Pfhrp2 Gene Deletions Research Articles

Histidine-rich protein (hrp) 2-based RDT false-negatives and Plasmodium falciparum hrp 2 and 3 gene deletions in low, seasonal and intense perennial transmission zones in Cameroon: a cross - sectional study.

False negative rapid diagnostic tests (RDTs) accruing to the non-detection of Plasmodium falciparum histidine-rich protein 2/3 (Pfhrp2/3) is threatening the diagnosis and management of malaria. Although regular monitoring is necessary to gauge the level of efficacy of the tool, studies in Cameroon remain limited. This study assessed Plasmodium spp. prevalence and Pfhrp2/3 gene deletions across ecological and transmission zones in Cameroon. This is a cross-sectional, multi-site, community- and hospital- based study, in 21 health facilities and 14 communities covering all five ecological settings in low seasonal (LS) and intense perennial (IPT) malaria transmission zones between 2019 and 2021. Participants were screened for malaria parasite using Pfhrp2 RDT and light microscopic examination of thick peripheral blood smears. DNA was extracted from dried blood spot using chelex®-100 and P. falciparum confirmed using varATS real-time quantitative Polymerase Chain Reaction (qPCR), P. malariae and P. ovale by real-time qPCR of Plasmepsin gene, and P. vivax using a commercial kit. Isolates with amplified Pfcsp and Pfama-1 genes were assayed for Pfhrp 2/3 gene deletions by conventional PCR. A total of 3,373 participants enrolled, 1,786 Plasmodium spp. infected, with 77.4% P. falciparum. Discordant RDT and qPCR results (False negatives) were reported in 191 (15.7%) P. falciparum mono-infected samples from LS (29%, 42) and IPT (13.9%, 149). The Pfhrp2+/Pfhrp3 + genotype was most frequent, similar between LS (5.5%, 8/145) and IPT (6.0%, 65/1,076). Single Pfhrp2 and Pfhrp3 gene deletions occurred in LS (0.7%, 1/145 each) and IPT (3.6%, 39/1,076 vs. 2.9%, 31/1,076), respectively. Whilst a single sample harboured Pfhrp2-/Pfhrp3- genotype in LS, 2.4% (26/1,076) were double deleted at IPT. Pfhrp2+/Pfhrp3- (0.3%, 3/1,076) and Pfhrp2-/Pfhrp3+ (1.2%, 13/1,076) genotypes were only observed in IPT. Pfhrp2, Pfhrp3 deletions and Pfhrp2-/Pfhrp3- genotype accounted for 78.8% (26), 69.7% (23) and 63.6% (21) RDT false negatives, respectively. Plasmodium falciparum remains the most dominant and widely distributed Plasmodium species across transmission and ecological zones in Cameroon. Although the low prevalence of Pfhrp2/3 gene deletions supports the continued use of HRP2-based RDTs for routine malaria diagnosis, the high proportion of false-negatives due to gene deleted parasites necessitates continued surveillance to inform control and elimination efforts.

High prevalence of asymptomatic and subpatent Plasmodium falciparum infections but no histidine-rich protein 2 gene deletion in Bouaké, Côte d’Ivoire

This study aimed to estimate the prevalence of asymptomatic and subpatent P. falciparum infections in the city of Bouaké, Central Côte d’Ivoire, to compare the performance of three tests, and to investigate potential P. falciparum histidine-rich protein 2 (pfhrp2) gene deletions. A cross-sectional survey was conducted in nine neighborhoods in Bouaké in 2016. Matched light microscopy (LM), rapid diagnostic test (RDT), and quantitative PCR (qPCR) data were used to determine the prevalence of P. falciparum infection and compare the performance of the three diagnostic tests. Pfhrp2/3 deletions were genotyped by digital PCR. Among 2313 individuals, 97.2% were asymptomatic and 2.8% were symptomatic. P. falciparum prevalence among symptomatic individuals was 25.8%, 30.3%, and 40.9% by LM, RDT, and varATS qPCR, respectively, and among asymptomatic individuals, it was 10.3%, 12.5%, and 34.9%. Asymptomatic infections comprised 96.4% of all malaria infections, with 58.2% detectable only by varATS qPCR. Although the prevalence of asymptomatic P. falciparum infections was higher in school-age children (5–14 years: 42.0%) compared to < 5 years (17.3%) and ≥ 15 years (35.9%), subpatent infections were more likely in ≥ 15 years (70.4%) than in < 5 years (39.7%) and school-age children (41.2%). LM and RDTs were reliable only at parasite densities > 10,000 parasites/µL. Individuals who were positive according to all three tests had significantly greater parasite density (856.8 parasites/µL; 95% CI 707.3–1,038) than did those who were positive by varATS qPCR only (13.7 parasites/µL; 95% CI 11.4–16.3) (p < 0.0001). No pfhrp2 deletions were observed. The high prevalence of asymptomatic and subpatent infections highlights the need for targeted strategies to reduce malaria in urban Côte d’Ivoire.

Malaria diagnosis challenges and pfhrp2 and pfhrp3 gene deletions using pregnant women as sentinel population in Nanoro region, Burkina Faso

ABSTRACT Malaria in pregnancy causes adverse consequences and prompt and accurate diagnosis is essential for case management. In malaria endemic countries, diagnosis is mainly based on rapid diagnostic tests (RDT) and microscopy. However, increasing reports of false negatives caused by low parasitemia and pfhrp2/3 deletions raise concerns about HRP2-based RDT usefulness. This study aimed to assess RDT and microscopy performance and to describe pfhrp2/3 deletions in a cohort of 418 pregnant women in Burkina Faso. Malaria was diagnosed using RDT and microscopy and blood samples were collected during antenatal care visits. Diagnostic results were compared to PCR as gold standard. Pfhrp2 and pfhrp3 deletions were characterized for patients with confirmed P. falciparum infection. RDT had better sensitivity (76%) but lower specificity (83%) than microscopy (sensitivity = 57%; specificity = 98%). Low parasitemia (<150 parasites/µL), especially in multigravidae, was the principal factor causing false negatives by both methods. Moreover, pfhrp2 deletion frequency among overall false negatives by RDT was 21.43%. Higher frequency of deletions was found among all samples, independently of RDT result, for example around 2% of samples had double deletions meaning that the majority of deletions had no effect on RDT testing. Finally, it was found higher pfhrp2 deletion in women with lower uterine height during the first trimester. Wider and National surveillance study of deletions is recommended among pregnant women and in Burkina Faso.

Molecular detection of sub-microscopic infections and Plasmodium falciparum histidine-rich protein-2 and 3 gene deletions in pre-elimination settings of South Africa

South Africa’s efforts toward eliminating malaria have positioned the country in the pre-elimination stage. Imported and sub-microscopic cases still contribute to the persistence of malaria in regions of low transmission as identified in this study where diagnostics is built largely on the use of Rapid Diagnostic Test (RDT). However, the presence of Pfhrp2/3 gene deletion is known to interfere with the accuracy of diagnosis with the use of RDT. Malaria elimination and detection of Pfhrp2/3 gene deletion in the pre-elimination setting requires accurate molecular surveillance. With the core objective of this study being the determination of the presence sub-microscopic malaria cases and deleted Pfhrp2/3 gene markers, a total of 354 samples were collected from five districts of KwaZulu Natal, South Africa. These samples were prepared for molecular analysis using primers and PCR conditions specific for amplification of 18S rRNA and msp-1gene. Positive amplicons were analysed for the presence of Pfhrp2/3 and flanking genes, along with Sanger sequencing and phylogenetic studies. Out of 354 samples collected 339 were tested negative with PfHRP2 based RDTs. Of these Pfhrp2 and Pfhrp3 gene deletions were confirmed in 94.7% (18/19) and 100% (19/19) respectively. High migration rate (75%) among the study participants was noted and phylogenetic analysis of sequenced isolates showed close evolutionary relatedness with India, United Kingdom, Iran, and Myanmar and China isolates. Molecular-based test is recommended as an essential surveillance tool for malaria management programs as the target focuses on elimination.

Plasmodium falciparum pfhrp2 and pfhrp3 gene deletions among patients enrolled at 100 health facilities throughout Tanzania: February to July 2021

Plasmodium falciparum with the histidine rich protein 2 gene (pfhrp2) deleted from its genome can escape diagnosis by HRP2-based rapid diagnostic tests (HRP2-RDTs). The World Health Organization (WHO) recommends switching to a non-HRP2 RDT for P. falciparum clinical case diagnosis when pfhrp2 deletion prevalence causes ≥ 5% of RDTs to return false negative results. Tanzania is a country of heterogenous P. falciparum transmission, with some regions approaching elimination and others at varying levels of control. In concordance with the current recommended WHO pfhrp2 deletion surveillance strategy, 100 health facilities encompassing 10 regions of Tanzania enrolled malaria-suspected patients between February and July 2021. Of 7863 persons of all ages enrolled and providing RDT result and blood sample, 3777 (48.0%) were positive by the national RDT testing for Plasmodium lactate dehydrogenase (pLDH) and/or HRP2. A second RDT testing specifically for the P. falciparum LDH (Pf-pLDH) antigen found 95 persons (2.5% of all RDT positives) were positive, though negative by the national RDT for HRP2, and were selected for pfhrp2 and pfhrp3 (pfhrp2/3) genotyping. Multiplex antigen detection by laboratory bead assay found 135/7847 (1.7%) of all blood samples positive for Plasmodium antigens but very low or no HRP2, and these were selected for genotyping as well. Of the samples selected for genotyping based on RDT or laboratory multiplex result, 158 were P. falciparum DNA positive, and 140 had sufficient DNA to be genotyped for pfhrp2/3. Most of these (125/140) were found to be pfhrp2+/pfhrp3+, with smaller numbers deleted for only pfhrp2 (n = 9) or only pfhrp3 (n = 6). No dual pfhrp2/3 deleted parasites were observed. This survey found that parasites with these gene deletions are rare in Tanzania, and estimated that 0.24% (95% confidence interval: 0.08% to 0.39%) of false-negative HRP2-RDTs for symptomatic persons were due to pfhrp2 deletions in this 2021 Tanzania survey. These data provide evidence for HRP2-based diagnostics as currently accurate for P. falciparum diagnosis in Tanzania.

Prevalence of Asymptomatic Falciparum Malaria, Risk Factors, and Absence of PfHRP2 Gene Deletion in Makurdi, Nigeria

Background: Malaria remains a persistent public health challenge, particularly in sub-Saharan Africa, where asymptomatic falciparum malaria poses a significant threat. Asymptomatic cases serve as a crucial parasite reservoir, contributing to ongoing transmission.&#x0D; Aim: The study investigates the prevalence of asymptomatic falciparum malaria and assesses the occurrence of gene deletion in the Plasmodium falciparum Histidine Rich Protein 2 (Pf HRP2) gene in Makurdi, Benue State, Nigeria.&#x0D; Methodology: A cross-sectional study was conducted between September and October 2019, which involved 374 apparently healthy individuals from five communities. Malaria diagnosis utilized Rapid Diagnostic Test (RDT) kits, microscopy, and polymerase chain reaction (PCR) for Pf HRP2 gene assessment.&#x0D; Results: The study found a prevalence of 25.4% by RDT and 28.1% by microscopy. Asymptomatic falciparum malaria was significantly influenced by location, proximity to water bodies, bed net usage, and history of malaria treatment, while, age, and insecticide usage showed no significant impact. PCR results revealed amplified fragments with band sizes ranging from 600 to 900 base pairs in 40 positive isolates, eliminating Pf HRP2 gene deletion as a cause for false negatives observed between RDT and microscopy results.&#x0D; Discussion/Conclusion: The study highlights a high malaria transmission rate in Nigeria, emphasizing the role of location-specific factors and bed net usage in the proliferation of asymptomatic falciparum malaria. Importantly, no gene deletion was identified in the Pf HRP2 gene among the studied Plasmodium falciparum isolates.

Limited threat of Plasmodium falciparum pfhrp2 and pfhrp3 gene deletion to the utility of HRP2-based malaria RDTs in Northern Uganda

BackgroundRapid diagnostic tests (RDTs) that detect Plasmodium falciparum histidine-rich protein-2 (PfHRP2) are exclusively deployed in Uganda, but deletion of the pfhrp2/3 target gene threatens their usefulness as malaria diagnosis and surveillance tools.MethodsA cross-sectional survey was conducted at 40 sites across four regions of Uganda in Acholi, Lango, W. Nile and Karamoja from March 2021 to June 2023. Symptomatic malaria suspected patients were recruited and screened with both HRP2 and pan lactate dehydrogenase (pLDH) detecting RDTs. Dried blood spots (DBS) were collected from all patients and a random subset were used for genomic analysis to confirm parasite species and pfhrp2 and pfhrp3 gene status. Plasmodium species was determined using a conventional multiplex PCR while pfhrp2 and pfhrp3 gene deletions were determined using a real-time multiplex qPCR. Expression of the HRP2 protein antigen in a subset of samples was further assessed using a ELISA.ResultsOut of 2435 symptomatic patients tested for malaria, 1504 (61.8%) were positive on pLDH RDT. Overall, qPCR confirmed single pfhrp2 gene deletion in 1 out of 416 (0.2%) randomly selected samples that were confirmed of P. falciparum mono-infections.ConclusionThese findings show limited threat of pfhrp2/3 gene deletions in the survey areas suggesting that HRP2 RDTs are still useful diagnostic tools for surveillance and diagnosis of P. falciparum malaria infections in symptomatic patients in this setting. Periodic genomic surveillance is warranted to monitor the frequency and trend of gene deletions and its effect on RDTs.

Plasmodium falciparum with pfhrp2/3 Deletion Not Detected in a 2018-2021 Malaria Longitudinal Cohort Study in Kinshasa Province, Democratic Republic of the Congo.

Histidine-rich protein 2- (HRP2-) based rapid diagnostic tests (RDTs) are widely used to detect Plasmodium falciparum in sub-Saharan Africa. Reports of parasites with pfhrp2 and/or pfhrp3 (pfhrp2/3) gene deletions in Africa raise concerns about the long-term viability of HRP2-based RDTs. We evaluated changes in pfhrp2/3 deletion prevalence over time using a 2018-2021 longitudinal study of 1,635 enrolled individuals in Kinshasa Province, Democratic Republic of the Congo (DRC). Samples collected during biannual household visits with ≥ 100 parasites/µL by quantitative real-time polymerase chain reaction were genotyped using a multiplex real-time PCR assay. Among 2,726 P. falciparum PCR-positive samples collected from 993 participants during the study period, 1,267 (46.5%) were genotyped. No pfhrp2/3 deletions or mixed pfhrp2/3-intact and -deleted infections were identified in our study. Pfhrp2/3-deleted parasites were not detected in Kinshasa Province; ongoing use of HRP2-based RDTs is appropriate.

A Diagnostic Accuracy Study to Evaluate Standard Rapid Diagnostic Test (RDT) Alone to Safely Rule Out Imported Malaria in Children Presenting to UK Emergency Departments.

Microscopy is the gold standard for malaria diagnosis but is dependent on trained personnel. Rapid diagnostic tests (RDTs) form the mainstay of diagnosis in endemic areas without access to high-quality microscopy. We aimed to evaluate whether RDT alone could rule out imported malaria in children presenting to UK emergency departments (EDs). UK-based, multi-center, retrospective, diagnostic accuracy study. Included: any child <16 years presenting to ED with history of fever and travel to a malaria-endemic country, between 01/01/2016 and 31/12/2017. Diagnosis: microscopy for malarial parasites (clinical reference standard) and RDT (index test). UK Health Research Authority approval: 20/HRA/1341. There were 47 cases of malaria out of 1,414 eligible cases (prevalence 3.3%) in a cohort of children whose median age was 4 years (IQR 2-9), of whom 43% were female. Cases of Plasmodium falciparum totaled 36 (77%, prevalence 2.5%). The sensitivity of RDT alone to detect malaria infection due to any Plasmodium species was 93.6% (95% CI 82.5-98.7%), specificity 99.4% (95% CI 98.9-99.7%), positive predictive value 84.6% (95% CI 71.9-93.1%) and negative predictive value 99.8% (95% CI 99.4-100.0%). Sensitivity of RDT to detect P. falciparum infection was 100% (90.3-100%), specificity 98.8% (98.1-99.3%), positive predictive value 69.2% (54.9-81.2%, n = 46/52) and negative predictive value 100% (99.7-100%, n = 1,362/1,362). RDTs were 100% sensitive in detecting P. falciparum malaria. However, lower sensitivity for other malaria species and the rise of pfhrp2 and pfhrp3 (pfhrp2/3) gene deletions in the P. falciparum parasite mandate the continued use of microscopy for diagnosing malaria.

Plasmodium falciparum pfhrp2 and pfhrp3 Gene Deletions in Malaria-Hyperendemic Region, South Sudan

Pfhrp2 and pfhrp3 gene deletions threaten the use of Plasmodium falciparum malaria rapid diagnostic tests globally. In South Sudan, deletion frequencies were 15.6% for pfhrp2, 20.0% for pfhrp3, and 7.5% for double deletions. Deletions were approximately twice as prevalent in monoclonal infections than in polyclonal infections.

Geographical distribution and genetic characterization of pfhrp2 negative Plasmodium falciparum parasites in the Peruvian Amazon.

Malaria rapid diagnostic tests (RDTs) have been evaluated in the Peruvian Amazon region and their performance has been variable. This region is known for being the first with documented evidence of wild Plasmodium falciparum parasites lacking pfhrp2 and pfhrp3 genes, leading to false-positive results with HRP2-based RDTs. In our attempt to further characterize the deletion pattern of these genes and their evolutionary relationship, 93 P. falciparum samples, collected in different communities from the Peruvian Amazon region between 2009 and 2010, were analyzed in this study. Genomic DNA was used to amplify 18S rRNA, pfmsp2 and pfglurp to confirm the diagnosis and DNA quality, respectively; pfhrp2, pfhrp3, and their flanking genes were amplified by PCR to assess the pattern of the gene deletions. In addition, microsatellite analysis were performed using seven neutral microsatellites (MS) and five microsatellite loci flanking pfhrp2. The data showed the absence of pfhrp3 gene in 53.76% (50/93) of the samples, reflecting a higher frequency than the proportion of pfhrp2 gene deletions (33.33%; 31/93). Among the flanking genes, the highest frequency of deletion was observed in the PF3D7_0831900 gene (78.49%; 73/93) for pfhrp2. MS marker analysis showed the presence of 8 P. falciparum lineages. The lineage Bv1 was the most prevalent among parasites lacking pfhrp2 and pfhrp3 genes. Additionally, using MS flanking pfhrp2 gene, the haplotypes α and δ were found to be the most abundant in this region. This study confirms the presence in this area of field isolates with deletions in either pfhrp2, pfhrp3, or both genes, along with their respective flanking regions. Our data suggest that some pfhrp2/pfhrp3 deletion haplotypes, in special the lineage Bv1, are widely dispersed within the Peruvian Amazon. The persistence of these haplotypes ensures a proportion of P.falciparum parasites lacking the pfhrp2/pfhrp3 genes in this area, which ultimately leads to false-negative results on PfHRP2-detecting malaria RDTs. However, additional studies are needed to not only confirm this hypothesis but also to further delineate the origin and genetic basis for the pfhrp2- and pfhrp3 gene deletions in wild P. falciparum parasites.

Spatiotemporal dynamics of Plasmodium falciparum histidine-rich protein 2 and 3 deletions in Peru

Peru was the first country where pfhrp2 and pfhrp3 gene deletions were detected despite the fact that rapid diagnostics tests are not commonly used for confirmatory malaria diagnosis. This context provides a unique scenario to study the dynamics of pfhrp2 and pfhrp3 gene deletions without apparent RDTs selection pressure. In this study we characterized the presence of pfhrp2 and pfhrp3 genes on 325 P. falciparum samples collected in Iquitos and surrounding communities between 2011 and 2018 in order to understand the dynamics of gene deletion prevalence, potential associations with clinical symptomatology and parasite genetic background. P. falciparum presence was confirmed by microscopy and PCR of 18 s rRNA, pfmsp1 and pfmsp2. Gene deletions were assessed by amplification of exon1 and exon2 of pfhrp2 and pfhrp3 using gene specific PCRs. Confirmation of absence of HRP2 expression was assessed by ELISA of HRP2 and pLDH. Genotyping of 254 samples were performed using a panel of seven neutral microsatellite markers. Overall, pfhrp2 and pfhrp3 dual gene deletions were detected in 67% (217/324) parasite samples. Concordance between pfhrp2 deletion and negligible HRP2 protein levels was observed (Cohen's Kappa = 0.842). Prevalence of gene deletions was heterogeneous across study sites (adjusted p < 0.005) but there is an overall tendency towards increase through time in the prevalence of dual pfhrp2/3-deleted parasites between 2011 (14.3%) and 2016 (88.39%) stabilizing around 65% in 2018. Dual deletions increase was associated with dominance of a single new parasite haplotype (H8) which rapidly spread to all study sites during the 8 study years. Interestingly, participants infected with dual pfhrp2/3-deleted parasites had a significantly lower parasitemias than those without gene deletions in this cohort. Our study showed the increase of pfhrp2/3 deletions in the absence of RDTs pressure and a clonal replacement of circulating lines in the Peruvian Amazon basin. These results suggest that other factors linked to the pfhrp2/3 deletion provide a selective advantage over non-deleted strains and highlight the need for additional studies and continuing surveillance.

Investigation of Plasmodium falciparum pfhrp2 and pfhrp3 gene deletions and performance of a rapid diagnostic test for identifying asymptomatic malaria infection in northern Ethiopia, 2015

BackgroundRapid diagnostic tests (RDTs) are widely used for malaria diagnosis of both symptomatic and asymptomatic infections. Although RDTs are a reliable and practical diagnostic tool, the sensitivity of histidine-rich protein 2 (HRP2)-based RDTs can be reduced if pfhrp2 or pfhrp3 (pfhrp2/3) gene deletions exist in the Plasmodium falciparum parasite population. This study evaluated dried blood spot (DBS) samples collected from a national household survey to investigate the presence of pfhrp2/3 deletions and the performance of the RDT used in the cross-sectional survey in a low transmission setting.MethodsThe 2015 Ethiopia Malaria Indicator Survey tested household members by RDT and collected DBS samples. DBS (n = 2648) from three regions in northern Ethiopia were tested by multiplex bead-based antigen detection assay after completion of the survey. The multiplex assay detected pan-Plasmodium lactate dehydrogenase (LDH), pAldolase, and HRP2 antigens in samples. Samples suspected for pfhrp2/3 gene deletions (pLDH and/or pAldolase positive but low or absent HRP2) were further investigated by molecular assays for gene deletions. Antigen results were also compared to each individual’s RDT results. Dose–response logistic regression models were fit to estimate RDT level of detection (LOD) antigen concentrations at which 50, 75, 90, and 95% of the RDTs returned a positive result during this survey.ResultsOut of 2,648 samples assayed, 29 were positive for pLDH or pAldolase antigens but low or absent for HRP2 signal, and 15 of these samples (51.7%) were successfully genotyped for pfhrp2/3. Of these 15 P. falciparum infections, eight showed single deletions in pfhrp3, one showed a single pfhrp2 deletion, and six were pfhrp2/3 double-deletions. Six pfhrp2 deletions were observed in Tigray and one in Amhara. Twenty-five were positive for HRP2 by the survey RDT while the more sensitive bead assay detected 30 HRP2-positive samples. A lower concentration of HRP2 antigen generated a positive test result by RDT compared to pLDH (95% LOD: 16.9 ng/mL vs. 319.2 ng/mL, respectively).ConclusionsThere is evidence of dual pfhrp2/3 gene deletions in the Tigray and Amhara regions of Ethiopia in 2015. As the prevalence of malaria was very low (< 2%), it is difficult to make strong conclusions on RDT performance, but these results challenge the utility of biomarkers in household surveys in very low transmission settings.

Plasmodium falciparum histidine-rich protein 2 and 3 genes deletion in global settings (2010\u20132021): a systematic review and meta-analysis

BackgroundThe usefulness of histidine-rich protein-2/3 (HRP2/3)-based rapid diagnostic tests of malaria due to Plasmodium falciparum has been threatened by the appearance of mutant PfHRP2/3 genes. This study was undertaken to determine the global pooled estimates of PfHRP2/3gene deletions.MethodsRelevant publications were identified from electronic databases such as; PubMed, EMBASE, and MEDLINE online. Besides, all the relevant literatures were retrieved through Google and Google Scholar. STATA software was used for data analysis. The pooled estimates were calculated using random effect model. The summary estimates were presented using forest plots and tables.ResultsA total of 27 studies were included in the systematic review. However, only 24 and 17 studies were included for PfHRP2 and 3 gene deletion meta-analysis, respectively. The prevalence of PfHRP2 gene deletion across the individual studies ranged from the highest 100% to the lowest 0%. However, the meta-analysis result showed that the global pooled prevalence of PfHRP2 and PfHRP3 gene deletions were 21.30% and 34.50%, respectively. The pooled proportion of PfHRP2 gene deletion among false negative PfHRP2-based RDTs results was found to be 41.10%. The gene deletion status was higher in South America and followed by Africa. The pooled estimate of PfHRP2 gene deletion among studies, which did not follow the WHO PfHRP2/3 gene deletion analysis protocol was higher than their counter parts (21.3% vs 10.5%).ConclusionsThis review showed that there is a high pooled prevalence of PfHRP2/3 gene deletions in Plasmodium falciparum confirmed isolates and also a high proportion of their deletions among false-negative malaria cases using PfHRP2-based RDT results. Hence, malaria diagnosis based on PfHRP2-based rapid tests seems to be less sensitive and warrants further evaluation of PfHRP2/3 gene deletions.

Analysis of nucleic acids extracted from rapid diagnostic tests reveals a significant proportion of false positive test results associated with recent malaria treatment

BackgroundSurveillance programmes often use malaria rapid diagnostic tests (RDTs) to determine the proportion of the population carrying parasites in their peripheral blood to assess the malaria transmission intensity. Despite an increasing number of reports on false-negative and false-positive RDT results, there is a lack of systematic quality control activities for RDTs deployed in malaria surveillance programmes.MethodsThe diagnostic performance of field-deployed RDTs used for malaria surveys was assessed by retrospective molecular analysis of the blood retained on the tests.ResultsOf the 2865 RDTs that were collected in 2018 on Bioko Island and analysed in this study, 4.7% had a false-negative result. These false-negative RDTs were associated with low parasite density infections. In 16.6% of analysed samples, masked pfhrp2 and pfhrp3 gene deletions were identified, in which at least one Plasmodium falciparum strain carried a gene deletion. Among all positive RDTs analysed, 28.4% were tested negative by qPCR and therefore considered to be false-positive. Analysing the questionnaire data collected from the participants, this high proportion of false-positive RDTs could be explained by P. falciparum histidine rich protein 2 (PfHRP2) antigen persistence after recent malaria treatment.ConclusionMalaria surveillance depending solely on RDTs needs well-integrated quality control procedures to assess the extent and impact of reduced sensitivity and specificity of RDTs on malaria control programmes.

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

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.

Accuracy of SD Malaria Ag P.f/Pan\xae as a rapid diagnostic test in French Amazonia

BackgroundFrench Guiana (FG) is a French overseas territory where malaria is endemic. The current incidence rate is 0.74‰ inhabitants, and Plasmodium vivax is widely predominating even though Plasmodium falciparum is still present due to imported cases mainly from Africa. In FG, rapid diagnostic test (SD Malaria Ag P.f/Pan®) is based on the detection of pan-pLDH, PfHRP2, and PfHRP3 antigens, while in South America, the share of deletion of PfHRP2 gene is significantly increasing. Accordingly, the study questions the reliability of RDTs in the Amazonian context.MethodsThe study is retrospective. It is conducted over 4 years and analysed 12,880 rapid diagnostic tests (RDTs) compared to concomitant Blood Film Tests (BFTs) sampled for malaria diagnosis.ResultsThe global assessment of the accuracy of SD Malaria Ag P.f/Pan® in the diagnostic of malaria shows both Positive and Negative Predictive Values (PPV and NPV) higher than 95%, except for PPV in the diagnosis of malaria to P. falciparum (88%). Overall, the concordance rate between RDT and BFT (positive/positive; negative/negative) was 99.5%. The PPV of the RDT in the follow-up of patients diagnosed with P. falciparum was the lowest during the first 28 days. The PPV of the RDT in the follow-up of patients diagnosed with P. vivax was the lowest during the first 21 days. The global sensitivity of SD Malaria Ag P.f/Pan® test was, on average, 96% (88.2–100) for P. falciparum and 93% (90.6–94.2) for P. vivax. The global specificity was 99.8% (99.5–100) for all included species.ConclusionSD Malaria Ag P.f/Pan® is a reliable rapid test used for the first-line diagnosis in remote healthcare centres. The test results should be interpreted in the light of patient’s recent medical history and the date of arrival to FG.

Prevalence of Pfhrp2 and Pfhrp3 Gene Deletions in Plasmodium Falciparum Isolates and their Performance of Hrp2 Based Malaria Rapid Diagnostic Tests in Three Districts of Ghana

Malaria rapid diagnostic tests (MRDTs) are important for malaria disease management. However, the performance of the RDTs is affected when the targeted antigens in the parasite have a variation or are altogether absent. The most common parasite target antigen in RDTs, Plasmodium falciparum histidine-rich protein 2 (HRP2), has been reported to be absent in some P. falciparum parasites. 371 patient samples, from Akuapem North (58.5%), Atiwa East (21.3%), and from New Juaben (20.2%), were used in the study. PCR provided the highest number, 14.8% (55/371), of positive detections for falciparum infections. Microscopy detected parasites in 20/261 (7.7%) samples, and the minimum parasite density by microscopy was 430 parasites/µL. Out of the 371 samples, 27 (7.3%) were positive by RDT. The highest RDT positivity rate, 13.3% (10/75), was observed at New Juaben. False-negative RDT results were obtained in 43/55 (78.2%) of the negative branded RDT kits. Only two microscopies positive sample were RDT positive. Using 18SrDNA PCR, 55 (14.8%) samples were positive for P. falciparum. In Akuapem North, 79.2 % (19/24) of the PCR positive samples had P. falciparum parasites that lacked exon 2 of PFHRP2. An overall RDT positivity rate of 7.3% (27/371) and false-negative rate of 78.2% (43/55) were observed for the study sites. Plasmodium falciparum parasite populations with deletions of the PFHRP2 and PFHRP3 genes are present in Ghana. There is an urgent need to investigate the prevalence and geographic distribution of these parasites. Keywords: Histidine Rich Protien (HRP), Malaria Rapid Diagnostic Test (MRDT), Malaria, Rapid Diagnostic Test (RDT).

Impact of Plasmodium falciparum pfhrp2 and pfhrp3 gene deletions on malaria control worldwide: a systematic review and meta-analysis

BackgroundDeletion of pfhrp2 and/or pfhrp3 genes cause false negatives in malaria rapid diagnostic test (RDT) and threating malaria control strategies. This systematic review aims to assess the main methodological aspects in the study of pfhrp2 and pfhrp3 gene deletions and its global epidemiological status, with special focus on their distribution in Africa; and its possible impact in RDT.MethodsThe systematic review was conducted by examining the principal issues of study design and methodological workflow of studies addressing pfhrp2 deletion. Meta-analysis was applied to represent reported prevalences of pfhrp2 and pfhrp3 single and double deletion in the World Health Organization (WHO) region. Pooled-prevalence of deletions was calculated using DerSimonnian-Laird random effect model. Then, in-deep analysis focused on Africa was performed to assess possible variables related with these deletions. Finally, the impact of these deletions in RDT results was analysed combining reported information about RDT sensitivity and deletion prevalences.Results49 articles were included for the systematic review and 37 for the meta-analysis, 13 of them placed in Africa. Study design differs significantly, especially in terms of population sample and information reported, resulting in high heterogeneity between studies that difficulties comparisons and merged conclusions. Reported prevalences vary widely in all the WHO regions, significantly higher deletion were reported in South-Central America, following by Africa and Asia. Pfhrp3 deletion is more prevalent (43% in South-Central America; 3% in Africa; and 1% in Asia) than pfhrp2 deletion (18% in South-Central America; 4% in Africa; and 3% in Asia) worldwide. In Africa, there were not found differences in deletion prevalence by geographical or population origin of samples. The prevalence of deletion among false negatives ranged from 0 to 100% in Africa, but in Asia and South-Central America was only up to 90% and 48%, respectively, showing substantial relation between deletions and false negatives.ConclusionThe concerning prevalence of pfhrp2, pfhrp3 and pfhrp2/3 gene deletions, as its possible implications in malaria control, highlights the importance of regular and systematic surveillance of these deletions. This review has also outlined that a standardized methodology could play a key role to ensure comparability between studies to get global conclusions.

Investigation of HRPF2 Gene Deletion in Plasmodium Falciparum in Northwestern Nigeria

Abstract Malaria Rapid Diagnostic Tests (RDTs) plays an important role in malaria management and control. The Pf HRP2 based RDT kit is the most widely used RDT for malaria diagnosis in Nigeria but is affected by the deletion of HRP2 gene in Plasmodium falciparum parasites. Therefore, identifying the prevalence and distribution of Plasmodium falciparum parasites with deleted Pf HRP2 is important for malaria control. Pf HRP2 gene deletion was assessed in this study by first carrying out Giemsa stained thick blood film microscopy and Pf HRP2 RDT strip test. The samples were further analyzed for molecular examination by PCR assay for multiple single–copy genes (Pf Cox3, Pf HRP2, Pf HRP3 and Pf Beta tubulin). This study found the existence of eight (8) Plasmodium falciparum isolates lacking the HRP2 gene in the samples analyzed, this necessitates the need to develop a unique RDT Kit targeting other housekeeping genes unique for Plasmodium falciparum with far greater sensitivity than the current ones as to reduce the chances of false negative RDT result as well as developing unique RDT Kits targeting both PfHRP2 and PfHRP3 genes concomitantly in order to reduce the chances of having a false positive RDT results.