Plasmodium Falciparum Histidine-rich Protein Research Articles

Genetic variability of histidine-rich protein 2 repeat sequences: Misleading factor in true determination of Plasmodium falciparum in different population

PurposeGenetically diverse parasites enhances resistance against antimalarials, vaccines and host immune responses. The present study was designed to evaluate the role played by Plasmodium falciparum genetic diversity in predicting the real world malarial population. MethodsInitially, the incidence pattern of all four northern Indian malarial species was examined using 18S rRNA gene and performed principal component analysis (PCA) based on frequencies of Plasmodium species. Consequently, genetic variance of Plasmodium falciparum histidine-rich protein-2 (Pfhrp2) gene among different malarial populations were compared using phylogenetic analysis. Multi-dimensional scaling was performed to assess genetic similarities and distances among studied populations. ResultsOf total 2168 patients screened, 561 patients with fever of unknown origin were included. 18S rRNA and Pfhrp2 genes were amplified in 78 and 45 samples, respectively. Among them 13.9%(78/561) patients had Plasmodium infection. Infections by P. falciparum, P. vivax and mixed infections were diagnosed among 47(60.2%) and 28(35.9%) and 3(3.8%) patients, respectively. We found eight types of Pfhrp2 amino acid sequence repeats among northern Indian population. The PCA findings were in line with genetic diversity and phylogenetic data. Temporal analysis showed the proportion of total diversity present in total subpopulation (ΔS/ΔT) was maximum for P. falciparum. ConclusionsHigher incidence of Pfhrp2 sequence variation through genetic recombination among multiple strains during sexual reproduction is potentially correlated with high transmission activity. This sequence variation might alter RDT detection sensitivities for different parasites by modulating the structure and frequency of antigenic epitopes.

Evaluating Malaria Rapid Diagnostic Tests and Microscopy for Detecting Plasmodium Infection and Status of Plasmodium falciparum Histidine-Rich Protein 2/3 Gene Deletions in Southeastern Nigeria.

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/pfhrp3 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.

Meta-analytic review on the impact of factors that affect performance of malaria rapid diagnostic test in Africa

Timely, accurate diagnosis and treatment has improved malaria case management. Malaria Rapid Diagnostic Test (mRDT) kits are largely used in malaria diagnosis. Their performance is compromised by factors related to gene deletions, parasite density, quality of the kit, poor storage conditions and end-user inefficiencies hence diagnosis gives either positive, negative, false negative (FN) or false positive (FP) which defines consequent management strategies. This review assessed reports on prevalence of the Plasmodium falciparum histidine rich protein 2/3 (Pfhrp2/3) gene deletions in malaria infected populations in Africa and the risk of mRDT failure to identify malaria positive cases. Preferred Reporting Items for Systematic Meta-Analysis (PRISMA) statement was used for data collection. Literature search was done using Google and Mendel search for data published in a malaria journal, Journal of infectious diseases, scientific reports, Annals of Ibadan postgraduate medicine, and BMC journals published between 2019 and 2023. Fifty eight reports were identified were screened and tested for eligibility. 
 Majority of studies described the consistent use of Pfhrp2/3 mRDT for malaria diagnosis in rural health facilities in Africa and nine reports met inclusion criteria for review. Five of them certified the world health organization’s sample criteria of ‘more than 350 sample’ to estimate the prevalence of Pfhrp2/3 gene deletions leading to declaration of false negative results of which one study posted FN outcome resulting from these deletions. Four out of nine studies did not meet this WHO criterion. This review affirmed presence of Pfhrp2/3 gene deletions challenges in Africa though other countries recorded the converse. Data was pooled using random effect models with Odds ratio and 95% confidence limit. The prevalence of the gene deletions was heterogeneous, ranging from 0% to 78.1%. The review found that an average prevalence of Pfhrp2/3 deletion as 26.2%. This was above the WHO standard recommended declaration value of 5%.; a factor that demonstrated setback to the use of mRDT in malaria endemic regions. Therefore alternative methods should be used where aspersions are cast on outcome of mRDT for 
 it will help improve malaria treatment, tracking and management.

Rapid diagnosis and prognosis of malaria infection using a microfluidic point-of-care immunoassay

Malaria is a major cause of illness and death worldwide. Rapid diagnostic tests are the most widely used tool for detecting malaria infection, however, they only provide binary results and lack the sensitivity needed to detect many asymptomatic infections. Molecular assays for quantifying malaria biomarkers offer higher detection sensitivity, however, they are time-consuming, and require expert training and expensive equipment, making them unsuitable for use in most of Africa. To address the need for simple, accurate and field-deployable malaria diagnostic tests, we have developed a microfluidic point-of-care (mPOC) immunoassay for rapid quantification of Plasmodium falciparum histidine-rich protein 2 (PfHRP2), a malaria parasite biomarker, in whole blood. This device features two diagnostic modes for detecting PfHRP2 at low (100's pg/mL) and high (1,000's ng/mL) concentrations, making it useful for multiple diagnostic applications, including the detection of asymptomatic infection, prediction of disease outcomes and diagnosis of cerebral malaria. Measurements of PfHRP2 in blood samples from malaria patients demonstrates that this platform offers similar accuracy as an ultra-sensitive PfHRP2 enzyme-linked immunosorbent assay (ELISA) test, while being 12× faster and simpler to use. This mPOC immunoassay can be deployed in rural health centers to assist clinicians in diagnosing and triaging malaria patients, ultimately improving patient outcomes.

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.
 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.
 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.
 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.
 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.

Quantification of Plasmodium falciparum HRP-2 as an alternative method to [3H]hypoxanthine incorporation to measure the parasite reduction ratio in vitro

In the absence of a highly efficacious vaccine, chemotherapy remains the cornerstone to control malaria morbidity and mortality. The threat of the emergence of parasites resistant to artemisinin-based combination therapies highlights the need for new antimalarial drugs ideally with superior properties. The killing rate reflects the speed of action of antimalarial drugs that can be measured in vitro through the parasite reduction ratio (PRR) assay to shortlist interesting candidates. As a standard, the in vitro PRR assay is performed by measuring [3H]hypoxanthine incorporation of Plasmodium falciparum. This methodology is restricted to specialized laboratories due to the handling of radioactive material. In this work, we describe the sandwich enzyme-linked immunosorbent assay to detect P. falciparum histidine-rich protein 2 (HRP-2) as an alternative methodology to assess the PRR. We first validated the methodology with established antimalarial drugs (artesunate, chloroquine, pyrimethamine, and atovaquone) by comparing our results to previous results of the [3H]hypoxanthine incorporation readout provided by an expert lab and subsequently assessed the speed of action of four new antimalarial candidates (compound 22, chlorotonil A, boromycin, and ivermectin). The HRP-2 PRR assay obtained comparable results to the hypoxanthine incorporation readout in terms of parasite growth rate over time, lag phase, and parasite clearance time. In addition, parasite growth following drug exposure was quantified after 7, 14, 21, and 28 days of recovery time. In conclusion, the PRR assay based on HRP-2 is similar to [3H]hypoxanthine in determining a drug´s parasite-killing rate and can be widely used in all research laboratories.

Reports on Plasmodium falciparum Histidine Rich Protein 2/3 Gene Deletions among African Countries between January 2010 and December 2021 Employed Different Methodological Approaches: A Systematic Review

Background: Plasmodium falciparum histidine rich protein 2/3 gene deletions (Pfhrp2/3gd) threatens usefulness of mRDT in diagnosing malaria cases. This review was performed to assess the methods used to report findings on those deletions between January 2010 and December 2021.
 Methods: Present search was performed using Medline, Google Scholar and PubMed.
 Results: Out of 94 articles identified, 19% (n=18) were included. Reviewed articles varied in methods employed to report those deletions.
 Conclusion: Methodological approaches for the Reports between January 2010 and December 2021 on Pfhrp2/3gd are varying and this implies that the findings may not be generalized. Prospective studies aiming at assessing Pfhrp2/3gd need to consider WHO guidelines since such studies are important in assessing the magnitude of Pfhrp2/3gd and therefore provide information towards formulating and change of policies aiming at malaria control.

Low Prevalence of Plasmodium falciparum Histidine-Rich Protein 2 and 3 Gene Deletions-A Multiregional Study in Central and West Africa.

Plasmodium falciparum parasites carrying deletions of histidine-rich protein 2 and 3 genes, pfhrp2 and pfhrp3, respectively, are likely to escape detection via HRP2-based rapid diagnostic tests (RDTs) and, consequently, treatment, posing a major risk to both the health of the infected individual and malaria control efforts. This study assessed the frequency of pfhrp2- and pfhrp3-deleted strains at four different study sites in Central Africa (number of samples analyzed: Gabon N = 534 and the Republic of Congo N = 917) and West Africa (number of samples analyzed: Nigeria N = 466 and Benin N = 120) using a highly sensitive multiplex qPCR. We found low prevalences for pfhrp2 (1%, 0%, 0.03% and 0) and pfhrp3 single deletions (0%, 0%, 0.03% and 0%) at all study sites (Gabon, the Republic of Congo, Nigeria and Benin, respectively). Double-deleted P. falciparum were only found in Nigeria in 1.6% of all internally controlled samples. The results of this pilot investigation do not point towards a high risk for false-negative RDT results due to pfhrp2/pfhrp3 deletions in Central and West African regions. However, as this scenario can change rapidly, continuous monitoring is essential to ensure that RDTs remain a suitable tool for the malaria diagnostic strategy.

Low frequency of Plasmodium falciparum hrp2/3 deletions from symptomatic infections at a primary healthcare facility in Kilifi, Kenya.

There is a growing concern for malaria control in the Horn of Africa region due to the spread and rise in the frequency of Plasmodium falciparum Histidine-rich Protein (hrp) 2 and 3 deletions. Parasites containing these gene deletions escape detection by the major PfHRP2-based rapid diagnostic test. In this study, the presence of Pfhrp2/3 deletions was examined in uncomplicated malaria patients in Kilifi County, from a region of moderate-high malaria transmission. 345 samples were collected from the Pingilikani dispensary in 2019/2020 during routine malaria care for patients attending this primary health care facility. The Carestart™ RDT and microscopy were used to test for malaria. In addition, qPCR was used to confirm the presence of parasites. In total, 249 individuals tested positive for malaria by RDT, 242 by qPCR, and 170 by microscopy. 11 samples that were RDT-negative and microscopy positive and 25 samples that were qPCR-positive and RDT-negative were considered false negative tests and were examined further for Pfhrp2/3 deletions. Pfhrp2/3-negative PCR samples were further genotyped at the dihydrofolate reductase (Pfdhfr) gene which served to further confirm that parasite DNA was present in the samples. The 242 qPCR-positive samples (confirmed the presence of DNA) were also selected for Pfhrp2/3 genotyping. To determine the frequency of false negative results in low parasitemia samples, the RDT- and qPCR-negative samples were genotyped for Pfdhfr before testing for Pfhrp2/3. There were no Pfhrp2 and Pfhrp3 negative but positive for dhfr parasites in the 11 (RDT negative and microscopy positive) and 25 samples (qPCR-positive and RDT-negative). In the larger qPCR-positive sample set, only 5 samples (2.1%) were negative for both hrp2 and hrp3, but positive for dhfr. Of the 5 samples, there were 4 with more than 100 parasites/µl, suggesting true hrp2/3 deletions. These findings revealed that there is currently a low prevalence of Pfhrp2 and Pfhrp3 deletions in the health facility in Kilifi. However, routine monitoring in other primary health care facilities across the different malaria endemicities in Kenya is urgently required to ensure appropriate use of malaria RDTs.

Using Malarial Retinopathy to Improve the Diagnosis of Pediatric Cerebral Malaria.

In malaria endemic areas, a high proportion of children have detectable parasitemia but show no clinical symptoms. When comatose from a cause other than malaria, this group confounds the cerebral malaria (CM) definition, making accurate diagnosis challenging. One important biomarker of CM is malarial retinopathy, a set of specific features visible in the ocular fundus. In this study, we quantified the contribution of malarial retinopathy in discriminating malaria-caused coma from non-malaria-caused coma. We estimated that 10% of our study cohort of N = 1,192 patients who met the WHO clinical definition of CM in Malawi had non-malarial coma based on a Gaussian mixture model using the parasite protein Plasmodium falciparum histidine-rich protein-2. A classification based on platelets, white blood cells, and retinopathy significantly improved the discriminative power of a previously established model including only platelets plus white blood cells (area under the receiver operating characteristic curve: 0.89 versus 0.75, P value < 0.001). We conclude that malarial retinopathy is highly predictive of malaria-caused versus non-malaria-caused coma and recommend that an ocular funduscopic examination to determine malarial retinopathy status be included in the assessment of parasitemic comatose African children.

Prevalence of Malaria and COVID-19 Infection in Akure North Local Government Area of Ondo State, Nigeria.

The prevalence of malaria and coronavirus disease (COVID-19) is highly devastating and has led to a serious public health challenge worldwide. In order to ensure proper control and elimination, the State Ministry of Health (MoH) Ondo State, Nigeria conducted medical examinations in Iju/Itaogbolu, Igoba, and Ogbese Health centers to identify and confirm cases of COVID-19 and malaria infection. This study provides the outcome of the epidemiological investigation of the prevalence of COVID-19 and malaria in Akure North Local Government Area of Ondo State, Nigeria. The study was a hospital-based secondary data analysis comprising of 11,389 and 682 individuals who visited various health centers in Akure North Local Government Area (LGA) for medical examinations on malaria and COVID-19, respectively. The COVID-19 cases were investigated using the fluid sample collected with a nasal swab or a throat swab, or spit of saliva into a tube and confirmed by real-time polymerase chain reaction (RT-PCR). The Plasmodium falciparum histidine-rich protein 2 (PfHRP2) RDT was employed to detect the P.falciparum antigen among participants' blood samples. The total prevalence of malaria and COVID-19 were 67.6% and 12.4%, respectively. Meanwhile, the month of September recorded the highest malaria prevalence of 81.8% while the month of April recorded the least malaria prevalence of 56.4%. Similarly, the highest case of COVID-19 (18.8%) was recorded in the month of November while the least case (2.4%) was recorded in the month of April (p < 0.05). The age range of 12-59 months had the highest malaria prevalence of 74.9% while 0-24 days age range recorded the least prevalence of 15.2% (p > 0.05). Apparently, the children were more infected with malaria parasites while adults were more infected with COVID-19. Conclusively, malaria and COVID-19 infections were prevalent in the study area. Thus, the people should be enlightened on the deadly risk of malaria and COVID-19 through the health workers, social media, and the community leaders to ensure compliance with appropriate preventive measures.

Performance of antigen detection for HRP2-based malaria rapid diagnostic tests in community surveys: Tanzania, July–November 2017

BackgroundMalaria rapid diagnostic tests (RDTs) based on the detection of the Plasmodium falciparum histidine-rich protein 2 (HRP2) antigen are widely used for detection of active infection with this parasite and are the only practical malaria diagnostic test in some endemic settings. External validation of RDT results from field surveys can confirm appropriate RDT performance.MethodsA community-based cross-sectional survey was conducted between July and November 2017 enrolling participants of all ages in households from 15 villages in four border regions of Tanzania: Geita, Kigoma, Mtwara and Ruvuma. All participants had an RDT performed in the field and provided a blood sample for later laboratory multiplex antigen detection of HRP2. In assessing the continuous HRP2 levels in participant blood versus RDT result, dose–response logistic regression provided quantitative estimates for HRP2 limit of detection (LOD).ResultsFrom the 15 study villages, 6941 persons were enrolled that had a RDT at time of enrollment and provided a DBS for later laboratory antigen detection. RDT positive prevalence for the HRP2 band by village ranged from 20.0 to 43.6%, but the magnitude of this prevalence did not have an effect on the estimated LOD of RDTs utilized in different villages. Overall, HRP2 single-target tests had a lower LOD at the 95% probability of positive RDT (4.3 ng/mL; 95% CI 3.4–5.4) when compared to pLDH/HRP2 dual target tests (5.4 ng/mL; 4.5–6.3), though this difference was not significant. With the exception of one village, all other 14 villages (93.3%) showed RDT LOD estimates at 90% probability of positive RDT between 0.5 and 12.0 ng/mL.ConclusionsBoth HRP2-only and pLDH/HRP2 combo RDTs utilized in a 2017 Tanzania cross-sectional survey of border regions generally performed well, and reliably detected HRP2 antigen in the low ng/mL range. Though single target tests had lower levels of HRP2 detection, both tests were within similar ranges among the 15 villages. Comparison of quantitative HRP2 detection limits among study sites can help interpret RDT testing results when generating population prevalence estimates for malaria infection.

Detection of histidine-rich protein 2- and/or 3-deleted Plasmodium falciparum using the automated hematology analyzer XN-31: A proof-of-concept study.

Rapid diagnostic tests (RDTs) based on immunochromatographic detection of Plasmodium falciparum histidine-rich protein 2 (HRP2) have been frequently used for malaria diagnosis. The HRP2-based RDTs are highly sensitive and easy to use; however, their sensitivity may be low in detecting P. falciparum strains carrying deletion of the pfhrp2 and pfhrp3 genes encoding HRP2 and HRP3, respectively. The automated hematology analyzer XN-31, developed by Sysmex (Kobe, Japan) to aid in malaria diagnosis, has higher sensitivity than RDTs owing to a unique automated nucleic acid staining technology that has shown great potential in clinical settings. In this study, we compared the performance of the XN-31 analyzer and two RDTs to detect pfhrp2- and/or pfhrp3-deleted parasites cultured in vitro. The analyses showed that the analyzer was not only as sensitive to pfhrp2- and/or pfhrp3-deleted strains as it was to the wild-type strain but also had higher sensitivity than the RDTs. These results suggested that the XN-31 analyzer is useful for rapid and reliable detection of pfhrp2- and/or pfhrp3-deleted parasites in clinical settings.

Performance Evaluation of Nested Polymerase Chain Reaction (Nested PCR), Light Microscopy, and Plasmodium falciparum Histidine-Rich Protein 2 Rapid Diagnostic Test (PfHRP2 RDT) in the Detection of Falciparum Malaria in a High-Transmission Setting in Southwestern Nigeria.

Malaria remains a major public health challenge worldwide. In order to ensure a prompt and accurate malaria diagnosis, the World Health Organization recommended the confirmatory parasitological diagnosis of malaria by microscopy and malaria rapid diagnostic test (RDT) prior to antimalarial administration and treatment. This study was designed to evaluate the performance of nested polymerase chain reaction (nested PCR), light microscopy, and Plasmodium falciparum histidine-rich protein 2 rapid diagnostic test (PfHRP2 RDT) in the detection of falciparum malaria in Akure, Nigeria. A cross-sectional and hospital-based study involving 601 febrile volunteer participants was conducted in Akure, Nigeria. Approximately 2-3 mL venous blood samples were obtained from each study participant for parasitological confirmation by microscopy and PfHRP2-based malaria RDT. Thick and thin films were prepared and viewed under the light microscope for parasite detection, parasite density quantification, and species identification, respectively. Dry blood spot samples were prepared on 3MM Whatman filter paper for nested PCR. The overall prevalence of microscopy, PfHRP2 RDT, and nested PCR were 64.89% (390/601), 65.7% (395/601), and 67.39% (405/601), respectively. The estimates of sensitivity, specificity, positive predictive value, negative predictive value, accuracy, and Youden's j index of microscopy and RDT were 96.30, 100.00, 100.00, 92.89, 97.50, 0.963, and 95.06, 94.90, 97.47, 90.29, 95.01, and 0.899, respectively. Malaria RDT recorded higher false negativity, compared microscopy (4.94% vs. 3.70%). A near perfect agreement was reported between microscopy and nested PCR, and between PfHRP2 RDT and nested PCR with Cohen's kappa (k) values of 0.94 and 0.88, respectively. This study revealed that PfHRP2 RDT and microscopy continues to remain sensitive and specific for falciparum malaria diagnosis in the study area.

Genetic deletions and high diversity of Plasmodium falciparum histidine-rich proteins 2 and 3 genes in parasite populations in Ghana.

Rapid diagnostic tests (RDTs) are used to diagnose malaria in Ghana and other malaria endemic countries. Plasmodium falciparum histidine-rich protein 2 (PFHRP2) based RDTs are widely used, however the occurrence of deletions of the pfhrp2 gene in some parasites have resulted in false negative test results. Monoclonal antibodies of PFHRP2 cross reacts with PFHRP3 because they share structural similarities and this complements the detection of the parasites by RDT. These two genes were investigated in Ghanaian P. falciparum parasite population to detect deletions and the polymorphisms in exon 2 of the pfhrp2 and pfhrp3 genes. Parasite isolates (2,540) from children ≤ 12 years with uncomplicated malaria from 2015 to 2020 transmission seasons were used. Both genes were amplified using nested PCR and negative results indicated the presence of the deletion of genes. Amplified genes were sequenced for the detection of the amino acid repeats. Deletions were observed in 30.7% (780/2,540) and 17.2% (438/2,540) of the samples for pfhrp2 and pfhrp3 respectively with increasing trends over the three time periods (χ2 -10.305, p = 0.001). A total of 1,632 amplicons were sequenced for each gene, analysis was done on 1,124 and 1,307 good quality sequences for pfhrp2 and pfhrp3 respectively. Pfhrp2 repeat polymorphisms were dominantly of types 2 (AHHAHHAAD) and 7 (AHHAAD) with large numbers of variants. A novel variant of type 14 (AHHANHATD) was seen for pfhrp2. For the pfhrp3 repeat types, 16 (AHHAAN), 17 (AHHDG) and 18 (AHHDD) were the dominant types observed. Variants of type 16 (AHHAAH) and (AHHASH) were also dominant. Repeat types 1, 2, 3, 4, 5, 6, 7, 8, 11, 13, 15, 16, and 19 were observed be shared by both genes. The haplotype diversity of both genes ranged between 0.872 and 1 indicating high diversity of the polymorphisms in the isolates. The implication of the findings of the frequencies of the pfhrp2 and pfhrp3 deletions as well as the variants of the main epitopes of the monoclonal antibodies for the RDT (types 2 and 7) in our isolates is an indication of decreased sensitivity of the RDTs in diagnosing malaria infections in Ghana.

Malaria Diagnosis Using Paper-Based Immunoassay for Clinical Blood Sampling and Analysis by a Miniature Mass Spectrometer.

In this work, we have developed a paper-based microfluidic device capable of remote biofluid collection followed by an analysis of the dried clinical samples using a miniature mass spectrometer. We have evaluated a portable mass spectrometer as a possible surveillance platform by analyzing the clinical malaria samples (whole blood) collected from Ghana. We synthesized pH-sensitive ionic probes and coupled them with monoclonal antibodies specific to the Plasmodium falciparum histidine-rich protein 2 (PfHRP2) malaria antigen. We then used the antibody-ionic probe conjugates in a paper-based immunoassay to capture PfHRP2 antigen from untreated whole blood. After the immunoassay, the bound ionic probes were cleaved, and the released mass tags were analyzed through an on-chip paper spray mass spectrometry strategy. During process optimization, we determined the detection limit for PfHRP2 in untreated human serum to be 0.216 nmol/L when using the miniature mass spectrometer. This sensitivity is comparable to the World Health Organization's suggested threshold of 0.227 nmol/L for PfHRP2, proving that our method will be applicable to diagnose symptomatic malaria infection (≥200 parasites per μL blood). The paper device can be stored at room temperature for at least 25 days without affecting the clinical outcome, with each stored paper chip offering good repeatability and reproducibility (RSD = 4-12%). The stability and sensitivity of the developed paper-based immunoassay platform will allow miniature mass spectrometers to be used for point-of-care malaria detection as well as in large-scale surveillance screening to aid eradication programs.

Application of egg yolk IgY on carboxylated polypyrrole films for impedimetric detection of PfHRP2 antigen

This paper described an impedimetric immunosensor for detecting Plasmodium falciparum histidine-rich protein 2 (PfHRP2). Antibodies from egg yolk (Ab-PfHRP2, IgY type) were linked covalently to the screen-printed gold electrodes (SPGE) surface modified with a thin film of Poly-pyrrole-pyrrole 3 carboxylic acid (P(Py-Py3COOH) to develop the sensing platform. The fabrication steps were followed by microscopic (scanning electron microscopy), spectroscopic (RAMAN spectroscopy and Energy-dispersive X-ray spectroscopy), and electrochemical (electrochemical impedance spectroscopy (EIS) and cyclic voltammetry) techniques. The determination of Ag-PfHRP2 was performed by EIS, and the BSA(bovine serum albumin)/Ab-PfHRP2(IgY)/P(Py-Py3COOH)/SPGE immunosensor recorded a linear response at 100–1000 ng mL−1 concentration range, with a limit of detection (LOD) of 27.47 ng mL−1. Its performance was confirmed by Enzyme-Linked Immuno-Sorbent Assay. The fabricated device uses a simple strategy of IgY immobilization, showing high sensitivity and good selectivity, and can be considered an alternative for carrying out malaria tests.

Comparative Assessment of the Sensitivity of Ten Commercial Rapid Diagnostic Test Kits for the Detection of Plasmodium.

Malaria is one of the most common tropical diseases encountered by members of the French military who are deployed in operations under constrained conditions in malaria-endemic areas. Blood smear microscopy—the gold standard for malaria diagnosis—is often not available in such settings, where the detection of malaria relies on rapid diagnostic tests (RDTs). Ten RDTs (from Biosynex, Carestart, Humasis, SD Bioline, and CTK Biotech), based on the detection of the Plasmodium falciparum histidine-rich protein 2 (HRP2) or lactate dehydrogenase (pLDH, PfLDH, or PvLDH), were assessed against 159 samples collected from imported malaria cases, including 79 P. falciparum, 37 P. vivax, 22 P. ovale, and 21 P. malariae parasites. Samples had been previously characterised using microscopy and real-time PCR. The overall sensitivities for the Plasmodium test ranged from 69.8% (111/159) to 95% (151/159). There was no significant difference for the specific detection of P. falciparum (96.2% to 98.7%, p = 0.845). No significant difference was found between sensitivities to P. vivax by pan LDH or pvLDH (81.1% (30/37) to 94.6% (35/37) (p = 0.845)). Some of the RDTs missed most of P. ovale and P. malariae, with sensitivities for all RDTs ranging respectively from 4.5% (1/22) to 81.8% (18/22) and 14.3% (3/21) to 95.2% (20/21). Carestart Malaria Pf/Pan (pLDH) Ag G0121, a pLDH-based RDT (PfLDH and pLDH), showed the highest sensitivities to P. falciparum (98.7%, 78/79), P. vivax (94.6%, 35/37), P. ovale (81.8%, 18/22), and P. malariae (95.2%, 20/21) and meets the requirements for military deployments in malaria-endemic areas.

Detection of Malaria Parasite Protein in Urine of Patients with Acute Uncomplicated Malaria Using Rapid Diagnostic Test Kits

Objectives: The invasive nature of the current malaria diagnostic techniques impairs compliance to diagnosis, especially for on-field detection. Adapting non-invasive methods of biological sample collection for rapid diagnosis of malaria infections may provide a more efficient approach to case management and epidemiological studies of malaria. This study was designed to evaluate the detection of Plasmodium falciparum Histidine-rich Protein II (PfHRP-2) in urine samples and optimization as diagnostic markers for P. falciparum infection. &#x0D; Methods: One hundred (100) microscopically confirmed patients with Plasmodium falciparum infection and 25 P. falciparum negative controls were recruited for the study. Blood samples of all participants were tested for the presence of PfHRP-2 using Rapid Diagnostic Test (RDT) kits. In addition, urine samples of the confirmed malaria-infected patients were analyzed for PfHRP-2 using the CareStartTM and Global Devices (USA) Malaria kits. The diagnostic performances of the RDT kits were evaluated.&#x0D; Results: Overall, the two brands of malaria rapid diagnostics demonstrated 71% sensitivity (95%CI=62.1-79.9%) and 96% specificity (95%CI=88.3-103.7%) for PfHRP-2 detection in urine. &#x0D; The sensitivities of the tests in urine at asexual parasitemia ≤ 2000 μL-1 and asexual parasitemia &gt; 2000 μL-1 were 69.6% (95%CI=56.3-82.9%) and 72.2% (95%CI=60.3-84.2%) respectively. Global Devices and CareStartTM kits had individual sensitivities of 80% (95%CI= 65.7-94.3%) and 67.1% (95%CI= 56.1-78.1%) respectively for PfHRP-2 detection in urine (P= 0.072).&#x0D; Conclusion: Findings revealed that urine-based RDTs have limited capacities for malaria diagnosis due to their low sensitivity and require more optimizations to meet required diagnostic standards. J Microbiol Infect Dis 2022; 12(3):97-107.