Highly-sensitive detection of Salmonella typhi in clinical blood samples by magnetic nanoparticle-based enrichment and in-situ measurement of isothermal amplification of nucleic acids

Abstract

Enteric fever continues to be a major cause of mortality and morbidity globally, particularly in poor resource settings. Lack of rapid diagnostic assays is a major driving factor for the empirical treatment of enteric fever. In this work, a rapid and sensitive method ‘Miod’ ‘has been developed. Miod includes a magnetic nanoparticle-based enrichment of target bacterial cells, followed by cell lysis and loop-mediated isothermal amplification (LAMP) of nucleic acids for signal augmentation along with concurrent measurement of signal via an in–situ optical detection system. To identify positive/negative enteric fever infections in clinical blood samples, the samples were processed using Miod at time = 0 hours and time = 4 hours post-incubation in blood culture media. Primers specific for the STY2879 gene were used to amplify the nucleic acids isolated from S. typhi cells. A limit of detection of 5 CFU/mL was achieved. No cross-reactivity of the primers were observed against 106 CFU/mL of common pathogenic bacterial species found in blood such as E. coli, P. aeruginosa, S. aureus, A. baumanni, E. faecalis, S. Paratyphi A and K. pneumonia. Miod was tested on 28 human clinical blood samples. The detection of both pre-and post-four-hours incubation confirmed the presence of viable S. typhi cells and allowed clinical correlation of infection. The positive and negative samples were successfully detected in less than 6 hours with 100% sensitivity and specificity.

Occurrences and Virulence Genes Detection of Salmonella Typhi Isolates Among Patients With Typhoid Fever

“…Most often, the stool is inoculated into uid enrichment medium such as Tetrathionate or Selenite broth and then plated on Desoxy Cholate-Citrate Agar. It is possible to test for salmonella O antigens directly on culture plates using slide agglutination and sub-cultured to peptone water to determine the structure of the H antigen and do additional biochemical analysis [9].…”

Classification of Salmonella enterica of the (Para-)Typhoid Fever Group by Fourier-Transform Infrared (FTIR) Spectroscopy

“…Therefore, attempts towards more easy-to-apply and rapid diagnostic tools have been launched. These include PCR [ 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 ] and loop-mediated isothermal amplification methods [ 33 , 34 , 35 , 36 , 37 , 38 , 39 ], both exhibiting sensitivity and specificity issues [ 38 , 39 ]. In addition, cross-reactions with serovars with high sequence homology like S. Paratyphi A and S. Weltevreden, S. Paratyphi B and S. Abony, as well as S. Paratyphi C and S. Choleraesuis [ 30 , 40 ] have been observed.…”

Identification of aldehyde oxidase 3 as a binding protein for squid ink polysaccharides using magnetic nanoparticles

“…Magnetic nanoparticles were prepared according to the slightly modied methods. 25,26 Preparation of activated magnetic beads (AMB). 2 mg of superparamagnetic nanometer beads (purchased from Shanghai Jinpan Biotech.…”