A novel technique for detecting antibiotic-resistant typhoid from rapid diagnostic tests.
Bottom Line: Fluoroquinolone-resistant typhoid is increasing.A simple, inexpensive molecular technique performed with DNA from positive RDTs accurately identified gyrA mutations consistent with phenotypic susceptibility testing results.Field diagnosis combined with centralized molecular resistance testing could improve typhoid management and surveillance in low-resource settings.
Affiliation: Lao-Oxford-Mahosot Hospital-Wellcome Trust Research Unit, Microbiology Laboratory, Vientiane, Lao PDR Public Health England, London, England, United Kingdom.Show MeSH
Related in: MedlinePlus
Mentions: Negative blood culture bottles (7 days) were seeded with ∼150 cells of S. Typhi NCTC 8385, reincubated, and inspected daily. A Gram stain was performed on turbid bottles to confirm the presence of GNRs. Ten bottles containing GNRs were used to inoculate 10 One-Step Salmonella Typhi antigen rapid detection kits (Standard Diagnostics, South Korea), which were used to optimize DNA extraction protocols (Fig. 1). Detection of the gyrA gene and its mutations was performed using previously described primers under slightly modified PCR conditions, followed by restriction fragment length polymorphism (RFLP) analysis (13). DNA from each section and extraction method underwent PCR as neat and diluted (1:10, 1:100, and 1:1,000) samples, plus 40 μg of bovine serum albumin (BSA; New England BioLabs) per reaction mixture to overcome inhibitors (14, 15). Subsequently, the intensities of bands on an agarose gel were compared, and the bands giving the greatest intensities were chosen as indicators for the optimal processing method. Wild-type S. Typhi NCTC 8385 and well-characterized strains with known gyrA mutations, provided by Oxford University Clinical Research Unit, Ho Chi Minh City, Vietnam (16), were used as controls.
Affiliation: Lao-Oxford-Mahosot Hospital-Wellcome Trust Research Unit, Microbiology Laboratory, Vientiane, Lao PDR Public Health England, London, England, United Kingdom.