Histidine-rich Protein 2 Antigen Research Articles

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.

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

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

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

Rapid diagnostic tests (RDTs) detecting histidine-rich protein 2 (HRP2) and HRP3 are widely used throughout sub-Saharan Africa (SSA) to diagnose Plasmodium falciparum malaria. However, multiple SSA countries have reported pfhrp2 and pfhrp3 (pfhrp2/3) gene deletions. Blood samples (n = 1109) collected from patients with P. falciparum infection from six health facilities throughout the Democratic Republic of the Congo (DRC) from March 2017 to January 2018 were evaluated for pfhrp2/3 deletions. Samples were assayed for HRP2, pan-Plasmodium LDH (pLDH) and aldolase (pAldolase) antigens by bead-based multiplex antigen assay. Samples with low HRP2 concentration compared to pLDH and pAldolase antigens were selected for further pfhrp2/3 genotyping PCRs. The majority of blood samples (93.3%, 1035/1109) had high concentrations of the HRP2 antigen. Single deletions of pfhrp2 were identified in 0.27% (3/1109) of screened samples, with one sample from each of the Kapolowe, Mikalayi, and Rutshuru study sites. A pfhrp3 single deletion (0.09%, 1/1109) was found in the Kapolowe site. Dual pfhrp2 and pfhrp3 deletions were not observed. Due to, the low numbers of pfhrp2 deletions and the sporadic locations of these deletions, the use of HRP2-based RDTs appears to still be appropriate for these locations in DRC.

Purification of native histidine-rich protein 2 (nHRP2) from Plasmodium falciparum culture supernatant, infected RBCs, and parasite lysate

BackgroundDespite the widespread use of histidine-rich protein 2 (HRP2)-based rapid diagnostic tests (RDTs), purified native HRP2 antigen is not standardly used in research applications or assessment of RDTs used in the field.MethodsThis report describes the purification of native HRP2 (nHRP2) from the HB3 Plasmodium falciparum culture strain. As this culture strain lacks pfhrp3 from its genome, it is an excellent source of HRP2 protein only and does not produce the closely-related HRP3. The nHRP2 protein was isolated from culture supernatant, infected red blood cells (iRBCs), and whole parasite lysate using nickel-metal chelate chromatography. Biochemical characterization of nHRP2 from HB3 culture was conducted by SDS-PAGE and western blotting, and nHRP2 was assayed by RDT, ELISA, and bead-based immunoassay.ResultsPurified nHRP2 was identified by SDS-PAGE and western blot as a − 60 kDa protein that bound anti-HRP-2 monoclonal antibodies. Mouse anti-HRP2 monoclonal antibody was found to produce high optical density readings between dilutions of 1:100 and 1:3,200 by ELISA with assay signal observed up to a 1:200,000 dilution. nHRP2 yield from HB3 culture by bead-based immunoassay revealed that both culture supernatant and iRBC lysate were practical sources of large quantities of this antigen, producing a total yield of 292.4 µg of nHRP2 from two pooled culture preparations. Assessment of nHRP2 recognition by RDTs revealed that Carestart Pf HRP2 and HRP2/pLDH RDTs detected purified nHRP2 when applied at concentrations between 20.6 and 2060 ng/mL, performing within a log-fold dilution of commercially-available recombinant HRP2. The band intensity observed for the nHRP2 dilutions was equivalent to that observed for P. falciparum culture strain dilutions of 3D7 and US06 F Nigeria XII between 12.5 and 1000 parasites/µL.ConclusionsPurified nHRP2 could be a valuable reagent for laboratory applications as well as assessment of new and existing RDTs prior to their use in clinical settings. These results establish that it is possible to extract microgram quantities of the native HRP2 antigen from HB3 culture and that this purified protein is well recognized by existing monoclonal antibody lines and RDTs.Graphical

HRP2 and HRP3 cross-reactivity and implications for HRP2-based RDT use in regions with Plasmodium falciparum hrp2 gene deletions

BackgroundThe Plasmodium falciparum antigen histidine rich protein 2 (HRP2) is a preferred target for malaria rapid diagnostic tests (RDTs) because of its abundant production by the parasite and thermal stability. As a result, a majority of RDTs procured globally target this antigen. However, previous reports from South America and recent reports from sub-Saharan Africa and Asia indicate that certain P. falciparum parasites have deletions of the gene coding for HRP2. The HRP2 antigen is paralogous to another P. falciparum antigen HRP3 and some antibodies to HRP2 cross-react with HRP3. Multiple parasites have been described with deletions of one or both hrp2 and hrp3 genes. It is unclear how the various combinations of hrp2 and hrp3 deletion genotypes affect clinical sensitivity of HRP2-based RDTs.MethodsCross-reactivity between HRP2 and HRP3 was tested on malaria RDTs using culture-adapted P. falciparum parasites with both hrp2 and hrp3 intact or with one or both genes deleted. Ten-fold serial dilutions of four culture-adapted P. falciparum parasites [3D7 (hrp2+/hrp3+), Dd2 (hrp2−/hrp3+), HB3 (hrp2+/hrp3−) and 3BD5 (hrp2−/hrp3−)] ranging from 100,000 to 0.01 parasites/µL were prepared. HRP2, Plasmodium lactate dehydrogenase (pLDH) and aldolase concentrations were determined for the diluted samples using a multiplex bead assay. The samples were subsequently tested on three RDT products designed to detect P. falciparum by HRP2 alone or in combination with pLDH.ResultsAt parasite densities of approximately 1000 parasites/µL, parasites that expressed either hrp2 or hrp3 were detected by all three RDTs. Multiplex based antigen measurement using HRP2- conjugated beads demonstrated higher antigen concentration when both hrp2 and hrp3 genes were intact (3D7 parasites, 47.9 ng/ml) compared to HB3 (3.02 ng/mL) and Dd2 (0.20 ng/mL) strains that had one gene deleted. 3D7 at 10 parasites/µL (0.45 ng/mL) was reactive on all three RDT products whereas none of the other parasites were reactive at that density.ConclusionsAbove a certain antigen threshold, HRP3 cross-reactivity on HRP2-based RDTs is sufficient to mask the effects of deletions of hrp2 only. Studies of hrp2 deletion and its effects on HRP2-based RDTs must be studied alongside hrp3 deletions and include clinical sample reactivity on HRP2-based tests.

Field performance of ultrasensitive and conventional malaria rapid diagnostic tests in southern Mozambique

BackgroundAn ultrasensitive malaria rapid diagnostic test (RDT) was recently developed for the improved detection of low-density Plasmodium falciparum infections. This study aimed to compare the diagnostic performance of the PfHRP2-based Abbott Malaria Ag P. falciparum ultrasensitive RDT (uRDT) to that of the conventional SD-Bioline Malaria Ag P. falciparum RDT (cRDT) when performed under field conditions.MethodsFinger-prick blood samples were collected from adults and children in two cross-sectional surveys in May of 2017 in southern Mozambique. Using real-time quantitative PCR (RT-qPCR) as the reference method, the age-specific diagnostic performance indicators of the cRDT and uRDT were compared. The presence of histidine-rich protein 2 (HRP2) and Plasmodium lactate dehydrogenase (pLDH) antigens was evaluated in a subset from dried blood spots by a quantitative antigen assay. pfhrp2 and pfhrp3 gene deletions were assessed in samples positive by RT-qPCR and negative by both RDTs.ResultsAmong the 4,396 participants with complete test results, the sensitivity of uRDTs (68.2; 95% CI 60.8 to 74.9) was marginally better than that of cRDTs (61.5; 95% CI 53.9 to 68.6) (p-value = 0.004), while the specificities were similar (uRDT: 99.0 [95% CI 98.6 to 99.2], cRDT: 99.2 [95% CI 98.9 to 99.4], p-value = 0.02). While the performance of both RDTs was lowest in ≥ 15-year-olds, driven by the higher prevalence of low parasite density infections in this group, the sensitivity of uRDTs was significantly higher in this age group (54.9, 95% CI 40.3 to 68.9) compared to the sensitivity of cRDTs (39.2, 95% CI 25.8 to 53.9) (p-value = 0.008). Both RDTs detected P. falciparum infections at similar geometric mean parasite densities (112.9 parasites/μL for uRDTs and 145.5 parasites/μL for cRDTs). The presence of HRP2 antigen was similar among false positive (FP) samples of both tests (80.5% among uRDT-FPs and 84.4% among cRDT-FPs). Only one false negative sample was detected with a partial pfhrp2 deletion.ConclusionThis study showed that the uRDTs developed by Abbott do not substantially outperform SD-Bioline Pf malaria RDTs in the community and are still not comparable to molecular methods to detect P. falciparum infections in this study setting.

Malaria Parasite Density in Individuals with Different Rapid Diagnostic Test Results and Concentrations of HRP2 Antigen.

Low-density malaria infections are a source of human morbidity in endemic settings and potentially contribute to ongoing malaria transmission. Conventional rapid diagnostic tests (RDTs) were designed to detect clinically relevant parasite and antigen levels, but it is largely unknown what proportion of parasite (and antigen positive) infections are missed by conventional RDTs. Furthermore, RDTs can also provide false positives from lingering histidine-rich protein 2 (HRP2) antigenemia from a past infection. We analyzed 207 samples from Angolan outpatients with a bead-based HRP2 antigen assay and by qRT-PCR for the presence of parasite nucleic acids. Among patients HRP2 positive but negative by conventional RDT, the rate of quantitative reverse transcription-PCR (qRT-PCR) positivity was 45% (95% CI: 35-56%), with a median parasitemia of 3.4 parasites/µL (interquartile range: 0.14-4.8). Only 15% (7-26%) of HRP2-negative samples were found to have parasite nucleic acids. A substantial proportion of persons with blood HRP2 antigen concentrations not detected by the conventional RDT were found to have evidence of active infection, but at low parasite density levels.

Concurrence of CareStart\u2122 Malaria HRP2 RDT with microscopy in population screening for Plasmodium falciparum infection in the Mount Cameroon area: predictors for RDT positivity

BackgroundMalaria remains a diagnostic challenge in many endemic communities. Although rapid diagnostic tests (RDTs) are presently widely used for malaria diagnosis, there is a dearth of information on post-marketing surveillance on its efficacy in Cameroon. The present study evaluated the performance characteristics of CareStart™ Malaria HRP2 (histidine-rich protein 2) antigen (Ag) RDT in diagnosing Plasmodium falciparum infection in the Mount Cameroon area and predictors associated with RDT positivity.MethodsThe CareStart™ Malaria HRP2 Plasmodium falciparum (G0141) Ag RDT was evaluated in a cross-sectional community-based survey involving 491 children of both sexes aged 6 months to 14 years between April and May 2018. Malaria parasitaemia was confirmed by light microscopy. Sensitivity (Se), specificity (Sp), positive (PPV) and negative (NPV) predictive values of the RDT, and the corresponding accuracy and Kappa value (κ) were determined using microscopy as the gold standard. Haemoglobin (Hb) concentration was obtained using an auto-haematology analyser. Results were compared using the chi-square test and associations between predictor variables, and RDT results were assessed using logistic regression analysis.ResultsMicroscopically confirmed malaria parasite prevalence was 27.7%, and geometric mean density was 187 parasites/μL of blood (range 70–1162). Se, Sp, PPV, NPV and accuracy were 82.4, 76.6, 57.4, 91.9 and 78.2%, respectively. Sensitivity depended on parasitaemia and reached 96.1% at densities ≥ 200 parasites/μL of blood. The accuracy of malaria parasitaemia (as assessed by the area under the receiver operating characteristic curve) to predict malaria by RDT was 75.4% (95% CI 70.6–80.1). The agreement between microscopy and RDT was moderate (κ = 0.52). RDT positivity was significantly associated with fever (P < 0.001), children less than 5 years (P = 0.02), history of fever within a month (P < 0.001) and anaemia (P = 0.002).ConclusionThe overall concurrence of CareStart™ Malaria HRP2 pf Ag RDT with microscopy in the detection of P. falciparum infection is moderate and is most useful at parasitaemia ≥ 200 parasites/μL of blood and presentation with fever. While RDT is effective as a diagnostic test for confirmation of clinical cases of malaria, its applications in population screening with a higher proportion of asymptomatic cases are limited.

Challenges in optimizing the histidine rich protein 2-detecting rapid diagnostic test

Malaria programs aimed at eliminating Plasmodium falciparum (Pf) incorporate active infection detection strategies to target the subclinical transmission reservoir. Currently available immunochromatographic lateral flow tests lack the sensitivity required for Pf malaria elimination; the limit of detection (LOD) of these rapid diagnostic tests (RDTs) is above that which is required to identify all transmissible infections. Malaria RDTs with a significantly improved LOD would enable more effective elimination interventions while retaining the critical advantages of low cost, ease of use, and rural deployment capability. Histidinerich protein 2 (HRP2) is a high-priority target analyte for identifying individuals at risk of transmitting Pf. HRP2 protein is secreted by Pf parasites in relatively high concentrations, is widely conserved among Pf strains, and persists in human plasma for up to four weeks. These characteristics allow HRP2 antigen detection even in the absence of circulating parasites during cyclical sequestration. However, the highly polymorphic nature of HRP2 makes optimization of RDTs challenging. Current HRP2-detecting RDT reagents primarily target Type 2 (AHHAHHAAD) and Type 7 (AHHAAD) HRP2 motifs, and independent antibody discovery confirms the most common selection of a C1-13/PTL-3 antibody conjugate pair yields the best RDT performance for the majority of parasite strains. In scenarios where malaria transmission rapidly decreases, exclusive use of a diagnostic whose sensitivity varies with the number of HRP2 epitope repeats can result in single axis evolutionary pressure, increasing selection for less-detectable parasite strains. To evaluate the feasibility of developing an HRP2-based RDT that is highly effective in mass testing and treatment programs, we have assessed the technical characteristics of HRP2 reagents. Our aim is to facilitate the development of improved malaria HRP2 RDTs by: (1) incorporating an understanding of HRP2 structure-function relationships and their impact on epitope-antibody interactions and adaptation, (2) correlating HRP2 concentration levels (in pg/mL) with traditional malaria diagnostic metrics (parasites/mL) for comparative evaluation of tests, and (3) predicting optimal capture reagents to improve and sustain HRP2 RDT performance. Based on our ongoing analysis, we present a product development strategy that combines capture reagent and platform innovations that can be used to make highly sensitive Pf malaria RDTs. In addition, we present criteria for the rational selection of HRP2 protein and parasite culture controls to support the product lifecycle from development through commercialization and scale-up.

Investigation of the kinetics of histidine-rich protein 2 and of the antibody responses to this antigen, in a group of malaria patients from India

Although immunological tests based on the detection of histidine-rich protein 2 (HRP2) from the parasites permit the rapid diagnosis of Plasmodium falciparum malaria, such tests are not yet sufficiently sensitive to detect every bloodsmear-positive case. Some individuals infected with P. falciparum may appear test-negative because of the presence of anti-HRP2 antibodies in their sera. A longitudinal follow-up of HRP2 antigenaemia and antibody responses to this antigen has now been conducted in a group of 45, bloodsmear-positive malaria cases of various ages, both during acute infection with P. falciparum and after antimalarial treatment. Pre-treatment, 'day-0' samples of fingerprick blood were tested for HRP2 (in antigen-capture ELISA) and for antigen-specific IgM and IgG (in indirect ELISA). The patients were then treated, with standard doses of chloroquine, before being retested, for HRP2 and anti-HRP2 antibodies, on days 7, 15 and 28. The level of antigenaemia, which on day 0 was found to be positively correlated with parasitaemia (r = 0.741; P < 0.001), had only fallen by an insignificant amount by day 7 but showed further, significant falls between days 7 and 15 (P < 0.001) and between days 15 and 28 (P < 0.01). Although no significant relationship was observed between the blood concentrations of HRP2 and anti-HRP2 IgM or IgG on days 0 or 7, the level of HRP2 antigenaemia was found to be positively correlated with the concurrent titre of anti-HRP2 IgM on day 15 (r = 0.612; P < 0.001) and day 28 (r = 0.501; P < 0.001). The titres of HRP2-specific IgG gradually increased over the 28 days of follow-up but were not found to be significantly correlated with the decreasing levels of HRP2 antigenaemia. When the 45 day-0 samples of blood were tested for HRP2 in a rapid diagnostic test (RDT), three appeared negative, probably because of interference from the circulating, free, anti-HRP2 antibodies in the plasma. The three RDT-negative samples were significantly different from the 42 RDT-positive, having relatively low HRP2 antigenaemias (P < 0.001) and relatively high titres of anti-HRP2 IgM (P < 0.05) and IgG (P < 0.001). Control samples of blood, from four patients infected with P. vivax and five healthy, normal individuals, were considered ELISA-negative for HRP2 and anti-HRP2 IgM or IgG. It appears that, during human infection with P. falciparum, serum levels of HRP2 antigen remain elevated for at least 7 days post-treatment, despite the host's development of antigen-specific immune responses both before and after treatment.

Detection of histidine rich protein 2 and panmalarial ICT Malaria Pf/Pv test antigens after chloroquine treatment of uncomplicated falciparum malaria does not reliably predict treatment outcome in eastern Indonesia.

In regions with drug-resistant malaria, the ability to rapidly detect or predict treatment failure (TF) soon after a course of standard therapy for Plasmodium falciparum malaria would facilitate the prompt institution of second-line therapy. We thus evaluated longitudinally the ability of the ICT Malaria Pf/Pv immunochromatographic test to predict treatment outcome. Sixty-six Sumbanese Indonesians with uncomplicated falciparum malaria were treated with chloroquine and followed for 28 days by use of 1997 World Health Organization criteria for assessment of therapeutic efficacy of antimalarial drugs. The ICT Pf/Pv testing could be compared with microscopy in approximately half of the patients on each day of follow-up. Although strongly positive histidine rich protein 2 (HRP2) line intensities (equal to or greater than the control band) in convalescence were highly predictive of TF, any degree of positivity for the HRP2 and panmalarial antigens in convalescence was only moderately predictive of TE Positive predictive values of the HRP2 and panmalarial antigens for TF were 76.9% and 87.0%, respectively, on Day 3, 82.4% and 87.5% on Day 7, and 78.9% and 78.9% on Day 14. Negative HRP2 and panmalarial antigen results in convalescence were even less predictive of an adequate clinical response, and false-negative HRP2 and panmalarial antigen test results were found in one-sixth (6 of 37) of recrudescent infections diagnosed by microscopy among patients with late treatment failure. To reliably predict treatment outcome with rapid antigen tests, further development appears necessary to improve sensitivity for viable asexual parasites while avoiding detection of both gametocytes and persistent antigen in convalescence.

Evaluation of a new rapid diagnostic test "Determine Malaria Pf" against standard blood film, ICT Malaria P.f and ParaSight F.

A new rapid immunochromatographic test for malaria diagnosis, Determine™ Malaria Pf, was evaluated. This diagnostic kit is an antigen capture assay based on the detection of histidine-rich protein 2 (HRP-2) of Plasmodium falciparum. Positive test results are read visually, with the formation of red bands within 30 min. 438 patient samples were tested from malaria endemic areas in Palawan, Philippines and non-endemic areas, the Research Institute for Tropical Medicine in Metro Manila and the Komagome Hospital and Keio University Hospital in Tokyo. Of 136 malaria samples, 101 was P. falciparum, 33 P. vivax and 2 mixed infection. There were 302 other diseases, 26 of infectious nature, 201 non-infectious, and 75 working normals. Using microscopy as the gold standard, . sensitivity for Determine™ Malaria Pf, ICT Malaria P.f ™ and ParaSight™ F was 100%, 99% and 100%, respectively, and specificity was 96.7%, 99.1%, and 93.4%, respectively. The positive predictive value for Determine™ Malaria Pf was 90.4%, 97.1% for ICT Malaria P.f™ and 82.4% for ParaSight™ F. The negative predictive value was 100% for Determine™ Malaria Pf, 99.7% for ICT Malaria P.f™ and 100% for ParaSight™ F. Determine™ Malaria Pf had an analytical sensitivity for Parasite detection as low as 30-50 Parasites/μl. Antigenemia was seen to persist until about9 days in patients treated with the second line drug sulfadoxine-pyrimethamine (Fansidar ®) despite a negative blood film. It was longer in chloroquine-treated patients, 14-21 days, only to give a strong positive signal on the day some patients recrudesce, also with a positive blood film. Following of HRP-2 antigen clearance might be used to predict early treatment failures or drug resistance. Determine™ Malaria Pf is a very simple 2-step rapid and convenient procedure with excellent sensitivity and specificity. Its marked simplicity and reliability makes it ideal for field use where remote health centers are minimally equipped and staffed, and malaria diagnosis urgently needed.