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Background: Mixed infection with multiple strains of the same pathogen in a single host can present clinical and analytical challenges. Whole genome sequence (WGS) data can identify signals of multiple strains in samples, though the precision of previous methods can be improved. Here, we present MixInfect2, a new tool to accurately detect mixed samples from Mycobacterium tuberculosis short-read WGS data. We then evaluate three approaches for reconstructing the underlying mixed constituent strain sequences. This allows these samples to be included in downstream analysis to gain insights into the epidemiology and transmission of mixed infections., Methods: We employed a Gaussian mixture model to cluster allele frequencies at mixed sites (hSNPs) in each sample to identify signals of multiple strains. Building upon our previous tool, MixInfect, we increased the accuracy of classifying in vitro mixed samples through multiple improvements to the bioinformatic pipeline. Major and minor proportion constituent strains were reconstructed using three approaches and assessed by comparing the estimated sequence to the known constituent strain sequence. Lastly, mixed infections in a real-world Mycobacterium tuberculosis population from Moldova were detected with MixInfect2 and clusters of recent transmission that included major and minor constituent strains were built., Results: All 36/36 in vitro mixed and 12/12 non-mixed samples were correctly classified with MixInfect2, and major strain proportions were estimated with high accuracy (within 3% of the true strain proportion), outperforming previous tools. Reconstructed major strain sequences closely matched the true constituent sequence by taking the allele at the highest frequency at hSNPs, while the best-performing approach to reconstruct the minor proportion strain sequence was identifying the closest non-mixed isolate in the same population, though no approach was effective when the minor strain proportion was at 5%. Finally, fewer mixed infections were identified in Moldova than previous estimates (6.6% vs 17.4%) and we found multiple instances where the constituent strains of mixed samples were present in transmission clusters., Conclusions: MixInfect2 accurately detects samples with evidence of mixed infection from short-read WGS data and provides an excellent estimate of the mixture proportions. While there are limitations in reconstructing the constituent strain sequences of mixed samples, we present recommendations for the best approach to include these isolates in further analyses., Competing Interests: Declarations. Ethics approval and consent to participate: This study was approved by the Ethics Committee of Research of the Phthisiopneumology Institute in Moldova and the Yale University Human Investigation Committee (no. 2000023071). Consent for publication: Not applicable. Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

Background: Mycobacterium tuberculosis complex (MTBC) species evolve slowly, so isolates from individuals linked in transmission often have identical or nearly identical genomes, making it difficult to reconstruct transmission chains. Finding additional sources of shared MTBC variation could help overcome this problem. Previous studies have reported MTBC diversity within infected individuals; however, whether within-host variation improves transmission inferences remains unclear. Here, we aimed to quantify within-host MTBC variation and assess whether such information improves transmission inferences., Methods: We conducted a retrospective genomic epidemiology study in which we reanalysed publicly available sequence data from household transmission studies published in PubMed from database inception until Jan 31, 2024, for which both genomic and epidemiological contact data were available, using household membership as a proxy for transmission linkage. We quantified minority variants (ie, positions with two or more alleles each supported by at least five-fold coverage and with a minor allele frequency of 1% or more) outside of PE and PPE genes, within individual samples and shared across samples. We used receiver operator characteristic (ROC) curves to compare the performance of a general linear model for household membership that included shared minority variants and one that included only fixed genetic differences., Findings: We identified three MTBC household transmission studies with publicly available whole-genome sequencing data and epidemiological linkages: a household transmission study in Vitória, Brazil (Colangeli et al), a retrospective population-based study of paediatric tuberculosis in British Columbia, Canada (Guthrie et al), and a retrospective population-based study in Oxfordshire, England (Walker et al). We found moderate levels of minority variation present in MTBC sequence data from cultured isolates that varied significantly across studies: mean 168·6 minority variants (95% CI 151·4-185·9) for the Colangeli et al dataset, 5·8 (1·5-10·2) for Guthrie et al (p<0·0001, Wilcoxon rank sum test, vs Colangeli et al), and 7·1 (2·4-11·9) for Walker et al (p<0·0001, Wilcoxon rank sum test, vs Colangeli et al). Isolates from household pairs shared more minority variants than did randomly selected pairs of isolates: mean 97·7 shared minority variants (79·1-116·3) versus 9·8 (8·6-11·0) in Colangeli et al, 0·8 (0·1-1·5) versus 0·2 (0·1-0·2) in Guthrie et al, and 0·7 (0·1-1·3) versus 0·2 (0·2-0·2) in Walker et al (all p<0·0001, Wilcoxon rank sum test). Shared within-host variation was significantly associated with household membership (odds ratio 1·51 [95% CI 1·30-1·71], p<0·0001), for one standard deviation increase in shared minority variants. Models that included shared within-host variation versus models without within-host variation improved the accuracy of predicting household membership in all three studies: area under the ROC curve 0·95 versus 0·92 for the Colangeli et al study, 0·99 versus 0·95 for the Guthrie et al study, and 0·93 versus 0·91 for the Walker et al study., Interpretation: Within-host MTBC variation persists through culture of sputum and could enhance the resolution of transmission inferences. The substantial differences in minority variation recovered across studies highlight the need to optimise approaches to recover and incorporate within-host variation into automated phylogenetic and transmission inference., Funding: National Institutes of Health., Competing Interests: Declaration of interests JRA reports support from the US National Institutes of Health (NIH); receiving donated Cepheid disposable open cartridges from Cepheid to Stanford University for NIH-funded tuberculosis research; participation in two Data Safety Monitoring Boards for NIH-funded tuberculosis trials; and participation in the Science Advisory Board for an NIH-funded tuberculosis network. JC reports grants from the NIH, Valneva/Butantan Institution, Coalition for Epidemic Preparedness Innovations–Sabin Institute, MSD, and Sanofi Pasteur for clinical and epidemiological studies; honoraria from Pfizer; participation in the Brazil Advisory Board for the mRNA-1273 vaccine Moderna–Zodiac, the Latin America and Brazilian Advisory Board for paxlovid (nirmatrelvir–ritonavir; Pfizer), the Brazil Advisory Board for the Qdenga vaccine (Takeda), and the Takeda Global Dengue Steering Committee. All other authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Despite the increasing burden of dengue in Kenya and Africa, the introduction and expansion of the virus in the region remain poorly understood. The objective of this study is to examine the genetic diversity and evolutionary histories of dengue virus (DENV) serotypes 1 and 3 in Kenya and contextualize their circulation within circulation dynamics in the broader African region. Viral RNA was extracted from samples collected from a cohort of febrile patients recruited at clinical sites in Kenya from 2013 to 2022. Samples were tested by polymerase chain reaction (PCR) for DENV presence. Five DENV-positive samples were serotyped, and complete viral genomes for phylogenetic inference were obtained via sequencing on Illumina platforms. Sequences generated in our study were combined with global datasets of sequences, and Bayesian and maximum likelihood methods were used to infer phylogenetic trees and geographic patterns of spread with a focus on Kenya and Africa as a whole. Four new DENV-1 and one new DENV-3 genomes were successfully sequenced and combined with 328 DENV-1 and 395 DENV-3 genomes from elsewhere for phylogenetic analyses. The DENV-1 sequences from our study formed a monophyletic cluster with an inferred common ancestor in 2019 (most recent common ancestor 2019 and 95% high posterior density 2018-19), which was closely related to sequences from Tanzania. The single DENV-3 sequence clustered with sequences from Tanzania and Kenya, was collected between 2017 and 2019 and was related to recent outbreaks in the region. Phylogenetic trees resolved multiple clades of DENV-1 and DENV-3 concurrently circulating in Africa, introduced in the early-to mid-2000s. Three DENV-1 and four DENV-3 clades are highlighted, introduced between 2000 and 2015. Phylogeographic models suggest frequent, independent importations of DENV lineages into Kenya and Africa from East and South-East Asia via distinct geographic pathways. DENV-1 and DENV-3 evolutionary dynamics in Africa are characterized by the cocirculation of multiple recently introduced lineages. Circulating lineages are introduced via distinct geographic pathways that may be centered around regional nexus locations. Increased surveillance is required to identify key regional locations that drive spread, and dengue interventions should focus on interrupting spread at these locations., Competing Interests: None declared., (© The Author(s) 2025. Published by Oxford University Press.)

Despite the increasing burden of dengue, the regional emergence of the virus in Kenya has not been examined. This study investigates the genetic structure and regional spread of dengue virus-2 in Kenya. Viral RNA from acutely ill patients in Kenya was enriched and sequenced. Six new dengue-2 genomes were combined with 349 publicly available genomes and phylogenies used to infer gene flow between Kenya and other countries. Analyses indicate two dengue-2 Cosmopolitan genotype lineages circulating in Kenya, linked to recent outbreaks in coastal Kenya and Burkina Faso. Lineages circulating in Western, Southern, and Eastern Africa exhibiting similar evolutionary features are also reported. Phylogeography suggests importation of dengue-2 into Kenya from East and Southeast Asia and bidirectional geneflow. Additional lineages circulating in Africa are also imported from East and Southeast Asia. These findings underscore how intermittent importations from East and Southeast Asia drive dengue-2 circulation in Kenya and Africa more broadly., (© 2024. The Author(s).)

Importance: The COVID-19 pandemic has led to 775 million documented cases and over 7 million deaths worldwide as of March 2024 and is an ongoing health crisis. To limit viral spread within households and in the community, public health officials have recommended self-isolation, self-quarantine of exposed household contacts, and mask use. Yet, risk of household transmission (HHT) may be underestimated due to low frequency of sampling, and risk factors for HHT are not well understood., Objectives: To estimate the secondary attack rate of SARS-CoV-2 within households and to define the risk factors for new infections in household members who are in close contact with the index case., Design, Setting, and Participants: In this prospective cohort study, from March 2020-December 2021 we enrolled 60 households with index cases who tested positive for SARS-CoV-2. All household contacts and index cases were tested daily for SARS-CoV-2 via reverse transcription polymerase chain reaction (RT-PCR) using self-collected anterior nares specimens. Households were followed until all study participants in the household tested negative for SARS-CoV-2 for seven consecutive days. We collected sex, age, race/ethnicity, comorbidities, and relationship to index case for secondary contacts, household level characteristics including primary income, household density, and square feet per person on property. We compared the sociodemographic variables between COVID-19 positive and negative household members and between households where secondary transmission did and did not occur., Main Outcomes and Measures: Daily anterior nares swabs were tested for SARS-CoV-2 using RT-PCR, in order to assess duration of nasal shedding of SARS-CoV-2, as well as risk of transmission to secondary household contacts., Results: Of the 163 participants in this study, 84 (51.5%) were women; median age (IQR) was 36.0 (17.0-54.0) years of age; 78 (47.8%) were white and 48 (29.5%) were Hispanic/LatinX. Of the fifty households with household contacts, at least one secondary case occurred in twenty-six households (52.0%) and forty-five household contacts (43.7%) were infected. Secondary attack rate was lowest among children of index cases (6/23, 26.1%). Modified Poisson regression identified that the risk of transmission to household contacts increases significantly with age (Risk ratio for each increase in years of age = 1.01, 95% CI = 1.00-1.02). Mixed effects regression models identified that participants with chronic diseases, such as asthma, diabetes, cancer, or cardiac disease, had higher Cts at baseline when compared to participants without chronic diseases (6.62, 95% CI: 1.46-11.77, p = 0.02) and show a slower rate of increase in Ct over time (-0.43, 95% CI: -0.77 to -0.09, p = 0.02)., Conclusions and Relevance: This study suggests that HHT represents a key source of community-based infection of SARS-CoV-2. Allocation of resources for contact investigations and prevention interventions should focus on the individuals at highest risk of infection in households, especially those with higher density homes., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Altamirano et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

While incidence of tuberculosis (TB) has decreased globally, in Paraguay, considered a medium-incidence country by the WHO, TB incidence has increased slightly from 42 per 100,000 in 2010 to 46 per 100,000 in 2022. We conducted a retrospective study of TB cases notified to the Paraguay National Program for Tuberculosis Control (NPTC) from 2018 to 2022 and quantified trends in specific populations identified as vulnerable. Of the 13,725 TB cases notified in Paraguay from 2018 to 2022, 2,331 (17%) occurred among incarcerated individuals and 1,743 (12.7%) occurred among self-identified Indigenous individuals. In 2022, the relative risk of TB was 87 and 6.4 among the incarcerated and Indigenous populations, compared with the non-incarcerated and non-Indigenous populations respectively. We found significant heterogeneity in TB incidence across Paraguay's 17 departments. Our findings highlight the urgency of expanding access to TB diagnosis, treatment, and prevention in populations at heightened risk of TB in Paraguay.

Background: The increased risk of tuberculosis (TB) among people deprived of liberty (PDL) is due to individual and institution-level factors. We followed a cohort of PDL from 5 prisons in Paraguay to describe the risk of TB during incarceration and after they were released., Methods: We linked a 2013 national census of prisons with TB records from the TB Program from 2010 to 2021 to identify TB notifications among incarcerated and formerly incarcerated individuals. We used multivariable Cox regression models to quantify the risk of TB during and following incarceration and to identify risk factors associated with TB., Findings: Among 2996 individuals incarcerated, 451 (15.1%) were diagnosed with TB. Of these, 262 (58.1%) cases occurred during incarceration and 189 (41.9%) occurred in the community after release. In prison, the hazard ratio of developing TB was 1.97 (95% CI: 1.52-2.61) after six months of incarceration and increased to 2.78 (95% CI: 1.82-4.24) after 36 months compared with the first six months. The overall TB notification rate was 2940 per 100,000 person-years. This rate increased with the duration of incarceration from 1335 per 100,000 person-years in the first year to 8455 per 100,000 person-years after 8 years. Among former prisoners, the rate of TB decreased from 1717 in the first year after release to 593 per 100 000 person-years after 8 years of follow up., Interpretation: Our study shows the alarming risk of TB associated with prison environments in Paraguay, and how this risk persists for years following incarceration. Effective TB control measures to protect the health of people during and following incarceration are urgently needed., Funding: Paraguay National Commission of Science and Technology grant CONACYT PIN 15-705 (GS, GES, SA)., Competing Interests: All authors report no potential conflicts., (© 2023 Published by Elsevier Ltd.)

Background: Because M. tuberculosis evolves slowly, transmission clusters often contain multiple individuals with identical consensus genomes, making it difficult to reconstruct transmission chains. Finding additional sources of shared M. tuberculosis variation could help overcome this problem. Previous studies have reported M. tuberculosis diversity within infected individuals; however, whether within-host variation improves transmission inferences remains unclear., Methods: To evaluate the transmission information present in within-host M. tuberculosis variation, we re-analyzed publicly available sequence data from three household transmission studies, using household membership as a proxy for transmission linkage between donor-recipient pairs., Findings: We found moderate levels of minority variation present in M. tuberculosis sequence data from cultured isolates that varied significantly across studies (mean: 6, 7, and 170 minority variants above a 1% minor allele frequency threshold, outside of PE/PPE genes). Isolates from household members shared more minority variants than did isolates from unlinked individuals in the three studies (mean 98 shared minority variants vs. 10; 0.8 vs. 0.2, and 0.7 vs. 0.2, respectively). Shared within-host variation was significantly associated with household membership (OR: 1.51 [1.30,1.71], for one standard deviation increase in shared minority variants). Models that included shared within-host variation improved the accuracy of predicting household membership in all three studies as compared to models without within-host variation (AUC: 0.95 versus 0.92, 0.99 versus 0.95, and 0.93 versus 0.91)., Interpretation: Within-host M. tuberculosis variation persists through culture and could enhance the resolution of transmission inferences. The substantial differences in minority variation recovered across studies highlights the need to optimize approaches to recover and incorporate within-host variation into automated phylogenetic and transmission inference., Funding: NIAID: 5K01AI173385., Competing Interests: Declaration of interest The authors report no conflict of interest.

There is an increasing burden of circulating vaccine-derived polioviruses (cVDPVs) due to the continued use of oral poliovirus vaccine (OPV). However, the informativeness of routine OPV VP1 sequencing for the early identification of viruses carrying virulence-associated reversion mutations has not been directly evaluated in a controlled setting. We prospectively collected 15,331 stool samples to track OPV shedding from children receiving OPV and their contacts for ten weeks following an immunization campaign in Veracruz State, Mexico and sequenced VP1 genes from 358 samples. We found that OPV was genetically unstable and evolves at an approximately clocklike rate that varies across serotypes and by vaccination status. Overall, 61% (11/18) of OPV-1, 71% (34/48) OPV-2, and 96% (54/56) OPV-3 samples with available data had evidence of a reversion at the key 5' UTR attenuating position and 28% (13/47) of OPV-1, 12% (14/117) OPV-2, and 91% (157/173) OPV-3 of Sabin-like viruses had ≥1 known reversion mutations in the VP1 gene. Our results are consistent with previous work documenting rapid reversion to virulence of OPV and underscores the need for intensive surveillance following OPV use., (© 2023. Springer Nature Limited.)

There is an increasing burden of circulating vaccine-derived polioviruses (cVDPVs) due to the continued use of oral poliovirus vaccine (OPV). However, the informativeness of routine OPV VP1 sequencing for the early identification of viruses carrying virulence-associated reversion mutations has not been directly evaluated in a controlled setting. We prospectively collected 15,331 stool samples to track OPV shedding from vaccinated children and their contacts for ten weeks following an immunization campaign in Veracruz State, Mexico and sequenced VP1 genes from 358 samples. We found that OPV was genetically unstable and evolves at an approximately clocklike rate that varies across serotypes and by vaccination status. Alarmingly, 28% (13/47) of OPV-1, 12% (14/117) OPV-2, and 91% (157/173) OPV-3 of Sabin-like viruses had ≥1 known reversion mutation. Our results suggest that current definitions of cVDPVs may exclude circulating virulent viruses that pose a public health risk and underscore the need for intensive surveillance following OPV use.

Recent rises in incident tuberculosis (TB) cases in Paraguay and the increasing concentration of TB within prisons highlight the urgency of targeting strategies to interrupt transmission and prevent new infections. However, whether specific cities or carceral institutions play a disproportionate role in transmission remains unknown. We conducted prospective genomic surveillance, sequencing 471 Mycobacterium tuberculosis complex genomes, from inside and outside prisons in Paraguay's two largest urban areas, Asunción and Ciudad del Este, from 2016 to 2021. We found genomic evidence of frequent recent transmission within prisons and transmission linkages spanning prisons and surrounding populations. We identified a signal of frequent M. tuberculosis spread between urban areas and marked recent population size expansion of the three largest genomic transmission clusters. Together, our findings highlight the urgency of strengthening TB control programs to reduce transmission risk within prisons in Paraguay, where incidence was 70 times that outside prisons in 2021., (© 2023. The Author(s).)

Background: The limited variation observed among severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) consensus sequences makes it difficult to reconstruct transmission linkages in outbreak settings. Previous studies have recovered variation within individual SARS-CoV-2 infections but have not yet measured the informativeness of within-host variation for transmission inference., Methods: We performed tiled amplicon sequencing on 307 SARS-CoV-2 samples, including 130 samples from 32 individuals in 14 households and 47 longitudinally sampled individuals, from 4 prospective studies with household membership data, a proxy for transmission linkage., Results: Consensus sequences from households had limited diversity (mean pairwise distance, 3.06 single-nucleotide polymorphisms [SNPs]; range, 0-40). Most (83.1%, 255 of 307) samples harbored at least 1 intrahost single-nucleotide variant ([iSNV] median, 117; interquartile range [IQR], 17-208), above a minor allele frequency threshold of 0.2%. Pairs in the same household shared significantly more iSNVs (mean, 1.20 iSNVs; 95% confidence interval [CI], 1.02-1.39) than did pairs in different households infected with the same viral clade (mean, 0.31 iSNVs; 95% CI, .28-.34), a signal that decreases with increasingly stringent minor allele frequency thresholds. The number of shared iSNVs was significantly associated with an increased odds of household membership (adjusted odds ratio, 1.35; 95% CI, 1.23-1.49). However, the poor concordance of iSNVs detected across sequencing replicates (24.8% and 35.0% above a 0.2% and 1% threshold) confirms technical concerns that current sequencing and bioinformatic workflows do not consistently recover low-frequency within-host variants., Conclusions: Shared within-host variation may augment the information in consensus sequences for predicting transmission linkages. Improving sensitivity and specificity of within-host variant identification will improve the informativeness of within-host variation., (© The Author(s) 2023. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

Background: Favipiravir, an oral, RNA-dependent RNA polymerase inhibitor, has in vitro activity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Despite limited data, favipiravir is administered to patients with coronavirus disease 2019 (COVID-19) in several countries., Methods: We conducted a phase 2, double-blind, randomized controlled outpatient trial of favipiravir in asymptomatic or mildly symptomatic adults with a positive SARS-CoV-2 reverse-transcription polymerase chain reaction assay (RT-PCR) within 72 hours of enrollment. Participants were randomized to receive placebo or favipiravir (1800 mg twice daily [BID] day 1, 800 mg BID days 2-10). The primary outcome was SARS-CoV-2 shedding cessation in a modified intention-to-treat (mITT) cohort of participants with positive enrollment RT-PCRs. Using SARS-CoV-2 amplicon-based sequencing, we assessed favipiravir's impact on mutagenesis., Results: We randomized 149 participants with 116 included in the mITT cohort. The participants' mean age was 43 years (standard deviation, 12.5 years) and 57 (49%) were women. We found no difference in time to shedding cessation overall (hazard ratio [HR], 0.76 favoring placebo [95% confidence interval {CI}, .48-1.20]) or in subgroups (age, sex, high-risk comorbidities, seropositivity, or symptom duration at enrollment). We detected no difference in time to symptom resolution (initial: HR, 0.84 [95% CI, .54-1.29]; sustained: HR, 0.87 [95% CI, .52-1.45]) and no difference in transition mutation accumulation in the viral genome during treatment., Conclusions: Our data do not support favipiravir at commonly used doses in outpatients with uncomplicated COVID-19. Further research is needed to ascertain if higher favipiravir doses are effective and safe for patients with COVID-19., Clinical Trials Registration: NCT04346628., Competing Interests: Potential conflicts of interest. M. H. reports payment or honoraria for lectures, presentations, speaker’s bureaus, manuscript writing, or educational events from UpToDate. A. S. reports grants from Gilead Sciences, Regeneron Pharma, and Janssen Pharma. H. H. reports salary support from anonymous donors to Stanford University and grant support from the NIH (UL1 TR003142). C. E. reports financial support for the present manuscript from Fujifilm Pharmaceuticals USA (payments made to author’s consulting LLC). P. J. reports research support from anonymous donors to Stanford University to support clinical work. R. H. K. is currently the Chief Medical Officer for AiPharma Global Holdings LLC; is an unpaid consultant to Fujifilm Toyama Chemical Co Ltd and the Anti Viral Drug Development Alliance; and reports support for attending conferences and stock options from AiPharma Global Holdings LLC. J. P. reports grants from Heluna Health (seroepidemiological studies of SARS-CoV-2), Gauss Surgical (testing of an antigen test for SARS-CoV-2) and Ono Pharmaceuticals (clinical trial of Camostat for SARS-CoV-2). J. R. A. reports research support from anonymous donors to Stanford University. C. K. reports licenses from Clear Creek Bio and a patent assigned to Stanford University (“Use of a dihydroorotate dehydrogenase (DHODH) inhibitor in combination with an inhibitor of pyrimidine salvage,” US patent number 10,736,911; 2020). Y. M. reports grants from the NIH (U54 MD010724, U54 MD010724-05S1, R21AI148810, P30AG059307, 000522211-022), Pfizer (C3671008, C4591007), the Bill & Melinda Gates Foundation (OPP1113682), and the Chan Zuckerberg Foundation (12089sc); has received payment or honoraria for lectures, presentations, speaker’s bureaus, manuscript writing, or educational events from the American Academy of Pediatrics for the National Conference; and is on a Pfizer DSMB (non–COVID-19 vaccine trial). All other authors report no potential conflicts of interest. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Infectious Diseases Society of America.)

Tuberculosis (TB) remains a leading cause of infectious mortality globally, yet most cases cannot be epidemiologically linked even with extensive contact investigations and whole genome sequencing. Consequently, there remain major gaps in our understanding of where and when M. tuberculosis (Mtb) exposures occur. We aimed to investigate whether Mtb can be detected in environments where TB patients were recently present, which could serve as a tool for characterizing exposure risk. We collected 389 environment surface (ES) swabs from two high TB burden prisons in Brazil, sampling 41 (n = 340) cells occupied by individuals with active TB and 7 (n = 49) cells from individuals without TB. In a subset of pooled swabs (n = 6) and a swab from a cigarette lighter from the cell with active TB patients, we enriched Mtb DNA using RNA-bait hybrid capture assays and performed whole genome sequencing. In prison cells, Mtb DNA was detected in 55/340 (16 %) of ES swabs from cells occupied by active TB patients and none (0/49) from cells in which no active TB patients were present. Mtb was detected in 13/16 (81 %) prison cells occupied by the individuals with high/medium sputum Xpert Mtb load and 8/25 (32 %) with low/very low sputum Mtb load (p = 0.003). Seven hybrid capture samples had a median genomic coverage of 140×. rpoB mutations conferring high-level rifampin resistance were detected in 3/7 ES swabs. Mtb was frequently detectable in environments recently occupied by individuals with active TB. This approach could be applied in congregate environments to identify and characterize high-risk settings for Mtb exposure., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Jason R. Andrews reports financial support was provided by National Institutes of Health., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Chronic lung allograft dysfunction (CLAD) remains the primary cause of death in lung transplant recipients (LTRs) despite improvements in immunosuppression management. Despite advances in knowledge regarding the pathogenesis of CLAD, treatments that are currently available are usuallyineffective and delay progression of disease at best.There are currently no evidence-based guidelines and minimal publications regarding the optimal treatment ofCLAD., Objective: To complete a comprehensive review of the literature for the prevention and medical management of CLAD., Methods: We identified the major domains of the medical management of CLAD and conducted a comprehensive search of PubMed and Embase databases to identify articles published from inception to December 2021 related to CLAD in LTRs. Studies published in English pertaining to the pharmacologic prevention and treatment of CLAD were included; highest priority was given to prospective, randomized, controlled trials if available. Prospective observational and retrospective controlled trials were prioritized next, followed by retrospective uncontrolled studies, case series, and finally case reports if the information was deemed to be pertinent. Reference lists of qualified publications were also reviewed to find any other publications of interest that were not found on initial search.In the absence of literature published in the aforementioned databases, additional articles were identified by reviewing abstracts presented at the International Society for Heart and Lung Transplantation and American Transplant Congress annual meetings between 2010-2021., Conclusion: CLAD should be identified as early as possible along with prompt intervention to optimize the possibility of stabilizing or improving lung function. More robust clinical data is needed to validate the use of all currently available and investigational treatment options for CLAD to identify the optimal pharmacotherapy management for this patient population., (© 2022 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background: Globally, prisons are high-incidence settings for tuberculosis. Yet the role of prisons as reservoirs of M. tuberculosis , propagating epidemics through spillover to surrounding communities, has been difficult to measure directly., Methods: To quantify the role of prisons in driving wider community M. tuberculosis transmission, we conducted prospective genomic surveillance in Central West Brazil from 2014 to 2019. We whole genome sequenced 1152 M. tuberculosis isolates collected during active and passive surveillance inside and outside prisons and linked genomes to detailed incarceration histories. We applied multiple phylogenetic and genomic clustering approaches and inferred timed transmission trees., Findings: M. tuberculosis sequences from incarcerated and non-incarcerated people were closely related in a maximum likelihood phylogeny. The majority (70.8%; 46/65) of genomic clusters including people with no incarceration history also included individuals with a recent history of incarceration. Among cases in individuals with no incarceration history, 50.6% (162/320) were in clusters that included individuals with recent incarceration history, suggesting that transmission chains often span prisons and communities. We identified a minimum of 18 highly probable spillover events, M. tuberculosis transmission from people with a recent incarceration history to people with no prior history of incarceration, occurring in the state's four largest cities and across sampling years. We additionally found that frequent transfers of people between the state's prisons creates a highly connected prison network that likely disseminates M. tuberculosis across the state., Interpretation: We developed a framework for measuring spillover from high-incidence environments to surrounding communities by integrating genomic and spatial information. Our findings indicate that, in this setting, prisons serve not only as disease reservoirs, but also disseminate M. tuberculosis across highly connected prison networks, both amplifying and propagating M. tuberculosis risk in surrounding communities., Funding: Brazil's National Council for Scientific and Technological Development and US National Institutes of Health., Competing Interests: Declaration of interests The authors declare no conflicts of interest.

BACKGROUND: TB notifications in Latin American prisons have more than doubled over the past two decades; however, treatment outcomes and their determinants among incarcerated individuals in this region are not well understood. METHODS: Newly diagnosed drug-susceptible TB cases reported to Brazil´s Information System for Notifiable Diseases ( Sistema de Informação de Agravos de Notificação , SINAN) between January 2015 and December 2017 were included. Multivariate logistic regression was used to assess socio-economic and clinical factors associated with treatment success among incarcerated individuals. RESULTS: Incarcerated individuals ( n = 17,776) had greater treatment success than non-incarcerated individuals ( n = 160,728; 82.2% vs. 75.1%; P < 0.0001), including after adjusting for demographic and clinical risk factors (adjusted odds ratio aOR 1.27, 95% CI 1.19-1.34). These differences were partially mediated by increased use of directly observed therapy among incarcerated individuals (DOT) (61% vs. 47%; P < 0.001), which was associated with greater efficacy in the incarcerated population (aOR 2.56 vs. aOR 2.17; P < 0.001). DOT was associated with improved treatment success among incarcerated subpopulations at elevated risk of poor outcomes. CONCLUSION: TB treatment success among incarcerated individuals in Brazil is higher than non-incarcerated individuals, but both fall below WHO targets. Expanding the use of DOT and services for socially and medically vulnerable individuals may improve outcomes in carceral settings.

We developed a simple, noninvasive mask sampling method to quantify and sequence severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from exhaled breath. We found substantial variation between individuals in SARS-CoV-2 copies exhaled over a 15-minute period, which moderately correlated with nasal swab viral load. Talking was associated with a median of 2 log 10 greater exhaled viral copies. Exposure varies substantially between individuals but may be risk stratified by nasal swab viral load and whether the exposure involved conversation., (© The Author(s) 2021. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

Background: Mortality during and after incarceration is poorly understood in low- and middle-income countries (LMICs). The need to address this knowledge gap is especially urgent in South America, which has the fastest growing prison population in the world. In Brazil, insufficient data have precluded our understanding of all-cause and cause-specific mortality during and after incarceration., Methods and Findings: We linked incarceration and mortality databases for the Brazilian state of Mato Grosso do Sul to obtain a retrospective cohort of 114,751 individuals with recent incarceration. Between January 1, 2009 and December 31, 2018, we identified 3,127 deaths of individuals with recent incarceration (705 in detention and 2,422 following release). We analyzed age-standardized, all-cause, and cause-specific mortality rates among individuals detained in different facility types and following release, compared to non-incarcerated residents. We additionally modeled mortality rates over time during and after incarceration for all causes of death, violence, or suicide. Deaths in custody were 2.2 times the number reported by the national prison administration (n = 317). Incarcerated men and boys experienced elevated mortality, compared with the non-incarcerated population, due to increased risk of death from violence, suicide, and communicable diseases, with the highest standardized incidence rate ratio (IRR) in semi-open prisons (2.4; 95% confidence interval [CI]: 2.0 to 2.8), police stations (3.1; 95% CI: 2.5 to 3.9), and youth detention (8.1; 95% CI: 5.9 to 10.8). Incarcerated women experienced increased mortality from suicide (IRR = 6.0, 95% CI: 1.2 to 17.7) and communicable diseases (IRR = 2.5, 95% CI: 1.1 to 5.0). Following release from prison, mortality was markedly elevated for men (IRR = 3.0; 95% CI: 2.8 to 3.1) and women (IRR = 2.4; 95% CI: 2.1 to 2.9). The risk of violent death and suicide was highest immediately post-release and declined over time; however, all-cause mortality remained elevated 8 years post-release. The limitations of this study include inability to establish causality, uncertain reliability of data during incarceration, and underestimation of mortality rates due to imperfect database linkage., Conclusions: Incarcerated individuals in Brazil experienced increased mortality from violence, suicide, and communicable diseases. Mortality was heightened following release for all leading causes of death, with particularly high risk of early violent death and elevated all-cause mortality up to 8 years post-release. These disparities may have been underrecognized in Brazil due to underreporting and insufficient data., Competing Interests: The authors have declared that no competing interests exist.

We use mathematical modelling to examine how microbial strain communities are structured by the host specialisation traits and antigenic relationships of their members. The model is quite general and broadly applicable, but we focus on Borrelia burgdorferi, the Lyme disease bacterium, transmitted by ticks to mice and birds. In this system, host specialisation driven by the evasion of innate immunity has been linked to multiple niche polymorphism, while antigenic differentiation driven by the evasion of adaptive immunity has been linked to negative frequency dependence. Our model is composed of two host species, one vector, and multiple co-circulating pathogen strains that vary in their host specificity and their antigenic distances from one another. We explore the conditions required to maintain pathogen diversity. We show that the combination of host specificity and antigenic differentiation creates an intricate niche structure. Unequivocal rules that relate the stability of a strain community directly to the trait composition of its members are elusive. However, broad patterns are evident. When antigenic differentiation is weak, stable communities are typically composed entirely of generalists that can exploit either host species equally well. As antigenic differentiation increases, more diverse stable communities emerge, typically around trait compositions of generalists, generalists and very similar specialists, and specialists roughly balanced between the two host species.

Background: Prisons are recognised as high-risk environments for tuberculosis, but there has been little systematic investigation of the global and regional incidence and prevalence of tuberculosis, and its determinants, in prisons. We did a systematic review and meta-analysis to assess the incidence and prevalence of tuberculosis in incarcerated populations by geographical region., Methods: In this systematic review and meta-analysis, we searched MEDLINE, Embase, Web of Knowledge, and the LILACS electronic database from Jan 1, 1980, to Nov 15, 2020, for cross-sectional and cohort studies reporting the incidence of Mycobacterium tuberculosis infection, incidence of tuberculosis, or prevalence of tuberculosis among incarcerated individuals in all geographical regions. We extracted data from individual studies, and calculated pooled estimates of incidence and prevalence through hierarchical Bayesian meta-regression modelling. We also did subgroup analyses by region. Incidence rate ratios between prisons and the general population were calculated by dividing the incidence of tuberculosis in prisons by WHO estimates of the national population-level incidence., Findings: We identified 159 relevant studies; 11 investigated the incidence of M tuberculosis infection (n=16 318), 51 investigated the incidence of tuberculosis (n=1 858 323), and 106 investigated the prevalence of tuberculosis (n=6 727 513) in incarcerated populations. The overall pooled incidence of M tuberculosis infection among prisoners was 15·0 (95% credible interval [CrI] 3·8-41·6) per 100 person-years. The incidence of tuberculosis (per 100 000 person-years) among prisoners was highest in studies from the WHO African (2190 [95% CrI 810-4840] cases) and South-East Asia (1550 [240-5300] cases) regions and in South America (970 [460-1860] cases), and lowest in North America (30 [20-50] cases) and the WHO Eastern Mediterranean region (270 [50-880] cases). The prevalence of tuberculosis was greater than 1000 per 100 000 prisoners in all global regions except for North America and the Western Pacific, and highest in the WHO South-East Asia region (1810 [95% CrI 670-4000] cases per 100 000 prisoners). The incidence rate ratio between prisons and the general population was much higher in South America (26·9; 95% CrI 17·1-40·1) than in other regions, but was nevertheless higher than ten in the WHO African (12·6; 6·2-22·3), Eastern Mediterranean (15·6; 6·5-32·5), and South-East Asia (11·7; 4·1-27·1) regions., Interpretation: Globally, people in prison are at high risk of contracting M tuberculosis infection and developing tuberculosis, with consistent disparities between prisons and the general population across regions. Tuberculosis control programmes should prioritise preventive interventions among incarcerated populations., Funding: US National Institutes of Health., Competing Interests: Declaration of interests We declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY-NC-ND 4.0 license. Published by Elsevier Ltd.. All rights reserved.)

In the past decade, tuberculosis incidence has declined in much of the world, but has risen in central and South America. It is not yet clear what is driving this reversal of progress in tuberculosis control. Since 2000, the incarcerated population in central and South America has grown by 206%, the greatest increase in the world. Over the same period, notified tuberculosis cases among the incarcerated population (hereinafter termed persons deprived of their liberty [PDL], following the Inter-American Commission on Human Rights) have risen by 269%. In both central and South America, the rise of disease among PDL more than offsets tuberculosis control gains in the general population. Tuberculosis is increasingly concentrated among PDL; currently, 11% of all notified tuberculosis cases in central and South America occur among PDL who comprise less than 1% of the population. The extraordinarily high risk of acquiring tuberculosis within prisons creates a health and human rights crisis for PDL that also undermines wider tuberculosis control efforts. Controlling tuberculosis in this region will require countries to take urgent measures to prioritise the health of PDL., Competing Interests: Declaration of interests RAL-O serves as Tuberculosis Prevention, Control and Elimination Advisor at the HIV, Hepatitis, Tuberculosis and Sexually Transmitted Infections Unit of the PAHO. MAE is Director of the Department of Communicable Diseases and Environmental Determinants of Health at the PAHO. All other authors declare no competing interests., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

International migrants are at heightened risk for tuberculosis (TB) disease. Intensified incarceration at international borders may compound population-wide TB risk. However, few studies have investigated the contributions of migration, local transmission, or prisons in driving incident TB at international borders. We conducted prospective population-based genomic surveillance in 3 cities along Brazil's central western border from 2014-2017. Although most isolates (89/132; 67%) fell within genomic transmission clusters, genetically unique isolates disproportionately occurred among participants with recent international travel (17/42; 40.5%), suggesting that both local transmission and migration contribute to incident TB. Isolates from 40 participants with and 76 without an incarceration history clustered together throughout a maximum-likelihood phylogeny, indicating the close interrelatedness of prison and community epidemics. Our findings highlight the need for ongoing surveillance to control continued introductions of TB and reduce the disproportionate burden of TB in prisons at Brazil's international borders.

Pathogen genomic data are increasingly used to characterize global and local transmission patterns of important human pathogens and to inform public health interventions. Yet, there is no current consensus on how to measure genomic variation. To test the effect of the variant-identification approach on transmission inferences for Mycobacterium tuberculosis, we conducted an experiment in which five genomic epidemiology groups applied variant-identification pipelines to the same outbreak sequence data. We compared the variants identified by each group in addition to transmission and phylogenetic inferences made with each variant set. To measure the performance of commonly used variant-identification tools, we simulated an outbreak. We compared the performance of three mapping algorithms, five variant callers and two variant filters in recovering true outbreak variants. Finally, we investigated the effect of applying increasingly stringent filters on transmission inferences and phylogenies. We found that variant-calling approaches used by different groups do not recover consistent sets of variants, which can lead to conflicting transmission inferences. Further, performance in recovering true variation varied widely across approaches. While no single variant-identification approach outperforms others in both recovering true genome-wide and outbreak-level variation, variant-identification algorithms calibrated upon real sequence data or that incorporate local reassembly outperform others in recovering true pairwise differences between isolates. The choice of variant filters contributed to extensive differences across pipelines, and applying increasingly stringent filters rapidly eroded the accuracy of transmission inferences and quality of phylogenies reconstructed from outbreak variation. Commonly used approaches to identify M. tuberculosis genomic variation have variable performance, particularly when predicting potential transmission links from pairwise genetic distances. Phylogenetic reconstruction may be improved by less stringent variant filtering. Approaches that improve variant identification in repetitive, hypervariable regions, such as long-read assemblies, may improve transmission inference.

Much remains unknown about Mycobacterium tuberculosis transmission. Seminal experimental studies from the 1950s demonstrated that airborne expulsion of droplet nuclei from an infectious tuberculosis (TB) patient is the primary route of transmission. However, these findings did not rule out other routes of M. tuberculos is transmission. We reviewed historical scientific evidence from the late 19th/early 20th century and contemporary studies investigating the presence, persistence and infectiousness of environmental M. tuberculosis We found both experimental and epidemiological evidence supporting the presence and viability of M. tuberculosis in multiple natural and built environments for months to years, presumably following contamination by a human source. Furthermore, several studies confirm M. tuberculosis viability and virulence in the environment using guinea pig and mouse models. Most of this evidence was historical; however, several recent studies have reported consistent findings of M. tuberculosis detection and viability in the environment using modern methods. Whether M. tuberculosis in environments represents an infectious threat to humans requires further investigation; this may represent an untapped source of data with which to further understand M. tuberculosis transmission. We discuss potential opportunities for harnessing these data to generate new insights into TB transmission in congregate settings., Competing Interests: Conflict of interest: L. Martinez has nothing to disclose. Conflict of interest: R. Verma has nothing to disclose. Conflict of interest: J. Croda has nothing to disclose. Conflict of interest: C.R. Horsburgh Jr has nothing to disclose. Conflict of interest: K.S. Walter has nothing to disclose. Conflict of interest: N. Degner has nothing to disclose. Conflict of interest: K. Middelkoop has nothing to disclose. Conflict of interest: A. Koch has nothing to disclose. Conflict of interest: S. Hermans has nothing to disclose. Conflict of interest: D.F. Warner has nothing to disclose. Conflict of interest: R. Wood has nothing to disclose. Conflict of interest: F. Cobelens has nothing to disclose. Conflict of interest: J.R. Andrews has nothing to disclose., (Copyright ©ERS 2019.)

[This corrects the article DOI: 10.1371/journal.ppat.1007470.].

Tsetse flies (Glossina spp.) vector pathogenic trypanosomes (Trypanosoma spp.) in sub-Saharan Africa. These parasites cause human and animal African trypanosomiases, which are debilitating diseases that inflict an enormous socio-economic burden on inhabitants of endemic regions. Current disease control strategies rely primarily on treating infected animals and reducing tsetse population densities. However, relevant programs are costly, labor intensive and difficult to sustain. As such, novel strategies aimed at reducing tsetse vector competence require development. Herein we investigated whether Kosakonia cowanii Zambiae (Kco&#95;Z), which confers Anopheles gambiae with resistance to Plasmodium, is able to colonize tsetse and induce a trypanosome refractory phenotype in the fly. Kco&#95;Z established stable infections in tsetse's gut and exhibited no adverse effect on the fly's survival. Flies with established Kco&#95;Z infections in their gut were significantly more refractory to infection with two distinct trypanosome species (T. congolense, 6% infection; T. brucei, 32% infection) than were age-matched flies that did not house the exogenous bacterium (T. congolense, 36% infected; T. brucei, 70% infected). Additionally, 52% of Kco&#95;Z colonized tsetse survived infection with entomopathogenic Serratia marcescens, compared with only 9% of their wild-type counterparts. These parasite and pathogen refractory phenotypes result from the fact that Kco&#95;Z acidifies tsetse's midgut environment, which inhibits trypanosome and Serratia growth and thus infection establishment. Finally, we determined that Kco&#95;Z infection does not impact the fecundity of male or female tsetse, nor the ability of male flies to compete with their wild-type counterparts for mates. We propose that Kco&#95;Z could be used as one component of an integrated strategy aimed at reducing the ability of tsetse to transmit pathogenic trypanosomes., Competing Interests: The authors have declared that no competing interests exist.

Background: It has been hypothesized that prisons serve as amplifiers of general tuberculosis (TB) epidemics, but there is a paucity of data on this phenomenon and the potential population-level effects of prison-focused interventions. This study (1) quantifies the TB risk for prisoners as they traverse incarceration and release, (2) mathematically models the impact of prison-based interventions on TB burden in the general population, and (3) generalizes this model to a wide range of epidemiological contexts., Methods and Findings: We obtained individual-level incarceration data for all inmates (n = 42,925) and all reported TB cases (n = 5,643) in the Brazilian state of Mato Grosso do Sul from 2007 through 2013. We matched individuals between prisoner and TB databases and estimated the incidence of TB from the time of incarceration and the time of prison release using Cox proportional hazards models. We identified 130 new TB cases diagnosed during incarceration and 170 among individuals released from prison. During imprisonment, TB rates increased from 111 cases per 100,000 person-years at entry to a maximum of 1,303 per 100,000 person-years at 5.2 years. At release, TB incidence was 229 per 100,000 person-years, which declined to 42 per 100,000 person-years (the average TB incidence in Brazil) after 7 years. We used these data to populate a compartmental model of TB transmission and incarceration to evaluate the effects of various prison-based interventions on the incidence of TB among prisoners and the general population. Annual mass TB screening within Brazilian prisons would reduce TB incidence in prisons by 47.4% (95% Bayesian credible interval [BCI], 44.4%-52.5%) and in the general population by 19.4% (95% BCI 17.9%-24.2%). A generalized model demonstrates that prison-based interventions would have maximum effectiveness in reducing community incidence in populations with a high concentration of TB in prisons and greater degrees of mixing between ex-prisoners and community members. Study limitations include our focus on a single Brazilian state and our retrospective use of administrative databases., Conclusions: Our findings suggest that the prison environment, more so than the prison population itself, drives TB incidence, and targeted interventions within prisons could have a substantial effect on the broader TB epidemic., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: SB receives a stipend as a specialty consulting editor for PLOS Medicine and serves on the journal’s editorial board.

Lyme disease is the most prevalent vector-borne disease in North America and continues to spread. The disease was first clinically described in the 1970s in Lyme, Connecticut, but the origins and history of spread of the Lyme disease bacteria, Borrelia burgdorferi sensu stricto (s.s.), are unknown. To explore the evolutionary history of B. burgdorferi in North America, we collected ticks from across the USA and southern Canada from 1984 to 2013 and sequenced the, to our knowledge, largest collection of 146 B. burgdorferi s.s. genomes. Here, we show that B. burgdorferi s.s. has a complex evolutionary history with previously undocumented levels of migration. Diversity is ancient and geographically widespread, well pre-dating the Lyme disease epidemic of the past ~40 years, as well as the Last Glacial Maximum ~20,000 years ago. This means the recent emergence of human Lyme disease probably reflects ecological change-climate change and land use changes over the past century-rather than evolutionary change of the bacterium.

Climate change is likely to profoundly modulate the burden of infectious diseases. However, attributing health impacts to a changing climate requires being able to associate changes in infectious disease incidence with the potentially complex influences of climate. This aim is further complicated by nonlinear feedbacks inherent in the dynamics of many infections, driven by the processes of immunity and transmission. Here, we detail the mechanisms by which climate drivers can shape infectious disease incidence, from direct effects on vector life history to indirect effects on human susceptibility, and detail the scope of variation available with which to probe these mechanisms. We review approaches used to evaluate and quantify associations between climate and infectious disease incidence, discuss the array of data available to tackle this question, and detail remaining challenges in understanding the implications of climate change for infectious disease incidence. We point to areas where synthesis between approaches used in climate science and infectious disease biology provide potential for progress., (© 2017 The Authors.)

Background: Babesia microti is an emerging tick-borne apicomplexan parasite with increasing geographic range and incidence in the United States. The rapid expansion of B. microti into its current distribution in the northeastern USA has been due to the range expansion of the tick vector, Ixodes scapularis, upon which the causative agent is dependent for transmission to humans., Results: To reconstruct the history of B. microti in the continental USA and clarify the evolutionary origin of human strains, we used multiplexed hybrid capture of 25 B. microti isolates obtained from I. scapularis and human blood. Despite low genomic variation compared with other Apicomplexa, B. microti was strongly structured into three highly differentiated genetic clusters in the northeastern USA. Bayesian analyses of the apicoplast genomes suggest that the origin of the current diversity of B. microti in northeastern USA dates back 46 thousand years with a signature of recent population expansion in the last 1000 years. Human-derived samples belonged to two rarely intermixing clusters, raising the possibility of highly divergent infectious phenotypes in humans., Conclusions: Our results validate the multiplexed hybrid capture strategy for characterizing genome-wide diversity and relatedness of B. microti from ticks and humans. We find strong population structure in B. microti samples from the Northeast indicating potential barriers to gene flow.

Hosts including humans, other vertebrates, and arthropods, are frequently infected with heterogeneous populations of pathogens. Within-host pathogen diversity has major implications for human health, epidemiology, and pathogen evolution. However, pathogen diversity within-hosts is difficult to characterize and little is known about the levels and sources of within-host diversity maintained in natural populations of disease vectors. Here, we examine genomic variation of the Lyme disease bacteria, Borrelia burgdorferi (Bb), in 98 individual field-collected tick vectors as a model for study of within-host processes. Deep population sequencing reveals extensive and previously undocumented levels of Bb variation: the majority (~70%) of ticks harbor mixed strain infections, which we define as levels Bb diversity pre-existing in a diverse inoculum. Within-tick diversity is thus a sample of the variation present within vertebrate hosts. Within individual ticks, we detect signatures of positive selection. Genes most commonly under positive selection across ticks include those involved in dissemination in vertebrate hosts and evasion of the vertebrate immune complement. By focusing on tick-borne Bb, we show that vectors can serve as epidemiological and evolutionary sentinels: within-vector pathogen diversity can be a useful and unbiased way to survey circulating pathogen diversity and identify evolutionary processes occurring in natural transmission cycles.

Modelling the spatial spread of vector-borne zoonotic pathogens maintained in enzootic transmission cycles remains a major challenge. The best available spatio-temporal data on pathogen spread often take the form of human disease surveillance data. By applying a classic ecological approach-occupancy modelling-to an epidemiological question of disease spread, we used surveillance data to examine the latent ecological invasion of tick-borne pathogens. Over the last half-century, previously undescribed tick-borne pathogens including the agents of Lyme disease and human babesiosis have rapidly spread across the northeast United States. Despite their epidemiological importance, the mechanisms of tick-borne pathogen invasion and drivers underlying the distinct invasion trajectories of the co-vectored pathogens remain unresolved. Our approach allowed us to estimate the unobserved ecological processes underlying pathogen spread while accounting for imperfect detection of human cases. Our model predicts that tick-borne diseases spread in a diffusion-like manner with occasional long-distance dispersal and that babesiosis spread exhibits strong dependence on Lyme disease., (© 2016 The Author(s).)

Background: Rapid and accurate retrieval of whole genome sequences of human pathogens from disease vectors or animal reservoirs will enable fine-resolution studies of pathogen epidemiological and evolutionary dynamics. However, next generation sequencing technologies have not yet been fully harnessed for the study of vector-borne and zoonotic pathogens, due to the difficulty of obtaining high-quality pathogen sequence data directly from field specimens with a high ratio of host to pathogen DNA., Results: We addressed this challenge by using custom probes for multiplexed hybrid capture to enrich for and sequence 30 Borrelia burgdorferi genomes from field samples of its arthropod vector. Hybrid capture enabled sequencing of nearly the complete genome (~99.5 %) of the Borrelia burgdorferi pathogen with 132-fold coverage, and identification of up to 12,291 single nucleotide polymorphisms per genome., Conclusions: The proprosed culture-independent method enables efficient whole genome capture and sequencing of pathogens directly from arthropod vectors, thus making population genomic study of vector-borne and zoonotic infectious diseases economically feasible and scalable. Furthermore, given the similarities of invertebrate field specimens to other mixed DNA templates characterized by a high ratio of host to pathogen DNA, we discuss the potential applicabilty of hybrid capture for genomic study across diverse study systems.

Yellow fever virus is a reemerging infection responsible for widespread, sporadic outbreaks across Africa. Although Aedes aegypti (L.) is the most important vector globally, in East Africa, epidemics may be vectored by Aedes bromeliae (Theobald), a member of the Aedes simpsoni (Theobald) species complex. The Ae. simpsoni complex contains 10 subspecies, of which Ae. bromeliae alone has been incriminated as a vector of yellow fever virus. However, morphological markers cannot distinguish Ae. bromeliae from conspecifics, including the sympatric and non-anthropophilic Aedes lilii (Theobald). Here, we used three sequenced nuclear markers to examine the population structure of Ae. simpsoni complex mosquitoes collected from diverse habitats in Rabai, Kenya. Gene trees consistently show strong support for the existence of two clades in Rabai, with segregation by habitat. Domestic mosquitoes segregate separately from forest-collected mosquitoes, providing evidence of habitat partitioning on a small spatial scale (< 5 km). Although speculative, these likely represent what have been described as Ae. bromeliae and Ae. lilii, respectively. The observation of high levels of diversity within Rabai indicates that this species complex may exhibit significant genetic differentiation across East Africa. The genetic structure, ecology, and range of this important disease vector are surprisingly understudied and need to be further characterized.

All Helicobacter pylori strains tested produced extracellular siderophores, detected by a modified Universal Detection medium, but growth on this medium was poor. By using the iron chelating compound, 2,2'-dipyridyl, outer membrane proteins of 78 and 40 kDa were detected in some, but not all strains examined. No direct binding of lactoferrin or transferrin (the mechanism used by Neisseria to obtain iron) could be demonstrated for H. pylori. Some other techniques for the study of iron-limitation on bacteria were found to be unsuitable for H. pylori.