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Background: African American breast cancer survivors may be at high risk for reproductive health problems, including menopause symptoms, sexual dysfunction, and distress about cancer-related infertility. The authors partnered with Sisters Network Inc. to create the Sisters Peer Counseling in Reproductive Issues After Treatment (SPIRIT) program, a culturally sensitive intervention program that combined a written workbook and peer counseling.Methods: Three hundred women were randomized to receive either the workbook plus 3 in-person sessions with a trained peer counselor or the workbook plus ≤ 30 minutes of telephone counseling to be initiated by the participant. Questionnaires at baseline, post-treatment, and at 6-month and 12-month follow-up assessed emotional distress, sexual function, relationship satisfaction, spirituality, menopause symptoms, and knowledge. Satisfaction with the program and the use of medical care also were assessed.Results: Both groups of women improved significantly in knowledge, decreased in distress, and had decreased hot flashes. Sexually active women had improved sexual function at 6-month follow-up but not at 1 year. However, peer counseling had little incremental benefit over the telephone counseling.Conclusions: The SPIRIT program was rated very useful by 66% of women. Outcomes justify continued use of the workbook and further research to optimize the impact of peer counseling. [ABSTRACT FROM AUTHOR]

Chronic upper limb pain often causes work loss, and return to work after pain management is disappointingly low. This study aimed to identify patient characteristics and beliefs contributing to return to work or nonreturn. A total of 103 (66%) ex-patients with CULP, who had completed a pain management program, agreed to telephone interview. Participants were predominantly female and in middle years; 53.4% were working part- or full-time. Their responses were related to pre- and posttreatment psychological and disability variables. Those patients who had returned to work, compared to those who had not, were more likely to have been working shortly before treatment (chi 2 = 36.77, p < 0.00001). They were more psychologically robust and were more confident of managing pain (t = 4.55, p < 0.001), and catastrophized less (t = 2.21, p = 0.029). They were also more optimistic about being capable of work (u = 566, p < 0.0001) and of overcoming obstacles to work (u = 889, p = 0.0103). Workers and nonworkers were not differentiated by expectations of support from their immediate line manager, although nonworkers doubted support available from colleagues. Overall, despite generalization of pain management strategies in nonwork activity, return to work depended on specific beliefs concerning work-relevant strategies. [ABSTRACT FROM AUTHOR]

Background: Plasmodium vivax is the dominant Plasmodium spp. causing malaria throughout tropical and sub-tropical countries. Humoral immunity is induced during P. vivax infection. However, data on longevity of antibody and memory B cell (MBC) responses is lacking. Follicular helper T cells (Tfh) are drivers of high-affinity and long-lived antibody responses. Understanding of Tfh-mediated immunity against malaria is valuable for vaccine development., Methodology/principal Findings: We enrolled 31 acutely infected P. vivax patients in low malaria transmission areas of Thailand to detect frequencies, phenotypes and kinetics of different subsets of circulating Tfh (cTfh) and MBCs, and to evaluate their association with humoral immunity following natural P. vivax infection. Expansion of cTfh2 cells, activated and atypical MBCs were shown during acute malaria. To relate increased cTfh2 cells to humoral immunity, P. vivax-specific MBCs and antibodies were assessed. High anti-PvCSP and -PvDBPII seropositivity was detected and most subjects produced MBCs specific to these antigens. The increased cTfh2 cells were positively related to atypical MBCs, plasmablasts/plasma cells, and anti-PvDBPII IgM and IgG levels. Distributions of memory cTfh cell subsets were altered from central memory (CM) to effector memory (EM) during infection. The highest ratios of cTfh-EM/cTfh-CM were represented in cTfh2 cells. Positive correlation of cTfh17-EM with activated and atypical MBCs was observed, while cTfh2-CM and cTfh17-CM cells were positively related to PvDBPII-specific MBCs and IgM levels., Conclusions/significance: Present study demonstrated that P. vivax infection induced cTfh polarization into cTfh2 subset, and alteration of memory cTfh2 phenotype from CM to EM phase. These P. vivax-induced cTfh responses significantly associated with generation of MBCs and antibody responses. Therefore, cTfh2 cells might possibly influence humoral immunity by inducing expansion of activated and atypical MBCs, and by generating P. vivax-specific MBCs and antibody responses following natural infection., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Thawornpan et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

As exploration of ocean depths >1000 m is only possible by expensive remotely operated underwater vehicles, deep-sea invertebrates represent a largely untapped source of marine metabolites for potential applications in medicine. Our current study aims to investigate these deep-sea invertebrates in Ireland to discover new biological and chemical diversity. Here, we investigate the bubble gum coral, Paragorgia arborea , collected at 1500 m depth from Whittard canyon in the Northeastern Atlantic. This species was selected following chemical profiling and biological screening. The isolation and structure elucidation of the main metabolites yielded three new diterpenes, namely, miolenol ( 1 ) and epoxymiolenol ( 2 ) characterized by the rare bicyclo[7.2.0]undec-4-ene skeleton, and the xeniolide epoxycoraxeniolide A ( 3 ), together with five known diterpenes. The structures of the new compounds were identified through extensive NMR analysis with their absolute configurations assigned by comparison between experimental and TDDFT-calculated ECD. The eight compounds were screened for cytotoxicity and antimalarial activity, and none displayed noteworthy bioactivity., Competing Interests: The authors declare no competing financial interest., (© 2024 The Authors. Published by American Chemical Society.)

Background: Limited English proficiency is associated with worse health outcomes regardless of health literacy. Prior research suggests that using interpreter services for low English proficiency helps mitigate the language barrier, is associated with improved health outcomes, and patient satisfaction; however, obstetric and neonatal outcomes and pregnancy risks in this population are not well studied., Objectives: The primary purpose of this study was to determine if low English proficiency is an independent risk factor for small for gestational age infants by utilizing interpreter use as a proxy for low English proficiency. Due to the known challenges in communication with a language barrier and discrimination against people whose first language is not English, we hypothesized that this could result in an increase in high risk conditions in pregnancy such as SGA. Our hypothesis was that the need for an interpreter would be associated with having small for gestational age infants., Study Design: We performed a retrospective cohort study at a single center using data between 1/1/2016 and 12/31/2021; we included singleton, live births ≥ 21 weeks gestation. We excluded multiple gestations, intrauterine fetal demise, and delivery < 21 weeks. The primary outcome was rate of small for gestational age. Small for gestational age was defined as birthweight < 10th percentile for gestational age using the 2018 Fenton newborn growth curve. Multivariable logistic regression was performed to control for confounding variables., Results: Of the 26,260 patients included in the study, 71.3% were non-Hispanic White, 9.5% were Hispanic/Latino, and 7.9% were non-Hispanic Black. Overall, 1,662 (6.3%) patients utilized an interpreter. Over half (58.0%) of patients requesting interpreter services were Hispanic. In unadjusted analyses, the rate of small for gestational age was not different between patients who used interpreter services (n=106, 6.4%) and those who did not (n=1612, 6.6 %), P=.779. After adjusting for race/ethnicity, gravidity, gestational age, private insurance, diabetes, hypertension, and prepregnancy body mass index, the use of interpreter services was associated with decreased odds of small for gestational age (aOR 0.67, 95% CI 0.53-0.84)., Conclusions: Our findings suggest that use of an interpreter is associated with a lower incidence of small for gestational age when controlling for patient characteristics and social determinants of health. Additional research is required to explore this association, but our results indicate that recognizing demographic risk factors and providing patients with social resources such as access to interpreter services may positively impact obstetric and neonatal outcomes., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Most Plasmodium vivax infections contain genetically distinct parasites, but the consequences of this polyclonality on the development of asexual parasites, their sexual differentiation, and their transmission remain unknown. We describe infections of Saimiri monkeys with two strains of P. vivax and the analyses of 80,024 parasites characterized by single cell RNA sequencing and individually genotyped. In our model, consecutive inoculations fail to establish polyclonal infections. By contrast, simultaneous inoculations of two strains lead to sustained polyclonal infections, although without detectable differences in parasite regulation or sexual commitment. Analyses of sporozoites dissected from mosquitoes fed on coinfected monkeys show that all genotypes are successfully transmitted to mosquitoes. However, after sporozoite inoculation, not all genotypes contribute to the subsequent blood infections, highlighting an important bottleneck during pre-erythrocytic development. Overall, these studies provide new insights on the mechanisms regulating the establishment of polyclonal P. vivax infections and their consequences for disease transmission., (© 2024. The Author(s).)

In malaria parasites, the erythrocyte binding-like proteins (EBL) are a family of invasion proteins that are attractive vaccine targets. In the case of Plasmodium vivax, the widespread malaria parasite, blood-stage vaccines have been largely focused on a single EBL candidate, the Duffy binding-like domain (DBL) of the Duffy binding protein (DBPII), due to its well-characterized role in the reticulocyte invasion. A novel P. vivax EBL family member, the Erythrocyte binding protein (EBP2, also named EBP or DBP2), binds preferentially to reticulocytes and may mediate an alternative P. vivax invasion pathway. To gain insight into the natural genetic diversity of the DBL domain of EBP2 (region II; EBP2-II), we analyzed ebp2-II gene sequences of 71 P. vivax isolates collected in different endemic settings of the Brazilian Amazon rainforest, where P. vivax is the predominant malaria-associated species. Although most of the substitutions in the ebp2-II gene were non-synonymous and suggested positive selection, the results showed that the DBL domain of the EBP2 was much less polymorphic than that of DBPII. The predominant EBP2 haplotype in the Amazon region corresponded to the C127 reference sequence first described in Cambodia (25% C127-like haplotype). An overview of ebp2-II gene sequences available at GenBank (n = 352) from seven countries (Cambodia, Madagascar, Myanmar, PNG, South Korea, Thailand, Vietnam) confirmed the C127-like haplotype as highly prevalent worldwide. Two out of 43 haplotypes (5 to 20 inferred per country) showed a global frequency of 60%. The results presented here open new avenues of research pursuit while suggesting that a vaccine based on the DBL domain of EBP2 should target a few haplotypes for broad coverage., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

CelTOS is a malaria vaccine antigen that is conserved in Plasmodium and other apicomplexan parasites and plays a role in cell-traversal. The structural basis and mechanisms of CelTOS-induced protective immunity to parasites are unknown. Here, CelTOS-specific monoclonal antibodies (mAbs) 7g7 and 4h12 demonstrated multistage activity, protecting against liver infection and preventing parasite transmission to mosquitoes. Both mAbs demonstrated cross-species activity with sterile protection against in vivo challenge with transgenic parasites containing either P. falciparum or P. vivax CelTOS, and with transmission reducing activity against P. falciparum. The mAbs prevented CelTOS-mediated pore formation providing insight into the protective mechanisms. X-ray crystallography and mutant-library epitope mapping revealed two distinct broadly conserved neutralizing epitopes. 7g7 bound to a parallel dimer of CelTOS, while 4h12 bound to a novel antiparallel dimer architecture. These findings inform the design of antibody therapies and vaccines and raise the prospect of a single intervention to simultaneously combat P. falciparum and P. vivax malaria., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Ivermectin, a broad-spectrum anti-parasitic drug, has been proposed as a novel vector control tool to reduce malaria transmission by mass drug administration. Ivermectin and some metabolites have mosquito-lethal effect, reducing Anopheles mosquito survival. Ivermectin inhibits liver stage development in a rodent malaria model, but no inhibition was observed in a primate malaria model or in a human malaria challenge trial. In the liver, cytochrome P450 3A4 and 3A5 enzymes metabolize ivermectin, which may impact drug efficacy. Thus, understanding ivermectin metabolism and assessing this impact on Plasmodium liver stage development is critical. Using primary human hepatocytes (PHHs), we characterized ivermectin metabolism and evaluated the efficacy of ivermectin and its primary metabolites M1 (3″- O -demethyl ivermectin) and M3 (4-hydroxymethyl ivermectin) against Plasmodium falciparum liver stages. Two different modes of ivermectin exposure were evaluated: prophylactic mode (days 0-3 post-infection) and curative mode (days 3-5 post-infection). We used two different PHH donors and modes to determine the inhibitory concentration (IC 50 ) of ivermectin, M1, M3, and the known anti-malarial drug pyrimethamine, with IC 50 values ranging from 1.391 to 14.44, 9.95-23.71, 4.767-8.384, and 0.9073-5.416 µM, respectively. In our PHH model, ivermectin and metabolites M1 and M3 demonstrated inhibitory activity against P. falciparum liver stages in curative treatment mode (days 3-5) and marginal activity in prophylactic treatment mode (days 0-3). Ivermectin had improved efficacy when co-administered with ketoconazole, a specific inhibitor of cytochrome P450 3A4 enzyme. Further studies should be performed to examine ivermectin liver stage efficacy when co-administered with CYP3A4 inhibitors and anti-malarial drugs to understand the pharmacokinetic and pharmacodynamic drug-drug interactions that enhance efficacy against human malaria parasites in vitro ., Competing Interests: The authors declare no conflict of interest.

During May-July 2023, a cluster of 7 patients at local hospitals in Florida, USA, received a diagnosis of Plasmodium vivax malaria. Whole-genome sequencing of the organism from 4 patients and phylogenetic analysis with worldwide representative P. vivax genomes indicated probable single parasite introduction from Central/South America.

Malaria parasites have adaptive mechanisms to modulate their intracellular redox status to tolerate the enhanced oxidizing effects created by malaria fever, hemoglobinopathies and other stress conditions, including antimalaria drugs. Emerging artemisinin (ART) resistance in Plasmodium falciparum is a complex phenotype linked to the parasite's tolerance of the activated drug's oxidative damage along with changes in vesicular transport, lipid metabolism, DNA repair, and exported proteins. In an earlier study, we discovered that many of these metabolic processes are induced in P. falciparum to respond to the oxidative damage caused by artemisinin, which exhibited a highly significant overlap with the parasite's adaptive response mechanisms to survive febrile temperatures. In addition, there was a significant overlap with the parasite's survival responses to oxidative stress. In this study, we investigated these relationships further using an in vitro model to evaluate if oxidative stress and heat-shock conditions could alter the parasite's response to artemisinin. The results revealed that compared to ideal culture conditions, the antimalarial efficacy of artemisinin was significantly reduced in parasites growing in intraerythrocytic oxidative stress but not in heat-shock condition. In contrast, heat shock significantly reduced the efficacy of lumefantrine that is an important ART combination therapy partner drug. We propose that prolonged exposure to intraerythrocytic microenvironmental oxidative stress, as would occur in endemic regions with high prevalence for sickle trait and other hemoglobinopathies, can predispose malaria parasites to develop tolerance to the oxidative damage caused by antimalarial drugs like artemisinin., Importance: Emerging resistance to the frontline antimalarial drug artemisinin represents a significant threat to worldwide malaria control and elimination. The patterns of parasite changes associated with emerging resistance represent a complex array of metabolic processes evident in various genetic mutations and altered transcription profiles. Genetic factors identified in regulating P. falciparum sensitivity to artemisinin overlap with the parasite's responses to malarial fever, sickle trait, and other types of oxidative stresses, suggesting conserved inducible survival responses. In this study we show that intraerythrocytic stress conditions, oxidative stress and heat shock, can significantly decrease the sensitivity of the parasite to artemisinin and lumefantrine, respectively. These results indicate that an intraerythrocytic oxidative stress microenvironment and heat-shock condition can alter antimalarial drug efficacy. Evaluating efficacy of antimalarial drugs under ideal in vitro culture conditions may not accurately predict drug efficacy in all malaria patients., Competing Interests: The authors declare no conflict of interest.

In Plasmodium vivax, the most studied vaccine antigens are aimed at blocking merozoite invasion of erythrocytes and disease development. Very few studies have evaluated pre-erythrocytic (PE) stage antigens. The P. vivax circumsporozoite protein (CSP), is considered the leading PE vaccine candidate, but immunity to CSP is short-lived and variant specific. Thus, there is a need to identify other potential candidates to partner with CSP in a multivalent vaccine to protect against infection and disease. We hypothesize that sporozoite antigens important for host cell infection are considered potential targets. In this study, we evaluated the magnitude and quality of naturally acquired antibody responses to four P. vivax PE antigens: sporozoite surface protein 3 (SSP3), sporozoite protein essential for traversal 1 (SPECT1), cell traversal protein of ookinetes and sporozoites (CelTOS) and CSP in plasma of P. vivax infected patients from Thailand. Naturally acquired antibodies to these antigens were prevalent in the study subjects, but with significant differences in magnitude of IgG antibody responses. About 80% of study participants had antibodies to all four antigens and only 2% did not have antibodies to any of the antigens. Most importantly, these antibodies inhibited sporozoite infection of hepatocytes in vitro. Significant variations in magnitude of antigen-specific inhibitory antibody responses were observed with individual samples. The highest inhibitory responses were observed with anti-CelTOS antibodies, followed by anti-SPECT1, SSP3 and CSP antibodies respectively. These data highlight the vaccine potential of these antigens in protecting against hepatocyte infection and the need for a multi-valent pre-erythrocytic vaccine to prevent liver stage development of P. vivax sporozoites., (© 2024. The Author(s).)

CRISPR/Cas9 technology applied to Plasmodium falciparum offers the potential to greatly improve gene editing, but such expectations including large DNA fragment knock-ins and sequential gene editing have remained unfulfilled. Here, we achieved a major advance in addressing this challenge, especially for creating large DNA fragment knock-ins and sequential editing, by modifying our suicide-rescue-based system that has already been demonstrated to be highly efficient for conventional gene editing. This improved approach was confirmed to mediate efficient knock-ins of DNA fragments up to 6.3 kb, to produce "marker-free" genetically engineered parasites and to show potential for sequential gene editing. This represents an important advancement in establishing platforms for large-scale genome editing, which might gain a better understanding of gene function for the most lethal cause of malaria and contribute to adjusting synthetic biology strategies to live parasite malaria vaccine development. Site-directed knock-in of large DNA fragments is highly efficient using suicide-rescue-based CRISPR/Cas9 system, and sequential gene insertion is feasible but further confirmation is still needed., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: Acquired functional inhibitory antibodies are one of several humoral immune mechanisms used to neutralize foreign pathogens. In vitro bioassays are useful tools for quantifying antibody-mediated inhibition and evaluating anti-parasite immune antibodies. However, a gap remains in understanding of how antibody-mediated inhibition in vitro translates to inhibition in vivo. In this study, two well-characterized transgenic Plasmodium berghei parasite lines, PbmCh-luc and Pb-PfCSP(r), and murine monoclonal antibodies (mAbs) specific to P. berghei and Plasmodium falciparum circumsporozoite protein (CSP), 3D11 and 2A10, respectively, were used to evaluate antibody-mediated inhibition of parasite development in both in vitro and in vivo functional assays., Methods: IC 50 values of mAbs were determined using an established inhibition of liver-stage development assay (ILSDA). For the in vivo inhibition assay, mice were passively immunized by transfer of the mAbs and subsequently challenged with 5.0 × 10 3 sporozoites via tail vein injection. The infection burden in both assays was quantified by luminescence and qRT-PCR of P. berghei 18S rRNA normalized to host GAPDH., Results: The IC 50 values quantified by relative luminescence of mAbs 3D11 and 2A10 were 0.396 µg/ml and 0.093 µg/ml, respectively, against transgenic lines in vitro. Using the highest (> 90%) inhibitory antibody concentrations in a passive transfer, an IC 50 of 233.8 µg/ml and 181.5 µg/ml for mAbs 3D11 and 2A10, respectively, was observed in vivo. At 25 µg (250 µg/ml), the 2A10 antibody significantly inhibited liver burden in mice compared to control. Additionally, qRT-PCR of P. berghei 18S rRNA served as a secondary validation of liver burden quantification., Conclusions: Results from both experimental models, ILSDA and in vivo challenge, demonstrated that increased concentrations of the homologous anti-CSP repeat mAbs increased parasite inhibition. However, differences in antibody IC 50 values between parasite lines did not allow a direct correlation between the inhibition of sporozoite invasion in vitro by ILSDA and the inhibition of mouse liver stage burden. Further studies are needed to establish the conditions for confident predictions for the in vitro ILSDA to be a predictor of in vivo outcomes using this model system., (© 2023. The Author(s).)

The pathophysiology of severe falciparum malaria involves a complex interaction between the host, parasite, and gut microbes. In this review, we focus on understanding parasite-induced intestinal injury and changes in the human intestinal microbiota composition in patients with Plasmodium falciparum malaria. During the blood stage of P. falciparum infection, infected red blood cells adhere to the vascular endothelium, leading to widespread microcirculatory obstruction in critical tissues, including the splanchnic vasculature. This process may cause intestinal injury and gut leakage. Epidemiological studies indicate higher rates of concurrent bacteraemia in severe malaria cases. Furthermore, severe malaria patients exhibit alterations in the composition and diversity of the intestinal microbiota, although the exact contribution to pathophysiology remains unclear. Mouse studies have demonstrated that the gut microbiota composition can impact susceptibility to Plasmodium infections. In patients with severe malaria, the microbiota shows an enrichment of pathobionts, including pathogens that are known to cause concomitant bloodstream infections. Microbial metabolites have also been detected in the plasma of severe malaria patients, potentially contributing to metabolic acidosis and other clinical complications. However, establishing causal relationships requires intervention studies targeting the gut microbiota., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Sriboonvorakul et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Malaria remains one of the deadliest infectious diseases. Transcriptional regulation effects of noncoding variants in this unusual genome of malaria parasites remain elusive. We developed a sequence-based, ab initio deep learning framework, MalariaSED, for predicting chromatin profiles in malaria parasites. The MalariaSED performance was validated by published ChIP-qPCR and TF motifs results. Applying MalariaSED to ~ 1.3 million variants shows that geographically differentiated noncoding variants are associated with parasite invasion and drug resistance. Further analysis reveals chromatin accessibility changes at Plasmodium falciparum rings are partly associated with artemisinin resistance. MalariaSED illuminates the potential functional roles of noncoding variants in malaria parasites., (© 2023. BioMed Central Ltd., part of Springer Nature.)

Introduction: Zoonotic transmission is a challenge for the control and elimination of malaria. It has been recorded in the Atlantic Forest, outside the Amazon which is the endemic region in Brazil. However, only very few studies have assessed the antibody response, especially of IgM antibodies, in Neotropical primates (NP). Therefore, in order to contribute to a better understanding of the immune response in different hosts and facilitate the identification of potential reservoirs, in this study, naturally acquired IgM antibody responses against Plasmodium antigens were evaluated, for the first time, in NP from the Atlantic Forest., Methods: The study was carried out using 154 NP samples from three different areas of the Atlantic Forest. IgM antibodies against peptides of the circumsporozoite protein (CSP) from different Plasmodium species and different erythrocytic stage antigens were detected by ELISA., Results: Fifty-nine percent of NP had IgM antibodies against at least one CSP peptide and 87% against at least one Plasmodium vivax erythrocytic stage antigen. Levels of antibodies against PvAMA-1 were the highest compared to the other antigens. All families of NP showed IgM antibodies against CSP peptides, and, most strikingly, against erythrocytic stage antigens. Generalized linear models demonstrated that IgM positivity against PvCSP and PvAMA-1 was associated with PCR-detectable blood-stage malaria infection and the host being free-living. Interestingly, animals with IgM against both PvCSP and PvAMA-1 were 4.7 times more likely to be PCR positive than animals that did not have IgM for these two antigens simultaneously., Discussion: IgM antibodies against different Plasmodium spp. antigens are present in NP from the Atlantic Forest. High seroprevalence and antibody levels against blood-stage antigens were observed, which had a significant association with molecular evidence of infection. IgM antibodies against CSP and AMA-1 may be used as a potential marker for the identification of NP infected with Plasmodium, which are reservoirs of malaria in the Brazilian Atlantic Forest., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Assis, Alvarenga, Souza, Sánchez-Arcila, Silva, Pina-Costa, Gonçalves, Souza, Nunes, Pissinatti, Moreira, Torres, Costa, Tinoco, Pereira, Soares, Sousa, Ntumngia, Adams, Kano, Hirano, Pratt-Riccio, Daniel-Ribeiro, Ferreira, Carvalho and Alves de Brito.)

Plasmodium vivax pre-erythrocytic (PE) vaccine research has lagged far behind efforts to develop Plasmodium falciparum vaccines. There is a critical gap in our knowledge of PE antigen targets that can induce functionally inhibitory neutralizing antibody responses. To overcome this gap and guide the selection of potential PE vaccine candidates, we considered key characteristics such as surface exposure, essentiality to infectivity and liver stage development, expression as recombinant proteins, and functional immunogenicity. Selected P. vivax sporozoite antigens were surface sporozoite protein 3 (SSP3), sporozoite microneme protein essential for cell traversal (SPECT1), sporozoite surface protein essential for liver-stage development (SPELD), and M2 domain of MAEBL. Sequence analysis revealed little variation occurred in putative B-cell and T-cell epitopes of the PE candidates. Each antigen was tested for expression as refolded recombinant proteins using an established bacterial expression platform and only SPELD failed. The successfully expressed antigens were immunogenic in vaccinated laboratory mice and were positively reactive with serum antibodies of P. vivax-exposed residents living in an endemic region in Thailand. Vaccine immune antisera were tested for reactivity to native sporozoite proteins and for their potential vaccine efficacy using an in vitro inhibition of liver stage development assay in primary human hepatocytes quantified on day 6 post-infection by high content imaging analysis. The anti-PE sera produced significant inhibition of P. vivax sporozoite invasion and liver stage development. This report provides an initial characterization of potential new PE candidates for a future P. vivax vaccine., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Nicholas et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The implementation of artemisinin (ART) combination therapies (ACTs) has greatly decreased deaths caused by Plasmodium falciparum malaria, but increasing ACT resistance in Southeast Asia and Africa could reverse this progress. Parasite population genetic studies have identified numerous genes, single-nucleotide polymorphisms (SNPs), and transcriptional signatures associated with altered artemisinin activity with SNPs in the Kelch13 (K13) gene being the most well-characterized artemisinin resistance marker. However, there is an increasing evidence that resistance to artemisinin in P. falciparum is not related only to K13 SNPs, prompting the need to characterize other novel genes that can alter ART responses in P. falciparum . In our previous analyses of P. falciparum piggyBac mutants, several genes of unknown function exhibited increased sensitivity to artemisinin that was similar to a mutant of K13. Further analysis of these genes and their gene co-expression networks indicated that the ART sensitivity cluster was functionally linked to DNA replication and repair, stress responses, and maintenance of homeostatic nuclear activity. In this study, we have characterized PF3D7&#95;1136600, another member of the ART sensitivity cluster. Previously annotated as a conserved Plasmodium gene of unknown function, we now provide putative annotation of this gene as a Modulator of Ring Stage Translation (MRST). Our findings reveal that the mutagenesis of MRST affects gene expression of multiple translation-associated pathways during the early ring stage of asexual development via putative ribosome assembly and maturation activity, suggesting an essential role of MRST in protein biosynthesis and another novel mechanism of altering the parasite's ART drug response.IMPORTANCE Plasmodium falciparum malaria killed more than 600,000 people in 2021, though ACTs have been critical in reducing malaria mortality as a first-line treatment for infection. However, ACT resistance in Southeast Asia and emerging resistance in Africa are detrimental to this progress. Mutations to Kelch13 (K13) have been identified to confer increased artemisinin tolerance in field isolates, however, genes other than K13 are implicated in altering how the parasite responds to artemisinin prompts additional analysis. Therefore, in this study we have characterized a P. falciparum mutant clone with altered sensitivity to artemisinin and identified a novel gene (PF3D7&#95;1136600) that is associated with alterations to parasite translational metabolism during critical timepoints for artemisinin drug response. Many genes of the P. falciparum genome remain unannotated, posing a challenge for drug-gene characterizations in the parasite. Therefore, through this study, we have putatively annotated PF3D7&#95;1136600 as a novel MRST gene and have identified a potential link between MRST and parasite stress response mechanisms., Competing Interests: The authors declare no conflict of interest.

Background: Knowledge of the diversity of invasion ligands in malaria parasites in endemic regions is essential to understand how natural selection influences genetic diversity of these ligands and their feasibility as possible targets for future vaccine development. In this study the diversity of four genes for merozoite invasion ligands was studied in Ecuadorian isolates of Plasmodium vivax., Methods: Eighty-eight samples from P. vivax infected individuals from the Coast and Amazon region of Ecuador were obtained between 2012 and 2015. The merozoite invasion genes pvmsp-1-19, pvdbpII, pvrbp1a-2 and pvama1 were amplified, sequenced, and compared to the Sal-1 strain. Polymorphisms were mapped and genetic relationships between haplotypes were determined., Results: Only one nonsynonymous polymorphism was detected in pvmsp-1-19, while 44 nonsynonymous polymorphisms were detected in pvdbpII, 56 in pvrbp1a-2 and 33 in pvama1. While haplotypes appeared to be more related within each area of study and there was less relationship between parasites of the coastal and Amazon regions of the country, diversification processes were observed in the two Amazon regions. The highest haplotypic diversity for most genes occurred in the East Amazon of the country. The high diversity observed in Ecuadorian samples is closer to Brazilian and Venezuelan isolates, but lower than reported in other endemic regions. In addition, departure from neutrality was observed in Ecuadorian pvama1. Polymorphisms for pvdbpII and pvama1 were associated to B-cell epitopes., Conclusions: pvdbpII and pvama1 genetic diversity found in Ecuadorian P. vivax was very similar to that encountered in other malaria endemic countries with varying transmission levels and segregated by geographic region. The highest diversity of P. vivax invasion genes in Ecuador was found in the Amazonian region. Although selection appeared to have small effect on pvdbpII and pvrbp1a-2, pvama1 was influenced by significant balancing selection., (© 2023. BioMed Central Ltd., part of Springer Nature.)

Transmission of the deadly malaria parasite Plasmodium falciparum from humans to mosquitoes is achieved by specialized intraerythrocytic sexual forms called gametocytes. Though the crucial regulatory mechanisms leading to gametocyte commitment have recently come to light, networks of genes that control sexual development remain to be elucidated. Here, we report a pooled-mutant screen to identify genes associated with gametocyte development in P. falciparum. Our results categorized genes that modulate gametocyte progression as hypoproducers or hyperproducers of gametocytes, and the in-depth analysis of individual clones confirmed phenotypes in sexual commitment rates and putative functions in gametocyte development. We present a new set of genes that have not been implicated in gametocytogenesis before and demonstrate the potential of forward genetic screens in isolating genes impacting parasite sexual biology, an exciting step toward the discovery of new antimalarials for a globally significant pathogen. IMPORTANCE Blocking human-to-vector transmission is an essential step toward malaria elimination. Gametocytes are solely responsible for achieving this transmission and represent an opportunity for therapeutic intervention. While these falciform-shaped parasite stages were first discovered in the 1880s, our understanding of the genetic determinants responsible for their formation and molecular mechanisms that drive their development is limited. In this work, we developed a scalable screening methodology with piggyBac mutants to identify genes that influence the development of gametocytes in the most lethal human malaria parasite, P. falciparum. By doing so, we lay the foundation for large-scale functional genomic studies specifically designed to address remaining questions about sexual commitment, maturation, and mosquito infection in P. falciparum. Such functional genetic screens will serve to expedite the identification of essential pathways and processes for the development of novel transmission-blocking agents., Competing Interests: The authors declare no conflict of interest.

The antimalarial activity of the frontline drug artemisinin involves generation of reactive oxygen species (ROS) leading to oxidative damage of parasite proteins. To achieve homeostasis and maintain protein quality control in the overwhelmed parasite, the ubiquitin-proteasome system kicks in. Even though molecular markers for artemisinin resistance like pfkelch13 have been identified, the intricate network of mechanisms driving resistance remains to be elucidated. Here, we report a forward genetic screening strategy that enables a broader identification of genetic factors responsible for altering sensitivity to dihydroartemisinin (DHA) and a proteasome inhibitor, bortezomib (BTZ). Using a library of isogenic piggyBac mutants in P. falciparum, we defined phenotype-genotype associations influencing drug responses and highlighted shared mechanisms between the two processes, which mainly included proteasome-mediated degradation and the lipid metabolism genes. Additional transcriptomic analysis of a DHA/BTZ-sensitive piggyBac mutant showed it is possible to find differences between the two response mechanisms on the specific components for regulation of the exportome. Our results provide further insight into the molecular mechanisms of antimalarial drug resistance. IMPORTANCE Malaria control is seriously threatened by the emergence and spread of Plasmodium falciparum resistance to the leading antimalarial, artemisinin. The potent killing activity of artemisinin results from oxidative damage unleashed by free heme activation released by hemoglobin digestion. Although the ubiquitin-proteasome system is considered critical for parasite survival of this toxicity, the diverse genetic changes linked to artemisinin resistance are complex and, so far, have not included the ubiquitin-proteasome system. In this study, we use a systematic forward genetic approach by screening a library of P. falciparum random piggyBac mutants to decipher the genetic factors driving malaria parasite responses to the oxidative stress caused by antimalarial drugs. This study compares phenotype-genotype associations influencing dihydroartemisinin responses with the proteasome inhibitor bortezomib to delineate the role of ubiquitin-proteasome system. Our study highlights shared and unique pathways from the complex array of molecular processes critical for P. falciparum survival resulting from the oxidative damage of artemisinin., Competing Interests: The authors declare no conflict of interest.

Fever is a part of the human innate immune response that contributes to limiting microbial growth and development in many infectious diseases. For the parasite Plasmodium falciparum, survival of febrile temperatures is crucial for its successful propagation in human populations as well as a fundamental aspect of malaria pathogenesis. This review discusses recent insights into the biological complexity of the malaria parasite's heat-shock response, which involves many cellular compartments and essential metabolic processes to alleviate oxidative stress and accumulation of damaged and unfolded proteins. We highlight the overlap between heat-shock and artemisinin resistance responses, while also explaining how the malaria parasite adapts its fever response to fight artemisinin treatment. Additionally, we discuss how this systemic and essential fight for survival can also contribute to parasite transmission to mosquitoes., Competing Interests: Declaration of Competing Interest Nothing declared., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Background: Assessing the status of malaria transmission in endemic areas becomes increasingly challenging as countries approach elimination. Serology can provide robust estimates of malaria transmission intensities, and multiplex serological assays allow for simultaneous assessment of markers of recent and historical malaria exposure., Methods: Here, we evaluated different statistical and machine learning methods for analyzing multiplex malaria-specific antibody response data to classify recent and historical exposure to Plasmodium falciparum and Plasmodium vivax . To assess these methods, we utilized samples from a health-facility based survey (n = 9132) in the Philippines, where we quantified antibody responses against 8  P. falciparum and 6  P. vivax -specific antigens from 3 sites with varying transmission intensity., Findings: Measurements of antibody responses and seroprevalence were consistent with the 3 sites' known endemicity status. Among the models tested, a machine learning (ML) approach (Random Forest model) using 4 serological markers (PfGLURP R2, Etramp5.Ag1, GEXP18, and PfMSP1 19 ) gave better predictions for P. falciparum recent infection in Palawan (AUC: 0.9591, CI 0.9497-0.9684) than individual antigen seropositivity. Although the ML approach did not improve P. vivax infection predictions, ML classifications confirmed the absence of recent exposure to P. falciparum and P. vivax in both Occidental Mindoro and Bataan. For predicting historical P. falciparum and P. vivax transmission, seroprevalence and seroconversion rates based on cumulative exposure markers AMA1 and MSP1 19 showed reliable trends in the 3 sites., Interpretation: Our study emphasizes the utility of serological markers in predicting recent and historical exposure in a sub-national elimination setting, and also highlights the potential use of machine learning models using multiplex antibody responses to improve assessment of the malaria transmission status of countries aiming for elimination. This work also provides baseline antibody data for monitoring risk in malaria-endemic areas in the Philippines., Funding: Newton Fund, Philippine Council for Health Research and Development, UK Medical Research Council., Competing Interests: The authors declare no competing interests., (© 2023 Published by Elsevier Ltd.)

Plasmodium vivax Duffy Binding Protein region II (PvDBPII) is a leading vaccine candidate against blood-stage vivax malaria. Anti-PvDBPII antibodies potentially block parasite invasion by inhibition of erythrocyte binding. However, knowledge of PvDBPII-specific T cell responses is limited. Here, to assess the responses of PvDBPII-specific CD4 + T cells in natural P. vivax infection, three cross-sectional studies were conducted in recovered subjects. In silico analysis was used for potential T cell epitope prediction and selection. PBMCs from P. vivax subjects were stimulated with selected peptides and examined for cytokine production by ELISPOT or intracellular cytokine staining. Six dominant T cell epitopes were identified. Peptide-driven T cell responses showed effector memory CD4 + T cell phenotype, secreting both IFN-γ and TNF-α cytokines. Single amino acid substitutions in three T cell epitopes altered levels of IFN-γ memory T cell responses. Seropositivity of anti-PvDBPII antibodies were detected during acute malaria (62%) and persisted up to 12 months (11%) following P. vivax infection. Further correlation analysis showed four out of eighteen subjects had positive antibody and CD4 + T cell responses to PvDBPII. Altogether, PvDBPII-specific CD4 + T cells were developed in natural P. vivax infections. Data on their antigenicity could facilitate development of an efficacious vivax malaria vaccine., (© 2023. The Author(s).)