Evaluation of fliC-d based direct blood PCR assays for typhoid diagnosis

Introduction of PCR into routine diagnostics has rapidly gained a pivotal role for diagnosis of a wide range of diseases, supplanting other conventional microbiological methods. This is true for typhoid fever also, where low bacterial count in the peripheral blood due to their intracellular existence in the reticulo endothelial system does not allow easy diagnosis by blood culture [2, 18]. Recently loop-mediated isothermal DNA amplification (LAMP) assay has been introduced for typhoid diagnosis which is still under investiagtion [19].

Among PCR assays, the Q-PCR has been well recognized to offer several advantages over N-PCR including quantification of bacterial load. When both the assays were compared, the Q-PCR was reported to show comparable or better result than N-PCR in many infectious diseases, but no such data was available for typhoid fever [20–22].

In this study, the analytical and diagnostic performances of Q-PCR and N-PCR assays targeting the fliC-d gene of S. Typhi were evaluated for typhoid diagnosis. Since same experimental conditions were used for both the methods, discrepancies in results could be attributed to the differences in detection limits of the respective method. In earlier reports, Ct values of ?30 or ?28 were regarded as positive for bacterial culture, and Ct values of ?40 or ?38 were positive for clinical samples [21, 23]. Similarly, based on the analytical assay results we have considered Ct values of ?30 and ?37 as positive for bacterial cultures and blood samples respectively.

The analytical specificities of the three PCR assays were found to be 100 % (Table 1). Analytical sensitivities of both N-PCR and Q-PCR assays were similar (3 copies/reaction) (Fig. 2). But, there was a 10-fold reduction in detection limit when spiked blood sample was compared with the bacterial culture in both N-PCR and Q-PCR (Table 2). Presence of human DNA or potential PCR inhibitors might be responsible for the decreased sensitivity of the spiked blood PCR. Detection limit of N-PCR was reported 0.04 pg (corresponding to 10 organisms) using blood DNA spiked with bacterial DNA as samples in earlier study [9]. Reported detection limits of Q-PCR were 1–5 DNA copies/reaction cloned in a plasmid vector and 250 organisms/ml in spiked blood samples [15].

Low isolation rate (21.8 %, 24/110) of S. Typhi among the study population indicated that blood culture did no longer reflect the true burden of the disease in a region and therefore should not be used as standard method. Hence we have used CDTF cases positive by any of the diagnostic assay methods as true positives and the sensitivity of Q-PCR (91.4 %) was found to be higher than N-PCR (81.5 %), but the difference was not statistically significant (p??0.1). PCR-based molecular diagnostics has been adopted as gold standard not only for typhoid, but also for other infectious diseases [11, 24]. In this study, the LR+ and LR- of Q-PCR were ? and 0.09 which indicated the potential clinical utility of Q-PCR for typhoid diagnosis. Likelihood ratios (LRs) are used to measure and express diagnostic accuracy. LR+ value of ?10 means that a positive test is good at ruling in a diagnosis, and LR- value of ?0.1 indicates that a negative test is good at ruling out a diagnosis [25].

For typhoid diagnosis, fliC-d-based PCR assays has been evaluated by other researchers, who have reported ?80 % sensitivity and 100 % specificity of N-PCR [9, 11, 12]. The sensitivity and specificity of Q-PCR were found 87 and 100 % in a study from Indonesia [14]. One study from Nepal showed limited sensitivity (42 %) of Q-PCR targeting a fimbrial-like adhesin gene of S. Typhi [15]. In this study, few culture confirmed typhoid fever cases showed negative results by N-PCR (n?=?6) and Q-PCR (n?=?2). Similar observation was also reported earlier by other researchers [9, 12, 15]. This may be due to the low number of bacteria in the patient’s blood below the detection limit of PCR assays.

Although higher copy number (median, 3160 copies/ml) of bacterial DNA was observed by Q-PCR assay in blood culture negative cases (n?=?57), the higher rate (60 %) of antibiotic intake among the study population might lead to negative results in blood culture mehod (Fig. 3). In this study, the difference in DNA copy number obtained between blood culture positive and negative typhoid fever cases was not significant (p??0.5); whereas one earlier study reported significant difference (p??0.005) between the two categories (range, 1010 to 43,500 and 3.9 to 990 copies/ml) [14].