an area of high malaria transmission intensity in western Kenya. Methods In October 2020, 240 healthy children without any reported malaria symptoms were screened for the presence of P. falciparum parasitaemia; 120 children were randomly selected to participate in a follow-up visit at 6–10 weeks. Malaria parasitaemia was assessed by blood-smear microscopy, us-RDT, and qPCR of a conserved var gene sequence from genomic DNA extracted from dried blood spots. Sensitivity, specificity, and predictive values were calculated for field diagnostic methods using qPCR as the gold standard. Comparison of detectable parasite density distributions and area under the curve were also calculated to determine the effectiveness of the us-RDT in detecting asymptomatic infections with low parasite densities. Results The us-RDT detected significantly more asymptomatic P. falciparum infections than microscopy (42.5% vs. 32.2%, P = 0.002). The positive predictive value was higher for microscopy (92.2%) than for us-RDT (82.4%). However, false negative rates were high for microscopy and us-RDT, with negative predictive values of 53.7% and 54.6%, respectively. While us-RDT detected significantly more infections than microscopy overall, the density distribution of detectable infections did not differ (P = 0.21), and qPCR detected significantly more low-density infections than both field methods (P < 0.001, for both comparisons). Conclusions Us-RDT is more sensitive than microscopy for detecting asymptomatic malaria parasitaemia in children. Though the detectable parasite density distributions by us-RDT in our specific study did not significantly differ from microscopy, the additional sensitivity of the us-RDT resulted in more identified asymptomatic infections in this important group of the population and makes the use of the us-RDT advisable compared to other currently available malaria field detection methods.

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Section: Discussionmentioning

confidence: 99%

Evaluation of an ultrasensitive HRP2–based rapid diagnostic test for detection of asymptomatic Plasmodium falciparum parasitaemia among children in western Kenya

Turnbull

Ayodo

Knight

et al. 2022

Malar J

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“…For absolute quantification of parasite density, a standard curve derived from DNA from cultured 3D7 parasites and quantified by droplet digital PCR (ddPCR) was run along samples. Samples positive for P. falciparum at a density of approximately >5 parasites/μL were typed for hrp2 and hrp3 deletion by ddPCR [34]. In this assay, hrp2 or hrp3 and a control gene (serine-tRNA ligase) are quantified in a single tube with very high specificity, thus providing highly accurate data on deletion status [34].…”

Section: P Falciparum Qpcr and Hrp2/3 Deletion Typingmentioning

confidence: 99%

“…Samples positive for P. falciparum at a density of approximately >5 parasites/μL were typed for hrp2 and hrp3 deletion by ddPCR [34]. In this assay, hrp2 or hrp3 and a control gene (serine-tRNA ligase) are quantified in a single tube with very high specificity, thus providing highly accurate data on deletion status [34]. The assay amplifies parts of hrp2 exon 2 that encodes for the antigen detected by the RDT.…”

Section: P Falciparum Qpcr and Hrp2/3 Deletion Typingmentioning

confidence: 99%

Performance of highly sensitive and conventional rapid diagnostic tests for clinical and subclinical Plasmodium falciparum infections, and hrp2/3 deletion status in Burundi

Niyukuri

Sinzinkayo

Troth

et al. 2022

PLOS Glob Public Health

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Rapid diagnostic tests (RDTs) are a key tool for the diagnosis of malaria infections among clinical and subclinical individuals. Low-density infections, and deletions of the P. falciparum hrp2/3 genes (encoding the HRP2 and HRP3 proteins detected by many RDTs) present challenges for RDT-based diagnosis. The novel Rapigen Biocredit three-band Plasmodium falciparum HRP2/LDH RDT was evaluated among 444 clinical and 468 subclinical individuals in a high transmission setting in Burundi. Results were compared to the AccessBio CareStart HRP2 RDT, and qPCR with a sensitivity of <0.3 parasites/μL blood. Sensitivity compared to qPCR among clinical patients for the Biocredit RDT was 79.9% (250/313, either of HRP2/LDH positive), compared to 73.2% (229/313) for CareStart (P = 0.048). Specificity of the Biocredit was 82.4% compared to 96.2% for CareStart. Among subclinical infections, sensitivity was 72.3% (162/224) compared to 58.5% (131/224) for CareStart (P = 0.003), and reached 88.3% (53/60) in children <15 years. Specificity was 84.4% for the Biocredit and 93.4% for the CareStart RDT. No (0/362) hrp2 and 2/366 hrp3 deletions were observed. In conclusion, the novel RDT showed improved sensitivity for the diagnosis of P. falciparum.

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“…In this assay, either hrp2 or hrp3 is multiplexed with a control gene, serine-tRNA ligase. Both targets are amplified with very high sensitivity and specificity, thus providing highly accurate data on deletion status [34]. For ddPCR, samples were shipped to the University of Notre Dame.…”

Section: Diagnosis By Microscopy Rdt and Qpcrmentioning

confidence: 99%

qPCR in a suitcase for rapid Plasmodium falciparum and Plasmodium vivax surveillance in Ethiopia

Carlier

Baker

Huwe

et al. 2022

PLOS Glob Public Health

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Many Plasmodium spp. infections, both in clinical and asymptomatic patients, are below the limit of detection of light microscopy or rapid diagnostic test (RDT). Molecular diagnosis by qPCR can be valuable for surveillance, but is often hampered by absence of laboratory capacity in endemic countries. To overcome this limitation, we optimized and tested a mobile qPCR laboratory for molecular diagnosis in Ziway, Ethiopia, where transmission intensity is low. Protocols were optimized to achieve high throughput and minimize costs and weight for easy transport. 899 samples from febrile patients and 1021 samples from asymptomatic individuals were screened by local microscopy, RDT, and qPCR within a period of six weeks. 34/52 clinical Plasmodium falciparum infections were missed by microscopy and RDT. Only 4 asymptomatic infections were detected. No hrp2 deletions were observed among 25 samples typed, but 19/24 samples carried hrp3 deletions. The majority (25/41) of Plasmodium vivax infections (1371 samples screened) were found among asymptomatic individuals. All asymptomatic P. vivax infections were negative by microscopy and RDT. In conclusion, the mobile laboratory described here can identify hidden parasite reservoirs within a short period of time, and thus inform malaria control activities.

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High-throughput Plasmodium falciparum hrp2 and hrp3 gene deletion typing by digital PCR to monitor malaria rapid diagnostic test efficacy

Cited by 36 publications

References 50 publications

Evaluation of an ultrasensitive HRP2–based rapid diagnostic test for detection of asymptomatic Plasmodium falciparum parasitaemia among children in western Kenya

Evaluation of an ultrasensitive HRP2–based rapid diagnostic test for detection of asymptomatic Plasmodium falciparum parasitaemia among children in western Kenya

Performance of highly sensitive and conventional rapid diagnostic tests for clinical and subclinical Plasmodium falciparum infections, and hrp2/3 deletion status in Burundi

qPCR in a suitcase for rapid Plasmodium falciparum and Plasmodium vivax surveillance in Ethiopia

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