OBJECTIVES:In 2023, malaria infected 12 million pregnant women in Africa. Preventive treatment with sulphadoxine-pyrimethamine (SP-IPTp) is recommended, but its efficacy is threatened by parasite resistance, driven by mutations in the Plasmodium falciparum dhfr (N51I, C59R, S108N, I164L) and dhps (A437G, K540E, A581G) genes. We assessed the effect of resistant infections on birthweight and examined the effectiveness of SP-IPTp dose timing on parasite persistence. METHODS:Between July 2022 and October 2023, we conducted a facility-based prospective cohort study among pregnant women attending antenatal care (ANC) in three health facilities in Northern Uganda. Eligible participants were those with confirmed pregnancy, informed consent, and delivery planned at the study site. At each ANC visit, malaria screening by microscopy was performed, and blood samples from positive cases were analysed for dhfr/dhps resistance markers through PCR and Sanger sequencing. Confirmed infections were treated with standard antimalarial therapy, distinct from SP-IPTp. Only women with parasitaemia were included in the analysis, and outcomes were birthweight and parasite persistence. Multivariable modelling was guided by directed acyclic graphs, and inverse probability weighting was applied to correct for sequencing-related selection bias. RESULTS:Of 462 women with parasitaemia, 278 (60.1%) infections were successfully genotyped, of which 82% were quintuple and 14% were sextuple mutants. Adjusted analysis found no significant association between resistance haplotypes and birthweight (-77 g, p = 0.330) nor parasite persistence (adjusted odds ratio: 3.81, p = 0.166). Infants of primigravidae weighed 238 g less (95% CI: -38 to -96 g, p = 0.001) than those of multigravidae, despite near-universal SP-IPTp coverage and standard case management. In contrast, each additional SP-IPTp dose administered after a detected infection was associated with higher odds of persistent parasitaemia at follow-up ANC visits (adjusted odds ratio: 1.90, 95% CI: 1.28-2.82; p = 0.002). CONCLUSIONS:Resistant haplotypes affected neither birthweight nor persistence. However, birthweight was significantly inferior among primigravidae despite SP-IPTp, and postinfection dosing was associated with increased risk of persistence.

Malaria remains a significant global health challenge, disproportionately affecting Sub-Saharan Africa. Pregnant women represent one of the most vulnerable populations. Despite numerous advances in malaria control measures, lost-to-follow-up (LTFU) in antenatal care (ANC) programs poses a critical barrier to achieving optimal maternal and neonatal outcomes and the success of public health interventions. The factors driving LTFU, particularly in rural, high-burden settings, are not completely understood. This study investigates the determinants of LTFU among pregnant women receiving malaria screening in the context of the ERASE-Rise Against Malaria project in Northern Uganda. An observational retrospective cohort study was conducted on 1,558 women, recruited from July 2022 to June 2024, during the operational research held to assess the impact of antimalarial resistance on malaria care among pregnant women in three healthcare facilities in Oyam and Kole districts. Data on individual level (sociodemographic and clinical) and health care-related factors were analyzed using a multilevel logistic regression model to identify predictors of LTFU, defined as the absence of cohort outcome data 30 days after the expected delivery date. Efforts to recover missing data included delivery register consultation, active phone calls, and contact tracing by village health team workers. 871 (55.9%) of the 1,558 women were LTFU. Recovery strategies reduced the missing data rate to 29.1% (n=454). Protective factors against LTFU included higher education (aOR=0.75, 95% CI: 0.54-1.03, p=0.0798), being primigravida (aOR=0.73, 95% CI: 0.56-0.97, p=0.0275), and experiencing malaria during pregnancy (aOR=0.61, 95% CI: 0.48-0.78, p<0.0001). Women attending Aboke Health Center IV (n=385) were over five times more likely to be LTFU than those at Aber Hospital (n=955) (aOR=5.57, 95% CI: 4.08-7.71, p<0.0001), highlighting significant geographic and structural barriers. The high rate of LTFU in malaria screening programs underscores the need for targeted interventions addressing individual, systemic and structural barriers. Strengthening community-level support, improving healthcare infrastructures, and integrating malaria prevention into broader maternal health services are crucial for enhancing retention in care. Addressing determinants of LTFU, systematically, through further qualitative and quantitative research, is essential to improving maternal and neonatal health outcomes and achieving malaria eradication goals in high-burden settings.

BACKGROUND:Artemisinin-based combination therapy for Plasmodium falciparum malaria is threatened by the emergence of partial resistance. METHODS:Molecular characterization of antimalarial resistance-associated genes Pfk13, Pfcrt, Pfmdr1, Pfdhfr, Pfdhps and PfCytB was performed on blood samples collected from two patients with recurrent episodes of P. falciparum malaria. RESULTS:Two severe P. falciparum malaria in Italian travelers from sub-Saharan Africa showed recrudescence after standard treatment with intravenous artesunate and oral dihydroartemisinin-piperaquine. In one case, the validated marker R561H of artemisinin partial resistance was identified. In the second patient, no Pfkelch13 mutations were detected, and the recrudescent episode was probably the consequence of low blood drug concentration administered by nasogastric tube, although the possible role of Pfkelch13-independent pathways or reduced artemisinin susceptibility cannot be ruled out. CONCLUSION:The first imported case of artemisinin-resistant P. falciparum in Italy from sub-Saharan Africa underscores the need for continuous surveillance and eventually adapting treatment protocols.

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.

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.

BACKGROUND:Pregnancy is both a risk factor for P. falciparum infection and development of severe malaria. In low- and middle-income countries, the COVID-19 pandemic severely impacted health systems, including utilization of maternal services. This study aimed to assess trends in delivering malaria in pregnancy-related health-care services before and during COVID-19 in Northern Uganda. METHODS:An interrupted time-series study comparing pre-COVID-19 (January 2018 to April 2020) and COVID-19 (May to December 2021) periods, based on the date the first COVID case was detected. The study involved 30 health facilities in Northern Uganda with 22,650 estimated pregnancies per year, 14% of which took place in hospital. Monthly data were sourced from District routinely collected indicators. Trends were analyzed by joinpoint regression models. RESULTS:From the onset of the COVID pandemic in Uganda (May 2020), we found a significant reduction in the number of women accessing a fourth antenatal care visit (from APC + 183.5 to + 4.98; p < 0.001) and taking at least three doses of intermittent preventive treatment in pregnancy (IPTp, from APC + 84.28 to -63.12; p < 0.001). However, we found no significant change in the trend of the total number of pregnant women managed as outpatients or hospitalized for malaria, as well as in the number of women attending their first antenatal visit and in the number of institutional deliveries. CONCLUSIONS:In our study, the COVID-19 pandemic significantly reduced access to ANC visits and IPTp uptake. However, the healthcare system maintained its capacity for managing malaria cases, first antenatal visits, and institutional deliveries.Trial registration: This study has been registered on the ClinicalTrials.gov public website on 26 April 2022. ClinicalTrials.gov Identifier: NCT05348746.

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

Like most countries in sub-Saharan African countries, Benin continues to bear a heavy malaria burden. In 2014, the National Malaria Control Programme (NMCP) changed its treatment policy, and recommended the use of artemisinin-based combination therapy (ACT) as first-line treatment for uncomplicated Plasmodium falciparum cases. The study presented here was conducted to investigate the impact of current antimalarial drug resistance on the country. Molecular surveillance targeting the Pfcrt, Pfmdr1, Pfkelch13, dhfr, and dhps genes was carried out on samples from patients positive for P. falciparum malaria by microscopy, LAMP and PCR diagnostic test. Molecular analysis was performed using targeted amplicon deep sequencing (TADS). In addition, the frequency of parasites with dual deletion of the histidine-rich protein 2 and 3 genes (pfhrp2 and pfhrp3), known to be responsible of the performance of HRP-based malaria rapid diagnostic tests (HRP-RDT), was estimated. Fifty-three falciparum samples collected at the Saint Jean de Dieu hospital in Tanguiéta, Benin, were tested. No Pfkelch13 validated or candidate artemisinin partial resistant variants were identified. A marked prevalence of Asn51Ile (N51I), Cys59Arg (C59R), and Ser108Asn (S108N) mutant alleles was found in the dhfr gene, representing the most frequent genotype (64%). Five-point mutations were detected in dhps, Ile431Val (I431V), Ser436Ala (S436A), Ala437Gly (A437G), Ala581Gly (A581G), Ala613Ser (A613S) of which the third was the most common (92%). No mutation was identified in dhps Lys540Glu (K540E). The quintuple mutant genotype resulting from the combination of the dhfr triple mutant (51I/59R/108N) with the dhps double mutant 436A/437G was detected at a frequency of 30%. Low levels of mutations in Pfcrt and no mutation at codon 86 in the Pfmdr1 DNA fragment were observed, whereas a high level of Tyr184Phe (Y184F) polymorphism in the Pfmdr1 gene was found. These results could be indicative, over a decade after the implementation of ACT therapy, of the return of chloroquine-sensitive but artemether-lumefantrine resistant falciparum genotypes in Benin. There was no evidence of HRP2 and HRP3 deletions. Data from the present study support the need for routine monitoring of molecular markers of antimalarial drug resistance as part of surveillance activities aimed to make informed treatment policy decisions at the national level.

A 68-year-old man returning from Republic of Côte d'Ivoire (Ivory Coast) was diagnosed with severe Plasmodium falciparum malaria and treated with intravenous artesunate followed by oral dihydroartemisinin-piperaquine (DHA-PPQ). A month later the patient experienced a new P. falciparum episode; analysis of pfmsp-1 and pfmsp-2 revealed that the infection was caused by a genetic strain identical to the strain that caused the initial episode, indicating resurgence of the previous infection. No mutations in genes associated with resistance to artemisinin derivatives (pfk13) or piperaquine (pfexonuclease, pfplasmepsin 2/3) were detected, suggesting that treatment failure could have been caused by drug malabsorption or poor drug manufacturing practices. A second treatment with atovaquone-proguanil was successful in eliminating the infection, with no further relapses. To our knowledge, this is the first description of a treatment failure with both artesunate and DHA-PPQ in a traveler returning from a malaria-endemic region. Analysis of molecular markers of resistance to antimalarial drugs revealed mutations associated with resistance to sulfadoxine (pfdhps) and pyrimethamine (pfdhfr), highlighting the important contribution of surveillance of imported malaria cases to the monitoring of drug resistance globally.

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.

We report the molecular evidence of dengue virus (DENV) and chikungunya virus (CHIKV) infection in symptomatic individuals in Cameroon and Gabon, respectively. Arthropod-borne viruses (arboviruses) are distributed in the tropical or subtropical regions, with DENV having the highest burden. The morbidity and mortality related to arboviral diseases raise the concern of timely and efficient surveillance and care. Our aim was to assess the circulation of arboviruses [DENV, CHIKV, Zika virus (ZIKV)] among febrile patients in Dschang (West Cameroon) and Kyé-ossi (South Cameroon, border with Gabon and Equatorial Guinea). Dried blood spots were collected from 601 consenting febrile patients, and 194 Plasmodium spp.-negative samples were tested for the molecular detection of cases of DENV, CHIKV and ZIKV infection. Overall, no case of ZIKV infection was found, whereas one case of DENV infection and one case of CHIKV infection were detected in Dschang and Kyé-ossi, respectively, with the CHIKV-infected patient being resident in Gabon. Our findings suggest the need to establish an active surveillance of arbovirus transmission in Cameroon and bordering countries.

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.

The Duffy blood group is a critical receptor for Plasmodium vivax (P. vivax) invasion of red blood cells, and consequently, P. vivax infections were considered rare in sub-Saharan Africa where the prevalence of Duffy-negativity is high. However, recently, P. vivax infections have been found in Duffy-negative Africans throughout the malaria transmission area of sub-Saharan Africa, raising important questions concerning the molecular composition of these P. vivax clones and the red blood cell receptors that facilitate their invasion. Here, we describe an unusually high number of P. vivax infections in febrile Duffy-negative Africans in Dschang, Cameroon (177 of 500 outpatients), as compared with Santchou (two of 400 outpatients) and Kyé-ossi (two of 101 outpatients), in other areas in Cameroon. In the discussion, we speculate on the possible reasons why Dschang might account for the unusually large numbers of P. vivax infections in Duffy-negative individuals living there.

BackgroundRecent studies showed the first emergence of the R561H artemisinin-associated resistance marker in Africa, which highlights the importance of continued molecular surveillance to assess the selection and spread of this and other drug resistance markers in the region.MethodIn this study, we used targeted amplicon deep sequencing of 116 isolates collected in two areas of Cameroon to genotype the major drug resistance genes, k13, crt, mdr1, dhfr, and dhps, and the cytochrome b gene (cytb) in Plasmodium falciparum.ResultsNo confirmed or associated artemisinin resistance markers were observed in Pfk13. In comparison, both major and minor alleles associated with drug resistance were found in Pfcrt, Pfmdr1, Pfdhfr, and Pfdhps. Notably, a high frequency of other nonsynonymous mutations was observed across all the genes, except for Pfcytb, suggesting continued selection pressure.ConclusionsThe results from this study supported the continued use of artemisinin-based combination therapy and administration of sulfadoxine-pyrimethamine for intermittent preventive therapy in pregnant women, and for seasonal chemoprevention in these study sites in Cameroon.

Plasmodium vivax has been considered for years as responsible for a mild form of malaria, due to the absence in the majority of its infections of the severe form of the disease, typical instead of the deadly human parasite P. falciparum. In the last decade, studies on vivax malaria have had a partial step ahead especially after the completion of the whole genome project, but there is still a gap of knowledge in the biology of this parasite. Moreover, the emergence of P. vivax antimalarial resistance in 1980s and its subsequent spread in the Southeast Asia have indicated new concerns about the possibility to control this parasite. P. vivax drug resistance poses a major threat to endemic countries and without important international efforts, we could assist in a near future to the paradox of seeing different malaria co-endemic countries, that have successfully controlled/eliminated P. falciparum, still fighting against P. vivax.

BACKGROUND:Routine molecular surveillance for imported drug-resistant malaria parasites to the USA and European Union is an important public health activity. The obtained molecular data are used to help keep chemoprophylaxis and treatment guidelines up to date for persons traveling to malaria endemic countries. Recent advances in next-generation sequencing (NGS) technologies provide a new and effective way of tracking malaria drug-resistant parasites.METHODS:As part of a technology transfer arrangement between the CDC Malaria Branch and the Istituto Superiore di Sanità (ISS), Rome, Italy, the recently described Malaria Resistance Surveillance (MaRS) protocol was used to genotype 148 Plasmodium falciparum isolates from Eritrea for kelch 13 (k13) and cytochrome b (cytb) genes, molecular markers associated with resistance to artemisinin (ART) and atovaquone/proguanil (AP), respectively.RESULTS:Spanning the full-length k13 gene, seven non-synonymous single nucleotide polymorphisms (SNPs) were found (K189N, K189T, E208K, D281V, E401Q, R622I and T535M), of which none have been associated with artemisinin resistance. No mutations were found in cytochrome b.CONCLUSION:All patients successfully genotyped carried parasites susceptible to ART and AP treatment. Future studies between CDC Malaria Branch and ISS are planned to expand the MaRS system, including data sharing, in an effort to maintain up to date treatment guidelines for travelers to malaria endemic countries.

Drug-resistant Plasmodium falciparum is a major threat to global malaria control and elimination efforts. In Botswana, a southern African country approaching malaria elimination, P. falciparum molecular data are not available. Parasites were assessed through pollymerase chain reaction (PCR) for confirmation of positive rapid diagnostic tests, multiplicity of infection (MOI), and drug resistance markers among isolates from clinical uncomplicated malaria cases collected at health facilities. Of 211 dried blood spot samples selected for the study, 186 (88.2%) were PCR positive for P. falciparum. The mean MOI based on MSP1 genotyping was 2.3 and was not associated with age. A high prevalence of wild-type parasites for pfcrt and pfmdr1 was found, with a haplotype frequency (K76/N86) of 88.8% and 17.7% of the isolates having two copies of the pfmdr1 gene. For pfATPase6, all the parasites carried the wild-type S769 allele. Sequencing showed no evidence of non-synonymous mutations associated with reduced artemisinin derivative sensitivity in the P. falciparum k13 gene. In conclusion, we found that P. falciparum parasites in Botswana were mostly wild type for the drug resistance markers evaluated. Yet, there was a high rate of a molecular marker associated to reduced sensitivity to lumefantrine. Our results indicate the need for systematic drug efficacy surveillance to complement malaria elimination efforts.

Dihydroartemisinin–piperaquine (DHA–PPQ) is the artemisinin combination therapy that was recently introduced for the treatment of Plasmodium falciparum uncomplicated malaria, but emerging resistance in South-East Asia is threatening its use. This report describes a case of DHA–PPQ treatment failure in uncomplicated malaria occurring in an immigrant living in Italy, after a travel to Ethiopia. Thirty days after malaria recovery following DHA–PPQ therapy, the patient had malaria recrudescence. According to the genotyping analysis, the same P. falciparum was responsible for both episodes. Thus, it seems important to consider possible malaria recrudescence occurring after DHA–PPQ therapy in patients from African countries