Detection of Salmonella enterica serovar Typhi by nested PCR using different procedures of nucleic acid extraction

Abstract

Typhoid fever is one of the major health problems in Bangladesh. It is caused by the bacterium Salmonella enterica serover Typhi (S. Typhi). The infection rate of S. Typhi is higher in children. The conventional diagnostic methods of typhoid have limitations. Most commonly used Widal test gives a high rate of false positive results. As such PCR method was tried to diagnose typhoid fever in some selected patients of Bangladesh. Fifty three (53) patients were enrolled in this study from Dhaka hospital of ICDDR, B and Kamalapur field site. Among them 27 (51 %) persons are male. Blood was collected from all patients. Three different methods namely, a method by Haque et al, direct boiling method and commercial Qiagen kit method were tried to extract Salmonella Typhi DNA from blood and invitro samples. To optimize the DNA extraction from blood invitro grown S. Typhi bacteria was spiked into blood. From the three different methods, commercial Qiagen kit method did well comparably to extract DNA from spiked blood than the other methods. However, none of these methods did appear promising to extract DNA from patient's blood. Because low percentage of PCR positive were found from patient's blood culture positive samples. So another method was tried which was described earlier by Boom's et al. [1990] for detection of S. Typhi from blood of suspected typhoid fever patient. This method (Boom's et al.[1990]) showed comparably satisfactory results than the other methods to deal with patient's blood. In this method the percentage of PCR positive was 35.7 % among the blood culture confirmed patients. DNA extraction was also done in blood culture negative samples by this method. Here 30.8% positive for PCR was found among the culture negative samples. However there are some drawbacks in DNA extraction procedures especially in low concentrated samples. In the method by Haque et al. [20011 unexpected band was found and same phenomenon was observed in direct boiling method. This might be due to contamination with S. Typhi. It seems that, S. Typhi can be detected from patient's blood specimen by PCR method for diagnostic purpose. However, more study is needed to evaluate the efficacy of PCR method with some modifications for detecting S. Typhi at low concentration in blood.