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Background RTS,S/AS01 is the first malaria vaccine to be approved and recommended for widespread implementation by the World Health Organization (WHO). Trials reported lower vaccine efficacies in higher-incidence sites, potentially due to a “rebound” in malaria cases in vaccinated children. When naturally acquired protection in the control group rises and vaccine protection in the vaccinated wanes concurrently, malaria incidence can become greater in the vaccinated than in the control group, resulting in negative vaccine efficacies. Methods Using data from the 2009–2014 phase III trial (NCT00866619) in Lilongwe, Malawi; Kintampo, Ghana; and Lambaréné, Gabon, we evaluate this hypothesis by estimating malaria incidence in each vaccine group over time and in varying transmission settings. After estimating transmission intensities using ecological variables, we fit models with 3-way interactions between vaccination, time, and transmission intensity. Results Over time, incidence decreased in the control group and increased in the vaccine group. Three-dose efficacy in the lowest-transmission-intensity group (0.25 cases per person-year [CPPY]) decreased from 88.2% to 15.0% over 4.5 years, compared with 81.6% to −27.7% in the highest-transmission-intensity group (3 CPPY). Conclusions These findings suggest that interventions, including the fourth RTS,S dose, that protect vaccinated individuals during the potential rebound period should be implemented for high-transmission settings.

Background: RTS,S/AS01 has been recommended by WHO for widespread implementation in medium to high malaria transmission settings. Previous analyses have noted lower vaccine efficacies in higher transmission settings, possibly due to the more rapid development of naturally acquired immunity in the control group. Methods: To investigate a reduced immune response to vaccination as a potential mechanism behind lower efficacy in high transmission areas, we examine initial vaccine antibody (anti-CSP IgG) response and vaccine efficacy against the first case of malaria to exclude the delayed malaria effect using data from three study areas (Kintampo, Ghana; Lilongwe, Malawi; Lambaréné, Gabon) from the 2009-2014 phase III trial (NCT00866619). Our key exposures are parasitemia during the vaccination series and malaria transmission intensity. We calculate vaccine efficacy (one minus hazard ratio) using a cox-proportional hazards model and allowing for the time-varying effect of RTS,S/AS01. Results: We find that antibody responses to the primary three-dose vaccination series were higher in Ghana than in Malawi and Gabon, but that neither antibody levels nor vaccine efficacy against the first case of malaria varied by transmission intensity or parasitemia during the primary vaccination series. Conclusions: We find that vaccine efficacy is unrelated to infections during vaccination. Contributing to a conflicting literature, our results suggest that vaccine efficacy is also unrelated to infections before vaccination, meaning that delayed malaria is likely the main reason for lower efficacy in high transmission settings, not reduced immune responses. This may be reassuring for implementation in high transmission settings, though further studies are needed.

Drug resistance necessitates the development of new antimalarial drugs. It is important to assess the activities of compounds under development against parasites now causing disease in Africa, where most malaria cases occur, and to determine if mutations in these parasites may limit the efficacies of new agents.

BackgroundIncreasing reports suggest that non-falciparum species are an underappreciated cause of malaria in sub-Saharan Africa, but their epidemiology is not well-defined. This is particularly true in regions of highP. falciparumendemicity such as the Democratic Republic of Congo (DRC), where 12% of the world’s malaria cases and 13% of deaths occur.Methods and FindingsThe cumulative incidence and prevalence of P. malariae and P. ovale spp. infection detected by real-time PCR were estimated among children and adults within a longitudinal study conducted in seven rural, peri-urban, and urban sites from 2015-2017 in Kinshasa Province, DRC. Participants were sampled at biannual household survey visits (asymptomatic) and during routine health facility visits (symptomatic). Participant-level characteristics associated with non-falciparum infections were estimated for single- and mixed-species infections. Among 9,089 samples collected from 1,565 participants over a 3-year period, the incidence of P. malariae and P. ovale spp. infection was 11% (95% CI: 9%-12%) and 7% (95% CI: 5%-8%) by one year, respectively, compared to a 67% (95% CI: 64%-70%) one-year cumulative incidence of P. falciparum infection. Incidence continued to rise in the second year of follow-up, reaching 26% and 15% in school-age children (5-14yo) for P. malariae and P. ovale spp., respectively. Prevalence of P. malariae, P. ovale spp., and P. falciparum infections during household visits were 3% (95% CI: 3%-4%), 1% (95% CI: 1%-2%), and 35% (95% CI: 33%-36%), respectively. Non-falciparum malaria was more prevalent in rural and peri-urban vs. urban sites, in school-age children, and among those with P. falciparum co-infection. A crude association was detected between P. malariae and any anemia in the symptomatic clinic population, although this association did not hold when stratified by anemia severity. No crude associations were detected between non-falciparum infection and fever prevalence.ConclusionsP. falciparumremains the primary driver of malaria morbidity and mortality in the DRC. However, non-falciparum species also pose an infection risk across sites of varying urbanicity and malaria endemicity within Kinshasa, DRC, particularly among children under 15 years of age. AsP. falciparuminterventions gain traction in high-burden settings like the DRC, continued surveillance and improved understanding of non-falciparum infections are warranted.

The seroprevalence of Kaposi sarcoma-associated herpesvirus (KSHV) and the incidence of endemic Kaposi sarcoma (KS) overlap with regions of malaria endemicity in sub-Saharan Africa. Multiple studies have shown an increased risk of KSHV seroconversion in children from high malaria compared to low malaria regions; however, the impact of acute episodes of Plasmodium falciparum (P. falciparum) malaria on KSHV’s biphasic life cycle and lytic reactivation has not been determined. Here, we examined KSHV serological profiles and viral loads in 134 children with acute malaria and 221 healthy children from high malaria regions in Kisumu, as well as 77 healthy children from low malaria regions in Nandi. We assayed KSHV, Epstein–Barr virus (EBV), and P. falciparum malaria antibody responses in these three by multiplexed Luminex assay. We confirmed that KSHV seroprevalence was significantly associated with malaria endemicity (OR = 1.95, 1.18–3.24 95% CI, p = 0.01) with 71–77% seropositivity in high-malaria (Kisumu) compared to 28% in low-malaria (Nandi) regions. Furthermore, KSHV serological profiles during acute malaria episodes were distinct from age-matched non-malaria-infected children from the same region. Paired IgG levels also varied after malaria treatment, with significantly higher anti-ORF59 at day 0 but elevated ORF38, ORF73, and K8.1 at day 3. Acute malaria episodes is characterized by perturbation of KSHV latency in seropositive children, providing further evidence that malaria endemicity contributes to the observed increase in endemic KS incidence in sub-Saharan Africa.

All of the putative mutations detected in eBL and sBL tumors.

Detailed methods used in the study.

S1. Assessment of putative tumor purity levels using IRIS signature genes from Abbas et al. S2. Epstein-barr virus transcription and lytic activity level measurements within primary eBL biopsies. S3. Dimension reduction with principal component analysis (PCA) on gene expression profiles. S4. All expressed human gene expression comparison between eBL tumors carrying EBV type 1 and eBL tumors with type 2 in log2 scale. S5. Sample to sample correlation heatmap of eBL, sBL tumors, and long-term BL cultures after removing all unwanted covariates except tumor origins. Gene expression profiles group into two clusters based on endemic and sporadic origins. S6. A) Common SNP counts detected in each sample after variant calling with RNAseq data by subsampling to balance the detection power (dbSNP v146). B) Rare non-synonymous variant fractions detected in each sample. S7. Lollipop plot for the non-synonymous mutations detected in BL tumors.

The emergence and spread of drug- and diagnostic-resistantPlasmodium falciparumare major impediments to malaria control and elimination. We deep sequenced known drug resistance mutations and other informative loci across the genome of 609 samples collected during a study across three regions of Ethiopia. We found that 8.0% (95% CI 7.0-9.0) of malaria cases were caused byP. falciparumcarrying the candidate artemisinin partial-resistanceK13622I mutation, which occurred less commonly in diagnostic-resistantpfhrp2/3-deleted than normal non-deleted parasites (p=0.03). Identity-by-descent analysis showed that 622I parasites were significantly more related than wild-type (pPfhrp2/3-deleted parasites were also highly related, with evidence of clonal transmissions at the district level. Parasites carrying bothpfhrp2/3deletion and 622I mutation were observed in some sites. These findings raise concern for future spread of combined drug- and diagnostic-resistant parasites and warrant close monitoring.

The Zanzibar archipelago of Tanzania has become a low-transmission area forPlasmodium falciparum. Despite being considered an area of pre-elimination for years, achieving elimination has been difficult, likely due to a combination of imported infections from mainland Tanzania, and continued local transmission. To shed light on these sources of transmission, we applied highly multiplexed genotyping utilizing molecular inversion probes to characterize the genetic relatedness of 391P. falciparumisolates collected across Zanzibar and in Bagamoyo District on the coastal mainland from 2016-2018. Overall, parasite populations on the coastal mainland and Zanzibar archipelago remain highly related. However, in Zanzibar the parasite population exhibits microstructure due to rapid decay of parasite relatedness over very short distances. This, along with highly related pairs withinshehias, suggests ongoing low level local transmission. We also identified highly related parasites acrossshehiasthat reflect human mobility on the main island of Unguja and identified a cluster of highly related parasites, suggestive of an outbreak, in the Micheweni district on Pemba island. Parasites in asymptomatic infections demonstrated higher complexity of infection than those in symptomatic infections, but have similar core genomes. Our data support that importation remains a main source of genetic diversity and contribution to the parasite population on Zanzibar, but they also show local outbreak clusters where targeted interventions are essential to block local transmission. These results highlight the need for preventive measures against imported malaria and enhanced control measures in areas that remain receptive for malaria reemergence due to susceptible hosts and competent vectors.

Background Accurate variant calls from whole genome sequencing (WGS) of Plasmodium falciparum infections are crucial in malaria population genomics. Here we optimized a falciparum variant calling pipeline based on GATK version 4 (GATK4) and applied it to 6,626 public Illumina WGS samples. Methods We optimized parameters that control the heterozygosity, local assembly region size, ploidy, mapping and base quality in both GATK HaplotypeCaller and GenotypeGVCFs leveraging control WGS and accurate PacBio assemblies of 10 laboratory strains. From these controls we generated a high-quality training dataset to recalibrate the raw variant data. Results On current high-quality samples (read length = 250bp, insert size = 405 - 524 bp ), we show improved sensitivity (86.6 ± 1.7% for SNPs and 82.2 ± 5.9% for indels) compared to the default GATK4 pipeline (77.7 ± 1.3% for SNPs; and 73.1 ± 5.1% for indels, adjusted P < 0.001) and previous variant calling with GATK version 3 (GATK3, 70.3 ± 3.0% for SNPs and 59.7 ± 5.8% for indels, adjusted P < 0.001). The sensitivity of our pipeline on simulated mixed infection samples (80.8 ± 6.1% for SNPs and 78.3 ± 5.1% for indels) was again improved relative to default GATK4 (68.8 ± 6.0% for SNPs and 38.9 ± 0.7% for indels, adjusted P < 0.001). Precision was high and comparable across all pipelines on each type of data tested. We further show that using the combination of high-quality SNPs and indels increases the resolution of local population population structure detection in sub-Saharan Africa. We finally demonstrate that increasing ploidy improves the detection of drug resistance mutations and estimation of complexity of infection. Conclusions Overall, we provide an optimized GATK4 pipeline and resource for falciparum variant calling which should help improve genomic studies of malaria.

Most malaria rapid diagnostic tests (RDTs) detect histidine-rich protein 2 (PfHRP2) and PfHRP3.Pfhrp2andpfhrp3gene deletions make parasites undetectable by RDTs. However, deletions have emerged where RDT use is uncommon and can occur independently of each other withpfhrp3deletion more frequent in many parasite populations. To improve our understanding of geographic patterns and mechanisms ofpfhrp3deletion, we analyze 8,765 publicly available, whole-genome-sequencedP. falciparumfield samples. Telomere healing is the onlypfhrp2deletion mechanism observed among the few availablepfhrp2-deleted genomes. Threepfhrp3deletion patterns predominate, all having chromosome 13 loss centromeric topfhrp3extending to the chromosome end: 1) chromosome 13 deletion at varied locations centromeric topfhrp3with signs of telomere healing (Asia; Pattern 13-); 2) congruent duplication of a chromosome 5 segment which includes multidrug-resistant 1 (pfmdr1) gene (Asia; Pattern 13-5++); and 3) congruent duplication of chromosome 11’s subtelomeric region (Americas/Africa; Pattern 13-11++). The most frequently observed pattern 13-11++’s centromeric boundary falls within the largest (15kb) known segmental duplication in the core genome. Long-read sequencing ofpfhrp3-deleted isolates from Honduras (HB3) and Sudan (SD01) confirms 13-11 hybrid chromosomes. Our findings demonstrate thatpfhrp3deletion in the Americas and Africa often results from duplication-mediated non-allelic homologous recombination, likely a common occurrence in meiosis, while in Asia, deletion ofpfhrp3may be related to benefits of duplicatingpfmdr1. Furthermore, this suggests that specific conditions including low transmission are needed for emergence of these deletions, a finding that can inform refined surveillance strategies.

Background Endemic Burkitt lymphoma (eBL) is potentiated through the interplay of Epstein Barr virus (EBV) and holoendemic Plasmodium falciparum malaria. To better understand EBV’s biology and role in eBL, we characterized genome-wide recombination sites and patterns as a source of genetic diversity in EBV genomes in our well-defined population of eBL cases and controls from Western Kenya. Methods EBV genomes representing 54 eBL cases and 32 healthy children from the same geographic region in Western Kenya that we previously sequenced were analyzed. Whole-genome multiple sequence alignment, recombination analyses, and phylogenetic inference were made using multiple alignment with fast Fourier transform, recombination detection program 4, and molecular evolutionary genetics analysis. Results We identified 28 different recombination events and 71 (82.6%) of the 86 EBV genomes analyzed contained evidence of one or more recombinant segments. Associated recombination breakpoints were found to occur in a total of 42 different genes, with only 7 (16.67%) being latent genes. Recombination events were major drivers of clustering within genome-wide phylogenetic trees. The occurrence of recombination segments was comparable between genomes from male and female participants and across age groups. More recombinant segments were found in EBV type 1 genomes (p = 6.4e − 06) and the genomes from the eBLs (p = 0.037). Two recombination events were enriched in the eBLs; event 47 (OR = 4.07, p = 0.038) and event 50 (OR = 14.24, p = 0.012). Conclusions EBV genomes have extensive evidence of recombination likely acquired progressively and cumulatively over time. Recombination patterns display a heterogeneous occurrence rate across the genome with enrichment in lytic genes. Overall, recombination appears to be a major evolutionary force impacting EBV diversity and genome structure with evidence of the association of specific recombinants with eBL.

Histidine-rich protein 2- (HRP2-) based rapid diagnostic tests (RDTs) are widely used to detectPlasmodium falciparumin sub-Saharan Africa. Reports of parasites withpfhrp2and/orpfhrp3 (pfhrp2/3)gene deletions in Africa raise concerns about the long-term viability of HRP2-based RDTs. We evaluated changes inpfhrp2/3deletion prevalence over time using a 2018-2021 longitudinal study of 1,635 enrolled individuals in Kinshasa Province, Democratic Republic of the Congo (DRC). Samples collected during biannual household visits with ≥ 100 parasites/μL by quantitative real-time PCR were genotyped using a multiplex real-time PCR assay. Among 2,726 P.falciparumPCR-positive samples collected from 993 participants during the study period, 1,267 (46.5%) were genotyped. Nopfhrp2/3deletions or mixedpfhrp2/3-intact and -deleted infections were identified in our study.Pfhrp2/3-deleted parasites were not detected in Kinshasa Province; ongoing use of HRP2-based RDTs is appropriate.

The proteasome is a promising target for antimalarial chemotherapy. We assessed ex vivo susceptibilities of fresh Plasmodium falciparum isolates from eastern Uganda to seven proteasome inhibitors: two asparagine ethylenediamines, two macrocyclic peptides, and three peptide boronates; five had median IC(50) values

In Africa, most rapid diagnostic tests (RDTs) for falciparum malaria recognize histidine-rich protein 2 antigen. Plasmodium falciparum parasites lacking histidine-rich protein 2 (pfhrp2) and 3 (pfhrp3) genes escape detection by these RDTs, but it is not known whether these deletions confer sufficient selective advantage to drive rapid population expansion. By studying blood samples from a cohort of 12,572 participants enroled in a prospective, cross-sectional survey along Ethiopia’s borders with Eritrea, Sudan and South Sudan using RDTs, PCR, an ultrasensitive bead-based immunoassay for antigen detection and next-generation sequencing, we estimate that histidine-rich protein 2-based RDTs would miss 9.7% (95% confidence interval 8.5–11.1) of P. falciparum malaria cases owing to pfhrp2 deletion. We applied a molecular inversion probe-targeted deep sequencing approach to identify distinct subtelomeric deletion patterns and well-established pfhrp3 deletions and to uncover recent expansion of a singular pfhrp2 deletion in all regions sampled. We propose a model in which pfhrp3 deletions have arisen independently multiple times, followed by strong positive selection for pfhrp2 deletion owing to RDT-based test-and-treatment. Existing diagnostic strategies need to be urgently reconsidered in Ethiopia, and improved surveillance for pfhrp2 deletion is needed throughout the Horn of Africa., A prospective, cross-sectional survey of 12,572 participants in Ethiopia reveals that malaria diagnostics miss almost 10% of cases owing to a gene deletion in Plasmodium falciparum that is under positive selection.

Summary: Background: Treatment and control of malaria depends on artemisinin-based combination therapies (ACTs) and is challenged by drug resistance, but thus far resistance to artemisinins and partner drugs has primarily occurred in southeast Asia. The aim of this study was to characterise antimalarial drug susceptibility of Plasmodium falciparum isolates from Tororo and Busia districts in Uganda. Methods: In this prospective longitudinal study, P falciparum isolates were collected from patients aged 6 months or older presenting at the Tororo District Hospital (Tororo district, a site with relatively low malaria incidence) or Masafu General Hospital (Busia district, a high-incidence site) in eastern Uganda with clinical symptoms of malaria, a positive Giemsa-stained blood film for P falciparum, and no signs of severe disease. Ex-vivo susceptibilities to ten antimalarial drugs were measured using a 72-h microplate growth inhibition assay with SYBR Green detection. Relevant P falciparum genetic polymorphisms were characterised by molecular methods. We compared results with those from earlier studies in this region and searched for associations between drug susceptibility and parasite genotypes. Findings: From June 10, 2016, to July 29, 2019, 361 P falciparum isolates were collected in the Busia district and 79 in the Tororo district from 440 participants. Of 440 total isolates, 392 (89%) successfully grew in culture and showed excellent drug susceptibility for chloroquine (median half-maximal inhibitory concentration [IC50] 20·0 nM [IQR 12·0–26·0]), monodesethylamodiaquine (7·1 nM [4·3–8·9]), pyronaridine (1·1 nM [0·7–2·3]), piperaquine (5·6 nM [3·3–8·6]), ferroquine (1·8 nM [1·5–3·3]), AQ-13 (24·0 nM [17·0–32·0]), lumefantrine (5·1 nM [3·2–7·7]), mefloquine (9·5 nM [6·6–13·0]), dihydroartemisinin (1·5 nM [1·0–2·0]), and atovaquone (0·3 nM [0·2–0·4]). Compared with results from our study in 2010–13, significant improvements in susceptibility were seen for chloroquine (median IC50 288·0 nM [IQR 122·0–607·0]; p100 nM) was more common in isolates from the Tororo district (11 [15%] of 71), compared with those from the Busia district (12 [4%] of 320; p=0·0017). We showed significant increases between 2010–12 and 2016–19 in the prevalences of wild-type P falciparum multidrug resistance protein 1 (PfMDR1) Asn86Tyr from 60% (391 of 653) to 99% (418 of 422; p

Background Evaluating the performance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) serological assays and clearly articulating the utility of selected antigens, isotypes, and thresholds is crucial to understanding the prevalence of infection within selected communities. Methods This cross-sectional study, implemented in 2020, screened PCR–confirmed coronavirus disease 2019 patients (n = 86), banked prepandemic and negative samples (n = 96), healthcare workers and family members (n = 552), and university employees (n = 327) for anti–SARS-CoV-2 receptor-binding domain, trimeric spike protein, and nucleocapsid protein immunoglobulin (Ig)G and IgA antibodies with a laboratory-developed enzyme-linked immunosorbent assay and tested how antigen, isotype and threshold choices affected the seroprevalence outcomes. The following threshold methods were evaluated: (i) mean + 3 standard deviations of the negative controls; (ii) 100% specificity for each antigen-isotype combination; and (iii) the maximal Youden index. Results We found vastly different seroprevalence estimates depending on selected antigens and isotypes and the applied threshold method, ranging from 0.0% to 85.4%. Subsequently, we maximized specificity and reported a seroprevalence, based on more than one antigen, ranging from 9.3% to 25.9%. Conclusions This study revealed the importance of evaluating serosurvey tools for antigen-, isotype-, and threshold-specific sensitivity and specificity, to interpret qualitative serosurvey outcomes reliably and consistently across studies.

Endemic Burkitt lymphoma (eBL) is an aggressive pediatric B cell lymphoma, common in Equatorial Africa. Co-infections with Epstein-Barr virus (EBV) and Plasmodium falciparum, coupled with c-myc translocation are involved in eBL etiology. Infection-induced immune evasion mechanisms to avoid T cell cytotoxicity may increase the role of Natural killer (NK) cells in anti-tumor immunosurveillance. Killer immunoglobulin-like receptor (KIR) genes on NK cells exhibit genotypic and allelic variations and are associated with susceptibility to diseases and malignancies. However, their role in eBL pathogenesis remains undefined. This retrospective study genotyped sixteen KIR genes and compared their frequencies in eBL patients (n = 104) and healthy geographically-matched children (n = 104) using sequence-specific primers polymerase chain reaction (SSP-PCR) technique. The relationship between KIR polymorphisms with EBV loads and eBL pathogenesis was investigated. Possession of ≥ 4 activating KIRs predisposed individuals to eBL (OR = 3.340; 95% CI 1.530–7.825; p = 0.004). High EBV levels were observed in Bx haplogroup (p = 0.016) and AB genotypes (p = 0.042) relative to AA haplogroup and AA genotype respectively, in eBL patients but not in healthy controls. Our results suggest that KIR-mediated NK cell stimulation could mute EBV control, contributing to eBL pathogenesis.

Introduction The Military Health System serves to globally provide health services and trained medical forces. Military providers possess variable levels of deployment preparedness. The aim of the Clinical Readiness Program is to develop and assess the knowledge, skills, and abilities (KSAs) needed for combat casualty care. Methods The Clinical Readiness Program developed a KSA metric for general and orthopedic surgery. The KSA methodology underwent a proof of concept in six medical treatment facilities. Results The KSA metric feasibly quantifies the combat relevance of surgical practice. Orthopedic surgeons are more likely than general surgeons to meet the threshold. Medical treatment facilities do not provide enough demand for general surgery services to achieve readiness. Conclusion The Clinical Readiness Program identifies imbalances between the health care delivery and readiness missions. To close the readiness gap, the Military Health System needs to recapture high KSA value procedures, expand access to care, and/or partner with civilian institutions.

Cell-specific microRNA (miRNA) expression estimates are important in characterizing the localization of miRNA signaling within tissues. Much of this data is obtained from cultured cells, a process known to significantly alter miRNA expression levels. Thus, our knowledge of in vivo cell miRNA expression estimates is poor. We previously demonstrated expression microdissection-miRNA-sequencing (xMD-miRNA-seq) as a means to acquire in vivo estimates, directly from formalin fixed tissues, albeit with limited yield. Here we optimized each step of the xMD process including tissue retrieval, tissue transfer, film preparation, and RNA isolation to increase RNA yields and ultimately show strong enrichment for in vivo miRNA expression by qPCR array. These method improvements, including the development of a non-crosslinked ethylene vinyl acetate (EVA) membrane, resulted in a 23-45 fold increase in miRNA yield, depending on cell type. By qPCR, miR-200a was increased 14-fold in xMD-derived small intestine epithelial cells, with a concurrent 336-fold reduction in miR-143, relative to the matched non-dissected duodenal tissue. xMD is now an optimized method to obtain robust in vivo miRNA expression estimates from cells.

Artemisinin partial resistance may facilitate selection of Plasmodium falciparum resistant to combination therapy partner drugs. We evaluated 99 P. falciparum isolates collected in 2021 from northern Uganda, where resistance-associated PfK13 C469Y and A675V mutations have emerged, and eastern Uganda, where these mutations are uncommon. With the ex vivo ring survival assay, isolates with the 469Y mutation (median survival 7.3% for mutant, 2.5% mixed, and 1.4% wild type) and/or mutations in Pfcoronin or falcipain-2a, had significantly greater survival; all isolates with survival >5% had mutations in at least one of these proteins. With ex vivo growth inhibition assays, susceptibility to lumefantrine (median IC50 14.6 vs. 6.9 nM, p p = 0.003) was decreased in northern vs. eastern Uganda; 14/49 northern vs. 0/38 eastern isolates had lumefantrine IC50 > 20 nM (p = 0.0002). Targeted sequencing of 819 isolates from 2015–21 identified multiple polymorphisms associated with altered drug susceptibility, notably PfK13 469Y with decreased susceptibility to lumefantrine (p = 6 × 10−8) and PfCRT mutations with chloroquine resistance (p = 1 × 10−20). Our results raise concern regarding activity of artemether-lumefantrine, the first-line antimalarial in Uganda.

Knowledge of host associations of blood-feeding vectors may afford insights into managing disease systems and protecting public health. However, the ability of methods to distinguish bloodmeal sources varies widely. We used two methods—Sanger sequencing and amplicon deep sequencing—to target a 228 bp region of the vertebrate Cytochrome b gene and determine hosts fed upon by triatomines (n = 115) collected primarily in Texas, USA. Direct Sanger sequencing of PCR amplicons was successful for 36 samples (31%). Sanger sequencing revealed 15 distinct host species, which included humans, domestic animals (Canis lupus familiaris, Ovis aries, Gallus gallus, Bos taurus, Felis catus, and Capra hircus), wildlife (Rattus rattus, Incilius nebulifer, Sciurus carolinensis, Sciurus niger, and Odocoileus virginianus), and captive animals (Panthera tigris, Colobus spp., and Chelonoidis carbonaria). Samples sequenced by the Sanger method were also subjected to Illumina MiSeq amplicon deep sequencing. The amplicon deep sequencing results (average of 302,080 usable reads per sample) replicated the host community revealed using Sanger sequencing, and detected additional hosts in five triatomines (13.9%), including two additional blood sources (Procyon lotor and Bassariscus astutus). Up to four bloodmeal sources were detected in a single triatomine (I. nebulifer, Homo sapiens, C. lupus familiaris, and S. carolinensis). Enhanced understanding of vector-host-parasite networks may allow for integrated vector management programs focusing on highly-utilized and highly-infected host species.

Between conflicts, many of the combat casualty care lessons learned are lost as the nation shifts priorities and providers leave the military. Solutions are needed to bridge the knowledge gap created by interwar periods. One of the foremost solutions is partnerships between civilian trauma centers and the military health system. Over the past two decades, a myriad of military-civilian partnerships (MCPs), which vary in their composition, duration, and focus, was created. The objective of this report is to describe the initial attempt of the Department of Defense to catalog existing MCPs to inform both civilian and military stakeholders. This initial catalog is intended as a reference to aid in future MCP development and facilitate the synchronization of efforts to improve trauma care delivery and readiness.Using methodology from the Institute of Defense Analysis, the total number of eligible trauma centers in the United States was determined. The Institute of Defense Analysis determined eligibility-based American College of Surgeons Trauma Center verification or state trauma center designation. Each military service provided their list of MCPs, which were categorized. Military-civilian partnerships were cataloged by various characteristics and program components. Key variables include number and type of personnel trained, duration of training, and focus, for example, team versus individual focused and training versus maintaining proficiency focused.A total of 1,139 hospitals in the United States are potentially eligible for MCPs. There are at least 87 unique partnerships; the majority are part-time sustainment MCPs. The Air Force has the largest number of providers in MCPs. There are many challenges to maintain accurate and up to date data on MCPs.With the collated information, the Defense Health Agency, military services, special operations community, and civilian partners will be better empowered to optimize the readiness value of their programs and better prepare our military medical providers for the nation's and military's future needs.

Artemether–lumefantrine (AL) is a first-line agent for uncomplicated malaria caused by Plasmodium falciparum. The WHO recommends periodic therapeutic efficacy studies of antimalarial drugs for the detection of malaria parasite drug resistance and to inform national malaria treatment policies. We conducted a therapeutic efficacy study of AL in a high malaria transmission region of northern Zambia from December 2014 to July 2015. One hundred children of ages 6 to 59 months presenting to a rural health clinic with uncomplicated falciparum malaria were admitted for treatment with AL (standard 6-dose regimen) and followed weekly for 5 weeks. Parasite counts were taken every 6 hours during treatment to assess parasite clearance. Recurrent episodes during follow-up (n = 14) were genotyped to distinguish recrudescence from reinfection and to identify drug resistance single nucleotide polymorphisms (SNPs) and multidrug resistance protein 1 (mdr1) copy number variation. Day 7 lumefantrine concentrations were measured for correspondence with posttreatment reinfection. All children who completed the parasite clearance portion of the study (n = 94) were microscopy-negative by 72 hours. The median parasite elimination half-life was 2.7 hours (interquartile range: 2.1–3.3). Genotype-corrected therapeutic efficacy was 98.8% (95% CI: 97.6–100). Purported artemisinin and lumefantrine drug resistance SNPs in atp6, 3D7_1451200, and mdr1 were detected but did not correlate with parasite recurrence, nor did day 7 lumefantrine concentrations. In summary, AL was highly effective for the treatment of uncomplicated falciparum malaria in northern Zambia during the study period. The high incidence of recurrent parasitemia was consistent with reinfection due to high, perennial malaria transmission.

Background In Uganda, artemether-lumefantrine is recommended for malaria treatment and sulfadoxine-pyrimethamine for chemoprevention during pregnancy, but drug resistance may limit efficacies. Methods Genetic polymorphisms associated with sensitivities to key drugs were characterized in samples collected from 16 sites across Uganda in 2018 and 2019 by ligase detection reaction fluorescent microsphere, molecular inversion probe, dideoxy sequencing, and quantitative polymerase chain reaction assays. Results Considering transporter polymorphisms associated with resistance to aminoquinolines, the prevalence of Plasmodium falciparum chloroquine resistance transporter (PfCRT) 76T decreased, but varied markedly between sites (0–46% in 2018; 0–23% in 2019); additional PfCRT polymorphisms and plasmepsin-2/3 amplifications associated elsewhere with resistance to piperaquine were not seen. For P. falciparum multidrug resistance protein 1, in 2019 the 86Y mutation was absent at all sites, the 1246Y mutation had prevalence ≤20% at 14 of 16 sites, and gene amplification was not seen. Considering mutations associated with high-level sulfadoxine-pyrimethamine resistance, prevalences of P. falciparum dihydrofolate reductase 164L (up to 80%) and dihydropteroate synthase 581G (up to 67%) were high at multiple sites. Considering P. falciparum kelch protein propeller domain mutations associated with artemisinin delayed clearance, prevalence of the 469Y and 675V mutations has increased at multiple sites in northern Uganda (up to 23% and 41%, respectively). Conclusions We demonstrate concerning spread of mutations that may limit efficacies of key antimalarial drugs.