The comparison of pyrosequencing molecular Gram stain, culture, and conventional Gram stain for diagnosing orthopaedic infections

Abstract

ABSTRACT:We have developed a combined real-time PCR and pyrosequencing assay that successfully differentiated the vast majority of gram-positive and gram-negative bacteria when bacterial isolates were tested. The purpose of this study was to evaluate this assay on clinical specimens obtained from orthopedic surgeries, and to prospectively compare the results of ''molecular Gram stain'' with culture and conventional direct Gram stain. Forty-five surgical specimens were obtained from patients who underwent orthopedic surgery procedures. The DNA was extracted and a set of broad-range PCR primers that targeted a part of the 16S rDNA gene was used for pan-bacterial PCR. The amplicons were submitted for pyrosequencing and the resulting molecular Gram stain characteristics were recorded. Culture and direct Gram staining were performed using standard methods for all cases. Surgical specimens were reviewed histologically for all cases that had a discrepancy between culture and molecular results. There was an 86.7% (39/45) agreement between the traditional and molecular methods. In 12/14 (85.7%) culture-proven cases of bacterial infection, molecular Gram stain characteristics were in agreement with the culture results, while the conventional Gram stain result was in agreement only for five cases (35.7%). In the 31 culture negative cases, 27 cases were also PCR negative, whereas 4 were PCR positive. Three of these were characterized as gram negative and one as gram positive by this molecular method. Molecular determination of the Gram stain characteristics of bacteria that cause orthopedic infections may be achieved, in most instances, by this method. Further studies are necessary to understand the clinical importance of PCR-positive/culture-negative results. ß

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“…It is not yet known if the high prevalence of bacteria identified in those studies represents the cause of patients' infection or false-positive results. Our recent results 1 in which PCR was used to identify evidence of bacteria in apparently aseptically loose implants showed bacterial DNA in 12%, which is a smaller proportion to that of Tunney et al 5 or Mariani et al 6 However, in vitro studies have also shown that PCR can identify DNA from dead bacteria. 3 While the detection of dead bacteria is interesting, it carries less clinical importance than detecting viable bacteria.…”

“…One hundred microliters of DNA extract was obtained for each sample. Quantitative PCR assays targeted 16S rDNA 1 and tuf using a Rotorgene 3000 1 (Corbett Research, Sydney, Australia). Tuf primer sequences (BioChem, Salt Lake City, UT) were: forward primer, 5 0 -ATGCCACAAACTCGTGA-3 0 , reverse primer, 5…”

“…1,2 However, a major limitation is the lack of differentiation between viable and dead bacteria. 3,4 Several previous studies 5,6 that have evaluated synovial fluid or orthopedic implants after total hip or knee arthroplasty have reported evidence of bacteria in a high proportion of cases.…”

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“…Twenty-five microliters of DNA extract was obtained for each sample. qPCR assays targeted 16S rDNA [5] and tufA DNA using a Rotorgene 3000 1 (Corbett Research, Sydney, Australia). The tufA primer sequences (BioChem, Salt Lake City, UT) were 5 0 -ATGCCACAAACTCGTGA-3 0 (forward primer) and 5 0 -ACCACGACCAGTGATTGAGAA-3 0 (reverse primer).…”

“…Molecular methods, especially PCR, have been recognized as a sensitive tool for improving the diagnosis of prosthetic joint infections [5,15]. Mariani et al [7] suggested PCR may be more sensitive than conventional cultures and some cases of prosthetic joint infection may be misclassified as aseptic loosening using current culture methods.…”