Teaching students to R3eason, not merely to solve problem sets: The role of philosophy and visual data communication in accessible data science education.

Much guidance on statistical training in STEM fields has been focused largely on the undergraduate cohort, with graduate education often being absent from the equation. Training in quantitative methods and reasoning is critical for graduate students in biomedical and science programs to foster reproducible and responsible research practices. We argue that graduate student education should more center around fundamental reasoning and integration skills rather than mainly on listing 1 statistical test method after the other without conveying the bigger context picture or critical argumentation skills that will enable student to improve research integrity through rigorous practice. Herein, we describe the approach we take in a quantitative reasoning course in the R3 program at the Johns Hopkins Bloomberg School of Public Health, with an error-focused lens, based on visualization and communication competencies. Specifically, we take this perspective stemming from the discussed causes of irreproducibility and apply it specifically to the many aspects of good statistical practice in science, ranging from experimental design to data collection and analysis, and conclusions drawn from the data. We also provide tips and guidelines for the implementation and adaptation of our course material to various graduate biomedical and STEM science programs.

Decreased prevalence of the Plasmodium falciparum Pfcrt K76T and Pfmdr1 and N86Y mutations post-chloroquine treatment withdrawal in Katete District, Eastern Zambia.

BACKGROUND: In 2002, Zambia withdrew chloroquine as first-line treatment for Plasmodium falciparum malaria due to increased treatment failure and worldwide spread of chloroquine resistance. The artemisinin combination regimen, artemether-lumefantrine, replaced chloroquine (CQ) as first choice malaria treatment. The present study determined the prevalence of CQ resistance molecular markers in the Pfcrt and Pfmdr1 genes in Eastern Zambia at 9 and 13 years after the removal of drug pressure. METHODS: Samples collected from Katete District during the drug therapeutic efficacy assessments conducted in 2012 and 2016 were assayed by polymerase chain reaction (PCR) and restriction fragment length polymorphisms (RFLP) to determine the prevalence of genetic mutations, K76T on the Pfcrt gene and N86Y on the Pfmdr1 gene. A total of 204 P. falciparum-positive DBS samples collected at these two time points were further analysed. RESULTS: Among the samples analysed for Pfcrt K76T and Pfmdr1 N86Y in the present study, 112 (82.4%) P. falciparum-infected samples collected in 2012 were successfully amplified for Pfcrt and 94 (69.1%) for Pfmdr1, while 69 (65.7%) and 72 (68.6%) samples from 2016 were successfully amplified for Pfcrt and Pfmdr1, respectively. In 2012, the prevalence of Pfcrt 76K (sensitive) was 97.3%, 76T (resistant) was 1.8%, and 0.8% had both 76K and 76T codons (mixed). Similarly in 2012, the prevalence of Pfmdr1 86N (sensitive) was 97.9% and 86Y (resistant) was 2.1%. In the 2016 samples, the prevalence of the respective samples was 100% Pfcrt 76K and Pfmdr1 86N. CONCLUSION: This study shows that there was a complete recovery of chloroquine-sensitive parasites by 2016 in Katete District, Eastern Zambia, 13 years following the withdrawal of CQ in the country. These findings add to the body of evidence for a fitness cost in CQ-resistant P. falciparum in Zambia and elsewhere. Further studies are recommended to monitor resistance countrywide and explore the feasibility of integration of the former best anti-malarial in combination therapy in the future.

Performance evaluation of RDT, light microscopy, and PET-PCR for detecting Plasmodium falciparum malaria infections in the 2018 Zambia National Malaria Indicator Survey.

BACKGROUND: Zambia continues to advance on the path to elimination with significant reductions in malaria morbidity and mortality. Crucial components that have contributed to progress thus far and are necessary for achieving the national malaria elimination goals include properly identifying and treating all malaria cases through accurate diagnosis. This study sought to compare and assess the diagnostic performance of Rapid Diagnostic Tests (RDT) and Light Microscopy (LM) with photo-induced electron transfer polymerase chain reaction (PET-PCR) as the gold standard using 2018 Malaria Indicator Survey (MIS) data across Zambia to better understand diagnostic accuracy metrics and how these vary across a transmission gradient. METHODS: Cross-sectional samples collected in a nationally representative survey from 7 provinces in Zambia were tested for the presence of malaria parasites by light microscopy (LM), rapid diagnostic test (RDT) and the gold standard PET-PCR. Diagnostic performance was assessed including sensitivity, specificity, negative- and positive-predictive values across a wide malaria transmission spectrum. Diagnostic accuracy metrics were measured, and statistically significant differences were calculated between test methods for different outcome variables. RESULTS: From the individuals included in the MIS, the overall prevalence of Plasmodium falciparum malaria was 32.9% by RDT, 19.4% by LM, and 23.2% by PET-PCR. Herein, RDT and LM diagnostic performance was compared against gold standard PET-PCR with LM displaying a higher diagnostic accuracy than RDTs (91.3% vs. 84.6% respectively) across the transmission spectrum in Zambia. However, the performance of both diagnostics was significantly reduced in low parasitaemia samples. Consistent with previous studies, RDT diagnostic accuracy was predominantly affected by a high rate of false positives. CONCLUSIONS: RDTs and LM both perform well across a range of transmission intensities within their respective target applications, i.e., in the community, for the former, where ease of use and speed of result is critical, and at the health facility, for the latter, where accuracy is prioritized. However, the performance of both diagnostic methods is adversely affected by low parasitaemia infections. As Zambia moves towards elimination more sensitive tools may be required to identify the last cases.

Reflections: enhancing critical thinking in science education by implementing philosophy elements into training.

In this work, I describe the trajectory of philosophy and science as separate disciplines from their early days as quite overlapping fields to their clear divergence in the latest centuries. From personal experience, I discuss the benefits of exposure to philosophy and closely related courses in undergraduate studies and bring to the forefront the positive aspects of integrating philosophy of science courses in graduate science, technology, engineering, and mathematics (STEM) curriculum. I also briefly offer some additional steps institutions can take to foster unity between areas of science and philosophy by incorporating interdisciplinary activities.

Investigating the rise of Omicron variant through genomic surveillance of SARS-CoV-2 infections in a highly vaccinated university population.

Novel variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continue to emerge as the coronavirus disease 2019 (COVID-19) pandemic extends into its fourth year. Understanding SARS-CoV-2 circulation in university populations is vital for effective interventions in higher education settings and will inform public health policy during pandemics. In this study, we generated 793 whole-genome sequences collected over an entire academic year in a university population in Indiana, USA. We clearly captured the rapidity with which Delta variant was wholly replaced by Omicron variant across the West Lafayette campus over the length of two academic semesters in a community with high vaccination rates. This mirrored the emergence of Omicron throughout the state of Indiana and the USA. Further, phylogenetic analyses demonstrated that there was a more diverse set of potential geographic origins for Omicron viruses introduction into campus when compared to Delta. Lastly, statistics indicated that there was a more significant role for international and out-of-state migration in the establishment of Omicron variants at Purdue. This surveillance workflow, coupled with viral genomic sequencing and phylogeographic analyses, provided critical insights into SARS-CoV-2 transmission dynamics and variant arrival.

Diversity and selection analyses identify transmission-blocking antigens as the optimal vaccine candidates in Plasmodium falciparum.

Background: A highly effective vaccine for malaria remains an elusive target, at least in part due to the under-appreciated natural parasite variation. This study aimed to investigate genetic and structural variation, and immune selection of leading malaria vaccine candidates across the Plasmodium falciparum's life cycle. Methods: We analyzed 325 P. falciparum whole genome sequences from Zambia, in addition to 791 genomes from five other African countries available in the MalariaGEN Pf3k Rdatabase. Ten vaccine antigens spanning three life-history stages were examined for genetic and structural variations, using population genetics measures, haplotype network analysis, and 3D structure selection analysis. Findings: Among the ten antigens analyzed, only three in the transmission-blocking vaccine category display P. falciparum 3D7 as the dominant haplotype. The antigens AMA1, CSP, MSP119 and CelTOS, are much more diverse than the other antigens, and their epitope regions are under moderate to strong balancing selection. In contrast, Rh5, a blood stage antigen, displays low diversity yet slightly stronger immune selection in the merozoite-blocking epitope region. Except for CelTOS, the transmission-blocking antigens Pfs25, Pfs48/45, Pfs230, Pfs47, and Pfs28 exhibit minimal diversity and no immune selection in epitopes that induce strain-transcending antibodies, suggesting potential effectiveness of 3D7-based vaccines in blocking transmission. Interpretations: These findings offer valuable insights into the selection of optimal vaccine candidates against P. falciparum. Based on our results, we recommend prioritizing conserved merozoite antigens and transmission-blocking antigens. Combining these antigens in multi-stage approaches may be particularly promising for malaria vaccine development initiatives. Funding: Purdue Department of Biological Sciences; Puskas Memorial Fellowship; National Institute of Allergy and Infectious Diseases (U19AI089680).

Genomics reveals heterogeneous Plasmodium falciparum transmission and population differentiation in Zambia and bordering countries.

Genomic surveillance plays a critical role in monitoring malaria transmission and understanding how the parasite adapts in response to interventions. We conducted genomic surveillance of malaria by sequencing 241 Plasmodium falciparum genomes from regions with varying levels of malaria transmission across Zambia. We found genomic evidence of high levels of within-host polygenomic infections, regardless of epidemiological characteristics, underscoring the extensive and ongoing endemic malaria transmission in the country. We identified country-level clustering of parasites from Zambia and neighboring countries, and distinct clustering of parasites from West Africa. Within Zambia, our identity by descent (IBD) relatedness analysis uncovered spatial clustering of closely related parasite pairs at the local level and rare cases of long-distance sharing. Genomic regions with large shared IBD segments and strong positive selection signatures identified genes involved in sulfadoxine-pyrimethamine and artemisinin combination therapies drug resistance, but no signature related to chloroquine resistance. Together, our findings enhance our understanding of P. falciparum transmission nationwide in Zambia and highlight the urgency of strengthening malaria control programs and surveillance of antimalarial drug resistance.

Genomics reveals heterogeneous Plasmodium falciparum transmission and selection signals in Zambia.

BACKGROUND: Genomic surveillance is crucial for monitoring malaria transmission and understanding parasite adaptation to interventions. Zambia lacks prior nationwide efforts in malaria genomic surveillance among African countries. METHODS: We conducted genomic surveillance of Plasmodium falciparum parasites from the 2018 Malaria Indicator Survey in Zambia, a nationally representative household survey of children under five years of age. We whole-genome sequenced and analyzed 241 P. falciparum genomes from regions with varying levels of malaria transmission across Zambia and estimated genetic metrics that are informative about transmission intensity, genetic relatedness between parasites, and selection. RESULTS: We provide genomic evidence of widespread within-host polygenomic infections, regardless of epidemiological characteristics, underscoring the extensive and ongoing endemic malaria transmission in Zambia. Our analysis reveals country-level clustering of parasites from Zambia and neighboring regions, with distinct separation in West Africa. Within Zambia, identity by descent (IBD) relatedness analysis uncovers local spatial clustering and rare cases of long-distance sharing of closely related parasite pairs. Genomic regions with large shared IBD segments and strong positive selection signatures implicate genes involved in sulfadoxine-pyrimethamine and artemisinin combination therapies drug resistance, but no signature related to chloroquine resistance. Furthermore, differences in selection signatures, including drug resistance loci, are observed between eastern and western Zambian parasite populations, suggesting variable transmission intensity and ongoing drug pressure. CONCLUSIONS: Our findings enhance our understanding of nationwide P. falciparum transmission in Zambia, establishing a baseline for analyzing parasite genetic metrics as they vary over time and space. These insights highlight the urgency of strengthening malaria control programs and surveillance of antimalarial drug resistance.

Malaria is caused by a parasite that is spread to humans via mosquito bites. It is a leading cause of death in children under five years old in sub-Saharan Africa. Analysis of the malaria parasite's complete set of DNA (its genome) can help us to understand transmission of the disease and how this changes in response to different strategies to control the disease. We analyzed the genomes of malaria parasites from children across Zambia. Our study revealed that 77% of children harbored multiple parasite strains, which suggests that local transmission (transmission between people within the same local area) is high. Genetic evidence for long-distance transmission was rarer. Furthermore, our findings suggest parasites are evolving in response to antimalarial drugs. Our study enhances our understanding of malaria dynamics in Zambia and may help to inform strategies for improved surveillance and control.

Improving research integrity: a framework for responsible science communication.

Research integrity, an essential precept of scientific inquiry and discovery, comprises norms such as Rigor, Reproducibility, and Responsibility (the 3R's). Over the past decades, numerous issues have arisen that challenge the reliability of scientific studies, including irreproducibility crises, lack of good scientific principles, and erroneous communications, which have impacted the public's trust in science and its findings. Here, we highlight one important component of research integrity that is often overlooked in the discussion of proposals for improving research quality and promoting robust research; one that spans from the lab bench to the dissemination of scientific work: responsible science communication. We briefly outline the role of education and institutions of higher education in teaching the tenets of good scientific practice and within that, the importance of adequate communications training. In that context, we present our framework of responsible science communication that we live by and teach to our students in courses and workshops that are part of the Johns Hopkins Bloomberg School of Public Health R3 Center for Innovation in Science Education.

Genomic Surveillance of SARS-CoV-2 in a University Community: Insights Into Tracking Variants, Transmission, and Spread of Gamma (P.1) Variant.

Background: Using a combination of data from routine surveillance, genomic sequencing, and phylogeographic analysis, we tracked the spread and introduction events of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants focusing on a large university community. Methods: Here, we sequenced and analyzed 677 high-quality SARS-CoV-2 genomes from positive RNA samples collected from Purdue University students, faculty, and staff who tested positive for the virus between January 2021 and May 2021, comprising an average of 32% of weekly cases across the time frame. Results: Our analysis of circulating SARS-CoV-2 variants over time revealed periods when variants of concern (VOC) Alpha (B.1.1.7) and Iota (B.1.526) reached rapid dominance and documented that VOC Gamma (P.1) was increasing in frequency as campus surveillance was ending. Phylodynamic analysis of Gamma genomes from campus alongside a subsampling of >20 000 previously published P.1 genomes revealed 10 independent introductions of this variant into the Purdue community, predominantly from elsewhere in the United States, with introductions from within the state of Indiana and from Illinois, and possibly Washington and New York, suggesting a degree of domestic spread. Conclusions: We conclude that a robust and sustained active and passive surveillance program coupled with genomic sequencing during a pandemic offers important insights into the dynamics of pathogen arrival and spread in a campus community and can help guide mitigation measures.

Phylogenetic Complexity of Morphologically Identified Anopheles squamosus in Southern Zambia.

Despite dramatic reductions in malaria cases in the catchment area of Macha Hospital, Choma District, Southern Province in Zambia, prevalence has remained near 1-2% by RDT for the past several years. To investigate residual malaria transmission in the area, this study focuses on the relative abundance, foraging behavior, and phylogenetic relationships of Anopheles squamosus specimens. In 2011, higher than expected rates of anthropophily were observed among "zoophilic" An. squamosus, a species that had sporadically been found to contain Plasmodium falciparum sporozoites. The importance of An. squamosus in the region was reaffirmed in 2016 when P. falciparum sporozoites were detected in numerous An. squamosus specimens. This study analyzed Centers for Disease Control (CDC) light trap collections of adult mosquitoes from two collection schemes: one performed as part of a reactive-test-and-treat program and the second performed along a geographical transect. Morphological identification, molecular verification of anopheline species, and blood meal source were determined on individual samples. Data from these collections supported earlier studies demonstrating An. squamosus to be primarily exophagic and zoophilic, allowing them to evade current control measures. The phylogenetic relationships generated from the specimens in this study illustrate the existence of well supported clade structure among An. squamosus specimens, which further emphasizes the importance of molecular identification of vectors. The primarily exophagic behavior of An. squamosus in these collections also highlights that indoor vector control strategies will not be sufficient for elimination of malaria in southern Zambia.

Multiple Novel Clades of Anopheline Mosquitoes Caught Outdoors in Northern Zambia.

Residual vector populations that do not come in contact with the most frequently utilized indoor-directed interventions present major challenges to global malaria eradication. Many of these residual populations are mosquito species about which little is known. As part of a study to assess the threat of outdoor exposure to malaria mosquitoes within the Southern and Central Africa International Centers of Excellence for Malaria Research, foraging female anophelines were collected outside households in Nchelenge District, northern Zambia. These anophelines proved to be more diverse than had previously been reported in the area. In order to further characterize the anopheline species, sequencing and phylogenetic approaches were utilized. Anopheline mosquitoes were collected from outdoor light traps, morphologically identified, and sent to Johns Hopkins Bloomberg School of Public Health for sequencing. Sanger sequencing from 115 field-derived samples yielded mitochondrial COI sequences, which were aligned with a homologous 488 bp gene segment from known anophelines (n = 140) retrieved from NCBI. Nuclear ITS2 sequences (n = 57) for at least one individual from each unique COI clade were generated and compared against NCBI's nucleotide BLAST database to provide additional evidence for taxonomical identity and structure. Molecular and morphological data were combined for assignment of species or higher taxonomy. Twelve phylogenetic groups were characterized from the COI and ITS2 sequence data, including the primary vector species Anopheles funestus s.s. and An. gambiae s.s. An unexpectedly large proportion of the field collections were identified as An. coustani and An. sp. 6. Six phylogenetic groups remain unidentified to species-level. Outdoor collections of anopheline mosquitoes in areas frequented by people in Nchelenge, northern Zambia, proved to be extremely diverse. Morphological misidentification and underrepresentation of some anopheline species in sequence databases confound efforts to confirm identity of potential malaria vector species. The large number of unidentified anophelines could compromise the malaria vector surveillance and malaria control efforts not only in northern Zambia but other places where surveillance and control are focused on indoor-foraging and resting anophelines. Therefore, it is critical to continue development of methodologies that allow better identification of these populations and revisiting and cleaning current genomic databases.

Genetic Diversity of Anopheles coustani (Diptera: Culicidae) in Malaria Transmission Foci in Southern and Central Africa.

Despite ongoing malaria control efforts implemented throughout sub-Saharan Africa, malaria remains an enormous public health concern. Current interventions such as indoor residual spraying with insecticides and use of insecticide-treated bed nets are aimed at targeting the key malaria vectors that are primarily endophagic and endophilic. Anopheles coustani s.l., an understudied vector of malaria, is a species previously thought to exhibit mostly zoophilic behavior. Like many of these understudied species, An. coustani has greater anthropophilic tendencies than previously appreciated, is often both endophagic and exophagic, and carries Plasmodium falciparum sporozoites. The aim of this study was to explore genetic variation of An. coustani mosquitoes and the potential of this species to contribute to malaria parasite transmission in high transmission settings in Zambia and the Democratic Republic of the Congo (DRC). Morphologically identified An. coustani specimens that were trapped outdoors in these study sites were analyzed by PCR and sequencing for species identification and bloodmeal sources, and malaria parasite infection was determined by ELISA and qPCR. Fifty An. coustani s.s. specimens were confirmed by analysis of mitochondrial DNA cytochrome c oxidase subunit I (COI) and ribosomal internal transcribed spacer region 2 (ITS2). Maximum likelihood phylogenetic analysis of COI and ITS2 sequences revealed two distinct phylogenetic groups within this relatively small regional collection. Our findings indicate that both An. coustani groups have anthropophilic and exophagic habits and come into frequent contact with P. falciparum, suggesting that this potential alternative malaria vector might elude current vector control measures in northern Zambia and southern DRC.