TY - JOUR

T1 - Comparison of two commercial broadrange PCR and sequencing assays for identification of bacteria in culture-negative clinical samples

AU - Stavnsbjerg, Camilla

AU - Frimodt-Moller, Niels

AU - Moser, Claus Ernst

AU - Bjarnsholt, Thomas

PY - 2017/3/27

Y1 - 2017/3/27

N2 - BackgroundCulturing has long been the gold standard for detecting aetiologic agents in bacterial infections. In some cases, however, culturing fails to detect the infection. To further investigate culture-negative samples, amplification and subsequent sequencing of the 16S rRNA gene is often applied. The aim of the present study was to compare the current method used at our Department of Clinical Microbiology, based on the MicroSeq ID system (Applied Biosystems, USA) with the Universal Microbe Detection (UMD) SelectNA kit (Molzym, Germany).Methods76 culture-negative samples were first processed with the MicroSeq ID analysis, where total DNA was extracted and the 16S gene amplified and sequenced with the MicroSeq ID system. Samples were subsequently processed with the UMD SelectNA analysis, where human DNA was removed during the DNA extraction procedure and the 16S gene amplified in a real-time PCR and sequenced.Results22 of 76 samples (28.9%) were positive for bacteria with the UMD SelectNA, which was significantly more (p = 0.0055) than the MicroSeq ID where 11 of 76 samples (14.5%) were positive. The UMD SelectNA assay identified more relevant bacterial pathogens than the MicroSeq ID analysis (p = 0.0233), but also found a number of species that were considered contaminations.ConclusionsThe UMD SelectNA assay was valuable for the identification of pathogens in culture-negative samples; however, due to the sensitive nature of the assay, extreme care is suggested in order to avoid false positives.

AB - BackgroundCulturing has long been the gold standard for detecting aetiologic agents in bacterial infections. In some cases, however, culturing fails to detect the infection. To further investigate culture-negative samples, amplification and subsequent sequencing of the 16S rRNA gene is often applied. The aim of the present study was to compare the current method used at our Department of Clinical Microbiology, based on the MicroSeq ID system (Applied Biosystems, USA) with the Universal Microbe Detection (UMD) SelectNA kit (Molzym, Germany).Methods76 culture-negative samples were first processed with the MicroSeq ID analysis, where total DNA was extracted and the 16S gene amplified and sequenced with the MicroSeq ID system. Samples were subsequently processed with the UMD SelectNA analysis, where human DNA was removed during the DNA extraction procedure and the 16S gene amplified in a real-time PCR and sequenced.Results22 of 76 samples (28.9%) were positive for bacteria with the UMD SelectNA, which was significantly more (p = 0.0055) than the MicroSeq ID where 11 of 76 samples (14.5%) were positive. The UMD SelectNA assay identified more relevant bacterial pathogens than the MicroSeq ID analysis (p = 0.0233), but also found a number of species that were considered contaminations.ConclusionsThe UMD SelectNA assay was valuable for the identification of pathogens in culture-negative samples; however, due to the sensitive nature of the assay, extreme care is suggested in order to avoid false positives.

KW - Culture-negative samples

KW - Molecular diagnostics

KW - Universal Microbe Detection

KW - 16S PCR

U2 - 10.1186/s12879-017-2333-9

DO - 10.1186/s12879-017-2333-9

M3 - Journal article

C2 - 28347280

VL - 17

JO - B M C Infectious Diseases

JF - B M C Infectious Diseases

SN - 1471-2334

M1 - 233

ER -