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Pholwat S.,University of Virginia | Sakai F.,Emory University | Turner P.,Cambodia Oxford Medical Research Unit | Turner P.,University of Oxford | And 2 more authors.
Journal of Clinical Microbiology | Year: 2016

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.


Croucher N.J.,Imperial College London | Mostowy R.,Imperial College London | Wymant C.,Imperial College London | Turner P.,Cambodia Oxford Medical Research Unit | And 3 more authors.
PLoS Biology | Year: 2016

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. © 2016 Croucher et al.


Reynolds C.,Imperial College London | Goudet A.,CEA Saclay Nuclear Research Center | Jenjaroen K.,Mahidol University | Sumonwiriya M.,Mahidol University | And 18 more authors.
Journal of Immunology | Year: 2015

There is an urgent need for a better understanding of adaptive immunity to Burkholderia pseudomallei, the causative agent of melioidosis that is frequently associated with sepsis or death in patients in Southeast Asia and Northern Australia. The imperative to identify vaccine targets is driven both by the public health agenda in these regions and biological threat concerns. In several intracellular bacterial pathogens, alkyl hydroperoxidase reductases are upregulated as part of the response to host oxidative stress, and they can stimulate strong adaptive immunity. We show that alkyl hydroperoxidase reductase (AhpC) of B. pseudomallei is strongly immunogenic for T cells of 'humanized' HLA transgenic mice and seropositive human donors. Some T cell epitopes, such as p6, are able to bind diverse HLA class II heterodimers and stimulate strong T cell immunity in mice and humans. Importantly, patients with acute melioidosis who survive infection show stronger T cell responses to AhpC relative to those who do not. Although the sequence of AhpC is virtually invariant among global B. pseudomallei clinical isolates, a Cambodian isolate varies only in C-terminal truncation of the p6 T cell epitope, raising the possibility of selection by host immunity. This variant peptide is virtually unable to stimulate T cell immunity. For an infection in which there has been debate about centrality of T cell immunity in defense, these observations support a role for T cell immunity to AhpC in disease protection. Copyright © 2015 The Authors.


Lubell Y.,Mahidol University | Lubell Y.,University of Oxford | Blacksell S.D.,Mahidol University | Blacksell S.D.,University of Oxford | And 25 more authors.
BMC Infectious Diseases | Year: 2015

Background: Poor targeting of antimicrobial drugs contributes to the millions of deaths each year from malaria, pneumonia, and other tropical infectious diseases. While malaria rapid diagnostic tests have improved use of antimalarial drugs, there are no similar tests to guide the use of antibiotics in undifferentiated fevers. In this study we estimate the diagnostic accuracy of two well established biomarkers of bacterial infection, procalcitonin and C-reactive protein (CRP) in discriminating between common viral and bacterial infections in malaria endemic settings of Southeast Asia. Methods: Serum procalcitonin and CRP levels were measured in stored serum samples from febrile patients enrolled in three prospective studies conducted in Cambodia, Laos and, Thailand. Of the 1372 patients with a microbiologically confirmed diagnosis, 1105 had a single viral, bacterial or malarial infection. Procalcitonin and CRP levels were compared amongst these aetiological groups and their sensitivity and specificity in distinguishing bacterial infections and bacteraemias from viral infections were estimated using standard thresholds. Results: Serum concentrations of both biomarkers were significantly higher in bacterial infections and malaria than in viral infections. The AUROC for CRP in discriminating between bacterial and viral infections was 0.83 (0.81-0.86) compared with 0.74 (0.71-0.77) for procalcitonin (p < 0.0001). This relative advantage was evident in all sites and when stratifying patients by age and admission status. For CRP at a threshold of 10 mg/L, the sensitivity of detecting bacterial infections was 95 % with a specificity of 49 %. At a threshold of 20 mg/L sensitivity was 86 % with a specificity of 67 %. For procalcitonin at a low threshold of 0.1 ng/mL the sensitivity was 90 % with a specificity of 39 %. At a higher threshold of 0.5 ng/ul sensitivity was 60 % with a specificity of 76 %. Conclusion: In samples from febrile patients with mono-infections from rural settings in Southeast Asia, CRP was a highly sensitive and moderately specific biomarker for discriminating between viral and bacterial infections. Use of a CRP rapid test in peripheral health settings could potentially be a simple and affordable measure to better identify patients in need of antibacterial treatment and part of a global strategy to combat the emergence of antibiotic resistance. © 2015 Lubell et al.


Turner P.,Cambodia Oxford Medical Research Unit | Turner P.,Mahidol University | Turner P.,University of Oxford | Pol S.,Cambodia Oxford Medical Research Unit | And 12 more authors.
Pediatric Infectious Disease Journal | Year: 2016

Background: 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. Methods: 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. Results: 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. Conclusions: 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. © 2016 Wolters Kluwer Health, Inc.

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