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Proportion of different detection methods positive for malaria. Histidine-rich protein 2 (HRP2)based rapid diagnostic tests (RDTs) generated more positive samples compared to other methods. PCR methods produced 13-20% more positive results compared to microscopy.
Source publication
Deletions of the Plasmodium falciparum hrp2 and hrp3 genes can affect the performance of HRP2-based malaria rapid diagnostic tests (RDTs). Such deletions have been reported from South America, India and Eritrea. Whether these parasites are widespread in East Africa is unknown. A total of 274 samples from asymptomatic children in Mbita, western Keny...
Context in source publication
Context 1
... negative. Of the 196 microscopic negative samples, 93 (52%) were RDT positives). The minimum parasite density reported by microscopy was 80 parasites/µL, suggesting that this was the threshold for detection by the field microscopist. Using 18SrDNA PCR and qPCR assays, 131 (47.8%) and 114 (41.6%), respec- tively, were positive for P. falciparum ( Fig. ...
Citations
... Nonetheless, in the context of malaria control and elimination, even low-density infections can be infectious to mosquitoes, maintaining transmission in the population, and are thus important to identify and treat [13,38,56]. While majority of subpatent infections are due to low density parasitaemia; in situations where HRP2-based RDTs are used, deletion of the histidine-rich protein 2 and histidine-rich protein 3 genes (pfhrp2 and pfhrp3) has also been implicated as a reason for the failure of RDTs to detect P. falciparum even with higher density infections [57][58][59]. It is important to continue monitoring for the occurrence and spread of hrp2/3 gene deleted parasites. ...
Background
Subpatent Plasmodium falciparum infections, defined as infections with parasite density below the detection limit of routine malaria diagnostic tests, contribute to infectious reservoirs, sustain transmission, and cause the failure of elimination strategies in target areas. This study assessed the prevalence of subpatent P. falciparum infections and associated risk factors in 14 regions of Mainland Tanzania.
Methods
The study used samples randomly selected from RDT-negative dried blood spots (DBS) (n = 2685/10,101) collected in 2021 at 100 health facilities across 10 regions of Mainland Tanzania, and four communities in four additional regions. The regions were selected from four transmission strata; high (five regions), moderate (three regions), low (three regions), and very low (three regions). DNA was extracted by Tween-Chelex method, and the Pf18S rRNA gene was amplified by quantitative polymerase chain reaction (qPCR). Logistic regression analysis was used to assess the associations between age groups, sex, fever status, and transmission strata with subpatent infection status, while linear regression analysis was used to assess the association between these factors and subpatent parasite density.
Results
Of the selected samples, 525/2685 (19.6%) were positive by qPCR for P. falciparum, and the positivity rates varied across different regions. Under-fives (aOR: 1.4, 95% CI 1.04–1.88; p < 0.05) from health facilities had higher odds of subpatent infections compared to other groups, while those from community surveys (aOR: 0.33, 95% CI 0.15–0.72; p = 0.005) had lower odds. Participants from very low transmission stratum had significantly lower odds of subpatent infection compared to those from high transmission stratum (aOR = 0.53, 95% CI = 0.37–0.78; p < 0.01). The log-transformed median parasite density (interquartile range) was 6.9 (5.8–8.5) parasites/µL, with significantly higher parasitaemia in the low transmission stratum compared to a very low one (11.4 vs 7.0 parasites/µL, p < 0.001).
Conclusion
Even in very low transmission settings, the prevalence of subpatent infections was 13%, and in low transmission settings it was even higher at 29.4%, suggesting a substantial reservoir that is likely to perpetuate transmission but can be missed by routine malaria case management strategies. Thus, control and elimination programmes may benefit from adoption of more sensitive detection methods to ensure that a higher proportion of subpatent infections are detected.
... Most RDTs detect two antigens: Plasmodium lactate dehydrogenase (pLDH), which is present in all Plasmodium spp. that infect humans, and P. falciparum histidine-rich protein 2 (PfHRP2), which is present in P. falciparum only [7,8]. PfHRP2-based RDTs primarily detect the product of the pfhrp2 gene on chromosome 8 but can also crossreact with the product of the pfhrp3 gene on chromosome 13, due to extensive sequence homology between the two genes [9,10]. ...
... This has led to a preference for PfHRP2-based RDTs, which are reported to be more sensitive and heatstable than RDTs detecting other malaria antigens [7,21]. However, P. falciparum with pfhrp2 and/or pfhrp3 deletions has been reported in a number of countries, including Eritrea [13,22] and Kenya [9,23,24]. Most of the reports are based on samples obtained from symptomatic patients seeking care at health facilities. ...
... Infections with P. falciparum lacking pfhrp2 and/or pfhrp3 were mostly asymptomatic and submicroscopic, highlighting the potential for these parasites to spread undetected. A previous study [9] reported the presence of P. falciparum lacking either pfhrp2 or pfhrp3 in Kenya in 2014. We confirm previous findings and report the presence of P. falciparum lacking both pfhrp2 and pfhrp3 in asymptomatic infections from as early as 2018. ...
Malaria rapid diagnostic tests (RDTs) targeting the Plasmodium falciparum histidine-rich protein 2 ( Pf HRP2) are widely used to diagnose P. falciparum infection. However, reports of P. falciparum strains lacking Pf HRP2 and the structurally similar Pf HRP3 have raised concerns about the utility and reliability of Pf HRP2-based RDTs. This study investigated the presence of P. falciparum with pfhrp2 and/or pfhrp3 gene deletions among infected residents in the Lake Victoria region, Kenya. Four cross-sectional malaria, surveys were conducted in four sites (Suba South, Mfangano, Kibuogi, and Ngodhe) from September 2018 to January 2020. P. falciparum infections were detected using a Pf HRP2-based RDT, microscopy, and PCR on 9120 finger-prick blood samples. Samples negative by RDT but positive by PCR were selected for PCR amplification of pfmsp1 and pfmsp2 to confirm the quality and quantity of P. falciparum DNA. Samples positive for both pfmsp1 and pfmsp2 were included for detection of deletions of exons 1 and 2 in pfhrp2 and pfhrp3 PCR. The multiplicity of infection (MOI) was determined as the higher allele count between pfmsp1 and pfmsp2 . Logistic regression analysis was performed to analyze the association between pfhrp2 and/or pfhrp3 deletions and demographic and infection variables. Of the 445 RDT-negative and PCR-positive samples, 125 (28.1%) were analyzed for pfhrp2 and pfhrp3 deletions. Single pfhrp2 deletion, single pfhrp3 deletion, and pfhrp2/3 double deletions were detected in 13 (10.4%), 19 (15.2%), and 36 (28.8%) samples, respectively. Single pfhrp2 deletion was found in all sites while single pfhrp3 deletion was found in all sites except Kibuogi. The majority of samples with pfhrp2 and/or pfhrp3 deletions were submicroscopic (73.5%), asymptomatic (80.9%), and monoclonal (80.9%). Polyclonal infection was significantly ( p = 0.022) associated with a lower odds of pfhrp2/3 double deletion, suggesting detection of intact pfhrp2/3 in mixed infections. We report the presence of P. falciparum with pfhrp2/pfhrp3 double deletions among asymptomatic and submicroscopic infections in Kenya. Our findings highlight the need for active monitoring of pfhrp2 and pfhrp3 deletions at the community level to improve malaria detection and control in the region.
... 2 Microscopy has long been the gold standard for malaria diagnosis in low-resource settings, including rural India, as it is the most cost-effective option and allows for the direct detection of parasite density and stage by species. 3 The evaluation of stained smears using light microscopy, however, requires technical expertise and the thorough maintenance of infrastructure, with proper quality control. 4 Given the short timeline to diagnosis and the absence of need for additional equipment or expertise, rapid diagnostic tests (RDTs) have become an increasingly used diagnostic method. ...
Diagnostic escape via Plasmodium falciparum ( P. falciparum ) histidine-rich protein 2 ( pfhrp2 ) gene deletions is a major potential hurdle for global malaria elimination efforts. We investigated the prevalence of pfhrp2 gene deletions in 15 malaria-endemic villages in the state of Odisha, India, and modeled their impact on an ongoing in-country malaria intervention program. We found that 61.6% of subpatent P. falciparum infections (i.e., rapid diagnostic test [RDT]-negative and positive by polymerase chain reaction [PCR]) had pfhrp2 gene deletions, which were predominantly located in the exon 2 region (96.2%) and largely identified in samples from febrile individuals (82.6%). DNA sequencing and protein diversity features were characterized in a subset of samples from individuals with subpatent infections carrying intact pfhrp2 exon 2 loci. Our analyses revealed novel amino acid repeat motifs (231–293 amino acids), and these variant repeat sequences differed from those of RDT+/PCR+ samples. We also evaluated the state-sponsored mass screening and treatment intervention in the context of pfhrp2 gene deletions. We found that mass screening and treatment conducted alongside additional interventions (e.g., long-lasting insecticidal net distribution, indoor residual spraying) reduced the relative risk of infection for both P. falciparum parasites harboring a pfhrp2 deletion (adjusted relative risk ratio [aRRR] = 0.3; 95% CI = 0.1–1.0) and P. falciparum parasites with intact pfhrp2 genes (aRRR = 0.4; 95% CI = 0.2–1.1) when compared with the use of mass screening and treatment by RDT alone. Combined, our findings highlight the need for alternative diagnostic targets and tools as India moves toward its goal of malaria elimination by 2030.
... In this regard, we are not sure of the origin of deletion of Pfhrp2 gene in Ethiopia in our study. Given that both HRP2 and HRP3 antigens are detected in peripheral circulation of falciparum infected patients, and have structural motif similarity [12], RDTs positivity was reported in the presence of only Pfhrp3 regardless of Pfhrp2 gene [36,37]. ...
Background: Rapid diagnostic tests (RDTs) targeting pfhistidine-rich protein 2 (Pfhrp2) are widely used for diagnosis of Plasmodium falciparum infections in resource-limited malaria endemic countries. However, test results are affected by deletions of the Pfhrp2, Pfhrp3, and flanking genes and associated negative results from rapid diagnostic devices were previously reported. Therefore, the aim of this study was to reveal the existing genetic profile of Pfhrp2 and Pfhrp3 genes of P. falciparum-infected patients in northwestern Ethiopia.
Methods: A total number of 302 blood samples were collected from children at Chilga (Aykel, Negade Bahir), and Sanja health centers in northwestern Ethiopia. Thirty-three (10.9%) samples tested positive for P. falciparum malaria. The Pfhrp2, Pfhrp3, and flanking genes (MAL7P1_228 and MAL7P1_230 for Pfhrp2, and MAL13P1_475 and MAL13P1_485 for Pfhrp3) were amplified using standard nested-PCR.
Results: Pfhrp2 and both of its flanking genes were found to be present in 12 (36.4%) out of the 33 samples. Twenty-one (63.6%) samples tested negative for the Pfhrp2 gene and 19 samples (57.6%) tested positive for at least one of the flanking genes. Five (15.2%) samples gave positive results for the Pfhrp3 gene and both of its flanking genes, whereas 16 (48.5%) tested negative for all three.
Conclusions: Our study provides widespread deletions in the Pfhrp2 and Pfhrp3 genes in Ethiopia, thereby confirming anecdotal reports of diagnostic failure with Pfhrp2-based RDTs in the region. The implications of our finding for the current diagnostic paradigm, which relies on the detection of P. falciparum by Pfhrp2-based RDTs in remote areas, may need rethinking.
... It is, therefore, more plausible the hrp2 gene was absent or perhaps modified in the P. falciparum. Deletion and/or microvariations of the hrp2 gene in P. falciparum has been reported in some malaria-endemic countries in southern America, Asia and Africa including Ghana (Addai-Mensah et al., 2020;Amoah et al., 2020Amoah et al., , 2016Beshir et al., 2017;Bharti et al., 2016;Fontecha et al., 2018;Murillo Solano et al., 2015). The extent of the spread and impact of these hrp2 mutations on malaria control and management are, however, yet to be fully appraised in the country. ...
Light microscopy is the gold-standard for the diagnosis of malaria, but RDT and PCR assays are also used. RDTs widely used in Ghana are based on the hrp2 gene product of P. falciparum. Our study investigated the reliability of P. falciparum-based RDTs for malaria diagnosis in northern Ghana. Blood samples of 267 light microscopy-diagnosed malaria patients were tested by P. falciparum-specific RDT and semi-nested PCR. Diagnostic performance of the RDT assay was evaluated using PCR as the referenced standard. Overall, the tests revealed infection as follows: RDT, 158/267 (59.2%) and a 20.9% RDT false-positive rate, and PCR, 160/267 (59.9%) and a 21.9% RDT false-negative rate. The sensitivity and specificity of the RDT kit were 78.12% (95% CI = 70.91% to 84.27%) and 69.16% (95% CI = 59.50% to 77.73%), respectively. The PPV and NPV of the RDT assay were found to be 79.11% (95% CI = 73.82% to 83.58%) and 67.89% (95% CI = 60.58% to 74.42%) respectively. The results also indicate that close to 40% of microscopy-diagnosed malaria may be due to non-falciparum malaria species. Our results suggest an unexpectedly high prevalence of non-falciparum malaria parasites in northern Ghana. The high rates of false-negative and false-positive RDT results in this study raise important questions about the reliability of the used P. falciparum-based RDTs in the diagnosis of malaria in Ghana.
... This study had a potential limitation. There is the possibility of false negative results due to HRP2 gene deletions [22][23][24]. However, a previous study in Ghana, found a low prevalence of false negative malaria, indicating RDTs remain effective in diagnosis of malaria [25]. ...
Background
Malaria remains a major public health problem, especially among children in sub-Saharan Africa. Knowledge of malaria parasite prevalence informs targeted interventions and helps to monitor the effectiveness of those interventions. This study aimed to determine prevalence and factors associated with malaria in children aged 6 months to 10 years in the Greater Accra Region of Ghana.
Methods
A community-based cross-sectional study was conducted among 8,741 children aged 6–59 months and 8,292 children aged 5–10 years in all 29 districts of the Greater Accra Region of Ghana in October 2020. Systematic random sampling was used to select communities, households and study participants. A structured questionnaire was used to collect data from caregivers. Rapid diagnostic test kits were used to determine the presence of malaria parasites in blood samples collected by fingerprick. Factors associated with malaria RDT-positivity were determined using multivariate logistic regression analysis.
Results
Of 8727 children aged 6–59 months and 8279 aged 5–10 years from whom blood samples were obtained, positive results were obtained for 289 (3.3%; 95% CI 3.0–3.7) and 406 (4.9%; 95% CI 4.5–5.4) respectively. Malaria parasite prevalence in the districts ranged from 0.9 to 10.7% and 1.4–15.0% in children aged 6–59 months and 5–10 years respectively. Factors associated with increased odds of malaria included higher age (AOR = 1.43; 95% CI 1.14–1.71), and living in households without nets on the windows (AOR 1.64; 95% CI 1.10–2.45). On the other hand, living in households located in urban communities was associated with a lower risk of malaria (AOR 0.56; 95% CI 0.40–0.78).
Conclusion
The average prevalence of malaria in the Greater Accra Region is low compared with other regions. However, there are potential hotspots that need to be targeted with appropriate interventions to accelerate the drive towards malaria elimination.
... Consequently, RDTs have limited utility in assessing treatment efficacy, monitoring progression, and assessing their impact on transmission [5]. In addition, recent reports stated that the RDT cause false negatives in Plasmodium falciparum due to deletions of the gene encoding target histidine rich protein 2/3 (HRP2/3) [6][7][8]. Plasmodium lactate dehydrogenase (pLDH) detecting RDTs are also available, however they have lesser sensitivity than HRP2-detecting RDTs [9]. ...
Background
Malaria control depends primarily on rapid and accurate diagnosis followed by successful treatment. Light microscopy is still used as a gold standard method for the diagnosis of malaria. The Sysmex hematology analyzer is a novel method for malaria detection. Therefore, the aim of this review was to investigate the diagnostic accuracy of the Sysmex hematology analyzer for malaria diagnosis.
Methods
Electronic databases like PubMed, PubMed Central, Science Direct databases, Google Scholar, and Scopus were used to find relevant articles from April to June 14, 2023. The studies’ methodological quality was assessed using the Quality Assessment of Diagnostic Accuracy Studies-2 tool. Using Review Manager 5.4.1, the estimates of sensitivity and specificity, as well as their 95% confidence intervals, were shown in forest plots. Midas software in Stata 14.0 was utilized to calculate the summary estimates of sensitivity, specificity, positive likelihood ratio, negative likelihood ratio, and diagnostic odds ratio. Heterogeneity was assessed by using I² statistics. In addition, publication bias was assessed using a funnel plot and Deeks’ test. Sub-group and meta- regression analysis were also performed.
Results
A total of 15 studies were assessed for diagnostic accuracy. The sensitivity and specificity of Sysmex hematology analyzer for studies ranged from 46% to 100% and 81% to 100%, respectively. The summary estimate of sensitivity and specificity of Sysmex hematology analyzer were 95% (95% CI: 85%-99%) and 99% (95% CI: 97%-100%), respectively. It had excellent diagnostic accuracy. There were significant heterogeneity among the studies included in this meta-analysis. The summary estimate of sensitivity and specificity of Sysmex hematology analyzer using polymerase chain reaction as the gold standard was 97.6% (95% CI: 83.2, 99.7) and 99.4% (98.5, 99.8), respectively.
Conclusion
In this review, Sysmex hematology analyzer had excellent diagnostic accuracy. Therefore, it could be used as an alternate diagnostic tool for malaria diagnosis in the hospital and health center.
Trial registration
Systematic review registration PROSPERO (2023: CRD42023427713). https://www.crd.york.ac.uk/prospero/display_record.php?ID=CRD42023427713.
... Rondônia, and Bolivia (68%) [14]. Few studies in South America and Southeast Asia have observed a high prevalence of parasites with the genes pfhrp2 deleted and pfhrp3 present when compared to other genotypes: pfhrp2+/pfhrp3and pfhrp2-/pfhrp3- [33,39]. In the Amazon Region, the prevalence of pfhrpr3parasites is higher than those of pfhrp2- [9,14]. ...
Several countries are reporting natural populations of P. falciparum with deletions in the pfhrp2/3 genes that can lead to false-negative results in rapid diagnostic tests. To investigate the prevalence of deletion in the pfhrp2/3 genes in the Rio Negro basin in the Brazilian Amazon and identify whether there is clinical differentiation between individuals infected by these parasites, clinical samples collected from 2003 to 2016 were analyzed from symptomatic and asymptomatic P. falciparum-infected individuals. The molecular deletion of pfhrp2 and pfhrp3 genes was evaluated using the protocols recommended by the WHO. From 82 samples used, 28 (34.2%) had a single deletion in pfhrp2, 19 (23.2%) had a single deletion in pfhrp3, 15 (18.3%) had a double deletion (pfhrp2/3), and 20 (24.4%) did not have a deletion in either gene. In total, 29.3% of individuals had an asymptomatic plasmodial infection and were 3.64 times more likely to have parasites with a double deletion (pfhrp2/3) than patients with clinical malaria (p = 0.02). The high prevalence of parasites with pfhrp2/3 deletions shows the need to implement a surveillance program in this area. Deletions in parasites may be associated with the clinical pattern of the disease in this area. More studies must be carried out to elucidate these findings.
... Notably, studies that have harnessed NGS technology have effectively reported the presence of pfhrp2/3 deletions. These studies have found varying frequencies of pfhrp2/3 gene deletions, with some regions showing higher prevalence than others [63,64]. In a study by Flannery et al. (2015), NGS technology was employed to successfully identify the copy number of drug-resistance associated genes in Peruvian isolates [65]. ...
The eradication of Plasmodium parasites, responsible for malaria, is a daunting global public health task. It requires a comprehensive approach that addresses symptomatic, asymptomatic, and submicroscopic cases. Overcoming this challenge relies on harnessing the power of molecular diagnostic tools, as traditional methods like microscopy and rapid diagnostic tests fall short in detecting low parasitaemia, contributing to the persistence of malaria transmission. By precisely identifying patients of all types and effectively characterizing malaria parasites, molecular tools may emerge as indispensable allies in the pursuit of malaria elimination. Furthermore, molecular tools can also provide valuable insights into parasite diversity, drug resistance patterns, and transmission dynamics, aiding in the implementation of targeted interventions and surveillance strategies. In this review, we explore the significance of molecular tools in the pursuit of malaria elimination, shedding light on their key contributions and potential impact on public health.
... Overall, the prevalence of pfhrp2-deleted P. falciparum strains circulating in the study area is very low. 7 Deletions of pfhrp2/3 have been found predominantly in the Brazilian Amazon region, 41 but reports of pfhrp2 or pfhrp3 deletions in African countries such as Mali, 41 Kenya, 42 the Democratic Republic of the Congo, 43 Gabon, 29 Ethiopia, 3 and Eritrea 44 have surfaced. More recently, there have also been reports of pfhrp2 deletions in symptomatic patients who traveled from Nigeria to Australia. ...
Delays in malaria diagnosis increase treatment failures and deaths. In endemic regions, standard diagnostic methods are microscopy and malaria rapid diagnostic tests (mRDTs) detecting Plasmodium falciparum histidine-rich protein 2/3 (PFHRP2/PFHRP3), but gene deletions can allow certain parasites to remain undetected. We enlisted a cohort comprising 207 symptomatic individuals, encompassing both children and adults, at a hospital in Nnewi, Nigeria. The prevalence of parasites was determined using a highly sensitive, species-specific quantitative polymerase chain reaction (SS-qPCR). Within a subset of 132 participants, we assessed the sensitivity and specificity of microscopy and HRP2-mRDTs in comparison to SS-qPCR for the detection of P. falciparum . We also investigated the prevalence of pfhrp2 / pfhrp 3 gene deletions. Greater sensitivity was achieved with mRDTs (95%) compared with microscopy (77%). Also, mRDTs exhibited greater specificity (68%) than microscopy (44%). The positive predictive value of mRDTs (89%) surpassed that of microscopy (80%), suggesting a greater probability of accurately indicating the presence of infection. The negative predictive value of mRDTs (82%) was far greater than microscopy (39%). Of the 165 P. falciparum –positive samples screened for pfhrp2 / pfhrp3 gene deletions, one gene deletion was detected in one sample. Regarding infection prevalence, 84% were positive for Plasmodium spp. (by reverse transcription [RT]-qPCR), with P. falciparum responsible for the majority (97%) of positive cases. Thus, exclusive reliance on microscopy in endemic areas may impede control efforts resulting from false negatives, underscoring the necessity for enhanced training and advocating for high-throughput molecular testing such as RT-qPCR or qPCR at referral centers to address limitations.
