Anti-PfGARP activates programmed cell death of parasites and reduces severe malaria.

Malaria caused by Plasmodium falciparum remains the leading single-agent cause of mortality in children1, yet the promise of an effective vaccine has not been fulfilled. Here, using our previously described differential screening method to analyse the proteome of blood-stage P. falciparum parasites2, we identify P. falciparum glutamic-acid-rich protein (PfGARP) as a parasite antigen that is recognized by antibodies in the plasma of children who are relatively resistant-but not those who are susceptible-to malaria caused by P. falciparum. PfGARP is a parasite antigen of 80 kDa that is expressed on the exofacial surface of erythrocytes infected by early-to-late-trophozoite-stage parasites. We demonstrate that antibodies against PfGARP kill trophozoite-infected erythrocytes in culture by inducing programmed cell death in the parasites, and that vaccinating non-human primates with PfGARP partially protects against a challenge with P. falciparum. Furthermore, our longitudinal cohort studies showed that, compared to individuals who had naturally occurring anti-PfGARP antibodies, Tanzanian children without anti-PfGARP antibodies had a 2.5-fold-higher risk of severe malaria and Kenyan adolescents and adults without these antibodies had a twofold-higher parasite density. By killing trophozoite-infected erythrocytes, PfGARP could synergize with other vaccines that target parasite invasion of hepatocytes or the invasion of and egress from erythrocytes.

A Novel Antiparasitic Compound Kills Ring-Stage Plasmodium falciparum and Retains Activity Against Artemisinin-Resistant Parasites.

Spreading antimalarial resistance threatens effective treatment of malaria, an infectious disease caused by Plasmodium parasites. We identified a compound, BCH070, that inhibits asexual growth of multiple antimalarial-resistant strains of Plasmodium falciparum (half maximal inhibitory concentration [IC50] = 1-2 µM), suggesting that BCH070 acts via a novel mechanism of action. BCH070 preferentially kills early ring-form trophozoites, and, importantly, equally inhibits ring-stage survival of wild-type and artemisinin-resistant parasites harboring the PfKelch13:C580Y mutation. Metabolomic analysis demonstrates that BCH070 likely targets multiple pathways in the parasite. BCH070 is a promising lead compound for development of new antimalarial combination therapy that retains activity against artemisinin-resistant parasites.

Maternally-derived Antibodies to Schizont Egress Antigen-1 and Protection of Infants From Severe Malaria.

BACKGROUND: In holoendemic areas, children suffer the most from Plasmodium falciparum malaria, yet newborns and young infants express a relative resistance to both infection and severe malarial disease (SM). This relative resistance has been ascribed to maternally-derived anti-parasite immunoglobulin G; however, the targets of these protective antibodies remain elusive. METHODS: We enrolled 647 newborns at birth from a malaria-holoendemic region of Tanzania. We collected cord blood, measured antibodies to Plasmodium falciparum Schizont Egress Antigen-1 (PfSEA-1), and related these antibodies to the risk of severe malaria in the first year of life. In addition, we vaccinated female mice with PbSEA-1, mated them, and challenged their pups with P. berghei ANKA parasites to assess the impact of maternal PbSEA-1 vaccination on newborns' resistance to malaria. RESULTS: Children with high cord-blood anti-PfSEA-1 antibody levels had 51.4% fewer cases of SM compared to individuals with lower anti-PfSEA-1 levels over 12 months of follow-up (P = .03). In 3 trials, pups born to PbSEA-1-vaccinated dams had significantly lower parasitemia and longer survival following a P. berghei challenge compared to pups born to control dams. CONCLUSIONS: We demonstrate that maternally-derived, cord-blood anti-PfSEA-1 antibodies predict decreased risk of SM in infants and vaccination of mice with PbSEA-1 prior to pregnancy protects their offspring from lethal P. berghei challenge. These results identify, for the first time, a parasite-specific target of maternal antibodies that protect infants from SM and suggest that vaccination of pregnant women with PfSEA-1 may afford a survival advantage to their offspring.

Antibodies to PfsEGXP, an Early Gametocyte-Enriched Phosphoprotein, Predict Decreased Plasmodium falciparum Gametocyte Density in Humans.

Background: Antigametocyte-specific immune responses may regulate Plasmodium falciparum gametocyte density, providing the rationale for pursuing transmission-blocking vaccines (TBVs) that target gametocytes in the human host. Methods: To identify novel antigametocyte TBV antigens, we interrogated the gametocyte proteome with our whole proteome differential screening method using plasma from a treatment-reinfection study conducted in western Kenya. At the start of the high-transmission season, 144 males (12-35 years) were enrolled and treated with quinine and doxycycline, peripheral venous blood samples were obtained, volunteers were observed, and weekly blood films were obtained for 18 weeks to quantify gametocytemia. Using plasma pooled from individuals with low versus high gametocyte carriage, we differentially screened a P falciparum gametocyte stage complementary deoxyribonucleic acid expression library. Results: We identified 8 parasite genes uniquely recognized by gametocyte-resistant but not by gametocyte-susceptible individuals. Antibodies to one of these antigens, PfsEGXP, predicted lower gametocytemia measured over the 18-week transmission season (P = .021). When analyzed dichotomously, anti-PfsEGXP responders had 31% lower gametocyte density over 18 weeks of follow-up, compared with nonresponders (P = .04). Conclusions: PfsEGXP is one of the first reported gametocyte-specific target of antibodies that predict decreased gametocyte density in humans and supports our novel TBV antigen discovery platform.

Immunization with PfGBP130 generates antibodies that inhibit RBC invasion by P. falciparum parasites.

Background: Despite decades of effort, Plasmodium falciparum malaria remains a leading killer of children. The absence of a highly effective vaccine and the emergence of parasites resistant to both diagnosis as well as treatment hamper effective public health interventions. Methods and results: To discover new vaccine candidates, we used our whole proteome differential screening method and identified PfGBP130 as a parasite protein uniquely recognized by antibodies from children who had developed resistance to P. falciparum infection but not from those who remained susceptible. We formulated PfGBP130 as lipid encapsulated mRNA, DNA plasmid, and recombinant protein-based immunogens and evaluated the efficacy of murine polyclonal anti-PfGBP130 antisera to inhibit parasite growth in vitro. Immunization of mice with PfGBP130-A (aa 111-374), the region identified in our differential screen, formulated as a DNA plasmid or lipid encapsulated mRNA, but not as a recombinant protein, induced antibodies that inhibited RBC invasion in vitro. mRNA encoding the full ectodomain of PfGBP130 (aa 89-824) also generated parasite growth-inhibitory antibodies. Conclusion: We are currently advancing PfGBP130-A formulated as a lipid-encapsulated mRNA for efficacy evaluation in non-human primates.

A class of tricyclic compounds blocking malaria parasite oocyst development and transmission.

Malaria is a deadly infectious disease in many tropical and subtropical countries. Previous efforts to eradicate malaria have failed, largely due to the emergence of drug-resistant parasites, insecticide-resistant mosquitoes and, in particular, the lack of drugs or vaccines to block parasite transmission. ATP-binding cassette (ABC) transporters are known to play a role in drug transport, metabolism, and resistance in many organisms, including malaria parasites. To investigate whether a Plasmodium falciparum ABC transporter (Pf14_0244 or PfABCG2) modulates parasite susceptibility to chemical compounds or plays a role in drug resistance, we disrupted the gene encoding PfABCG2, screened the recombinant and the wild-type 3D7 parasites against a library containing 2,816 drugs approved for human or animal use, and identified an antihistamine (ketotifen) that became less active against the PfABCG2-disrupted parasite in culture. In addition to some activity against asexual stages and gametocytes, ketotifen was highly potent in blocking oocyst development of P. falciparum and the rodent parasite Plasmodium yoelii in mosquitoes. Tests of structurally related tricyclic compounds identified additional compounds with similar activities in inhibiting transmission. Additionally, ketotifen appeared to have some activity against relapse of Plasmodium cynomolgi infection in rhesus monkeys. Further clinical evaluation of ketotifen and related compounds, including synthetic new derivatives, in blocking malaria transmission may provide new weapons for the current effort of malaria eradication.

Protocol for Differential Biopanning of P. falciparum Phage Display cDNA Library to Identify Parasite Targets of Protective Antibodies.

Malaria remains a significant global health burden, killing hundreds of thousands of children annually (WHO, The world malaria report. WHO, Geneva, 2019). Despite decades of effort, no broadly effective vaccine exists. Differential screening of parasite phage display libraries is a promising approach to identify the targets of human antibodies expressed by resistant but not by susceptible individuals (Raj et al., Nature, 582, 104-108, 2020; Science, 344, 871-877, 2014). Our whole proteome differential screening (WPDS) approach consists of positive selection to capture phage that bind antibodies expressed by malaria-resistant individuals, followed by negative selection to remove phage that bind antibodies expressed by malaria-susceptible individuals, and amplification of differentially recognized clones.

A newly characterized malaria antigen on erythrocyte and merozoite surfaces induces parasite inhibitory antibodies.

We previously identified a Plasmodium falciparum (Pf) protein of unknown function encoded by a single-copy gene, PF3D7_1134300, as a target of antibodies in plasma of Tanzanian children in a whole-proteome differential screen. Here we characterize this protein as a blood-stage antigen that localizes to the surface membranes of both parasitized erythrocytes and merozoites, hence its designation as Pf erythrocyte membrane and merozoite antigen 1 (PfEMMA1). Mouse anti-PfEMMA1 antisera and affinity-purified human anti-PfEMMA1 antibodies inhibited growth of P. falciparum strains by up to 68% in growth inhibition assays. Following challenge with uniformly fatal Plasmodium berghei (Pb) ANKA, up to 40% of mice immunized with recombinant PbEMMA1 self-cured, and median survival of lethally infected mice was up to 2.6-fold longer than controls (21 vs. 8 d, P = 0.005). Furthermore, high levels of naturally acquired human anti-PfEMMA1 antibodies were associated with a 46% decrease in parasitemia over 2.5 yr of follow-up of Tanzanian children. Together, these findings suggest that antibodies to PfEMMA1 mediate protection against malaria.

Lower limb biomechanics before and after anterior cruciate ligament reconstruction: A systematic review.

This review aimed to synthesise the findings of literature that have assessed the changes in lower limb biomechanics following anterior cruciate ligament (ACL) reconstructive surgery. Systematic searches of CINHAL, MEDLINE, SCOPUS, and SPORTDiscus databases were run. All included studies had presented biomechanical variables pre- and post-surgery for the same participants. Articles were categorised by the analysed movement, and effect sizes were calculated. Fifty-four studies met the inclusion criteria, providing data on gait (n = 31), balance (n = 12), joint position sense (n = 5), stair ambulation (n = 4), pivoting (n = 6), and landing (n = 5). Measures of balance performance and joint position sense showed improvements from pre- to post-surgery. Changes in joint kinematics were inconsistent between studies, however increased knee flexion excursion, and reduced tibial anterior translation and internal rotation post reconstruction were identified. Joint kinetics reduced in magnitude in the early stages after surgery (≤5 weeks), then increased later in recovery (≥24 weeks). Risk of bias assessment identified most articles had a moderate or high risk (low = 5; moderate = 21; high = 11) resulting from participant retention and surgical intervention differences. The results of the review identified that although lower limb biomechanics did alter following reconstruction, few variables provided consistent results across studies and tasks. The low methodological quality of some articles may have contributed to these inconsistent findings. Alternatively, differences across studies may have resulted from individual coping strategies of participants that have previously been suggested to be present before reconstructive surgery, and future research should look to explore individual coping strategies to ACL reconstruction.

Cauda equina syndrome secondary to lumbar disc herniation.

The authors conducted a retrospective study of 8 cases of cauda equina syndrome (CES) due to a herniated lumbar intervertebral disc, in order to highlight the clinical presentation and outcome after urgent surgical decompression. Between 1995 and 1999, 8 patients (4 men and 4 women) with ages ranging from 43 to 64 years (mean 53 years) presented with CES. There were two modes of presentation: acute to subacute (4 patients) and insidious (4 patients). In 3 patients the precipitating factor was stooping, with or without heavy lifting. One patient gave a history of stooping and heavy lifting, followed by manipulation therapy. Urinary bladder involvement was present in all patients (100%). Perianal sensory involvement was present in four cases, bilateral in two and unilateral in the other two. Seven patients had complete recovery of bladder function. One patient had residual urinary symptoms, though she was continent; she had been operated upon within 33 hours, within the classical 48 hours suggested by larger series. Residual weakness of lower limb muscles was present in three cases. In this small series there was no distinct correlation between timing of operation and results. Conclusion : The classical presentation of CES is not obvious. Surgery as an emergency is recommended: within 48 hours, according to the old rule. Even if surgery is done late due to delayed presentation, significant improvement in the bladder function can still be expected.

Stem fracture of a collarless, polished, double-taper cemented femoral prosthesis: a case report.

We report a case of stem fracture of a collarless, polished, double-taper cemented stainless steel femoral prosthesis. Scanning electron microscopy showed fatigue striations, indicating failure by fatigue. This case study highlights that stem fracture can still occur with modern implants. Size 0 CPT stem should be avoided in patients with high body mass index. To our knowledge this is the first case report of a CPT stem fracture used for primary total hip replacement.

Introducing a New Junior Doctor Electronic Weekend Handover on an Orthopaedic Ward.

Junior Doctors working on the Orthopaedic wards at a district general hospital identified the lack of a formal weekend handover. The Royal Colleges,GMC and Foundation Programme curriculum all emphasise the importance of a safe and effective handover. Doctors found that the current system of using a written, paper-based handover was unreliable, un-legible, and inefficient. Baseline measurements were sought in the form of a questionnaire which allowed us to obtain the limitations to the current handover. After this and a focus group, a new electronic, 'Microsoft Word' based handover was created and a repeat surgery issued in 2 weeks. Further PDSA cycles over the course of 8 weeks helped to improve and implement the new handover. The overall rating, out of 10, of the new handover increased from 3.4 to 8. Doctors felt the new handover was safer for patients and could be used as a tool for reviewing or referring patients. This project describes the use of a simple, cost-effective intervention that helped to improve patient safety and staff satisfaction.

Identification of Protective B-Cell Epitopes within the Novel Malaria Vaccine Candidate Plasmodium falciparum Schizont Egress Antigen 1.

Naturally acquired antibodies to Plasmodium falciparum schizont egress antigen 1 (PfSEA-1A) are associated with protection against severe malaria in children. Vaccination of mice with SEA-1A from Plasmodium berghei (PbSEA-1A) decreases parasitemia and prolongs survival following P. berghei ANKA challenge. To enhance the immunogenicity of PfSEA-1A, we identified five linear B-cell epitopes using peptide microarrays probed with antisera from nonhuman primates vaccinated with recombinant PfSEA-1A (rPfSEA-1A). We evaluated the relationship between epitope-specific antibody levels and protection from parasitemia in a longitudinal treatment-reinfection cohort in western Kenya. Antibodies to three epitopes were associated with 16 to 17% decreased parasitemia over an 18-week high transmission season. We are currently designing immunogens to enhance antibody responses to these three epitopes.

An efficient PCR--SSCP-based method for detection of a chloroquine resistance marker in the PfCRT gene of Plasmodium falciparum.

The spread of chloroquine resistance throughout the world poses a major problem in combating malaria. In the present study, an efficient polymerase chain reaction-single strand conformational polymorphism (PCR--SSCP)-based assay detected the PfCRT K76T point mutation, which is a marker for chloroquine resistance. For the first time, we have used a PCR--SSCP-based technique to identify the mutation in a single-step labelling reaction during PCR and SSCP gel electrophoresis. This assay is 100% efficient, giving no false-positive or -negative results, and can be carried out within a short bench time. We have successfully analysed 120 natural isolates using the PCR-SSCP method for detection of the chloroquine resistance marker and found 91 of the 120 samples to show the PfCRT T76 mutation, and 71% (65 of the 91 samples) showed a positive correlation with chloroquine resistance from the clinical data of the patients. The PCR-SSCP technique can also be applied for the detection of new haplotypes of the PfCRT gene and surveillance of chloroquine-resistant malaria in malaria-endemic localities around the world.

Combined detection of Brugia malayi and Wuchereria bancrofti using single PCR.

A single step PCR method has been developed for the combined detection of the human filarial parasites, Brugia malayi and Wuchereria bancrofti. Parasites' DNA were isolated from filaria positive blood samples that were collected from endemic areas. The primers used were Hha1 and Ssp I, which amplified the DNA fragments of 322 bp and 188 bp specific to B. malayi and W. bancrofti, respectively. The sensitivity of the assay was tested with blood and mosquito samples having one W. bancrofti in a pool of 10 B. malayi. The assay was further evaluated on field collected blood and mosquito samples. Use of this assay as a diagnostic tool for the detection of filariasis being the most promising aspect of this study, offers scope for detection of both the parasites even at low levels of infection.

Genetic diversity in the merozoite surface protein 1 gene of Plasmodium falciparum in different malaria-endemic localities.

A number of stage-specific antigens have been characterized for vaccine development in Plasmodium falciparum malaria. The polymorphic merozoite surface protein 1 (MSP-1) of Plasmodium falciparum is a major asexual blood stage malaria vaccine candidate antigen. In the present study, we analyzed the impact of hyperendemic malaria transmission, mesoendemic malaria transmission, and multiple infection on allelic diversity. We have used a simple strategy of polymerase chain reaction amplification and slot-blot hybridization to analyze variable regions of block-2, block-4 and blocks 6-10 of the MSP-1 gene. The allelic types of isolates collected from regions of hyperendemic malaria transmission (RHEMT) and mesoendemic malaria transmission (RMEMT) were compared. In RHEMT, 20 of 24 possible gene types were found among 163 isolates and more than one allelic type was found in 82 (50.3%) of the isolates. Thirteen of 24 possible gene types were found among 125 isolates in RMEMT and 27 (21.6%) of them contained more than one allele type. Our results suggest for the first time that the allelic distribution or allelic diversity and chances of finding multi-strain parasites in isolates in an area vary with the rate of transmission. Analyses of isolates containing more than one strain of parasite suggest that allelic types are randomly distributed, no specific type of alleles predominately show multi-strain infection, and neither strain of the parasite affect the process of infection and development of another.

Antibodies to PfSEA-1 block parasite egress from RBCs and protect against malaria infection.

Novel vaccines are urgently needed to reduce the burden of severe malaria. Using a differential whole-proteome screening method, we identified Plasmodium falciparum schizont egress antigen-1 (PfSEA-1), a 244-kilodalton parasite antigen expressed in schizont-infected red blood cells (RBCs). Antibodies to PfSEA-1 decreased parasite replication by arresting schizont rupture, and conditional disruption of PfSEA-1 resulted in a profound parasite replication defect. Vaccination of mice with recombinant Plasmodium berghei PbSEA-1 significantly reduced parasitemia and delayed mortality after lethal challenge with the Plasmodium berghei strain ANKA. Tanzanian children with antibodies to recombinant PfSEA-1A (rPfSEA-1A) did not experience severe malaria, and Kenyan adolescents and adults with antibodies to rPfSEA-1A had significantly lower parasite densities than individuals without these antibodies. By blocking schizont egress, PfSEA-1 may synergize with other vaccines targeting hepatocyte and RBC invasion.

Disruption of a Plasmodium falciparum multidrug resistance-associated protein (PfMRP) alters its fitness and transport of antimalarial drugs and glutathione.

ATP-binding cassette transporters play an important role in drug resistance and nutrient transport. In the human malaria parasite Plasmodium falciparum, a homolog of the human p-glycoprotein (PfPgh-1) was shown to be involved in resistance to several drugs. More recently, many transporters were associated with higher IC(50) levels in responses to chloroquine (CQ) and quinine (QN) in field isolates. Subsequent studies, however, could not confirm the associations, although inaccuracy in drug tests in the later studies could contribute to the lack of associations. Here we disrupted a gene encoding a putative multidrug resistance-associated protein (PfMRP) that was previously shown to be associated with P. falciparum responses to CQ and QN. Parasites with disrupted PfMRP (W2/MRPDelta) could not grow to a parasitemia higher than 5% under normal culture conditions, possibly because of lower efficiency in removing toxic metabolites. The W2/MRPDelta parasite also accumulated more radioactive glutathione, CQ, and QN and became more sensitive to multiple antimalarial drugs, including CQ, QN, artemisinin, piperaquine, and primaquine. PfMRP was localized on the parasite surface membrane, within membrane-bound vesicles, and along the straight side of the D-shaped stage II gametocytes. The results suggest that PfMRP plays a role in the efflux of glutathione, CQ, and QN and contributes to parasite responses to multiple antimalarial drugs, possibly by pumping drugs outside the parasite.

The development and evaluation of a single step multiplex PCR method for simultaneous detection of Brugia malayi and Wuchereria bancrofti.

A single step novel multiplex polymerase chain reaction (PCR) has been developed for simultaneous detection of human filarial parasites, Brugia malayi and Wuchereria bancrofti, from blood samples and mosquitoes. The primers used were novel and have been tested with the parasite DNA amplifying 188bp (BM) and 129bp (WB) DNA fragments, specific to B. malayi and W. bancrofti, respectively, in a single reaction. The specificity of the PCR product was confirmed by DNA sequencing and slot blot hybridization assay. The test was found highly sensitive for both B. malayi and W. bancrofti by detecting the parasitaemia up to the level of one microfilaria per reaction. The assay was further evaluated on 98 blood samples and 144 mosquito samples collected from filarial endemic areas. The PCR was found to be more efficient in comparison to microscopy by detecting 8% and 5% more filarial parasites in field-collected blood and mosquito samples, respectively. This novel PCR that offers scope for simultaneous detection of both the parasites may be used as a diagnostic tool for the detection of filariasis in population and can be adopted for rapid surveillance and monitoring of mosquitoes for use in the effective control of filariasis.