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Background Pathogen genotyping via polymerase chain reaction (PCR) amplicon sequencing (AmpSeq) is an informative disease surveillance tool. Several large AmpSeq panels containing > 100 multiplexed PCR amplicons have been developed as alternatives to whole-genome sequencing (WGS) methods for the Plasmodium spp. parasites that cause malaria, especially for parasite drug resistance tracking and relatedness analysis. However, these large multiplexes typically yield sparse data for samples with parasitemia below 10 parasites/μl. Smaller multiplexes optimized for low-parasitemia genotyping have received insufficient methodological work but have the potential to serve multiple important use cases. Managing contamination risk during PCR steps represents another key methodological gap that requires attention in the AmpSeq field. Methods Here we describe a new 6-locus Plasmodium falciparum AmpSeq ‘miniplex’ (SIMPLseq) optimized for high-sensitivity analyses that also integrates a contamination detection system based on well-specific inline barcodes applied during first-round PCR (PCR1; in addition to conventional indexing steps during second-round PCR). We assess panel diversity using publicly available WGS and use mock samples to estimate sensitivity and precision relative to 4CAST, a previously described miniplex. We also create deliberate contamination events to assess contamination detection rigor and estimate unintentional contamination rates during assay application to malaria-infected dried blood spots collected in Mali. Results SIMPLseq shows high haplotypic diversity in silico, distinguishing 96.0% of sample pairs drawn randomly from 12 subnational sample sets. SIMPLseq outperforms 4CAST in sensitivity analyses, achieving 100% average locus detection at ≥ 0.5 parasites/μl and ≥ 50% average locus detection at 0.25 and 0.125 parasites/μl, with zero false-positive haplotypes at a 1% detection limit across 25 replicates. Inline barcoding does not significantly affect yield when using a ‘sentinel’ design, whereby one of the six multiplexed PCR1 primer pairs contains the well-specific sequence pair. Sentinel barcoding correctly identified all 24 contaminations introduced deliberately during PCR1 product handling and identified 39 unintentional contaminations in the 1420-sample Malian run. Conclusions SIMPLseq significantly extends the malaria genomic epidemiology toolkit, coupling high-sensitivity P. falciparum genotyping with PCR contamination detection in a simple laboratory protocol that uses only open-source reagents and does not require a costly pre-amplification step. Key prospective use cases for SIMPLseq include recurrent infection classification, polyclonality estimation, and genotypic infection endpoint application to intervention efficacy trials.

Plasmodium vivax is the main cause of malaria outside of sub-Saharan Africa, and in many settings it presents significant challenges to malaria elimination efforts. Despite some control successes in the Americas, regional annual case counts of malaria have increased by over 25% between 2014 and 2023, largely driven by P. vivax. Genomic surveillance can play a key role in understanding the extent to which disease persistence represents indigenous transmission as opposed to introduction of new strains through migration, and whether specific variants evade control measures. Efforts to make P. vivax genomic surveillance more cost-effective have led to the development of targeted sequencing-based methods, which strike a varying balance between assay sensitivity and breadth/informativeness. We introduce two new highly sensitive multiplexed amplicon sequencing panels for P. vivax : PvGTSeq and PvCRiSP. PvGTSeq requires selective whole-genome amplification (sWGA) and contains 249 amplicons—36 for antimalarial resistance and 213 for population structure—optimized for Latin America but applicable to all continents. PvCRiSP features four highly polymorphic amplicons that operate without sWGA and is designed to estimate complexity of infection (COI), identify instances of clonal transmission, and characterize recurrent episodes. Both panels use a single multiplex PCR with non-proprietary reagents, achieve ≥75% amplicon recovery at parasitemias as low as five parasites/μL, and PvCRiSP remains effective with low quality DNA. PvGTSeq showed high sequencing accuracy (error rate 3.85e-4% - 2.87e-3%), and both panels efficiently detected alleles from minority clones in simulated polyclonal infections. We validated both panels with samples from Colombia, Guyana, Honduras, Panama, and Venezuela, and performed in-silico assessments using data from 16 countries worldwide, confirming that these two panels have high power to discriminate samples and assign global geographic origin to imported cases. These panels will therefore be useful tools for P. vivax molecular surveillance in diverse geographic settings.

Malaria in South America remains a serious public health problem. ( ) is the most important malaria vector across tropical Latin America. Vector-targeted disease control efforts require a thorough understanding of mosquito demographic and evolutionary patterns. We present and analyze whole genomes of 1094 (median depth 18x) from six South American countries. We observe deep geographic population structure, high genetic diversity including thirteen putative segregating inversions, and no evidence for cryptic sympatric taxa despite high interpopulation divergence. Strong signals of selection are plausibly driven by insecticides, especially on cytochrome P450 genes, one of which we validated experimentally. Our results will facilitate effective mosquito surveillance and control, while highlighting ongoing challenges that a diverse vector poses for malaria elimination in the western hemisphere.

Doxycycline post-exposure prophylaxis (doxy-PEP) reduces bacterial sexually transmitted infections among men who have sex with men and transgender women. Although poised for widespread clinical implementation, the impact of doxy-PEP on antimicrobial resistance remains a primary concern as its effects on the gut microbiome and resistome, or the antimicrobial resistance genes (ARGs) present in the gut microbiome, are unknown. To investigate these effects, we studied participants from the DoxyPEP trial, a randomized clinical trial comparing doxy-PEP use, a one-time doxycycline 200-mg dose taken after condomless sex (DP arm, n  = 100), to standard of care (SOC arm, n  = 50) among men who have sex with men and transgender women. From self-collected rectal swabs at enrollment (day-0) and after 6 months (month-6), we performed metagenomic DNA sequencing (DNA-seq) or metatranscriptomic RNA sequencing (RNA-seq). DNA-seq data were analyzable from 127 samples derived from 89 participants, and RNA-seq data were analyzable from 86 samples derived from 70 participants. We compared the bacterial microbiome and resistome between the two study arms and over time. The median number of doxycycline doses taken since enrollment by participants with DNA-seq data was zero (interquartile range (IQR): 0–7 doses) for the SOC arm and 42 (IQR: 27–64 doses) for the DP arm. Tetracycline ARGs were detected in all day-0 DNA-seq samples and in 85% of day-0 RNA-seq samples. The proportional mass of tetracycline ARGs in the resistome increased between day-0 and month-6 in DP participants from 46% to 51% in the metagenome ( P  = 2.3 × 10 −2 ) and from 4% to 15% in the metatranscriptome ( P  = 4.5 × 10 −6 ), but no statistically significant increases in other ARG classes were observed. Exposure to a higher number of doxycycline doses correlated with proportional enrichment of tetracycline ARGs in the metagenome (Spearman’s ρ  = 0.23, P  = 9.0 × 10 −3 ) and metatranscriptome (Spearman’s ρ  = 0.55, P  = 3.7 × 10 −8 ). Bacterial microbiome alpha diversity, beta diversity and total bacterial mass did not differ between day-0 and month-6 samples from DP participants when assessed by either DNA-seq or RNA-seq. In an abundance-based correlation analysis, we observed an increase over time in the strength of the correlation between tetracycline ARGs and specific bacterial taxa, including some common human pathogens. In sum, doxy-PEP use over a 6-month period was associated with an increase in the proportion of tetracycline ARGs comprising the gut resistome and an increase in the expression of tetracycline ARGs. At 6 months of doxy-PEP use, no residual differences were observed in alpha and beta diversity or taxonomic composition of the gut microbiome. As doxy-PEP is implemented as a public health strategy, further studies and population-level surveillance of doxycycline-resistant pathogens are needed to understand the implications of these findings. ClinicalTrials.gov registration number: NCT03980223 . In participants from a randomized controlled trial, doxy-PEP use over 6 months minimally affected the gut microbiome’s taxonomic composition but increased the abundance and active expression of tetracycline antibiotic resistance genes.

Purpose Clinical sequencing emerging in health care may result in secondary findings (SFs). Methods Seventy-four of 6240 (1.2%) participants who underwent genome or exome sequencing through the Clinical Sequencing Exploratory Research (CSER) Consortium received one or more SFs from the original American College of Medical Genetics and Genomics (ACMG) recommended 56 gene–condition pair list; we assessed clinical and psychosocial actions. Results The overall adjusted prevalence of SFs in the ACMG 56 genes across the CSER consortium was 1.7%. Initially 32% of the family histories were positive, and post disclosure, this increased to 48%. The average cost of follow-up medical actions per finding up to a 1-year period was $128 (observed, range: $0–$678) and $421 (recommended, range: $141–$1114). Case reports revealed variability in the frequency of and follow-up on medical recommendations patients received associated with each SF gene–condition pair. Participants did not report adverse psychosocial impact associated with receiving SFs; this was corroborated by 18 participant (or parent) interviews. All interviewed participants shared findings with relatives and reported that relatives did not pursue additional testing or care. Conclusion Our results suggest that disclosure of SFs shows little to no adverse impact on participants and adds only modestly to near-term health-care costs; additional studies are needed to confirm these findings.

Both vaccination and seasonal chemoprevention are effective interventions against malaria, but both morbidity and mortality due to malaria remain high, particularly in sub-Saharan Africa. A recent clinical trial showed that the combination of these interventions provided substantial protection against malaria over a five-year period. We performed amplicon-based genotyping of highly polymorphic parasite antigens within 1530 samples from this clinical trial. We evaluated the complexity of infection within these samples, finding that participants who received both interventions had less complex infections (fewer genetically distinct parasite strains) than participants who received either intervention given alone. We also evaluated the prevalence of parasites matching the vaccine construct CSP, at the level of haplotypes, epitopes, and individual amino acid changes. We found no significant differences in the prevalence of vaccine-matching alleles between vaccinated and unvaccinated groups. For context, this study genotyped about half as many non-vaccinated participants as the original phase 3 trial for RTS,S. While a comparable number of vaccinated individuals were genotyped in both studies, the infections in this study had less complex infections, leading to an overall lower number of parasite strains to analyze. These results suggest that the combination of vaccination with seasonal chemoprevention provides protection from infection, in addition to clinical disease, and they highlight the importance of continued molecular surveillance as malaria interventions are deployed.

We examined 3,046,538 acute respiratory infection (ARI) encounters with 6,103 national telehealth physicians from January 2019 to October 2021. The antibiotic prescribing rates were 44% for all ARIs; 46% were antibiotic appropriate; 65% were potentially appropriate; 19% resulted from inappropriate diagnoses; and 10% were related to coronavirus disease 2019 (COVID-19) diagnosis.

Background This is a post-hoc secondary analysis of a randomized controlled trial whose purpose was to analyze the effect of a supervised-combined aerobic and resistance exercise (concurrent exercise) training program during pregnancy on 1-month infant neuromotor skills based on maternal pre-pregnancy body mass index (BMI). Methods Ninety-four pregnant women participated in this study, which was conducted at East Carolina University (Greenville, North Carolina, USA) between 2015 and 2018, and were allocated into concurrent exercise ( n  = 42) or stretching and breathing group ( n  = 52). The exercise group followed a 50-min 3 days/week moderate intensity concurrent exercise training program from the 16th gestational week until birth. Infant neuromotor skills were assessed with the Peabody Developmental Motor Scales, 2nd edition (PDMS-2). Results Per protocol analyses showed that infants from the exercise group and whose mothers were normal-weight, had higher PDMS-2 Stationary, Locomotion (borderline) and Gross Motor Quotient (GMQ) percentiles than infants of stretching and breathing participants ( p  = 0.022, ηp2 = 0.20; p  = 0.054, ηp2 = 0.15; p  = 0.022, ηp2 = 0.20, respectively). No differences between groups were found in infants whose mothers were normal-weight in reflexes percentiles in the adjusted model, or those infants whose mothers were overweight/obese in any of the outcomes (all p  ≥ 0.05). Conclusions Concurrent exercise training during pregnancy improves neuromotor skills in infants at 1 month of age in normal-weight women. Further studies are needed to understand the influence of concurrent exercise training during pregnancy on infants whose mothers are overweight or obese, and the potential mechanism behind the role of maternal BMI in the development of infant neuromotor skills. Clinical Trial Registration ClinicalTrials.gov Identifier: NCT03838146. Key Points Infants from normal-weight (but not overweight/obese) women who performed concurrent exercise during pregnancy presented better neuromotor skills than infants of stretching and breathing participants. The results of this study encourage the implementation of strategies to keep pregnant women active throughout pregnancy, to improve the neuromotor skills of future generations. Further studies are needed to understand the influence of concurrent exercise training during pregnancy on infants whose mothers are overweight or obese, which provides future lines of research necessary for society.

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