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1.  Reassortment in segmented RNA viruses: mechanisms and outcomes 
Nature reviews. Microbiology  2016;14(7):448-460.
Segmented RNA viruses are widespread in nature and include important human, animal and plant pathogens, such as influenza viruses and rotaviruses. Although the origin of RNA virus genome segmentation remains elusive, a major consequence of this genome structure is the capacity for reassortment to occur during co-infection, whereby segments are exchanged among different viral strains. Therefore, reassortment can create viral progeny that contain genes that are derived from more than one parent, potentially conferring important fitness advantages or disadvantages to the progeny virus. However, for segmented RNA viruses that package their multiple genome segments into a single virion particle, reassortment also requires genetic compatibility between parental strains, which occurs in the form of conserved packaging signals, and the maintenance of RNA and protein interactions. In this Review, we discuss recent studies that examined the mechanisms and outcomes of reassortment for three well-studied viral families — Cystoviridae, Orthomyxoviridae and Reoviridae — and discuss how these findings provide new perspectives on the replication and evolution of segmented RNA viruses.
PMCID: PMC5119462  PMID: 27211789
2.  A retrospective analysis of melioidosis in Cambodian children, 2009–2013 
BMC Infectious Diseases  2016;16:688.
Melioidiosis, infection by Burkholderia pseudomallei, is an important but frequently under-recognised cause of morbidity and mortality in Southeast Asia and elsewhere in the tropics. Data on the epidemiology of paediatric melioidosis in Cambodia are extremely limited.
Culture-positive melioidosis cases presenting to Angkor Hospital for Children, a non-governmental paediatric hospital located in Siem Reap, Northern Cambodia, between 1st January 2009 and 31st December 2013 were identified by searches of hospital and laboratory databases and logbooks.
One hundred seventy-three evaluable cases were identified, presenting from eight provinces. For Siem Reap province, the median commune level incidence was estimated to be 28-35 cases per 100,000 children <15 years per year. Most cases presented during the wet season, May to October. The median age at presentation was 5.7 years (range 8 days–15.9 years). Apart from undernutrition, co-morbidities were rare. Three quarters (131/173) of the children had localised infection, most commonly skin/soft tissue infection (60 cases) or suppurative parotitis (51 cases). There were 39 children with B. pseudomallei bacteraemia: 29 (74.4%) of these had clinical and/or radiological evidence of pneumonia. Overall mortality was 16.8% (29/173) with mortality in bacteraemic cases of 71.8% (28/39). At least seven children did not receive an antimicrobial with activity against B. pseudomallei prior to death.
This retrospective study demonstrated a considerable burden of melioidosis in Cambodian children. Given the high mortality associated with bacteraemic infection, there is an urgent need for greater awareness amongst healthcare professionals in Cambodia and other countries where melioidosis is known or suspected to be endemic. Empiric treatment guidelines should ensure suspected cases are treated early with appropriate antimicrobials.
Electronic supplementary material
The online version of this article (doi:10.1186/s12879-016-2034-9) contains supplementary material, which is available to authorized users.
PMCID: PMC5117556  PMID: 27871233
Cambodia; Melioidosis; Paediatric
3.  Understanding pneumococcal serotype 1 biology through population genomic analysis 
BMC Infectious Diseases  2016;16:649.
Pneumococcus kills over one million children annually and over 90 % of these deaths occur in low-income countries especially in Sub-Saharan Africa (SSA) where HIV exacerbates the disease burden. In SSA, serotype 1 pneumococci particularly the endemic ST217 clone, causes majority of the pneumococcal disease burden. To understand the evolution of the virulent ST217 clone, we analysed ST217 whole genomes from isolates sampled from African and Asian countries.
We analysed 226 whole genome sequences from the ST217 lineage sampled from 9 African and 4 Asian countries. We constructed a whole genome alignment and used it for phylogenetic and coalescent analyses. We also screened the genomes to determine presence of antibiotic resistance conferring genes.
Population structure analysis grouped the ST217 isolates into five sequence clusters (SCs), which were highly associated with different geographical regions and showed limited intracontinental and intercontinental spread. The SCs showed lower than expected genomic sequence, which suggested strong purifying selection and small population sizes caused by bottlenecks. Recombination rates varied between the SCs but were lower than in other successful clones such as PMEN1. African isolates showed higher prevalence of antibiotic resistance genes than Asian isolates. Interestingly, certain West African isolates harbored a defective chloramphenicol and tetracycline resistance-conferring element (Tn5253) with a deletion in the loci encoding the chloramphenicol resistance gene (cat pC194), which caused lower chloramphenicol than tetracycline resistance. Furthermore, certain genes that promote colonisation were absent in the isolates, which may contribute to serotype 1’s rarity in carriage and consequently its lower recombination rates.
The high phylogeographic diversity of the ST217 clone shows that this clone has been in circulation globally for a long time, which allowed its diversification and adaptation in different geographical regions. Such geographic adaptation reflects local variations in selection pressures in different locales. Further studies will be required to fully understand the biological mechanisms which makes the ST217 clone highly invasive but unable to successfully colonise the human nasopharynx for long durations which results in lower recombination rates.
Electronic supplementary material
The online version of this article (doi:10.1186/s12879-016-1987-z) contains supplementary material, which is available to authorized users.
PMCID: PMC5100261  PMID: 27821148
Pneumococcal serotype 1; ST217; Phylogeography; Evolution; Antibiotic resistance
5.  The Emperor Has No Symptoms: The Risks of a Blanket Approach to Using Epinephrine Autoinjectors for All Allergic Reactions 
Fatal anaphylaxis in humans is rare and unpredictable. We note a trend to provide allergic individuals with care plans that recommend immediate use of epinephrine autoinjectors if allergen ingestion is suspected, even in the absence of any allergic symptoms, without any supporting evidence base. Instructions to use an autoinjector device, irrespective of reaction severity and especially when symptoms are actually absent, are likely to add to parental and patient anxiety. Of greater concern is the possibility of epinephrine being administered “too early” to treat initial, mild symptoms that then progress to severe anaphylaxis. It is not hard to visualize a scenario where one or both epinephrine autoinjectors have been deployed for mild symptoms, yet the reaction progresses to a severe reaction and no further epinephrine is available for administration. Epinephrine needs to be available as a rescue treatment for anaphylaxis, potentially buying valuable minutes while emergency medical services are activated to attend. Food-allergic individuals and their carers need to be provided with more constructive strategies and support than merely being told to “use your pen.”
PMCID: PMC5123619  PMID: 27283056
Food allergy; Anaphylaxis; Epinephrine; Autoinjector; Emergency management; ED, Emergency departments; EMS, Emergency medical services
6.  Rate of novel host invasion affects adaptability of evolving RNA virus lineages 
Although differing rates of environmental turnover should be consequential for the dynamics of adaptive change, this idea has been rarely examined outside of theory. In particular, the importance of RNA viruses in disease emergence warrants experiments testing how differing rates of novel host invasion may impact the ability of viruses to adaptively shift onto a novel host. To test whether the rate of environmental turnover influences adaptation, we experimentally evolved 144 Sindbis virus lineages in replicated tissue-culture environments, which transitioned from being dominated by a permissive host cell type to a novel host cell type. The rate at which the novel host ‘invaded’ the environment varied by treatment. The fitness (growth rate) of evolved virus populations was measured on each host type, and molecular substitutions were mapped via whole genome consensus sequencing. Results showed that virus populations more consistently reached high fitness levels on the novel host when the novel host ‘invaded’ the environment more gradually, and gradual invasion resulted in less variable genomic outcomes. Moreover, virus populations that experienced a rapid shift onto the novel host converged upon different genotypes than populations that experienced a gradual shift onto the novel host, suggesting a strong effect of historical contingency.
PMCID: PMC4632612  PMID: 26246544
adaptation; Sindbis virus; alphavirus; experimental evolution; epistasis; contingency
7.  The Use of TKM-100802 and Convalescent Plasma in 2 Patients With Ebola Virus Disease in the United States 
Kraft, Colleen S. | Hewlett, Angela L. | Koepsell, Scott | Winkler, Anne M. | Kratochvil, Christopher J. | Larson, LuAnn | Varkey, Jay B. | Mehta, Aneesh K. | Lyon, G. Marshall | Friedman-Moraco, Rachel J. | Marconi, Vincent C. | Hill, Charles E. | Sullivan, James N. | Johnson, Daniel W. | Lisco, Steven J. | Mulligan, Mark J. | Uyeki, Timothy M. | McElroy, Anita K. | Sealy, Tara | Campbell, Shelley | Spiropoulou, Christina | Ströher, Ute | Crozier, Ian | Sacra, Richard | Connor, Michael J. | Sueblinvong, Viranuj | Franch, Harold A. | Smith, Philip W. | Ribner, Bruce S. | Smith, Philip | Hewlett, Angela | Schwedhelm, Shelly | Boulter, Kate | Beam, Elizabeth | Gibbs, Shawn | Lowe, John | Kratochvil, Chris | Sullivan, James | Johnson, Dan | Lisco, Steve | Piquette, Craig | Bailey, Kristina | Auxier, Joseph | Boer, Brian | Hanson, Travis | Kaseman, Julia | Khan, M. Salman | Rhee, Ji Hyun | Wells, Adam | Florescu, Diana | Kalil, Andre | Rupp, Mark | Becker, Valerie | Boeckman, Bridget | Elder, Erica | Fitch, Abby | Flood, Elizabeth | Freml, Meagan | Frigge, Roman | Hanzlik, Kelly Ann | Jensen, Lois | Kraus, Nicole | Molacek, Drew | Nevins, Jerry | Parker, Alicia | Peters, Jeff | Rand, Cheryl | Roesler, Karen | Ryalls, Kendall | Shradar, Morgan | Strain, Amanda | Sunderman, Timothy | Sundermeier, Jennifer | Swanhorst, John | Vasa, Angela | Bellinghausen, Jean | Freihaut, Frank | Denny, Susan | Mainelli, Lauren | Pinkney, Dee | Ray, Deborah | Jevne, Jay | Knight, Kalen | McCroy, Derrick | Nadeau, Ralph | Nightser, Anna | Iwen, Pete | Sambol, Tony | Herrera, Vicki | Morgan, David | Trotter, Sarah | Kerby, Amy | Peters, Sue | Southern, Timothy | Murphy, Caitlin | Morris, Rosanna | Nuss, Sue | Franco, Theresa | Ogden, Connie | Lazure, Julie | Straub, Dawn | Koepsell, Scott | Hinrichs, Steve | Plumb, Troy | Florescu, Marius | Becker, John | Souchek, Jenna | Beck, Jon | Larson, Luann | Heires, Peggy | Gordon, Bruce | Paulsen, Gail | Wolford, Barb | Petersen, Jill | Marion, Nedra | Hayes, Kim | Tyner, Kate | Wilson, Taylor | Baltes, Paul | Nowatzke, Jenny | Nguyen, Jonathan | Turner, Paul | Boonstra, Barb | Jelden, Katelyn | Portrey, Randy | Stringfield, Doug | Scofield, Bryan | Svanda, Gary | Horihan, Jolene | Dahl, Chris | Bruno, Megan | Malm, Kelsey | Litz, Ron | Fehringer, Jessica | Paladino, Katie | Opp, Tammy | Bell, Sonia | Adams, Anne | Allen, Mary Beth | Bachman, Robert | Bornstein, William | Cantrell, Dee | Cosper, Pam | Feistritzer, Nancye | Fox, John | Gartland, Bryce | Goodman, Jen | Grant, Susan | Howard-Crow, Dallis | Horowitz, Ira | Pugh, David | Ritenour, Chad | Ash, Toni | Barnes, Christopher | Calhoun, Jason | Chapman, Lauren | Daye, Tracey | Durr, Haley | Evans, Shunasee | Gentry, Janice | Ginnane, Jan | Grant, Susan | Haynes, Chris | Hill, Carolyn | Hillis, Dustin | Johnson, Crystal | Loomis, Jessica | Mamora, Josia | Mitchell, Laura | Morgan, Jill | Osakwe, Nancy | Owen, Jacqueline | Piazza, Sarah | Shirley, Kristina | Siddens, Jodi | Silas, Carrie | Slabach, Jason | Tirador, Elaina | Todd, Donnette | Vanairsdale, Sharon | Brammer, Nicole | Buchanan, Juli | Burd, Eileen | Cardella, John | Eaves, Brenda | Evans, Crystal | Hostetler, Krista | Jenkins, Karen | Lindsey, Maureen | Magee, Jordan | Powers, Randall | Ritchie, James | Ryan, Emily | Bonds, Shannon | Emamifar, Amir | Kuban, Tish | Pack, Jan | Rogers, Susan | Golston, George | Kaufman, Sean | Olinger, Patricia | Olinger, Sean | Rengarajan, Kalpana | Thomaston, Scott | Beck, Emily | Desroches, Paula | Hall, Cynthia | Walker, Celeste | Bryant, Connie | Hackman, Betsy | Howard, Regina | Jones, Marolyn | Broughton, Jeff | Frisle, Brian | Jackson, Robert | Lewis, Jerry | Brown-Haithco, Robin | Gartin, Miranda Lynn | Geralds-Washington, Erica | James-Jones, Rhonda | Miller, Donald | Stark, Dan | McGee, Gentrice | Jones, Porcia | Scott-Harris, Linda | Cain, James | Davis, Roderick | Johnson, Tyrone | Pickett, Tyrone | Shaw, Anthony | Truesdale, Tenina | Blum, Jim | Hill, Laureen | Sevransky, Jon | Zivot, Joel | Walker, Seth | Klopman, Matthew | Matkins, Ricky | Meechan, Cathy | Meechan, Paul | Schwock, Kathy | Schuck, Jen | Stack, Kathy | Wolf, Francis | Bray, Bruce | Isakov, Alex | Shartar, Sam | Miles, Wade | Jamison, Aaro | Arevalo, John | Stallings, Gail | Christenbury, Janet | Dollard, Vince | De Gennaro, Melanie | Korschun, Holly | Seideman, Nancy | Ziegler, Tom | Griffith, Daniel P. | Dave, Nisha | Wall, Susan | Hall, Melida | McGhee, Dwania | Clarke, Tim | Vaught, Rachel | Peterson-Pileri, Katrina
Two healthcare workers were repatriated from West Africa and received TKM-Ebola and convalescent plasma for treatment of Ebola virus disease. Both of these individuals recovered, and it is not clear what role these medical countermeasures played in their recovery.
Background. The current West Africa Ebola virus disease (EVD) outbreak has resulted in multiple individuals being medically evacuated to other countries for clinical management.
Methods. We report two patients who were transported from West Africa to the United States for treatment of EVD. Both patients received aggressive supportive care measures, as well as an investigational therapeutic (TKM-100802) and convalescent plasma.
Results. While one patient experienced critical illness with multi-organ failure requiring mechanical ventilation and renal replacement therapy, both patients recovered without serious long-term sequelae to date.
Conclusions. It is unclear what role the experimental drug and convalescent plasma had in the recovery of these patients. Prospective clinical trials are needed to delineate the role of investigational therapies in the care of patients with EVD.
PMCID: PMC4542597  PMID: 25904375
TKM-Ebola; convalescent plasma; convalescent serum; Ebola virus; Ebola virus disease
8.  South Asia as a Reservoir for the Global Spread of Ciprofloxacin-Resistant Shigella sonnei: A Cross-Sectional Study 
PLoS Medicine  2016;13(8):e1002055.
Antimicrobial resistance is a major issue in the Shigellae, particularly as a specific multidrug-resistant (MDR) lineage of Shigella sonnei (lineage III) is becoming globally dominant. Ciprofloxacin is a recommended treatment for Shigella infections. However, ciprofloxacin-resistant S. sonnei are being increasingly isolated in Asia and sporadically reported on other continents. We hypothesized that Asia is a primary hub for the recent international spread of ciprofloxacin-resistant S. sonnei.
Methods and Findings
We performed whole-genome sequencing on a collection of 60 contemporaneous ciprofloxacin-resistant S. sonnei isolated in four countries within Asia (Vietnam, n = 11; Bhutan, n = 12; Thailand, n = 1; Cambodia, n = 1) and two outside of Asia (Australia, n = 19; Ireland, n = 16). We reconstructed the recent evolutionary history of these organisms and combined these data with their geographical location of isolation. Placing these sequences into a global phylogeny, we found that all ciprofloxacin-resistant S. sonnei formed a single clade within a Central Asian expansion of lineage III. Furthermore, our data show that resistance to ciprofloxacin within S. sonnei may be globally attributed to a single clonal emergence event, encompassing sequential gyrA-S83L, parC-S80I, and gyrA-D87G mutations. Geographical data predict that South Asia is the likely primary source of these organisms, which are being regularly exported across Asia and intercontinentally into Australia, the United States and Europe. Our analysis was limited by the number of S. sonnei sequences available from diverse geographical areas and time periods, and we cannot discount the potential existence of other unsampled reservoir populations of antimicrobial-resistant S. sonnei.
This study suggests that a single clone, which is widespread in South Asia, is likely driving the current intercontinental surge of ciprofloxacin-resistant S. sonnei and is capable of establishing endemic transmission in new locations. Despite being limited in geographical scope, our work has major implications for understanding the international transfer of antimicrobial-resistant pathogens, with S. sonnei acting as a tractable model for studying how antimicrobial-resistant Gram-negative bacteria spread globally.
Stephen Baker and colleagues use whole-genome sequencing and phylogenetic charactarization of a global collection of ciprofloxacin-resistant Shigella sonnei isolates to explore the origins of the recent spread of the antimicrobial-resistant infection.
Author Summary
Why Was This Study Done?
Antimicrobial resistance is a major issue in Shigella, and ciprofloxacin is a recommended treatment for Shigella infections.
Ciprofloxacin-resistant Shigella sonnei are being increasingly isolated globally.
What Did the Researchers Do and Find?
We performed genome sequencing on 60 ciprofloxacin-resistant S. sonnei isolated in six countries within and outside of Asia.
Placing these genome sequences in context with other strains, we found that all ciprofloxacin-resistant S. sonnei formed a single clade, with South Asia as the likely primary source of these organisms.
These organisms are also being regularly exported across Asia and intercontinentally into Australia, the United States, and Europe.
The number of S. sonnei sequences available from other locations limited our analysis.
What Do These Findings Mean?
Our work suggests that a single clone, which is widespread in South Asia, is driving the current global surge of ciprofloxacin-resistant S. sonnei.
We argue that S. sonnei acts as a tractable model for studying how antimicrobial-resistant, Gram-negative bacteria spread globally.
PMCID: PMC4970813  PMID: 27483136
9.  Time to abandon the hygiene hypothesis: new perspectives on allergic disease, the human microbiome, infectious disease prevention and the role of targeted hygiene 
Perspectives in Public Health  2016;136(4):213-224.
To review the burden of allergic and infectious diseases and the evidence for a link to microbial exposure, the human microbiome and immune system, and to assess whether we could develop lifestyles which reconnect us with exposures which could reduce the risk of allergic disease while also protecting against infectious disease.
Using methodology based on the Delphi technique, six experts in infectious and allergic disease were surveyed to allow for elicitation of group judgement and consensus view on issues pertinent to the aim.
Key themes emerged where evidence shows that interaction with microbes that inhabit the natural environment and human microbiome plays an essential role in immune regulation. Changes in lifestyle and environmental exposure, rapid urbanisation, altered diet and antibiotic use have had profound effects on the human microbiome, leading to failure of immunotolerance and increased risk of allergic disease. Although evidence supports the concept of immune regulation driven by microbe–host interactions, the term ‘hygiene hypothesis’ is a misleading misnomer. There is no good evidence that hygiene, as the public understands, is responsible for the clinically relevant changes to microbial exposures.
Evidence suggests a combination of strategies, including natural childbirth, breast feeding, increased social exposure through sport, other outdoor activities, less time spent indoors, diet and appropriate antibiotic use, may help restore the microbiome and perhaps reduce risks of allergic disease. Preventive efforts must focus on early life. The term ‘hygiene hypothesis’ must be abandoned. Promotion of a risk assessment approach (targeted hygiene) provides a framework for maximising protection against pathogen exposure while allowing spread of essential microbes between family members. To build on these findings, we must change public, public health and professional perceptions about the microbiome and about hygiene. We need to restore public understanding of hygiene as a means to prevent infectious disease.
PMCID: PMC4966430  PMID: 27354505
allergy; infectious disease; hygiene; cleaning; antibiotics; diet
10.  Characterisation of Invasive Streptococcus pneumoniae Isolated from Cambodian Children between 2007 – 2012 
PLoS ONE  2016;11(7):e0159358.
The 13-valent pneumococcal vaccine (PCV13) was introduced in Cambodia in January 2015. There are limited data concerning the common serotypes causing invasive pneumococcal disease (IPD). Knowledge of the circulating pneumococcal serotypes is important to monitor epidemiological changes before and after vaccine implementation.
All episodes of IPD defined by the isolation of Streptococcus pneumoniae from blood, cerebrospinal fluid or other sterile site in Cambodian children admitted to the Angkor Hospital for Children in Siem Reap, Northwestern Cambodia, between 1st January 2007 and 1st July 2012 were retrospectively studied. Streptococcus pneumoniae isolates that could be retrieved underwent phenotypic typing and whole genome sequencing.
There were 90 Cambodian children hospitalized with IPD with a median (IQR) age of 2.3 years (0.9–6.2). The case fatality was 15.6% (95% CI 8–23). Of 50 Streptococcus pneumoniae isolates available for further testing, 46% were penicillin non-susceptible and 8% were ceftriaxone non-susceptible, 78% were cotrimoxazole resistant, 30% were erythromycin resistant and 30% chloramphenicol resistant. There were no significant changes in resistance levels over the five-year period. The most common serotypes were 1 (11/50; 22%), 23F (8/50; 16%), 14 (6/50; 12%), 5 (5/50; 10%) and 19A (3/50; 6%). Coverage by PCV7, PCV10 and PCV13 was 44%, 76% and 92% respectively. We identified novel multilocus sequence types and resistotypes using whole genome sequencing.
This study suggests IPD is an important disease in Cambodian children and can have a significant mortality. PCV13 coverage of the serotypes determined in studied strains was high and consistent with another recent study. The phenotypic resistance patterns observed were similar to other regional studies. The use of whole genome sequencing in the present study provides additional typing and resistance information together with the description of novel sequence types and resistotypes.
PMCID: PMC4957771  PMID: 27448096
11.  High Prevalence of Antimicrobial-resistant Gram-negative Colonization in Hospitalized Cambodian Infants 
Supplemental Digital Content is available in the text.
Antimicrobial-resistant Gram-negative infections are a significant cause of mortality in young infants. We aimed to determine characteristics of, and risk factors for, colonization and invasive infection caused by 3rd generation cephalosporin (3GC) or carbapenem-resistant organisms in outborn infants admitted to a neonatal unit (NU) in Cambodia.
During the first year of operation, patients admitted to the Angkor Hospital for Children NU, Siem Reap, Cambodia, underwent rectal swabbing on admission and twice weekly until discharge. Swabs were taken also from 7 environmental sites. Swabs were cultured to identify 3GC or carbapenem-resistant Acinetobacter sp., Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa.
The study included 333 infants with a median age at NU admission of 10 days (range, 0–43). Colonization by ≥1 3GC-resistant organism was detected in 85.9% (286/333). Admission swabs were collected in 289 infants: 61.9% were colonized by a 3GC-resistant organism at the time of admission, and a further 23.2% were colonized during hospitalization, at a median of 4 days [95% confidence interval: 3–5]. Probiotic treatment (hazard ratio: 0.58; 95% confidence interval: 0.35–0.98) was associated with delayed colonization. Colonization by a carbapenem-resistant organism occurred in 25 (7.5%) infants. Six infants had NU-associated K. pneumoniae bacteremia; phenotypically identical colonizing strains were found in 3 infants. Environmental colonization occurred early.
Colonization by antimicrobial-resistant Gram-negative organisms occurred early in hospitalized Cambodian infants and was associated with subsequent invasive infection. Trials of potential interventions such as probiotics are needed.
PMCID: PMC4957964  PMID: 27124686
colonization; neonate; infant; Gram-negative; antimicrobial resistance
12.  Improving the safety of oral immunotherapy for food allergy 
Pediatric Allergy and Immunology  2015;27(2):117-125.
Food allergy is a major public health problem in children, impacting upon the affected individual, their families and others charged with their care, for example educational establishments, and the food industry. In contrast to most other paediatric diseases, there is no established cure: current management is based upon dietary avoidance and the provision of rescue medication in the event of accidental reactions, which are common. This strategy has significant limitations and impacts adversely on health‐related quality of life. In the last decade, research into disease‐modifying treatments for food allergy has emerged, predominantly for peanut, egg and cow's milk. Most studies have used the oral route (oral immunotherapy, OIT), in which increasing amounts of allergen are given over weeks–months. OIT has proven effective to induce immune modulation and ‘desensitization’ – that is, an increase in the amount of food allergen that can be consumed, so long as regular (typically daily) doses are continued. However, its ability to induce permanent tolerance once ongoing exposure has stopped seems limited. Additionally, the short‐ and long‐term safety of OIT is often poorly reported, raising concerns about its implementation in routine practice. Most patients experience allergic reactions and, although generally mild, severe reactions have occurred. Long‐term adherence is unclear, which rises concerns given the low rates of long‐term tolerance induction. Current research focuses on improving current limitations, especially safety. Strategies include alternative routes (sublingual, epicutaneous), modified hypoallergenic products and adjuvants (anti‐IgE, pre‐/probiotics). Biomarkers of safe/successful OIT are also under investigation.
PMCID: PMC4950028  PMID: 26593873
children; food allergy; oral immunotherapy; quality of life; safety
13.  Phylogeographical analysis of the dominant multidrug-resistant H58 clade of Salmonella Typhi identifies inter- and intracontinental transmission events 
Wong, Vanessa K | Baker, Stephen | Pickard, Derek J | Parkhill, Julian | Page, Andrew J | Feasey, Nicholas A | Kingsley, Robert A | Thomson, Nicholas R | Keane, Jacqueline A | Weill, François-Xavier | Edwards, David J | Hawkey, Jane | Harris, Simon R | Mather, Alison E | Cain, Amy K | Hadfield, James | Hart, Peter J | Thieu, Nga Tran Vu | Klemm, Elizabeth J | Glinos, Dafni A | Breiman, Robert F | Watson, Conall H | Kariuki, Samuel | Gordon, Melita A | Heyderman, Robert S | Okoro, Chinyere | Jacobs, Jan | Lunguya, Octavie | Edmunds, W John | Msefula, Chisomo | Chabalgoity, Jose A | Kama, Mike | Jenkins, Kylie | Dutta, Shanta | Marks, Florian | Campos, Josefina | Thompson, Corinne | Obaro, Stephen | MacLennan, Calman A | Dolecek, Christiane | Keddy, Karen H | Smith, Anthony M | Parry, Christopher M | Karkey, Abhilasha | Mulholland, E Kim | Campbell, James I | Dongol, Sabina | Basnyat, Buddha | Dufour, Muriel | Bandaranayake, Don | Naseri, Take Toleafoa | Singh, Shalini Pravin | Hatta, Mochammad | Newton, Paul | Onsare, Robert S | Isaia, Lupeoletalalei | Dance, David | Davong, Viengmon | Thwaites, Guy | Wijedoru, Lalith | Crump, John A | De Pinna, Elizabeth | Nair, Satheesh | Nilles, Eric J | Thanh, Duy Pham | Turner, Paul | Soeng, Sona | Valcanis, Mary | Powling, Joan | Dimovski, Karolina | Hogg, Geoff | Farrar, Jeremy | Holt, Kathryn E | Dougan, Gordon
Nature genetics  2015;47(6):632-639.
The emergence of multidrug-resistant (MDR) typhoid is a major global health threat affecting many countries where the disease is endemic. Here whole-genome sequence analysis of 1,832 Salmonella enterica serovar Typhi (S. Typhi) identifies a single dominant MDR lineage, H58, that has emerged and spread throughout Asia and Africa over the last 30 years. Our analysis identifies numerous transmissions of H58, including multiple transfers from Asia to Africa and an ongoing, unrecognized MDR epidemic within Africa itself. Notably, our analysis indicates that H58 lineages are displacing antibiotic-sensitive isolates, transforming the global population structure of this pathogen. H58 isolates can harbor a complex MDR element residing either on transmissible IncHI1 plasmids or within multiple chromosomal integration sites. We also identify new mutations that define the H58 lineage. This phylogeographical analysis provides a framework to facilitate global management of MDR typhoid and is applicable to similar MDR lineages emerging in other bacterial species.
PMCID: PMC4921243  PMID: 25961941
14.  Development of a TaqMan Array Card for Pneumococcal Serotyping on Isolates and Nasopharyngeal Samples 
Journal of Clinical Microbiology  2016;54(7):1842-1850.
Streptococcus pneumoniae is both a commensal and a major pathogen that causes invasive disease in people of all ages. The introduction of serotype-specific pneumococcal vaccines has reduced the burden of disease but has also led to replacement with new strains; thus, serotyping remains important for vaccine-related disease surveillance. Conventional serotyping methods are laborious and expensive. We developed an easy-to-perform genotypic TaqMan array card (TAC) to identify S. pneumoniae strains, including lytA-based sequences, and 53 sequence-specific PCRs to identify 74 serotypes/serogroups covering all current vaccine types as well as prevalent nonvaccine types. The TAC method was evaluated on 146 clinical S. pneumoniae isolates and 13 nonpneumococcal species that naturally inhabit the upper respiratory tract and yielded 97% (142/146) sensitivity and 100% (13/13) specificity versus results of standard Quellung serotyping. The calculated limit of detection was 20 to 200 fg (∼8 to 84 genome equivalents) per reaction. On 23 blinded nasopharyngeal specimens that were pneumococcus culture positive, the TAC pan-pneumococcus lytA assay was positive in 21 (91% sensitivity versus culture). On TAC lytA-positive specimens, a serotype result was obtained on 86%, and the result was 95% accurate versus the subsequent culture's Quellung result. TAC also detected mixed serotypes in two specimens where Quellung detected only the predominant serotype. This TAC method yields fast and comprehensive serotyping compared to the standard method and may be useful on direct specimens.
PMCID: PMC4922116  PMID: 27170020
15.  The Molecular and Spatial Epidemiology of Typhoid Fever in Rural Cambodia 
PLoS Neglected Tropical Diseases  2016;10(6):e0004785.
Typhoid fever, caused by the bacterium Salmonella Typhi, is an endemic cause of febrile disease in Cambodia. The aim of this study was to better understand the epidemiology of pediatric typhoid fever in Cambodia. We accessed routine blood culture data from Angkor Hospital for Children (AHC) in Siem Reap province between 2007 and 2014, and performed whole genome sequencing (WGS) on the isolated bacteria to characterize the S. Typhi population. The resulting phylogenetic information was combined with conventional epidemiological approaches to investigate the spatiotemporal distribution of S. Typhi and population-level risk factors for reported disease. During the study period, there were 262 cases of typhoid within a 100 km radius of AHC, with a median patient age of 8.2 years (IQR: 5.1–11.5 years). The majority of infections occurred during the rainy season, and commune incidences as high as 11.36/1,000 in children aged <15 years were observed over the study period. A population-based risk factor analysis found that access to water within households and increasing distance from Tonle Sap Lake were protective. Spatial mapping and WGS provided additional resolution for these findings, and confirmed that proximity to the lake was associated with discrete spatiotemporal disease clusters. We confirmed the dominance of MDR H58 S. Typhi in this population, and found substantial evidence of diversification (at least seven sublineages) within this single lineage. We conclude that there is a substantial burden of pediatric typhoid fever in rural communes in Cambodia. Our data provide a platform for additional population-based typhoid fever studies in this location, and suggest that this would be a suitable setting in which to introduce a school-based vaccination programme with Vi conjugate vaccines.
Author Summary
Typhoid fever is an infectious disease caused by the bacterium Salmonella Typhi. The disease is generally restricted to those living in low-income settings with poor sanitation. Typhoid fever is a common cause of fever requiring hospital treatment in Cambodia, but limited data is available on the epidemiology of the disease. To better understand typhoid fever in Cambodia, we accessed routine hospital data for typhoid fever from a single healthcare facility treating sick children in Siem Reap in the central Cambodia between 2007 and 2014. We mapped the location of these cases and examined population-based risk factors for reported disease. Additionally, we decoded the genomes of the S. Typhi isolated from children attending the hospital to understand how the organism has evolved and spread throughout the population. We found a large burden of typhoid fever in children in this largely rural setting in central Cambodia. We also found that disease was associated with the rainy season and that living close to Tonle Sap Lake increased the risk of disease. The genomes of the sequenced bacteria showed that a diverse range of strains were circulating during the study, and allowed us to identify signatures of location- and time-specific outbreaks. Our work provides baseline data for additional typhoid fever studies in the population living in this location and findings suggest that rural Cambodia would be a suitable setting in which to introduce a school-based vaccination program with new typhoid vaccines.
PMCID: PMC4917249  PMID: 27331909
16.  Molecular Epidemiology of Group A Streptococcus Infections in Cambodian Children, 2007 - 2012 
PMCID: PMC4643749  PMID: 26780027
Group A Streptococcus; emm-type; vaccine; children; epidemiology
17.  Phage selection restores antibiotic sensitivity in MDR Pseudomonas aeruginosa 
Scientific Reports  2016;6:26717.
Increasing prevalence and severity of multi-drug-resistant (MDR) bacterial infections has necessitated novel antibacterial strategies. Ideally, new approaches would target bacterial pathogens while exerting selection for reduced pathogenesis when these bacteria inevitably evolve resistance to therapeutic intervention. As an example of such a management strategy, we isolated a lytic bacteriophage, OMKO1, (family Myoviridae) of Pseudomonas aeruginosa that utilizes the outer membrane porin M (OprM) of the multidrug efflux systems MexAB and MexXY as a receptor-binding site. Results show that phage selection produces an evolutionary trade-off in MDR P. aeruginosa, whereby the evolution of bacterial resistance to phage attack changes the efflux pump mechanism, causing increased sensitivity to drugs from several antibiotic classes. Although modern phage therapy is still in its infancy, we conclude that phages, such as OMKO1, represent a new approach to phage therapy where bacteriophages exert selection for MDR bacteria to become increasingly sensitive to traditional antibiotics. This approach, using phages as targeted antibacterials, could extend the lifetime of our current antibiotics and potentially reduce the incidence of antibiotic resistant infections.
PMCID: PMC4880932  PMID: 27225966
18.  Evolution of parasitism and mutualism between filamentous phage M13 and Escherichia coli 
PeerJ  2016;4:e2060.
Background. How host-symbiont interactions coevolve between mutualism and parasitism depends on the ecology of the system and on the genetic and physiological constraints of the organisms involved. Theory often predicts that greater reliance on horizontal transmission favors increased costs of infection and may result in more virulent parasites or less beneficial mutualists. We set out to understand transitions between parasitism and mutualism by evolving the filamentous bacteriophage M13 and its host Escherichia coli.
Results. The effect of phage M13 on bacterial fitness depends on the growth environment, and initial assays revealed that infected bacteria reproduce faster and to higher density than uninfected bacteria in 96-well microplates. These data suggested that M13 is, in fact, a facultative mutualist of E. coli. We then allowed E. coli and M13 to evolve in replicated environments, which varied in the relative opportunity for horizontal and vertical transmission of phage in order to assess the evolutionary stability of this mutualism. After 20 experimental passages, infected bacteria from treatments with both vertical and horizontal transmission of phage had evolved the fastest growth rates. At the same time, phage from these treatments no longer benefited the ancestral bacteria.
Conclusions. These data suggest a positive correlation between the positive effects of M13 on E. coli hosts from the same culture and the negative effects of the same phage toward the ancestral bacterial genotype. The results also expose flaws in applying concepts from the virulence-transmission tradeoff hypothesis to mutualism evolution. We discuss the data in the context of more recent theory on how horizontal transmission affects mutualisms and explore how these effects influence phages encoding virulence factors in pathogenic bacteria.
PMCID: PMC4888304  PMID: 27257543
Transmission mode; Pleiotropy; Tolerance; Experimental evolution; Symbiosis
19.  Capsular Typing Method for Streptococcus agalactiae Using Whole-Genome Sequence Data 
Journal of Clinical Microbiology  2016;54(5):1388-1390.
Group B streptococcus (GBS) capsular serotypes are major determinants of virulence and affect potential vaccine coverage. Here we report a whole-genome-sequencing-based method for GBS serotype assignment. This method shows strong agreement (kappa of 0.92) with conventional methods and increased serotype assignment (100%) to all 10 capsular types.
PMCID: PMC4844738  PMID: 26962081
20.  Repeatable Population Dynamics among Vesicular Stomatitis Virus Lineages Evolved under High Co-infection 
Parasites and hosts can experience oscillatory cycles, where the densities of these interacting species dynamically fluctuate through time. Viruses with different replication strategies can also interact to produce cyclical dynamics. Frequent cellular co-infection can select for defective-interfering particles (DIPs): “cheater” viruses with shortened genomes that interfere with intracellular replication of full-length (ordinary) viruses. DIPs are positively selected when rare because they out-replicate ordinary viruses during co-infection, but DIPs are negatively selected when common because ordinary viruses become unavailable for intracellular exploitation via cheating. Here, we tested whether oscillatory dynamics of ordinary viruses were similar across independently evolved populations of vesicular stomatitis virus (VSV). Results showed identical cyclical dynamics across populations in the first 10 experimental passages, which transitioned to repeatable dampened oscillations by passage 20. Genomic analyses revealed parallel molecular substitutions across populations, particularly novel mutations that became dominant by passage 10. Our study showed that oscillatory dynamics and molecular evolution of interacting viruses were highly repeatable in VSV populations passaged under frequent co-infection. Furthermore, our data suggested that frequent co-infection with DIPs caused lowered performance of full-length viruses, by reducing their population densities by orders of magnitude compared to reproduction of ordinary viruses during strictly clonal infections.
PMCID: PMC4815288  PMID: 27065953
cheating behavior; co-evolution; defective interfering particle; ecological dynamics; experimental evolution; RNA virus; virus ecology
22.  Evolutionary History of the Global Emergence of the Escherichia coli Epidemic Clone ST131 
mBio  2016;7(2):e02162-15.
Escherichia coli sequence type 131 (ST131) has emerged globally as the most predominant extraintestinal pathogenic lineage within this clinically important species, and its association with fluoroquinolone and extended-spectrum cephalosporin resistance impacts significantly on treatment. The evolutionary histories of this lineage, and of important antimicrobial resistance elements within it, remain unclearly defined. This study of the largest worldwide collection (n = 215) of sequenced ST131 E. coli isolates to date demonstrates that the clonal expansion of two previously recognized antimicrobial-resistant clades, C1/H30R and C2/H30Rx, started around 25 years ago, consistent with the widespread introduction of fluoroquinolones and extended-spectrum cephalosporins in clinical medicine. These two clades appear to have emerged in the United States, with the expansion of the C2/H30Rx clade driven by the acquisition of a blaCTX-M-15-containing IncFII-like plasmid that has subsequently undergone extensive rearrangement. Several other evolutionary processes influencing the trajectory of this drug-resistant lineage are described, including sporadic acquisitions of CTX-M resistance plasmids and chromosomal integration of blaCTX-M within subclusters followed by vertical evolution. These processes are also occurring for another family of CTX-M gene variants more recently observed among ST131, the blaCTX-M-14/14-like group. The complexity of the evolutionary history of ST131 has important implications for antimicrobial resistance surveillance, epidemiological analysis, and control of emerging clinical lineages of E. coli. These data also highlight the global imperative to reduce specific antibiotic selection pressures and demonstrate the important and varied roles played by plasmids and other mobile genetic elements in the perpetuation of antimicrobial resistance within lineages.
Escherichia coli, perennially a major bacterial pathogen, is becoming increasingly difficult to manage due to emerging resistance to all preferred antimicrobials. Resistance is concentrated within specific E. coli lineages, such as sequence type 131 (ST131). Clarification of the genetic basis for clonally associated resistance is key to devising intervention strategies. We used high-resolution genomic analysis of a large global collection of ST131 isolates to define the evolutionary history of extended-spectrum beta-lactamase production in ST131. We documented diverse contributory genetic processes, including stable chromosomal integrations of resistance genes, persistence and evolution of mobile resistance elements within sublineages, and sporadic acquisition of different resistance elements. Both global distribution and regional segregation were evident. The diversity of resistance element acquisition and propagation within ST131 indicates a need for control and surveillance strategies that target both bacterial strains and mobile genetic elements.
PMCID: PMC4807372  PMID: 27006459
23.  Horizontal DNA Transfer Mechanisms of Bacteria as Weapons of Intragenomic Conflict 
PLoS Biology  2016;14(3):e1002394.
Horizontal DNA transfer (HDT) is a pervasive mechanism of diversification in many microbial species, but its primary evolutionary role remains controversial. Much recent research has emphasised the adaptive benefit of acquiring novel DNA, but here we argue instead that intragenomic conflict provides a coherent framework for understanding the evolutionary origins of HDT. To test this hypothesis, we developed a mathematical model of a clonally descended bacterial population undergoing HDT through transmission of mobile genetic elements (MGEs) and genetic transformation. Including the known bias of transformation toward the acquisition of shorter alleles into the model suggested it could be an effective means of counteracting the spread of MGEs. Both constitutive and transient competence for transformation were found to provide an effective defence against parasitic MGEs; transient competence could also be effective at permitting the selective spread of MGEs conferring a benefit on their host bacterium. The coordination of transient competence with cell–cell killing, observed in multiple species, was found to result in synergistic blocking of MGE transmission through releasing genomic DNA for homologous recombination while simultaneously reducing horizontal MGE spread by lowering the local cell density. To evaluate the feasibility of the functions suggested by the modelling analysis, we analysed genomic data from longitudinal sampling of individuals carrying Streptococcus pneumoniae. This revealed the frequent within-host coexistence of clonally descended cells that differed in their MGE infection status, a necessary condition for the proposed mechanism to operate. Additionally, we found multiple examples of MGEs inhibiting transformation through integrative disruption of genes encoding the competence machinery across many species, providing evidence of an ongoing “arms race.” Reduced rates of transformation have also been observed in cells infected by MGEs that reduce the concentration of extracellular DNA through secretion of DNases. Simulations predicted that either mechanism of limiting transformation would benefit individual MGEs, but also that this tactic’s effectiveness was limited by competition with other MGEs coinfecting the same cell. A further observed behaviour we hypothesised to reduce elimination by transformation was MGE activation when cells become competent. Our model predicted that this response was effective at counteracting transformation independently of competing MGEs. Therefore, this framework is able to explain both common properties of MGEs, and the seemingly paradoxical bacterial behaviours of transformation and cell–cell killing within clonally related populations, as the consequences of intragenomic conflict between self-replicating chromosomes and parasitic MGEs. The antagonistic nature of the different mechanisms of HDT over short timescales means their contribution to bacterial evolution is likely to be substantially greater than previously appreciated.
Modelling and high-resolution genomic data demonstrate the evolutionary significance of an ongoing arms race between bacterial uptake of environmental DNA ("transformation") and horizontal DNA transfer driven by mobile genetic elements.
Author Summary
Bacteria are able to rapidly change their characteristics, such as antibiotic resistance, by acquiring genes from surrounding cells. Some sets of genes, called mobile genetic elements (MGEs), drive their own movement between bacterial cells by transmitting in viral or viral-like manners. These selfish genes spread themselves, even if harmful to their bacterial host. Some bacteria also actively absorb DNA from the environment, a process known as transformation. Why bacteria employ transformation is controversial. Importing all DNA molecules at the same rate means cells acquire detrimental and beneficial mutations at equal frequencies. However, transformation does exhibit a known bias: it preferentially imports shorter DNA molecules, meaning it tends to delete, rather than insert, genes. By incorporating this bias into computer simulations, we show that the spread of a selfish mobile genetic element between bacteria is inhibited when cells use transformation to delete it from their genomes. We hypothesised that this ability to delete selfish mobile genetic elements is an important function of transformation. To test this hypothesis, we analysed longitudinal bacterial samples from individuals colonised by S. pneumoniae and found the bacterial cells often differed in the mobile genes they contained while still in the same host and are, therefore, able to exchange DNA by transformation. Additionally, transformable bacteria had fewer selfish mobile genes than related nontransformable bacteria. We also identified examples of mobile elements that employ various tactics to prevent their host cells undergoing transformation, indicating that some selfish mobile genes are able to circumvent deletion-by-transformation. Hence, we conclude bacterial evolution is strongly influenced by an ongoing arms race between cells and selfish mobile genes.
PMCID: PMC4774983  PMID: 26934590
24.  Lack of Utility of Nasopharyngeal Swabs for Diagnosis of Burkholderia pseudomallei Pneumonia in Paediatric Patients 
Journal of Tropical Pediatrics  2016;62(4):328-330.
Diagnosis of Burkholderia pseudomallei pneumonia in children is challenging. We investigated the utility of nasopharyngeal swabs taken from 194 paediatric patients on admission with radiologically proven pneumonia. Melioidosis was proven in 0.5% of samples tested and only in a third of those known to be bacteraemic with B. pseudomallei. It appears unlikely that culture of nasopharyngeal secretions is helpful to confirm B. pseudomallei pneumonia in paediatric patients.
PMCID: PMC4998522  PMID: 26874977
Burkholderia pseudomallei; melioidosis; paediatric; nasopharyngeal swab
25.  Use of Blood Smears and Dried Blood Spots for Polymerase Chain Reaction–Based Detection and Quantification of Bacterial Infection and Plasmodium falciparum in Severely Ill Febrile African Children 
Molecular approaches offer a means of testing archived samples stored as dried blood spots in settings where standard blood cultures are not possible. Peripheral blood films are one suggested source of material, although the sensitivity of this approach has not been well defined. Thin blood smears and dried blood spots from a severe pediatric malaria study were assessed using specific polymerase chain reaction (PCR) primers to detect non-typhoidal Salmonella (NTS; MisL gene), Streptococcus pneumoniae (lytA), and Plasmodium falciparum (18S rRNA). Of 16 cases of NTS and S. pneumoniae confirmed on blood culture, none were positive by PCR using DNA extracts from blood films or dried blood spots. In contrast, four of 36 dried blood spots and two of 178 plasma samples were PCR positive for S. pneumoniae, despite negative bacterial blood cultures, suggesting false positives. Quantitative assessment revealed that the effective concentration of P. falciparum DNA in blood films was three log orders of magnitude lower than for dried blood spots. The P. falciparum kelch13 gene could not be amplified from blood films. These findings question the value of blood PCR-based approaches for detection of NTS and S. pneumoniae, and show that stored blood films are an inefficient method of studying P. falciparum.
PMCID: PMC4751960  PMID: 26711525

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