Detection of Typhoidal and Paratyphoidal Salmonella in Blood by Real-time Polymerase Chain Reaction

Abstract

Background: The gold standard for diagnosis of enteric fever caused by Salmonella Typhi or Salmonella Paratyphi A or B is bone marrow culture. However, because bone marrow aspiration is highly invasive, many hospitals and large health centers perform blood culture instead. As blood culture has several limitations, there is a need for novel typhoid diagnostics with improved sensitivity and more rapid time to detection.

Methods: We developed a clyA-based real-time polymerase chain reaction (qPCR) method to detect Salmonella Typhi and Salmonella Paratyphi A simultaneously in blood. The sensitivity and specificity of this probeset was first evaluated in vitro in the laboratory and then in a typhoid-endemic population, in Karachi, Pakistan, and in healthy US volunteers.

Results: We optimized a DNA extraction and real-time PCR-based method that could reliably detect 1 colony-forming unit/mL of Salmonella Typhi. The probe set was able to detect clinical Salmonella Typhi and Salmonella Paratyphi A strains and also diarrheagenic Escherichia coli, but not invasive E. coli or other invasive bacteria. In the field, the clyA qPCR diagnostic was 40% as sensitive as blood culture. However, when qPCR-positive specimens were considered to be true positives, blood culture only exhibited 28.57% sensitivity. Specificity was ≥90% for all comparisons and in the healthy US volunteers. qPCR was significantly faster than blood culture in terms of detection of typhoid and paratyphoid.

Conclusions: Based on lessons learned, we recommend that future field trials of this and other novel diagnostics that detect typhoidal and nontyphoidal Salmonella employ multiple methodologies to define a "positive" sample.

Keywords: PCR; Salmonella; blood; diagnostic; typhoid.

Figure 1.

In vitro sensitivity of the Salmonella real-time polymerase chain reaction (qPCR) assay. Lymphocytes were isolated from 2–3 mL whole human blood by lysing red blood cells. Cell pellets were spiked with various concentrations of Salmonella Typhi Ty2, and DNA was isolated using a QIAamp Blood DNA Mini kit and tested using the clyA qPCR. The colored symbols represent 2 different users and the various symbols represent experiments performed on different occasions. Data are expressed as mean ± standard deviation from 3 replica wells. The maximum cycle threshold (Ct) value is 50 cycles. Wells with an undetermined Ct were assigned a Ct of 51.

Figure 2.

Cycle threshold (Ct) values of real-time polymerase chain reaction–positive specimens according to blood culture result. Data are expressed as a box-and-whisker plot with whiskers representing the minimum and maximum value.

Figure 3.

Age (A) and volume of blood tested (B) of specimens that were blood culture negative and real-time polymerase chain reaction (qPCR) positive; blood culture positive and qPCR negative; and positive for both blood culture and qPCR. Age data are expressed as a box-and-whisker plot with whiskers representing the minimum and maximum value. Blood volumes are expressed as a scatter plot with the bar indicating the mean. Abbreviations: -ve, negative; +ve, positive; Cx, culture.

Figure 4.

Time to identification for blood culture or real-time polymerase chain reaction (qPCR) positivity. Data are expressed as a box-and-whisker plot with whiskers representing the minimum and maximum value. Data were analyzed using Mann–Whitney test, 2-tailed.