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Abstract Vector control in the Bijagós Archipelago of Guinea-Bissau currently relies on pyrethroid insecticide-treated nets. However, data on insecticide resistance in Guinea-Bissau is limited. This study identified deltamethrin resistance in the Anopheles gambiae sensu lato complex on Bubaque island using WHO tube tests in November 2022. Whole genome sequencing of An. gambiae sensu stricto mosquitoes identified six single nucleotide polymorphisms (SNPs) previously associated with, or putatively associated with, insecticide resistance: T791M, L995F, N1570Y, A1746S and P1874L in the vgsc gene, and L119V in the gste2 gene. Twenty additional non-synonymous SNPs were identified in insecticide-resistance associated genes. Four of these SNPs were present at frequencies over 5% in the population: T154S, I126F and G26S in the vgsc gene and A65S in ace1. Genome wide selection scans using Garud’s H12 statistic identified two selective sweeps: one in chromosome X and one in chromosome 2R. Both selective sweeps overlap with metabolic genes previously associated with insecticide resistance, including cyp9k1 and the cyp6aa/cyp6p gene cluster. This study presents the first phenotypic testing for deltamethrin resistance and the first whole genome sequence data for Anopheles gambiae mosquitoes from the Bijagós, contributing data of significance for vector control policy in this region.

Abstract Background Anopheles melas is an understudied malaria vector with a potential role in malaria transmission on the Bijagós Archipelago of Guinea-Bissau. This study presents the first whole-genome sequencing and population genetic analysis for this species from the Bijagós. To our knowledge, this also represents the largest population genetic analysis using WGS data from non-pooled An. melas mosquitoes. Methods WGS was conducted for 30 individual An. melas collected during the peak malaria transmission season in 2019 from six different islands on the Bijagós Archipelago. Bioinformatics tools were used to investigate the population structure and prevalence of insecticide resistance markers in this mosquito population. Results Insecticide resistance mutations associated with pyrethroid resistance in Anopheles gambiae s.s. from the Bijagós were absent in the An. melas population, and no signatures of selective sweeps were identified in insecticide resistance-associated genes. Analysis of structural variants identified a large duplication encompassing the cytochrome-P450 gene cyp9k1. Phylogenetic analysis using publicly available mitochondrial genomes indicated that An. melas from the Bijagós split into two phylogenetic groups because of differentiation on the mitochondrial genome attributed to the cytochrome C oxidase subunits COX I and COX II and the NADH dehydrogenase subunits 1, 4, 4L and 5. Conclusions This study identified an absence of insecticide-resistant SNPs common to An. gambiae in the An. melas population, but did identify structural variation over insecticide resistance-associated genes. Furthermore, this study presents novel insights into the population structure of this malaria vector using WGS analysis. Additional studies are required to further understand the role of this vector in malaria transmission. Graphical abstract

Abstract Background Severe malaria is a life-threatening infection, particularly affecting children under the age of 5 years in Africa. Current treatment with parenteral artemisinin derivatives is highly efficacious. However, artemisinin partial resistance is widespread in Southeast Asia, resulting in delayed parasite clearance after therapy, and has emerged independently in South America, Oceania, and Africa. Hence, new treatments for severe malaria are needed, and it is prudent to define their characteristics now. This manuscript focuses on the target product profile (TPP) for new treatments for severe malaria. It also highlights preparedness when considering ways of protecting the utility of artemisinin-based therapies. Target product profile Severe malaria treatments must be highly potent, with rapid onset of antiparasitic activity to clear the infection as quickly as possible to prevent complications. They should also have a low potential for drug resistance selection, given the high parasite burden in patients with severe malaria. Combination therapies are needed to deter resistance selection and dissemination. Partner drugs which are approved for uncomplicated malaria treatment would provide the most rapid development pathway for combinations, though new candidate molecules should be considered. Artemisinin combination approaches to severe malaria would extend the lifespan of current therapy, but ideally, completely novel, non-artemisinin-based combination therapies for severe malaria should be developed. These should be advanced to at least phase 2 clinical trials, enabling rapid progression to patient use should current treatment fail clinically. New drug combinations for severe malaria should be available as injectable formulations for rapid and effective treatment, or as rectal formulations for pre-referral intervention in resource-limited settings. Conclusion Defining the TPP is a key step to align responses across the community to proactively address the potential for clinical failure of artesunate in severe malaria. In the shorter term, artemisinin-based combination therapies should be developed using approved or novel drugs. In the longer term, novel combination treatments should be pursued. Thus, this TPP aims to direct efforts to preserve the efficacy of existing treatments while improving care and outcomes for individuals affected by this life-threatening disease.

BackgroundPoint of care hemoglobin meters play key roles in increasing access to anemia screening in antenatal care especially in settings with limited access to laboratories. We aimed to determine the diagnostic accuracy of a non-invasive spot-check hemoglobin (SpHb) meter, Masimo Rad-67® Pulse CO-Oximeter®, in the diagnosis of anemia in pregnant women attending antenatal care clinics in Kilifi, Kenya.MethodsThis was a diagnostic accuracy study that retrospectively evaluated SpHb against a validated reference standard of laboratory assessed hemoglobin (Lab Hb) by a SYSMEX XN-330 automated hematology analyzer. The study was nested within a prospective pregnancy cohort study that recruited unselected pregnant women from antenatal care clinics in two public hospitals in Kilifi County, coastal Kenya. Records with both SpHb and Lab Hb were selected from pregnancy visits between May 2021 and December 2022. Linear regression and Bland-Altman analysis were performed to compare the two tests and diagnostic accuracy parameters obtained for the diagnosis of anemia.ResultsA total of 2,975 records (from 2,203 unique participants), with paired SpHb and Lab Hb were analyzed. Linear regression showed a significant but weak positive correlation, a proportional bias of 0.44 (95% CI 0.41–0.47) and a constant of 7.59 (95% CI 7.30–7.87, p

Introduction The burden of severe maternal morbidity is highest in sub-Saharan Africa, and its relative contribution to maternal (ill) health may increase as maternal mortality continues to fall. Women’s perspective of their long-term recovery following severe morbidity beyond the standard 42-day postpartum period remains largely unexplored.Methods This woman-centred, grounded theory study was nested within the Pregnancy Care Integrating Translational Science Everywhere (PRECISE) study in Kilifi, Kenya. Purposive and theoretical sampling was used to recruit 20 women who experienced either a maternal near-miss event (n=11), potentially life-threatening condition (n=6) or no severe morbidity (n=3). Women were purposively selected between 6 and 36 months post partum at the time of interview to compare recovery trajectories. Using a constant comparative approach of line-by-line open codes, focused codes, super-categories and themes, we developed testable hypotheses of women’s postpartum recovery trajectories after severe maternal morbidity.Results Grounded in women’s accounts of their lived experience, we identify three phases of recovery following severe maternal morbidity: ‘loss’, ‘transition’ and ‘adaptation to a new normal’. These themes are supported by multiple, overlapping super-categories: loss of understanding of own health, functioning and autonomy; transition in women’s identity and relationships; and adaptation to a new physical, psychosocial and economic state. This recovery process is multidimensional, potentially cyclical and extends far beyond the standard 42-day postpartum period.Conclusion Women’s complex needs following severe maternal morbidity require a reconceptualisation of postpartum recovery as extending far beyond the standard 42-day postpartum period. Women’s accounts expose major deficiencies in the provision of postpartum and mental healthcare. Improved postpartum care provision at the primary healthcare level, with reach extended through community health workers, is essential to identify and treat chronic mental or physical health problems following severe maternal morbidity.

Abstract Background Insecticide resistance is reducing the efficacy of vector control interventions, consequently threatening efforts to control vector-borne diseases, including malaria. Investigating the prevalence of molecular markers of resistance is a useful tool for monitoring the spread of insecticide resistance in disease vectors. The Bijagós Archipelago (Bijagós) in Guinea-Bissau is a region of stable malaria transmission where insecticide-treated nets are the mainstay for malaria control. However, the prevalence of molecular markers of insecticide resistance in malaria vectors is not well understood. Methods A total of 214 Anopheles mosquitoes were analysed from 13 islands across the Bijagós. These mosquitoes were collected using CDC light traps in November 2019, during the peak malaria transmission season. High-throughput multiplex amplicon sequencing was used to investigate the prevalence of 17 different molecular markers associated with insecticide resistance in four genes: vgsc, rdl, ace1 and gste2. Results Of the 17 screened mutations, four were identified in mosquitoes from the Bijagós: vgsc L995F (12.2%), N1570Y (6.2%) and A1746S (0.7%) and rdl A269G (1.1%). This study is the first to report the L995F knock-down resistance (kdr)-west allele in Anopheles melas on the Archipelago. An additional eight non-synonymous single-nucleotide polymorphisms were identified across the four genes which have not been described previously. The prevalences of the vgsc L995F and N1570Y mutations were higher on Bubaque Island than on the other islands in this study; Bubaque is the most populous island in the archipelago, with the greatest population mobility and connection to continental Guinea-Bissau. Conclusions This study provides the first surveillance data for genetic markers present in malaria vectors from islands across the Bijagós Archipelago. Overall prevalence of insecticide resistance mutations was found to be low. However, the identification of the vgsc L995F and N1570Y mutations associated with pyrethroid resistance warrants further monitoring. This is particularly important as the mainstay of malaria control on the islands is the use of pyrethroid insecticide-treated nets. Graphical Abstract

Abstract Accurate and rapid point-of-care (PoC) diagnostics are critical to the control of the COVID-19 pandemic. The current standard for accurate diagnosis of SARS-CoV-2 is laboratory-based reverse transcription polymerase chain reaction (RT-PCR) assays. Here, a preliminary prospective performance evaluation of the QuantuMDx Q-POC SARS-CoV-2 RT-PCR assay is reported. Between November 2020 and March 2021, 49 longitudinal combined nose/throat (NT) swabs from 29 individuals hospitalised with RT-PCR confirmed COVID-19 were obtained at St George’s Hospital, London. In addition, 101 mid-nasal (MN) swabs were obtained from healthy volunteers in June 2021. These samples were used to evaluate the Q-POC SARS-CoV-2 RT-PCR assay. The primary analysis was to compare the sensitivity and specificity of the Q-POC test against a reference laboratory-based RT-PCR assay. The overall sensitivity of the Q-POC test compared with the reference test was 96.88% (83.78– 99.92% CI) for a cycle threshold (Ct) cut-off value for the reference test of 35 and 80.00% (64.35–90.95% CI) without altering the reference test’s Ct cut-off value of 40. The Q-POC test is a sensitive, specific and rapid PoC test for SARS-CoV-2 at a reference Ct cut-off value of 35. The Q-POC test provides an accurate option for RT-PCR at PoC without the need for sample pre-processing and laboratory handling, enabling rapid diagnosis and clinical triage in acute care and other settings.

Low vaginal self-sampling has been pioneered as an important development to improve uptake of cervical screening globally. Limited research is available in specific patient groups in the UK exploring views around self-sampling to detect high-risk human papillomavirus (hrHPV) DNA. Therefore, we explored patient views to support development of a novel point-of-care self-sampling cervical cancer screening device, by undertaking a cross-sectional semi-structured questionnaire survey to explore preferences, acceptability, barriers and facilitators around self-sampling. Patients attending a colposcopy clinic, 25-64 years old, were invited to participate after having carried out a low vaginal self-sample using a regular flocked swab. Participants self-completed an anonymous 12-point questionnaire. Quantitative data were analysed in MS Excel and Graphpad Prism, and qualitative data with Nvivo. We recruited 274 patients with a questionnaire response rate of 76%. Acceptability of self-sampling was high (95%, n = 187/197; Cronbachs-α = 0.778). Participants were asked their choice of future screening method: a) low vaginal self-sampling, b) healthcare professional collected vaginal swab, c) cervical brush sample with healthcare professional speculum examination, or d) no preference. Preferences were: a) 37% (n = 74/198), b) 19% (n = 37/198); c) 9% (n = 17/198), and d) 35% (n = 70/198), showing no single option as a strong preference. Key motivators were: Test simplicity (90%, n = 170/190), speed (81%, n = 153/190) and less pain (65%, n = 123/190). Barriers included lack of confidence taking the sample (53%, n = 10/19), resulting in preference for a healthcare professional sample (47%, n = 9/19). Whilst self-sampling showed high acceptability, lack of strong preference for screening method may reflect that respondents attending colposcopy are already engaged with screening and have differing perception of cervical cancer risk. This group appear less likely to 'switch' to self-sampling, and it may be better targeted within primary and community care, focusing on under-screened populations. Any shift in this paradigm in the UK requires comprehensive education and support for patients and providers.

BackgroundSeroprevalence studies are an alternative approach to estimating the extent of transmission of SARS-CoV-2 and the evolution of the pandemic in different geographical settings. We aimed to determine the SARS-CoV-2 seroprevalence from March 2020 to March 2022 in a rural and urban setting in Kilifi County, Kenya.MethodsWe obtained representative random samples of stored serum from a pregnancy cohort study for the period March 2020 to March 2022 and tested for antibodies against the spike protein using a qualitative SARS-CoV-2 ELISA kit (Wantai, total antibodies). All positive samples were retested for anti-SARS-CoV-2 anti-nucleocapsid antibodies (Euroimmun, ELISA kits, NCP, qualitative, IgG) and anti-spike protein antibodies (Euroimmun, ELISA kits, QuantiVac; quantitative, IgG).ResultsA total of 2,495 (of 4,703 available) samples were tested. There was an overall trend of increasing seropositivity from a low of 0% [95% CI 0–0.06] in March 2020 to a high of 89.4% [95% CI 83.36–93.82] in Feb 2022. Of the Wantai test-positive samples, 59.7% (95% CI 57.06–62.34) tested positive by the Euroimmun anti-SARS-CoV-2 NCP test and 75.9% (95% CI 73.55–78.17) tested positive by the Euroimmun anti-SARS-CoV-2 QuantiVac test. No differences were observed between the urban and rural hospital but villages adjacent to the major highway traversing the study area had a higher seroprevalence.ConclusionAnti-SARS-CoV-2 seroprevalence rose rapidly, with most of the population exposed to SARS-CoV-2 within 23 months of the first cases. The high cumulative seroprevalence suggests greater population exposure to SARS-CoV-2 than that reported from surveillance data.

Abstract Following integrated malaria control interventions, malaria burden on the Bijagós Archipelago has significantly decreased. Understanding the genomic diversity of circulating Plasmodium falciparum malaria parasites can assist infection control, through identifying drug resistance mutations and characterising the complexity of population structure. This study presents the first whole genome sequence data for P. falciparum isolates from the Bijagós Archipelago. Amplified DNA from P. falciparum isolates sourced from dried blood spot samples of 15 asymptomatic malaria cases were sequenced. Using 1.3 million SNPs characterised across 795 African P. falciparum isolates, population structure analyses revealed that isolates from the archipelago cluster with samples from mainland West Africa and appear closely related to mainland populations; without forming a separate phylogenetic cluster. This study characterises SNPs associated with antimalarial drug resistance on the archipelago. We observed fixation of the PfDHFR mutations N51I and S108N, associated with resistance to sulphadoxine-pyrimethamine, and the continued presence of PfCRT K76T, associated with chloroquine resistance. These data have relevance for infection control and drug resistance surveillance; particularly considering expected increases in antimalarial drug use following updated WHO recommendations, and the recent implementation of seasonal malaria chemoprevention and mass drug administration in the region.

Abstract The World Health Organization (WHO) recommends surveillance of molecular markers of resistance to anti-malarial drugs. This is particularly important in the case of mass drug administration (MDA), which is endorsed by the WHO in some settings to combat malaria. Dihydroartemisinin-piperaquine (DHA-PPQ) is an artemisinin-based combination therapy which has been used in MDA. This review analyses the impact of MDA with DHA-PPQ on the evolution of molecular markers of drug resistance. The review is split into two parts. Section I reviews the current evidence for different molecular markers of resistance to DHA-PPQ. This includes an overview of the prevalence of these molecular markers in Plasmodium falciparum Whole Genome Sequence data from the MalariaGEN Pf3k project. Section II is a systematic literature review of the impact that MDA with DHA-PPQ has had on the evolution of molecular markers of resistance. This systematic review followed PRISMA guidelines. This review found that despite being a recognd surveillance tool by the WHO, the surveillance of molecular markers of resistance following MDA with DHA-PPQ was not commonly performed. Of the total 96 papers screened for eligibility in this review, only 20 analysed molecular markers of drug resistance. The molecular markers published were also not standardized. Overall, this warrants greater reporting of molecular marker prevalence following MDA implementation. This should include putative pfcrt mutations which have been found to convey resistance to DHA-PPQ in vitro.

Abstract Severe acute respiratory coronavirus 2 (SARS-CoV-2) has spread globally since its emergence in 2019. Most SARS-CoV-2 infections generate immune responses leading to rising levels of immunoglobulins (Ig) M, A and G which can be detected using diagnostic tests including enzyme-linked immunosorbent assays (ELISA). Whilst implying previous SARS-CoV-2 infection, the detection of Ig by ELISA does not guarantee the presence of neutralising antibodies (NAb) that can prevent the virus infecting cells. Plaque reduction neutralisation tests (PRNT) detect NAb, but are not amenable to mass testing as they take several days and require use of SARS-CoV-2 in high biocontainment laboratories. We evaluated the ability of IgG and IgM ELISAs targeting SARS-CoV-2 spike subunit 1 receptor binding domain (S1-RBD), and spike subunit 2 (S2) and nucleocapsid protein (NP), at predicting the presence and magnitude of NAb determined by PRNT. IgG S2 + NP ELISA was 96.8% [95% CI 83.8–99.9] sensitive and 88.9% [95% CI 51.8–99.7] specific at predicting the presence of NAbs (PRNT80 > 1:40). IgG and IgM S1-RBD ELISAs correlated with PRNT titre, with higher ELISA results increasing the likelihood of a robust neutralising response. The IgM S1-RBD assay can be used as a rapid, high throughput test to approximate the magnitude of NAb titre.

Introduction: Use of hydroxychloroquine in patients with coronavirus disease 2019 (COVID-19) was widespread and uncontrolled until recently. Patients vulnerable to severe COVID-19 are at risk of hydroxychloroquine interactions with co-morbidities and co-medications contributing to detrimental, including fatal, adverse treatment effects. Methods: A retrospective survey was undertaken of health conditions and co-medications of patients with COVID-19 who were pre-screened for enrolment in a randomized, double-blind, placebo-controlled hydroxychloroquine multi-centre trial. Results: The survey involved 305 patients [median age 71 (interquartile range 59–81) years]. The majority of patients (n = 279, 92%) considered for inclusion in the clinical trial were not eligible, mainly due to safety concerns caused by health conditions or co-medications. The most common were QT-prolonging drugs (n = 188, 62%) and haematologic/haemato-oncologic diseases (n = 39, 13%) which prohibited the administration of hydroxychloroquine. In addition, 165 (54%) patients had health conditions and 167 (55%) patients were on co-medications that did not prohibit the use of hydroxychloroquine but had a risk of adverse interactions with hydroxychloroquine. The most common were diabetes (n = 86, 28%), renal insufficiency (n = 69, 23%) and heart failure (n = 58, 19%). Conclusion: The majority of hospitalized patients with COVID-19 had health conditions or took co-medications precluding safe treatment with hydroxychloroquine. Therefore, hydroxychloroquine should be administered with extreme caution in elderly patients with COVID-19, and only in clinical trials.

Abstract Serological testing is emerging as a powerful tool to progress our understanding of COVID-19 exposure, transmission and immune response. Large-scale testing is limited by the need for in-person blood collection by staff trained in venepuncture, and the limited sensitivity of lateral flow tests. Capillary blood self-sampling and postage to laboratories for analysis could provide a reliable alternative. Two-hundred and nine matched venous and capillary blood samples were obtained from thirty nine participants and analysed using a COVID-19 IgG ELISA to detect antibodies against SARS-CoV-2. Thirty eight out of thirty nine participants were able to self-collect an adequate sample of capillary blood (≥ 50 µl). Using plasma from venous blood collected in lithium heparin as the reference standard, matched capillary blood samples, collected in lithium heparin-treated tubes and on filter paper as dried blood spots, achieved a Cohen’s kappa coefficient of > 0.88 (near-perfect agreement, 95% CI 0.738–1.000). Storage of capillary blood at room temperature for up to 7 days post sampling did not affect concordance. Our results indicate that capillary blood self-sampling is a reliable and feasible alternative to venepuncture for serological assessment in COVID-19.

We investigated the dynamics of seroconversion in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. During March 29–May 22, 2020, we collected serum samples and associated clinical data from 177 persons in London, UK, who had SARS-CoV-2 infection. We measured IgG against SARS-CoV-2 and compared antibody levels with patient outcomes, demographic information, and laboratory characteristics. We found that 2.0%–8.5% of persons did not seroconvert 3–6 weeks after infection. Persons who seroconverted were older, were more likely to have concurrent conditions, and had higher levels of inflammatory markers. Non-White persons had higher antibody concentrations than those who identified as White; these concentrations did not decline during follow-up. Serologic assay results correlated with disease outcome, race, and other risk factors for severe SARS-CoV-2 infection. Serologic assays can be used in surveillance to clarify the duration and protective nature of humoral responses to SARS-CoV-2 infection.

The aim of this study was to assess pathogen DNA extraction with a new spin column-based method (DNA-XT). DNA from either whole-blood samples spiked with Plasmodium falciparum or Leishmania donovani amastigote culture was extracted with DNA-XT and compared with that produced by a commercial extraction kit (DNeasy®). Eluates from large and small sample volumes were assessed by PCR and spectroscopy. Using a small volume (5 μl) of blood, the DNA-XT and DNeasy methods produced eluates with similar DNA concentrations (0.63 vs 1.06 ng/μl, respectively). The DNA-XT method produced DNA with lower PCR inhibition than DNeasy. The new technique was also twice as fast and required fewer plastics and manipulations but had reduced total recovered DNA compared with DNeasy.

Abstract We read with interest the publication on malaria treatment by Obonyo et al. (Malaria J 21:30, 2022). This commentary questions the methodology, especially the chosen time points of treatment outcome measures.

The COVID-19 pandemic caused by the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) poses an unprecedented challenge to humanity. SARS-CoV-2 infections range from asymptomatic to severe courses of COVID-19 with acute respiratory distress syndrome (ARDS), multiorgan involvement and death. Risk factors for disease severity include older age, male sex, increased BMI and pre-existing comorbidities. Ethnicity is also relevant to COVID-19 susceptibility and severity. Host genetic predisposition to COVID-19 is now increasingly recognized and whole genome and candidate gene association studies regarding COVID-19 susceptibility have been performed. Several common and rare variants in genes related to inflammation or immune responses have been identified. We summarize research on COVID-19 host genetics and compile genetic variants associated with susceptibility to COVID-19 and disease severity. We discuss candidate genes that should be investigated further to understand such associations and provide insights relevant to pathogenesis, risk classification, therapy response, precision medicine, and drug repurposing.

BackgroundIndividuals infected with SARS-CoV-2 develop neutralising antibodies. We investigated the proportion of individuals with SARS-CoV-2 neutralising antibodies after infection and how this proportion varies with selected covariates.Methodology/principal findingsThis systematic review and meta-analysis examined the proportion of individuals with SARS-CoV-2 neutralising antibodies after infection and how these proportions vary with selected covariates. Three models using the maximum likelihood method assessed these proportions by study group, covariates and individually extracted data (protocol CRD42020208913). A total of 983 reports were identified and 27 were included. The pooled (95%CI) proportion of individuals with neutralising antibodies was 85.3% (83.5-86.9) using the titre cut off >1:20 and 83.9% (82.2-85.6), 70.2% (68.1-72.5) and 54.2% (52.0-56.5) with titres >1:40, >1:80 and >1:160, respectively. These proportions were higher among patients with severe COVID-19 (e.g., titres >1:80, 84.8% [80.0-89.2], >1:160, 74.4% [67.5-79.7]) than those with mild presentation (56.7% [49.9-62.9] and 44.1% [37.3-50.6], respectively) and lowest among asymptomatic infections (28.6% [17.9-39.2] and 10.0% [3.7-20.1], respectively). IgG and neutralising antibody levels correlated poorly.Conclusions/significance85% of individuals with proven SARS-CoV-2 infection had detectable neutralising antibodies. This proportion varied with disease severity, study setting, time since infection and the method used to measure antibodies.

Background Technological advances and high throughput biological assays can facilitate discovery science in biobanks from population cohorts, including pregnant women. Biological pathways associated with health outcomes differ depending on geography, and high-income country data may not generalise to low-resource settings. We conducted a systematic review to identify prospective pregnancy cohorts in sub-Saharan Africa (SSA) that include biobanked samples with potential to enhance discovery science opportunity.Methods Inclusion criteria were prospective data collection during pregnancy, with associated biobanking in SSA. Data sources included: scientific databases (with comprehensive search terms), grey literature, hand searching applicable reference lists and expert input. Results were screened in a three-stage process based on title, abstract and full text by two independent reviewers. The review is registered on PROSPERO (CRD42019147483).Results Fourteen SSA studies met the inclusion criteria from database searches (n=8), reference list searches (n=2) and expert input (n=4). Three studies have ongoing data collection. The most represented countries were South Africa and Mozambique (Southern Africa) (n=3), Benin (Western Africa) (n=4) and Tanzania (Eastern Africa) (n=4); including an estimated 31 763 women. Samples commonly collected were blood, cord blood and placenta. Seven studies collected neonatal samples. Common clinical outcomes included maternal and perinatal mortality, malaria and preterm birth.Conclusions Increasingly numerous pregnancy cohorts in SSA that include biobanking are generating a uniquely valuable resource for collaborative discovery science, and improved understanding of the high regional risks of maternal, fetal and neonatal morbidity and mortality. Future studies should align protocols and consider their added value and distinct contributions.

Abstract Managing multidrug resistant malaria can be problematic if currently available artemisinin-containing anti-malarial combination treatments are not used appropriately. Here, I debate that the best way to manage multidrug resistant malaria is to make best use of existing treatments and to develop new classes of anti-malarial drugs and not to make ‘triple combination therapies’ when there is already resistance to one or more proposed components.

Predicting vaccine efficacy remains a challenge. We used a systems vaccinology approach to identify early innate immune correlates of antibody induction in humans receiving the Ebola vaccine rVSV-ZEBOV. Blood samples from days 0, 1, 3, 7, and 14 were analyzed for changes in cytokine levels, innate immune cell subsets, and gene expression. Integrative statistical analyses with cross-validation identified a signature of 5 early innate markers correlating with antibody titers on day 28 and beyond. Among those, IP-10 on day 3 and MFI of CXCR6 on NK cells on day 1 were independent correlates. Consistently, we found an early gene expression signature linked to IP-10. This comprehensive characterization of early innate immune responses to the rVSV-ZEBOV vaccine in humans revealed immune signatures linked to IP-10. These results suggest correlates of vaccine-induced antibody induction and provide a rationale to explore strategies for augmenting the effectiveness of vaccines through manipulation of IP-10.

Background: The recent West African Ebola epidemic led to accelerated efforts to test Ebola vaccine candidates. As part of the World Health Organisation-led VSV Ebola Consortium (VEBCON), we performed a phase I clinical trial investigating rVSV-ZEBOV (a recombinant vesicular stomatitis virus-vectored Ebola vaccine), which has recently demonstrated protection from Ebola virus disease (EVD) in phase III clinical trials and is currently in advanced stages of licensing. So far, correlates of immune protection are incompletely understood and the role of cell-mediated immune responses has not been comprehensively investigated to date. Methods: We recruited 30 healthy subjects aged 18–55 into an open-label, dose-escalation phase I trial testing three doses of rVSV-ZEBOV (3 × 105 plaque-forming units (PFU), 3 × 106 PFU, 2 × 107 PFU) (ClinicalTrials.gov; NCT02283099). Main study objectives were safety and immunogenicity, while exploratory objectives included lymphocyte dynamics, cell-mediated immunity and cytokine networks, which were assessed using flow cytometry, ELISpot and LUMINEX assay. Findings: Immunization with rVSV-ZEBOV was well tolerated without serious vaccine-related adverse events. Ebola virus-specific neutralizing antibodies were induced in nearly all individuals. Additionally, vaccinees, particularly within the highest dose cohort, generated Ebola glycoprotein (GP)-specific T cells and initiated a cascade of signaling molecules following stimulation of peripheral blood mononuclear cells with Ebola GP peptides. Interpretation: In addition to a benign safety and robust humoral immunogenicity profile, subjects immunized with 2 × 107 PFU elicited higher cellular immune responses and stronger interlocked cytokine networks compared to lower dose groups. To our knowledge these data represent the first detailed cell-mediated immuneprofile of a clinical trial testing rVSV-ZEBOV, which is of particular interest in light of its potential upcoming licensure as the first Ebola vaccine. VEBCON trial Hamburg, Germany (NCT02283099).

Abstract Background The Plasmodium falciparum multidrug resistance 1 (PfMDR1), P. falciparum Ca2+-ATPase (PfATP6) and Kelch-13 propeller domain (PfK13) loci are molecular markers of parasite susceptibility to anti-malarial drugs. Their frequency distributions were determined in the isolates collected from children with severe malaria originating from three African countries. Methods Samples from 287 children with severe malaria [(Gabon: n = 114); (Ghana: n = 89); (Kenya: n = 84)] were genotyped for pfmdr1, pfatp6 and pfk13 loci by DNA sequencing and assessing pfmdr1 copy number variation (CNV) by real-time PCR. Results Pfmdr1-N86Y mutation was detected in 48, 10 and 10% in Lambaréné, Kumasi and Kisumu, respectively. At codon 184, the prevalence of the mutation was 73% in Lambaréné, 63% in Kumasi and 49% Kisumu. The S1034C and N1042D variants were absent at all three sites, while the frequency of the D1246Y mutation was 1, 3 and 13% in Lambaréné, Kumasi and Kisumu, respectively. Isolates with two pfmdr1 gene copy number predominantly harboured the N86Y wild-type allele and were mostly found in Kumasi (10%) (P

Iron is an essential nutrient but, in high concentrations, it is also toxic to cells. Here, Slavic et al. identify an iron transporter in malaria parasites that plays a major role in iron detoxification and is required for the parasite’s normal development.

Antimalarial drug resistance is a major constraint for malaria control and elimination efforts. Artemisinin-based combination therapy is now the mainstay for malaria treatment. However, delayed parasite clearance following treatment with artemisinin derivatives has now spread in the Greater Mekong Sub region and may emerge or spread to other malaria endemic regions. This spread is of great concern for malaria control programmes, as no alternatives to artemisinin-based combination therapies are expected to be available in the near future. There is a need to strengthen surveillance systems for early detection and response to the antimalarial drug resistance threat. Current surveillance is mainly done through therapeutic efficacy studies; however these studies are complex and both time- and resource-intensive. For multiple common antimalarials, parasite drug resistance has been correlated with specific genetic mutations, and the molecular markers associated with antimalarial drug resistance offer a simple and powerful tool to monitor the emergence and spread of resistant parasites. Different techniques to analyse molecular markers associated with antimalarial drug resistance are available, each with advantages and disadvantages. However, procedures are not adequately harmonized to facilitate comparisons between sites. Here we describe the target product profiles for tests to analyse molecular markers associated with antimalarial drug resistance, discuss how use of current techniques can be standardised, and identify the requirements for an ideal product that would allow malaria endemic countries to provide useful spatial and temporal information on the spread of resistance.

Background: Artesunate is an antimalarial agent with broad anti-cancer activity in in vitro and animal experiments and case reports. Artesunate has not been studied in rigorous clinical trials for anticancer effects. Aim: To determine the anticancer effect and tolerability of oral artesunate in colorectal cancer (CRC). Methods: This was a single centre, randomised, double-blind, placebo-controlled trial. Patients planned for curative resection of biopsy confirmed single primary site CRC were randomised (n = 23) by computer-generated code supplied in opaque envelopes to receive preoperatively either 14 daily doses of oral artesunate (200 mg; n = 12) or placebo (n = 11). The primary outcome measure was the proportion of tumour cells undergoing apoptosis (significant if >7% showed Tunel staining). Secondary immunohistochemical outcomes assessed these tumour markers: VEGF, EGFR, c-MYC, CD31, Ki67 and p53, and clinical responses. Findings: 20 patients (artesunate = 9, placebo = 11) completed the trial per protocol. Randomization groups were comparable clinically and for tumour characteristics. Apoptosis in >7% of cells was seen in 67% and 55% of patients in artesunate and placebo groups, respectively. Using Bayesian analysis, the probabilities of an artesunate treatment effect reducing Ki67 and increasing CD31 expression were 0.89 and 0.79, respectively. During a median follow up of 42 months 1 patient in the artesunate and 6 patients in the placebo group developed recurrent CRC. Interpretation: Artesunate has anti-proliferative properties in CRC and is generally well tolerated.

BackgroundThe rVSVΔG-ZEBOV-GP vaccine prevented Ebola virus disease when used at 2 × 107 plaque-forming units (PFU) in a trial in Guinea. This study provides further safety and immunogenicity data.Methods and findingsA randomised, open-label phase I trial in Lambaréné, Gabon, studied 5 single intramuscular vaccine doses of 3 × 103, 3 × 104, 3 × 105, 3 × 106, or 2 × 107 PFU in 115 adults and a dose of 2 × 107 PFU in 20 adolescents and 20 children. The primary objective was safety and tolerability 28 days post-injection. Immunogenicity, viraemia, and shedding post-vaccination were evaluated as secondary objectives. In adults, mild-to-moderate adverse events were frequent, but there were no serious or severe adverse events related to vaccination. Before vaccination, Zaire Ebola virus (ZEBOV)-glycoprotein (GP)-specific and ZEBOV antibodies were detected in 11% and 27% of adults, respectively. In adults, 74%-100% of individuals who received a dose 3 × 104, 3 × 105, 3 × 106, or 2 × 107 PFU had a ≥4.0-fold increase in geometric mean titres (GMTs) of ZEBOV-GP-specific antibodies at day 28, reaching GMTs of 489 (95% CI: 264-908), 556 (95% CI: 280-1,101), 1,245 (95% CI: 899-1,724), and 1,503 (95% CI: 931-2,426), respectively. Twenty-two percent of adults had a ≥4-fold increase of ZEBOV antibodies, with GMTs at day 28 of 1,015 (647-1,591), 1,887 (1,154-3,085), 1,445 (1,013-2,062), and 3,958 (2,249-6,967) for the same doses, respectively. These antibodies persisted up to day 180 for doses ≥3 × 105 PFU. Adults with antibodies before vaccination had higher GMTs throughout. Neutralising antibodies were detected in more than 50% of participants at doses ≥3 × 105 PFU. As in adults, no serious or severe adverse events related to vaccine occurred in adolescents or children. At day 2, vaccine RNA titres were higher for adolescents and children than adults. At day 7, 78% of adolescents and 35% of children had recombinant vesicular stomatitis virus RNA detectable in saliva. The vaccine induced high GMTs of ZEBOV-GP-specific antibodies at day 28 in adolescents, 1,428 (95% CI: 1,025-1,989), and children, 1,620 (95% CI: 806-3,259), and in both groups antibody titres increased up to day 180. The absence of a control group, lack of stratification for baseline antibody status, and imbalances in male/female ratio are the main limitations of this study.ConclusionsOur data confirm the acceptable safety and immunogenicity profile of the 2 × 107 PFU dose in adults and support consideration of lower doses for paediatric populations and those who request boosting.Trial registrationPan African Clinical Trials Registry PACTR201411000919191.

Objective: The purpose of this study was to determine the impact of a revised curriculum on medical student academic motivation, burnout, and quality of life. Methods: This cross-sectional comparative study involved 2 medical school cohorts of second year and fourth year medical students at The University of Auckland: a cohort under a traditional curriculum (n = 437) and a cohort under a revised curriculum (n = 446). Participants completed self-reported questionnaires measuring academic motivation, burnout, and quality of life. Two multivariate analyses of covariance (MANCOVAs) were conducted. Results: The response rate was 48%. No statistically significant differences were found between curriculum cohorts for mean scores of academic motivation, personal burnout, and quality of life. However, differences were found when comparing preclinical medical students and students in their clinical years of training. In comparison with Year 2 medical students, the MANCOVA for Year 4 students showed a significant main effect for the revised curriculum with respect to both physical and environmental quality of life. Conclusions: A revised medical curriculum had a differential effect on quality of life for Year 4 students in the latter years of medical school who are based in a clinical learning environment.

BackgroundCurrent artesunate (ARS) regimens for severe malaria are complex. Once daily intramuscular (i.m.) injection for 3 d would be simpler and more appropriate for remote health facilities than the current WHO-recommended regimen of five intravenous (i.v.) or i.m. injections over 4 d. We compared both a three-dose i.m. and a three-dose i.v. parenteral ARS regimen with the standard five-dose regimen using a non-inferiority design (with non-inferiority margins of 10%).Methods and findingsThis randomized controlled trial included children (0.5-10 y) with severe malaria at seven sites in five African countries to assess whether the efficacy of simplified three-dose regimens is non-inferior to a five-dose regimen. We randomly allocated 1,047 children to receive a total dose of 12 mg/kg ARS as either a control regimen of five i.m. injections of 2.4 mg/kg (at 0, 12, 24, 48, and 72 h) (n = 348) or three injections of 4 mg/kg (at 0, 24, and 48 h) either i.m. (n = 348) or i.v. (n = 351), both of which were the intervention arms. The primary endpoint was the proportion of children with ≥ 99% reduction in parasitemia at 24 h from admission values, measured by microscopists who were blinded to the group allocations. Primary analysis was performed on the per-protocol population, which was 96% of the intention-to-treat population. Secondary analyses included an analysis of host and parasite genotypes as risks for prolongation of parasite clearance kinetics, measured every 6 h, and a Kaplan-Meier analysis to compare parasite clearance kinetics between treatment groups. A post hoc analysis was performed for delayed anemia, defined as hemoglobin ≤ 7 g/dl 7 d or more after admission. The per-protocol population was 1,002 children (five-dose i.m.: n = 331; three-dose i.m.: n = 338; three-dose i.v.: n = 333); 139 participants were lost to follow-up. In the three-dose i.m. arm, 265/338 (78%) children had a ≥ 99% reduction in parasitemia at 24 h compared to 263/331 (79%) receiving the five-dose i.m. regimen, showing non-inferiority of the simplified three-dose regimen to the conventional five-dose regimen (95% CI -7, 5; p = 0.02). In the three-dose i.v. arm, 246/333 (74%) children had ≥ 99% reduction in parasitemia at 24 h; hence, non-inferiority of this regimen to the five-dose control regimen was not shown (95% CI -12, 1; p = 0.24). Delayed parasite clearance was associated with the N86YPfmdr1 genotype. In a post hoc analysis, 192/885 (22%) children developed delayed anemia, an adverse event associated with increased leukocyte counts. There was no observed difference in delayed anemia between treatment arms. A potential limitation of the study is its open-label design, although the primary outcome measures were assessed in a blinded manner.ConclusionsA simplified three-dose i.m. regimen for severe malaria in African children is non-inferior to the more complex WHO-recommended regimen. Parenteral ARS is associated with a risk of delayed anemia in African children.Trial registrationPan African Clinical Trials Registry PACTR201102000277177.

Plasmodium knowlesi is a newly described zoonosis that causes malaria in the human population that can be severe and fatal. The study of P. knowlesi parasites from human clinical isolates is relatively new and, in order to obtain maximum information from patient sample collections, we explored the possibility of generating P. knowlesi genome sequences from archived clinical isolates. Our patient sample collection consisted of frozen whole blood samples that contained excessive human DNA contamination and, in that form, were not suitable for parasite genome sequencing. We developed a method to reduce the amount of human DNA in the thawed blood samples in preparation for high throughput parasite genome sequencing using Illumina HiSeq and MiSeq sequencing platforms. Seven of fifteen samples processed had sufficiently pure P. knowlesi DNA for whole genome sequencing. The reads were mapped to the P. knowlesi H strain reference genome and an average mapping of 90% was obtained. Genes with low coverage were removed leaving 4623 genes for subsequent analyses. Previously we identified a DNA sequence dimorphism on a small fragment of the P. knowlesi normocyte binding protein xa gene on chromosome 14. We used the genome data to assemble full-length Pknbpxa sequences and discovered that the dimorphism extended along the gene. An in-house algorithm was developed to detect SNP sites co-associating with the dimorphism. More than half of the P. knowlesi genome was dimorphic, involving genes on all chromosomes and suggesting that two distinct types of P. knowlesi infect the human population in Sarawak, Malaysian Borneo. We use P. knowlesi clinical samples to demonstrate that Plasmodium DNA from archived patient samples can produce high quality genome data. We show that analyses, of even small numbers of difficult clinical malaria isolates, can generate comprehensive genomic information that will improve our understanding of malaria parasite diversity and pathobiology.

Emerging pathogens undermine initiatives to control the global health impact of infectious diseases. Zoonotic malaria is no exception. Plasmodium knowlesi, a malaria parasite of Southeast Asian macaques, has entered the human population. P. knowlesi, like Plasmodium falciparum, can reach high parasitaemia in human infections, and the World Health Organization guidelines for severe malaria list hyperparasitaemia among the measures of severe malaria in both infections. Not all patients with P. knowlesi infections develop hyperparasitaemia, and it is important to determine why. Between isolate variability in erythrocyte invasion, efficiency seems key. Here we investigate the idea that particular alleles of two P. knowlesi erythrocyte invasion genes, P. knowlesi normocyte binding protein Pknbpxa and Pknbpxb, influence parasitaemia and human disease progression. Pknbpxa and Pknbpxb reference DNA sequences were generated from five geographically and temporally distinct P. knowlesi patient isolates. Polymorphic regions of each gene (approximately 800 bp) were identified by haplotyping 147 patient isolates at each locus. Parasitaemia in the study cohort was associated with markers of disease severity including liver and renal dysfunction, haemoglobin, platelets and lactate, (r = ≥ 0.34, p =

Abstract Accurate stage determination is crucial in the choice of treatment for patients suffering from sleeping sickness, also known as human African trypanosomiasis (HAT). Current staging methods, based on the counting of white blood cells (WBC) and the detection of parasites in the cerebrospinal fluid (CSF) have limited accuracy. We hypothesized that immune mediators reliable for staging T. b. gambiense HAT could also be used to stratify T. b. rhodesiense patients, the less common form of HAT. A population comprising 85 T. b. rhodesiense patients, 14 stage 1 (S1) and 71 stage 2 (S2) enrolled in Malawi and Uganda, was investigated. The CSF levels of IgM, MMP‐9, CXCL13, CXCL10, ICAM‐1, VCAM‐1, neopterin and B2MG were measured and their staging performances evaluated using receiver operating characteristic (ROC) analyses. IgM, MMP‐9 and CXCL13 were the most accurate markers for stage determination (partial AUC 88%, 86% and 85%, respectively). The combination in panels of three molecules comprising CXCL13‐CXCL10‐MMP‐9 or CXCL13‐CXCL10‐IgM significantly increased their staging ability to partial AUC 94% (p value < 0.01). The present study highlighted new potential markers for stage determination of T. b. rhodesiense patients. Further investigations are needed to better evaluate these molecules, alone or in panels, as alternatives to WBC to make reliable choice of treatment.

Ca(2+) contributes to a myriad of important cellular processes in all organisms, including the apicomplexans, Plasmodium and Toxoplasma. Due to its varied and essential roles, free Ca(2+) is tightly regulated by complex mechanisms. These mechanisms are therefore of interest as putative drug targets. One pathway in Ca(2+) homeostatic control in apicomplexans uses a Ca(2+)/H(+) exchanger (a member of the cation exchanger family, CAX). The P. falciparum CAX (PfCAX) has recently been characterised in asexual blood stage parasites. To determine the physiological importance of apicomplexan CAXs, tagging and knock-out strategies were undertaken in the genetically tractable T. gondii and P. berghei parasites. In addition, a yeast heterologous expression system was used to study the function of apicomplexan CAXs. Tagging of T. gondii and P. berghei CAXs (TgCAX and PbCAX) under control of their endogenous promoters could not demonstrate measureable expression of either CAX in tachyzoites and asexual blood stages, respectively. These results were consistent with the ability of parasites to tolerate knock-outs of the genes for TgCAX and PbCAX at these developmental stages. In contrast, PbCAX expression was detectable during sexual stages of development in female gametocytes/gametes, zygotes and ookinetes, where it was dispersed in membranous networks within the cytosol (with minimal mitochondrial localisation). Furthermore, genetically disrupted parasites failed to develop further from "round" form zygotes, suggesting that PbCAX is essential for ookinete development and differentiation. This impeded phenotype could be rescued by removal of extracellular Ca(2+). Therefore, PbCAX provides a mechanism for free living parasites to multiply within the ionic microenvironment of the mosquito midgut. Ca(2+) homeostasis mediated by PbCAX is critical and suggests plasmodial CAXs may be targeted in approaches designed to block parasite transmission.

BACKGROUND: Post-therapeutic follow-up is essential to confirm cure and to detect early treatment failures in patients affected by sleeping sickness (HAT). Current methods, based on finding of parasites in blood and cerebrospinal fluid (CSF) and counting of white blood cells (WBC) in CSF, are imperfect. New markers for treatment outcome evaluation are needed. We hypothesized that alternative CSF markers, able to diagnose the meningo-encephalitic stage of the disease, could also be useful for the evaluation of treatment outcome. METHODOLOGY/PRINCIPAL FINDINGS: Cerebrospinal fluid from patients affected by Trypanosoma brucei gambiense HAT and followed for two years after treatment was investigated. The population comprised stage 2 (S2) patients either cured or experiencing treatment failure during the follow-up. IgM, neopterin, B2MG, MMP-9, ICAM-1, VCAM-1, CXCL10 and CXCL13 were first screened on a small number of HAT patients (n = 97). Neopterin and CXCL13 showed the highest accuracy in discriminating between S2 cured and S2 relapsed patients (AUC 99% and 94%, respectively). When verified on a larger cohort (n = 242), neopterin resulted to be the most efficient predictor of outcome. High levels of this molecule before treatment were already associated with an increased risk of treatment failure. At six months after treatment, neopterin discriminated between cured and relapsed S2 patients with 87% specificity and 92% sensitivity, showing a higher accuracy than white blood cell numbers. CONCLUSIONS/SIGNIFICANCE: In the present study, neopterin was highlighted as a useful marker for the evaluation of the post-therapeutic outcome in patients suffering from sleeping sickness. Detectable levels of this marker in the CSF have the potential to shorten the follow-up for HAT patients to six months after the end of the treatment.

BackgroundSleeping sickness, or human African trypanosomiasis (HAT), is a protozoan disease that affects rural communities in sub-Saharan Africa. Determination of the disease stage, essential for correct treatment, represents a key issue in the management of patients. In the present study we evaluated the potential of CXCL10, CXCL13, ICAM-1, VCAM-1, MMP-9, B2MG, neopterin and IgM to complement current methods for staging Trypanosoma brucei gambiense patients.Methods and findingsFive hundred and twelve T. b. gambiense HAT patients originated from Angola, Chad and the Democratic Republic of the Congo (D.R.C.). Their classification as stage 2 (S2) was based on the number of white blood cells (WBC) (>5/µL) or presence of parasites in the cerebrospinal fluid (CSF). The CSF concentration of the eight markers was first measured on a training cohort encompassing 100 patients (44 S1 and 56 S2). IgM and neopterin were the best in discriminating between the two stages of disease with 86.4% and 84.1% specificity respectively, at 100% sensitivity. When a validation cohort (412 patients) was tested, neopterin (14.3 nmol/L) correctly classified 88% of S1 and S2 patients, confirming its high staging power. On this second cohort, neopterin also predicted both the presence of parasites, and of neurological signs, with the same ability as IgM and WBC, the current reference for staging.ConclusionsThis study has demonstrated that neopterin is an excellent biomarker for staging T. b. gambiense HAT patients. A rapid diagnostic test for detecting this metabolite in CSF could help in more accurate stage determination.

A major obstacle to effectively treat and control tuberculosis is the absence of an accurate, rapid, and low-cost diagnostic tool. A new approach for the screening of patients for tuberculosis is the use of rapid diagnostic classification algorithms.We tested a previously published diagnostic algorithm based on four biomarkers as a screening tool for tuberculosis in a Central European patient population using an assessor-blinded cross-sectional study design. In addition, we developed an improved diagnostic classification algorithm based on a study population at a tertiary hospital in Vienna, Austria, by supervised computational statistics.The diagnostic accuracy of the previously published diagnostic algorithm for our patient population consisting of 206 patients was 54% (CI: 47%-61%). An improved model was constructed using inflammation parameters and clinical information. A diagnostic accuracy of 86% (CI: 80%-90%) was demonstrated by 10-fold cross validation. An alternative model relying solely on clinical parameters exhibited a diagnostic accuracy of 85% (CI: 79%-89%).Here we show that a rapid diagnostic algorithm based on clinical parameters is only slightly improved by inclusion of inflammation markers in our cohort. Our results also emphasize the need for validation of new diagnostic algorithms in different settings and patient populations.

Modelling is widely used to inform decisions about management of malaria and acute febrile illnesses. Most models depend on estimates of the probability that untreated patients with malaria or bacterial illnesses will progress to severe disease or death. However, data on these key parameters are lacking and assumptions are frequently made based on expert opinion. Widely diverse opinions can lead to conflicting outcomes in models they inform.A Delphi survey was conducted with malaria experts aiming to reach consensus on key parameters for public health and economic models, relating to the outcome of untreated febrile illnesses. Survey questions were stratified by malaria transmission intensity, patient age, and HIV prevalence. The impact of the variability in opinion on decision models is illustrated with a model previously used to assess the cost-effectiveness of malaria rapid diagnostic tests. Some consensus was reached around the probability that patients from higher transmission settings with untreated malaria would progress to severe disease (median 3%, inter-quartile range (IQR) 1-5%), and the probability that a non-malaria illness required antibiotics in areas of low HIV prevalence (median 20%). Children living in low transmission areas were considered to be at higher risk of progressing to severe malaria (median 30%, IQR 10-58%) than those from higher transmission areas (median 13%, IQR 7-30%). Estimates of the probability of dying from severe malaria were high in all settings (medians 60-73%). However, opinions varied widely for most parameters, and did not converge on resurveying.This study highlights the uncertainty around potential consequences of untreated malaria and bacterial illnesses. The lack of consensus on most parameters, the wide range of estimates, and the impact of variability in estimates on model outputs, demonstrate the importance of sensitivity analysis for decision models employing expert opinion. Results of such models should be interpreted cautiously. The diversity of expert opinion should be recognised when policy options are debated.

Plasmodium knowlesi has entered the human population of Southeast Asia. Naturally acquired knowlesi malaria is newly described with relatively little available data, including data on the host response to infection. Therefore pre-treatment cytokine and chemokine profiles were determined for 94 P. knowlesi, and for comparison, 20, P. vivax and 22 P. falciparum, patients recruited in Malaysian Borneo. Nine, five and one patient with P. knowlesi, P. falciparum and P. vivax respectively had complicated malaria as defined by World Health Organisation. Patients with uncomplicated P. knowlesi had lower levels of the pro-inflammatory cytokines IL-8 and TNFα than those with complicated disease (both p

BACKGROUND:The analysis of complex proteomic and genomic profiles involves the identification of significant markers within a set of hundreds or even thousands of variables that represent a high-dimensional problem space. The occurrence of noise, redundancy or combinatorial interactions in the profile makes the selection of relevant variables harder. METHODOLOGY/PRINCIPAL FINDINGS:Here we propose a method to select variables based on estimated relevance to hidden patterns. Our method combines a weighted-kernel discriminant with an iterative stochastic probability estimation algorithm to discover the relevance distribution over the set of variables. We verified the ability of our method to select predefined relevant variables in synthetic proteome-like data and then assessed its performance on biological high-dimensional problems. Experiments were run on serum proteomic datasets of infectious diseases. The resulting variable subsets achieved classification accuracies of 99% on Human African Trypanosomiasis, 91% on Tuberculosis, and 91% on Malaria serum proteomic profiles with fewer than 20% of variables selected. Our method scaled-up to dimensionalities of much higher orders of magnitude as shown with gene expression microarray datasets in which we obtained classification accuracies close to 90% with fewer than 1% of the total number of variables. CONCLUSIONS:Our method consistently found relevant variables attaining high classification accuracies across synthetic and biological datasets. Notably, it yielded very compact subsets compared to the original number of variables, which should simplify downstream biological experimentation.

Intra-rectal artesunate has been developed as a potentially life-saving treatment of severe malaria in rural village settings where administration of parenteral antimalarial drugs is not possible. We studied the population pharmacokinetics of intra-rectal artesunate and the relationship with parasitological responses in patients with moderately severe falciparum malaria.Adults and children in Africa and Southeast Asia with moderately severe malaria were recruited in two Phase II studies (12 adults from Southeast Asia and 11 children from Africa) with intensive sampling protocols, and three Phase III studies (44 children from Southeast Asia, and 86 children and 26 adults from Africa) with sparse sampling. All patients received 10 mg/kg artesunate as a single intra-rectal dose of suppositories. Venous blood samples were taken during a period of 24 h following dosing. Plasma artesunate and dihydroartemisinin (DHA, the main biologically active metabolite) concentrations were measured by high-performance liquid chromatography with electrochemical detection. The pharmacokinetic properties of DHA were determined using nonlinear mixed-effects modelling. Artesunate is rapidly hydrolysed in vivo to DHA, and this contributes the majority of antimalarial activity. For DHA, a one-compartment model assuming complete conversion from artesunate and first-order appearance and elimination kinetics gave the best fit to the data. The mean population estimate of apparent clearance (CL/F) was 2.64 (l/kg/h) with 66% inter-individual variability. The apparent volume of distribution (V/F) was 2.75 (l/kg) with 96% inter-individual variability. The estimated DHA population mean elimination half-life was 43 min. Gender was associated with increased mean CL/F by 1.14 (95% CI: 0.36-1.92) (l/kg/h) for a male compared with a female, and weight was positively associated with V/F. Larger V/Fs were observed for the patients requiring early rescue treatment compared with the remainder, independent of any confounders. No associations between the parasitological responses and the posterior individual estimates of V/F, CL/F, and AUC0-6h were observed.The pharmacokinetic properties of DHA were affected only by gender and body weight. Patients with the lowest area under the DHA concentration curve did not have slower parasite clearance, suggesting that rectal artesunate is well absorbed in most patients with moderately severe malaria. However, a number of modelling assumptions were required due to the large intra- and inter-individual variability of the DHA concentrations.

BACKGROUND: The degree of volume depletion in severe malaria is currently unknown, although knowledge of fluid compartment volumes can guide therapy. To assist management of severely ill children, and to test the hypothesis that volume changes in fluid compartments reflect disease severity, we measured body compartment volumes in Gabonese children with malaria. METHODS AND FINDINGS: Total body water volume (TBW) and extracellular water volume (ECW) were estimated in children with severe or moderate malaria and in convalescence by tracer dilution with heavy water and bromide, respectively. Intracellular water volume (ICW) was derived from these parameters. Bioelectrical impedance analysis estimates of TBW and ECW were calibrated against dilution methods, and bioelectrical impedance analysis measurements were taken daily until discharge. Sixteen children had severe and 19 moderate malaria. Severe childhood malaria was associated with depletion of TBW (mean [SD] of 37 [33] ml/kg, or 6.7% [6.0%]) relative to measurement at discharge. This is defined as mild dehydration in other conditions. ECW measurements were normal on admission in children with severe malaria and did not rise in the first few days of admission. Volumes in different compartments (TBW, ECW, and ICW) were not related to hyperlactataemia or other clinical and laboratory markers of disease severity. Moderate malaria was not associated with a depletion of TBW. CONCLUSIONS: Significant hypovolaemia does not exacerbate complications of severe or moderate malaria. As rapid rehydration of children with malaria may have risks, we suggest that fluid replacement regimens should aim to correct fluid losses over 12-24 h.