Evaluation of fliC-d based direct blood PCR assays for typhoid diagnosis.
Typhoid cases need to be diagnosed accurately for early antibiotic therapy and reducing mortality. Identification of Salmonella Typhi (S. Typhi) in blood culture is conclusive, but has poor sensitivity. Detection of S. Typhi by PCR from blood sample has shown promise. Real-time quantitative PCR (Q-PCR) has been widely used in diagnostics for its rapidity and reliability. In the present study, the performance of molecular methods like conventional PCR (C-PCR), nested PCR (N-PCR) and Q-PCR were investigated and compared by targeting S. Typhi specific flagellar fliC-d gene directly in blood samples for typhoid diagnosis.



Analytical sensitivities and specificities of the PCR assays were determined under laboratory condition followed by diagnostic performances were demonstrated in 110 clinically diagnosed typhoid fever (CDTF) cases included as study subjects. The DNA detection limit of C-PCR was observed 3 × 10(4) copies/reaction; those of N-PCR and Q-PCR (cutoff Ct value, ≤37) were 3 copies/reaction. The C-PCR was not further evaluated since it showed negative results with all clinical samples due to low sensitivity. Low isolation rate (21.8 %, 24/110) of S. Typhi by blood culture did not reflect the true burden of typhoid fever among the study subjects. Hence diagnostic performances of N-PCR and Q-PCR were determined considering CDTF cases positive by any of the diagnostic assay methods (n = 81) as true positives. Laboratory confirmed non-typhoidal cases (n = 29) were included as true negatives. On comparison, although both the assays were 100 % specific; sensitivity (91.4 % vs. 81.5 %) and efficiency (93.6 % vs. 86.4 %) of Q-PCR were better, but statistically not significant (p > 0.1) than N-PCR. The positive and negative likelihood ratios of Q-PCR were ∞ and 0.09 which indicated the potential clinical utility of Q-PCR for typhoid diagnosis. Q-PCR was more rapid than N-PCR (2 h vs. 6 h) in obtaining test results.



This study demonstrates for the first time that TaqMan-based Q-PCR assay performs more favorably than N-PCR for direct detection of S. Typhi DNA in blood samples. Direct and quantitative blood Q-PCR is a rapid and reliable method for diagnosis of typhoid fever.
Bacteriology Division, National Institute of Cholera and Enteric Diseases, P-33, CIT Road, Scheme XM, Beliaghata, P.O. Box 177, Kolkata, 700010, West Bengal, India.
Surojit
S
Das
Microbiology Division, Apollo Gleneagles Hospitals, Kolkata, West Bengal, India.
Ujjwayini
U
Ray
Clinical Division, Dr. B. C. Roy Memorial Post Graduate Institute of Pediatric Sciences, Kolkata, West Bengal, India.
Irfaan
I
Akhter
Department of Instrumentation and Electronics Engineering, Jadavpur University, Kolkata, West Bengal, India.
Arka
A
Chattopadhyay
Clinical Division, Dr. B. C. Roy Memorial Post Graduate Institute of Pediatric Sciences, Kolkata, West Bengal, India.
Dilip Kumar
DK
Paul
Bacteriology Division, National Institute of Cholera and Enteric Diseases, P-33, CIT Road, Scheme XM, Beliaghata, P.O. Box 177, Kolkata, 700010, West Bengal, India. shanta1232001@yahoo.co.in.
Shanta
S
Dutta
D016470:Bacteremia / Q000097:blood / Q000175:diagnosis / Q000235:genetics; D015373:Bacterial Typing Techniques; D001483:Base Sequence; D002648:Child; D002675:Child, Preschool; D017931:DNA Primers; D004269:DNA, Bacterial / Q000097:blood*; D005260:Female; D005408:Flagellin / Q000235:genetics*; D005798:Genes, Bacterial; D006801:Humans; D007223:Infant; D008297:Male; D016133:Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D012485:Salmonella typhi / Q000235:genetics / Q000302:isolation & purification*; D014435:Typhoid Fever / Q000097:blood* / Q000175:diagnosis / Q000235:genetics*
D017931:DNA Primers; D004269:DNA, Bacterial; D005408:Flagellin
Diagnostics; PCR; Typhoid; fliC-d gene