Multiplex PCR for differential diagnosis of emerging typhoidal pathogens directly from blood samples

Abstract

Classically Salmonella enterica serovar Typhi (S. Typhi) is associated with typhoid, a major health problem in developing countries. However, in recent years S. Paratyphi A and Vi-negative variants of S. Typhi have emerged rapidly. We have developed a nested multiplex PCR targeting five different genes for differential diagnosis of typhoidal pathogens which has been optimized to be directly applicable on clinical blood samples. Of 42 multiplex PCR-positive blood samples, 26, nine, and two were Vi-positive S. Typhi, Vi-negative S. Typhi and S. Paratyphi A, respectively, and five patients were found to have mixed infection. Seventeen patients grew Salmonella from blood culture and the remaining 25 were positive in the Salmonella-specific PCR. Tests with several common pathogens confirmed the specificity of the assay. We conclude that the proposed multiplex PCR is rapid, sensitive and specific for the diagnosis of typhoidal pathogens directly from blood samples.

Mix-infection of S. Typhi and ParaTyphi A in Typhoid Fever and Chronic Typhoid Carriers: A Nested PCR Based Study in North India

“…TH and AH [22]. The other study, based on nucleic acid amplification from blood samples of enteric fever patient's has also shown 12% positivity for both the serotypes [11]. Interestingly, 16.4% of the blood samples in the present study were also found positive for the mixed infection.…”

“…The possibility of co-infection has already been suggested by the population based serological study from Nigeria [4] and nucleic acid based detection in blood from Pakistan in acute enteric fever cases [11]. Moreover, S. ParaTyphi A causes indistinguishable clinical features [8] and may be associated with more complications [12][13].…”

Laboratory Diagnosis of Enteric Fever: A Review Update

“…These results show that the nested PCR has good potential to be a rapid tool for the definitive, differential diagnosis of typhoid and is superior to conventional methods 57 .…”

Comment on: development of Vi conjugate – a new generation of typhoid vaccine

“…During analysis of 60 stored strains of serovar Typhi from the Faisalabad region of Pakistan by PCR for the presence of SPI-7 and two genes essential for Vi production: tviA and tviB, 9 (15%) were tested negative for both tviA and tviB, while only two of such strains lacked SPI-7 [3]. Furthermore, the 42 multiplex PCR-positive isolates from the Faisalabad region of Pakistan included 9 Vi negatives [4].…”

“…The expanded usage of conjugated Vi vaccines [1] in areas with frequent circulation of Vi-negative isolates [3,4] could aid in selection and dissemination of the Vi-negative strains in the community. The role of circulating Vi-negative strains could be tremendous following extended usage of Vi vaccines.…”