Detection of Periprosthetic Infections With Use of Ribosomal RNA-Based Polymerase Chain Reaction

Bergin, Patrick E.; Doppelt, Jason D.; Hamilton, William G.; Mirick, Gudrun E.; Jones, Angela E.; Sritulanondha, Supatra; Helm, Jeannine M.; Tuan, Rocky S.
March 2010
Journal of Bone & Joint Surgery, American Volume;Mar2010, Vol. 92-A Issue 3, p654
Academic Journal
Background: Previously described molecular biology techniques used to detect periprosthetic infections have been complicated by false-positive results. We have reported the development of a messenger RNA (mRNA)-based procedure to reduce these false-positive results. The limitations of this procedure are the lack of a universal target and reduced sensitivity due to a low concentration of bacterial mRNAs in test samples. The objective of the present study was to determine whether reverse transcription-quantitative polymerase chain reaction (RT-qPCR) using universal primers can be used to detect the more abundant bacterial ribosomal RNA (rRNA) as an indicator of periprosthetic infection. Methods: Serial dilutions of simulated synovial fluid infections were analyzed with rRNA RT-qPCR to determine the detection limit of this assay. Escherichia coil cultures treated with gentamicin were analyzed with RT-qPCR over a twentyday time course to determine the degradation of rRNA as compared with the decrease in the viable cell count as determined by means of cell plating. As a proof of concept, group-specific polymerase chain reaction primers were developed for Streptococcus species and were tested against fifteen orthopaedically relevant organisms to show the potential for speciation with this assay. Sixty-four patients with a symptomatic effusion at the site of a total knee arthroplasty were enrolled, and complete patient information was documented in a prospective manner. Synovial fluid analysis with rRNA RT-qPCR was performed in a blind fashion. Results: The rRNA RT-qPCR assay was able to detect as few as 590 colony forming units/mL of Staphylococcus aureus and 2900 colony forming units/mL of Escherichia coil. The rRNA RT-qPCR signal closely followed cell death, pointing to its potential use as a viability marker. Three group-specific primer sets correctly identified their intended targets without amplifying closely related species. Clinically, the test correctly identified all six patients with a confirmed infection and all fifty patients who clearly did not have an infection. Eight patients had some laboratory or clinical signs of infection, but their status could not be confirmed. Infection was indicated by rRNA RT-qPCR in three of these patients who had elevated synovial fluid white blood-cell counts but negative results on culture. For statistical purposes, all patients who were categorized as indeterminate were considered to have an infection for the purpose of analysis, for a prevalence of 22% in this cohort. Conclusions: With respect to current diagnostic tests, rRNA-based RT-qPCR demonstrated 100% specificity and positive predictive value with a sensitivity equivalent to that of intraoperative culture. The RT-qPCR signal followed bacterial culture trends but exhibited detectable level for seven days after sterilization, allowing for the detection of infection after the antibiotic administration. These findings indicate that rRNA RT-qPCR is a sensitive and reliable test that retains the universal detection and speciation of DNA-based methods while functioning as a viability indicator.


Related Articles

  • Application of Multiplex PCR for Detection and Differentiation of Entamoeba histolytica, Entamoeba dispar and Entamoeba moshkovskii. ZEBARDAST, Nozhat; HAGHIGHI, Ali; YEGANEH, Farshid; SEYYED TABAEI, Seyyed Javad; GHARAVI, Mohammad Javad; FALLAHI, Shirzad; LASJERDI, Zohreh; SALEHI, Nima; TAGHIPOUR, Niloofar; KOHANSAL, Cobra; NADERI, Farideh // Iranian Journal of Parasitology;Oct-Dec2014, Vol. 9 Issue 4, p466 

    Background: Entamoeba moshkovskii and E. dispar are impossible to differentiate microscopically from the pathogenic species E. histolytica. Multiplex polymerase chain reaction (Multiplex PCR) is a widespread molecular biology technique for amplification of multiple targets in a single PCR...

  • Bioinformatics and molecular biology for the quantification of closely related bacteria. Nagarajan, Karthiga; Loh, Kai-Chee; Swarup, Sanjay // Applied Microbiology & Biotechnology;Jul2013, Vol. 97 Issue 14, p6489 

    Molecular biological methods for mixed culture analysis outshine conventional culture-based techniques in terms of better sensitivity and reliability. The majority of these methods exploit the 16S rRNA sequences of the community DNA, which often fall short for the analysis of closely related...

  • Quantitative Molecular Detection of Putative Periodontal Pathogens in Clinically Healthy and Periodontally Diseased Subjects. Göhler, André; Hetzer, Adrian; Holtfreter, Birte; Geisel, Marie Henrike; Schmidt, Carsten Oliver; Steinmetz, Ivo; Kocher, Thomas // PLoS ONE;Jul2014, Vol. 9 Issue 7, p1 

    Periodontitis is a multi-microbial oral infection with high prevalence among adults. Putative oral pathogens are commonly found in periodontally diseased individuals. However, these organisms can be also detected in the oral cavity of healthy subjects. This leads to the hypothesis, that...

  • 16S rRNA PCR-Denaturing Gradient Gel Electrophoresis of Oral Lactobacillus casei Group and Their Phenotypic Appearances. Piwat, S.; Teanpaisan, R. // ISRN Microbiology;2013, p1 

    This study aimed to develop a 16S rRNA PCR-denaturing gradient gel electrophoresis (DGGE) to identify the species level of Lactobacillus casei group and to investigate their characteristics of acid production and inhibitory effect. PCR-DGGE has been developed based on the 16S rRNA gene, and a...

  • High Abundance of Escherichia During the Establishment of Fecal Microbiota in Brazilian Children. Taddei, Carla; Oliveira, Fernanda; Duarte, Rubens; Talarico, Silvia; Takagi, Elizabeth; Ramos Carvalho, Isabel; Gomes, Filumena; Brandt, Kátia; Martinez, Marina // Microbial Ecology;Apr2014, Vol. 67 Issue 3, p624 

    The sequence of bacterial events that occurs during the colonization of the gastrointestinal tract may affect the future health of the host. A clear understanding of the colonization process of the human neonatal gut in developing countries is lacking because the few available studies were...

  • Candida sanyaensis sp. nov., an ascomycetous yeast species isolated from soil. Hui, Feng-Li; Niu, Qiu-Hong; Ke, Tao; Li, Ying-Xia; Lee, Ching-Fu // Antonie van Leeuwenhoek;Jan2013, Vol. 103 Issue 1, p47 

    Strains representing a novel ascomycetous yeast species, Candida sanyaensis, were isolated from soil samples collected on Hainan Island and Taiwan Island in China. Analysis of the D1/D2 domains of the large subunit (LUS) rRNA gene and internal transcribed spacer (ITS) regions of these strains...

  • Identification and molecular typing of Streptococcus agalactiae isolated from pond-cultured tilapia in China. Xing Ye; Jiong Li; Maixin Lu; Guocheng Deng; Xiaoyan Jiang; Yuanyuan Tian; Yingchun Quan; Qian Jian // Fisheries Science;Jul2011, Vol. 77 Issue 4, p623 

    Bacteria strains with strong virulence were isolated from pond-cultured tilapia in China. They were identified as Streptococcus agalactiae by biochemical assays, and confirmed by 16S ribosomal RNA (rRNA) and group B Streptococcus (GBS)-specific gene cfb analyses. Multiplex polymerase chain...

  • Plant and Fungal Diversity in Gut Microbiota as Revealed by Molecular and Culture Investigations. Gouba, Nina; Raoult, Didier; Drancourt, Michel // PLoS ONE;Mar2013, Vol. 8 Issue 3, p1 

    Background: Few studies describing eukaryotic communities in the human gut microbiota have been published. The objective of this study was to investigate comprehensively the repertoire of plant and fungal species in the gut microbiota of an obese patient. Methodology/Principal Findings: A...

  • Urgent need for authentic (derived from type or typified material) ITS sequence database for all fungi. Sharma, Rahul // Current Science (00113891);12/10/2012, Vol. 103 Issue 11, p1270 

    The author discusses aspects of the identification of fungal species with molecular methods. He highlights the prevalence of usage of Polymerase Chain Reaction (PCR) for the detection of fungal species. Also investigated is the Internal Transcribed Spacer (ITS) region of nuclear ribosomal DNA...


Read the Article


Sorry, but this item is not currently available from your library.

Try another library?
Sign out of this library

Other Topics