title: Development and Evaluation of a Blood Culture PCR Assay for Rapid Detection of Salmonella Paratyphi A in Clinical Samples.
abstract: BACKGROUND
  Enteric fever remains an important cause of morbidity in many low-income countries and Salmonella Paratyphi A has emerged as the aetiological agent in an increasing proportion of cases. Lack of adequate diagnostics hinders early diagnosis and prompt treatment of both typhoid and paratyphoid but development of assays to identify paratyphoid has been particularly neglected. Here we describe the development of a rapid and sensitive blood culture PCR method for detection of Salmonella Paratyphi A from blood, potentially allowing for appropriate diagnosis and antimicrobial treatment to be initiated on the same day.
  
  
  METHODS
  Venous blood samples from volunteers experimentally challenged orally with Salmonella Paratyphi A, who subsequently developed paratyphoid, were taken on the day of diagnosis; 10 ml for quantitative blood culture and automated blood culture, and 5 ml for blood culture PCR. In the latter assay, bacteria were grown in tryptone soy broth containing 2.4% ox bile and micrococcal nuclease for 5 hours (37°C) before bacterial DNA was isolated for PCR detection targeting the fliC-a gene of Salmonella Paratyphi A.
  
  
  RESULTS
  An optimized broth containing 2.4% ox bile and micrococcal nuclease, as well as a PCR test was developed for a blood culture PCR assay of Salmonella Paratyphi A. The volunteers diagnosed with paratyphoid had a median bacterial burden of 1 (range 0.1-6.9) CFU/ml blood. All the blood culture PCR positive cases where a positive bacterial growth was shown by quantitative blood culture had a bacterial burden of ≥ 0.3 CFU/ ml blood. The blood culture PCR assay identified an equal number of positive cases as automated blood culture at higher bacterial loads (≥0.3 CFU/ml blood), but utilized only half the volume of specimens.
  
  
  CONCLUSIONS
  The blood culture PCR method for detection of Salmonella Paratyphi A can be completed within 9 hours and offers the potential for same-day diagnosis of enteric fever. Using 5 ml blood, it exhibited a lower limit of detection equal to 0.3 CFU/ml blood, and it performed at least as well as automated blood culture at higher bacterial loads (≥0.3 CFU/ml blood) of clinical specimens despite using half the volume of blood. The findings warrant its further study in endemic populations with a potential use as a novel diagnostic which fills the present gap of paratyphoid diagnostics.
authors.affiliation: Oxford Vaccine Group, Department of Paediatrics, University of Oxford and the NIHR Oxford Biomedical Research Centre, Oxford, United Kingdom.
authors.forename: Liqing
authors.initials: L
authors.lastname: Zhou
authors.affiliation: Oxford Vaccine Group, Department of Paediatrics, University of Oxford and the NIHR Oxford Biomedical Research Centre, Oxford, United Kingdom.
authors.forename: Claire
authors.initials: C
authors.lastname: Jones
authors.affiliation: Oxford Vaccine Group, Department of Paediatrics, University of Oxford and the NIHR Oxford Biomedical Research Centre, Oxford, United Kingdom.
authors.forename: Malick M
authors.initials: MM
authors.lastname: Gibani
authors.affiliation: Oxford Vaccine Group, Department of Paediatrics, University of Oxford and the NIHR Oxford Biomedical Research Centre, Oxford, United Kingdom.
authors.forename: Hazel
authors.initials: H
authors.lastname: Dobinson
authors.affiliation: Oxford Vaccine Group, Department of Paediatrics, University of Oxford and the NIHR Oxford Biomedical Research Centre, Oxford, United Kingdom.
authors.forename: Helena
authors.initials: H
authors.lastname: Thomaides-Brears
authors.affiliation: Oxford Vaccine Group, Department of Paediatrics, University of Oxford and the NIHR Oxford Biomedical Research Centre, Oxford, United Kingdom.
authors.forename: Sonu
authors.initials: S
authors.lastname: Shrestha
authors.affiliation: Oxford Vaccine Group, Department of Paediatrics, University of Oxford and the NIHR Oxford Biomedical Research Centre, Oxford, United Kingdom.
authors.forename: Christoph J
authors.initials: CJ
authors.lastname: Blohmke
authors.affiliation: Oxford Vaccine Group, Department of Paediatrics, University of Oxford and the NIHR Oxford Biomedical Research Centre, Oxford, United Kingdom.
authors.forename: Thomas C
authors.initials: TC
authors.lastname: Darton
authors.affiliation: Oxford Vaccine Group, Department of Paediatrics, University of Oxford and the NIHR Oxford Biomedical Research Centre, Oxford, United Kingdom.
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Pollard
mesh_terms: D001322:Autoanalysis / Q000379:methods; D004269:DNA, Bacterial / Q000235:genetics; D006801:Humans; D010284:Paratyphoid Fever / Q000097:blood / Q000175:diagnosis*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012482:Salmonella paratyphi A / Q000235:genetics; D012680:Sensitivity and Specificity
chemical_list: D004269:DNA, Bacterial