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Background Plasmodium malariae is the most neglected of the six human malaria species and it is still unknown which is the mechanism underlying the long latency of this Plasmodium . Case presentation A case of PCR-confirmed P. malariae recurrence in a 52-year old Italian man was observed 5 months after a primary attack. In the interval between the two observed episodes of malaria the patient denied any further stay in endemic areas except for a visit to Libya, a country considered malaria-free. Genomic DNA of the P. malariae strain using five microsatellites (PM2, PM9, PM11, PM25, PM34) and the antigen marker of circumsporozoite ( csp ) was amplified and sequenced. Analysis of polymorphisms of the P. malariae csp central repeat region showed differences between the strains responsible of the first and second episode of malaria. A difference in the allele size was also observed for the sequence analysis of PM2 microsatellites. Conclusions Plasmodium malariae is a challenging human malaria parasite and even with the use of molecular techniques the pathogenesis of recurrent episodes cannot be precisely explained.

We report 3 cases of babesiosis in Italy caused by Babesia species that are rarely reported in humans. The circulation of Babesia spp. among vectors, animals, and humans might be more common than previously thought, and babesiosis might be an underdiagnosed and emerging disease in Italy and Europe.

We conducted a hospital-based cross-sectional study of Plasmodium falciparum in 2017 in southeastern Sudan. Among 257 P. falciparum samples, we found 22% harbored the pfkelch13 R622I mutation and 10.7% showed hrp2/3 gene deletions. Our findings highlight the urgent need for enhanced surveillance of drug- and diagnostic-resistant parasites in the Horn of Africa.

Plasmodium vivax is the predominant malaria parasite in Latin America. Its colonization history in the region is rich and complex, and is still highly debated, especially about its origin(s). Our study employed cutting-edge population genomic techniques to analyze whole genome variation from 620 P . vivax isolates, including 107 newly sequenced samples from West Africa, Middle East, and Latin America. This sampling represents nearly all potential source populations worldwide currently available. Analyses of the genetic structure, diversity, ancestry, coalescent-based inferences, including demographic scenario testing using Approximate Bayesian Computation, have revealed a more complex evolutionary history than previously envisioned. Indeed, our analyses suggest that the current American P . vivax populations predominantly stemmed from a now-extinct European lineage, with the potential contribution also from unsampled populations, most likely of West African origin. We also found evidence that P . vivax arrived in Latin America in multiple waves, initially during early European contact and later through post-colonial human migration waves in the late 19 th -century. This study provides a fresh perspective on P . vivax ’s intricate evolutionary journey and brings insights into the possible contribution of West African P . vivax populations to the colonization history of Latin America.

Background The production of Plasmodium gametocytes in vitro is a real challenge. Many protocols have been described, but few have resulted in the production of viable and infectious gametocytes in sufficient quantities to conduct research on—but not limited to—transmission-blocking drug and vaccine development. The aim of this review was to identify and discuss gametocyte production protocols that have been developed over the last two decades. Methods We analyzed the original gametocyte production protocols published from 2000 onwards based on a literature search and a thorough review. A systematic review was performed of relevant articles identified in the PubMed, Web of Sciences and ScienceDirect databases. Results A total 23 studies on the production of Plasmodium gametocytes were identified, 19 involving in vitro Plasmodium falciparum , one involving Plasmodium knowlesi and three involving ex vivo Plasmodium vivax . Of the in vitro studies, 90% used environmental stressors to trigger gametocytogenesis. Mature gametocytemia of up to 4% was reported. Conclusions Several biological parameters contribute to an optimal production in vitro of viable and infectious mature gametocytes. The knowledge gained from this systematic review on the molecular mechanisms involved in gametocytogenesis enables reproducible gametocyte protocols with transgenic parasite lines to be set up. This review highlights the need for additional gametocyte production protocols for Plasmodium species other than P. falciparum . Graphical abstract

Background ETRAMP11.2 (PVX_003565) is a well-characterized protein with antigenic potential. It is considered to be a serological marker for diagnostic tools, and it has been suggested as a potential vaccine candidate. Despite its immunological relevance, the polymorphism of the P. vivax ETRAMP11.2 gene ( pvetramp11.2 ) remains undefined. The genetic variability of an antigen may limit the effectiveness of its application as a serological surveillance tool and in vaccine development and, therefore, the aim of this study was to investigate the genetic diversity of pvetramp11.2 in parasite populations from Amazonian regions and worldwide. We also evaluated amino acid polymorphism on predicted B-cell epitopes. The low variability of the sequence encoding PvETRAMP11.2 protein suggests that it would be a suitable marker in prospective serodiagnostic assays for surveillance strategies or in vaccine design against P. vivax malaria. Methods The pvetramp11.2 of P. vivax isolates collected from Brazil ( n  = 68) and Peru ( n  = 36) were sequenced and analyzed to assess nucleotide polymorphisms, allele distributions, population differentiation, genetic diversity and signature of selection. In addition, sequences ( n  = 104) of seven populations from different geographical regions were retrieved from the PlasmoDB database and included in the analysis to study the worldwide allele distribution. Potential linear B-cell epitopes and their polymorphisms were also explored. Results The multiple alignments of 208 pvetramp11.2 sequences revealed a low polymorphism and a marked geographical variation in allele diversity. Seven polymorphic sites and 11 alleles were identified. All of the alleles were detected in isolates from the Latin American region and five alleles were detected in isolates from the Southeast Asia/Papua New Guinea (SEA/PNG) region. Three alleles were shared by all Latin American populations (H1, H6 and H7). The H1 allele (reference allele from Salvador-1 strain), which was absent in the SEA/PNG populations, was the most represented allele in populations from Brazil (54%) and was also detected at high frequencies in populations from all other Latin America countries (range: 13.0% to 33.3%). The H2 allele was the major allele in SEA/PNG populations, but was poorly represented in Latin America populations (only in Brazil: 7.3%). Plasmodium vivax populations from Latin America showed a marked inter-population genetic differentiation (fixation index [Fst]) in contrast to SEA/PNG populations. Codon bias measures (effective number of codons [ENC] and Codon bias index [CBI]) indicated preferential use of synonymous codons, suggesting selective pressure at the translation level. Only three amino acid substitutions, located in the C-terminus, were detected. Linear B-cell epitope mapping predicted two epitopes in the Sal-1 PvETRAMP11.2 protein, one of which was fully conserved in all of the parasite populations analyzed. Conclusions We provide an overview of the allele distribution and genetic differentiation of ETRAMP11.2 antigen in P. vivax populations from different endemic areas of the world. The reduced polymorphism and the high degree of protein conservation supports the application of PvETRAMP11.2 protein as a reliable antigen for application in serological assays or vaccine design. Our findings provide useful information that can be used to inform future study designs. Graphical abstract

Plasmodium vivax is the most prevalent human malaria parasite, causing serious public health problems in malaria-endemic countries. Until recently the Duffy-negative blood group phenotype was considered to confer resistance to vivax malaria for most African ethnicities. We and others have reported that P. vivax strains in African countries from Madagascar to Mauritania display capacity to cause clinical vivax malaria in Duffy-negative people. New insights must now explain Duffy-independent P. vivax invasion of human erythrocytes. Through recent whole genome sequencing we obtained ≥ 70× coverage of the P. vivax genome from five field-isolates, resulting in ≥ 93% of the Sal I reference sequenced at coverage greater than 20×. Combined with sequences from one additional Malagasy field isolate and from five monkey-adapted strains, we describe here identification of DNA sequence rearrangements in the P. vivax genome, including discovery of a duplication of the P. vivax Duffy binding protein (PvDBP) gene. A survey of Malagasy patients infected with P. vivax showed that the PvDBP duplication was present in numerous locations in Madagascar and found in over 50% of infected patients evaluated. Extended geographic surveys showed that the PvDBP duplication was detected frequently in vivax patients living in East Africa and in some residents of non-African P. vivax-endemic countries. Additionally, the PvDBP duplication was observed in travelers seeking treatment of vivax malaria upon returning home. PvDBP duplication prevalence was highest in west-central Madagascar sites where the highest frequencies of P. vivax-infected, Duffy-negative people were reported. The highly conserved nature of the sequence involved in the PvDBP duplication suggests that it has occurred in a recent evolutionary time frame. These data suggest that PvDBP, a merozoite surface protein involved in red cell adhesion is rapidly evolving, possibly in response to constraints imposed by erythrocyte Duffy negativity in some human populations.

Background Uganda accounts for 5% of all malaria cases and deaths reported globally and, in endemic countries, pregnancy is a risk factor for both acquisition of P. falciparum infection and development of severe malaria. In recent years, malaria control has been threatened by COVID-19 pandemic and by the emergence, in Northern Uganda, of both resistance to artemisinin derivatives and to sulfadoxine-pyrimethamine. Methods In this facility-based, prospective, observational study, pregnant women will be recruited at antenatal-care visits and followed-up until delivery. Collected data will explore the incidence of asymptomatic parasitemia and malaria-related outcomes, as well as the attitudes towards malaria prevention, administration of intermittent preventive treatment, healthcare seeking behavior and use of insecticide-treated nets. A subpopulation of women diagnosed with malaria will be recruited and their blood samples will be analyzed for detection of genetic markers of resistance to artemisinin derivatives and sulfadoxine-pyrimethamine. Also, to investigate the impact of COVID-19 on malaria care among pregnant women, a retrospective, interrupted-time series will be conducted on at the study sites for the period January 2018 to December 2021. Discussion The present study will explore the impact of COVID-19 pandemic on incidence of malaria and malaria-related adverse outcomes, along with the prevalence of resistance to artemisinin derivatives and to sulfadoxine-pyrimethamine. To our knowledge, this is the first study aiming to explore the combined effect of these factors on a cohort of pregnant women. Trial registration : This study has been registered on the ClinicalTrials.gov public website on 26th April, 2022. ClinicalTrials.gov Identifier: NCT05348746.

Plasmodium vivax malaria is a neglected disease, particularly during pregnancy. Severe vivax malaria is associated with inflammatory responses but in pregnancy immune alterations make it uncertain as to what cytokine signatures predominate, and how the type and quantity of blood immune mediators influence delivery outcomes. We measured the plasma concentrations of a set of thirty-one biomarkers, comprising cytokines, chemokines and growth factors, in 987 plasma samples from a cohort of 572 pregnant women from five malaria-endemic tropical countries and related these concentrations to delivery outcomes (birth weight and hemoglobin levels) and malaria infection. Samples were collected at recruitment (first antenatal visit) and at delivery (periphery, cord and placenta). At recruitment, we found that P. vivax-infected pregnant women had higher plasma concentrations of proinflammatory (IL-6, IL-1β, CCL4, CCL2, CXCL10) and TH1-related cytokines (mainly IL-12) than uninfected women. This biomarker signature was essentially lost at delivery and was not associated with birth weight nor hemoglobin levels. Antiinflammatory cytokines (IL-10) were positively associated with infection and poor delivery outcomes. CCL11 was the only biomarker to show a negative association with P. vivax infection and its concentration at recruitment was positively associated with hemoglobin levels at delivery. Birth weight was negatively associated with peripheral IL-4 levels at delivery. Our multi-biomarker multicenter study is the first comprehensive one to characterize the immunological signature of P. vivax infection in pregnancy thus far. In conclusion, data show that while TH1 and pro-inflammatory responses are dominant during P. vivax infection in pregnancy, antiinflammatory cytokines may compensate excessive inflammation avoiding poor delivery outcomes, and skewness toward a TH2 response may trigger worse delivery outcomes. CCL11, a chemokine largely neglected in the field of malaria, emerges as an important marker of exposure or mediator in this condition.

Abstract Background Plasmodium falciparum (P. falciparum) malaria is a significant public health problem in returning travellers, and artemisinin combination therapy (ACT) remains the first choice for treatment. Several single nucleotide polymorphisms (SNPs) in the P. falciparum kelch 13 (Pfk13) gene have been associated with artemisinin (ART) resistance. Moreover, the increase in the P. falciparum plasmepsin 2 (Pfpm2) gene copy number was shown to be linked with reduced susceptibility of P. falciparum to piperaquine (PPQ), a partner drug in an ACT regimen. Active molecular surveillance for imported drug-resistant malaria parasites is a pivotal activity to provide adequate chemoprophylaxis and treatment guidelines. Methods A retrospective study to review imported P. falciparum malaria in patients admitted to Spallanzani Institute between 2014 and 2015 was conducted. Information collected included clinic and epidemiological characteristics such as age, gender, country of origin, time since arrival to our country, travel history. All P.falciparum isolates were analysed for SNPs in the Pfk13 gene and for copy number variations in the Pfpm2 gene. Results P. falciparum malaria was identified in 54 travellers. The mean age was 37 years, 44 were males. All cases were imported from non-EU countries. In the Pfk13 gene two mutations (R561R and F673L) were detected. Six P. falciparum isolates carried two copies of Pfpm2 gene, and one three copies, representing ≈16% of the analysed isolates. Conclusions None of the SNPs known to be associated with ART resistance were detected in the examined parasites. Our results provide evidence that Pfpm2 duplications (associated with piperaquine resistance) occur in Africa, emphasizing the necessity to better decode the genetic background associated with PPQ resistance. Further epidemiological investigations in Pfpm2 amplification along with mutations in the Pfk13 gene will be useful for developing and updating anti-malarial guidance in travellers.