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The investigation of factors associated with susceptibility to severe malaria is best achieved using case-control studies. The presence of a history of severe malaria in controls could affect the quality of their phenotype and study findings and hence should be rigorously determined. Here, we assessed the performance of a qualitative questionnaire to identify a history of cerebral malaria in controls in a case-control study of severe malaria in Mali. We evaluated the archived medical records of 220 children diagnosed with severe diseases at health care centers in rural and urban settings in Mali from 2018 to 2019. Parents of enrolled children were then identified and interviewed using a structured questionnaire by an investigator blinded to the diagnosis. The diagnosis derived from the interview was then compared with the diagnosis from the medical records as the reference diagnosis. The sensitivity and specificity of the questionnaire to detect cerebral malaria in history were, respectively, 84% and 76%. The questionnaire was concordant with the medical record diagnosis in 60% (95% CI: 50-71%) of cases. For other clinical phenotypes of malaria (severe malaria anemia, uncomplicated malaria, and severe malaria anemia concurrent with cerebral malaria), sensitivity and specificity ranged from 42% to 85% and 88% to 96%, respectively. Positive and negative predictive values were, respectively, 75% and 85%. The questionnaire demonstrated suitable sensitivity and specificity to identify cerebral malaria in a participant's history. In sub-Saharan Africa, a region with suboptimal medical record archives, such a tool could be used in case-control studies of severe malaria to select controls.

Plasmodium parasites, the causative organism of malaria, caused over 600,000 deaths in 2022. In Mali, Plasmodium falciparum causes the majority of malaria cases and deaths and is transmitted seasonally. Anti-malarial immunity develops slowly over repeated exposures to P. falciparum and some aspects of this immunity (e.g., antibody titers) wane during the non-transmission, dry season. Here, we sequenced RNA from 33 pediatric blood samples collected during P. falciparum infections at the beginning or end of a transmission season, and characterized the host and parasite gene expression profiles for paired, consecutive infections. We found that human gene expression changes more over the course of one transmission season than between seasons, with signatures of partial development of an adaptive immune response during one transmission season and stability in gene expression during the dry season. Additionally, we found that P. falciparum gene expression did not vary with timing during the season and remained stable both across and between seasons, despite varying human immune pressures. Our results provide insights into the dynamics of anti-malarial immune response development over short time frames that could be exploited by future vaccine and prevention efforts., Importance: Our work seeks to understand how the immune response to Plasmodium falciparum malaria changes between infections that occur during low and high malaria transmission seasons, and highlights that immune gene expression changes more during the high transmission season. This provides important insight into the dynamics of the anti-malarial immune response that are important to characterize over these short time frames to better understand how to exploit this immune response with future vaccine efforts., Competing Interests: The authors declare no conflict of interest.

In Bandiagara, Mali, children experience on average two clinical malaria episodes per year. However, even in the same transmission area, the number of uncomplicated symptomatic infections, and their parasitemia, can vary dramatically among children. We simultaneously characterize host and parasite gene expression profiles from 136 Malian children with symptomatic falciparum malaria and examine differences in the relative proportion of immune cells and parasite stages, as well as in gene expression, associated with infection and or patient characteristics. Parasitemia explains much of the variation in host and parasite gene expression, and infections with higher parasitemia display proportionally more neutrophils and fewer T cells, suggesting parasitemia-dependent neutrophil recruitment and/or T cell extravasation to secondary lymphoid organs. The child's age also strongly correlates with variations in gene expression: Plasmodium falciparum genes associated with age suggest that older children carry more male gametocytes, while variations in host gene expression indicate a stronger innate response in younger children and stronger adaptive response in older children. These analyses highlight the variability in host responses and parasite regulation during P. falciparum symptomatic infections and emphasize the importance of considering the children's age when studying and treating malaria infections., (© 2024. The Author(s).)

In Bandiagara, Mali, children experience on average two clinical malaria episodes per season. However, even in the same transmission area, the number of uncomplicated symptomatic infections, and their parasitemia, vary dramatically among children. To examine the factors contributing to these variations, we simultaneously characterized the host and parasite gene expression profiles from 136 children with symptomatic falciparum malaria and analyzed the expression of 9,205 human and 2,484 Plasmodium genes. We used gene expression deconvolution to estimate the relative proportion of immune cells and parasite stages in each sample and to adjust the differential gene expression analyses. Parasitemia explained much of the variation in both host and parasite gene expression and revealed that infections with higher parasitemia had more neutrophils and fewer T cells, suggesting parasitemia-dependent neutrophil recruitment and/or T cell extravasation to secondary lymphoid organs. The child's age was also strongly correlated with gene expression variations. Plasmodium falciparum genes associated with age suggested that older children carried more male gametocytes, while host genes associated with age indicated a stronger innate response (through TLR and NLR signaling) in younger children and stronger adaptive immunity (through TCR and BCR signaling) in older children. These analyses highlight the variability in host responses and parasite regulation during P. falciparum symptomatic infections and emphasize the importance of considering the children's age when studying and treating malaria infections., Competing Interests: Competing interests: Authors declare that they have no competing interests.

Antibody responses to variant surface antigens (VSAs) produced by the malaria parasite Plasmodium falciparum may contribute to age-related natural immunity to severe malaria. One VSA family, P. falciparum erythrocyte membrane protein-1 (PfEMP1), includes a subset of proteins that binds endothelial protein C receptor (EPCR) in human hosts and potentially disrupts the regulation of inflammatory responses, which may lead to the development of severe malaria. We probed peptide microarrays containing segments spanning five PfEMP1 EPCR-binding domain variants with sera from 10 Malian adults and 10 children to determine the differences between adult and pediatric immune responses. We defined serorecognized peptides and amino acid residues as those that elicited a significantly higher antibody response than malaria-naïve controls. We aimed to identify regions consistently serorecognized among adults but not among children across PfEMP1 variants, potentially indicating regions that drive the development of immunity to severe malaria. Adult sera consistently demonstrated broader and more intense serologic responses to constitutive PfEMP1 peptides than pediatric sera, including peptides in EPCR-binding domains. Both adults and children serorecognized a significantly higher proportion of EPCR-binding peptides than peptides that do not directly participate in receptor binding, indicating a preferential development of serologic responses at functional residues. Over the course of a single malaria transmission season, pediatric serological responses increased between the start and the peak of the season, but waned as the transmission season ended. IMPORTANCE Severe malaria and death related to malaria disproportionately affect sub-Saharan children under 5 years of age, commonly manifesting as cerebral malaria and/or severe malarial anemia. In contrast, adults in malaria-endemic regions tend to experience asymptomatic or mild disease. Our findings indicate that natural immunity to malaria targets specific regions within the EPCR-binding domain, particularly peptides containing EPCR-binding residues. Epitopes containing these residues may be promising targets for vaccines or therapeutics directed against severe malaria. Our approach provides insight into the development of natural immunity to a binding target linked to severe malaria by characterizing an "adult-like" response as recognizing a proportion of epitopes within the PfEMP1 protein, particularly regions that mediate EPCR binding. This "adult-like" response likely requires multiple years of malaria exposure, as increases in pediatric serologic response over a single malaria transmission season do not appear significant., Competing Interests: The authors declare no conflict of interest.

Plasmodium parasites caused 241 million cases of malaria and over 600,000 deaths in 2020. Both P. falciparum and P. ovale are endemic to Mali and cause clinical malaria, with P. falciparum infections typically being more severe. Here, we sequenced RNA from nine pediatric blood samples collected during infections with either P. falciparum or P. ovale, and characterized the host and parasite gene expression profiles. We found that human gene expression varies more between individuals than according to the parasite species causing the infection, while parasite gene expression profiles cluster by species. Additionally, we characterized DNA polymorphisms of the parasites directly from the RNA-seq reads and found comparable levels of genetic diversity in both species, despite dramatic differences in prevalence. Our results provide unique insights into host-pathogen interactions during malaria infections and their variations according to the infecting Plasmodium species, which will be critical to develop better elimination strategies against all human Plasmodium parasites., Competing Interests: None, (Copyright: © 2023 Tebben 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.)

Purpose: This study describes factors associated with COVID-19 precautions (i.e., self-isolation and the use of personal protective equipment) among a sample of adults with intellectual and developmental disabilities (IDD) in Ontario, Canada., Methods: The sample included 756 home care recipients with IDD who did not test positive for COVID-19 between March 2020 and July 2021. Among these, some received COVID-19 precautions. Precaution data were obtained from a large metropolitan organization serving persons with IDD in Ontario, and linked to home care assessment data. Unadjusted and adjusted odds ratios with 95% confidence intervals were calculated using logistic regression models to examine the association between COVID-19 cautions and demographic and clinical factors. Effect modification and interactions were explored., Results: One hundred twenty-seven (16.8%) home care clients experienced precautions. After adjustment, congregate setting, aggression, and limited mobility were significantly associated with COVID-19 precautions. Age modified the relationship between congregate setting and precautions., Conclusions: Pandemic responses need to recognize the impact on subgroups of adults with IDD, such as those living in congregate settings (including younger individuals) or engaging in responsive behaviors. How these precautions impacted individuals-in the short and long term-warrants further investigation., (Copyright © 2022 Elsevier Inc. All rights reserved.)

var genes encode Plasmodium falciparum erythrocyte membrane protein-1 (PfEMP1) antigens. These highly diverse antigens are displayed on the surface of infected erythrocytes and play a critical role in immune evasion and sequestration of infected erythrocytes. Studies of var expression using non-leukocyte-depleted blood are challenging because of the predominance of host genetic material and lack of conserved var segments. Our goal was to enrich for parasite RNA, allowing de novo assembly of var genes and detection of expressed novel variants. We used two overall approaches: (i) enriching for total mRNA in the sequencing library preparations and (ii) enriching for parasite RNA with a custom capture array based on Roche's SeqCap EZ enrichment system. The capture array was designed with probes based on the whole 3D7 reference genome and an additional >4,000 full-length var gene sequences from other P. falciparum strains. We tested each method on RNA samples from Malian children with severe or uncomplicated malaria infections. All reads mapping to the human genome were removed, the remaining reads were assembled de novo into transcripts, and from these, var -like transcripts were identified and annotated. The capture array produced the longest maximum length and largest numbers of var gene transcripts in each sample, particularly in samples with low parasitemia. Identifying the most-expressed var gene sequences in whole-blood clinical samples without the need for extensive processing or generating sample-specific reference genome data is critical for understanding the role of PfEMP1s in malaria pathogenesis. IMPORTANCE Malaria parasites display antigens on the surface of infected red blood cells in the human host that facilitate attachment to blood vessels, contributing to the severity of infection. These antigens are highly variable, allowing the parasite to evade the immune system. Identifying these expressed antigens is critical to understanding the development of severe malarial disease. However, clinical samples contain limited amounts of parasite genetic material, a challenge for sequencing efforts further compounded by the extreme diversity of the parasite surface antigens. We present a method that enriches for these antigen sequences in clinical samples using a custom capture array, requiring minimal processing in the field. While our results are focused on the malaria parasite Plasmodium falciparum, this approach has broad applicability to other highly diverse antigens from other parasites and pathogens such as those that cause giardiasis and leishmaniasis.

Objective: The goal of this pilot study was to assess the feasibility of a 9-day, smartphone-based ecological momentary assessment (EMA) protocol for tracking the frequency of Canadian adolescents' exposures to cannabis marketing, their reactions to such exposures, and the context in which exposures occur in the real-world and in real-time., Method: Participants were n = 18 adolescents between the ages of 14 and 18 years of age. They used an EMA application to capture and describe cannabis marketing exposures through photographs and brief questionnaires assessing marketing channel and context. Participants also rated their reactions to each exposure in real-time., Results: Results showed that participants were generally compliant with the protocol. Participants recorded 40 total exposures to cannabis marketing, representing an average of 2.2 ( SD 2.3) exposures per participant during the 9-day study. Exposures tended to occur in the afternoon (45.0%) or evening (37.5%), and while participants were at home (70%) and alone (52.5%). Most exposures occurred through promotion by public figures (27.5%) or explicitly marked internet ads (27.5%)., Conclusion: This is the first study to demonstrate the feasibility and utility of EMA to capture adolescent exposures to cannabis marketing as it occurs in participants' natural environments. Our research offers an early look at the predictable wave of cannabis advertising targeting youth and a promising approach for studying its impacts in a post-legalization context, as well as a strategy for assessing policies, such as advertising restrictions, intending to mitigate the harms of early cannabis use among youth., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2021 The Authors.)

Bacillus velezensis, a species first described in 2005, has been mostly associated with plants and the environment. To date, there is no genome available for this species from human samples. In this announcement, we present the genome of Bacillus velezensis strain Marseille-Q1230, which was isolated from a stool sample from a child suffering from severe acute malnutrition. The genome assembled into 15 contigs and had a size of 3,861,152 bp, with a GC content of 46.6%. A total of 3,716 protein-coding genes, including 3 antibiotic resistance genes and 92 RNAs, were predicted.

Children are highly susceptible to clinical malaria, and in regions where malaria is endemic, their immune systems must face successive encounters with Plasmodium falciparum parasites before they develop immunity, first against severe disease and later against uncomplicated malaria. Understanding cellular and molecular interactions between host and parasites during an infection could provide insights into the processes underlying this gradual acquisition of immunity, as well as to how parasites adapt to infect hosts that are successively more malaria experienced. Here, we describe methods to analyze the host and parasite gene expression profiles generated simultaneously from blood samples collected from five consecutive symptomatic P. falciparum infections in three Malian children. We show that the data generated enable statistical assessment of the proportions of (i) each white blood cell subset and (ii) the parasite developmental stages, as well as investigations of host-parasite gene coexpression. We also use the sequences generated to analyze allelic variations in transcribed regions and determine the complexity of each infection. While limited by the modest sample size, our analyses suggest that host gene expression profiles primarily clustered by individual, while the parasite gene expression profiles seemed to differentiate early from late infections. Overall, this study provides a solid framework to examine the mechanisms underlying acquisition of immunity to malaria infections using whole-blood transcriptome sequencing (RNA-seq). IMPORTANCE We show that dual RNA-seq from patient blood samples allows characterization of host/parasite interactions during malaria infections and can provide a solid framework to study the acquisition of antimalarial immunity, as well as the adaptations of P. falciparum to malaria-experienced hosts., (Copyright © 2020 Bradwell et al.)

Blastocystis is the most common protozoan colonizing the gut of vertebrates. It modulates the human digestive microbiota in the absence of inflammation and gastrointestinal disease. Although it has been associated with human diseases, including inflammatory bowel disease, its pathogenicity remains controversial. This study aimed to assess the influence of Blastocystis on the gut bacterial communities in healthy children. We conducted a cross-sectional study on 147 Blastocystis - colonized and 149 Blastocystis - noncolonized Malian children, with Blastocystis colonization assessed by real-time PCR and gut microbial communities characterized via 16S rRNA gene (Illumina MiSeq) sequencing and bioinformatics analysis. The gut microbiota diversity was higher in Blastocystis-colonized compared to Blastocystis - noncolonized children. The phyla Firmicutes, Elusimicrobia, Lentisphaerae, and Euryarchaeota were higher in Blastocystis - colonized children, whereas Actinobacteria, Proteobacteria, unassigned bacteria, and Deinococcus-Thermus were higher in Blastocystis - noncolonized children. Moreover, Faecalibacterium prausnitzii (family Ruminococcaceae) and Roseburia sp. (family Lachnospiraceae) abundance was higher in Blastocystis-colonized children. We conclude that Blastocystis colonization is significantly associated with a higher diversity of the gut bacterial communities in healthy children, while it is not associated with the presence of potentially pathogenic bacteria in the human gut., Competing Interests: The authors declare that they have no conflict of interest.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been recently described as an innovative and effective tool for identifying arthropods and mosquito blood meal sources. To test this approach in the context of an entomological survey in the field, mosquitoes were collected from five ecologically distinct areas of Mali. We successfully analysed the blood meals from 651 mosquito abdomens crushed on Whatman filter paper (WFPs) in the field using MALDI-TOF MS. The legs of 826 mosquitoes were then submitted for MALDI-TOF MS analysis in order to identify the different mosquito species. Eight mosquito species were identified, including Anopheles gambiae Giles, Anopheles coluzzii, Anopheles arabiensis, Culex quinquefasciatus, Culex neavei, Culex perexiguus, Aedes aegypti and Aedes fowleri in Mali. The field mosquitoes for which MALDI-TOF MS did not provide successful identification were not previously available in our database. These specimens were subsequently molecularly identified. The WFP blood meal sources found in this study were matched against human blood (n = 619), chicken blood (n = 9), cow blood (n = 9), donkey blood (n = 6), dog blood (n = 5) and sheep blood (n = 3). This study reinforces the fact that MALDI-TOF MS is a promising tool for entomological surveys.

Ticks are considered the second vector of human and animal diseases after mosquitoes. Therefore, identification of ticks and associated pathogens is an important step in the management of these vectors. In recent years, Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been reported as a promising method for the identification of arthropods including ticks. The objective of this study was to improve the conditions for the preparation of tick samples for their identification by MALDI-TOF MS from field-collected ethanol-stored Malian samples and to evaluate the capacity of this technology to distinguish infected and uninfected ticks. A total of 1,333 ticks were collected from mammals in three distinct sites from Mali. Morphological identification allowed classification of ticks into 6 species including Amblyomma variegatum, Hyalomma truncatum, Hyalomma marginatum rufipes, Rhipicephalus (Boophilus) microplus, Rhipicephalus evertsi evertsi and Rhipicephalus sanguineus sl. Among those, 471 ticks were randomly selected for molecular and proteomic analyses. Tick legs submitted to MALDI-TOF MS revealed a concordant morpho/molecular identification of 99.6%. The inclusion in our MALDI-TOF MS arthropod database of MS reference spectra from ethanol-preserved tick leg specimens was required to obtain reliable identification. When tested by molecular tools, 76.6%, 37.6%, 20.8% and 1.1% of the specimens tested were positive for Rickettsia spp., Coxiella burnetii, Anaplasmataceae and Borrelia spp., respectively. These results support the fact that MALDI-TOF is a reliable tool for the identification of ticks conserved in alcohol and enhances knowledge about the diversity of tick species and pathogens transmitted by ticks circulating in Mali.

Background: Mosquitoes transmit a wide range of human parasitic and viral diseases. In recent years, new techniques such as MALDI-TOF MS have been developed to identify mosquitoes at the species level, which is key for entomological surveys. Additionally, there is increasing interest in the mosquito microbiota and its role in vector capacity., Methods: The culturomics approach previously used in our laboratory to study human gut microbiota was applied to evaluate the midgut bacterial diversity of Anopheles gambiae (wild and laboratory strains), Aedes albopictus (wild and laboratory strains) and Culex quinquefasciatus (wild strains) in order to determine the influence of the environmental status on the midgut microbiota of the mosquitoes., Results: Mosquitoes collected in the field were accurately identified by MALDI-TOF MS analysis of their legs. Adult mosquito midgut microbiota was composed of four phyla, including Proteobacteria, Bacteroidetes, Actinobacteria and Firmicutes. The majority of the bacteria detected in the microbiota of mosquitoes were gram-negative and belong to the phylum Proteobacteria. MALDI-TOF MS identified for the first time a new bacterial species from An. gambiae midgut microbiota., Conclusion: In this study, the culturomics approach was found to be a reliable technique for exploring the diversity of the mosquito microbiota. MALDI-TOF MS was confirmed as a promising technique to identify mosquitoes collected in the field. Culturomics allowed the isolation of a new bacterial species not previously associated with mosquito vectors. The environment plays a role in the bacterial diversity of the microbiota, which could enable the development of new control strategies for mosquito-borne disease.

Objective: The objective of this work was to describe the epidemiology of schistosomiasis 10 years after mass administration of praziquantel began in Sotuba, Mali., Methodology/results: This observational cross-sectional survey in Sotuba, a periurban village in the Bamako district, took place from July to September 2010 and collected stool and urine samples from residents at least one year old. Kato-Katz (for stool) and urine filtration techniques were used to detect Schistosoma mansoni and S. hæmatobium eggs, respectively. Overall, 335 urine samples and 300 stool samples were examined. The prevalence rate was 5.4% (18/335) for S. hæmatobium and 8.7% (26/300) for S. mansoni. Excretion of these eggs was most frequent in the 6-15 year-old group: 4.8% (16/335) for S. hæmatobium and 7.7% (23/300) for S. mansoni. Snails of the Biomphalaria pfeifferi and Bulinus truncatus species were the intermediate hosts, captured in the fields at water contact points. The principal clinical symptoms reported by participants were abdominal pain 27.2% (61/169) and headaches 23.2% (52/169)., Conclusion: Despite the implementation of mass drug administration in Mali a decade ago, our results show that schistosomiasis transmission continues in Sotuba. Assessment of the risk factors for this persistent transmission is strongly needed.

Background: Hemoglobin C trait, like hemoglobin S trait, protects against severe malaria in children, but it is unclear whether hemoglobin C trait also protects against uncomplicated malaria. We hypothesized that Malian children with hemoglobin C trait would have a lower risk of clinical malaria than children with hemoglobin AA., Methods: Three hundred children aged 0-6 years were enrolled in a cohort study of malaria incidence in Bandiagara, Mali, with continuous passive and monthly active follow-up from June 2009 to June 2010., Results: Compared to hemoglobin AA children (n = 242), hemoglobin AC children (n = 39) had a longer time to first clinical malaria episode (hazard ratio [HR], 0.19; P = .001; 364 median malaria-free days vs 181 days), fewer episodes of clinical malaria, and a lower cumulative parasite burden. Similarly, hemoglobin AS children (n = 14) had a longer time to first clinical malaria episode than hemoglobin AA children (HR, 0.15; P = .015; 364 median malaria-free days vs 181 days), but experienced the most asymptomatic malaria infections of any group., Conclusions: Both hemoglobin C and S traits exerted a protective effect against clinical malaria episodes, but appeared to do so by mechanisms that differentially affect the response to infecting malaria parasites., (© The Author 2015. Published by Oxford University Press on behalf of the Infectious Diseases Society of America.)

Background: Novel treatments against for tinea capitis are needed, and the natural aminosterol squalamine is a potential topical antidermatophyte drug candidate., Objectives: This phase II randomized double-blind placebo-controlled clinical trial aimed at testing the efficacy and safety of a three-week squalamine ointment regimen for the treatment of tinea capitis., Patients: Males aged 6-15 years presenting with tinea capitis were treated with either topical squalamine ointment or placebo for 3 weeks. The primary endpoint was complete clinical cure. The secondary endpoints were the occurrence of local and/or systemic adverse events, mycological cure, and partial clinical response. Prospective follow-up of clinical adverse events was performed daily., Results: Five patients were treated with 1% squalamine ointment and 15 with placebo. No complete cure was observed. No clinical or biological adverse event was recorded. A significantly (p = 0.03) better hair-growth score, indicating a partial clinical improvement of the tinea capitis lesion, was observed in the patients treated with squalamine compared to those treated with placebo., Conclusion: This three-week squalamine ointment regimen was well tolerated and showed an encouraging partial clinical activity for the treatment of tinea capitis. Further studies are needed to evaluate the efficacy of topical squalamine alone against tinea corporis or in combination with a systemic antidermatophyte drug against tinea capitis.

Objectives: Because lice-transmitted infections are a real public health problem, epidemiological studies in different ecoclimatic zones of Africa are useful. This article aims to describe the frequency of lice infestation, their genotypes, and their infection by pathogens in the regions of Koulikoro and Mopti., Methodology: A cross-sectional survey allowed us to collect lice from rural populations. Techniques of molecular biology (real-time PCR, standard PCR, and genotyping) were used for analysis of lice samples., Results: Infestation rates were 57% (12/21) among subjects in Diankabou, in the Sahelian zone; 91% (39/43) in Doneguebougou, and 86% (59/69) in Zorocoro, in a savanna zone. The overall lice infestation rate in the samples in the three localities was 83% (110/133). Real-time PCR showed 3% (4/92) of Acinetobacter baumanii but no B. quintana in Diankabou. Phylogenetic analysis of the mitochondrial gene (Cytb) showed that head lice in Mali belong to genotype C., Conclusions: The high frequency of lice infestation in the study population indicates that it would be useful to conduct national epidemiological surveys to estimate the magnitude of this public health problem.

Background: Leishmania major complex is the main causative agent of zoonotic cutaneous leishmaniasis (ZCL) in the Old World. Phlebotomus papatasi and Phlebotomus duboscqi are recognized vectors of L. major complex in Northern and Southern Sahara, respectively. In Mali, ZCL due to L. major is an emerging public health problem, with several cases reported from different parts of the country. The main objective of the present study was to identify the vectors of Leishmania major in the Bandiagara area, in Mali., Methodology/principal Findings: An entomological survey was carried out in the ZCL foci of Bandiagara area. Sandflies were collected using CDC miniature light traps and sticky papers. In the field, live female Phlebotomine sandflies were identified and examined for the presence of promastigotes. The remaining sandflies were identified morphologically and tested for Leishmania by PCR in the ITS2 gene. The source of blood meal of the engorged females was determined using the cyt-b sequence. Out of the 3,259 collected sandflies, 1,324 were identified morphologically, and consisted of 20 species, of which four belonged to the genus Phlebotomus and 16 to the genus Sergentomyia. Leishmania major DNA was detected by PCR in 7 of the 446 females (1.6%), specifically 2 out of 115 Phlebotomus duboscqi specimens, and 5 from 198 Sergentomyia darlingi specimens. Human DNA was detected in one blood-fed female S. darlingi positive for L. major DNA., Conclusion: Our data suggest the possible involvement of P. duboscqi and potentially S. darlingi in the transmission of ZCL in Mali.

Plasmodium falciparum rosetting (the spontaneous binding of infected erythrocytes to uninfected erythrocytes) is a well-recognized parasite virulence factor. However, it is currently unclear whether rosetting is associated with all clinical forms of severe malaria, or only with specific syndromes such as cerebral malaria. We investigated the relationship between rosetting and clinical malaria in 209 Malian children enrolled in a case-control study of severe malaria. Rosetting was significantly higher in parasite isolates from severe malaria cases compared with non-severe hyperparasitemia and uncomplicated malaria controls (F(2,117) = 8.15, P < 0.001). Analysis of sub-categories of severe malaria (unrousable coma, severe anemia, non-comatose neurological impairment, repeated seizures or a small heterogeneous group with signs of renal failure or jaundice) showed high levels of rosetting in all sub-categories, and no statistically significant differences in rosetting between sub-categories (F(4,67) = 1.28, P = 0.28). Thus rosetting may contribute to the pathogenesis of all severe malaria syndromes in African children, and interventions to disrupt rosetting could be potential adjunctive therapies for all forms of severe malaria in Africa.

The Plasmodium falciparum variant erythrocyte surface antigens known as PfEMP1, encoded by the var gene family, are thought to play a crucial role in malaria pathogenesis because they mediate adhesion to host cells and immuno-modulation. Var genes have been divided into three major groups (A, B and C) and two intermediate groups (B/A and B/C) on the basis of their genomic location and upstream sequence. We analysed expressed sequence tags of the var gene DBLalpha domain to investigate var gene transcription in relation to disease severity in Malian children. We found that P. falciparum isolates from children with cerebral malaria (unrousable coma) predominantly transcribe var genes with DBLalpha1-like domains that are characteristic of Group A or B/A var genes. In contrast, isolates from children with equally high parasite burdens but no symptoms or signs of severe malaria (hyperparasitaemia patients) predominantly transcribe var genes with DBLalpha0-like domains that are characteristic of the B and C-related var gene groups. These results suggest that var genes with DBLalpha1-like domains (Group A or B/A) may be implicated in the pathogenesis of cerebral malaria, while var genes with DBLalpha0-like domains promote less virulent malaria infections.

Treating malaria before immunizing has been standard in malaria vaccine field trials. To assess the impact of this practice on subsequent infection and disease incidence, we conducted a randomized cohort study in Bandiagara, Mali. Subjects received a treatment dose of sulfadoxine-pyrimethamine (SP) or no treatment at the beginning of the transmission season. Cumulative and age-specific incidence of clinical episodes was similar between the 2 groups, but SP treatment delayed the median time to first clinical episode from 38.5 to 68 days, and after this initial period of protection, disease incidence in the SP group quickly surpassed the incidence in the untreated group. Parasite densities during disease episodes were lower in the SP group. SP was chosen as the drug for initial parasite clearance for the following reasons: 1) it has been used in previous vaccine trials; 2) our studies have found it to have >99% efficacy in treating uncomplicated malaria in Mali compared to 85-90% efficacy for chloroquine in this area; 3) SP is the approved second-line antimalarial agent in Mali; and 4) its single-dose regimen ensures compliance when treatment is directly observed.