Diabetes mellitus is associated with increased prevalence of latent tuberculosis infection: Results from the National Health and Nutrition Examination Survey (preprint)
Barron MM , Shaw KM , McKeever Bullard K , Ali MK , Magee MJ . bioRxiv 2017 204461 Aims We aimed to determine the association between prediabetes and diabetes with latent TB using National Health and Nutrition Examination Survey data.Methods We performed a cross-sectional analysis of 2011-2012 National Health and Nutrition Examination Survey data. Participants ≥20 years were eligible. Diabetes was defined by glycated hemoglobin (HbA1c) as no diabetes (≤5.6% [38 mmol/mol]), prediabetes (5.7-6.4% [3946mmol/mol]), and diabetes (≥6.5% [48 mmol/mol]) combined with self-reported diabetes. Latent TB infection was defined by the QuantiFERON®-TB Gold In Tube (QFT-GIT) test. Adjusted odds ratios (aOR) of latent TB infection by diabetes status were calculated using logistic regression and accounted for the stratified probability sample.Results Diabetes and QFT-GIT measurements were available for 4,958 (89.2%) included participants. Prevalence of diabetes was 11.4% (95%CI 9.8-13.0%) and 22.1% (95%CI 20.523.8%) had prediabetes. Prevalence of latent TB infection was 5.9% (95%CI 4.9-7.0%). After adjusting for age, sex, smoking status, history of active TB, and foreign born status, the odds of latent TB infection were greater among adults with diabetes (aOR 1.90, 95%CI 1.15-3.14) compared to those without diabetes. The odds of latent TB in adults with prediabetes (aOR 1.15, 95%CI 0.90-1.47) was similar to those without diabetes.Conclusions Diabetes is associated with latent TB infection among adults in the United States, even after adjusting for confounding factors. Given diabetes increases the risk of active TB, patients with co-prevalent diabetes and latent TB may be targeted for latent TB treatment. |
Modeling missing cases and transmission links in networks of extensively drug-resistant tuberculosis in KwaZulu-Natal, South Africa (preprint)
Nelson KN , Gandhi NR , Mathema B , Lopman BA , Brust JCM , Auld SC , Ismail N , Omar SV , Brown TS , Allana S , Campbell A , Moodley P , Mlisana K , Shah NS , Jenness SM . bioRxiv 2019 655969 The transmission patterns of drug-resistant tuberculosis (TB) remain poorly understood, despite over half a million incident cases in 2017. Modeling TB transmission networks can provide insight into the nature and drivers of transmission, but incomplete and non-random sampling of TB cases can pose challenges to making inferences from epidemiologic and molecular data. We conducted a quantitative bias analysis to assess the effect of missing cases on a transmission network inferred from Mtb sequencing data on extensively drug-resistant (XDR) TB cases in South Africa. We tested scenarios in which cases were missing at random, differentially by clinical characteristics, or differentially by transmission (i.e., cases with many links were under or over-sampled). Under the assumption cases were missing at random, cases in the complete, modeled network would have had a mean of 20 or more transmission links, which is far higher than expected, in order to reproduce the observed, partial network. Instead, we found that the most likely scenario involved undersampling of high-transmitting cases, and further models provided evidence for superspreading behavior. This is, to our knowledge, the first study to define and assess the support for different mechanisms of missingness in a study of TB transmission. Our findings should caution interpretation of results of future studies of TB transmission in high-incidence settings, given the potential for biased sampling, and should motivate further research aimed at identifying the specific host, pathogen, or environmental factors contributing to superspreading. |
Hepatitis E as a cause of adult hospitalization in Bangladesh: Results from an acute jaundice surveillance study in six tertiary hospitals, 2014-2017 (preprint)
Paul RC , Nazneen A , Banik KC , Sumon SA , Paul KK , Akram A , Uzzaman MS , Iqbal T , Tejada-Strop A , Kamili S , Luby SP , Gidding HF , Hayen A , Gurley ES . bioRxiv 2019 688721 In the absence of reliable data on the burden of hepatitis E virus (HEV) in high endemic countries, we established a hospital-based acute jaundice surveillance program in six tertiary hospitals in Bangladesh to estimate the burden of HEV infection among hospitalized acute jaundice patients aged ≥14 years, identify seasonal and geographic patterns in the prevalence of hepatitis E, and examine factors associated with death.We collected blood specimens from enrolled acute jaundice patients, defined as new onset of either yellow eyes or skin during the past three months of hospital admission, and tested for immunoglobulin M (IgM) antibodies against HEV, HBV and HAV. The enrolled patients were followed up three months after hospital discharge to assess their survival status; pregnant women were followed up three months after their delivery to assess pregnancy outcomes.From December’2014 to September’2017, 1925 patients with acute jaundice were enrolled; 661 (34%) had acute hepatitis E, 48 (8%) had hepatitis A, and 293 (15%) had acute hepatitis B infection. Case fatality among hepatitis E patients was 5% (28/589). Most of the hepatitis E cases were males (74%; 486/661), but case fatality was higher among females—12% (8/68) among pregnant and 8% (7/91) among non-pregnant women. Half of the patients who died with acute hepatitis E had co-infection with HAV or HBV. Of the 62 HEV infected mothers who were alive until the delivery, 9 (15%) had miscarriage/stillbirth, and of those children who were born alive, 19% (10/53) died, all within one week of birth.This study confirms that hepatitis E is the leading cause of acute jaundice, leads to hospitalizations in all regions in Bangladesh, occurs throughout the year, and is associated with considerable morbidity and mortality. Effective control measures should be taken to reduce the risk of HEV infections including improvements in water quality, sanitation and hygiene practices and the introduction of HEV vaccine to high-risk groups.Author summary In the absence of reliable surveillance data on the burden of hepatitis E in endemic countries, we conducted a hospital-based acute jaundice surveillance study over a two and a half year period in six tertiary hospitals in Bangladesh. The study confirms that HEV infections occur throughout the year, and is a major (34%) cause of acute jaundice in tertiary hospitals in Bangladesh. Three-quarters of the acute hepatitis E cases were male, and HEV infection was higher among patients residing in urban areas than patients in rural areas (41% vs 32%). The overall case fatality rate of acute HEV infections in hospitals was 5%, but was higher among pregnant women (12%). Hepatitis E patients who died were more likely to have co-infection with HAV or HBV than the HEV infected patients who did not die. Fifteen percent of HEV infected mothers had miscarriage/stillbirth. Of the children who were born alive, 19% died, all within one week of birth. Considering the high burden of hepatitis E among hospitalized acute jaundice patients, Bangladesh could take control measures to reduce this risk including improvements in water quality, sanitation and hygiene practices and the introduction of hepatitis E vaccine in high-risk areas. |
Hospitalizations associated with respiratory syncytial virus (RSV) and influenza in children, including children having a diagnosis of asthma (preprint)
Goldstein E , Finelli L , O'Halloran A , Liu P , Karaca Z , Steiner CA , Viboud C , Lipsitch M . bioRxiv 2019 161067 Background There is uncertainty about the burden of hospitalization associated with RSV and influenza in children, including those with underlying medical conditions.Methods We applied previously developed methodology (Goldstein et al., Epidemiology 2012) to HealthCare Cost and Utilization Project (HCUP) hospitalization data and additional data related to asthma diagnosis/previous history in hospitalized children to estimate RSV and influenza-associated hospitalization rates in different subpopulations of US children between 2003-2010.Results The estimated average annual rates (per 100,000 children) of RSV-associated hospitalization with a respiratory cause (ICD-9 codes 460-519) present anywhere in the discharge diagnosis were 2381 (95% CI(2252,2515)) in age <1y; 710.6(609.1,809.2) (age 1y); 395(327.7,462.4) (age 2y); 211.3(154.6,266.8) (age 3y); 111.1(62.4,160.1) (age 4y); 72.3(29.3,116.4) (ages 5-6y); 35.6(9.9,62.2) (ages 7-11y); and 39(17.5,60.6) (ages 12-17y).The corresponding rates of influenza-associated hospitalization were lower, ranging from 181(142.5,220.3) in age <1y to 17.9(11.7,24.2) in ages 12-17y. The relative risks for RSV-related hospitalization associated with a prior diagnosis of asthma in age groups under 5y ranged between 3.1(2.1,4.7) (age <1y) to 6.7(4.2,11.8) (age 2y); the corresponding risks for influenza-related hospitalization ranged from 2.8(2.1,4) (age <1y) to 4.9(3.8,6.4) (age 3y).Conclusions RSV-associated hospitalization rates in young children are high and decline rapidly with age. Young children with an asthma diagnosis should be target groups for RSV and influenza-related mitigation efforts, possibly including RSV prophylaxis for the youngest children. |
A Collaborative Multi-Model Ensemble for Real-Time Influenza Season Forecasting in the U.S (preprint)
Reich NG , McGowan CJ , Yamana TK , Tushar A , Ray EL , Osthus D , Kandula S , Brooks LC , Crawford-Crudell W , Gibson GC , Moore E , Silva R , Biggerstaff M , Johansson MA , Rosenfeld R , Shaman J . bioRxiv 2019 566604 Seasonal influenza results in substantial annual morbidity and mortality in the United States and worldwide. Accurate forecasts of key features of influenza epidemics, such as the timing and severity of the peak incidence in a given season, can inform public health response to outbreaks. As part of ongoing efforts to incorporate data and advanced analytical methods into public health decision-making, the United States Centers for Disease Control and Prevention (CDC) has organized seasonal influenza forecasting challenges since the 2013/2014 season. In the 2017/2018 season, 22 teams participated. A subset of four teams created a research consortium called the FluSight Network in early 2017. During the 2017/2018 season they worked together to produce a collaborative multi-model ensemble that combined 21 separate component models into a single model using a machine learning technique called stacking. This approach creates a weighted average of predictive densities where the weight for each component is based on that component’s forecast accuracy in past seasons. In the 2017/2018 influenza season, one of the largest seasonal outbreaks in the last 15 years, this multi-model ensemble performed better on average than all individual component models and placed second overall in the CDC challenge. It also outperformed the baseline multi-model ensemble created by the CDC that took a simple average of all models submitted to the forecasting challenge. This project shows that collaborative efforts between research teams to develop ensemble forecasting approaches can bring measurable improvements in forecast accuracy and important reductions in the variability of performance from year to year. Efforts such as this, that emphasize real-time testing and evaluation of forecasting models and facilitate the close collaboration between public health officials and modeling researchers, are essential to improving our understanding of how best to use forecasts to improve public health response to seasonal and emerging epidemic threats. |
Transcriptome Analysis of Neisseria gonorrhoeae During Natural Infection Reveals Differential Expression of Antibiotic Resistance Determinants Between Men and Women (preprint)
Nudel K , McClure R , Moreau M , Briars E , Abrams AJ , Tjaden B , Su XH , Trees D , Rice PA , Massari P , Genco CA . bioRxiv 2018 319970 Neisseria gonorrhoeae is a bacterial pathogen responsible for the sexually transmitted infection, gonorrhea. Emergence of antimicrobial resistance (AMR) of N. gonorrhoeae worldwide has resulted in limited therapeutic choices for this infection. Men who seek treatment often have symptomatic urethritis; in contrast, gonococcal cervicitis in women is usually minimally symptomatic, but may progress to pelvic inflammatory disease. Previously, we reported the first analysis of gonococcal transcriptome expression determined in secretions from women with cervical infection. Here, we defined gonococcal global transcriptional responses in urethral specimens from men with symptomatic urethritis and compared these with transcriptional responses in specimens obtained from women with cervical infections, and of in vitro-grown N. gonorrhoeae isolates. This is the first comprehensive comparison of gonococcal gene expression in infected men and women. RNA sequencing analysis revealed that 9.4% of gonococcal genes showed increased expression exclusively in men and included genes involved in host immune cell interactions and 4.3% showed increased expression exclusively in women and included phage-associated genes. Infected men and women displayed comparable antibiotic resistant-genotypes and in vitro –phenotypes, but a 4-fold higher expression of the Mtr efflux pump-related genes was observed in men. These results suggest that expression of AMR genes is programmed genotypically, and also driven by sex-specific environments. Collectively, our results indicate that distinct N. gonorrhoeae gene expression signatures are detected during genital infection in men and women. We propose that therapeutic strategies could target sex specific differences in expression of antibiotic resistance genes. |
The PrEP Care Continuum and Racial Disparities in HIV Incidence among Men Who Have Sex with Men (preprint)
Jenness SM , Maloney KM , Smith DK , Hoover KW , Goodreau SM , Rosenberg ES , Weiss KM , Liu AY , Rao DW , Sullivan PS . bioRxiv 2018 249540 Background HIV preexposure prophylaxis (PrEP) could reduce the disparities in HIV incidence among black men who have sex with men (BMSM) compared to white MSM (WMSM), but this may depend on progression through the PrEP care continuum.Methods We expanded our network-based mathematical model of HIV transmission for MSM, which simulates PrEP based on CDC’s clinical practice guidelines, to include race-stratified transitions through the PrEP continuum steps of awareness, access, prescription, adherence, and retention. Continuum parameters were estimated based on published incidence cohorts and PrEP open-label studies. Counterfactuals included a no-PrEP reference scenario, and intervention scenarios in which the BMSM continuum step parameters were modified.Results Implementing PrEP according to the observed BMSM continuum was projected to result in 8.4% of all BMSM on PrEP at year 10, yielding a 23% decline in incidence (HR = 0.77). On an absolute scale, the racial disparity in incidence in this scenario was 4.95 per 100 person-years at risk (PYAR), a 19% decline from the 6.08 per 100 PYAR disparity in the reference scenario. If BMSM continuum parameters were equal to WMSM values, 17.7% of BMSM would be on PrEP, yielding a 47% decline in incidence (HR = 0.53) and a disparity of 3.30 per 100 PYAR (a 46% decline in the disparity).Conclusions PrEP could lower HIV incidence overall and reduce absolute racial disparities between BMSM and WMSM. Interventions addressing the racial gaps in the PrEP continuum will be needed to further decrease these HIV disparities. |
Forecasting seasonal influenza in the U.S.: A collaborative multi-year, multi-model assessment of forecast performance (preprint)
Reich NG , Brooks LC , Fox SJ , Kandula S , McGowan CJ , Moore E , Osthus D , Ray EL , Tushar A , Yamana TK , Biggerstaff M , Johansson MA , Rosenfeld R , Shaman J . bioRxiv 2018 397190 Influenza infects an estimated 9 to 35 million individuals each year in the United States and is a contributing cause for between 12,000 and 56,000 deaths annually. Seasonal outbreaks of influenza are common in temperate regions of the world, with highest incidence typically occurring in colder and drier months of the year. Real-time forecasts of influenza transmission can inform public health response to outbreaks. We present the results of a multi-institution collaborative effort to standardize the collection and evaluation of forecasting models for influenza in the US for the 2010/2011 through 2016/2017 influenza seasons. For these seven seasons, we assembled weekly real-time forecasts of 7 targets of public health interest from 22 different models. We compared forecast accuracy of each model relative to a historical baseline seasonal average. Across all regions of the US, over half of the models showed consistently better performance than the historical baseline when forecasting incidence of influenza-like illness 1, 2 and 3 weeks ahead of available data and when forecasting the timing and magnitude of the seasonal peak. In some regions, delays in data reporting were strongly and negatively associated with forecast accuracy. More timely reporting and an improved overall accessibility to novel and traditional data sources are needed to improve forecasting accuracy and its integration with real-time public health decision-making. |
Interim impact evaluation of the hepatitis C virus elimination program in Georgia (preprint)
Walker JG , Fraser H , Lim AG , Gvinjilia L , Hagan L , Kuchuloria T , Martin NK , Nasrullah M , Shadaker S , Aladashvili M , Asatiani A , Baliashvili D , Butsashvili M , Chikovani I , Khonelidze I , Kirtadze I , Kuniholm MH , Otiashvili D , Stvilia K , Tsertsvadze T , Hickman M , Morgan J , Gamkrelidze A , Kvaratskhelia V , Averhoff F , Vickerman P . bioRxiv 2018 270579 Background and Aims Georgia has one of the highest hepatitis C virus (HCV) prevalence rates in the world, with >5% of the adult population (~150,000 people) chronically infected. In April 2015, the Georgian government, in collaboration with CDC and other partners, launched a national program to eliminate HCV through scaling up HCV treatment and prevention interventions, with the aim of achieving a 90% reduction in prevalence by 2020. We evaluate the interim impact of the HCV treatment program as of 31 October 2017, and assess the feasibility of achieving the elimination goal by 2020.Method We developed a dynamic HCV transmission model to capture the current and historical epidemic dynamics of HCV in Georgia, including the main drivers of transmission. Using the 2015 national sero-survey and prior surveys conducted among people who inject drugs (PWID) from 1997-2015, the model was calibrated to data on HCV prevalence by age, gender and PWID status, and the age distribution of PWID. We use the model to project the interim impact of treatment strategies currently being undertaken as part of the ongoing Georgia HCV elimination program, while accounting for treatment failure/loss to follow up, in order to determine whether they are on track to achieving their HCV elimination target by 2020, or whether strategies need to be modified to ensure success.Results A treatment rate of 2,050 patients/month was required from the beginning of the national program to achieve a 90% reduction in prevalence by the end of 2020, with equal treatment rates of PWID and the general population. From May 2015 to October 2017, 40,420 patients were treated, an average of ~1,350 per month; although the treatment rate has recently declined from a peak of 4,500/month in September 2016 to 2100/month in November-December 2016, and 1000/month in August-October 2017, with a sustained virological response rate (SVR) of 98% per-protocol or 78% intent to treat. The model projects that the treatments undertaken up to October 2017 have reduced adult chronic prevalence by 26% (18-35%) to 3.7% (2.9-5.1%), reduced total incidence by 25% (15-35%), and prevented 1845 (751-3969) new infections and 93 (31-177) HCV-related deaths. If the treatment rate of 1000 patients initiated per month continues, prevalence will have halved by 2020, and reduce by 90% by 2026. In order to reach a 90% reduction by 2020, the treatment rate must increase 3.5-fold to 4000/month.Conclusion The Georgia HCV elimination program has accomplished an impressive scale up of treatment, which has already impacted on prevalence and incidence, and averted deaths due to HCV. However, extensive scale up is needed to achieve a 90% reduction in prevalence by 2020. |
Co-circulating mumps lineages at multiple geographic scales (preprint)
Wohl S , Metsky HC , Schaffner SF , Piantadosi A , Burns M , Lewnard JA , Chak B , Krasilnikova LA , Siddle KJ , Matranga CB , Bankamp B , Hennigan S , Sabina B , Byrne EH , McNall RJ , Park DJ , Gharib S , Fitzgerald S , Barreira P , Fleming S , Lett S , Rota PA , Madoff LC , Yozwiak NL , MacInnis BL , Smole S , Grad YH , Sabeti PC . bioRxiv 2018 343897 Despite widespread vaccination, eleven thousand mumps cases were reported in the United States (US) in 2016–17, including hundreds in Massachusetts, primarily in college settings. We generated 203 whole genome mumps virus (MuV) sequences from Massachusetts and 15 other states to understand the dynamics of mumps spread locally and nationally, as well as to search for variants potentially related to vaccination. We observed multiple MuV lineages circulating within Massachusetts during 2016–17, evidence for multiple introductions of the virus to the state, and extensive geographic movement of MuV within the US on short time scales. We found no evidence that variants arising during this outbreak contributed to vaccine escape. Combining epidemiological and genomic data, we observed multiple co-circulating clades within individual universities as well as spillover into the local community. Detailed data from one well-sampled university allowed us to estimate an effective reproductive number within that university significantly greater than one. We also used publicly available small hydrophobic (SH) gene sequences to estimate migration between world regions and to place this outbreak in a global context, but demonstrate that these short sequences, historically used for MuV genotyping, are inadequate for tracing detailed transmission. Our findings suggest continuous, often undetected, circulation of mumps both locally and nationally, and highlight the value of combining genomic and epidemiological data to track viral disease transmission at high resolution. |
Dust or disease? Perceptions of influenza in rural Southern Malawi (preprint)
Phiri M , Gooding K , Peterson I , Mambule I , Nundwe S , McMorrow M , Desmond N . bioRxiv 2018 470856 Background Influenza virus infections cause between 291 243 and 645 832 deaths annually, with the highest burden in low-income settings. Research in high-income countries has examined public understanding of influenza, but there is little information on views and behaviours about influenza in low-income countries. We explored communities’ ideas about the severity, causes, prevention and treatment of influenza in Chikwawa district, Malawi.Methods We conducted 64 in-depth interviews with parents of children aged <5 years, and 7 focus groups with community health workers, parents, and traditional healers. Data were analysed thematically and using a framework matrix to compare views between groups.Results Respondents held varied ideas about influenza, and many were uncertain about its causes and treatment. Some parents, traditional healers and health workers thought influenza was not severe because they felt it did not cause death or limit activities, but others disagreed. Many saw influenza as a symptom of other conditions, especially malaria and pneumonia, rather than as a disease of its own. Most mentioned dust as the main cause of influenza and believed influenza could be prevented by cleaning the home thoroughly. Treatment seeking for influenza followed different stages, usually starting with home remedies followed by purchasing drugs from groceries and then visiting a health centre. Seeking a clinician tended to be triggered by severe symptoms like high fever or difficulty breathing, and suspicions of malaria or pneumonia. Community health workers provide health education for communities, but some lacked understanding of influenza.Conclusion Our findings suggest uncertainty about the causes and control of influenza among parents and varied levels of understanding among health providers. Strengthening the capacity of community health workers to provide relevant information about influenza prevention and treatment could address parents’ interest in further information and support informed health seeking and engagement with future influenza interventions. |
Mechanisms associated with pyrethroid resistance in populations of Aedes aegypti (Diptera: Culicidae) from the Caribbean coast of Colombia (preprint)
Pareja-Loaiza PX , Santacoloma Varon L , Rey Vega G , Gómez-Camargo D , Maestre-Serrano R , Lenhart A . bioRxiv 2020 2020.01.23.916577 Aedes aegypti is the main vector of dengue, chikungunya, and Zika viruses, which are of great public health importance in Colombia. Aedes control strategies in Colombia rely heavily on the use of organophosphate and pyrethroid insecticides, providing constant selection pressure and the emergence of resistant populations. In recent years, insecticide use has increased due to the increased incidence of dengue and recent introductions of chikungunya and Zika. In the present study, pyrethroid resistance was studied across six populations of A. aegypti from the Caribbean coast of Colombia. Susceptibility to λ-cyhalothrin, deltamethrin, and permethrin was assessed, and resistance intensity was determined. Activity levels of enzymes associated with resistance were measured, and the frequencies of three kdr alleles (V1016I, F1534C, V410L) were calculated. Results showed variations in pyrethroid susceptibility across A. aegypti populations and altered enzyme activity levels were detected. The kdr alleles were detected in all populations, with high variations in frequencies: V1016I (frequency ranging from 0.15–0.70), F1534C (range 0.94–1.00), and V410L (range 0.05–0.72). In assays of phenotyped individuals, associations were observed between the presence of V1016I, F1534C, and V410L alleles and resistance to the evaluated pyrethroids, as well as between the VI1016/CC1534/VL410 tri-locus genotype and λ-cyhalothrin and permethrin resistance. The results of the present study contribute to the knowledge of the mechanisms underlying the resistance to key pyrethroids used to control A. aegypti along the Caribbean coast of Colombia. |
Bats are key hosts in the radiation of mammal-associated Bartonella bacteria (preprint)
McKee CD , Bai Y , Webb CT , Kosoy MY . bioRxiv 2020 2020.04.03.024521 Bats are notorious reservoirs of several zoonotic diseases and may be uniquely tolerant of infection among mammals. Broad sampling has revealed the importance of bats in the diversification and spread of viruses and eukaryotes to other animal hosts. Vector-borne bacteria of the genus Bartonella are prevalent and diverse in mammals globally and recent surveys have revealed numerous Bartonella lineages in bats. We assembled a sequence database of Bartonella strains, consisting of nine genetic loci from 209 previously characterized lineages and 121 new cultured strains from bats, and used these data to perform the most comprehensive phylogenetic analysis of Bartonella to date. This analysis included estimation of divergence dates using a molecular clock and ancestral reconstruction of host associations and geography. We estimate that Bartonella began infecting mammals 62 million years ago near the Cretaceous-Paleogene boundary. Additionally, the radiation of particular Bartonella clades correlate strongly to the timing of diversification and biogeography of mammalian hosts. Bats were inferred to be the ancestral hosts of all mammal-associated Bartonella and appear to be responsible for the early geographic expansion of the genus. We conclude that bats have had a deep influence on the evolutionary radiation of Bartonella bacteria and their spread to other mammalian orders. These results support a ‘bat seeding’ hypothesis that could explain similar evolutionary patterns in other mammalian parasite taxa. Application of such phylogenetic tools as we have used to other taxa may reveal the general importance of bats in the ancient diversification of mammalian parasites.Significance statement Discovering the evolutionary history of infectious agents in animals is important for understanding the process of host adaptation and the origins of human diseases. To clarify the evolution of the Bartonella genus, which contains important human pathogens, we performed phylogenetic analysis on a broad diversity of Bartonella strains, including novel strains from bats. Our results indicate that Bartonella clades diversified along with their mammal hosts over millions of years. Bats appear to be especially important in the early radiation and geographic dispersal of Bartonella lineages. These patterns are consistent with research indicating a chiropteran origin of important human viruses and eukaryotic parasites, suggesting that bats may play a unique role as historical sources of infections to other hosts. |
Climate and urbanization drive mosquito preference for humans (preprint)
Rose NH , Sylla M , Badolo A , Lutomiah J , Ayala D , Aribodor OB , Ibe N , Akorli J , Otoo S , Mutebi JP , Kriete AL , Ewing EG , Sang R , Gloria-Soria A , Powell JR , Baker RE , White BJ , Crawford JE , McBride CS . bioRxiv 2020 2020.02.12.939041 The majority of mosquito-borne illness is spread by a few mosquito species that have evolved to specialize in biting humans, yet the precise causes of this behavioral shift are poorly understood. We address this gap in the arboviral vector Aedes aegypti. We first characterize the behaviour of mosquitoes from 27 sites scattered across the species’ ancestral range in sub-Saharan Africa, revealing previously unrecognized diversity in female preference for human versus animal odor. We then use modelling to show that this diversity can be almost fully predicted by two ecological factors – dry season intensity and human population density. Finally we integrate this information with whole genome sequence data from 345 individual mosquitoes to identify a single underlying ancestry component linked to human preference, with genetic changes concentrated in a few key chromosomal regions. Our findings strongly suggest that human-biting in this important disease vector originally evolved as a by-product of breeding in human-stored water in areas where doing so provided the only means to survive the long, hot dry season. Our model also predicts that changes in human population density are likely to drive future mosquito evolution. Rapid urbanization may drive a shift to human-biting in many cities across Africa by 2050. |
An assessment of adult mosquito collection techniques for studying species abundance and diversity in Maferinyah, Guinea (preprint)
Cansado-Utrilla C , Jeffries CL , Kristan M , Brugman VA , Heard P , Camara G , Sylla M , Beavogui AH , Messenger LA , Walker T . bioRxiv 2019 772822 Background Guinea is a West African country with a high prevalence of vector-borne diseases where few entomological studies have been undertaken. Although several mosquito collection methods are routinely used for surveillance in vector control programmes, they target different behaviours causing bias in species diversity and abundance. Given the paucity of mosquito trap data in West Africa, we compared the performance of five trap-lure combinations and Human Landing Catches (HLCs) in Guinea.Methods Five mosquito traps were compared in a 5×5 Latin Square design for 15 days in three villages in Guinea between June and July 2018. CDC light traps, BG sentinel 2 traps (with BG and MB5 lures), gravid traps and Stealth traps were deployed for 24-hour intervals with mosquitoes collected every 12 hours (day and night collections). HLCs were also performed for 15 nights. A Generalised Linear Mixed Model was applied to compare the effect of the traps, sites and collection times on the mosquito abundance. Species identification was confirmed using PCR-based analysis and Sanger sequencing.Results In total, 10,610 mosquitoes were captured across all five traps. Significantly more mosquitoes (P<0.005) were collected by Stealth traps (7,096) compared to the rest of the traps. Stealth traps and BG sentinel 2 traps were the best at capturing An. gambiae and Ae. aegypti mosquitoes respectively. HLCs captured predominantly An. coluzzii (41%) and hybrids of An. gambiae s.s. / An. coluzzii (36%) in contrast to the five adult traps, which captured predominantly An. melas (83%). Senguelen (rural) presented the highest abundance of mosquitoes and overall diversity in comparison with Fandie (semi-rural) and Maferinyah Centre One (semi-urban). To our knowledge, four species are reported for the first time in Guinea.Conclusions Stealth traps presented the best performance overall, suggesting that this trap may play an important role for mosquito surveillance in Guinea and similar sites in West Africa. We recommend the incorporation of molecular tools in entomological studies since it has helped to reveal, together with morphological identification, the presence of 25 mosquito species in this area.BG2BG sentinel 2 trapBG2-BGBG sentinel 2 trap with BG lureBG2-MB5BG sentinel 2 trap with MB5 lureGLMMgeneralized linear mixed modelGTGravid trapHLCHuman Landing CatchLTCDC light trapSTStealth trap |
Characterizing the molecular and metabolic mechanisms of insecticide resistance in Anopheles gambiae s.l. in Faranah, Guinea (preprint)
Stica C , Jeffries CL , Irish SR , Barry Y , Camara D , Yansane I , Kristan M , Walker T , Messenger LA . bioRxiv 2019 610998 Background In recent years, the scale-up of long-lasting insecticidal nets (LLINs) and indoor residual spraying (IRS) has greatly reduced malaria transmission. However, malaria remains a global public health concern with the majority of disease burden in sub-Saharan Africa. Insecticide resistance is a growing problem among Anopheles vector populations, with potential implications for the continued effectiveness of available control interventions. Improved understanding of current resistance levels and underlying mechanisms is essential to design appropriate management strategies and to mitigate future selection for resistance.Methods Anopheles gambiae s.l. mosquitoes were collected from three villages in Faranah Prefecture, Guinea and their levels of susceptibility to seven insecticides were measured using CDC resistance intensity bioassays. Synergist assays with piperonyl butoxide (PBO) were also undertaken to assess the role of elevated mixed-function oxidases in resistance. RNA was extracted from 563 individuals and PCR was performed on cDNA to determine vector species, presence of target site mutations (L1014F kdr, N1575Y and G119S Ace-1), Plasmodium falciparum infection, and relative expression of three metabolic genes (CYP6M2, CYP6P3 and GSTD3).Results In Faranah, resistance to permethrin and deltamethrin was observed, as well as possible resistance to bendiocarb. All assayed vector populations were fully susceptible to alpha-cypermethrin, pirimiphos-methyl, clothianidin and chlorfenapyr. Plasmodium falciparum infection was detected in 7.3% (37/508) mosquitoes tested. The L1014F kdr mutation was found in 100% of a sub-sample of 60 mosquitoes, supporting its fixation in the region. The N1575Y mutation was identified in 20% (113/561) of individuals, with ongoing selection evidenced by significant deviations from Hardy-Weinberg equilibrium. The G119S Ace-1 mutation was detected in 62.1% (18/29) of mosquitoes tested and was highly predictive of bendiocarb bioassay survival. The metabolic resistance genes, CYP6M2, CYP6P3 and GSTD3, were found to be overexpressed in wild resistant and susceptible An. gambiae s.s. populations, compared to a susceptible G3 colony. Furthermore, CYP6P3 was significantly overexpressed in bendiocarb survivors, implicating its potential role in carbamate resistance in Faranah.Conclusions Identification of intense resistance to permethrin and deltamethrin in Faranah, is of concern, as the Guinea National Malaria Control Program (NMCP) relies exclusively on the distribution of pyrethroid-treated LLINs for vector control. Study findings will be used to guide current and future control strategies in the region. |
Minimum Information for Reusable Arthropod Abundance Data (MIReAAD) (preprint)
Rund SSC , Braak K , Cator L , Copas K , Emrich SJ , Giraldo-Calderon GI , Johansson MA , Heydari N , Hobern D , Kelly SA , Lawson D , Lord C , MacCallum RM , Roche DG , Ryan SJ , Schigel D , Vandegrift K , Watts M , Zaspel JM , Pawar S . bioRxiv 2018 429142 Arthropods play a dominant role in natural and human-modified terrestrial ecosystem dynamics. Spatially-explicit population time-series are crucial for statistical or mathematical models of these dynamics and assessment of their veterinary, medical, agricultural, and ecological impacts. Arthropod data have been collected world-wide for over a century, but remain scattered and largely inaccessible. With the ever-present and growing threat of arthropod vectors of infectious diseases and pest species, there are enormous amounts of historical and ongoing surveillance. These data are currently reported in a wide variety of formats, typically lacking sufficient metadata to make reuse and re-analysis possible. We present the first minimum information standard for arthropod abundance. Developed with broad stakeholder collaboration, it balances sufficiency for reuse with the practicality of preparing the data for submission. It is designed to optimize data (re-)usability from the “FAIR,” (Findable, Accessible, Interoperable, and Reusable) principles of public data archiving (PDA). This standard will facilitate data unification across research initiatives and communities dedicated to surveillance for detection and control of vector-borne diseases and pests. |
Novel Wolbachia strains in Anopheles malaria vectors from Sub-Saharan Africa (preprint)
Jeffries CL , Lawrence GG , Golovko G , Kristan M , Orsborne J , Spence K , Hurn E , Bandibabone J , Tantely LM , Raharimalala FN , Keita K , Camara D , Barry Y , Wat'senga F , Manzambi EZ , Afrane YA , Mohammed AR , Abeku TA , Hedge S , Khanipov K , Pimenova M , Fofanov Y , Boyer S , Irish SR , Hughes GL , Walker T . bioRxiv 2018 338434 Anopheles (An.) mosquitoes contain bacteria that can influence Plasmodium parasites. Wolbachia, a common insect endosymbiont, has historically been considered absent from Anopheles but has recently been found in An. gambiae populations. Here, we assessed a range of Anopheles species from five malaria-endemic countries for Wolbachia and Plasmodium infection. Strikingly, we found Wolbachia infections in An. coluzzii, An. gambiae s.s, An. arabiensis, An. moucheti and An. species ‘A’ increasing the number of Anopheles species known to be naturally infected by this endosymbiont. Molecular analysis suggests the presence of phylogenetically diverse novel strains, while qPCR and 16S rRNA sequencing indicates that Wolbachia is the dominant member of the microbiota in An. moucheti and An. species ‘A’. We found no evidence of Wolbachia/Asaia co-infections, and presence of these endosymbionts did not have significant effects on malaria prevalence. We discuss the importance of novel Wolbachia strains in Anopheles and potential implications for disease control. |
Investigating the relationship between insecticide resistance, underlying molecular mechanisms and malaria prevalence in Anopheles gambiae s.l. from Guinea (preprint)
Collins E , Vaselli NM , Sylla M , Beavogui AH , Orsborne J , Walker T , Messenger LA . bioRxiv 2018 434688 The threat of insecticide resistance across sub-Saharan Africa is anticipated to severely impact the continued effectiveness of malaria vector control. We investigated the effect of carbamate and pyrethroid resistance on Anopheles gambiae s.l age, Plasmodium falciparum infection and characterized molecular resistance mechanisms in Guinea. Pyrethroid resistance was intense, with survivors of ten times the insecticidal concentration required to kill susceptible individuals. The L1014F kdr allele was significantly associated with mosquito survival following deltamethrin or permethrin treatment (p=0.003 and p=0.04, respectively). N1575Y and I1527T mutations were identified in 13% and 10% of individuals, respectively, but neither conferred increased pyrethroid tolerance. Partial restoration of pyrethroid susceptibility following synergist pre-exposure suggest a role for mixed-function oxidases. Carbamate resistance was lower and significantly associated with the G119S Ace-1 mutation (p=0.001). Oocyst rates were 6.8% and 4.2% among resistant and susceptible mosquitoes, respectively; survivors of bendiocarb exposure were significantly more likely to be infected (p=0.03). Resistant mosquitoes had significantly lower parity rates; however, a subset of intensely pyrethroid-resistant vectors were more likely to be parous (p=0.042 and p=0.045, for survivors of five and ten times the diagnostic dose of insecticides, respectively). Our findings emphasize the need for additional studies directly assessing the influence of insecticide resistance on mosquito fitness. |
A chromosome-scale assembly of the major African malaria vector Anopheles funestus (preprint)
Ghurye J , Koren S , Small ST , Redmond S , Howell P , Phillippy AM , Besansky NJ . bioRxiv 2018 492777 Background Anopheles funestus is one of the three most consequential and widespread vectors of human malaria in tropical Africa. However, the lack of a high-quality reference genome has hindered the association of phenotypic traits with their genetic basis in this important mosquito.Findings Here we present a new high-quality An. funestus reference genome (AfunF3) assembled using 240x coverage of long-read single-molecule sequencing for contigging, combined with 100x coverage of short-read Hi-C data for chromosome scaffolding. The assembled contigs total 446 Mbp of sequence and contain substantial duplication due to alternative alleles present in the sequenced pool of mosquitos from the FUMOZ colony. Using alignment and depth-of-coverage information, these contigs were deduplicated to a 211 Mbp primary assembly, which is closer to the expected haploid genome size of 250 Mbp. This primary assembly consists of 1,053 contigs organized into 3 chromosome-scale scaffolds with an N50 contig size of 632 kbp and an N50 scaffold size of 93.811 Mbp, representing a 100-fold improvement in continuity versus the current reference assembly, AfunF1.Conclusion This highly contiguous and complete An. funestus reference genome assembly will serve as an improved basis for future studies of genomic variation and organization in this important disease vector. |
African nonhuman primates are infected with the yaws bacterium Treponema pallidum subsp. pertenue (preprint)
Knauf S , Gogarten JF , Schuenemann VJ , De Nys HM , Dux A , Strouhal M , Mikalova L , Bos KI , Armstrong R , Batamuzi EK , Chuma IS , Davoust B , Diatta G , Fyumagwa RD , Kazwala RR , Keyyu JD , Lejora IAV , Levasseur A , Liu H , Mayhew MA , Mediannikov O , Raoult D , Wittig RM , Roos C , Leendertz FH , Smajs D , Nieselt K , Krause J , Calvignac-Spencer S . bioRxiv 2017 135491 Treponema pallidum subsp. pertenue (TPE) is the causative agent of yaws. The disease was subject to global eradication efforts in the mid 20th century but reemerged in West Africa, Southern Asia, and the Pacific region. Despite its importance for eradication, detailed data on possible nonhuman disease reservoirs are missing. A number of African nonhuman primates (NHPs) have been reported to show skin ulcerations suggestive of treponemal infection in humans. Furthermore antibodies against Treponema pallidum (TP) have been repeatedly detected in wild NHP populations. While genetic studies confirmed that NHPs are infected with TP strains, subspecies identification was only possible once for a strain isolated in 1966, pinpointing the involvement of TPE. We therefore collected a number of recently isolated simian TP strains and determined eight whole genome sequences using hybridization capture or long-range PCR combined with next-generation sequencing. These new genomes were compared with those of known human TP isolates. Our results show that naturally occurring simian TP strains circulating in three African NHP species all cluster with human TPE strains and show the same genomic structure as human TPE strains. These data indicate that humans are not the exclusive host for the yaws bacterium and that a One Health approach is required to achieve sustainable eradication of human yaws. |
Nowcasting by Bayesian Smoothing: A flexible, generalizable model for real-time epidemic tracking (preprint)
McGough SF , Johansson MA , Lipsitch M , Menzies NA . bioRxiv 2019 663823 Delays in case reporting are common to disease surveillance systems, making it difficult to track diseases in real-time. “Nowcast” approaches attempt to estimate the complete case counts for a given reporting date, using a time series of case reports that is known to be incomplete due to reporting delays. Modeling the reporting delay distribution is a common feature of nowcast approaches. However, many nowcast approaches ignore a crucial feature of infectious disease transmission—that future cases are intrinsically linked to past reported cases—and are optimized to a single application, which may limit generalizability. Here, we present a Bayesian approach, NobBS (Nowcasting by Bayesian Smoothing) capable of producing smooth and accurate nowcasts in multiple disease settings. We test NobBS on dengue in Puerto Rico and influenza-like illness (ILI) in the United States to examine performance and robustness across settings exhibiting a range of common reporting delay characteristics (from stable to time-varying), and compare this approach with a published nowcasting package. We show that introducing a temporal relationship between cases considerably improves performance when the reporting delay distribution is time-varying, and we identify trade-offs in the role of moving windows to accurately capture changes in the delay. We present software implementing this new approach (R package “NobBS”) for widespread application.Significance Achieving accurate, real-time estimates of disease activity is challenged by delays in case reporting. However, approaches that seek to estimate cases in spite of reporting delays often do not consider the temporal relationship between cases during an outbreak, nor do they identify characteristics of robust approaches that generalize to a wide range of surveillance contexts with very different reporting delays. Here, we present a smooth Bayesian nowcasting approach that produces accurate estimates that capture the time evolution of the epidemic curve and outperform a previous approach in the literature. We assess the performance for two diseases to identify important features of the reporting delay distribution that contribute to the model’s performance and robustness across surveillance settings. |
Heterogeneous local dynamics revealed by classification analysis of spatially disaggregated time series data (preprint)
Perkins TA , Rodriguez-Barraquer I , Manore C , Siraj AS , Espana G , Barker CM , Johansson MA , Reiner RCJr . bioRxiv 2019 276006 Time series data provide a crucial window into infectious disease dynamics, yet their utility is often limited by the spatially aggregated form in which they are presented. When working with time series data, violating the implicit assumption of homogeneous dynamics below the scale of spatial aggregation could bias inferences about underlying processes. We tested this assumption in the context of the 2015-2016 Zika epidemic in Colombia, where time series of weekly case reports were available at national, departmental, and municipal scales. First, we performed a descriptive analysis, which showed that the timing of departmental-level epidemic peaks varied by three months and that departmental-level estimates of the time-varying reproduction number, R(t), showed patterns that were distinct from a national-level estimate. Second, we applied a classification algorithm to six features of proportional cumulative incidence curves, which showed that variability in epidemic duration, the length of the epidemic tail, and consistency with a cumulative normal density curve made the greatest contributions to distinguishing groups. Third, we applied this classification algorithm to data simulated with a stochastic transmission model, which showed that group assignments were consistent with simulated differences in the basic reproduction number, R0. This result, along with associations between spatial drivers of transmission and group assignments based on observed data, suggests that the classification algorithm is capable of detecting differences in temporal patterns that are associated with differences in underlying drivers of incidence patterns. Overall, this diversity of temporal patterns at local scales underscores the value of spatially disaggregated time series data. |
Emergence of a novel Salmonella enterica serotype Reading clone is linked to its expansion in commercial turkey production, resulting in unanticipated human illness in North America (preprint)
Miller EA , Elnekave E , Flores-Figueroa C , Johnson A , Kearney A , Munoz-Aguayo J , Tagg KA , Tschetter L , Weber BP , Nadon CA , Boxrud D , Singer RS , Folster JP , Johnson TJ . bioRxiv 2019 855734 Concurrent separate human outbreaks of Salmonella enterica serotype Reading occurred in 2017-2019 in the United States and Canada, which were both linked to the consumption of raw turkey products. In this study, a comprehensive genomic investigation was conducted to reconstruct the evolutionary history of S. Reading from turkeys, and to determine the genomic context of outbreaks involving this rarely isolated Salmonella serotype. A total of 988 isolates of U.S. origin were examined using whole genome-based approaches, including current and historical isolates from humans, meat, and live food animals. Broadly, isolates clustered into three major clades, with one apparently highly adapted turkey clade. Within the turkey clade isolates clustered into three subclades, including an “emergent” clade that only contained isolates dated 2016 or later, including many of the isolates from these outbreaks. Genomic differences were identified between emergent and other turkey subclades suggesting that the apparent success of currently circulating subclades clade is, in part, attributable to plasmid acquisitions conferring antimicrobial resistance, gain of phage-like sequences with cargo virulence factors, and mutations in systems that may be involved in beta-glucuronidase activity and resistance towards colicins. U.S. and Canadian outbreak isolates were found interspersed throughout the emergent subclade and the other circulating subclade. The emergence of a novel S. Reading turkey subclade, coinciding temporally with expansion in commercial turkey production and with U.S. and Canadian human outbreaks, indicates that emergent strains with higher potential for niche success were likely vertically transferred and rapidly disseminated from a common source.Importance Increasingly, outbreak investigations involving foodborne pathogens are confounded by the inter-connectedness of food animal production and distribution, necessitating high-resolution genomic investigations to determine their basis. Fortunately, surveillance and whole genome sequencing, combined with the public availability of these data, enable comprehensive queries to determine underlying causes of such outbreaks. Utilizing this pipeline, it was determined that a novel clone of Salmonella Reading has emerged that coincides with increased abundance in raw turkey products and two outbreaks of human illness in North America. The rapid dissemination of this highly adapted and conserved clone indicates that it was likely obtained from a common source and rapidly disseminated across turkey production. Key genomic changes may have contributed to its apparent continued success in the barn environment, and ability to cause illness in humans. |
Rapid, sensitive, full genome sequencing of Severe Acute Respiratory Syndrome Virus Coronavirus 2 (SARS-CoV-2) (preprint)
Paden CR , Tao Y , Queen K , Zhang J , Li Y , Uehara A , Tong S . bioRxiv 2020 2020.04.22.055897 SARS-CoV-2 recently emerged, resulting a global pandemic. Rapid genomic information is critical to understanding transmission and pathogenesis. Here, we describe validated protocols for generating high-quality full-length genomes from primary samples. The first employs multiplex RT-PCR followed by MinION or MiSeq sequencing. The second uses singleplex, nested RT-PCR and Sanger sequencing.Competing Interest StatementThe authors have declared no competing interest. |
A Vibrio cholerae Core Genome Multilocus Sequence Typing Scheme to Facilitate the Epidemiological Study of Cholera (preprint)
Liang KYH , Orata FD , Islam MT , Nasreen T , Alam M , Tarr CL , Boucher YF . bioRxiv 2020 2020.01.27.919118 Core genome multilocus sequence typing (cgMLST) has gained popularity in recent years in epidemiological research and subspecies level classification. cgMLST retains the intuitive nature of traditional MLST but offers much greater resolution by utilizing significantly larger portions of the genome. Here, we introduce a cgMLST scheme for Vibrio cholerae, a bacterium abundant in marine and freshwater environments and the etiologic agent of cholera. A set of 2,443 core genes ubiquitous in V. cholerae were used to analyze a comprehensive dataset of 1,262 clinical and environmental strains collected from 52 countries, including 65 newly sequenced genomes in this study. We established a sublineage threshold based on 133 allelic differences that creates clusters nearly identical to traditional MLST types, providing backwards compatibility to new cgMLST classifications. We also defined an outbreak threshold based on seven allelic differences that is capable of identifying strains from the same outbreak and closely related isolates which could give clues on outbreak origin. Using cgMLST, we confirmed the South Asian origin of modern epidemics and identified clustering affinity among sublineages of environmental isolates from the same geographic origin. Advantages of this method are highlighted by direct comparison with existing classification methods, such as MLST and single nucleotide polymorphism-based methods. cgMLST outperforms all existing methods in terms of resolution, standardization, and ease-of-use. We anticipate this scheme will serve as a basis for a universally applicable and standardized classification system for V. cholerae research and epidemiological surveillance in the future. This cgMLST scheme is publicly available on PubMLST (https://pubmlst.org/vcholerae/).IMPORTANCE Toxigenic Vibrio cholerae of the O1 and O139 serogroups are the causative agent of cholera, an acute diarrheal disease that plagued the world for centuries, if not millennia. Here, we introduce a core genome multilocus sequence typing (cgMLST) scheme for V. cholerae. Using cgMLST, we established an outbreak threshold that can efficiently identify outbreak related strains and potential sources of introduction. We also defined a sublineage threshold that is similar to traditional MLST sequence type which will provide context to this new typing method by relating it to previous MLST results. cgMLST outperforms all existing methods in terms of resolution, standardization, and ease-of-use, making this scheme the most suitable method for V. cholerae typing and surveillance worldwide. |
Novel quinolone resistance determinant, qepA8, in Shigella flexneri isolated in the United States, 2016 (preprint)
Webb HE , Tagg KA , Chen JC , Kim J , Lindsey R , Francois Watkins LK , Karp BE , Sugawara Y , Folster JP . bioRxiv 2019 726950 A qepA8+ Shigella flexneri was cultured from the stool of a traveler returning from India and East Asia. This chromosomally encoded qepA variant, has a six-base insertion, and may have been mobilized as part of a complex IS1-mediated composite transposon including catA1, aadA1, and blaOXA-1. In laboratory E. coli, qepA8 alone only conferred decreased ciprofloxacin susceptibility; however, it may work in combination with additional mechanisms to confer clinical resistance. |
Preadaptation of pandemic GII.4 noroviruses in hidden virus reservoirs years before emergence (preprint)
Ruis C , Lindesmith LC , Mallory ML , Brewer-Jensen PD , Bryant JM , Costantini V , Monit C , Vinjé J , Baric RS , Goldstein RA , Breuer J . bioRxiv 2019 658765 The control of pandemic pathogens depends on early prediction of pandemic variants and, more generally, understanding origins of such variants and factors that drive their global spread. This is especially important for GII.4 norovirus, where vaccines under development offer promise to prevent hundreds of millions of annual gastroenteritis cases. Previous studies have suggested that new GII.4 pandemic viruses evolve from previous pandemic variants through substitutions in the antigenic region of the VP1 protein that enable evasion of host population immunity, leading to global spread. In contrast, we show here that the acquisition of new genetic and antigenic characteristics is not the proximal driver of new pandemics. Instead, pandemic GII.4 viruses circulate undetected for years before causing a new pandemic, during which time they diversify and spread over wide geographical areas. Serological data demonstrate that by 2003, some nine years before it emerged as a new pandemic, the ancestral 2012 pandemic strain had already acquired the antigenic characteristics that allowed it to evade prevailing population immunity against the previous 2009 pandemic variant. These results provide strong evidence that viral genetic changes are necessary but not sufficient for GII.4 pandemic spread. Instead, we suggest that it is changes in host population immunity that enable pandemic spread of an antigenically-preadapted GII.4 variant. These results indicate that predicting future GII.4 pandemic variants will require surveillance of currently unsampled reservoir populations. Furthermore, a broadly acting GII.4 vaccine will be critical to prevent future pandemics.Significance Norovirus pandemics and their associated public health and economic costs could be prevented by effective vaccines. However, vaccine development and distribution will require identification of the sources and drivers of new pandemics. We here use phylogenetics and serological experiments to develop and test a new hypothesis of pandemic norovirus emergence. We find that pandemic noroviruses preadapt, diversify and spread worldwide years prior to emergence, strongly indicating that genetic changes are necessary but not sufficient to drive a new pandemic. We instead suggest that changes in population immunity enable pandemic emergence of a pre-adapted low-level variant. These findings indicate that prediction of new pandemics will require surveillance of under-sampled virus reservoirs and that norovirus vaccines will need to elicit broad immunity. |
The effect of variant interference on de novo assembly for viral deep sequencing (preprint)
Castro CJ , Marine RL , Ramos E , Ng TFF . bioRxiv 2019 815480 Viruses have high mutation rates and generally exist as a mixture of variants in biological samples. Next-generation sequencing (NGS) approach has surpassed Sanger for generating long viral sequences, yet how variants affect NGS de novo assembly remains largely unexplored. Our results from >15,000 simulated experiments showed that presence of variants can turn an assembly of one genome into tens to thousands of contigs. This “variant interference” (VI) is highly consistent and reproducible by ten most used de novo assemblers, and occurs independent of genome length, read length, and GC content. The main driver of VI is pairwise identities between viral variants. These findings were further supported by in silico simulations, where selective removal of minor variant reads from clinical datasets allow the “rescue” of full viral genomes from fragmented contigs. These results call for careful interpretation of contigs and contig numbers from de novo assembly in viral deep sequencing. |
Fast estimation of genetic relatedness between members of heterogeneous populations of closely related genomic variants (preprint)
Tsyvina V , Campo DS , Sims S , Zelikovsky A , Khudyakov Y , Skums P . bioRxiv 2018 324418 Many biological analysis tasks require extraction of families of genetically similar sequences from large datasets produced by Next-generation Sequencing (NGS). Such tasks include detection of viral transmissions by analysis of all genetically close pairs of sequences from viral datasets sampled from infected individuals or studying of evolution of viruses or immune repertoires by analysis of network of intra-host viral variants or antibody clonotypes formed by genetically close sequences. The most obvious naϊeve algorithms to extract such sequence families are impractical in light of the massive size of modern NGS datasets. In this paper, we present fast and scalable k-mer-based framework to perform such sequence similarity queries efficiently, which specifically targets data produced by deep sequencing of heterogeneous populations such as viruses. The tool is freely available for download at https://github.com/vyacheslav-tsivina/signature-sj |
Genome-wide patterns of gene expression in a wild primate indicate species-specific mechanisms associated with tolerance to natural simian immunodeficiency virus infection (preprint)
Simons ND , Eick GN , Ruiz-Lopez MJ , Hyeroba D , Omeja PA , Weny G , Chapman CA , Goldberg TL , Zheng H , Shankar A , Switzer WM , Frost SDW , Jones JH , Sterner KN , Ting N . bioRxiv 2018 395152 Over 40 species of nonhuman primates host simian immunodeficiency viruses (SIVs). In natural hosts, infection is generally assumed to be nonpathogenic due to a long coevolutionary history between host and virus, although pathogenicity is difficult to study in wild nonhuman primates. We used whole-blood RNA-seq and SIV prevalence from 29 wild Ugandan red colobus (Piliocolobus tephrosceles) to assess the effects of SIV infection on host gene expression in wild, naturally SIV-infected primates. We found no evidence for chronic immune activation in infected individuals, suggesting that SIV is not immunocompromising in this species, in contrast to HIV in humans. Notably, an immunosuppressive gene, CD101, was upregulated in infected individuals. This gene has not been previously described in the context of nonpathogenic SIV infection. This expands the known variation associated with SIV infection in natural hosts, and may suggest a novel mechanism for tolerance of SIV infection in the Ugandan red colobus. |
Genomic basis of multidrug-resistance, mating, and virulence in Candida auris and related emerging species (preprint)
Munoz JF , Gade L , Chow NA , Loparev VN , Juieng P , Berkow EL , Farrer RA , Litvintseva AP , Cuomo CA . bioRxiv 2018 299917 Candida auris is an emergent fungal pathogen of rising public health concern due to increasing reports of outbreaks in healthcare settings and resistance to multiple classes of antifungal drugs. While distantly related to the more common pathogens C. albicans and C. glabrata, C. auris is closely related to three rarely observed and often multidrug-resistant species, C. haemulonii, C. duobushaemulonii and C. pseudohaemulonii. Here, we generated and analyzed near complete genome assemblies and RNA-Seq-guided gene predictions for isolates from each of the four major C. auris clades and for C. haemulonii, C. duobushaemulonii and C. pseudohaemulonii. Our analyses mapped seven chromosomes and revealed chromosomal rearrangements between C. auris clades and related species. We found conservation of genes involved in mating and meiosis and identified both MTLa and MTLα C. auris isolates, suggesting the potential for mating between clades. Gene conservation analysis highlighted that many genes linked to drug resistance and virulence in other pathogenic Candida species are conserved in C. auris and related species including expanded families of transporters and lipases, as well as mutations and copy number variants in ERG11 that confer drug resistance. In addition, we found genetic features of the emerging species that likely underlie differences in virulence and drug response between these and other Candida species, including genes involved in cell wall structure. To begin to characterize the species-specific genes important for antifungal response, we profiled the gene expression of C. auris in response to voriconazole and amphotericin B and found induction of several transporters and metabolic regulators that may play a role in drug resistance. This study provides a comprehensive view of the genomic basis of drug resistance, potential for mating, and virulence in this emerging fungal clade. |
The contribution of parent-to-offspring transmission of telomeres to the heritability of telomere length in humans (preprint)
Delgado DA , Zhang C , Demanelis K , Chen LS , Gao J , Roy S , Shinkle J , Sabarinathan M , Argos M , Tong L , Ahmed A , Islam T , Rakibuz-Zaman M , Sarwar G , Shahriar H , Rahman M , Yunus M , Doherty JA , Jasmine F , Kibriya MG , Ahsan H , Pierce BL . bioRxiv 2018 276030 Leukocyte telomere length (LTL) is a heritable trait with two potential sources of heritability (h2): inherited variation in non-telomeric regions (e.g., SNPs that influence telomere maintenance) and variability in the lengths of telomeres in gametes that produce offspring zygotes (i.e., “direct” inheritance). Prior studies of LTL h2 have not attempted to disentangle these two sources. Here, we use a novel approach for detecting the direct inheritance of telomeres by studying the association between identity-by-descent (IBD) sharing at chromosome ends and phenotypic similarity in LTL. We measured genome-wide SNPs and LTL for a sample of 5,069 Bangladeshi adults with substantial relatedness. For each of the 7,254 relative pairs identified, we used SNPs near the telomeres to estimate the number of chromosome ends shared IBD, a proxy for the number of telomeres shared IBD (Tshared). We then estimated the association between Tshared and the squared pairwise difference in LTL ((ΔLTL)2) within various classes of relatives (siblings, avuncular, cousins, and distant), adjusting for overall genetic relatedness (ϕ). The association between Tshared and (ΔLTL)2 was inverse among all relative pair types. In a meta-analysis including all relative pairs (ϕ >0.05), the association between Tshared and (ΔLTL)2 (P=0.002) was stronger than the association between ϕ and (ΔLTL)2 (P=0.45). Our results provide strong evidence that telomere length (TL) in parental germ cells impacts TL in offspring cells and contributes to LTL h2 despite telomere “reprogramming” during embryonic development. Applying our method to larger studies will enable robust estimation of LTL h2 attributable to direction transmission. |
Direct RNA Sequencing of the Complete Influenza A Virus Genome (preprint)
Keller MW , Rambo-Martin BL , Wilson MM , Ridenour CA , Shepard SS , Stark TJ , Neuhaus EB , Dugan VG , Wentworth DE , Barnes JR . bioRxiv 2018 300384 For the first time, a complete genome of an RNA virus has been sequenced in its original form. Previously, RNA was sequenced by the chemical degradation of radiolabelled RNA, a difficult method that produced only short sequences. Instead, RNA has usually been sequenced indirectly by copying it into cDNA, which is often amplified to dsDNA by PCR and subsequently analyzed using a variety of DNA sequencing methods. We designed an adapter to short highly conserved termini of the influenza virus genome to target the (-) sense RNA into a protein nanopore on the Oxford Nanopore MinION sequencing platform. Utilizing this method and total RNA extracted from the allantoic fluid of infected chicken eggs, we demonstrate successful sequencing of the complete influenza virus genome with 100% nucleotide coverage, 99% consensus identity, and 99% of reads mapped to influenza. By utilizing the same methodology we can redesign the adapter in order to expand the targets to include viral mRNA and (+) sense cRNA, which are essential to the viral life cycle. This has the potential to identify and quantify splice variants and base modifications, which are not practically measurable with current methods. |
Quantifying the Risk and Cost of Active Monitoring for Infectious Diseases (preprint)
Reich NG , Lessler J , Varma JK , Vora NM . bioRxiv 2017 156497 During outbreaks of deadly emerging pathogens (e.g., Ebola, MERS-CoV) and bioterror threats (e.g., smallpox), actively monitoring potentially infected individuals aims to limit disease transmission and morbidity. Guidance issued by CDC on active monitoring was a cornerstone of its response to the West Africa Ebola outbreak. There are limited data on how to balance the costs and performance of this important public health activity. We present a framework that estimates the risks and costs of specific durations of active monitoring for pathogens of significant public health concern. We analyze data from New York City’s Ebola active monitoring program over a 16-month period in 2014-2016. For monitored individuals, we identified unique durations of active monitoring that minimize expected costs for those at “low (but not zero) risk” and “some or high risk”: 21 and 31 days, respectively. Extending our analysis to smallpox and MERS-CoV, we found that the optimal length of active monitoring relative to the median incubation period was reduced compared to Ebola due to less variable incubation periods. Active monitoring can save lives but is expensive. Resources can be most effectively allocated by using exposure-risk categories to modify the duration or intensity of active monitoring. |
Tracing the evolutionary history and global expansion of Candida auris using population genomic analyses (preprint)
Chow NA , Munoz JF , Gade L , Berkow EL , Li X , Welsh RM , Forsberg K , Lockhart SR , Adam R , Alanio A , Alastruey-Izquierdo A , Althawadi S , Arauz AB , Ben-Ami R , Bharat A , Calvo B , Desnos-Ollivier M , Escandon P , Gardam D , Gunturu R , Heath CH , Kurzai O , Martin R , Litvintseva AP , Cuomo CA . bioRxiv 2020 2020.01.06.896548 Candida auris has emerged globally as a multidrug-resistant yeast that can spread via nosocomial transmission. An initial phylogenetic study of isolates from Japan, India, Pakistan, South Africa, and Venezuela revealed four populations (Clades I, II, III, and IV) corresponding to these geographic regions. Since this description, C. auris has been reported in over 30 additional countries. To trace this global emergence, we compared the genomes of 304 C. auris isolates from 19 countries on six continents. We found that four predominant clades persist across wide geographic locations. We observed phylogeographic mixing in most clades; Clade IV, with isolates mainly from South America, demonstrated the strongest phylogeographic substructure. C. auris isolates from two clades with opposite mating types were detected contemporaneously in a single healthcare facility in Kenya. We estimated a Bayesian molecular clock phylogeny and dated the origin of each clade within the last 339 years; outbreak-causing clusters from Clades I, III, and IV originated 34-35 years ago. We observed high rates of antifungal resistance in Clade I, including four isolates resistant to all three major classes of antifungals. Mutations that contribute to resistance varied between the clades, with Y132F in ERG11 as the most widespread mutation associated with azole resistance and S639P in FKS1 for echinocandin resistance. Copy number variants in ERG11 predominantly appeared in Clade III and were associated with fluconazole resistance. These results provide a global context for the phylogeography, population structure, and mechanisms associated with antifungal resistance in C. auris.Importance In less than a decade, C. auris has emerged in healthcare settings worldwide; this species is capable of colonizing skin and causing outbreaks of invasive candidiasis. In contrast to other Candida species, C. auris is unique in its ability to spread via nosocomial transmission and its high rates of drug resistance. As part of the public health response, whole-genome sequencing has played a major role in characterizing transmission dynamics and detecting new C. auris introductions. Through a global collaboration, we assessed genome evolution of isolates of C. auris from 19 countries. Here, we described estimated timing of the expansion of each C. auris clade and of fluconazole resistance, characterized discrete phylogeographic population structure of each clade, and compared genome data to sensitivity measurements to describe how antifungal resistance mechanisms vary across the population. These efforts are critical for a sustained, robust public health response that effectively utilizes molecular epidemiology. |
Mutations in TAC1B: a novel genetic determinant of clinical fluconazole resistance in C. auris (preprint)
Rybak JM , Munoz JF , Barker KS , Parker JE , Esquivel BD , Berkow EL , Lockhart SR , Gade L , Palmer GE , White TC , Kelly SL , Cuomo CA , Rogers PD . bioRxiv 2020 2020.02.18.955534 Candida auris has emerged as a multidrug-resistant pathogen of great clinical concern. Approximately 90% of clinical C. auris isolates are resistant to fluconazole, the most commonly prescribed antifungal agent, yet it remains unknown what mechanisms underpin this fluconazole resistance. To identify novel mechanisms contributing to fluconazole resistance in C. auris, the fluconazole-susceptible C. auris clinical isolate AR0387 was passaged in media supplemented with fluconazole to generate derivative strains which had acquired increased fluconazole resistance in vitro. Comparative analysis of comprehensive sterol profiles, [3H]-fluconazole uptake, sequencing of C. auris genes homologous to genes known to contribute to fluconazole resistance in other species of Candida, and the relative expression of C. auris ERG11, CDR1, and MDR1 were performed. All fluconazole-evolved derivative strains were found to have acquired mutations in the zinc-cluster transcription factor-encoding gene, TAC1B, and a corresponding increase in CDR1 expression relative to the parental clinical isolate, AR0387. Mutations in TAC1B were also identified in a set of 304 globally distributed C. auris clinical isolates representing each of the four major clades. Introduction of the most common mutation found among fluconazole-resistant clinical isolates of C. auris into the fluconazole-susceptible isolate AR0387, was confirmed to increase fluconazole resistance by 8-fold, and the correction of the same mutation in a fluconazole-resistant isolate, AR0390, decreased fluconazole MIC by 16-fold. Taken together, these data demonstrate that C. auris can rapidly acquire resistance to fluconazole in-vitro, and that mutations in TAC1B significantly contribute to clinical fluconazole resistance.IMPORTANCE Candida auris is an emerging multidrug-resistant pathogen of global concern, known to be responsible for outbreaks on six continents and commonly resistant to antifungals. While the vast majority of clinical C. auris isolates are highly resistant to fluconazole, an essential part of the available antifungal arsenal, very little is known about the mechanisms contributing to resistance. In this work, we show that mutations in the transcription factor TAC1B significantly contribute to clinical fluconazole resistance. These studies demonstrate that mutations in TAC1B can arise rapidly in vitro upon exposure to fluconazole, and that a multitude of resistance-associated TAC1B mutations are present among the majority of fluconazole-resistant C. auris isolates from a global collection and appear specific to a subset of lineages or clades. Thus, identification of this novel genetic determinant of resistance significantly adds to the understanding of clinical antifungal resistance in C. auris. |
Laboratory Analysis of an Outbreak of Candida auris in New York from 2016 to 2018-Impact and Lessons Learned (preprint)
Zhu Y , O'Brien B , Leach L , Clark A , Bates M , Adams E , Ostrowsky B , Quinn M , Dufort E , Southwick K , Erazo R , Haley VB , Bucher C , Chaturvedi V , Limberger RJ , Blog D , Lutterloh E , Chaturvedi S . bioRxiv 2019 760090 Candida auris is a multidrug-resistant yeast which has emerged in healthcare facilities worldwide, however little is known about identification methods, patient colonization, spread, environmental survival, and drug resistance. Colonization on both biotic and abiotic surfaces, along with travel, appear to be the major factors for the spread of this pathogen across the globe. In this investigation, we present laboratory findings from an ongoing C. auris outbreak in NY from August 2016 through 2018. A total of 540 clinical isolates, 11,035 patient surveillance specimens, and 3,672 environmental surveillance samples were analyzed. Laboratory methods included matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for yeast isolate identification, real-time PCR for rapid surveillance sample screening, culture on selective/non-selective media for recovery of C. auris and other yeasts from surveillance samples, antifungal susceptibility testing to determine the C. auris resistance profile, and Sanger sequencing of ribosomal genes for C. auris genotyping. Results included: a) identification and confirmation of C. auris in 413 clinical isolates and 931 patient surveillance isolates, as well as identification of 277 clinical cases and 350 colonized cases from 151 healthcare facilities including 59 hospitals, 92 nursing homes, 1 long-term acute care hospital (LTACH), and 2 hospices, b) successful utilization of an in-house developed C. auris real-time PCR assay for the rapid screening of patient and environmental surveillance samples, c) demonstration of relatively heavier colonization of C. auris in nares compared to the axilla/groin, and d) predominance of the South Asia Clade I with intrinsic resistance to fluconazole and elevated minimum inhibitory concentration (MIC) to voriconazole (81%), amphotericin B (61%), 5-FC (3%) and echinocandins (1%). These findings reflect greater regional prevalence and incidence of C. auris and the deployment of better detection tools in an unprecedented outbreak. |
Quantifying the roles of vomiting, diarrhea, and residents vs. staff in norovirus transmission in U.S. nursing home outbreaks (preprint)
Adams C , Young D , Gastanaduy PA , Paul P , Marsh Z , Hall AJ , Lopman BA . bioRxiv 2019 707356 The role of individual case characteristics, such as symptoms or demographics, in norovirus transmissibility is poorly understood. Six nursing home norovirus outbreaks occurring in South Carolina, U.S. from 2014 to 2016 were examined. We aimed to quantify the contribution of symptoms and other case characteristics in norovirus transmission using the reproduction number (REi) as an estimate of individual case infectivity and to examine how transmission changes over the course of an outbreak. Individual estimates of REi were calculated using a maximum likelihood procedure to infer the average number of secondary cases generated by each case. The associations between case characteristics and REi were estimated using a multivariate mixed linear model. Outbreaks began with one to three index case(s) with large estimated REi’s (range: 1.48 to 8.70) relative to subsequent cases. Of the 209 cases, 155 (75%) vomited, 164 (79%) had diarrhea, and 158 (76%) were nursing home residents (vs. staff). Cases who vomited infected 2.74 (95% CI: 1.90, 3.94) more individuals than non-vomiters, cases with diarrhea infected 1.62 (95% CI: 1.09, 2.41) more individuals than cases without diarrhea, and resident-cases infected 1.69 (95% CI: 1.18, 2.42) more individuals than staff-cases. Index cases tended to be residents (vs. staff) who vomited and infected considerably more secondary cases compared to non-index cases. Results suggest that individuals, particularly residents, who vomit are more infectious and tend to drive norovirus transmission in U.S. nursing home norovirus outbreaks. While diarrhea also plays a role in norovirus transmission, it is to a lesser degree than vomiting in these settings. Results lend support for prevention and control measures that focus on cases who vomit, particularly if those cases are residents.Author summary The majority of all norovirus outbreaks reported to the CDC occur in long-term care facilities (LTCFs), including nursing homes, where older residents are at risk for more severe or prolonged infection. Because there is currently no publicly available norovirus vaccine, sound control measures are key to controlling norovirus outbreaks, but there is little evidence that standard control measures are effective in reducing the size and/or duration of LTCF norovirus outbreaks. Hence, studies leading to a better understanding of disease spread and prevention of additional cases, and thus more effective control measures, are needed. To this end, we aimed to quantify factors associated with norovirus transmission and to examine how transmission changes over the course of an outbreak. We show that vomiting and, to a lesser extent, diarrhea are critical in initiating and sustaining norovirus transmission in U.S. nursing home norovirus outbreaks. We also show that nursing home residents, rather than staff, are the primary drivers of transmission. Results suggest that control measures focusing on cases who vomit, particularly if those cases are residents, would be most effective at curtailing norovirus transmission in these settings. |
In Silico Identification of Three Types of Integrative and Conjugative Elements (ICEs) in Elizabethkingia anophelis Strains Isolated from Around the World (preprint)
Xu J , Pei D , Nicholson A , Lan Y , Xia Q . bioRxiv 2018 402107 Elizabethkingia anophelis is an emerging global multidrug-resistant opportunistic pathogen. We assessed the diversity among 13 complete genomes and 23 draft genomes of E. anophelis derived from various environmental settings and human infections from different geographic regions around the world over past decades from 1950s. Thirty-one of these 36 strains harbor integrative and conjugative elements (ICEs). A total of 52 ICEs were identified, and categorized into three ICE types based on the architecture of signature genes in the conjugation module. The type II and III ICEs were found to integrate into regions adjacent to tRNA genes, while type I ICEs used a variety of integration sites, inserting into intergenic regions or even directly into a gene, sometimes disrupting gene function. Integrases such as tyrosine recombinases, serine recombinases and DDE transposases were found in most ICEs. The ICEs carry various cargo genes including transcription regulators and those involved in antibiotic resistance. The CRISPR-Cas system was found in nine strains, including four strains in which CRISPR-Cas machinery and ICEs co-exist. ICE distribution in the strains showed no geographic or temporal patterns. The ICEs in E. anophelis differ in gene structure and sequence from CTnDOT, a well-studied ICE prevalent in Bacteroides spp. This is the first set of ICEs identified in the family Flavobacteriaceae. As a prevalent type of mobile genetic elements in various strains of E. anophelis around the world, the categorization of ICEs will facilitate further investigations such as virulence, genome epidemiology and adaptation genomics of E. anophelis.Importance Elizabethkingia anophelis is an opportunistic human pathogen, and the genetic diversity between strains from around the world becomes apparent as more genomes are sequenced. The Integrative Conjugative Element (ICE), found in many bacterial species, contains genes for transfer via conjugation and integration into the chromosome, along with various cargo genes. ICEs are identified in 31 of 36 strains and categorized into three types based on architecture of modular genes, integrases, and integration sites. ICE distribution in different strains displays no spatial and temporal patterns. Several ICE-containing strains also possessed CRISPR-Cas units, considered to be the bacterial adaptive immune system providing protection against phage and predatory mobile genetic elements. This co-existence suggests that ICEs are beneficial or at least not harmful to the bacterial cells they inhabit. ICEs as a component of the mobile genetic repertoire enable recipients to resist antibiotics, survive disinfecting agents, and adapt to various ecological niches. |
Droplet rather than Aerosol Mediated Dispersion is the Primary Mechanism of Bacterial transmission from Contaminated Hand Washing Sink Traps (preprint)
Kotay S , Donlan RM , Ganim C , Barry K , Christensen BE , Mathers AJ . bioRxiv 2018 392431 An alarming rise in hospital outbreaks implicating hand-washing sinks has led to widespread acknowledgement that sinks are a major reservoir of antibiotic resistant pathogens in patient-care areas. An earlier study using a GFP-expressing Escherichia coli (GFP-E. coli) as a model organism demonstrated dispersal from drain biofilm in contaminated sinks. The present study further characterizes the dispersal of microorganisms from contaminated sinks. Replicate hand-washing sinks were inoculated with GFP-E. coli, and dispersion was measured using qualitative (settle plates) and quantitative (air sampling) methods. Dispersal caused by faucet water was captured with settle plates and air sampling methods when bacteria were present on the drain. In contrast, no dispersal was captured without or in between faucet events amending earlier theory that bacteria aerosolize from P-trap and disperse. Numbers of dispersed GFP-E. coli diminished substantially within 30 minutes after faucet usage, suggesting that the organisms were associated with larger droplet-sized particles that are not suspended in the air for long periods.IMPORTANCE Among the possible environmental reservoirs in a patient care environment, sink drains are increasingly recognized as potential reservoir of multidrug resistant healthcare-associated pathogens to hospitalized patients. With increasing antimicrobial resistance limiting therapeutic options for patients, better understanding of how pathogens disseminate from sink drains is urgently needed. Once this knowledge gap has decreased, interventions can be engineered to decrease or eliminate transmission from hospital sink drains to patients. The current study further defines the mechanisms of transmission for bacteria colonizing sink drains. |
Integrating genotypes and phenotypes improves long-term forecasts of seasonal influenza A/H3N2 evolution (preprint)
Huddleston J , Barnes JR , Rowe T , Xu X , Kondor R , Wentworth DE , Whittaker L , Ermetal B , Daniels RS , McCauley JW , Fujisaki S , Nakamura K , Kishida N , Watanabe S , Hasegawa H , Barr I , Subbarao K , Neher RA , Bedford T . bioRxiv 2020 2020.06.12.145151 Seasonal influenza virus A/H3N2 is a major cause of death globally. Vaccination remains the most effective preventative. Rapid mutation of hemagglutinin allows viruses to escape adaptive immunity. This antigenic drift necessitates regular vaccine updates. Effective vaccine strains need to represent H3N2 populations circulating one year after strain selection. Experts select strains based on experimental measurements of antigenic drift and predictions made by models from hemagglutinin sequences. We developed a novel influenza forecasting framework that integrates phenotypic measures of antigenic drift and functional constraint with previously published sequence-only fitness estimates. Forecasts informed by phenotypic measures of antigenic drift consistently outperformed previous sequence-only estimates, while sequence-only estimates of functional constraint surpassed more comprehensive experimentally-informed estimates. Importantly, the best models integrated estimates of both functional constraint and either antigenic drift phenotypes or recent population growth.Competing Interest StatementThe authors have declared no competing interest. |
Modeling cholera transmission and vaccination in a refugee camp (preprint)
Havumaki J , Meza R , Phares CR , Date K , Eisenberg MC . bioRxiv 2019 514406 Background Cholera remains a major public health concern, particularly in refugee camps, which may contend with overcrowding and scarcity of resources. Maela, the largest long-standing refugee camp in Thailand, experienced four cholera outbreaks between 2005 and 2010. In 2013, a cholera vaccine campaign was implemented in the camp. To assist in the evaluation of the campaign, we developed a mathematical model of cholera in Maela.Methodology/Principal Findings We formulated a Susceptible-Infectious-Water-Recovered-based cholera transmission model and estimated parameters using incidence data from 2010. We next evaluated the reduction in cases conferred by several immunization strategies, varying timing, effectiveness, and resources (i.e., vaccine availability). Finally, we generated post-campaign case forecasts, to determine whether a booster campaign was needed. Using parameters from our calibrated model, our analyses suggest that preexposure vaccination can substantially reduce the risk of cholera even when the < 50% of the population is given the full two-dose series. Additionally, the preferred number of doses per person should be considered in the context of one vs. two dose effectiveness and vaccine availability. For reactive vaccination, a trade-off between timing and effectiveness was revealed, indicating that it may be beneficial to give one dose to more people rather than two doses to fewer people, which would incur a delay in administration of the second dose. Forecasting using realistic coverage levels indicated that there was no need for a booster campaign in 2014.Conclusions/Significance Our analyses suggest that vaccination in conjunction with water sanitation and hygiene improvements provides an effective strategy for cholera outbreaks in refugee camps. Effective preexposure vaccination depends on timing and effectiveness. If a camp is facing an ongoing outbreak, delayed distribution of vaccines can substantially alter the effectiveness of reactive vaccination, suggesting that quick distribution of vaccines may be more important than ensuring every individual receives both vaccine doses.Author summary We developed an age-structured Susceptible-Infectious-Water-Recovered (SIWR)-based transmission model to consider different cholera vaccination strategies in Maela, the largest long-standing refugee camp in Thailand. Our model was fit to cholera incidence data from 2010 and was in part parameterized by demographic data collected from the camp. We considered multiple scenarios, including both a theoretical exploration of the effects of variation in timing, effectiveness and supply, as well as the real-world coverage of vaccine in Maela. The preferred number of doses per person and timing of vaccination campaigns should be considered in the context of one vs. two dose effectiveness and logistical constraints. Importantly, our analysis coincided with an actual cholera vaccination campaign in the camp and was used to evaluate the campaign and to help determine that there was no need for a follow-up booster campaign. The setting of our analysis is particularly relevant given the recent worldwide increase in total numbers of refugees. Results from our model highlight the utility of vaccination to prevent cholera. Vaccination campaigns can be combined with more permanent water, sanitation, and hygiene infrastructure improvements to reduce the risk of cholera and other enteric disease epidemics. Overall, this study demonstrates that mathematical modeling can generate useful insights into real-world policy decisions. |
Combining serological and contact data to derive target immunity levels for achieving and maintaining measles elimination (preprint)
Funk S , Knapp JK , Lebo E , Reef SE , Dabbagh AJ , Kretsinger K , Jit M , Edmunds WJ , Strebel PM . bioRxiv 2019 201574 Background Vaccination has reduced the global incidence of measles to the lowest rates in history. However, local interruption of measles virus transmission requires sustained high levels of population immunity that can be challenging to achieve and maintain. The herd immunity threshold for measles is typically stipulated at 90–95%. This figure does not easily translate into age-specific immunity levels required to interrupt transmission. Previous estimates of such levels were based on speculative contact patterns based on historical data from high-income countries. The aim of this study was to determine age-specific immunity levels that would ensure elimination of measles when taking into account empirically observed contact patterns.Methods We combined estimated immunity levels from serological data in 17 countries with studies of age-specific mixing patterns to derive contact-adjusted immunity levels. We then compared these to case data from the 10 years following the seroprevalence studies to establish a contact-adjusted immunity threshold for elimination. We lastly combined a range of hypothetical immunity profiles with contact data from a wide range of socioeconomic and demographic settings to determine whether they would be sufficient for elimination.Results We found that contact-adjusted immunity levels were able to predict whether countries would experience outbreaks in the decade following the serological studies in about 70% of countries. The corresponding threshold level of contact-adjusted immunity was found to be 93%, corresponding to an average basic reproduction number of approximately 14. Testing different scenarios of immunity with this threshold level using contact studies from around the world, we found that 95% immunity would have to be achieved by the age of five and maintained across older age groups to guarantee elimination. This reflects a greater level of immunity required in 5–9 year olds than established previously.Conclusions The immunity levels we found necessary for measles elimination are higher than previous guidance. The importance of achieving high immunity levels in 5–9 year olds presents both a challenge and an opportunity. While such high levels can be difficult to achieve, school entry provides an opportunity to ensure sufficient vaccination coverage. Combined with observations of contact patterns, further national and sub-national serological studies could serve to highlight key gaps in immunity that need to be filled in order to achieve national and regional measles elimination.ESEN2European Sero-Epidemiology Network 2EUROEuropean RegionMCEMisclassification errorWHOWorld Health Organization |
Early signals of vaccine driven perturbation seen in pneumococcal carriage population genomic data (preprint)
Chaguza C , Heinsbroek E , Gladstone RA , Tafatatha T , Alaerts M , Peno C , Cornick JE , Musicha P , Bar-Zeev N , Kamng'ona A , Kadioglu A , McGee L , Hanage WP , Breiman RF , Heyderman RS , French N , Everett DB , Bentley SD . bioRxiv 2018 459693 Pneumococcal conjugate vaccines (PCV) have reduced pneumococcal diseases globally. Despite this, much remains to be learned about their effect on pathogen population structure. Here we undertook whole genome sequencing of 660 pneumococcal strains from asymptomatic carriers to investigate population restructuring in pneumococcal strains sampled before and after PCV13 introduction in a previously vaccine-naïve setting. We show substantial decreasing frequency of vaccine-type (VT) strains and their strain diversity post-vaccination in the vaccinated but not unvaccinated age groups indicative of direct but limited or delayed indirect effect of vaccination. Clearance of identical VT serotypes associated with multiple lineages occurred regardless of their genetic background. Interestingly, despite the increasing frequency of non-vaccine type (NVT) strains through serotype replacement, the serotype diversity was not fully restored to the levels observed prior to vaccination implying limited serotype replacement. The frequency of antibiotic resistant strains was low and remained largely unchanged post-vaccination but intermediate-penicillin-resistant lineages were reduced in the post vaccine population. Significant perturbations marked by changing frequency of accessory genes associated with diverse functions especially mobile genetic elements and bacteriocin activity were detected. This phylogenomic analysis demonstrates early vaccine-induced pneumococcal population restructuring not only at serotype but also accessory genome level.Author summary Different formulations of PCVs have been effective in reducing the invasive pneumococcal disease burden globally. Clinical trials have started to indicate high impact and effectiveness of PCV13 in Sub Saharan Africa (SSA) but there is limited understanding of how the introduction of PCVs alters the population structure of pneumococcal strains at serotype and genomic level. Here we investigated this using pneumococcal strains sampled pre‐ and post-PCV13 introduction from a previously vaccine naïve setting in Northern Malawi. Our findings reveal decrease in frequency of VT serotypes and their associated lineages in the largely vaccinated under-five population but not older individuals indicating a direct but limited or delayed indirect protection. The diversity of serotypes also decreased post-vaccination in VT strains in the under-fives but there was no change in NVT strains suggesting incomplete serotype replacement. At the genomic level, logistic regression revealed changing frequency of accessory genes largely associated with mobile genetic elements but such changes did not include any antibiotic resistance genes. These findings show significant perturbations at serotype and accessory genome level in carried pneumococcal population after two years from PCV13 introduction but the pneumococcal population was still perturbed and had not returned to a new equilibrium state. |
Evaluation of correlates of protection against influenza A(H3N2) and A(H1N1)pdm09 infection: Applications to the hospitalized patient population (preprint)
Petrie JG , Martin ET , Truscon R , Johnson E , Cheng CK , McSpadden EJ , Malosh RE , Lauring AS , Lamerato LE , Eichelberger MC , Ferdinands JM , Monto AS . bioRxiv 2018 416628 Background Influenza vaccines are important for prevention of influenza-associated hospitalization. Assessments of serologic correlates of protection can support interpretation of influenza vaccine effectiveness evaluations in hospitalized populations.Methods Serum specimens collected at admission from adults hospitalized for treatment of acute respiratory illnesses during two influenza seasons were tested in hemagglutination-inhibition (HAI) and neuraminidase-inhibition (NAI) assays. We evaluated the suitability of these specimens as proxies for pre-infection immune status, and measured associations between antibody titers and influenza vaccination and infectionResults Specimens were collected within 3 days of illness onset from 65% of participants; geometric mean titers (GMTs) did not vary by day of collection. In both seasons, vaccinated participants had higher HAI and NAI GMTs than unvaccinated participants. HAI titers against the 2014-2015 A(H3N2) vaccine strain did not correlate with protection from infection with antigenically-drifted A(H3N2) viruses that circulated that season. In contrast, higher HAI titers against the A(H1N1)pdm09 vaccine strain were associated with reduced odds of A(H1N1)pdm09 infection in 2015-2016.Conclusions Serum collected after hospital admission can be used to assess correlates of protection against influenza infection. Broader implementation of similar studies would provide an opportunity to understand the successes and shortcomings of current influenza vaccines.We thank Jin Gao and Laura Couzens for technical support and are indebted to St Jude Children’s Research Hospital for plasmids that were used to generate reassortant influenza viruses. |
Guiding Vaccine Efficacy Trial Design During Public Health Emergencies: An interactive web-based decision support tool (preprint)
Bellan SE , Eggo RM , Gsell PS , Kucharski AJ , Dean NE , Donohue R , Zook M , Odhiambo F , Longini IM Jr , Brisson M , Mahon BE , Henao-Restrepo AM . bioRxiv 2018 252783 The design and execution of rigorous, fast, and ethical vaccine efficacy trials can be challenging during epidemics of emerging pathogens, such as the 2014-2016 Ebola virus and 2015-2016 Zika virus epidemics. Response to an urgent public health crisis requires accelerated research even as emerging epidemics themselves change rapidly and are inherently less well understood than well-established diseases. As part of the World Health Organization Research and Development Blueprint, we designed a web-based interactive decision support system (InterVax-Tool) to help diverse stakeholders navigate the epidemiological, logistical, and ethical decisions involved in designing a vaccine efficacy trial during a public health emergency. In contrast to existing literature on trial design, InterVax-Tool offers high-level visual and interactive assistance through a set of four decision trees, guiding users through selection of 1) the Primary Endpoint, (2) the Target Population, (3) Randomization, and (4) the Comparator. Guidance is provided on how each of fourteen key considerations–grouped as Epidemiological, Vaccine-related, Infrastructural, or Sociocultural–should be used to inform each decision in the trial design process. The tool is not intended to provide a black box decision framework for identifying an optimal trial design, but rather to facilitate transparent, collaborative and comprehensive discussion of the relevant decisions, while recording the decision process. The tool can also assist capacity building by providing a cross-disciplinary picture of trial design using concepts from epidemiology, study design, vaccinology, biostatistics, mathematical modeling and clinical research ethics. Here, we describe the goals and features of InterVax-Tool as well as its application to the design of a Zika vaccine efficacy trial.One Sentence Summary An interactive web-based decision support tool was developed to assist in the design of vaccine efficacy trials during emerging outbreaks. |
Heterogeneous susceptibility to rotavirus infection and gastroenteritis in two birth cohort studies: parameter estimation and epidemiological implications (preprint)
Lewnard JA , Lopman BA , Parashar UD , Bennett A , Bar-Zeev N , Cunliffe NA , Samuel P , Guerrero ML , Ruiz-Palacios G , Kang G , Pitzer VE . bioRxiv 2018 242172 Variation in susceptibility is a known contributor to bias in studies estimating immune protection acquired from vaccination or natural infection. However, difficulty measuring this heterogeneity hinders assessment of its influence on estimates. Cohort studies, randomized trials, and post-licensure studies have reported reduced natural and vaccine-derived protection against rotavirus gastroenteritis in low- and middle-income countries (LMICs). We sought to understand differences in susceptibility among children enrolled in two birth-cohort studies of rotavirus in LMICs, and to explore the implications for estimation of immune protection. We re-analyzed data from studies conducted in Mexico City, Mexico and Vellore, India. Cumulatively, 573 unvaccinated children experienced 1418 rotavirus infections and 371 episodes of rotavirus gastroenteritis (RVGE) over 17,636 child-months. We developed a model characterizing susceptibility to rotavirus infection and RVGE among children, accounting for aspects of the natural history of rotavirus and differences in transmission rates between settings, and tested whether modelgenerated susceptibility measurements were associated with demographic and anthropometric factors. We identified greater variation in susceptibility to rotavirus infection and RVGE in Vellore than in Mexico City. In both cohorts, susceptibility to rotavirus infection and RVGE were associated with male sex, lower birth weight, lower maternal education, and having fewer siblings; within Vellore, susceptibility was also associated with lower socioeconomic status. Children who were more susceptible to rotavirus also experienced higher rates of diarrhea due to other causes. Simulations suggest that discrepant estimates of naturally-acquired immunity against RVGE can be attributed, in part, to between-setting differences in transmission intensity and susceptibility of children. We found that more children in Vellore than in Mexico City belong to a high-risk group for rotavirus infection and RVGE, and demonstrate that bias owing to differences in rotavirus transmission intensity and population susceptibility may hinder comparison of estimated immune protection against RVGE.Author summary Differences in susceptibility can help explain why some individuals, and not others, acquire infection and exhibit symptoms when exposed to infectious disease agents. However, it is difficult to distinguish between differences in susceptibility versus exposure in epidemiological studies. We developed a modeling approach to distinguish transmission intensity and susceptibility in data from cohort studies of rotavirus infection among children in Mexico City, Mexico, and Vellore, India, and evaluated how these factors may have contributed to differences in estimates of naturally-acquired immune protection between the studies. We found that more children were at “high risk” of acquiring rotavirus infection, and of experiencing gastroenteritis when infected, in Vellore versus Mexico City. The probability of belonging to this high-risk stratum was associated with recognized risk factors such as lower socioeconomic status, lower birth weight, and risk of diarrhea due to other causes. We also found the risk for rotavirus infections to cause symptoms declined with age, and was independent of acquired immunity. Together, these findings can account for estimates of lower protective efficacy of acquired immunity against rotavirus gastroenteritis in high-incidence settings, which mirrors estimates of reduced effectiveness of live oral rotavirus vaccines in low- and middle-income countries. |
Antibodies against egg- and cell-grown influenza A(H3N2) viruses in adults hospitalized during the 2017-2018 season (preprint)
Levine MZ , Martin ET , Petrie JG , Lauring AS , Holiday C , Jefferson S , Fitzsimmons WJ , Johnson E , Ferdinands JM , Monto AS . bioRxiv 2018 439471 Background The 2017-2018 US influenza season was severe with low vaccine effectiveness. Circulating A(H3N2) viruses from multiple genetic groups were antigenically similar to cell-grown vaccine strains. However, most influenza vaccines are egg-propagated.Methods Serum was collected shortly after illness onset from 15 PCR confirmed A(H3N2) infected cases and 15 uninfected (controls) hospitalized adults enrolled in an influenza vaccine effectiveness study.Geometric mean titers against egg- and cell-grown A/Hong Kong/4801/2014 A(H3N2) vaccine strains and representative circulating viruses (including A/Washington/16/2017) were determined by microneutralization (MN) assays. Independent effects of strain-specific titers on susceptibility were estimated by logistic regression.Results MN titers against egg-A/Hong Kong were significantly higher among those who were vaccinated (MN GMT: 173 vs 41; P = 0.01). However, antibody titers to cell-grown viruses were much lower in all individuals (P>0.05) regardless of vaccination. In unadjusted models, a 2-fold increase in MN titers against egg-A/Hong Kong was not significantly protective against infection (29% reduction; p=0.09), but a similar increase in cell-A/Washington titer (3C.2a2) was protective (60% reduction; p=0.02). A similar increase in egg-A/Hong Kong titer was not significantly associated with odds of infection when adjusting for MN titers against A/Washington (15% reduction; P=0.61). A 54% reduction of odds of infection was observed with a 2-fold increase in A/Washington (not significant; P=0.07), adjusted for egg-A/Hong Kong titer.Conclusion Although individuals vaccinated in 2017-2018 had high antibody titers against the egg-adapted vaccine strain, antibody responses to cell-grown circulating viruses may not be sufficient to provide protection, likely due to egg-adaptation in the vaccine.We thank Maryna Eichelberger, Hongquan Wan, Jin Gao, and Laura Couzens (Food and Drug Administration) for technical support and providing reassortant influenza viruses for use in the enzyme-linked lectin assays. St Jude Children’s Research Hospital provided plasmids that were used to generate these reassortant influenza viruses. We thank Mrs F Liaini Gross, Lauren Horner and Makeda Kay from Influenza Division, Centers for Disease Control and Prevention for technical support for virus propagation and specimen management. |
Testing hypotheses about the microbiome using the linear decomposition model (LDM) (preprint)
Hu YJ , Satten GA . bioRxiv 2020 229831 Motivation Methods for analyzing microbiome data generally fall into one of two groups: tests of the global hypothesis of any microbiome effect, which do not provide any information on the contribution of individual operational taxonomic units (OTUs); and tests for individual OTUs, which do not typically provide a global test of microbiome effect. Without a unified approach, the findings of a global test may be hard to resolve with the findings at the individual OTU level. Further, many tests of individual OTU effects do not preserve the false discovery rate (FDR).Results We introduce the linear decomposition model (LDM), that provides a single analysis path that includes global tests of any effect of the microbiome, tests of the effects of individual OTUs while accounting for multiple testing by controlling the FDR, and a connection to distance-based ordination. The LDM accommodates both continuous and discrete variables (e.g., clinical outcomes, environmental factors) as well as interaction terms to be tested either singly or in combination, allows for adjustment of confounding covariates, and uses permutation-based p-values that can control for correlation. The LDM can also be applied to transformed data, and an “omnibus” test can easily combine results from analyses conducted on different transformation scales. We also provide a new implementation of PERMANOVA based on our approach. For global testing, our simulations indicate the LDM provided correct type I error and can have comparable power to existing distance-based methods. For testing individual OTUs, our simulations indicate the LDM controlled the FDR well. In contrast, DESeq2 often had inflated FDR; MetagenomeSeq generally had the lowest sensitivity. The flexibility of the LDM for a variety of microbiome studies is illustrated by the analysis of data from two microbiome studies. We also show that our implementation of PERMANOVA can outperform existing implementations. |
Optical microscopy reveals the dynamic nature of B. pseudomallei morphology during β-lactam antimicrobial susceptibility testing (preprint)
McLaughlin HP , Bugrysheva J , Sue D . bioRxiv 2020 2020.01.13.904995 Background In Gram-negative species, β-lactam antibiotics target penicillin binding proteins (PBPs) resulting in morphological alterations of bacterial cells. Observations of antibiotic-induced cell morphology changes can rapidly and accurately differentiate drug susceptible from resistant bacterial strains; however, resistant cells do not always remain unchanged. Burkholderia pseudomallei is a Gram-negative, biothreat pathogen and the causative agent of melioidosis, an often fatal infectious disease for humans.Results Here, we identified β-lactam targets in B. pseudomallei by in silico analysis. Ten genes encoding putative PBPs, including PBP-1, PBP-2, PBP-3 and PBP-6, were detected in the genomes of susceptible and resistant strains. Real-time, live-cell imaging of B. pseudomallei strains demonstrated dynamic morphological changes in broth containing clinically relevant β-lactam antibiotics. At sub-inhibitory concentrations of ceftazidime (CAZ), amoxicillin-clavulanic acid (AMC), and imipenem (IPM), filamentation, varying in length and proportion, was an initial response of the multidrug-resistant strain Bp1651 in exponential phase. However, a dominant morphotype reemerged during stationary phase that resembled cells unexposed to antibiotics. Similar morphology dynamics were observed for AMC-resistant strains, MSHR1655 and 724644, when exposed to sub-inhibitory concentrations of AMC. For all B. pseudomallei strains evaluated, increased exposure time and exposure to increased concentrations of AMC at and above minimal inhibitory concentrations (MICs) in broth resulted in cell morphology shifts from filaments to spheroplasts and/or cell lysis. B. pseudomallei morphology changes were more consistent in IPM. Spheroplast formation followed by cell lysis was observed for all strains in broth containing IPM at concentrations greater than or equal to MICs, however, the time to cell lysis was variable. Length of B. pseudomallei cells was strain-, drug- and drug concentration-dependent.Conclusions Both resistant and susceptible B. pseudomallei strains exhibited filamentation during early exposure to AMC and CAZ at concentrations used to interpret susceptibility (based on CLSI guidelines). While developing a rapid β-lactam antimicrobial susceptibility test based on cell-shape alone requires more extensive analyses, optical microscopy detected B. pseudomallei growth attributes that lend insight into antibiotic response and antibacterial mechanisms of action.(AST)Antimicrobial susceptibility test,(BMD)broth microdilution,(CLSI)Clinical and Laboratory Standards Institute,(IPM)imipenem,(CAZ)ceftazidime,(AMC)amoxicillin-clavulanic acid,(MDR)multidrug-resistant,(R)resistant,(I)intermediate,(S)susceptible,(SBA)Trypticase Soy Agar II with 5% sheep blood,(CAMHB)cation-adjusted Mueller Hinton broth,(MIC)minimal inhibitory concentrations,(TES)N-tris(hydroxymethyl) methyl-2-aminoethanesulfonic acid,(SESA)Segmentation and Extraction Surface Area,(SD)standard deviation,(PBPs)penicillin-binding proteins. |
An orally bioavailable broad-spectrum antiviral inhibits SARS-CoV-2 and multiple endemic, epidemic and bat coronavirus (preprint)
Sheahan TP , Sims AC , Zhou S , Graham RL , Hill CS , Leist SR , Schafer A , Dinnon KH 3rd , Montgomery SA , Agostini ML , Pruijssers AJ , Chappell JD , Brown AJ , Bluemling GR , Natchus MG , Saindane M , Kolykhalov AA , Painter G , Harcourt J , Tamin A , Thornburg NJ , Swanstrom R , Denison MR , Baric RS . bioRxiv 2020 2020.03.19.997890 Coronaviruses (CoVs) traffic frequently between species resulting in novel disease outbreaks, most recently exemplified by the newly emerged SARS-CoV-2. Herein, we show that the ribonucleoside analog β-D-N4-hydroxycytidine (NHC, EIDD-1931) has broad spectrum antiviral activity against SARS-CoV 2, MERS-CoV, SARS-CoV, and related zoonotic group 2b or 2c Bat-CoVs, as well as increased potency against a coronavirus bearing resistance mutations to another nucleoside analog inhibitor. In mice infected with SARS-CoV or MERS-CoV, both prophylactic and therapeutic administration of EIDD-2801, an orally bioavailable NHC-prodrug (b-D-N4-hydroxycytidine-5’-isopropyl ester), improved pulmonary function, and reduced virus titer and body weight loss. Decreased MERS-CoV yields in vitro and in vivo were associated with increased transition mutation frequency in viral but not host cell RNA, supporting a mechanism of lethal mutagenesis. The potency of NHC/EIDD-2801 against multiple coronaviruses, its therapeutic efficacy, and oral bioavailability in vivo, all highlight its potential utility as an effective antiviral against SARS-CoV-2 and other future zoonotic coronaviruses. |
Quantitative differences between intra-host HCV populations from persons with recently established and persistent infections (preprint)
Icer Baykal PB , Lara J , Khudyakov Y , Zelikovsky A , Skums P . bioRxiv 2020 2020.06.17.157792 Background Detection of incident hepatitis C virus (HCV) infections is crucial for identification of outbreaks and development of public health interventions. However, there is no single diagnostic assay for distinguishing recent and persistent HCV infections. HCV exists in each infected host as a heterogeneous population of genomic variants, whose evolutionary dynamics remain incompletely understood. Genetic analysis of such viral populations can be applied to the detection of incident HCV infections and used to understand intra-host viral evolution.Methods We studied intra-host HCV populations sampled using next-generation sequencing from 98 recently and 256 persistently infected individuals. Genetic structure of the populations was evaluated using 245,878 viral sequences from these individuals and a set of selected parameters measuring their diversity, topological structure, complexity, strength of selection, epistasis, evolutionary dynamics, and physico-chemical properties.Findings Distributions of the viral population parameters differ significantly between recent and persistent infections. A general increase in viral genetic diversity from recent to persistent infections is frequently accompanied by decline in genomic complexity and increase in structuredness of the HCV population, likely reflecting a high level of intra-host adaptation at later stages of infection. Using these findings, we developed a Machine Learning classifier for the infection staging, which yielded a detection accuracy of 95.22%, thus providing a higher accuracy than other genomic-based models.Interpretation The detection of a strong association between several HCV genetic factors and stages of infection suggests that intra-host HCV population develops in a complex but regular and predictable manner in the course of infection. The proposed models may serve as a foundation of cyber-molecular assays for staging infection, that could potentially complement and/or substitute standard laboratory assays.Funding AZ and PS were supported by NIH grant 1R01EB025022. PIB was supported by GSU MBD fellowship.Competing Interest StatementThe authors have declared no competing interest. |
Differential pathogenesis of Usutu virus isolates in mice (preprint)
Kuchinsky SC , Hawks SA , Mossel EC , Coutermarsh-Ott S , Duggal NK . bioRxiv 2020 2020.08.31.275149 Usutu virus (USUV; Flavivirus), a close phylogenetic and ecological relative of West Nile virus, is a zoonotic virus that can cause neuroinvasive disease in humans. USUV is maintained in an enzootic cycle between Culex mosquitoes and birds. Since the first isolation in 1959 in South Africa, USUV has spread throughout Africa and Europe. Reported human cases have increased over the last few decades, primarily in Europe, with symptoms ranging from mild febrile illness to severe neurological effects. In this study, we investigated whether USUV has become more pathogenic during emergence in Europe. Interferon α/β receptor knockout (Ifnar1-/-) mice were inoculated with recent USUV isolates from Africa and Europe, as well as the historic 1959 South African strain. The three tested African strains and one European strain from Spain caused 100% mortality in inoculated mice, with similar survival times and histopathology in tissues. Unexpectedly, a European strain from the Netherlands caused only 12% mortality and significantly less histopathology in tissues from mice compared to mice inoculated with the other strains. Viremia was highest in mice inoculated with the recent African strains and lowest in mice inoculated with the Netherlands strain. Based on phylogenetics, the USUV isolates from Spain and the Netherlands were derived from separate introductions into Europe, suggesting that disease outcomes may differ for USUV strains circulating in Europe. These results also suggest that while more human USUV disease cases have been reported in Europe recently, circulating African USUV strains are still a potential major health concern.Author Summary Usutu virus (USUV) is an emerging mosquito-borne virus that causes severe neuroinvasive disease in humans. USUV was first detected in Africa in 1959, and cases of human disease have increased in recent years. Most USUV disease cases are now reported in Europe, where the virus currently circulating. One possibility for the increase in case numbers is that USUV strains have become more pathogenic over time during its spread from Africa into Europe. We compared the pathogenesis of five USUV isolates from Africa and Europe in a mouse model. Three isolates from Africa and one isolate from Europe caused 100% mortality in mice. Surprisingly, one isolate from Netherlands caused only 12% motality. Significantly less histopathology was observed in tissues from mice inoculated with the Netherlands strain compared to mice inoculated with the other four strains. Our results suggest that, even though more human USUV disease cases have been reported in Europe recently, African USUV strains are also highly pathogenic.Competing Interest StatementThe authors have declared no competing interest. |
Rapid Detection of Genetic Engineering, Structural Variation, and Antimicrobial Resistance Markers in Bacterial Biothreat Pathogens by Nanopore Sequencing (preprint)
Gargis AS , Cherney B , Conley AB , McLaughlin HP , Sue D . bioRxiv 2019 730093 Widespread release of Bacillus anthracis (anthrax) or Yersinia pestis (plague) would prompt a public health emergency. During an exposure event, high-quality whole genome sequencing (WGS) can identify genetic engineering, including the introduction of antimicrobial resistance (AMR) genes. Here, we developed rapid WGS laboratory and bioinformatics workflows using a long-read nanopore sequencer (MinION) for Y. pestis (6.5h) and B. anthracis (8.5h) and sequenced strains with different AMR profiles. Both salt-precipitation and silica-membrane extracted DNA were suitable for MinION WGS using both rapid and field library preparation methods. In replicate experiments, nanopore quality metrics were defined for genome assembly and mutation analysis. AMR markers were correctly detected and >99% coverage of chromosomes and plasmids was achieved using 100,000 raw sequencing reads. While chromosomes and large and small plasmids were accurately assembled, including novel multimeric forms of the Y. pestis virulence plasmid, pPCP1, MinION reads were error-prone, particularly in homopolymer regions. MinION sequencing holds promise as a practical, front-line strategy for on-site pathogen characterization to speed the public health response during a biothreat emergency. |
Strong population structure in Venezuelan populations of Coccidioides posadasii (preprint)
Teixeira MM , Alvarado P , Roe CC , Thompson GR 3rd , Patane JSL , Sahl JW , Keim P , Galgiani JN , Litvintseva AP , Matute DR , Barker BM . bioRxiv 2019 719328 Coccidioides posadasii is a pathogenic fungus that causes coccidioidomycosis in many arid regions of the Americas. One of these regions is bordered by the Caribbean Sea, and the surrounding landscape may play an important role in the dispersion of C. posadasii across South America through southeastern Mexico, Honduras, Guatemala and Venezuela. Comparative phylogenomic analyses of C. posadasii reveal that clinical strains from Venezuela are genetically distinct from the North American populations found in Arizona (AZ), Texas, Mexico, and the rest of South America (TX/MX/SA). We find evidence for admixture between the Venezuela and the North American populations of C. posadasii in Central America. As expected, the proportion of Venezuelan alleles in the admixed population decreases as latitude (and distance from Venezuela) increases. Our results indicate that the population in Venezuela may have been subjected to a recent bottleneck, and shows strong population structure. This analysis provides insight into potential for Coccidioides spp. to invade new regions. |
Structure-function analysis reveals a DNA polymerization-independent role for mitochondrial DNA polymerase IC in African trypanosomes (preprint)
Miller JC , Delzell SB , Concepcion-Acevedo J , Boucher MJ , Klingbeil MM . bioRxiv 2019 730523 The mitochondrial DNA of Trypanosoma brucei and related parasites is a catenated network containing thousands of minicircles and tens of maxicircles called kinetoplast DNA (kDNA). Replication of the single nucleoid requires at least three DNA polymerases (POLIB, POLIC, and POLID) each having discrete localization near the kDNA during S phase. POLIB and POLID have roles in minicircle replication while the specific role of POLIC in kDNA maintenance is less clear. Here, we use an RNAi-complementation system to dissect the functions of the distinct POLIC domains: the conserved family A DNA polymerase domain (POLA) and the uncharacterized N-terminal region (UCR). While RNAi complementation with wild-type POLIC restored kDNA content and cell cycle localization, active site point mutations in the POLA domain impaired minicircle replication similarly to POLIB and POLID depletions. Complementation with the POLA domain alone abolished POLIC foci formation and partially rescued the RNAi phenotype. Furthermore, we provide evidence of a crucial role for the UCR in cell cycle localization and segregation of kDNA daughter networks. This is the first report of a DNA polymerase that impacts DNA segregation.Summary statement Mitochondrial DNA segregation in African trypanosomes is supported by a dual-functioning DNA polymerase. |
Using the NCBI AMRFinder Tool to Determine Antimicrobial Resistance Genotype-Phenotype Correlations Within a Collection of NARMS Isolates (preprint)
Feldgarden M , Brover V , Haft DH , Prasad AB , Slotta DJ , Tolstoy I , Tyson GH , Zhao S , Hsu CH , McDermott PF , Tadesse DA , Morales C , Simmons M , Tillman G , Wasilenko J , Folster JP , Klimke W . bioRxiv 2019 550707 Antimicrobial resistance (AMR) is a major public health problem that requires publicly available tools for rapid analysis. To identify acquired AMR genes in whole genome sequences, the National Center for Biotechnology Information (NCBI) has produced a high-quality, curated, AMR gene reference database consisting of up-to-date protein and gene nomenclature, a set of hidden Markov models (HMMs), and a curated protein family hierarchy. Currently, the Bacterial Antimicrobial Resistance Reference Gene Database contains 4,579 antimicrobial resistance gene proteins and more than 560 HMMs.Here, we describe AMRFinder, a tool that uses this reference dataset to identify AMR genes. To assess the predictive ability of AMRFinder, we measured the consistency between predicted AMR genotypes from AMRFinder against resistance phenotypes of 6,242 isolates from the National Antimicrobial Resistance Monitoring System (NARMS). This included 5,425 Salmonella enterica, 770 Campylobacter spp., and 47 Escherichia coli phenotypically tested against various antimicrobial agents. Of 87,679 susceptibility tests performed, 98.4% were consistent with predictions.To assess the accuracy of AMRFinder, we compared its gene symbol output with that of a 2017 version of ResFinder, another publicly available resistance gene database. Most gene calls were identical, but there were 1,229 gene symbol differences between them, with differences due to both algorithmic differences and database composition. AMRFinder missed 16 loci that Resfinder found, while Resfinder missed 1,147 loci AMRFinder identified. Two missing drug classes from the 2017 version of ResFinder contributed 81% of missed loci. Based on these results, AMRFinder appears to be a highly accurate AMR gene detection system.Importance Antimicrobial resistance is a major public health problem. Traditionally, antimicrobial resistance has been identified using phenotypic assays. With the advent of genome sequencing, we now can identify resistance genes and deduce if an isolate could be resistant to antibiotics. We describe a database of 4,579 acquired antimicrobial resistance genes, the largest publicly available, and a software tool to identify genes in bacterial genomes, AMRFinder. Unlike other tools, AMRFinder uses a gene hierarchy to prevent overpredicting what the correct gene call should be, enabling more accurate assessment. To assess these resources, we determined the resistance gene content of over 6,200 bacterial isolates from the National Antimicrobial Resistance Monitoring System that have been assayed using traditional methods and that also have had their genomes sequenced. We also compared our gene assessments to those of a popularly used tool. We found that AMRFinder has a high overall consistency between genotypes and phenotypes. |
Neck Musculoskeletal Model Generation through Anthropometric Scaling (preprint)
Roos PE , Vasavada A , Zheng L , Zhou X . bioRxiv 2019 695833 A new methodology was developed to quickly generate whole body models with detailed neck musculoskeletal architecture that are properly scaled in terms of anthropometry and muscle strength. This method was implemented in an anthropometry model generation software that allows users to interactively generate any new male or female musculoskeletal models with adjustment of anthropometric parameters (such as height, weight, neck circumference, and neck length) without the need of subject-specific motion capture or medical images. 50th percentile male and female models were developed based on the 2012 US Army Anthropometric Survey (ANSUR II) database and optimized with a novel bilevel optimization method to have strengths comparable to experimentally measured values in the literature. Other percentile models (ranging from the 1st to 99th percentile) were generated based on anthropometric scaling of the 50th percentile models and compared. The resultant models are reasonably accurate in terms of both musculoskeletal geometry and neck strength, demonstrating the effectiveness of the developed methodology for interactive neck model generation with anthropometric scaling. |
A novel mosaic tetracycline resistance gene tet(S/M) detected in a multidrug-resistant pneumococcal CC230 lineage that underwent capsular switching in South Africa (preprint)
Lo SW , Gladstone RA , van Tonder AJ , Du Plessis M , Cornick JE , Hawkins PA , Madhi SA , Nzenze SA , Kandasamy R , Ravikumar KL , Elmdaghri N , Kwambana-Adams B , Almeida SCG , Skoczynska A , Egorova E , Titov L , Saha SK , Paragi M , Everett DB , Antonio M , Klugman KP , Li Y , Metcalf BJ , Beall B , McGee L , Breiman RF , Bentley SD , von Gottberg A . bioRxiv 2019 718460 Objective We reported a novel tetracycline-resistant gene in Streptococcus pneumoniae and investigated its temporal spread in relation to nationwide clinical interventions.Methods We whole genome sequenced 12,254 pneumococcal isolates from twenty-nine countries on an Illumina HiSeq Sequencer. Serotypes, sequence types and antibiotic resistance were inferred from genomes. Phylogeny was built based on single-nucleotide variants. Temporal changes of spread were reconstructed using a birth-death model.Results We identified tet(S/M) in 131 pneumococcal isolates, 97 (74%) caused invasive pneumococcal diseases among young children (59% HIV-positive, where HIV status was available) in South Africa. A majority of tet(S/M)-positive isolates (129/131) belong to clonal complex (CC)230. A global phylogeny of CC230 (n=389) revealed that tet(S/M)-positive isolates formed a sub-lineage that exhibited multidrug-resistance. Using the genomic data and a birth-death model, we detected an unrecognised outbreak of this sub-lineage in South Africa between 2000 and 2004 with an expected secondary infections (R) of ~2.5. R declined to ~1.0 in 2005 and <1.0 in 2012. The declining epidemic coincided and could be related to the nationwide implementation of anti-retroviral treatment (ART) for HIV-infected individuals in 2004 and PCVs in late 2000s. Capsular switching from vaccine serotype 14 to non-vaccine serotype 23A was observed within the sub-lineage.Conclusions The prevalence of tet(S/M) in pneumococci was low and its dissemination was due to an unrecognised outbreak of CC230 in South Africa prior to ART and PCVs. However, capsular switching in this multidrug-resistant sub-lineage highlighted its potential to continue to cause disease in the post-PCV13 era. |
Oral tenofovir disoproxil fumarate/emtricitabine for HIV pre-exposure prophylaxis increases expression of type I/III interferon-stimulated factors in the gastrointestinal tract but not in the blood (preprint)
Hughes SM , Levy CN , Calienes FL , Stekler JD , Pandey U , Vojtech L , Berard AR , Birse K , Noël-Romas L , Richardson B , Golden JB , Cartwright M , Collier AC , Stevens CE , Curlin ME , Holtz TH , Mugo N , Irungu E , Katabira E , Muwonge T , Lama JR , Baeten JM , Burgener A , Lingappa JR , McElrath MJ , Mackelprang R , McGowan I , Cranston RD , Cameron MJ , Hladik F . bioRxiv 2019 701961 Tenofovir disoproxil fumarate and emtricitabine are used for HIV treatment and pre-exposure prophylaxis. Previously, we found that topical rectal application of tenofovir 1% gel caused many gene expression changes. Here, we measured RNA and protein expression in several clinical trials of oral administration in HIV-uninfected individuals (using microarrays, RNAseq, droplet digital PCR, mass spectrometry, and microscopy). We found tens to hundreds of differentially expressed genes in the gastrointestinal tract, but none in the blood or female reproductive tract. In rectal samples from one trial, most of the 13 upregulated genes were related to type I/III interferon signaling. Similar changes were seen at the protein level in the same trial and in the duodenum and rectum in another trial. We conclude that tenofovir disoproxil fumarate and emtricitabine have little effect on gene expression in the blood or female reproductive tract but increase type I/III interferon signaling in the gut. This effect may enhance their anti-viral efficacy when used as pre-exposure prophylaxis, in particular to prevent rectal HIV transmission. However, it may also contribute to chronic immune activation and HIV reservoir maintenance in chronically treated people living with HIV. |
Pragmatic selection of larval mosquito diets for insectary rearing of Anopheles gambiae and Aedes aegypti (preprint)
Benedict MQ , Hunt CM , Vella MG , Gonzalez KM , Dotson EM , Collins CM . bioRxiv 2019 740142 Larval mosquitoes are aquatic omnivorous scavengers which scrape food from submerged surfaces and collect suspended food particles with their mouth brushes. The composition of diets that have been used in insectaries varies widely though necessarily provides sufficient nutrition to allow colonies to be maintained. Issues such as cost, availability and experience influence which diet is selected. One component of larval diets, essential fatty acids, appears to be necessary for normal flight though deficiencies may not be evident in laboratory cages and are likely more important when mosquitoes are reared for release into the field in e.g. mark-release-recapture and genetic control activities.In this study, four diets were compared for rearing Anopheles gambiae and Aedes aegypti, all of which provide these essential fatty acids. Two diets were custom formulations specifically designed for mosquitoes (Damiens) and two were commercially available fish foods: Doctors Foster and Smith Koi Staple Diet and TetraMin Plus Flakes. Development rate, survival, dry weight and adult longevity of mosquitoes reared with these four diets were measured. The method of presentation of one diet, Koi pellets, was additionally fed in two forms, pellets or a slurry, to determine any effect of food presentation on survival and development rate.While various criteria might be selected to choose ‘the best’ food, the readily-available Koi pellets resulted in development rates and adult longevity equal to the other diets, high survival to the adult stage and, additionally, this is available at low cost. |
Pyrethroid exposure alters Anopheles albimanus microbiota and resistant mosquitoes harbor more insecticide-metabolizing bacteria (preprint)
Dada N , Lol JC , Benedict AC , Lopez F , Sheth M , Dzuris N , Padilla N , Lenhart A . bioRxiv 2019 537480 A deeper understanding of the mechanisms underlying insecticide resistance is needed to mitigate its threat to malaria vector control. Building upon our earlier identified associations between mosquito microbiota and insecticide resistance, we demonstrate for the first time, type-specific effects of pyrethroid exposure on internal and cuticle surface bacteria in adult progeny of field-collected Anopheles albimanus. In contrast, larval cuticle surface—but not internal—bacteria were affected by pyrethroid exposure. Being over five-folds more abundant in pyrethroid resistant adults, as compared to susceptible or non-insecticide-exposed mosquitoes, Klebsiella (alphacypermethrin), Pantoea and Asaia (permethrin) were identified as potential markers of pyrethroid resistance in An. albimanus. We also show for the first time that An. albimanus larvae and adult cuticles harbor more diverse bacterial communities than their internal microbial niches. Our findings indicate insecticide selection pressures on mosquito microbiota, and support the hypothesis of an undescribed microbe-mediated mechanism of insecticide metabolism in mosquitoes. |
Enteropathogen antibody dynamics and force of infection among children in low-resource settings (preprint)
Arnold BF , Martin DL , Juma J , Mkocha H , Ochieng JB , Cooley GM , Omore R , Goodhew EB , Morris JF , Costantini V , Vinje J , Lammie PJ , Priest JW . bioRxiv 2019 522920 Little is known about enteropathogen seroepidemiology among children in low-resource settings. We measured serological IgG responses to eight enteropathogens (Giardia intestinalis, Cryptosporidium parvum, Entamoeba histolytica, Salmonella enterica, enterotoxigenic Escherichia coli, Vibrio cholerae, Campylobacter jejuni, norovirus) in cohorts from Haiti, Kenya, and Tanzania. We studied antibody dynamics and force of infection across pathogens and cohorts. Enteropathogens shared common seroepidemiologic features that enabled between-pathogen comparisons of transmission. Overall, exposure was intense: for most pathogens the window of primary infection was <3 years old; for highest transmission pathogens primary infection occurred within the first year. Longitudinal profiles revealed significant IgG boosting and waning above seropositivity cutoffs, underscoring the value of longitudinal designs to estimate force of infection. Seroprevalence and force of infection were rank-preserving across pathogens, illustrating the measures provide similar information about transmission heterogeneity. Our findings suggest multiplex antibody assays are a promising approach to measure population-level enteropathogen transmission in serologic surveillance. |
The ER chaperone PfGRP170 is essential for asexual development and is linked to stress response in malaria parasites (preprint)
Kudyba HM , Cobb DW , Fierro MA , Florentin A , Ljolje D , Singh B , Lucchi NW , Muralidharan V . bioRxiv 2019 406181 The vast majority of malaria mortality is attributed to one parasite species: Plasmodium falciparum. Asexual replication of the parasite within the red blood cell is responsible for the pathology of the disease. In Plasmodium, the endoplasmic reticulum (ER) is a central hub for protein folding and trafficking as well as stress response pathways. In this study, we tested the role of an uncharacterized ER protein, PfGRP170, in regulating these key functions by generating conditional mutants. Our data show that PfGRP170 localizes to the ER and is essential for asexual growth, specifically required for proper development of schizonts. PfGRP170 is essential for surviving heat shock, suggesting a critical role in cellular stress response. The data demonstrate that PfGRP170 interacts with the Plasmodium orthologue of the ER chaperone, BiP. Finally, we found that loss of PfGRP170 function leads to the activation of the Plasmodium eIF2α kinase, PK4, suggesting a specific role for this protein in this parasite stress response pathway. |
Evolutionary superscaffolding and chromosome anchoring to improve Anopheles genome assemblies (preprint)
Waterhouse RM , Aganezov S , Anselmetti Y , Lee J , Ruzzante L , Reijnders Mjmf , Feron R , Berard S , George P , Hahn MW , Howell PI , Kamali M , Koren S , Lawson D , Maslen G , Peery A , Phillippy AM , Sharakhova MV , Tannier E , Unger MF , Zhang SV , Alekseyev MA , Besansky NJ , Chauve C , Emrich SJ , Sharakhov IV . bioRxiv 2019 434670 Background New sequencing technologies have lowered financial barriers to whole genome sequencing, but resulting assemblies are often fragmented and far from ‘finished’. Updating multi-scaffold drafts to chromosome-level status can be achieved through experimental mapping or re-sequencing efforts. Avoiding the costs associated with such approaches, comparative genomic analysis of gene order conservation (synteny) to predict scaffold neighbours (adjacencies) offers a potentially useful complementary method for improving draft assemblies.Results We employed three gene synteny-based methods applied to 21 Anopheles mosquito assemblies to produce consensus sets of scaffold adjacencies. For subsets of the assemblies we integrated these with additional supporting data to confirm and complement the synteny-based adjacencies: six with physical mapping data that anchor scaffolds to chromosome locations, 13 with paired-end RNA sequencing (RNAseq) data, and three with new assemblies based on re-scaffolding or Pacific Biosciences long-read data. Our combined analyses produced 20 new superscaffolded assemblies with improved contiguities: seven for which assignments of non-anchored scaffolds to chromosome arms span more than 75% of the assemblies, and a further seven with chromosome anchoring including an 88% anchored Anopheles arabiensis assembly and, respectively, 73% and 84% anchored assemblies with comprehensively updated cytogenetic photomaps for Anopheles funestus and Anopheles stephensi.Conclusions Experimental data from probe mapping, RNAseq, or long-read technologies, where available, all contribute to successful upgrading of draft assemblies. Our comparisons show that gene synteny-based computational methods represent a valuable alternative or complementary approach. Our improved Anopheles reference assemblies highlight the utility of applying comparative genomics approaches to improve community genomic resources.ADADSEQAGOAGOUTI-basedAGOUTIannotated genome optimization using transcriptome information toolALNalignment-basedCAMSAcomparative analysis and merging of scaffold assemblies toolDPdynamic programmingFISHfluorescence in situ hybridizationGAGOS-ASMGOS-ASMGene order scaffold assemblerKbpkilobasepairsMbpmegabasepairsOSORTHOSTITCHPacBioPacific BiosciencesPBPacBio-basedPHYphysical-mapping-basedRNAseqRNA sequencingQTLquantitative trait lociSYNsynteny-based. |
Identification of CP77 as the third orthopoxvirus SAMD9L inhibitor with a unique specificity for a rodent SAMD9L (preprint)
Zhang F , Meng X , Townsend MB , Satheshkumar PS , Xiang Y . bioRxiv 2019 551556 Orthopoxviruses (OPXVs) have a broad host range in mammalian cells, but Chinese hamster ovary (CHO) cells are non-permissive for vaccinia virus (VACV). Here, we revealed a species-specific difference in host restriction factor SAMD9L as the cause for the restriction and identified orthopoxvirus CP77 as a unique inhibitor capable of antagonizing Chinese hamster SAMD9L (chSAMD9L). Two known VACV inhibitors of SAMD9 and SAMD9L (SAMD9&L), K1 and C7, can bind human and mouse SAMD9&L, but neither can bind chSAMD9L. CRISPR-Cas9 knockout of chSAMD9L from CHO cells removed the restriction for VACV, while ectopic expression of chSAMD9L imposed the restriction for VACV in a human cell line, demonstrating that chSAMD9L is a potent restriction factor for VACV. Contrary to K1 and C7, cowpox virus CP77 can bind chSAMD9L and rescue VACV replication in cells expressing chSAMD9L, indicating that CP77 is yet another SAMD9L inhibitor but has a unique specificity for chSAMD9L. Binding studies showed that the N-terminal 382 amino acids of CP77 were sufficient for binding chSAMD9L and that both K1 and CP77 target a common internal region of SAMD9L. Growth studies with nearly all OPXV species showed that the ability of OPXVs in antagonizing chSAMD9L correlates with CP77 gene status and that a functional CP77 ortholog was maintained in many OPXVs, including monkeypox virus. Our data suggest that species-specific difference in rodent SAMD9L poses a barrier for cross-species OPXV infection and that OPXVs have evolved three SAMD9L inhibitors with different specificities to overcome this barrier.IMPORTANCE Several OPXV species, including monkeypox virus and cowpox virus, cause zoonotic infection in humans. They are believed to use wild rodents as the reservoir or intermediate hosts, but the host or viral factors that are important for OPXV host range in rodents are unknown. Here, we showed that the abortive replication of several OPXV species in a Chinese hamster cell line was caused by a species-specific difference in the host antiviral factor SAMD9L, indicating that SAMD9L divergence in different rodent species poses a barrier for cross-species OPXV infection. While the Chinese hamster SAMD9L could not be inhibited by two previously identified OPXV inhibitors of human and mouse SAMD9L, it can be inhibited by cowpox virus CP77, indicating that OPXVs encode three SAMD9L inhibitors with different specificity. Our data suggest that OPXV host range in broad rodent species depends on three SAMD9L inhibitors with different specificities. |
Comparison of Illumina MiSeq and the Ion Torrent PGM and S5 platforms for whole-genome sequencing of picornaviruses and caliciviruses (preprint)
Marine RL , Magana LC , Castro CJ , Zhao K , Montmayeur AM , Schmidt A , Diez-Valcarce M , Fan Ng TF , Vinje J , Burns CC , Allan Nix W , Rota PA , Oberste MS . bioRxiv 2019 705632 Next-generation sequencing is a powerful tool for virological surveillance. While Illumina® and Ion Torrent® sequencing platforms are used extensively for generating viral RNA genome sequences, there is limited data comparing different platforms. We evaluated the Illumina MiSeq, Ion Torrent PGM and Ion Torrent S5 platforms using a panel of sixteen specimens containing picornaviruses and human caliciviruses (noroviruses and sapoviruses). The specimens were processed, using combinations of three library preparation and five sequencing kits, to assess the quality and completeness of assembled viral genomes, and an estimation of cost per sample to generate the data was calculated. The choice of library preparation kit and sequencing platform was found to impact the breadth of genome coverage and accuracy of consensus viral genomes. The Ion Torrent S5 outperformed the older Ion Torrent PGM platform in data quality and cost, and generated the highest proportion of reads for enterovirus D68 samples. However, indels at homopolymer regions impacted the accuracy of consensus genome sequences. For lower throughput sequencing runs (i.e., Ion Torrent 510 or Illumina MiSeq Nano V2), the cost per sample was lower on the MiSeq platform, whereas with higher throughput runs (Ion Torrent 530 or Illumina MiSeq V2) the cost per sample was comparable. These findings suggest that the Ion Torrent S5 and Illumina MiSeq platforms are both viable options for genomic sequencing of RNA viruses, each with specific advantages and tradeoffs. |
Detection and discrimination of influenza B Victoria lineage deletion variant viruses by real-time RT-PCR (preprint)
Shu B , Kirby MK , Warnes C , Sessions WM , Davis WG , Liu J , Wilson MM , Wentworth DE , Barnes JR . bioRxiv 2019 818617 Influenza B viruses have two genetically and antigenically distinct lineages, B/Victoria/2/1987-like (VIC) and B/Yamagata/16/1988-like (YAM) viruses, that emerged in the 1980s and co-circulate annually during the influenza season. During the 2016-2017 influenza season, influenza B/VIC lineage variant viruses emerged with two (K162N163) or three (K162N163D164) amino acid (AA) deletions in the hemagglutinin protein. Hemagglutination inhibition assays demonstrate that these deletion variant influenza B/VIC viruses are antigenically distinct from each other and from the progenitor B/VIC virus that lacks the deletion. Therefore, there are currently four antigenically distinct HA proteins expressed by influenza B co-circulating: B/YAM, B/VIC V1A (no deletion), B/VIC V1A.1 (two-AA deletion), and B/VIC V1A.2 and V1A.3 (three-AA deletion). The prevalence of these viruses differs across geographic regions, making it critical to have a sensitive, rapid diagnostic assay(s) that detect and distinguish these Influenza B variant viruses during surveillance. Here, we present a real time RT-PCR assay that targets the influenza B/VIC deletion region in the HA gene and detects and distinguishes the influenza B/VIC V1A, B/VIC V1A.1, B/VIC V1A.2 and B/VIC V1A.3 variant viruses, with no cross-reactivity. This assay can be run as a multiplex reaction, allowing for increased testing efficiency and reduced cost. Coupling this assay with the CDC Human Influenza Virus Real-Time RT-PCR Diagnostic Panel Influenza B Lineage Genotyping Kit results in rapid detection and characterization of circulating influenza B viruses. Having accurate and detailed surveillance information on these distinct Influenza B variant viruses will provide insight into the prevalence and geographic distribution and could aid in vaccine recommendations. |
Detection of classic and cryptic Strongyloides genotypes by deep amplicon sequencing: A preliminary survey of dog and human specimens collected from remote Australian communities (preprint)
Beknazarova M , Barratt JLN , Bradbury RS , Lane M , Whiley H , Ross K . bioRxiv 2019 549535 Strongyloidiasis is caused by the human infective nematodes Strongyloides stercoralis, Strongyloides fuelleborni subsp. fuelleborni and Strongyloides fuelleborni subsp. kellyi. The zoonotic potential of S. stercoralis and the potential role of dogs in the maintenance of strongyloidiasis transmission has been a topic of interest and discussion for many years. In Australia, strongyloidiasis is prevalent in remote socioeconomically disadvantaged communities in the north of the continent. Being an isolated continent that has been separated from other regions for a long geological period, description of the diversity of Australian Strongyloides genotypes adds to our understanding of the genetic diversity within the genus. Using PCR enrichment combined with Illumina sequencing technology, we sequenced the Strongyloides SSU 18S rDNA hyper-variable I and hyper-variable IV regions using Strongyloides-specific primers, and a fragment of the mtDNA cox1 gene using primers that are broadly specific for Strongyloides sp. and hookworms. These loci were amplified from DNA extracted from Australian human and dog faeces, and one human sputum sample. Using this approach, we confirm for the first time that potentially zoonotic S. stercoralis genotypes are present in Australia, suggesting that dogs represent a potential reservoir of human strongyloidiasis in remote Australian communities.Author summary Strongyloides stercoralis is a soil-transmitted nematode that causes the disease strongyloidiasis. Due to the autoinfective nature of this parasite, it can re-infect a host causing chronic infection. If not diagnosed and treated it can be highly detrimental to human health and has a high mortality rate. Strongyloidiasis is common in remote communities in the north of Australia and has been an issue for decades. Despite various successful intervention programs to treat human strongyloidiasis, the disease remains endemic in those communities. Here for the first time we looked at the Australian dogs’ potential to infect humans and found that they carry two genetically distinct strains of Strongyloides spp., one of which also infects humans. This supports the hypothesis that dogs are a potential source for human strongyloidiasis. We also found that dogs in Australia might be carrying unique haplotypes. Whether these new haplotypes are also human infective is to be confirmed by further research. |
Schistosomiasis Induces Persistent DNA Methylation and Tuberculosis-specific Immune Changes (preprint)
DiNardo AR , Nishiguchi T , Mace EM , Rajapakshe K , Mtetwa G , Kay A , Maphalala G , Secor WE , Mejia R , Orange JS , Coarfa C , Bhalla KN , Graviss EA , Mandalakas AM , Makedonas G . bioRxiv 2018 255125 Epigenetic mechanisms, like DNA methylation, determine immune cell phenotype. To understand the epigenetic alterations induced by helminth co-infections, we evaluated the longitudinal effect of ascariasis and schistosomiasis infection on CD4+ T cell DNA methylation and the downstream tuberculosis (TB)-specific and BCG-induced immune phenotype. All experiments were performed on human primary immune cells from a longitudinal cohort of recently TB-exposed children. Compared to age-matched uninfected controls, children with active Schistosoma haematobium and Ascaris lumbricoides infection had 751 differentially DNA methylated genes with 72% hyper-methylated. Gene ontology pathway analysis identified inhibition of IFN-γ signaling, cellular proliferation, and the Th1 pathway. Targeted RT-PCR after methyl-specific endonuclease digestion confirmed DNA hyper-methylation of the transcription factors BATF3, ID2, STAT5A, IRF5, PPARg, RUNX2, IRF4 and NFATC1 and cytokines or cytokine receptors IFNGR1, TNFS11, RELT (TNF receptor), IL12RB2 and IL12B (p< 0.001; Sidak-Bonferroni). Functional blockage of the IFN-γ signaling pathway was confirmed with helminth-infected individuals having decreased up-regulation of IFN-γ-inducible genes (Mann-Whitney p < 0.05). Hypo-methylation of the IL-4 pathway and DNA hyper-methylation of the Th1 pathway was confirmed by antigen-specific multidimensional flow cytometry demonstrating decreased TB-specific IFN-γ and TNF and increased IL-4 production by CD4+ T cells (Wilcoxon signed rank P <0.05). In S.haematobium infected individuals, these DNA methylation and immune phenotypic changes persisted at least six months after successful deworming. This work demonstrates that helminth infection induces DNA methylation and immune perturbations that inhibit TB-specific immune control and that the duration of these changes are helminth-specific. |
Validation of novel Mycobacterium tuberculosis isoniazid resistance mutations not detectable by common molecular tests (preprint)
Kandler JL , Mercante AD , Dalton TL , Ezewudo MN , Cowan LS , Burns SP , Metchock B , Cegielski P , Posey JE . bioRxiv 2018 322750 Resistance to the first-line anti-tuberculosis (TB) drug, isoniazid (INH), is widespread, and the mechanism of resistance is unknown in approximately 15% of INH-resistant (INH-R) strains. To improve molecular detection of INH-R TB, we used whole genome sequencing (WGS) to analyze 52 phenotypically INH-R Mycobacterium tuberculosis complex (MTBC) clinical isolates that lacked the common katG S315T or inhA promoter mutations. Approximately 94% (49/52) of strains had mutations at known INH-associated loci that were likely to confer INH resistance. All such mutations would be detectable by sequencing more DNA adjacent to existing target regions. Use of WGS minimized the chances of missing infrequent INH resistance mutations outside commonly targeted hotspots. We used recombineering to generate 12 observed clinical katG mutations in the pansusceptible H37Rv reference strain and determined their impact on INH resistance. Our functional genetic experiments have confirmed the role of seven suspected INH resistance mutations and discovered five novel INH resistance mutations. All recombineered katG mutations conferred resistance to INH at a minimum inhibitory concentration of ≥0.25 μg/mL and should be added to the list of INH resistance determinants targeted by molecular diagnostic assays. We conclude that WGS is a superior method for detection of INH-R MTBC compared to current targeted molecular testing methods and could provide earlier diagnosis of drug-resistant TB. |
Mechanistic basis for decreased antimicrobial susceptibility in a clinical isolate of Neisseria gonorrhoeae possessing a mosaic-like mtr efflux pump locus (preprint)
Rouquette-Loughlin CE , Reimche JL , Balthazar JT , Dhulipala V , Gernert KM , Kersh EN , Pham CD , Pettus K , Abrams AJ , Trees DL , St Cyr S , Shafer WM . bioRxiv 2018 448712 Recent reports suggest that mosaic-like sequences within the mtr (multiple transferable resistance) efflux pump locus of Neisseria gonorrhoeae likely originating from commensal Neisseria sp. by transformation can increase the ability of gonococci to resist structurally diverse antimicrobials. Thus, acquisition of numerous nucleotide changes within the mtrR gene encoding the transcriptional repressor (MtrR) of the mtrCDE efflux pump-encoding operon or overlapping promoter region for both along with those that cause amino acid changes in the MtrD transporter protein were recently reported to decrease gonococcal susceptibility to numerous antimicrobials, including azithromycin (Azi) (Wadsworth et al. 2018. MBio. doi.org/10.1128/mBio.01419-18). We performed detailed genetic and molecular studies to define the mechanistic basis for why such strains can exhibit decreased susceptibility to MtrCDE antimicrobial substrates including Azi. We report that a strong cis-acting transcriptional impact of a single nucleotide change within the -35 hexamer of the mtrCDE promoter as well gain-of-function amino acid changes at the C-terminal region of MtrD can mechanistically account for the decreased antimicrobial susceptibility of gonococci with a mosaic-like mtr locus.IMPORTANCE Historically, after introduction of an antibiotic for treatment of gonorrhea, strains of N. gonorrhoeae emerge that display clinical resistance due to spontaneous mutation or acquisition of resistance genes. Genetic exchange between members of the Neisseria genus occurring by transformation can cause significant changes in gonococci that impact the structure of an antibiotic target or expression of genes involved in resistance. The results presented herein provide a framework for understanding how mosaic-like DNA sequences from commensal Neisseria that recombine within the gonococcal mtr efflux pump locus function to decrease bacterial susceptibility to antimicrobials including antibiotics used in therapy of gonorrhea. |
The NDV-3A vaccine protects mice from multidrug resistant Candida auris infection (preprint)
Singh S , Uppuluri P , Alqarihi A , Elhassan H , French S , Lockhart SR , Chiller T , Edwards JE Jr , Ibrahim AS . bioRxiv 2018 465096 Candida auris is an emerging, multi-drug resistant, health care-associated fungal pathogen. Its predominant prevalence in hospitals and nursing homes indicates its ability to adhere to and colonize the skin, or persist in an environment outside the host - a trait unique from other Candida species. Besides being associated globally with life-threatening disseminated infections, C. auris also poses significant clinical challenges due to its ability to adhere to polymeric surfaces and form highly drug-resistant biofilms. Here, we performed bioinformatic studies to identify the presence of adhesin proteins in C. auris, with sequence as well as 3-D structural homologies to the major adhesin/invasin of C. albicans, Als3. Anti-Als3p antibodies generated by vaccinating mice with NDV-3A (a vaccine based on the N-terminus of Als3 protein formulated with alum) recognized C. auris in vitro, blocked its ability to form biofilms and enhanced macrophage-mediated killing of the fungus. Furthermore, NDV-3A vaccination induced significant levels of C. auris cross-reactive humoral and cellular immune responses, and protected immunosuppressed mice from lethal C. auris disseminated infection, compared to the control alum-vaccinated mice. Finally, NDV-3A potentiated the protective efficacy of the antifungal drug micafungin, against C. auris candidemia. Identification of Als3-like adhesins in C. auris makes it a target for immunotherapeutic strategies using NDV-3A, a vaccine with known efficacy against other Candida species and safety as well as efficacy in clinical trials. Considering that C. auris can be resistant to almost all classes of antifungal drugs, such an approach has profound clinical relevance.Author Summary Candida auris has emerged as a major health concern to hospitalized patients and nursing home subjects. C. auris strains display multidrug resistance to current antifungal therapy and cause lethal infections. We have determined that C. auris harbors homologs of C. albicans Als cell surface proteins. The C. albicans NDV-3A vaccine, harboring the N-terminus of Als3p formulated with alum, generates cross-reactive antibodies against C. auris clinical isolates and protects neutropenic mice from hematogenously disseminated C. auris infection. Importantly, the NDV-3A vaccine displays an additive protective effect in neutropenic mice when combined with micafungin. Due to its proven safety and efficacy in humans against C. albicans infection, our studies support the expedited testing of the NDV-3A vaccine against C. auris in future clinical trials. |
Proficiency of WHO Global Foodborne Infections Network External Quality Assurance System participants in the identification and susceptibility testing of thermo-tolerant Campylobacter spp. from 2003-2012 (preprint)
Pedersen SK , Wagenaar JA , Vigre H , Roer L , Mikoleit M , Aidara-Kane A , Cawthorne AL , Aarestrup FM , Hendriksen RS . bioRxiv 2018 359794 Campylobacter spp. are food- and water borne pathogens. While rather accurate estimates for these pathogens are available in industrialized countries, a lack of diagnostic capacity in developing countries limits accurate assessments of prevalence in many regions. Proficiency in the identification and susceptibility testing of these organisms is critical for surveillance and control efforts. The aim of the study was to assess performance for identification and susceptibility testing of thermo-tolerant Campylobacter among laboratories participating in the World Health Organization (WHO) Global Foodborne Infections Network (GFN) External Quality Assurance System (EQAS) over a nine year period.Participants (primarily national level laboratories) were encouraged to self-evaluate performance as part of continuous quality improvement.The ability to correctly identify Campylobacter spp. varied by year and ranged from 61.9 % (2008) to 90.7 % (2012), and the ability to correctly perform antimicrobial susceptibility testing (AST) for Campylobacter spp. appeared to steadily increase from 91.4 % to 93.6 % in the test period (2009-2012).Poorest performance (60.0 % correct identification and 86.8 % correct AST results) was observed in African laboratories.Overall, approximately 10 % of laboratories reported either an incorrect identification or antibiogramme. As most participants were (supra)-national reference laboratories, these data raise significant concerns regarding capacity and proficiency at the local, clinical level. Addressing these diagnostic challenges is critical for both patient level management and broader surveillance and control efforts. |
An epitope-resurfaced virus-like particle can induce broad neutralizing antibody against four serotypes of dengue virus (preprint)
Shen WF , Galula JU , Liu JH , Liao MY , Huang CH , Wang YC , Wu HC , Liang JJ , Lin YL , Whitney MT , Chang GJ , Chen SR , Wu SR , Chao DY . bioRxiv 2018 351700 Dengue fever is caused by four different serotypes of dengue virus (DENV) which is the leading cause of worldwide arboviral diseases in humans. Virus-like particles (VLPs) containing flavivirus prM/E proteins have been demonstrated to be a potential vaccine candidate; however, the structure of dengue VLP is poorly understood. Herein we show for the first time that mD2VLP particles possess a T=1 icosahedral symmetry with a groove located within the E-protein dimers near the 2-fold vertices that exposed highly overlapping, cryptic neutralizing epitopes through cryo-electron microscopy reconstruction. Mice vaccinated with highly matured virus-like particles derived from DENV serotype 2 (mD2VLP) can generate higher cross reactive (CR) neutralization antibodies (NtAbs) and were protected against all 4 serotypes of DENV through clonal expansion supported by hybridoma and B-cell repertoire analysis. Our results revealed that a “epitope-resurfaced” mature-form dengue VLP has the potential to induce quaternary structure-recognizing broad CR NtAbs. |
Global emergence and population dynamics of divergent serotype 3 CC180 pneumococci (preprint)
Azarian T , Mitchell PK , Georgieva M , Thompson CM , Ghouila A , Pollard AJ , von Gottberg A , du Plessis M , Antonio M , Kwambana-Adams BA , Clarke SC , Everett D , Cornick J , Sadowy E , Hryniewicz W , Skoczynska A , Moisi JC , McGee L , Beall B , Metcalf BJ , Breiman RF , Ho PL , Reid R , O'Brien KL , Gladstone RA , Bentley SD , Hanage WP . bioRxiv 2018 314880 Streptococcus pneumoniae serotype 3 remains a significant cause of morbidity and mortality worldwide, despite inclusion in the 13-valent pneumococcal conjugate vaccine (PCV13). Serotype 3 increased in carriage since the implementation of PCV13 in the United States, while invasive disease rates remain unchanged. We investigated the persistence of serotype 3 in carriage and disease, through genomic analyses of a global sample of 301 serotype 3 isolates of the Netherlands3–31 (PMEN31) clone CC180, combined with associated patient data and PCV utilization among countries of isolate collection. We assessed phenotypic variation between dominant clades in capsule charge (zeta potential), capsular polysaccharide shedding, and susceptibility to opsonophagocytic killing, which have previously been associated with carriage duration, invasiveness, and vaccine escape. We identify a recent shift in the CC180 population attributed to a lineage termed Clade II, which was estimated by Bayesian coalescent analysis to have first appeared in 1968 [95% HPD: 1939–1989] and increased in prevalence and effective population size thereafter. Clade II isolates are divergent from the pre-PCV13 serotype 3 population in non-capsular antigenic composition, competence, and antibiotic susceptibility, the last resulting from the acquisition of a Tn916-like conjugative transposon. Differences in recombination rates among clades correlated with variations in the ATP-binding subunit of Clp protease as well as amino acid substitutions in the comCDE operon. Opsonophagocytic killing assays elucidated the low observed efficacy of PCV13 against serotype 3. Variation in PCV13 use among sampled countries was not independently correlated with the CC180 population shift; therefore, genotypic and phenotypic differences in protein antigens and, in particular, antibiotic resistance may have contributed to the increase of Clade II. Our analysis emphasizes the need for routine, representative sampling of isolates from disperse geographic regions, including historically under-sampled areas. We also highlight the value of genomics in resolving antigenic and epidemiological variations within a serotype, which may have implications for future vaccine development.Author Summary Streptococcus pneumoniae is a leading cause of bacterial pneumoniae, meningitis, and otitis media. Despite inclusion in the most recent pneumococcal conjugate vaccine, PCV13, serotype 3 remains epidemiologically important globally. We investigated the persistence of serotype 3 using whole-genome sequencing data form 301 isolates collected among 24 countries from 1993–2014. Through phylogenetic analysis, we identified three distinct lineages within a single clonal complex, CC180, and found one has recently emerged and grown in prevalence. We then compared genomic difference among lineages as well as variations in pneumococcal vaccine use among sampled countries. We found that the recently emerged lineage, termed Clade II, has a higher prevalence of antibiotic resistance compared to other lineages, diverse surface protein antigens, and a higher rate of recombination, a process by which bacteria can uptake and incorporate genetic material from its surroundings. Differences in vaccine use among sampled countries did not appear to be associated with the emergence of Clade II. We highlight the need to routine, representative sampling of bacterial isolates from diverse geographic areas and show the utility of genomic data in resolving epidemiological differences within a pathogen population. |
Geographical distribution of Anopheles stephensi in eastern Ethiopia (preprint)
Balkew M , Mumba P , Dengela D , Yohannes G , Getachew D , Yared S , Chibsa S , Murphy M , George K , Lopez K , Janies D , Choi SH , Spear J , Irish SR , Carter TE . bioRxiv 2019 802587 Background The recent detection of the South Asian malaria vector An. stephensi in Ethiopia and other regions in the Horn of Africa has raised concerns about its potential impact on malaria transmission. We report here findings of survey for this species in eastern Ethiopia using both morphological and molecular methods for species identification.Methods Adult and larval/pupal collections were conducted at ten sites in eastern Ethiopia and Anopheles specimens’ species were determined using standard morphological keys and genetic analysis.Results In total, 2,231 morphologically identified An. stephensi were collected. A molecular approach incorporating both PCR endpoint assay and sequencing of portions of the internal transcribed spacer 2 (ITS2) and cytochrome oxidase I (COI) loci confirmed the identity of the An. stephensi in most cases (119/124 of the morphologically identified An. stephensi confirmed molecularly). Additionally, we observed Aedes aegypti larvae and pupae at many of the An. stephensi larval habitats.Conclusions Our findings show that An. stephensi is widely distributed in eastern Ethiopia and highlight the need for further surveillance in the southern, western and northern parts of the country and throughout the Horn of Africa. |
Peripheral blood DNA methylation and autism spectrum disorder (preprint)
Andrews SV , Sheppard B , Windham GC , Schieve LA , Schendel DE , Croen LA , Chopra P , Alisch RS , Newschaffer CJ , Warren ST , Feinberg AP , Fallin MD , Ladd-Acosta C . bioRxiv 2018 320622 BackgroundSeveral reports have suggested a role for epigenetic mechanisms in ASD etiology. Epigenome-wide association studies (EWAS) in autism spectrum disorder (ASD) may shed light on particular biological mechanisms. However, studies of ASD cases versus controls have been limited by post-mortem timing and severely small sample sizes. Reports from in-life sampling of blood or saliva have also been very limited in sample size, and/or genomic coverage. We present the largest case-control EWAS for ASD to date, combining data from population-based case-control and case-sibling pair studies.MethodsDNA from 968 blood samples from children in the Study to Explore Early Development (SEED 1) was used to generate epigenome-wide array DNA methylation (DNAm) data at 485,512 CpG sites for 453 cases and 515 controls, using the Illumina 450K Beadchip. The Simons Simplex Collection (SSC) provided 450K array DNAm data on an additional 343 cases and their unaffected siblings. We performed EWAS meta-analysis across results from the two data sets, with adjustment for sex and surrogate variables that reflect major sources of biological variation and technical confounding such as cell type, batch, and ancestry. We compared top EWAS results to those from a previous brain-based analysis. We also tested for enrichment of ASD EWAS CpGs for being targets of meQTL associations using available SNP genotype data in the SEED sample.FindingsIn this meta-analysis of blood-based DNA from 796 cases and 858 controls, no single CpG met a Bonferroni discovery threshold of p < 1.12×10−7. Seven CpGs showed differences at p < 1×10−5 and 48 at 1×10−4. Of the top 7, 5 showed brain-based ASD associations as well, often with larger effect sizes, and the top 48 overall showed modest concordance (r = 0.31) in direction of effect with cerebellum samples. Finally, we observed suggestive evidence for enrichment of CpG sites controlled by SNPs (meQTL targets) among the EWAS CpGs hits, which was consistent across EWAS and meQTL discovery p-value thresholds.ConclusionsWe report the largest case-control EWAS study of ASD to date. No single CpG site showed a large enough DNAm difference between cases and controls to achieve epigenome-wide significance in this sample size. However, our results suggest the potential to observe disease associations from blood-based samples. Among the 7 sites achieving suggestive statistical significance, we observed consistent, and stronger, effects at the same sites among brain samples. Discovery-oriented EWAS for ASD using blood samples will likely need even larger samples and unified genetic data to further understand DNAm differences in ASD. |
Reduced long-lasting insecticidal net efficacy and pyrethroid insecticide resistance are associated with over-expression of CYP6P4, CYP6P3 and CYP6Z1 in populations of Anopheles coluzzii from South-East Côte d’Ivoire (preprint)
Meiwald A , Clark E , Kristan M , Edi C , Jeffries CL , Pelloquin B , Irish SR , Walker T , Messenger LA . bioRxiv 2020 2020.09.24.311639 Background Resistance to major public health insecticides in Côte d’Ivoire has intensified and now threatens the long-term effectiveness of malaria vector control interventions.Methods This study evaluated the bioefficacy of conventional and next-generation long-lasting insecticidal nets (LLINs), determined resistance profiles, and characterized molecular and metabolic mechanisms in wild Anopheles coluzzii from South-East Côte d’Ivoire in 2019.Results Phenotypic resistance was intense: more than 25% of mosquitoes survived exposure to ten times the doses of pyrethroids required to kill susceptible populations. Similarly, 24-hour mortality to deltamethrin-only LLINs was very low and not significantly different to an untreated net. Sub-lethal pyrethroid exposure did not induce significant delayed vector mortality 72 hours later. In contrast, LLINs containing the synergist piperonyl butoxide (PBO), or new insecticides, clothianidin and chlorfenapyr, were highly toxic to An. coluzzii. Pyrethroid-susceptible An. coluzzii were significantly more likely to be infected with malaria, compared to those that survived insecticidal exposure. Pyrethroid resistance was associated with significant over-expression of CYP6P4, CPY6Z1 and CYP6P3.Conclusions Study findings raise concerns regarding the operational failure of standard LLINs and support the urgent deployment of vector control interventions incorporating PBO, chlorfenapyr or clothianidin in areas of high resistance intensity in Côte d’Ivoire.Competing Interest StatementThe authors have declared no competing interest. |
A search for snail-related answers to explain differences in response of Schistosoma mansoni to praziquantel treatment among responding and persistent hotspot villages along the Kenyan shore of Lake Victoria (preprint)
Mutuku MW , Laidemitt MR , Beechler BR , Mwangi IN , Otiato FO , Agola EL , Ochanda H , Kamel B , Mkoji GM , Steinauer ML , Loker ES . bioRxiv 2019 394031 Following a four-year annual praziquantel treatment campaign the resulting prevalence of S. mansoni was seen to differ among individual villages along the Kenyan shore of Lake Victoria. We have investigated possible inherent differences in snail-related aspects of transmission among such 10 villages, including six persistent hotspot (PHS) villages (≤30% reduction in prevalence following repeated treatments) located along the west-facing shore of the lake, and four PZQ-responding (RESP) villages (>30% prevalence reduction following repeated treatment) along Winam Gulf. When taking into account all sampling sites and times and water hyacinth presence/absence, shoreline-associated B. sudanica from PHS and RESP villages did not differ in relative abundance or prevalence of S. mansoni infection. Water hyacinth intrusions were associated with increased B. sudanica abundance. The deeper water snail Biomphalaria choanomphala was significantly more abundant in the PHS villages and prevalence of S. mansoni among villages both before and after control was positively correlated with B. choanomphala abundance. Worm recoveries from sentinel mice did not differ between PHS and RESP villages, and abundance of non-schistosome trematode species was not associated with S. mansoni abundance. Biomphalaria choanomphala provides an alternative, deepwater mode of transmission that may favor greater persistence of S. mansoni in PHS villages. As we found evidence for ongoing S. mansoni transmission in all 10 villages, we conclude conditions conducive for transmission and reinfection occur ubiquitously. This argues for an integrated, basin-wide plan for schistosomiasis control to counteract rapid reinfections facilitated by large snail populations and movements of infected people around the lake. |
Multiplex serologic testing within a cross-sectional lymphatic filariasis sentinel site survey in coastal Kenya reveals community-level differences in IgG antibody responses to parasitic diseases and vaccines (preprint)
Njenga SM , Kanyi HM , Arnold BF , Matendechero SH , Onsongo JK , Won KY , Priest JW . bioRxiv 2019 604181 Accurate, cost-effective measurement of the burden of co-endemic infections would enable public health managers to identify opportunities for implementation of integrated control programs. Dried blood spots (DBS) collected during a cross-sectional lymphatic filariasis sentinel site survey in the Kenyan coastal counties of Lamu, Tana River, Kilifi, Kwale, and Taita-Taveta were used for the integrated detection of serologic IgG antibodies against antigens from several parasitic infections (Wuchereria bancrofti, Schistosoma mansoni, Plasmodium spp, Ascaris lumbricoides, and Strongyloides stercoralis) as well as markers for immunity to vaccine-preventable diseases (measles, diphtheria, and tetanus) on a multiplex bead assay (MBA) platform. High heterogeneity was observed in antibody responses by pathogen and antigen across the sentinel sites. Antibody seroprevalence against Wb123, Bm14, and Bm33 recombinant filarial antigens were generally higher in Ndau Island (p<0.0001), which also had the highest prevalence of filarial antigenemia compared to other communities. Antibody responses to the Plasmodium species antigens CSP and MSP-119 were higher in Kilifi and Kwale counties, with Jaribuni community showing higher overall mean seroprevalence (p<0.0001). Kimorigo community in Taita-Taveta County was the only area where antibody responses against Schistosoma mansoni Sm25 recombinant antigen were detected. Seroprevalence rates to Strongyloides antigen NIE ranged between 3% and 26%, and there was high heterogeneity in immune responses against an Ascaris antigen among the study communities. Differences were observed between communities in terms of seroprevalence to vaccine-preventable diseases. Seroprotection to tetanus was lower in all 3 communities in Kwale County compared to the rest of the communities. This study has demonstrated that the MBA platform holds promise for rapid integrated monitoring of trends of infections of public health importance in endemic areas, and assessing the effectiveness of control and elimination programs.Author Summary Establishment of successful private-public partnerships in the recent past has led to an increase in resources available for control and elimination of malaria and Neglected Tropical Diseases (NTDs). Implementation of control and elimination programs and their subsequent monitoring and evaluation would be greatly facilitated by development of new tools and strategies for rapid identification of areas of transmission so that interventions could be prioritized to regions where they were most needed. Since development of antibody responses in a host depend on exposure to an infectious agent, assessment of such serologic markers provides a sensitive way to measure differences between populations in pathogen exposure. Our study applied a state-of-the-art multiplex bead assay platform to perform integrated measurement of antibody responses to multiple parasitic diseases and immunizing antigens for vaccine-preventable diseases (VPDs) in ten lymphatic filariasis sentinel sites across the Kenyan coastal region. A community-level analysis of age-specific and overall mean seroprevalence fit using a flexible model ensemble provided an improved understanding about the distributions of the various parasitic infections and seroprotection to VPDs. This study provides an important proof of concept for how we could dramatically increase the value of existing surveillance activities using small volumes of blood collected on filter paper and analyzed using a single multiplex laboratory assay and novel data analysis techniques. |
The Impact of Antimalarial Resistance on the Genetic Structure of Plasmodium falciparum in the DRC (preprint)
Verity R , Aydemir O , Brazeau NF , Watson OJ , Hathaway NJ , Mwandagalirwa MK , Marsh PW , Thwai K , Fulton T , Denton M , Morgan AP , Parr JB , Tumwebaze PK , Conrad M , Rosenthal PJ , Ishengoma DS , Ngondi J , Gutman J , Mulenga M , Norris DE , Moss WJ , Mensah BA , Myers-Hansen JL , Ghansah A , Tshefu AK , Ghani AC , Meshnick SR , Bailey JA , Juliano JJ . bioRxiv 2019 656561 The Democratic Republic of the Congo (DRC) harbors 11% of global malaria cases, yet little is known about the spatial and genetic structure of the parasite population in that country. We sequenced 2537 Plasmodium falciparum infections, including a nationally representative population sample from DRC and samples from surrounding countries, using molecular inversion probes - a novel high-throughput genotyping tool. We identified an east-west divide in haplotypes known to confer resistance to chloroquine and sulfadoxine-pyrimethamine. Furthermore, we identified highly related parasites over large geographic distances, indicative of gene flow and migration. Our results were consistent with a background of isolation by distance combined with the effects of selection for antimalarial drug resistance. This study provides a high-resolution view of parasite genetic structure across a large country in Africa and provides a baseline to study how implementation programs may impact parasite populations. |
Field evaluation of malachite green loop-mediated isothermal amplification as a malaria parasite detection tool in a health post in Roraima state, Brazil (preprint)
Kudyba HM , Louzada J , Ljolje D , Kudyba KA , Muralidharan V , Oliveira-Ferreira J , Lucchi NW . bioRxiv 2018 408609 Malaria is a debilitating parasitic disease that causes significant morbidity and mortality. Microscopic detection of parasites is currently the “gold standard” diagnostic. This technique is limited in its ability to detect low-density infections, is time consuming, and requires a highly trained microscopist. Malaria epidemiological surveillance studies especially aimed at the detection of low-density infection and asymptomatic cases will require more sensitive and user-friendly tools. We have shown previously that the molecular-based, colorimetric malachite green loop-mediated isothermal amplification (MG-LAMP) assay is a valuable tool for diagnosing malaria infection in a laboratory setting. In this study, we field evaluated this assay in a malaria diagnostic post in Roraima, Brazil. We prospectively collected 91 patient samples and performed microscopy, MG-LAMP, and real-time PCR (PET-PCR) to detect Plasmodium infection. Two independent readers were used to score the MG-LAMP tests to assess whether the sample was positive (blue/green) or negative (clear). There was 100% agreement between the two readers (Kappa=1). All tests detected 33 positive samples, but both the MG-LAMP and PET-PCR detected 6 and 7 more positive samples, respectively. The PET-PCR assay detected 6 mixed infections (defined as infection with both P. falciparum and P. vivax) while microscopy detected one and MG-LAMP detected two of these mixed infections. Microscopy did not detect any Plasmodium infection in 26 of the enrolled asymptomatic cases while MG-LAMP detected five and PET-PCR assay three positive cases. Overall, MG-LAMP provided a simpler and user-friendly molecular method for malaria diagnosis that is more sensitive than microscopy. Additionally, MG- LAMP has the capacity to test 38 samples per run (one hour), allowing for the screening of large number of samples which is appealing when large-scale studies are necessary e.g. in community surveillance studies. The current MG-LAMP assay was limited in its ability to detect mixed infection when compared to the PET-PCR, but otherwise proved to be a powerful tool for malaria parasite detection in the field and opens new perspectives in the implementation of surveillance studies in malaria elimination campaigns. |
%svy_logistic_regression: A generic SAS® macro for simple and multiple logistic regression and creating quality publication-ready tables using survey or non-survey data (preprint)
Muthusi J , Mwalili S , Young P . bioRxiv 2019 575605 Introduction Reproducible research is increasingly gaining interest in the research community. Automating the production of research manuscript tables from statistical software can help increase the reproducibility of findings. Logistic regression is used in studying disease prevalence and associated factors in epidemiological studies and can be easily performed using widely available software including SAS, SUDAAN, Stata or R. However, output from these software must be processed further to make it readily presentable. There exists a number of procedures developed to organize regression output, though many of them suffer limitations of flexibility, complexity, lack of validation checks for input parameters, as well as inability to incorporate survey design.Methods We developed a SAS macro, %svy_logistic_regression, for fitting simple and multiple logistic regression models. The macro also creates quality publication-ready tables using survey or non-survey data which aims to increase transparency of data analyses. It further significantly reduces turn-around time for conducting analysis and preparing output tables while also addressing the limitations of existing procedures.Results We demonstrate the use of the macro in the analysis of the 2013-2014 National Health and Nutrition Examination Survey (NHANES), a complex survey designed to assess the health and nutritional status of adults and children in the United States. The output presented here is directly from the macro and is consistent with how regression results are often presented in the epidemiological and biomedical literature, with unadjusted and adjusted model results presented side by side.Conclusions The SAS code presented in this macro is comprehensive, easy to follow, manipulate and to extend to other areas of interest. It can also be incorporated quickly by the statistician for immediate use. It is an especially valuable tool for generating quality, easy to review tables which can be incorporated directly in a publication. |
Mapping the Host-Pathogen Space to Link Longitudinal and Cross-sectional Biomarker Data: Leptospira Infection in California Sea Lions (Zalophus californianus) as a Case Study (preprint)
Prager KC , Buhnerkempe MG , Greig DJ , Orr AJ , Jensen ED , Gomez F , Galloway RL , Wu Q , Gulland FMD , Lloyd-Smith JO . bioRxiv 2019 819532 Confronted with the challenge of understanding population-level processes, disease ecologists and epidemiologists often simplify quantitative data into distinct physiological states (e.g. susceptible, exposed, infected, recovered). However, data defining these states often fall along a spectrum rather than into clear categories. Hence, the host-pathogen relationship is more accurately defined using quantitative data, often integrating multiple diagnostic measures, just as clinicians do to assess their patients. We use quantitative data on a bacterial infection (Leptospira interrogans) in California sea lions (Zalophus californianus) to improve both our individual-level and population-level understanding of this host-pathogen system. We create a “host-pathogen space” by mapping multiple biomarkers of infection (e.g. serum antibodies, pathogen DNA) and disease state (e.g. serum chemistry values) from 13 longitudinally sampled, severely ill individuals to visualize and characterize changes in these values through time. We describe a clear, unidirectional trajectory of disease and recovery within this host-pathogen space. Remarkably, this trajectory also captures the broad patterns in larger cross-sectional datasets of 1456 wild sea lions in all states of health. This mapping framework enables us to determine an individual’s location in their time-course since initial infection, and to visualize the full range of clinical states and antibody responses induced by pathogen exposure, including severe acute disease, chronic subclinical infection, and recovery. We identify predictive relationships between biomarkers and outcomes such as survival and pathogen shedding, and in certain cases we can impute values for missing data, thus increasing the size of the useable dataset. Mapping the host-pathogen space and using quantitative biomarker data provides more nuanced approaches for understanding and modeling disease dynamics in a system, yielding benefits for the clinician who needs to triage patients and prevent transmission, and for the disease ecologist or epidemiologist wishing to develop appropriate risk management strategies and assess health impacts on a population scale.Author Summary A pathogen can cause a range of disease severity across different host individuals, and these presentations change over the time-course from infection to recovery. These facts complicate the work of epidemiologists and disease ecologists seeking to understand the factors governing disease spread, often working with cross-sectional data. Recognizing these facts also highlights the shortcomings of classical approaches to modeling infectious disease, which typically rely on discrete and well-defined disease states. Here we show that by analyzing multiple biomarkers of health and infection simultaneously, treating these values as quantitative rather than binary indicators, and including a modest amount of longitudinal sampling of hosts, we can create a map of the host-pathogen interaction that shows the full spectrum of disease presentations and opens doors for new insights and predictions. By accounting for individual variation and capturing changes through time since infection, this mapping framework enables more robust interpretation of cross-sectional data; e.g., to detect predictive relationships between biomarkers and key outcomes such as survival, or to assess whether observed disease is associated with the pathogen of interest. This approach can help epidemiologists, ecologists and clinicians to better study and manage the many infectious diseases that exhibit complex relationships with their hosts. |
From people to Panthera: Natural SARS-CoV-2 infection in tigers and lions at the Bronx Zoo (preprint)
McAloose D , Laverack M , Wang L , Killian ML , Caserta LC , Yuan F , Mitchell PK , Queen K , Mauldin MR , Cronk BD , Bartlett SL , Sykes JM , Zec S , Stokol T , Ingerman K , Delaney MA , Fredrickson R , Ivančić M , Jenkins-Moore M , Mozingo K , Franzen K , Bergeson NH , Goodman L , Wang H , Fang Y , Olmstead C , McCann C , Thomas P , Goodrich E , Elvinger F , Smith DC , Tong S , Slavinski S , Calle PP , Terio K , Torchetti MK , Diel DG . bioRxiv 2020 2020.07.22.213959 We describe the first cases of natural SARS-CoV-2 infection detected in animals in the United States. In March 2020, four tigers and three lions at the Bronx Zoo developed mild respiratory signs. SARS-CoV-2 RNA was detected by rRT-PCR in respiratory secretions and/or feces from all seven affected animals; viral RNA and/or antibodies were detected in their keepers. SARS-CoV-2 was isolated from respiratory secretions or feces from three affected animals; in situ hybridization co-localized viral RNA with cellular damage. Whole genome sequence and haplotype network analyses showed tigers and lions were infected with two different SARS-CoV-2 strains, suggesting independent viral introductions. The source of SARS-CoV-2 infection in the lions is unknown. Epidemiological data and genetic similarities between keeper and tiger viruses indicate human to animal transmission.Competing Interest StatementThe authors have declared no competing interest. |
A systematic review and evaluation of Zika virus forecasting and prediction research during a public health emergency of international concern (preprint)
Kobres PY , Chretien JP , Johansson MA , Morgan JJ , Whung PY , Mukundan H , Del Valle SY , Forshey BM , Quandelacy TM , Biggerstaff M , Viboud C , Pollett S . bioRxiv 2019 634832 INTRODUCTION Epidemic forecasting and prediction tools have the potential to provide actionable information in the midst of emerging epidemics. While numerous predictive studies were published during the 2016-2017 Zika Virus (ZIKV) pandemic, it remains unknown how timely, reproducible and actionable the information produced by these studies was.METHODS To improve the functional use of mathematical modeling in support of future infectious disease outbreaks, we conducted a systematic review of all ZIKV prediction studies published during the recent ZIKV pandemic using the PRISMA guidelines. Using MEDLINE, EMBASE and grey literature review, we identified studies that forecasted, predicted or simulated ecological or epidemiological phenomenon related to the Zika pandemic that were published as of March 01, 2017. Eligible studies underwent evaluation of objectives, data sources, methods, timeliness, reproducibility, accessibility and clarity by independent reviewers.RESULTS 2034 studies were identified, of which n = 73 met eligibility criteria. Spatial spread, R0 (basic reproductive number) and epidemic dynamics were most commonly predicted, with few studies predicting Guillain-Barré Syndrome burden (4%), sexual transmission risk (4%) and intervention impact (4%). Most studies specifically examined populations in the Americas (52%), with few African-specific studies (4%). Case count (67%), vector (41%) and demographic data (37%) were the most common data sources. Real-time internet data and pathogen genomic information were used in 7% and 0% of studies, respectively, and social science and behavioral data were typically absent in modeling efforts. Deterministic models were favored over stochastic approaches. Forty percent of studies made model data entirely available, 29% provided all relevant model code, 43% presented uncertainty in all predictions and 54% provided sufficient methodological detail allowing complete reproducibility. Fifty-one percent of predictions were published after the epidemic peak in the Americas. While the use of preprints improved the accessibility of ZIKV predictions by a median 119 days sooner than journal publication dates, they were used in only 30% of studies.CONCLUSIONS Many ZIKV predictions were published during the 2016-2017 pandemic. The accessibility, reproducibility, timeliness, and incorporation of uncertainty in these published predictions varied and indicates that there is substantial room for improvement. To enhance the utility of analytical tools for outbreak response, it is essential to improve the sharing of model data, code, and preprints for future outbreaks, epidemics and pandemics.Author summary Researchers published many studies which sought to predict and forecast important features of Zika virus (ZIKV) infections and their spread during the 2016-2017 ZIKV pandemic. We conducted a comprehensive review of such ZIKV prediction studies and evaluated their aims, the data sources they used, which methods were used, how timely they were published, and whether they provided sufficient information to be used or reproduced by others. Of the 73 studies evaluated, we found that the accessibility, reproducibility, timeliness, and incorporation of uncertainty in these published predictions varied and indicates that there is substantial room for improvement. We identified that the release of study findings before formal journal publication (‘pre-prints’) increased the timeliness of Zika prediction studies, but note they were infrequently used during this public health emergency. Addressing these areas can improve our understanding of Zika and other outbreaks and ensure that forecasts can inform preparedness and response to future outbreaks, epidemics and pandemics. |
Mitigating Pandemic Risk with Influenza A Virus Field Surveillance at a Swine-Human Interface (preprint)
Rambo-Martin BL , Keller MW , Wilson MM , Nolting JM , Anderson TK , Vincent AL , Bagal UR , Jang Y , Neuhaus EB , Davis CT , Bowman AS , Wentworth DE , Barnes JR . bioRxiv 2019 585588 Working overnight at a large swine exhibition, we identified an influenza A virus (IAV) outbreak in swine, nanopore-sequenced 13 IAV genomes from samples collected, and in real-time, determined that these viruses posed a novel risk to humans due to genetic mismatches between the viruses and current pre-pandemic candidate vaccine viruses (CVV). We developed and used a portable IAV sequencing and analysis platform called Mia (Mobile Influenza Analysis) to complete and characterize full-length consensus genomes approximately 18 hours after unpacking the mobile lab. Swine are important animal IAV reservoirs that have given rise to pandemic viruses via zoonotic transmission. Genomic analyses of IAV in swine are critical to understanding pandemic risk of viruses in this reservoir, and characterization of viruses circulating in exhibition swine enables rapid comparison to current seasonal influenza vaccines and CVVs. The Mia system rapidly identified three genetically distinct swine IAV lineages from three subtypes: A(H1N1), A(H3N2) and A(H1N2). Additional analysis of the HA protein sequences of the A(H1N2) viruses identified >30 amino acid differences between the HA1 portion of the hemagglutinin of these viruses and the most closely related pre-2009 CVV. All virus sequences were emailed to colleagues at CDC who initiated development of a synthetically derived CVV designed to provide an optimal antigenic match with the viruses detected in the exhibition. In subsequent months, this virus caused 13 infections in humans, and was the dominant variant virus in the US detected in 2018. Had this virus caused a severe outbreak or pandemic, our proactive surveillance efforts and CVV derivation would have provided an approximate 8 week time advantage for vaccine manufacturing. This is the first report of the use of field-derived nanopore sequencing data to initiate a real-time, actionable public health countermeasure. |
Comprehensive evaluation of differential serodiagnosis between Zika and dengue viral infection (preprint)
Chao DY , Whitney MT , Davis BS , Medina FA , Munoz JL , Chang GJ . bioRxiv 2018 421628 Diagnostic testing for Zika virus (ZIKV) or dengue virus (DENV) infection can be accomplished by a nucleic acid detection method; however, a negative result does not exclude infection due to the low virus titer during infection depending on the timing of sample collection. Therefore, a ZIKV- or DENV-specific serological assay is essential for the accurate diagnosis of patients and to prevent potential severe health outcomes. A retrospective study design with dual approaches of collecting human serum samples for testing was developed. All serum samples were extensively evaluated by using both non-infectious virus-like particles (VLPs) and soluble non-structural protein 1 (NS1) in the standard immunoglobulin M (IgM) antibody-capture enzyme-linked immunosorbent assay (MAC-ELISA). Both VLP- and NS1-MAC-ELISAs were found to have similar sensitivity for detecting anti-premembrane/envelope and NS1 antibodies from ZIKV-infected patient sera. Group cross reactive (GR)-antibody-ablated homologous fusion peptide-mutated (FP)-VLPs consistently showed higher P/N values than homologous wild-type VLPs. Therefore, FP-VLPs were used to develop the algorithm for differentiating ZIKV from DENV infection. Overall, the sensitivity and specificity of the FP-VLP-MAC-ELISA and the NS1-MAC-ELISA were each higher than 80% with no statistical significance. A novel approach to differentiate ZIKV from DENV infection serologically has been developed. The accuracy can reach up to 95% when combining both VLP and NS1 assays. In comparison to current guidelines using neutralization tests to measure ZIKV antibody, this approach can facilitate laboratory screening for ZIKV infection, especially in regions where DENV infection is endemic and capacity for neutralization testing does not exist. |
Downgrading disease transmission risk estimates using terminal importations (preprint)
Fox SJ , Bellan SE , Perkins TA , Johansson MA , Meyers LA . bioRxiv 2018 265942 As emerging and re-emerging infectious diseases like dengue, Ebola, chikungunya, and Zika threaten new populations worldwide, officials scramble to assess local severity and transmissibility, with little to no epidemiological history to draw upon. Standard methods for assessing autochthonous (local) transmission risk make either indirect estimates based on ecological suitability or direct estimates only after local cases accumulate. However, an overlooked source of epidemiological data that can meaningfully inform risk assessments prior to outbreak emergence is the absence of transmission by imported cases. Here, we present a method for updating a priori ecological estimates of transmission risk using real-time importation data. We demonstrate our method using Zika importation and transmission data from Texas in 2016, a high-risk region in the southern United States. Our updated risk estimates are lower than previously reported, with only six counties in Texas likely to sustain a Zika epidemic, and consistent with the number of autochthonous cases detected in 2017. Importation events can thereby provide critical, early insight into local transmission risks as infectious diseases expand their global reach. |
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