title: Detection of Typhoidal and Paratyphoidal Salmonella in Blood by Real-time Polymerase Chain Reaction.
abstract: BACKGROUND
  The gold standard for diagnosis of enteric fever caused by Salmonella Typhi or Salmonella Paratyphi A or B is bone marrow culture. However, because bone marrow aspiration is highly invasive, many hospitals and large health centers perform blood culture instead. As blood culture has several limitations, there is a need for novel typhoid diagnostics with improved sensitivity and more rapid time to detection.
  
  
  METHODS
  We developed a clyA-based real-time polymerase chain reaction (qPCR) method to detect Salmonella Typhi and Salmonella Paratyphi A simultaneously in blood. The sensitivity and specificity of this probeset was first evaluated in vitro in the laboratory and then in a typhoid-endemic population, in Karachi, Pakistan, and in healthy US volunteers.
  
  
  RESULTS
  We optimized a DNA extraction and real-time PCR-based method that could reliably detect 1 colony-forming unit/mL of Salmonella Typhi. The probe set was able to detect clinical Salmonella Typhi and Salmonella Paratyphi A strains and also diarrheagenic Escherichia coli, but not invasive E. coli or other invasive bacteria. In the field, the clyA qPCR diagnostic was 40% as sensitive as blood culture. However, when qPCR-positive specimens were considered to be true positives, blood culture only exhibited 28.57% sensitivity. Specificity was ≥90% for all comparisons and in the healthy US volunteers. qPCR was significantly faster than blood culture in terms of detection of typhoid and paratyphoid.
  
  
  CONCLUSIONS
  Based on lessons learned, we recommend that future field trials of this and other novel diagnostics that detect typhoidal and nontyphoidal Salmonella employ multiple methodologies to define a "positive" sample.
authors.affiliation: Center for Vaccine Development Department of Medicine, University of Maryland, Baltimore.
authors.forename: Sharon M
authors.initials: SM
authors.lastname: Tennant
authors.affiliation: Center for Vaccine Development Department of Medicine, University of Maryland, Baltimore.
authors.forename: Deanna
authors.initials: D
authors.lastname: Toema
authors.affiliation: Department of Paediatrics and Child Health.
authors.forename: Farah
authors.initials: F
authors.lastname: Qamar
authors.affiliation: Department of Paediatrics and Child Health Department of Biological and Biomedical Sciences, Aga Khan University, Karachi, Pakistan.
authors.forename: Najeeha
authors.initials: N
authors.lastname: Iqbal
authors.affiliation: Center for Vaccine Development Department of Pediatrics, University of Maryland, Baltimore.
authors.forename: Mary Adetinuke
authors.initials: MA
authors.lastname: Boyd
authors.affiliation: Center for Vaccine Development Department of Medicine, University of Maryland, Baltimore.
authors.forename: Joanna M
authors.initials: JM
authors.lastname: Marshall
authors.affiliation: Center for Vaccine Development Department of Medicine, University of Maryland, Baltimore.
authors.forename: William C
authors.initials: WC
authors.lastname: Blackwelder
authors.affiliation: Center for Vaccine Development Department of Medicine, University of Maryland, Baltimore.
authors.forename: Yukun
authors.initials: Y
authors.lastname: Wu
authors.affiliation: Department of Paediatrics and Child Health.
authors.forename: Farheen
authors.initials: F
authors.lastname: Quadri
authors.affiliation: Department of Paediatrics and Child Health.
authors.forename: Asia
authors.initials: A
authors.lastname: Khan
authors.affiliation: Department of Paediatrics and Child Health.
authors.forename: Fatima
authors.initials: F
authors.lastname: Aziz
authors.affiliation: Department of Paediatrics and Child Health.
authors.forename: Kumail
authors.initials: K
authors.lastname: Ahmad
authors.affiliation: Department of Paediatrics and Child Health.
authors.forename: Adil
authors.initials: A
authors.lastname: Kalam
authors.affiliation: Department of Paediatrics and Child Health.
authors.forename: Ehtisham
authors.initials: E
authors.lastname: Asif
authors.affiliation: Department of Paediatrics and Child Health.
authors.forename: Shahida
authors.initials: S
authors.lastname: Qureshi
authors.affiliation: Department of Pathology and Microbiology, Aga Khan University, Karachi, Pakistan.
authors.forename: Erum
authors.initials: E
authors.lastname: Khan
authors.affiliation: Department of Paediatrics and Child Health.
authors.forename: Anita K
authors.initials: AK
authors.lastname: Zaidi
authors.affiliation: Center for Vaccine Development Department of Medicine, University of Maryland, Baltimore Department of Pediatrics, University of Maryland, Baltimore.
authors.forename: Myron M
authors.initials: MM
authors.lastname: Levine
mesh_terms: D000293:Adolescent; D002648:Child; D002675:Child, Preschool; D004926:Escherichia coli / Q000145:classification / Q000235:genetics; D005260:Female; D064368:Healthy Volunteers; D006801:Humans; D008297:Male; D010154:Pakistan; D010284:Paratyphoid Fever / Q000097:blood / Q000175:diagnosis* / Q000382:microbiology; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012482:Salmonella paratyphi A / Q000235:genetics / Q000302:isolation & purification*; D012485:Salmonella typhi / Q000235:genetics / Q000302:isolation & purification*; D012680:Sensitivity and Specificity; D014435:Typhoid Fever / Q000097:blood / Q000175:diagnosis* / Q000382:microbiology
keywords: PCR; Salmonella; blood; diagnostic; typhoid