Portable and cost-effective genetic detection and characterization of Plasmodium falciparum hrp2 using the MinION sequencer.

The prevalence of Plasmodium falciparum hrp2 (pfhrp2)-deleted parasites threatens the efficacy of the most used and sensitive malaria rapid diagnostic tests and highlights the need for continued surveillance for this gene deletion. While PCR methods are adequate for determining pfhrp2 presence or absence, they offer a limited view of its genetic diversity. Here, we present a portable sequencing method using the MinION. Pfhrp2 amplicons were generated from individual samples, barcoded, and pooled for sequencing. To overcome potential crosstalk between barcodes, we implemented a coverage-based threshold for pfhrp2 deletion confirmation. Amino acid repeat types were then counted and visualized with custom Python scripts following de novo assembly. We evaluated this assay using well-characterized reference strains and 152 field isolates with and without pfhrp2 deletions, of which 38 were also sequenced on the PacBio platform to provide a standard for comparison. Of 152 field samples, 93 surpassed the positivity threshold, and of those samples, 62/93 had a dominant pfhrp2 repeat type. PacBio-sequenced samples with a dominant repeat-type profile from the MinION sequencing data matched the PacBio profile. This field-deployable assay can be used alone for surveilling pfhrp2 diversity or as a sequencing-based addition to the World Health Organization's existing deletion surveillance protocol.

The Anglo-Saxon migration and the formation of the early English gene pool.

The history of the British Isles and Ireland is characterized by multiple periods of major cultural change, including the influential transformation after the end of Roman rule, which precipitated shifts in language, settlement patterns and material culture1. The extent to which migration from continental Europe mediated these transitions is a matter of long-standing debate2-4. Here we study genome-wide ancient DNA from 460 medieval northwestern Europeans-including 278 individuals from England-alongside archaeological data, to infer contemporary population dynamics. We identify a substantial increase of continental northern European ancestry in early medieval England, which is closely related to the early medieval and present-day inhabitants of Germany and Denmark, implying large-scale substantial migration across the North Sea into Britain during the Early Middle Ages. As a result, the individuals who we analysed from eastern England derived up to 76% of their ancestry from the continental North Sea zone, albeit with substantial regional variation and heterogeneity within sites. We show that women with immigrant ancestry were more often furnished with grave goods than women with local ancestry, whereas men with weapons were as likely not to be of immigrant ancestry. A comparison with present-day Britain indicates that subsequent demographic events reduced the fraction of continental northern European ancestry while introducing further ancestry components into the English gene pool, including substantial southwestern European ancestry most closely related to that seen in Iron Age France5,6.

The impact of frequently neglected model violations on bacterial recombination rate estimation: a case study in Mycobacterium canettii and Mycobacterium tuberculosis.

Mycobacterium canettii is a causative agent of tuberculosis in humans, along with the members of the Mycobacterium tuberculosis complex. Frequently used as an outgroup to the M. tuberculosis complex in phylogenetic analyses, M. canettii is thought to offer the best proxy for the progenitor species that gave rise to the complex. Here, we leverage whole-genome sequencing data and biologically relevant population genomic models to compare the evolutionary dynamics driving variation in the recombining M. canettii with that in the nonrecombining M. tuberculosis complex, and discuss differences in observed genomic diversity in the light of expected levels of Hill-Robertson interference. In doing so, we highlight the methodological challenges of estimating recombination rates through traditional population genetic approaches using sequences called from populations of microorganisms and evaluate the likely mis-inference that arises owing to a neglect of common model violations including purifying selection, background selection, progeny skew, and population size change. In addition, we compare performance when full within-host polymorphism data are utilized, versus the more common approach of basing analyses on within-host consensus sequences.

Design and Implementation of a COVID-19 Case Investigation Program: An Academic-Public Health Partnership, Arizona, 2020.

From May through July 2020, Arizona was a global hotspot for new COVID-19 cases. In response to the surge of cases, local public health departments looked for innovative ways to form external partnerships to address their staffing needs. In collaboration with the Maricopa County Department of Public Health, the Arizona State University Student Outbreak Response Team (SORT) created and implemented a virtual call center to conduct public health case investigations for COVID-19. SORT officially launched a dedicated COVID-19 case investigation program after 3 weeks of program design and training. From June 29 through November 8, 2020, SORT recruited and trained 218 case investigators, completed 5000 case patient interviews, and closed 10 000 cases. Our team also developed process improvements to address disparities in case investigation timeliness. A strong infrastructure designed to accommodate remote case investigations, paired with a large workforce, enabled SORT to provide additional surge capacity for the county's high volume of cases. University-driven multidisciplinary case investigator teams working in partnership with state, tribal, and local public health staff members can be an effective tool for supporting a diverse and growing public health workforce. We discuss the essential design factors involved in building a university program to complement local COVID-19 response efforts, including workflows for case management, volunteer case investigator recruitment and training, secure technology platforms for conducting case investigations remotely, and robust data-tracking procedures for maintaining quality control and timely case reporting.

Accuracy of Case-Based Seroprevalence of SARS-CoV-2 Antibodies in Maricopa County, Arizona.

We conducted a community seroprevalence survey in Arizona, from September 12 to October 1, 2020, to determine the presence of antibodies to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We used the seroprevalence estimate to predict SARS-CoV-2 infections in the jurisdiction by applying the adjusted seroprevalence to the county's population. The estimated community seroprevalence of SARS-CoV-2 infections was 4.3 times greater (95% confidence interval = 2.2, 7.5) than the number of reported cases. Field surveys with representative sampling provide data that may help fill in gaps in traditional public health reporting. (Am J Public Health. 2022;112(1):38-42. https://doi.org/10.2105/AJPH.2021.306568).

Ten millennia of hepatitis B virus evolution.

Hepatitis B virus (HBV) has been infecting humans for millennia and remains a global health problem, but its past diversity and dispersal routes are largely unknown. We generated HBV genomic data from 137 Eurasians and Native Americans dated between ~10,500 and ~400 years ago. We date the most recent common ancestor of all HBV lineages to between ~20,000 and 12,000 years ago, with the virus present in European and South American hunter-gatherers during the early Holocene. After the European Neolithic transition, Mesolithic HBV strains were replaced by a lineage likely disseminated by early farmers that prevailed throughout western Eurasia for ~4000 years, declining around the end of the 2nd millennium BCE. The only remnant of this prehistoric HBV diversity is the rare genotype G, which appears to have reemerged during the HIV pandemic.

The population genomics of within-host Mycobacterium tuberculosis.

Recent progress in genomic sequencing from patient samples has allowed for the first detailed insight into the within-host genetic diversity of Mycobacterium tuberculosis (M.TB), revealing remarkably low levels of variation. While this has often been attributed to low mutation rates, other factors have been described, including resistance evolution (i.e., selective sweeps), widespread purifying and background selection, and, more recently, progeny skew. Here we review recent findings pertaining to the processes governing the evolutionary dynamics of M.TB, discuss their implications for improving our understanding of this important human pathogen, and make recommendations for future work. Significantly, this emerging evolutionary framework involving the joint estimation of demographic, selective, and reproductive processes is forming a new paradigm for the study of within-host pathogen evolution that will be widely applicable across organisms.

Estimating molecular preservation of the intestinal microbiome via metagenomic analyses of latrine sediments from two medieval cities.

Ancient latrine sediments, which contain the concentrated collective biological waste of past whole human communities, have the potential to be excellent proxies for human gastrointestinal health on the population level. A rich body of literature explores their use to detect the presence of gut-associated eukaryotic parasites through microscopy, immunoassays and genetics. Despite this interest, a lack of studies have explored the whole genetic content of ancient latrine sediments through consideration not only of gut-associated parasites, but also of core community gut microbiome signals that remain from the group that used the latrine. Here, we present a metagenomic analysis of bulk sediment from medieval latrines in Riga (Latvia) and Jerusalem. Our analyses reveal survival of microbial DNA representative of intestinal flora as well as numerous parasites. These data are compared against parasite taxon identifications obtained via microscopy and ELISA techniques. Together, these findings provide a first glimpse into the rich prokaryotic and eukaryotic intestinal flora of pre-industrial agricultural populations, which may give a better context for interpreting the health of modern microbiomes. This article is part of the theme issue 'Insights into health and disease from ancient biomolecules'.

A seventeenth-century Mycobacterium tuberculosis genome supports a Neolithic emergence of the Mycobacterium tuberculosis complex.

BACKGROUND: Although tuberculosis accounts for the highest mortality from a bacterial infection on a global scale, questions persist regarding its origin. One hypothesis based on modern Mycobacterium tuberculosis complex (MTBC) genomes suggests their most recent common ancestor followed human migrations out of Africa approximately 70,000 years before present. However, studies using ancient genomes as calibration points have yielded much younger dates of less than 6000 years. Here, we aim to address this discrepancy through the analysis of the highest-coverage and highest-quality ancient MTBC genome available to date, reconstructed from a calcified lung nodule of Bishop Peder Winstrup of Lund (b. 1605-d. 1679). RESULTS: A metagenomic approach for taxonomic classification of whole DNA content permitted the identification of abundant DNA belonging to the human host and the MTBC, with few non-TB bacterial taxa comprising the background. Genomic enrichment enabled the reconstruction of a 141-fold coverage M. tuberculosis genome. In utilizing this high-quality, high-coverage seventeenth-century genome as a calibration point for dating the MTBC, we employed multiple Bayesian tree models, including birth-death models, which allowed us to model pathogen population dynamics and data sampling strategies more realistically than those based on the coalescent. CONCLUSIONS: The results of our metagenomic analysis demonstrate the unique preservation environment calcified nodules provide for DNA. Importantly, we estimate a most recent common ancestor date for the MTBC of between 2190 and 4501 before present and for Lineage 4 of between 929 and 2084 before present using multiple models, confirming a Neolithic emergence for the MTBC.

A treponemal genome from an historic plague victim supports a recent emergence of yaws and its presence in 15th century Europe.

Developments in techniques for identification of pathogen DNA in archaeological samples can expand our resolution of disease detection. Our application of a non-targeted molecular screening tool for the parallel detection of pathogens in historical plague victims from post-medieval Lithuania revealed the presence of more than one active disease in one individual. In addition to Yersinia pestis, we detected and genomically characterized a septic infection of Treponema pallidum pertenue, a subtype of the treponemal disease family recognised as the cause of the tropical disease yaws. Our finding in northern Europe of a disease that is currently restricted to equatorial regions is interpreted within an historical framework of intercontinental trade and potential disease movements. Through this we offer an alternative hypothesis for the history and evolution of the treponemal diseases, and posit that yaws be considered an important contributor to the sudden epidemic of late 15th century Europe that is widely ascribed to syphilis.

Emergence of human-adapted Salmonella enterica is linked to the Neolithization process.

It has been hypothesized that the Neolithic transition towards an agricultural and pastoralist economy facilitated the emergence of human-adapted pathogens. Here, we recovered eight Salmonella enterica subsp. enterica genomes from human skeletons of transitional foragers, pastoralists and agropastoralists in western Eurasia that were up to 6,500 yr old. Despite the high genetic diversity of S. enterica, all ancient bacterial genomes clustered in a single previously uncharacterized branch that contains S. enterica adapted to multiple mammalian species. All ancient bacterial genomes from prehistoric (agro-)pastoralists fall within a part of this branch that also includes the human-specific S. enterica Paratyphi C, illustrating the evolution of a human pathogen over a period of 5,000 yr. Bacterial genomic comparisons suggest that the earlier ancient strains were not host specific, differed in pathogenic potential and experienced convergent pseudogenization that accompanied their downstream host adaptation. These observations support the concept that the emergence of human-adapted S. enterica is linked to human cultural transformations.

Paleomicrobiology: Diagnosis and Evolution of Ancient Pathogens.

The last century has witnessed progress in the study of ancient infectious disease from purely medical descriptions of past ailments to dynamic interpretations of past population health that draw upon multiple perspectives. The recent adoption of high-throughput DNA sequencing has led to an expanded understanding of pathogen presence, evolution, and ecology across the globe. This genomic revolution has led to the identification of disease-causing microbes in both expected and unexpected contexts, while also providing for the genomic characterization of ancient pathogens previously believed to be unattainable by available methods. In this review we explore the development of DNA-based ancient pathogen research, the specialized methods and tools that have emerged to authenticate and explore infectious disease of the past, and the unique challenges that persist in molecular paleopathology. We offer guidelines to mitigate the impact of these challenges, which will allow for more reliable interpretations of data in this rapidly evolving field of investigation.

Differential preservation of endogenous human and microbial DNA in dental calculus and dentin.

Dental calculus (calcified dental plaque) is prevalent in archaeological skeletal collections and is a rich source of oral microbiome and host-derived ancient biomolecules. Recently, it has been proposed that dental calculus may provide a more robust environment for DNA preservation than other skeletal remains, but this has not been systematically tested. In this study, shotgun-sequenced data from paired dental calculus and dentin samples from 48 globally distributed individuals are compared using a metagenomic approach. Overall, we find DNA from dental calculus is consistently more abundant and less contaminated than DNA from dentin. The majority of DNA in dental calculus is microbial and originates from the oral microbiome; however, a small but consistent proportion of DNA (mean 0.08 ± 0.08%, range 0.007-0.47%) derives from the host genome. Host DNA content within dentin is variable (mean 13.70 ± 18.62%, range 0.003-70.14%), and for a subset of dentin samples (15.21%), oral bacteria contribute > 20% of total DNA. Human DNA in dental calculus is highly fragmented, and is consistently shorter than both microbial DNA in dental calculus and human DNA in paired dentin samples. Finally, we find that microbial DNA fragmentation patterns are associated with guanine-cytosine (GC) content, but not aspects of cellular structure.

Salmonella enterica genomes from victims of a major sixteenth-century epidemic in Mexico.

Indigenous populations of the Americas experienced high mortality rates during the early contact period as a result of infectious diseases, many of which were introduced by Europeans. Most of the pathogenic agents that caused these outbreaks remain unknown. Through the introduction of a new metagenomic analysis tool called MALT, applied here to search for traces of ancient pathogen DNA, we were able to identify Salmonella enterica in individuals buried in an early contact era epidemic cemetery at Teposcolula-Yucundaa, Oaxaca in southern Mexico. This cemetery is linked, based on historical and archaeological evidence, to the 1545-1550 CE epidemic that affected large parts of Mexico. Locally, this epidemic was known as 'cocoliztli', the pathogenic cause of which has been debated for more than a century. Here, we present genome-wide data from ten individuals for Salmonella enterica subsp. enterica serovar Paratyphi C, a bacterial cause of enteric fever. We propose that S. Paratyphi C be considered a strong candidate for the epidemic population decline during the 1545 cocoliztli outbreak at Teposcolula-Yucundaa.