Blood culture is currently the gold standard for diagnosis of typhoid fever, but it takes several days for the isolation and identification of causative organisms. As early diagnosis of the disease and prompt treatment are essential for optimal management, especially in children, a rapid sensitive detection method for typhoid fever is urgently needed. PCR is sensitive and rapid, and could possibly replace blood culture as the new "gold standard". However, initial research indicated similar sensitivity to blood culture and lower specificity .
Given the problems associated with conventional methods for diagnosis of typhoid fever, the present study was carried out in an attempt to develop a rapid, reliable, specific, and sensitive method for detection of Salmonella serovar Typhi. Artificially spiked blood samples were used throughout the study to mimic clinical specimens in the development and evaluation of a blood culture PCR method for detection of Salmonella serovar Typhi.
Early studies found that the number of Salmonella bacteria circulating in the blood of patients with Salmonella bacteremia is small, e.g. 0.5-22 bacteria per millilitre of blood found in 15 patients with typhoid fever , fewer than 2 bacteria per millilitre of blood found in 90% of 80 positive blood cultures from patients with Salmonella bacteremia , and a median of 0.3 (IQR, 0.1-10) bacteria per millilitre of blood in further 81 patients with typhoid fever . Therefore, it is very important to use small bacterial inocula when developing and evaluating a system for detection of Salmonella serovar Typhi in clinical specimens. In the present study, blood samples were spiked with Salmonella serovar Typhi Quailes strain at a ratio of 1 ± 0.5 CFU per millilitre of blood, which closely mimics clinical specimens taken from patients with typhoid fever.
The initial evaluation study, in which 2-6 CFU of Salmonella serovar Typhi Quailes strain were mixed with 4 ml of blood and then cultured in TSB medium, found that the bacteria did not grow during 5 hours of incubation. The results remained the same when blood samples taken from different healthy donors were used. Moreover, when blood was artificially inoculated with a small number of Salmonella bacteria, followed by plating, fewer bacteria were recovered from the inoculated blood samples than the input (data not shown). This, consistent with other studies [11, 19, 24, 25], clearly demonstrates that human blood has bactericidal activity against Salmonella serovar Typhi. Furthermore, one study found that the defibrinated whole blood from patients with Salmonella bacteremia was bactericidal for small inocula of three of five Salmonella strains isolated from the patient's blood . This suggests that the blood or serum of patients with Salmonella bacteremia is also frequently bactericidal against the Salmonella isolated from their blood. Therefore, a culture medium which inhibits the bactericidal activity of blood may be beneficial in the development of a blood culture-PCR assay system.
Since bile was first used for blood culture of patients with Salmonella bacteremia in 1906 , bile containing media have repeatedly been found superior for isolation of enteric fever pathogens (Salmonella serovars, Typhi and Paratyphi) from whole blood . Conradi attributed the effectiveness of bile as a culture medium to its effect in preventing coagulation of blood , and further demonstrated that bile decreases the bactericidal activity of serum . Although Kaye et al. did not find that the anticoagulant effect was an important factor in the superiority of bile over TSB as a blood culture medium for Salmonella, they confirmed and expanded the observation that bile decreases the bactericidal activity of serum . They found that bile inactivated the complement activity of blood, and also disrupted leukocytes in which Salmonella may be present. They argued that in patients with Salmonella bacteremia rapid release of Salmonella from leukocytes by bile might be a very important factor contributing to the advantage of bile as a culture medium. The intracellular presence of Salmonella serovar Typhi was demonstrated by the study  showing that the number of CFU of Salmonella serovar Typhi per milillitre sample increased by 3.3 fold and 1.9 fold respectively when bone marrow and blood cells were lyzed. Therefore, a blood culture medium could increase bacterial growth if it contains blood cell lyzing agents. Having examined further the effect of bile on the growth of Salmonella serovar Typhi, the present study confirmed the observation that bile does not have a growth-promoting effect on Salmonella . In fact, Salmonella serovar Typhi Quailes strain did not survive in 10% bile in the absence of TSB (data not shown). Furthermore, the present study also demonstrated that bile, in absence of blood, inhibits the growth of Salmonella serovar Typhi Quailes strain at high concentrations of bile in TSB-bile medium. In order to determine the concentration of bile for use in a TSB-bile blood culture system, the lysis of blood cells by bile was studied. The results showed that the lysis of blood cells depends on the bile concentration and incubation time. At a concentration of 2.4%, bile not only lyzes blood cells completely within 1.5 hours so that the intracellular bacteria could be released, but also has no inhibiting effect on the growth of Salmonella serovar Typhi Quailes strain. Therefore, 2.4% bile seems an optimal concentration for use in the TSB-bile blood culture system.
It has been asserted that PCR is sensitive and rapid, and is a better alternative than conventional methods for pathogen detection. In the last decade, PCR has been widely researched for early diagnosis of typhoid fever. PCR as a diagnostic tool for detection of Salmonella serovar Typhi was first studied by Song et al.  who developed a nested PCR for amplification of the fliC-d gene of Salmonella serovar Typhi which could detect 5 bacteria/ml, compared to106 bacteria/ml by regular PCR. Recently, Ali et al.  reported a nested multiplex PCR for detection of both Salmonella serovars, Typhi and Paratyphi, with a sensitivity of 10 bacteria/ml. However, initial research indicated that PCR has similar sensitivity to blood culture and lower specificity . This, at least in part, can be attributed to the low number of Salmonella bacteria circulating in the blood of typhoid patients and the inadequate method of DNA preparation. In normal clinical samples, there is a median of 0.3 CFU per millilitre of blood which is far below the sensitivity of PCR so far developed, and therefore, it is not surprising that PCR has not been used for diagnosis of typhoid fever. To overcome the difficulties caused by the low numbers of Salmonella bacteria present in typhoid patient blood samples, pre-enrichment of bacteria is necessary prior to PCR detection. A recent study demonstrated that the 5 hour broth culture enrichment improved PCR sensitivity by 10 times for spiked blood, and 100 times for spiked stool samples . The present study has used the optimized TSB-bile blood culture system, in which 0.75 bacteria per ml of blood was used to mimic real clinical samples, and 4 ml blood for the culture with 3 CFU in total, for enrichment of bacteria prior to the PCR detection of Salmonella serovar Typhi. At the bacterial level used in present study, it was very difficult to detect the presence of Salmonella serovar Typhi by regular PCR without bacterial enrichment and/or removal of human DNA. However, three hour enrichment of Salmonella serovar Typhi in the TSB-bile medium could increase the number of CFUs to the level at which detection is possible using regular PCR. The sensitivity of this novel blood culture PCR method was equivalent to 0.75 CFU per millilitre of blood which is comparable to the bacterial number present in clinical typhoid samples. Moreover, the turnaround time of the assay was less than 8 hours rather than several days for conventional blood culture. Therefore, this new TSB-bile blood culture PCR system is superior (speed and sensitivity) to conventional blood culture and PCR methods and could potentially make early detection of Salmonella serovar Typhi possible for prompt treatment of patients with typhoid fever.