Plasmodium Falciparum Histidine-rich Protein Research Articles

A new highly sensitive enzyme-linked immunosorbent assay for the detection of Plasmodium falciparum histidine-rich protein 2 in whole blood

BackgroundThe detection of submicroscopic infections in low prevalence settings has become an increasingly important challenge for malaria elimination strategies. The current field rapid diagnostic tests (RDTs) for Plasmodium falciparum malaria are inadequate to detect low-density infections. Therefore, there is a need to develop more sensitive field diagnostic tools. In parallel, a highly sensitive laboratory reference assay will be essential to evaluate new diagnostic tools. Recently, the highly sensitive Alere™ Malaria Ag P.f ELISA (HS ELISA) was developed to detect P. falciparum histidine-rich protein 2 (HRP2) in clinical whole blood specimens. In this study, the analytical and clinical performance of the HS ELISA was determined using recombinant P. falciparum HRP2, P. falciparum native culture parasites, and archived highly pedigreed clinical whole blood specimens from Karen village, Myanmar and Nagongera, Uganda.ResultsThe HS ELISA has an analytical sensitivity of less than 25 pg/mL and shows strong specificity for P. falciparum HRP2 when tested against P. falciparum native culture strains with pfhrp2 and pfhrp3 gene deletions. Additionally, the Z′-factor statistic of 0.862 indicates the HS ELISA as an excellent, reproducible assay, and the coefficients of variation for inter- and intra-plate testing, 11.76% and 2.51%, were acceptable. Against clinical whole blood specimens with concordant microscopic and PCR results, the HS ELISA showed 100% (95% CI 96.4–100) diagnostic sensitivity and 97.9% (95% CI 94.8–99.4) diagnostic specificity. For P. falciparum positive specimens with HRP2 concentrations below 400 pg/mL, the sensitivity and specificity were 100% (95% CI 88.4–100) and 88.9% (95% CI 70.8–97.6), respectively. The overall sensitivity and specificity for all 352 samples were 100% (CI 95% 96–100%) and 97.3% (CI 95% 94–99%).ConclusionsThe HS ELISA is a robust and reproducible assay. The findings suggest that the HS ELISA may be a useful tool as an affordable reference assay for new ultra-sensitive HRP2-based RDTs.

Efficacy of Histochemical Staining Techniques in the Detection of Plasmodium falciparum Histidine-Rich Proteins in Blood of Children with Malaria

Objective: Falciparum malaria predominates in sub-Saharan Africa and children below
 five years are the most vulnerable. Giemsa-stained microscopy is the gold
 standard in malaria diagnosis. Diagnosis with rapid diagnostic test (RDT) kit
 is also common and over 80% of available malaria RDT kits is Plasmodium falciparum histidine-rich
 protein 2-based (Pfhrp2). However, these histidine-rich protein 2-based kits
 have been observed to give false positive and negative results due to
 persistent antigenemia and low parasitaemia respectively. Thus, the methods of
 Pauly, Perls, and Means & Feeney were adopted to explore the advantage of
 using microscopy for specific detection of these histidine-rich proteins and
 their usefulness in detecting low parasitemia in children. 
 
 Methods: Children
 aged 0-5 years (n=200) visiting three hospitals and private laboratories in
 Calabar were recruited. Whole blood samples were tested with CareStart Malaria
 HRP2-based kit, and blood films were made and stained with Giemsa, Pauly, Perls
 and Means & Feeney for microscopy.
 
 Results: The
 sensitivity and specificity were Giemsa (56.4%, 79.8%), Means & Feeney
 (52.5%, 77.8%), Perls (47.5%, 85.9), Pauly (45.5%, 86.9%), and RDT (23.8%, 96%).
 Pauly method had the highest area under the curve of 0.830 while RDT method had
 the lowest of 0.661. Among the positive cases low parasitemia detected by the
 histochemical methods was Perls 36 (75%), Pauly 32 (69.6%), and Means &
 Feeney 34 (64.2%), and for Giemsa method 40 (70.2%).
 
 Conclusion: Pauly method was the most accurate. All three methods
 were sensitive in detecting low parasitemia. These diagnostic methods are
 useful in malaria diagnosis in this endemic population. J Microbiol Infect Dis 2018; 8(2):55-60

Global analysis of Plasmodium falciparum histidine-rich protein-2 (pfhrp2) and pfhrp3 gene deletions using whole-genome sequencing data and meta-analysis

Many rapid diagnostic tests (RDT) used on suspected malaria cases are based on the detection of the protein encoded by the Plasmodium falciparum histidine-rich protein-2 (pfhrp2) gene, which shares a high sequence homology with pfhrp3 in the 3D7 reference genome. Parasite isolates showing pfhrp2 and pfhrp3 gene deletions have been emerging over the years, but a comprehensive genetic analysis of these variants is still lacking. With this purpose, genomic data from experimental P. falciparum genetic crosses between different laboratory lines (3D7, HB3, DD2, 7G8 and GB4) were first analysed (n = 98). The frequency of pfhrp2 deletions was consistent with a Mendelian prediction in HB3 × DD2 (56.7%; 95%CI = (39.5%–72.9%)). Moreover, the pfhrp2 and pfhrp3 deletions segregated independently of each other in the same genetic cross. Analysis of 3D7 × HB3 and 7G8 × GB4 estimated the probability of spontaneously generating a pfhrp2 deletion during sexual recombination to be up to 6.2%. Next, whole genome sequence data from 1970 P. falciparum isolates collected globally were analysed. Nine samples displayed depth of coverage consistent with pfhrp2 deletions (0.5%), but the corresponding split-read analysis could not confirm deletions in seven of these samples. Twenty-eight isolates had evidence of pfhrp3 deletions (1.4%), which are widespread in Southeast Asia. Finally, a meta-analysis of published data revealed a positive mean association between the frequencies of pfhrp2 and pfhrp3 deletions in Africa and South America. This result suggested a shared selective pressure acting on these genetic variants. In conclusion, evidence of genetic selection on both pfhrp2 and pfhrp3 deletions was presented, but experimental crosses do not provide evidence of a fitness cost of these variants. Further work is urgently needed to accurately determine the prevalence and the degree of association between these genetic variants, and the respective impact on diagnostic accuracy of many in-use RDT.

Cerebrospinal fluid Plasmodium falciparum histidine-rich protein-2 in pediatric cerebral malaria

BackgroundCerebral malaria (CM) causes a rapidly developing coma, and remains a major contributor to morbidity and mortality in malaria-endemic regions. This study sought to determine the relationship between cerebrospinal fluid (CSF) Plasmodium falciparum histidine rich protein-2 (PfHRP-2) and clinical, laboratory and radiographic features in a cohort of children with retinopathy-positive CM.MethodsPatients included in the study were admitted (2009–2013) to the Pediatric Research Ward (Queen Elizabeth Central Hospital, Blantyre, Malawi) meeting World Health Organization criteria for CM with findings of malarial retinopathy. Enzyme-linked immunosorbent assay was used to determine plasma and CSF PfHRP-2 levels. Wilcoxon rank-sum tests and multivariable logistic regression analysis assessed the association of clinical and radiographic characteristics with the primary outcome of death during hospitalization.ResultsIn this cohort of 94 patients, median age was 44 (interquartile range 29–62) months, 53 (56.4%) patients were male, 6 (7%) were HIV-infected, and 10 (11%) died during hospitalization. Elevated concentrations of plasma lactate (p = 0.005) and CSF PfHRP-2 (p = 0.04) were significantly associated with death. On multivariable analysis, higher PfHRP-2 in the CSF was associated with death (odds ratio 9.00, 95% confidence interval 1.44–56.42) while plasma PfHRP-2 was not (odds ratio 2.05, 95% confidence interval 0.45–9.35).ConclusionsElevation of CSF, but not plasma PfHRP-2, is associated with death in this paediatric CM cohort. PfHRP-2 egress into the CSF may represent alteration of blood brain barrier permeability related to the sequestration of parasitized erythrocytes in the cerebral microvasculature.

Characterization of Plasmodium Lactate Dehydrogenase and Histidine-Rich Protein 2 Clearance Patterns via Rapid On-Bead Detection from a Single Dried Blood Spot.

.A rapid, on-bead enzyme-linked immunosorbent assay for Plasmodium lactate dehydrogenase (pLDH) and Plasmodium falciparum histidine-rich protein 2 (HRP2) was adapted for use with dried blood spot (DBS) samples. This assay detected both biomarkers from a single DBS sample with only 45 minutes of total incubation time and detection limits of 600 ± 500 pM (pLDH) and 69 ± 30 pM (HRP2), corresponding to 150 and 24 parasites/μL, respectively. This sensitive and reproducible on-bead detection method was used to quantify pLDH and HRP2 in patient DBS samples from rural Zambia collected at multiple time points after treatment. Biomarker clearance patterns relative to parasite clearance were determined; pLDH clearance followed closely with parasite clearance, whereas most patients maintained detectable levels of HRP2 for 35–52 days after treatment. Furthermore, weak-to-moderate correlations between biomarker concentration and parasite densities were found for both biomarkers. This work demonstrates the utility of the developed assay for epidemiological study and surveillance of malaria.

Insights into the Performance of SD Bioline Malaria Ag P.f/Pan Rapid Diagnostic Test and Plasmodium falciparum Histidine-Rich Protein 2 Gene Variation in Madagascar.

Plasmodium falciparum histidine-rich protein 2 (PfHRP2) forms the basis of many current malaria rapid diagnostic tests (RDTs). However, the parasites lacking part or all of the pfhrp2 gene do not express the PfHRP2 protein and are, therefore, not identifiable by PfHRP2-detecting RDTs. We evaluated the performance of the SD Bioline Malaria Ag P.f/Pan RDT together with pfhrp2 variation in Madagascar. Genomic DNA isolated from 260 patient blood samples were polymerase chain reaction (PCR)-amplified for the parasite 18S rRNA and pfhrp2 genes. Post-PCR ligation detection reaction-fluorescent microsphere assay (LDR-FMA) was performed for the identification of parasite species. Plasmodium falciparum histidine-rich protein 2 amplicons were sequenced. Polymerase chain reaction diagnosis of patient samples showed that 29% (75/260) were infected and P. falciparum was present in 95% (71/75) of these PCR-positive samples. Comparing RDT and P. falciparum detection by LDR-FMA, eight samples were RDT negative but P. falciparum positive (false negatives), all of which were pfhrp2 positive. The sensitivity and specificity of the RDT were 87% and 90%, respectively. Seventy-three samples were amplified for pfhrp2, from which nine randomly selected amplicons were sequenced, yielding 13 sequences. Amplification of pfhrp2, combined with RDT analysis and P. falciparum detection by LDR-FMA, showed that there was no indication of pfhrp2 deletion. Sequence analysis of pfhrp2 showed that the correlation between pfhrp2 sequence structure and RDT detection rates was unclear. Although the observed absence of pfhrp2 deletion from the samples screened here is encouraging, continued monitoring of the efficacy of the SD Bioline Malaria Ag P.f/Pan RDT for malaria diagnosis in Madagascar is warranted.

Major Threat to Malaria Control Programs by Plasmodium falciparum Lacking Histidine-Rich Protein 2, Eritrea.

False-negative results for Plasmodium falciparum histidine-rich protein (HRP) 2–based rapid diagnostic tests (RDTs) are increasing in Eritrea. We investigated HRP gene 2/3 (pfhrp2/pfhrp3) status in 50 infected patients at 2 hospitals. We showed that 80.8% (21/26) of patients at Ghindae Hospital and 41.7% (10/24) at Massawa Hospital were infected with pfhrp2-negative parasites and 92.3% (24/26) of patients at Ghindae Hospital and 70.8% (17/24) at Massawa Hospital were infected with pfhrp3-negative parasites. Parasite densities between pfhrp2-positive and pfhrp2-negative patients were comparable. All pfhrp2-negative samples had no detectable HRP2/3 antigen and showed negative results for HRP2-based RDTs. pfhrp2-negative parasites were genetically less diverse and formed 2 clusters with no close relationships to parasites from Peru. These parasites probably emerged independently by selection in Eritrea. High prevalence of pfhrp2-negative parasites caused a high rate of false-negative results for RDTs. Determining prevalence of pfhrp2-negative parasites is urgently needed in neighboring countries to assist case management policies.

Posttreatment HRP2 Clearance in Patients with Uncomplicated Plasmodium falciparum Malaria.

The response to antimalarial treatment is assessed using serial microscopy. New techniques for accurate measurement of the Plasmodium falciparum histidine-rich protein 2 (HRP2) antigen have allowed for monitoring of the antigen concentration over time, offering a potential alternative for assessing treatment response. Posttreatment HRP2 concentrations were measured in samples obtained longitudinally from 537 individuals with P. falciparum malaria who were participating in efficacy trials in Angola, Tanzania, and Senegal. The HRP2 half-life was estimated using a first-order kinetics clearance model. The association between the HRP2 concentration 3 days after treatment and recrudescence of infection was assessed. Despite substantial variation in HRP2 concentrations among participants at baseline, concentrations consistently showed a first-order exponential decline. The median half-life of HRP2 was estimated to be 4.5 days (interquartile range [IQR], 3.3-6.6 days) in Angola, 4.7 days (IQR, 4.0-5.9 days) in Tanzania, and 3.0 days (IQR, 2.1-4.5 days) in Senegal. The day 3 HRP2 concentration was predictive of eventual recrudescence, with an area under the receiver operating characteristic curve of 0.86 (95% confidence interval, .73-.99). Consistent HRP2 clearance dynamics following successful antimalarial treatment imply a common underlying mechanism of biological clearance. Patients who ultimately did not respond to treatment did not exhibit this same pattern of clearance, even in the absence of other indications of inadequate response to treatment.

Evaluation of rapid PfHRP-2/pLDH-based tests in diagnosing microscopy-confirmed falciparum malaria in Hodeidah governorate, Yemen

Along with the determination of malaria infection rate among suspected patients attending hospitals in Hodeidah governorate, the present study evaluated the accuracy of Plasmodium falciparum histidine-rich protein-2 (PfHRP-2)/parasite-specific lactate dehydrogenase (pLDH)-based rapid diagnostic test (RDT) for the diagnosis of microscopy-confirmed falciparum malaria. An overall malaria infection rate of 19.3% (57/295) among suspected patients attending hospitals was microscopically confirmed. The sensitivity of thin blood films for the detection of malaria parasites was 79.0% compared to thick films and was greatly affected by the parasite density, being 65.0% or less at parasite densities of ≤1000 parasites/μl of blood. Compared to light microscopy, the present study revealed sensitivity levels of 100.0% (95% CI: 92.0–100.0) vs. 94.7% (95% CI: 84.2–98.6), specificity levels of 97.3% (95% CI: 89.8–99.5) vs. 100.0% (95% CI: 93.9–100.0), positive predictive values of 89.9% (95% CI: 88.3–99.0) vs. 100.0 (95% CI: 91.6–100.0) and negative predictive values of 100.0% (95% CI: 93.9–100.0) vs. 98.7% (95% CI: 89.3–98.7) for the PfHRP-2 and pLDH components of SD BIOLINE® RDT, respectively, for falciparum malaria diagnosis. Therefore, the overall accuracy levels of the PfHRP-2 and pLDH components of the investigated RDT for the diagnosis of microscopy-confirmed falciparum malaria are 98.5% (95% CI: 94.6–99.6) and 97.7% (95% CI: 93.5–99.2), respectively.

Malaria surveys using rapid diagnostic tests and validation of results using post hoc quantification of Plasmodium falciparum histidine-rich protein 2

BackgroundRapid diagnostic test (RDT) positivity is supplanting microscopy as the standard measure of malaria burden at the population level. However, there is currently no standard for externally validating RDT results from field surveys.MethodsIndividuals’ blood concentration of the Plasmodium falciparum histidine rich protein 2 (HRP2) protein were compared to results of HRP2-detecting RDTs in participants from field surveys in Angola, Mozambique, Haiti, and Senegal. A logistic regression model was used to estimate the HRP2 concentrations corresponding to the 50 and 90% level of detection (LOD) specific for each survey.ResultsThere was a sigmoidal dose–response relationship between HRP2 concentration and RDT positivity for all surveys. Variation was noted in estimates for field RDT sensitivity, with the 50% LOD ranging between 0.076 and 6.1 ng/mL and the 90% LOD ranging between 1.1 and 53 ng/mL. Surveys conducted in two different provinces of Angola using the same brand of RDT and same study methodology showed a threefold difference in LOD.ConclusionsMeasures of malaria prevalence estimated using population RDT positivity should be interpreted in the context of potentially large variation in RDT LODs between, and even within, surveys. Surveys based on RDT positivity would benefit from external validation of field RDT results by comparing RDT positivity and antigen concentration.

Molecular surveillance of pfhrp2 and pfhrp3 deletions in Plasmodium falciparum isolates from Mozambique

BackgroundMalaria programmes use Plasmodium falciparum histidine-rich protein-2 (PfHRP2) based rapid diagnostic tests (RDTs) for malaria diagnosis. The deletion of this target antigen could potentially lead to misdiagnosis, delayed treatment and continuation of active transmission.MethodsPlasmodium falciparum isolates (n = 1162) collected in Southern Mozambique were assessed by RDTs, microscopy and/or 18SrRNA qPCR. pfhrp2 and pfhrp3 deletions were investigated in isolates from individuals who were negative by RDT but positive by microscopy and/or qPCR (n = 69) using gene-specific PCRs, with kelch13 PCR as the parasite DNA control.ResultsLack of pfhrp2 PCR amplification was observed in one of the 69 isolates subjected to molecular analysis [1.45% (95% CI 0.3–7.8%)].ConclusionsThe low prevalence of pfhrp2 deletions suggests that RDTs will detect the vast majority of the P. falciparum infections. Nevertheless, active surveillance for changing deletion frequencies is required.

Plasmodium falciparum HRP2 ELISA for analysis of dried blood spot samples in rural Zambia

BackgroundDried blood spots are commonly used for sample collection in clinical and non-clinical settings. This method is simple, and biomolecules in the samples remain stable for months at room temperature. In the field, blood samples for the study and diagnosis of malaria are often collected on dried blood spot cards, so development of a biomarker extraction and analysis method is needed.MethodsA simple extraction procedure for the malarial biomarker Plasmodium falciparum histidine-rich protein 2 (HRP2) from dried blood spots was optimized to achieve maximum extraction efficiency. This method was used to assess the stability of HRP2 in dried blood spots. Furthermore, 328 patient samples made available from rural Zambia were analysed for HRP2 using the developed method. These samples were collected at the initial administration of artemisinin-based combination therapy and at several points following treatment.ResultsAn average extraction efficiency of 70% HRP2 with a low picomolar detection limit was achieved. In specific storage conditions HRP2 was found to be stable in dried blood spots for at least 6 months. Analysis of patient samples showed the method to have a sensitivity of 94% and a specificity of 89% when compared with microscopy, and trends in HRP2 clearance after treatment were observed.ConclusionsThe dried blood spot ELISA for HRP2 was found to be sensitive, specific and accurate. The method was effectively used to assess biomarker clearance characteristics in patient samples, which prove it to be ideal for gaining further insight into the disease and epidemiological applications.

High-Sensitivity Assays for Plasmodium falciparum Infection by Immuno-Polymerase Chain Reaction Detection of PfIDEh and PfLDH Antigens.

Rapid diagnostic tests based on Plasmodium falciparum histidine-rich protein II (PfHRP-II) and P. falciparum lactate dehydrogenase (PfLDH) antigens are widely deployed for detection of P. falciparum infection; however, these tests often miss cases of low-level parasitemia, and PfHRP-II tests can give false-negative results when P. falciparum strains do not express this antigen. We screened proteomic data for highly expressed P. falciparum proteins and compared their features to those of PfHRP-II and PfLDH biomarkers. Search criteria included high levels of expression, conservation in all parasite strains, and good correlation of antigen levels with parasitemia and its clearance after drug treatment. Different assay methods were compared for sensitive detection of parasitemia in P. falciparum cultures. Among potential new biomarkers, a P. falciparum homolog of insulin-degrading enzyme (PfIDEh) met our search criteria. Comparative enzyme-linked immunosorbent assays with monoclonal antibodies against PfLDH or PfIDEh showed detection limits of 100-200 parasites/µL and 200-400 parasites/µL, respectively. Detection was dramatically improved by use of real-time immuno-polymerase chain reaction (PCR), to parasitemia limits of 0.02 parasite/µL and 0.78 parasite/µL in PfLDH- and PfIDEh-based assays, respectively. The ability of PfLDH- or PfIDEh-based immuno-PCR assays to detect <1 parasite/µL suggests that improvements of bound antibody sensor technology may greatly increase the sensitivity of malaria rapid diagnostic tests.

Estimating the Added Utility of Highly Sensitive Histidine-Rich Protein 2 Detection in Outpatient Clinics in Sub-Saharan Africa.

Most malaria testing is by rapid diagnostic tests (RDTs) that detect Plasmodium falciparum histidine-rich protein 2 (HRP2). Recently, several RDT manufacturers have developed highly sensitive RDTs (hsRDTs), promising a limit of detection (LOD) orders of magnitude lower than conventional RDTs. To model the added utility of hsRDTs, HRP2 concentration in Angolan outpatients was measured quantitatively using an ultrasensitive bead-based assay. The distribution of HRP2 concentration was bimodal in both afebrile and febrile patients. The conventional RDT was able to detect 81% of all HRP2-positive febrile patients and 52-77% of HRP2-positive afebrile patients. The added utility of hsRDTs was estimated to be greater in afebrile patients, where an hsRDT with a LOD of 200 pg/mL would detect an additional 50-60% of HRP2-positive persons compared with a conventional RDT with a LOD of 3,000 pg/mL. In febrile patients, the hsRDT would detect an additional 10-20% of cases. Conventional RDTs already capture the vast majority of symptomatic HRP2-positive individuals, and hsRDTs would have to reach a sufficiently low LOD approaching 200 pg/mL to provide added utility in identifying HRP2-positive, asymptomatic individuals.

Development of an Immunosensor for PfHRP 2 as a Biomarker for Malaria Detection

Plasmodium falciparum histidine-rich protein 2 (PfHRP 2) was selected in this work as the biomarker for the detection and diagnosis of malaria. An enzyme-linked immunosorbent assay (ELISA) was first developed to evaluate the immunoreagent’s suitability for the sensor’s development. A gold-based sensor with an integrated counter and an Ag/AgCl reference electrode was first selected and characterised and then used to develop the immunosensor for PfHRP 2, which enables a low cost, easy to use, and sensitive biosensor for malaria diagnosis. The sensor was applied to immobilise the anti-PfHRP 2 monoclonal antibody as the capture receptor. A sandwich ELISA assay format was constructed using horseradish peroxidase (HRP) as the enzyme label, and the electrochemical signal was generated using a 3, 3′, 5, 5′tetramethyl-benzidine dihydrochloride (TMB)/H2O2 system. The performance of the assay and the sensor were optimised and characterised, achieving a PfHRP 2 limit of detection (LOD) of 2.14 ng·mL−1 in buffer samples and 2.95 ng∙mL−1 in 100% spiked serum samples. The assay signal was then amplified using gold nanoparticles conjugated detection antibody-enzyme and a detection limit of 36 pg∙mL−1 was achieved in buffer samples and 40 pg∙mL−1 in serum samples. This sensor format is ideal for malaria detection and on-site analysis as a point-of-care device (POC) in resource-limited settings where the implementation of malaria diagnostics is essential in control and elimination efforts.

Biolayer interferometry predicts ELISA performance of monoclonal antibody pairs for Plasmodium falciparum histidine-rich protein 2

Predicting antibody pair performance in a sandwich format streamlines development of antibody-based diagnostics and laboratory research tools, such as enzyme-linked immunosorbent assays (ELISAs) and lateral flow immunoassays (LFAs). We have evaluated panels of monoclonal antibodies against the malarial parasite biomarker Plasmodium falciparum histidine rich protein 2 (HRP2), including 9 new monoclonal antibodies, using biolayer interferometry (BLI) and screened antibody pairs in a checkerboard ELISA. This study showed BLI predicts antibody pair ELISA performance for HRP2. Pairs that included capture antibodies with low off-rate constants and detection antibodies with high on-rate constants performed best in an ELISA format.

Serum Lipids and Lipoproteins During Uncomplicated Malaria: A Cohort Study in Lambaréné, Gabon.

AbstractThe serum lipid profile in malaria patients has been found to differ from that of healthy controls. We investigated serum lipid profile changes in malaria patients over time compared with patients with other febrile diseases. In total, 217 patients were included in the study (111 malaria patients and 106 symptomatic controls, defined as malaria-negative febrile patients). Serum lipid levels (mmol/L) were significantly lower in malaria patients compared with those with other febrile diseases (total cholesterol [TC] = 3.26 [standard deviation = 0.94] versus 3.97 [1.22; P < 0.001]; high-density lipoprotein cholesterol [HDL-C] = 0.43 [0.47] versus 1.05 [0.67; P < 0.001], low-density lipoprotein cholesterol [LDL-C] = 2.05 [0.76] versus 2.42 [0.90; P < 0.001]. Triglycerides (TGs) levels were higher in malaria patients (1.81 [1.02] versus 1.11 [0.82; P < 0.001]). No significant differences were found for apolipoprotein A1, apolipoprotein B, and lipoprotein(a). Cholesterol levels increased toward reference values on day 28 (TC = 3.26-3.98, P < 0.001; HDL-C = 0.43-0.96, P < 0.001; LDL-C = 2.05-2.60, P < 0.001). TG levels decreased from 1.81 on admission to 1.76 (day 3) and 0.88 (day 28; P = 0.130). Lipid profile changes were not correlated with parasitemia or Plasmodium falciparum histidine-rich protein 2 levels. This study confirms characteristic temporary lipid profile changes in malaria. Lipid profile changes demonstrated a good accuracy to discriminate between malaria and other febrile diseases (area under the curve = 0.80 (95% confidence interval = 0.742-0.863, P < 0.001). Several plausible hypotheses exist regarding the pathophysiology of lipid profile changes in malaria. Further studies to elucidate the precise pathways may lead to improved understanding of the underlying pathophysiology.

Rapid concentration and elution of malarial antigen histidine-rich protein II using solid phase Zn(II) resin in a simple flow-through pipette tip format.

Rapid diagnostic tests (RDTs) designed to function at the point of care are becoming more prevalent in malaria diagnostics because of their low cost and simplicity. While many of these tests function effectively with high parasite density samples, their poor sensitivity can often lead to misdiagnosis when parasitemia falls below 100 parasites/μl. In this study, a flow-through pipette-based column was explored as a cost-effective means to capture and elute more Plasmodium falciparum histidine-rich protein II (HRPII) antigen, concentrating the biomarker available in large-volume lysed whole blood samples into volumes compatible with Plasmodium falciparum-specific RDTs. A systematic investigation of immobilized metal affinity chromatography divalent metal species and solid phase supports established the optimal design parameters necessary to create a flow-through column incorporated into a standard pipette tip. The bidirectional flow inherent to this format maximizes mixing efficiency so that in less than 5 min of sample processing, the test band signal intensity was increased up to a factor of twelve from HRPII concentrations as low as 25 pM. In addition, the limit of detection per sample was decreased by a factor of five when compared to the RDT manufacturer's suggested protocol. Both the development process and commercial viability of this application are explored, serving as a potential model for future applications.

Analytical sensitivity of current best-in-class malaria rapid diagnostic tests

BackgroundRapid diagnostic tests (RDTs) are today the most widely used method for malaria diagnosis and are recommended, alongside microscopy, for the confirmation of suspected cases before the administration of anti-malarial treatment. The diagnostic performance of RDTs, as compared to microscopy or PCR is well described but the actual analytical sensitivity of current best-in-class tests is poorly documented. This value is however a key performance indicator and a benchmark value needed to developed new RDTs of improved sensitivity.MethodsThirteen RDTs detecting either the Plasmodium falciparum histidine rich protein 2 (HRP2) or the plasmodial lactate dehydrogenase (pLDH) antigens were selected from the best performing RDTs according to the WHO–FIND product testing programme. The analytical sensitivity of these products was evaluated using a range of reference materials including P. falciparum and Plasmodium vivax whole parasite samples as well as recombinant proteins.ResultsThe best performing HRP2-based RDTs could detect all P. falciparum cultured samples at concentrations as low as 0.8 ng/mL of HRP2. The limit of detection of the best performing pLDH-based RDT specifically detecting P. vivax was 25 ng/mL of pLDH.ConclusionThe analytical sensitivity of P. vivax and Pan pLDH-based RDTs appears to vary considerably from product to product, and improvement of the limit-of-detection for P. vivax detecting RDTs is needed to match the performance of HRP2 and Pf pLDH-based RDTs for P. falciparum. Different assays using different reference materials produce different values for antigen concentration in a given specimen, highlighting the need to establish universal reference assays.

Implications of Parasites Lacking Plasmodium falciparum Histidine-Rich Protein 2 on Malaria Morbidity and Control When Rapid Diagnostic Tests Are Used for Diagnosis.

Rapid diagnostic tests (RDTs) are an important tool for malaria diagnosis, with most using antibodies against Plasmodium falciparum histidine-rich protein 2 (PfHRP2). Reports of P. falciparum lacking this protein are increasing, creating a problem for diagnosis of falciparum malaria in locations without quality-assured microscopy. An agent-based stochastic simulation model of P. falciparum transmission was used to investigate the selective pressure exerted on parasite populations by use of RDTs for diagnosis of symptomatic cases. The model considered parasites with normal, reduced, or no PfHRP2, and diagnosis using PfHRP2-only or combination RDTs. Use of PfHRP2-only RDTs in communities where a PfHRP2-negative parasite was introduced during the simulation resulted in transmission of the parasite in >80% of cases, compared with <30% for normal or PfHRP2-reduced parasites. Using PfHRP2-only RDTs in the presence of PfHRP2-negative parasites caused an increase in prevalence, reduced RDT positivity within symptomatic patients but no change in the number of antimalarial treatments due to false-negative RDT results. Diagnosis with PfHRP2/Pf-Plasmodium lactate dehydrogenase combination RDTs did not select for PfHRP2-negative parasites. The use of PfHRP2-only RDTs is sufficient to select P. falciparum parasites lacking this protein, thus posing a significant public health problem, which could be moderated by using PfHRP2/Pf-Plasmodium lactate dehydrogenase combination RDTs.