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The prevalence of Plasmodium falciparum hrp2 (pfhrp2)-deleted parasites threatens the efficacy of the most used and sensitive malaria rapid diagnostic tests and highlights the need for continued surveillance for this gene deletion. While PCR methods are adequate for determining pfhrp2 presence or absence, they offer a limited view of its genetic diversity. Here, we present a portable sequencing method using the MinION. Pfhrp2 amplicons were generated from individual samples, barcoded, and pooled for sequencing. To overcome potential crosstalk between barcodes, we implemented a coverage-based threshold for pfhrp2 deletion confirmation. Amino acid repeat types were then counted and visualized with custom Python scripts following de novo assembly. We evaluated this assay using well-characterized reference strains and 152 field isolates with and without pfhrp2 deletions, of which 38 were also sequenced on the PacBio platform to provide a standard for comparison. Of 152 field samples, 93 surpassed the positivity threshold, and of those samples, 62/93 had a dominant pfhrp2 repeat type. PacBio-sequenced samples with a dominant repeat-type profile from the MinION sequencing data matched the PacBio profile. This field-deployable assay can be used alone for surveilling pfhrp2 diversity or as a sequencing-based addition to the World Health Organization's existing deletion surveillance protocol., (© 2023. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Deletions of pfhrp2 and paralogue pfhrp3 (pfhrp2/3) genes threaten Plasmodium falciparum diagnosis by rapid diagnostic test. We examined 1,002 samples from suspected malaria patients in Djibouti City, Djibouti, to investigate pfhrp2/3 deletions. We performed assays for Plasmodium antigen carriage, pfhrp2/3 genotyping, and sequencing for 7 neutral microsatellites to assess relatedness. By PCR assay, 311 (31.0%) samples tested positive for P. falciparum infection, and 296 (95.2%) were successfully genotyped; 37 (12.5%) samples were pfhrp2+/pfhrp3+, 51 (17.2%) were pfhrp2+/pfhrp3-, 5 (1.7%) were pfhrp2-/pfhrp3+, and 203 (68.6%) were pfhrp2-/pfhrp3-. Histidine-rich protein 2/3 antigen concentrations were reduced with corresponding gene deletions. Djibouti P. falciparum is closely related to Ethiopia and Eritrea parasites (pairwise G ST 0.68 [Ethiopia] and 0.77 [Eritrea]). P. falciparum with deletions in pfhrp2/3 genes were highly prevalent in Djibouti City in 2019-2020; they appear to have arisen de novo within the Horn of Africa and have not been imported.

Background: Despite 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., Methods: This 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., Results: Purified 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., Conclusions: Purified 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., (© 2021. The Author(s).)

Background: The 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., Methods: Cross-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., Results: At 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., Conclusions: Above 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.

Background: The use of rapid diagnostic tests (RDTs) to diagnose malaria is common in sub-Saharan African laboratories, remote primary health facilities and in the community. Currently, there is a lack of reliable methods to ascertain health worker competency to accurately use RDTs in the testing and diagnosis of malaria. Dried tube specimens (DTS) have been shown to be a consistent and useful method for quality control of malaria RDTs; however, its application in National Quality Management programmes has been limited., Methods: A Plasmodium falciparum strain was grown in culture and harvested to create DTS of varying parasite density (0, 100, 200, 500 and 1000 parasites/µL). Using the dried tube specimens as quality control material, a proficiency testing (PT) programme was carried out in 80 representative health centres in Togo. Health worker competency for performing malaria RDTs was assessed using five blinded DTS samples, and the DTS were tested in the same manner as a patient sample would be tested by multiple testers per health centre., Results: All the DTS with 100 parasites/µl and 50% of DTS with 200 parasites/µl were classified as non-reactive during the pre-PT quality control step. Therefore, data from these parasite densities were not analysed as part of the PT dataset. PT scores across all 80 facilities and 235 testers was 100% for 0 parasites/µl, 63% for 500 parasites/µl and 93% for 1000 parasites/µl. Overall, 59% of the 80 healthcare centres that participated in the PT programme received a score of 80% or higher on a set of 0, 500 and 1000 parasites/ µl DTS samples. Sixty percent of health workers at these centres recorded correct test results for all three samples., Conclusions: The use of DTS for a malaria PT programme was the first of its kind ever conducted in Togo. The ease of use and stability of the DTS illustrates that this type of samples can be considered for the assessment of staff competency. The implementation of quality management systems, refresher training and expanded PT at remote testing facilities are essential elements to improve the quality of malaria diagnosis.

Background: The World Health Organization recommends confirmatory diagnosis by microscopy or malaria rapid diagnostic test (RDT) in patients with suspected malaria. In recent years, mobile medical applications (MMAs), which can interpret RDT test results have entered the market. To evaluate the performance of commercially available MMAs, an evaluation was conducted by comparing RDT results read by MMAs to RDT results read by the human eye., Methods: Five different MMAs were evaluated on six different RDT products using cultured Plasmodium falciparum blood samples at five dilutions ranging from 20 to 1000 parasites (p)/microlitre (µl) and malaria negative blood samples. The RDTs were performed in a controlled, laboratory setting by a trained operator who visually read the RDT results. A second trained operator then used the MMAs to read the RDT results. Sensitivity (Sn) and specificity (Sp) for the RDTs were calculated in a Bayesian framework using mixed models., Results: The RDT Sn of the P. falciparum (Pf) test line, when read by the trained human eye was significantly higher compared to when read by MMAs (74% vs. average 47%) at samples of 20 p/µl. In higher density samples, the Sn was comparable to the human eye (97%) for three MMAs. The RDT Sn of test lines that detect all Plasmodium species (Pan line), when read by the trained human eye was significantly higher compared to when read by MMAs (79% vs. average 56%) across all densities. The RDT Sp, when read by the human eye or MMAs was 99% for both the Pf and Pan test lines across all densities., Conclusions: The study results show that in a laboratory setting, most MMAs produced similar results interpreting the Pf test line of RDTs at parasite densities typically found in patients that experience malaria symptoms (> 100 p/µl) compared to the human eye. At low parasite densities for the Pf line and across all parasite densities for the Pan line, MMAs were less accurate than the human eye. Future efforts should focus on improving the band/line detection at lower band intensities and evaluating additional MMA functionalities like the ability to identify and classify RDT errors or anomalies.

Background: Malaria rapid diagnostic tests (RDTs) have greatly improved access to diagnosis in endemic countries. Most RDTs detect Plasmodium falciparum histidine-rich protein 2 (HRP2), but their sensitivity is seriously threatened by the emergence of pfhrp2-deleted parasites. RDTs detecting P. falciparum or pan-lactate dehydrogenase (Pf- or pan-LDH) provide alternatives. The objective of this study was to systematically assess the performance of malaria RDTs against well-characterized pfhrp2-deleted P. falciparum parasites., Methods: Thirty-two RDTs were tested against 100 wild-type clinical isolates (200 parasites/µL), and 40 samples from 10 culture-adapted and clinical isolates of pfhrp2-deleted parasites. Wild-type and pfhrp2-deleted parasites had comparable Pf-LDH concentrations. Pf-LDH-detecting RDTs were also tested against 18 clinical isolates at higher density (2,000 parasites/µL) lacking both pfhrp2 and pfhrp3., Results: RDT positivity against pfhrp2-deleted parasites was highest (> 94%) for the two pan-LDH-only RDTs. The positivity rate for the nine Pf-LDH-detecting RDTs varied widely, with similar median positivity between double-deleted (pfhrp2/3 negative; 63.9%) and single-deleted (pfhrp2-negative/pfhrp3-positive; 59.1%) parasites, both lower than against wild-type P. falciparum (93.8%). Median positivity for HRP2-detecting RDTs against 22 single-deleted parasites was 69.9 and 35.2% for HRP2-only and HRP2-combination RDTs, respectively, compared to 96.0 and 92.5% for wild-type parasites. Eight of nine Pf-LDH RDTs detected all clinical, double-deleted samples at 2,000 parasites/µL., Conclusions: The pan-LDH-only RDTs evaluated performed well. Performance of Pf-LDH-detecting RDTs against wild-type P. falciparum does not necessarily predict performance against pfhrp2-deleted parasites. Furthermore, many, but not all HRP2-based RDTs, detect pfhrp2-negative/pfhrp3-positive samples, with implications for the HRP2-based RDT screening approach for detection and surveillance of HRP2-negative parasites.

Background: Malaria rapid diagnostic tests (RDTs) are largely responsible for the gains made in the proportion of malaria cases confirmed with a parasitological test. However, quality assurance programs to support their use remain a challenge. A dried tube specimen (DTS) method was developed that showed potential for use as a stable source of quality control (QC) sample for RDTs and for use in external quality assessments or proficiency testing (PT). DTS was further assessed with focus on sample stability under field settings in Benin and Liberia., Methods: DTS were prepared using Plasmodium falciparum 3D7 or W2 strains at concentrations of 1000, 500 or 0 parasites/µL and tested for baseline reactivity at the Centers for Disease Control and Prevention, Atlanta before shipping. In Benin and Liberia, DTS were stored under refrigeration in a reference laboratory (RL) or in health centres under ambient temperatures. Seven rounds of testing were performed at 4-week intervals during which DTS were tested on RDTs stored at the RL or at health centres. Observed DTS reactivity at the RL and health centres were compared to expected reactivity to determine DTS stability. DTS were also assembled into a PT panel and tested by health facility staff at the mid and end time-points of the study. Daily maximum and minimum storage temperatures for RDTs and DTS were recorded., Results: In Benin, DTS, irrespective of storage conditions, produced the expected reactivity at all time points. However, evidence of degradation was observed at weeks 20 and 24 for DTS stored at ambient temperatures at the health centres and not those stored under refrigeration at the RL. In Liberia, sample degradation was observed starting at week 8 especially among DTS stored at the health facilities. The degradation was associated with prolonged storage of DTS under ambient temperature prior to study commencement and less than optimal storage temperatures at the RL. Use of DTS in a PT enabled identification of health worker errors in performing the tests., Conclusion: DTS is a feasible tool for use as QC material and for PT under field conditions. Long-term (> 5 months) storage of DTS requires refrigeration.

Histidine-rich protein 2 (HRP2) detecting rapid diagnostic tests (RDTs) have played an important role in enabling prompt malaria diagnosis in remote locations. However, emergence of pfhrp2 deleted parasites is threatening the efficacy of RDTs, and the World Health Organization (WHO) has highlighted surveillance of these deletions as a priority. Nested PCR is used to confirm pfhrp2 deletion but is costly and laborious. Due to spurious amplification of paralogue pfhrp3, the identity of nested exon 1 PCR product must be confirmed by sequencing. Here we describe a new one-step PCR method for detection of pfhrp2. To determine sensitivity and specificity, all PCRs were performed in triplicate. Using photo-induced electron transfer (PET) PCR detecting 18srRNA as true positive, one-step had comparable sensitivity of 95.0% (88.7-98.4%) to nested exon 1, 99.0% (94.6-99.9%) and nested exon 2, 98.0% (93.0-99.8%), and comparable specificity 93.8% (69.8-99.8%) to nested exon 1 100.0% (79.4-100.0%) and nested exon 2, 100.0% (74.4-100.0%). Sequencing revealed that one step PCR does not amplify pfhrp3. Logistic regression models applied to measure the 95% level of detection of the one-step PCR in clinical isolates provided estimates of 133p/μL (95% confidence interval (CI): 3-793p/μL) for whole blood (WB) samples and 385p/μL (95% CI: 31-2133 p/μL) for dried blood spots (DBSs). When considering protocol attributes, the one-step PCR is less expensive, faster and more suitable for high throughput. In summary, we have developed a more accurate PCR method that may be ideal for the application of the WHO protocol for investigating pfhrp2 deletions in symptomatic individuals presenting to health care facilities., Competing Interests: Authors Gireesh Subramaniam and Jasmine Padilla are employed by the Oak Ridge Institute for Science and Education (ORISE). ORISE provided support in the form of salary for authors GS and JP, but did not have any additional role role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests statement: Authors Sophie Jones and Dhruviben Patel are employed by Williams Consulting LLC. There are no patents, products in development or marketed products to declare. This does not alter our adherence to all the PLOS ONE policies on sharing data and materials. Authors Gireesh Subramaniam and Jasmine Padilla are employed by the Oak Ridge Institute for Science and Education (ORISE). There are no patents, products in development or marketed products to declare. This does not alter our adherence to all the PLOS ONE policies on sharing data and materials.

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.

Background: Detection of Plasmodium antigens provides evidence of malaria infection status and is the basis for most malaria diagnosis., Methods: We developed a sensitive bead-based multiplex assay for laboratory use, which simultaneously detects pan-Plasmodium aldolase (pAldo), pan-Plasmodium lactate dehydrogenase (pLDH), and P. falciparum histidine-rich protein 2 (PfHRP2) antigens. The assay was validated against purified recombinant antigens, monospecies malaria infections, and noninfected blood samples. To test against samples collected in an endemic setting, Angolan outpatient samples (n = 1267) were assayed., Results: Of 466 Angolan samples positive for at least 1 antigen, the most common antigen profiles were PfHRP2+/pAldo+/pLDH+ (167, 36%), PfHRP2+/pAldo-/pLDH- (163, 35%), and PfHRP2+/pAldo+/pLDH- (129, 28%). Antigen profile was predictive of polymerase chain reaction (PCR) positivity and parasite density. Eight Angolan samples (1.7%) had no or very low PfHRP2 but were positive for 1 or both of the other antigens. PCR analysis confirmed 3 (0.6%) were P. ovale infections and 2 (0.4%) represented P. falciparum parasites lacking Pfhrp2 and/or Pfhrp3., Conclusions: These are the first reports of Pfhrp2/3 deletion mutants in Angola. High-throughput multiplex antigen detection can inexpensively screen for low-density P. falciparum, non-falciparum, and Pfhrp2/3-deleted parasites to provide population-level antigen estimates and identify specimens requiring further molecular characterization.

Background: Rapid 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., Methods: Individuals' 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., Results: There 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., Conclusions: Measures 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.

Background: Rapid diagnostic tests (RDTs) are now widely used for laboratory confirmation of suspected malaria cases to comply with the World Health Organization recommendation for universal testing before treatment. However, many malaria programmes lack quality control (QC) processes to assess RDT use under field conditions. Prior research showed the feasibility of using the dried tube specimen (DTS) method for preserving Plasmodium falciparum parasites for use as QC samples for RDTs. This study focused on the use of DTS for RDT QC and proficiency testing under field conditions., Methods: DTS were prepared using cultured P. falciparum at densities of 500 and 1,000 parasites/μL; 50 μL aliquots of these along with parasite negative human blood controls (0 parasites/μL) were air-dried in specimen tubes and reactivity verified after rehydration. The DTS were used in a field study in the Oromia Region of Ethiopia. Replicate DTS samples containing 0, 500 and 1,000 parasites/μL were stored at 4°C at a reference laboratory and at ambient temperatures at two nearby health facilities. At weeks 0, 4, 8, 12, 16, 20, and 24, the DTS were rehydrated and tested on RDTs stored under manufacturer-recommended temperatures at the RL and on RDTs stored under site-specific conditions at the two health facilities. Reactivity of DTS stored at 4°C at the reference laboratory on RDTs stored at the reference laboratory was considered the gold standard for assessing DTS stability. A proficiency-testing panel consisting of one negative and three positive samples, monitored with a checklist was administered at weeks 12 and 24., Results: At all the seven time points, DTS stored at both the reference laboratory and health facility were reactive on RDTs stored under the recommended temperature and under field conditions, and the DTS without malaria parasites were negative. At the reference laboratory and one health facility, a 500 parasites/μL DTS from the proficiency panel was falsely reported as negative at week 24 due to errors in interpreting faint test lines., Conclusions: The DTS method can be used under field conditions to supplement other RDT QC methods and health worker proficiency in Ethiopia and possibly other malaria-endemic countries.

Background: Rapid diagnostic tests (RDTs) are central to fulfilling the WHO's recommendation for parasitologic confirmation of all suspected cases of malaria. RDT performance may be compromised when exposed to the high temperature conditions typical of most malaria endemic regions. However, a systematic method to monitor RDT quality and performance in endemic countries is lacking at the present time. Current methods to monitor RDT performance in the field include comparing results from RDTs to diagnoses made by light microscopy and observing health workers perform tests. These methods are not substitutes for direct quality control. In this study, the suitability of dried Plasmodium falciparum-infected blood as quality control samples for malaria RDTs was evaluated., Methods: Three cultured strains of P. falciparum at 200 and 2,000 parasites/μl were tested on 10 brands of RDT. After baseline testing to determine initial reactivity, aliquots of parasite-infected blood were air dried, stored at 35°C, room temperature (~25°C) or 4°C for one, four and 12 weeks and were then tested on the 10 RDTs after rehydration. Extended stability testing of dried blood stored at 4°C was done using P. falciparum strain 3D7 at 1,000 and 2,000 parasites/μl., Results: All dried blood samples at 2,000 parasites/μl retained reactivity (100% sensitivity) at all three temperatures and time points for all nine RDT brands that detect histidine-rich protein-2 (HRP2). The dried blood samples with 200 parasites/μl were detected by six of the nine HRP2-based RDTs at all storage temperatures and time points. The sensitivity for two of the three remaining HRP2-based RDTs was 100% up to four weeks of storage at all temperatures but dropped to 87.5% at week 12. Of the four RDTs that detect plasmodium lactate dehydrogenase (pLDH) in a pan-specific manner, alone or in combination with HRP2, the detection of pLDH in samples with 2,000 parasites/μL was 100% for two RDTs and 80% for the other two RDTs. The mean level for detection of pLDH at 200 parasites/μl was low (29%), with a range of 0% to100%, which was partly attributable to weak initial baseline reactivity. Reactivity of dried 3D7 at 1,000 and 2,000 parasites/μl stored at 4°C was retained at 100% for up to 52 weeks for both HRP2 and pLDH., Conclusions: In the absence of native or recombinant positive control antigens, well-standardized P. falciparum-infected dried blood samples can be used as positive control samples for monitoring RDT performance, particularly with HRP2-detecting tests.

Two chimpanzees were vaccinated intramuscularly against malaria using plasmid DNA expressing the pre-erythrocytic antigens thrombospondin related adhesion protein (PfTRAP) and liver stage specific antigen-1 (PfLSA-1) of Plasmodium falciparum together with GM-CSF protein. A recombinant modified vaccinia virus Ankara (MVA) expressing PfTRAP was injected intramuscularly 6 weeks later to boost the immune response. This sequence of antigen delivery induced a specific and long-lasting T cell and antibody response to PfTRAP as detected by ELISPOT assay and ELISA. Antibody responses were detected after four DNA injections, and were boosted by injection of recombinant MVA expressing PfTRAP. Interferon-gamma secreting antigen-specific T cells were detected in both animals, but only after boosting with recombinant MVA. By screening a panel of PfTRAP-derived peptides, an epitope was identified that was recognized by cytotoxic T lymphocytes in one of the chimpanzees studied. T cells specific for this epitope were present in PBMCs and liver-infiltrating lymphocytes at a frequency of between 1 in 200 and 1 in 500. The high immunogenicity of this prime-boost regimen in chimpanzees supports further assessment of this delivery strategy for the induction of protection against P. falciparum malaria in humans.

The search for a cytotoxic T lymphocyte (CTL)-inducing malaria vaccine has moved forward from epitope identification to planning stages of safety and immunogenicity trials of candidate vaccines. Development of CTL-inducing vaccine candidates has taken center stage based on the observation that CTL-mediated protection might be the dominant mechanism by which sterile immunity is achieved in irradiated sporozoite immunization experiments in humans and laboratory animals. However, studies in naturally infected individuals living in endemic areas, as reviewed here by Michael Aidoo and Venkatachalam Udhayakumar, have revealed that CTL induction might be influenced by factors such as parasite variants, host genes, other infections and transmission patterns. The influence of these factors on CTL induction has been demonstrated individually and in various combinations in controlled animal experiments. However, in naturally infected humans, they are presented in a complex host-parasite-environment interaction, in a manner that is not easily achieved in laboratory-based experiments. Understanding these interactions is crucial for the development and testing of CTL-inducing vaccines for humans.

The development of an effective preerythrocytic vaccine against Plasmodium falciparum malaria is likely to require inclusion of components from several preerythrocytic antigens. The association of HLA-B53 with resistance to severe malaria in West Africa provided evidence that HLA class I-restricted CD8(+) T-cell responses play a role in protective immunity in African children, supporting data from rodent models of malaria. Previously, a single epitope from liver-stage-specific antigen 1 (LSA-1) has been shown to be recognized by HLA-B53-specific cytotoxic T lymphocytes (CTL), but HLA-B53 epitopes were not found in four other antigens. In this study we measured CTL responses to peptides from the recently sequenced antigen liver-stage antigen 3 (LSA-3) and identified in it a new epitope restricted by HLA-B53. Several CTL epitopes restricted by other class I types were also identified within LSA-3 in studies in The Gambia and Tanzania. CTL were also identified to an additional P. falciparum antigen, exported protein 1 (Exp-1), the homologue of which is a protective antigen in a rodent model of malaria. These findings emphasize the diversity of P. falciparum antigens recognized by CD8(+) T cells in humans and support the inclusion of components from several antigens in new CTL-inducing vaccines against malaria.

Host-parasite coevolution has been likened to a molecular arms race, with particular parasite genes evolving to evade specific host defenses. Study of the variants of an antigenic epitope of Plasmodium falciparum that induces a cytotoxic T cell response supports this view. In African children with malaria, the variants present are influenced by the presence of a human leukocyte antigen (HLA) type that restricts the immune response to this epitope. The distribution of parasite variants may be further influenced by the ability of cohabiting parasite strains to facilitate each other's survival by down-regulating cellular immune responses, using altered peptide ligand antagonism.

In this study, we have investigated the extent of natural polymorphism in the CD8+ cytotoxic T lymphocyte (CTL) determinant (amino acids 368-390) of circumsporozoite (CS) protein of Plasmodium falciparum field isolates from a holoendemic region of Kenya, and determined how this variation affects the CTL reactivities in clinically immune adults and binding specificities to human histocompatibility leukocyte antigen (HLA)-B35. Among the eight variant sequences that were found in this region, four were new and not seen in parasites from other geographical regions. When synthetic peptides corresponding to the eight variants were used to test the presence of CTL response in different donors, a different spectrum of CTL reactivity to these variants was noticed. While CTL from some donors recognized the P1 sequence (the most prevalent type of sequence) but not P8 (another major variant), other donors showed a reverse pattern of reactivity. Although none of the donors was able to recognize all the variants, CTL responses to all the eight variant sequences were found in this population. An octamer peptide with P1 sequence KPKDELDY in this polymorphic determinant was known to bind HLA-B35. When we tested the effect of natural variation in this octamer sequence on HLA-B35 binding, it became evident that SP13 with D --> N substitution retained its binding specificity to HLA-B35. On the other hand, the SP12 octamer sequence which had two substitutions did not bind HLA-B35. The most interesting finding was the observation that a D --> G substitution at position 374 rescued the binding ability of SP14, which otherwise could not bind to this HLA molecule due to E --> Q amino acid substitution at position 372. To our knowledge, this is the first demonstration showing that a natural polymorphism can rescue the binding specificity to an HLA-class I molecule that was lost due to another natural amino acid substitution. Altogether, these results demonstrate that natural polymorphism in the CS protein affects both the CTL reactivity and the ability to bind to HLA-B35.

Cytotoxic T lymphocytes (CTL) have been implicated in immunity to Plasmodium falciparum infection and disease. We have previously described the use of peptides to define malaria-specific CTL epitopes. To determine whether these peptide epitopes are processed intracellularly from the whole antigen we have developed recombinant vaccinia viruses (rVV) expressing three malaria antigens: thrombospondin-related adhesive protein (TRAP), Pfs16 and the C-terminal half of liver-stage antigen (LSA)-1. Target cells infected with recombinant viruses were lysed by malaria-specific CTL from semi-immune African donors. We also tested the ability of cells infected with these recombinant vaccinia viruses to re-stimulate malaria-specific CTL in peripheral blood lymphocytes from malaria immune adults. Two other pox virus recombinants, NYVAC, an attenuated vaccinia virus, and ALVAC, a canarypox virus, both expressing malaria antigens were also evaluated for their ability to stimulate malaria-specific CTL in contrast to peptide, none of these viruses successfully re-stimulated CTL from the peripheral blood lymphocytes of semi-immune donors. The ability of human CTL from naturally exposed individuals to recognize processed antigen supports the relevance of these cells in protective immunity to malaria.

Some individuals living in malaria-endemic areas have CTL to Plasmodium falciparum liver stage Ags. We have quantified these CTL responses using limiting dilution analysis studies on the peripheral blood cells of naturally exposed Gambian donors. CTL precursor frequencies were determined to a wide range of epitopes derived from different liver stage Ags (liver stage protein 1, circumsporozoite protein, thrombospondin-related anonymous protein, and sporozoite threonine/asparagine-rich protein) restricted through common HLA alleles present in this population (HLA-A2.1, -A2.2, -B7, -B8, -B35, and B53). Precursor frequencies were between 17 and 98/million PBMC and correlated with the levels of specific lysis in parallel bulk cultures. The quantitative nature of limiting dilution assay analysis revealed varying degrees of immunodominance in the CTL responses to different epitopes within single proteins (thrombospondin related anonymous protein: tr42, tr43, tr26, tr29, and tr39; circumsporozoite protein: cp6, cp26, and cp29) and within individual donors. The temporal stability of some of these CTL responses was determined over a 4-yr period. This is the first quantitative study of CTL specific for any plasmodial species or nonviral pathogen in humans and provides a basis for a multiepitope approach to malaria vaccination.

Studies in The Gambia have provided indirect evidence that cytotoxic T lymphocytes (CTL) play a protective role against malaria in humans and recently, using allele-specific HLA class I peptide motifs, several peptide epitopes for CTL in four pre-erythrocytic Plasmodium falciparum antigens have been identified in naturally exposed Gambians. However, CTL levels were low, suggesting that boosting these low levels by immunization might provide substantial protection. In the Kilombero valley of Tanzania, malaria transmission is holoendemic and 300 times more intense than in The Gambia. We report here that several of the epitopes identified in The Gambia are also recognized in naturally exposed, partially immune Tanzanian adults and that levels of CTL are similar to or slightly higher than in Gambian subjects, despite the much higher inoculation rate. We report a new HLA-A2.1-restricted epitope from the thrombospondin-related anonymous protein (TRAP) and we demonstrate that peptide epitopes in TRAP are naturally processed for recognition by CTL from naturally exposed humans. The common allele of a variable HLA-B7-restricted epitope in the circumsporozoite protein behaved as an altered peptide ligand (APL) with respect to CTL cognate for a rarer allelic variant of this epitope, suggesting that APL antagonism may occur in natural CTL responses to P. falciparum. The moderate levels of CTL observed, even in this area of intense malaria transmission, points to the need to assess candidate vaccines aimed at increasing CTL levels.

The protective association between the human leukocyte antigen HLA-B53 and severe malaria was investigated by sequencing of peptides eluted from this molecule followed by screening of candidate epitopes from pre-erythrocytic-stage antigens of Plasmodium falciparum in biochemical and cellular assays. Among malaria-immune Africans, HLA-B53-restricted cytotoxic T lymphocytes recognized a conserved nonamer peptide from liver-stage-specific antigen-1 (LSA-1), but no HLA-B53-restricted epitopes were identified in other antigens. These findings indicate a possible molecular basis for this HLA-disease association and support the candidacy of liver-stage-specific antigen-1 as a malaria vaccine component.

Clin Microbiol Infect 2011; 17: 1624-1631 Abstract Rapid diagnostic tests (RDTs) for malaria have improved the availability of parasite-based diagnosis throughout the malaria-endemic world. Accurate malaria diagnosis is essential for malaria case management, surveillance, and elimination. RDTs are inexpensive, simple to perform, and provide results in 15-20 min. Despite high sensitivity and specificity for Plasmodium falciparum infections, RDTs have several limitations that may reduce their utility in low-transmission settings: they do not reliably detect low-density parasitaemia (≤200 parasites/μL), many are less sensitive for Plasmodium vivax infections, and their ability to detect Plasmodium ovale and Plasmodium malariae is unknown. Therefore, in elimination settings, alternative tools with higher sensitivity for low-density infections (e.g. nucleic acid-based tests) are required to complement field diagnostics, and new highly sensitive and specific field-appropriate tests must be developed to ensure accurate diagnosis of symptomatic and asymptomatic carriers. As malaria transmission declines, the proportion of low-density infections among symptomatic and asymptomatic persons is likely to increase, which may limit the utility of RDTs. Monitoring malaria in elimination settings will probably depend on the use of more than one diagnostic tool in clinical-care and surveillance activities, and the combination of tools utilized will need to be informed by regular monitoring of test performance through effective quality assurance. [ABSTRACT FROM AUTHOR]