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Funding Information: The study was funded by the U.S. President’s Malaria Initiative and the Advanced Molecular Detection program at CDC. Funding Information: We thank all study staff and participants, Venceslau Mambi Pelenda, José Bumba da Cunha, Oliveira Kiatoko, Felismina Caquece, Luzala Elisabeth Armando Garcia, Djos Kialanda, Garcia Pembele, Domingos Jandondo, and Belmira José Bondo. The findings and conclusions in this report are those of the author(s) and do not necessarily represent the official position of the U.S. Centers for Disease Control and Prevention or the U.S. Agency for International Development. The study was funded by the U.S. President’s Malaria Initiative and the Advanced Molecular Detection program at CDC. P.R.D., C.M.F., B.N.A., S.L., R.H., J.F.M.M., F.F., J.F.M., and M.M.P. designed and participated in the specimen collection. P.R.D., A.L.M.C., S.R.R., and J.K. performed the laboratory analysis. P.R.D., A.L.M.C., J.K., S.R.R., J.-H.M.O., and E.T. analyzed the sequencing data. S.R.R. and M.M.P. wrote the manuscript. All authors read and approved the final version of the manuscript. Publisher Copyright: Copyright © 2023 Rosillo et al. Sulfadoxine-pyrimethamine (SP) is used for prevention of malaria in pregnant women in Angola. We sequenced the Plasmodium falciparum dihydrofolate reductase (pfdhfr) and dihydropteroate synthase (pfdhps) genes, implicated in SP resistance, in samples collected during a 2019 study of artemisinin-based combination therapy efficacy in Benguela, Lunda Sul, and Zaire provinces. A total of 90 day 0 and day of failure samples were individually sequenced, while 508 day 0 samples from participants without recurrent parasitemia were pooled after DNA extraction into 61 pools. The N51I, C59R, and S108N pfdhfr mutations and A437G pfdhps mutations were present at high proportions in all provinces (weighted allele frequencies, 62% to 100%). The K540E pfdhps mutation was present at lower proportions (10% to 14%). The A581G pfdhps mutation was only observed in Zaire, at a 4.6% estimated prevalence. The I431V and A613S mutations were also only observed in Zaire, at a prevalence of 2.8% to 2.9%. The most common (27% to 66%) reconstructed haplotype in all three provinces was the canonical quadruple pfdhfr pfdhps mutant. The canonical quintuple mutant was absent in Lunda Sul and Benguela and present in 7.9% of samples in Zaire. A single canonical sextuple (2.6%) mutant was observed in Zaire Province. Proportions of the pfdhps K540E and A581G mutations were well below the World Health Organization thresholds for meaningful SP resistance (prevalence of 95% for K540E and 10% for A581G). Samples from therapeutic efficacy studies represent a convenient source of samples for monitoring SP resistance markers. publishersversion published

Background Recent studies showed the first emergence of the R561H artemisinin-associated resistance marker in Africa, which highlights the importance of continued molecular surveillance to assess the selection and spread of this and other drug resistance markers in the region. Method In this study, we used targeted amplicon deep sequencing of 116 isolates collected in two areas of Cameroon to genotype the major drug resistance genes, k13, crt, mdr1, dhfr, and dhps, and the cytochrome b gene (cytb) in Plasmodium falciparum. Results No confirmed or associated artemisinin resistance markers were observed in Pfk13. In comparison, both major and minor alleles associated with drug resistance were found in Pfcrt, Pfmdr1, Pfdhfr, and Pfdhps. Notably, a high frequency of other nonsynonymous mutations was observed across all the genes, except for Pfcytb, suggesting continued selection pressure. Conclusions The results from this study supported the continued use of artemisinin-based combination therapy and administration of sulfadoxine-pyrimethamine for intermittent preventive therapy in pregnant women, and for seasonal chemoprevention in these study sites in Cameroon.

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

The spread of drug resistance to antimalarial treatments poses a serious public health risk globally. To combat this risk, molecular surveillance of drug resistance is imperative. We report the prevalence of mutations in the Plasmodium falciparum kelch 13 propeller domain associated with partial artemisinin resistance, which we determined by using Sanger sequencing samples from patients enrolled in therapeutic efficacy studies from 9 sub-Saharan countries during 2014-2018. Of the 2,865 samples successfully sequenced before treatment (day of enrollment) and on the day of treatment failure, 29 (1.0%) samples contained 11 unique nonsynonymous mutations and 83 (2.9%) samples contained 27 unique synonymous mutations. Two samples from Kenya contained the S522C mutation, which has been associated with delayed parasite clearance; however, no samples contained validated or candidate artemisinin-resistance mutations.

The leading cause of genetic dilated cardiomyopathy (DCM) is due to mutations in the TTN gene, impacting approximately 15-20% of familial and 18% of sporadic DCM cases. Currently, there is potential for a personalized RNA-based therapeutic approach in titin-based DCM, utilizing antisense oligonucleotide (AON) mediated exon-skipping, which attempts to reframe mutated titin transcripts, resulting in shortened, functional protein. However, the TTN gene is massive with 363 exons; each newly identified TTN exon mutation provides a challenge to address when considering the potential application of AON mediated exon skipping. In the initial phase of this strategy, the mutated TTN exon requires specific AON design and evaluation to assess the exon skipping effectiveness for subsequent experiments. Here, we present a detailed protocol to effectively assemble and evaluate AONs for efficient exon-skipping in targeted TTN exons. We chose a previously identified TTN 1-bp deletion mutation in exon 335 as an exemplary target exon, which causes a frameshift mutation leading to truncated A-band titin in DCM. We designed two specific AONs to mask the Ttn exon 335 and confirmed successfully mediated exon skipping without disrupting the Ttn reading frame. In addition, we evaluated and confirmed AON-treated HL-1 cells show maintained store-operated calcium entry, fractional shortening as well as preserved sarcomeric formation in comparison to control samples, indicating the treated cardiomyocytes retain adequate, essential cell function and structure, proving the treated cells can compensate for the loss of exon 335. These results indicate our method offers the first systematic protocol in designing and evaluating AONs specifically for mutated TTN target exons, expanding the framework of future advancements in the therapeutic potential of antisense-mediated exon skipping in titin-based DCM.

Die dilatative Kardiomyopathie (DCM) ist eine multifaktorielle Erkrankung; ca. 15-20% der familiären und 18% der sporadischen Formen lassen sich allerdings auf Mutationen im Titin, einem Sarkomerprotein mit wichtigen Funktionen für den kontraktilen Apparat, zurückführen. Im Moment steht keine spezifische Therapie für Patienten mit fortgeschrittener DCM zur Verfügung, sodass neben symptomatischen Maßnahmen mit Behandlung der Herzinsuffizienz lediglich die Option einer Herztransplantation existiert (Hahn et al.,2019). Ein potentieller Ansatz einer genotyp-spezifischen Therapie ist das sogenannte „Exon skipping“. Hierfür werden RNA-basierte Antisense-Oligonucleotide (AON) verwendet, um ein mutiertes Titin-Exon gezielt zu überspringen. Durch Wiederherstellung des Leserahmens entsteht ein verkürztes, aber funktionell intaktes Titin-Protein. In einer vorausgegangenen Machbarkeitsstudie haben wir das Prinzip des Exon-skippings an Zellkultur- und Mausmodell demonstrieren können. Das TTN-Gen besteht jedoch aus 363 Exonen und ist somit massiv; alle AON Sequenzen müssen deshalb für jedes einzelne Exon spezifisch entwickelt werden(Gramlich et al., 2015). Diese Arbeit beschreibt ein Protokoll, welches für das Design und die Evaluierung von AONs für ein effizientes Exon-Skipping spezifischer TTN-Exone angewandt werden kann. Zunächst werden verschiedene in silico Algorithmen beschrieben, die putative AON Sequenzen zum Überspringen eines Titin-Exons erstellen. Anschließend werden diese AONs in transfizierten HL-1 Zellen, einer murinen Kardiomyozytenlinie, getestet. Neben dem Nachweis eines korrekten Überspringens des Target- Exons mittels RT- PCR und Sanger-Sequenzierung werden die funktionellen Auswirkungen des skippings durch Immunhistochemie, Store-Operate-Calcium-Entry (SOCE) und Kontraktilitätsmessungen untersucht(Hahn et al.,2019). Diese Arbeit ist somit das erste systematische Protokoll zum AON-Design und deren Evaluation, um spezifische TTN Exons durch Antisense-Therapie zu überspringen. Dies ist ein erster Schritt in Richtung einer individualisierten Therapie für die titin-basierten dilatativen Kardiomyopathie(Hahn et al.,2019).

We thank Rasmussen and Ringwald for further highlighting the importance of routine monitoring of antimalarial drug efficacy in sub-Saharan Africa, including Angola.….

During our transthoracic echocardiography (TTE) courses, medical students showed difficulty in spatial orientation. We implemented the use of 3D printed cardiac models of standard TTE views PLAX, PSAX, and A4C and assessed their efficacy in TTE-teaching.One hundred fifty-three participants were split into two groups. A pre-test-retest of anatomy, 2D -, and 3D orientation was conducted. The intervention group (n = 77) was taught using 3D models; the control group (n = 76) without. Both were comparable with respect to baseline parameters. Besides test-scores, a Likert scale recorded experiences, difficulties, and evaluation of teaching instruments.From the 153 students evaluated, 123 improved, 20 did worse, and ten achieved the same result after the course. The median overall pre-test score was 29 of 41 points, and the retest score was 35 (p 0.001). However, the intervention group taught with the 3D models, scored significantly better overall (p = 0.016), and in 2D-thinking (p = 0.002) and visual thinking (p = 0.006) subtests. A backward multivariate linear regression model revealed that the 3D models are a strong individual predictor of an excellent visual thinking score. In addition, our study showed that students with difficulty in visual thinking benefited considerably from the 3D models.Students taught using the 3D models significantly improved when compared with conventional teaching. Students regarded the provided models as most helpful in their learning process. We advocate the implementation of 3D-printed heart models featuring the standard views for teaching echocardiography. These findings may be transferable to other evidence based medical and surgical teaching interventions.

Biennial therapeutic efficacy monitoring is a crucial activity for ensuring the efficacy of currently used artemisinin-based combination therapy in Angola. Children with acute uncomplicated Plasmodium falciparum infection in sentinel sites in the Benguela, Zaire, and Lunda Sul Provinces were treated with artemether-lumefantrine (AL) or artesunate-amodiaquine (ASAQ) and monitored for 28 days to assess clinical and parasitological responses. Molecular correction was performed using seven microsatellite markers., Biennial therapeutic efficacy monitoring is a crucial activity for ensuring the efficacy of currently used artemisinin-based combination therapy in Angola. Children with acute uncomplicated Plasmodium falciparum infection in sentinel sites in the Benguela, Zaire, and Lunda Sul Provinces were treated with artemether-lumefantrine (AL) or artesunate-amodiaquine (ASAQ) and monitored for 28 days to assess clinical and parasitological responses. Molecular correction was performed using seven microsatellite markers. Samples from treatment failures were genotyped for the pfk13, pfcrt, and pfmdr1 genes. Day 3 clearance rates were ≥95% in all arms. Uncorrected day 28 Kaplan-Meier efficacy estimates ranged from 84.2 to 90.1% for the AL arms and 84.7 to 100% for the ASAQ arms. Corrected day 28 estimates were 87.6% (95% confidence interval [CI], 81 to 95%) for the AL arm in Lunda Sul, 92.2% (95% CI, 87 to 98%) for AL in Zaire, 95.6% (95% CI, 91 to 100%) for ASAQ in Zaire, 98.4% (95% CI, 96 to 100%) for AL in Benguela, and 100% for ASAQ in Benguela and Lunda Sul. All 103 analyzed samples had wild-type pfk13 sequences. The 76T pfcrt allele was found in most (92%; 11/12) ASAQ late-failure samples but in only 16% (4/25) of AL failure samples. The N86 pfmdr1 allele was found in 97% (34/35) of treatment failures. The AL efficacy in Lunda Sul was below the 90% World Health Organization threshold, the third time in four rounds that this threshold was crossed for an AL arm in Angola. In contrast, the observed ASAQ efficacy has not been below 95% to date in Angola, including this latest round.

The number of Asian migrants working in sub-Saharan developing countries like Angola has been increasing. Their malaria risk, prevention, and care-seeking practices have not been characterized. A cross-sectional survey was conducted in 733 Chinese and Southeast Asian migrants in Angola. Respondents were interviewed and provided blood samples. Samples were analyzed to detect Plasmodium antigen and characterize host anti-Plasmodium response. Positive samples were genotyped using the pfs47 marker. Most respondents (72%; 95% CI: 68-75) reported using bed nets, but less than 1% reported using chemoprophylaxis. Depending on the assay, 1-4% of respondents had evidence of active malaria infection. By contrast, 55% (95% CI: 52-59) were seropositive for Plasmodium antibodies. Most infections were Plasmodium falciparum, but infection and/or exposure to Plasmodium vivax and Plasmodium malariae was also detected. Seroprevalence by time in Angola showed most exposure occurred locally. One respondent had sufficiently high parasitemia for pfs47 genotyping, which showed that the infection was likely locally acquired despite recent travel to home country. Asian migrants to Angola are at substantial risk of malaria. Employers should consider enhanced malaria prevention programs, including chemoprophylaxis; embassies should encourage prevention practices. Angolan healthcare workers should be aware of high malaria exposure in Asian migrants.

Background Anti-malarial resistance is a threat to recent gains in malaria control. This study aimed to assess the efficacy and safety of artesunate–amodiaquine (ASAQ) and artemether–lumefantrine (AL) in the management of uncomplicated malaria and to measure the prevalence of molecular markers of resistance of Plasmodium falciparum in sentinel sites in Maferinyah and Labé Health Districts in Guinea in 2016. Methods This was a two-arm randomised controlled trial of the efficacy of AL and ASAQ among children aged 6–59 months with uncomplicated Plasmodium falciparum malaria in two sites. Children were followed for 28 days to assess clinical and parasitological response. The primary outcome was the Kaplan–Meier estimate of Day 28 (D28) efficacy after correction by microsatellite-genotyping. Pre-treatment (D0) and day of failure samples were assayed for molecular markers of resistance in the pfk13 and pfmdr1 genes. Results A total of 421 participants were included with 211 participants in the Maferinyah site and 210 in Labé. No early treatment failure was observed in any study arms. However, 22 (5.3%) participants developed a late treatment failure (8 in the ASAQ arm and 14 in the AL arm), which were further classified as 2 recrudescences and 20 reinfections. The Kaplan–Meier estimate of the corrected efficacy at D28 was 100% for both AL and ASAQ in Maferinyah site and 99% (95% Confidence Interval: 97.2–100%) for ASAQ and 99% (97.1–100%) for AL in Labé. The majority of successfully analysed D0 (98%, 380/389) and all day of failure (100%, 22/22) samples were wild type for pfk13. All 9 observed pfk13 mutations were polymorphisms not associated with artemisinin resistance. The NFD haplotype was the predominant haplotype in both D0 (197/362, 54%) and day of failure samples (11/18, 61%) successfully analysed for pfmdr1. Conclusion This study observed high efficacy and safety of both ASAQ and AL in Guinea, providing evidence for their continued use to treat uncomplicated malaria. Continued monitoring of ACT efficacy and safety and molecular makers of resistance in Guinea is important to detect emergence of parasite resistance and to inform evidence-based malaria treatment policies.

We report a case of a 3-week-old infant who presented a heart murmur and low oxygen saturation. An echocardiography was performed and presented a common arterial trunk type B4 with an interrupted aortic arch and intact ventricular septum. We describe the surgical management and short-term follow-up.

Background: Antimalarial resistance is a threat to recent gains in malaria control. This study aimed to assess the efficacy and safety of artesunate-amodiaquine (ASAQ) and artemether-lumefantrine (AL) in the management of uncomplicated malaria and to measure the prevalence of molecular markers of resistance of Plasmodium falciparum in sentinel sites in Maferinyah and Labé Health Districts in Guinea in 2016.Methods: This was a two-arm randomized controlled trial of the efficacy of AL and ASAQ in children aged 6-59 months with P. falciparum monoinfection in two sites. Children were followed for 28 days to assess clinical and parasitological response. The primary outcome was the Kaplan-Meier estimate of Day 28 (D28) efficacy after correction by microsatellite-genotyping. Pre-treatment (D0) and day of failure samples were assayed for molecular markers of resistance in the pfk13 and pfmdr1 genes.Results: A total of 421 participants were included with 211 participants in the Maferinyah site and 210 in Labé. No early treatment failure was observed in any study arms. However, 22 (5.3%) participants developed a late treatment failure, which were further classified as 2 recrudescences and 20 reinfections. The Kaplan-Meier estimate of the corrected efficacy at D28 was 100% for both AL and ASAQ in Maferinyah site and 99% (95% Confidence Interval: 97.2-100%) for ASAQ and 99% (97.1-100%) for AL in Labé. The majority of successfully analyzed D0 (98%, 380/389) and all day of failure (100%, 22/22) samples were wild type for pfk13. All 9 observed pfk13 mutations were polymorphisms not associated with artemisinin resistance. The NFD haplotype was the predominant haplotype in both D0 (197/362, 54%) and day of failure samples (11/18, 61%) successfully analyzed for pfmdr1.Conclusions: This study observed high efficacy and safety of both ASAQ and AL in Guinea, providing evidence for their continued use to treat uncomplicated malaria. Continued monitoring of ACT efficacy and safety and molecular makers of resistance in Guinea is important to detect emergence of parasite resistance and to inform evidence-based malaria treatment policies.

Outbreaks of cyclosporiasis, a food-borne illness caused by the coccidian parasite Cyclospora cayetanensis have increased in the USA in recent years, with approximately 2300 laboratory-confirmed cases reported in 2018. Genotyping tools are needed to inform epidemiological investigations, yet genotyping Cyclospora has proven challenging due to its sexual reproductive cycle which produces complex infections characterized by high genetic heterogeneity. We used targeted amplicon deep sequencing and a recently described ensemble-based distance statistic that accommodates heterogeneous (mixed) genotypes and specimens with partial genotyping data, to genotype and cluster 648 C. cayetanensis samples submitted to CDC in 2018. The performance of the ensemble was assessed by comparing ensemble-identified genetic clusters to analogous clusters identified independently based on common food exposures. Using these epidemiologic clusters as a gold standard, the ensemble facilitated genetic clustering with 93.8% sensitivity and 99.7% specificity. Hence, we anticipate that this procedure will greatly complement epidemiologic investigations of cyclosporiasis.

Dilated cardiomyopathy (DCM) is the result of maladaptive cardiac remodeling, which involves microRNA regulation. In turn, microRNAs can contribute to the remodeling process by post-transcriptional modulation of gene expression networks. The exact role of microRNAs in the pathogenesis of DCM is largely unknown. Here, we used an inducible DCM mouse model that carries a human truncation mutation in the sarcomeric protein titin to dissect microRNA pathways in DCM development.MicroRNA microarray studies revealed up-regulation of microRNA-208b in the myocardium of DCM mice and DCM patients (p0.05 compared to controls). In order to investigate the effect of microRNA-208b on cardiac remodeling, loss-of-function and gain-of-function studies were performed by repetitive injections of LNA-modified microRNA-208b mimics and antimiR-208b. MiR-208b overexpression resulted in cardiac hypertrophy, whereas miR-208b antagonisation prevented transition of adaptive to maladaptive remodeling in the DCM mouse model. In vitro studies identified several pro-hypertrophic transcription factors as potential targets of miR-208b, suggesting that microRNA-208b plays an important role in cardiac development and growth. MiR-208b was also upregulated in DCM patients, but not in heart failure patients due to ischemic heart disease or myocarditis.Our data suggests that miR-208b is involved in the remodeling process and pathogenesis of DCM by post-transcriptional gene expression modulation. MicroRNA-208b might be a novel therapeutic target for DCM.

Mutations in the Plasmodium falciparumk13 (Pfk13) gene are linked to delayed parasite clearance in response to artemisinin-based combination therapies (ACTs) in Southeast Asia. To explore the evolutionary rate and constraints acting on this gene, k13 orthologs from species sharing a recent common ancestor with P. falciparum and Plasmodium vivax were analyzed. These comparative studies were followed by genetic polymorphism analyses within P. falciparum using 982 complete Pfk13 sequences from public databases and new data obtained by next-generation sequencing from African and Haitian isolates. Although k13 orthologs evolve at heterogeneous rates, the gene was conserved across the genus, with only synonymous substitutions being found at residues where mutations linked to the delayed parasite clearance phenotype have been reported. This suggests that those residues were under constraint from undergoing nonsynonymous changes during evolution of the genus. No fixed nonsynonymous differences were found between Pfk13 and its orthologs in closely related species found in African apes. This indicates that all nonsynonymous substitutions currently found in Pfk13 are younger than the time of divergence between P. falciparum and its closely related species. At the population level, no mutations linked to delayed parasite clearance were found in our samples from Africa and Haiti. However, there is a high number of single Pfk13 mutations segregating in P. falciparum populations, and two predominant alleles are distributed worldwide. This pattern is discussed in terms of how changes in the efficacy of natural selection, affected by population expansion, may have allowed for the emergence of mutations tolerant to ACTs.

The recent advances in next-generation sequencing technologies provide a new and effective way of tracking malaria drug-resistant parasites. To take advantage of this technology, an end-to-end Illumina targeted amplicon deep sequencing (TADS) and bioinformatics pipeline for molecular surveillance of drug resistance in P. falciparum , called ma laria r esistance s urveillance (MaRS), was developed. TADS relies on PCR enriching genomic regions, specifically target genes of interest, prior to deep sequencing. MaRS enables researchers to simultaneously collect data on allele frequencies of multiple full-length P. falciparum drug resistance genes ( crt , mdr1 , k13 , dhfr , dhps , and the cytochrome b gene), as well as the mitochondrial genome. Information is captured at the individual patient level for both known and potential new single nucleotide polymorphisms associated with drug resistance. The MaRS pipeline was validated using 245 imported malaria cases that were reported to the Centers for Disease Control and Prevention (CDC). The chloroquine resistance crt CV IET genotype (mutations underlined) was observed in 42% of samples, the highly pyrimethamine-resistant dhps IRN triple mutant in 92% of samples, and the sulfadoxine resistance dhps mutation S GE AA in 26% of samples. The mdr1 N F SND genotype was found in 40% of samples. With the exception of two cases imported from Cambodia, no artemisinin resistance k13 alleles were identified, and 99% of patients carried parasites susceptible to atovaquone-proguanil. Our goal is to implement MaRS at the CDC for routine surveillance of imported malaria cases in the United States and to aid in the adoption of this system at participating state public health laboratories, as well as by global partners.