title: The sensitivity of real-time PCR amplification targeting invasive Salmonella serovars in biological specimens.
abstract: BACKGROUND
  PCR amplification for the detection of pathogens in biological material is generally considered a rapid and informative diagnostic technique. Invasive Salmonella serovars, which cause enteric fever, can be commonly cultured from the blood of infected patients. Yet, the isolation of invasive Salmonella serovars from blood is protracted and potentially insensitive.
  
  
  METHODS
  We developed and optimised a novel multiplex three colour real-time PCR assay to detect specific target sequences in the genomes of Salmonella serovars Typhi and Paratyphi A. We performed the assay on DNA extracted from blood and bone marrow samples from culture positive and negative enteric fever patients.
  
  
  RESULTS
  The assay was validated and demonstrated a high level of specificity and reproducibility under experimental conditions. All bone marrow samples tested positive for Salmonella, however, the sensitivity on blood samples was limited. The assay demonstrated an overall specificity of 100% (75/75) and sensitivity of 53.9% (69/128) on all biological samples. We then tested the PCR detection limit by performing bacterial counts after inoculation into blood culture bottles.
  
  
  CONCLUSIONS
  Our findings corroborate previous clinical findings, whereby the bacterial load of S. Typhi in peripheral blood is low, often below detection by culture and, consequently, below detection by PCR. Whilst the assay may be utilised for environmental sampling or on differing biological samples, our data suggest that PCR performed directly on blood samples may be an unsuitable methodology and a potentially unachievable target for the routine diagnosis of enteric fever.
authors.affiliation: Oxford University Clinical Research Unit, Hospital for Tropical Diseases, Ho Chi Minh City, Vietnam.
authors.forename: Tran Vu Thieu
authors.initials: TV
authors.lastname: Nga
authors.forename: Abhilasha
authors.initials: A
authors.lastname: Karkey
authors.forename: Sabina
authors.initials: S
authors.lastname: Dongol
authors.forename: Hang Nguyen
authors.initials: HN
authors.lastname: Thuy
authors.forename: Sarah
authors.initials: S
authors.lastname: Dunstan
authors.forename: Kathryn
authors.initials: K
authors.lastname: Holt
authors.forename: Le Thi Phuong
authors.initials: le TP
authors.lastname: Tu
authors.forename: James I
authors.initials: JI
authors.lastname: Campbell
authors.forename: Tran Thuy
authors.initials: TT
authors.lastname: Chau
authors.forename: Nguyen Van Vinh
authors.initials: NV
authors.lastname: Chau
authors.forename: Amit
authors.initials: A
authors.lastname: Arjyal
authors.forename: Samir
authors.initials: S
authors.lastname: Koirala
authors.forename: Buddha
authors.initials: B
authors.lastname: Basnyat
authors.forename: Christiane
authors.initials: C
authors.lastname: Dolecek
authors.forename: Jeremy
authors.initials: J
authors.lastname: Farrar
authors.forename: Stephen
authors.initials: S
authors.lastname: Baker
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D001431:Bacteriological Techniques / Q000379:methods*; D001769:Blood / Q000382:microbiology; D001853:Bone Marrow / Q000382:microbiology; D006801:Humans; D008875:Middle Aged; D010284:Paratyphoid Fever / Q000175:diagnosis*; D016133:Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D012482:Salmonella paratyphi A / Q000235:genetics / Q000302:isolation & purification*; D012485:Salmonella typhi / Q000235:genetics / Q000302:isolation & purification*; D012680:Sensitivity and Specificity; D014435:Typhoid Fever / Q000175:diagnosis*; D055815:Young Adult