Aims Evaluation of 16S PCR in addition to the standard culture to improve the pathogen detection rate in clinical specimens. Methods and Results Microbiological culture and direct 16S PCR was performed on specimens from suspected prosthetic joint infection patients (cohort-1) and on tissues and fluids from other normally sterile body sites (cohort-2). Based on clinical and microbiological data, the detection rate for both methods was assessed, assuming no superiority of either 16S PCR or culture. In cohort-1, 469 specimens were obtained. Culture was positive in 170 (36 center dot 2%) specimens, 16S PCR detected 70 (41 center dot 2%) of those pathogens. Additionally, 16S PCR detected pathogens in 13 of 299 (4 center dot 3%) culture-negative specimens. In cohort-2, pathogens were cultured in 52 of 430 (12 center dot 1%) specimens and 16S PCR revealed those pathogens in 32 (61 center dot 5%) specimens. 16S PCR detected pathogens in 31 of 378 (8 center dot 2%) culture-negative specimens. Conclusions Overall, the yield with 16S PCR was low. For cohort-1 16S PCR detected pathogens in 4 center dot 3% of culture-negative specimens, where this was 8 center dot 2% for cohort-2. Significance and Impact of the Study Although direct 16S PCR cannot replace culture, it may offer a valuable additional diagnostic option for detection of difficult to culture micro-organisms in culture-negative clinical specimens.