Propidium monoazide–polymerase chain reaction for detection of residual periprosthetic joint infection in two-stage revision

Askar, Mohamed; Sajid, Mariam; Nassif, Yassar; Ashraf, Waheed; Scammell, Brigitte E.; Bayston, Roger

doi:10.1007/s11033-019-05092-z

Cited by 12 publications

(12 citation statements)

References 33 publications

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“…Therefore, to study their potential microbiota, further developments in bioinformatics and more targeted methods, such as qPCR, might be required. Future studies may consider use of propidium monoazide-polymerase chain reaction, which prevents amplification of nucleic acids from free or dead microorganisms [ 49 ]. To undertake such assays, we must identify taxa of interest.…”

Section: Discussionmentioning

confidence: 99%

Deciphering the low abundance microbiota of presumed aseptic hip and knee implants

et al. 2021

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16S rRNA gene sequencing of DNA extracted from clinically uninfected hip and knee implant samples has revealed polymicrobial populations. However, previous studies assessed 16S rRNA gene sequencing as a technique for the diagnosis of periprosthetic joint infections, leaving the microbiota of presumed aseptic hip and knee implants largely unstudied. These communities of microorganisms might play important roles in aspects of host health, such as aseptic loosening. Therefore, this study sought to characterize the bacterial composition of presumed aseptic joint implant microbiota using next generation 16S rRNA gene sequencing, and it evaluated this method for future investigations. 248 samples were collected from implants of 41 patients undergoing total hip or knee arthroplasty revision for presumed aseptic failure. DNA was extracted using two methodologies—one optimized for high throughput and the other for human samples—and amplicons of the V4 region of the 16S rRNA gene were sequenced. Sequencing data were analyzed and compared with ancillary specific PCR and microbiological culture. Computational tools (SourceTracker and decontam) were used to detect and compensate for environmental and processing contaminants. Microbial diversity of patient samples was higher than that of open-air controls and differentially abundant taxa were detected between these conditions, possibly reflecting a true microbiota that is present in clinically uninfected joint implants. However, positive control-associated artifacts and DNA extraction methodology significantly affected sequencing results. As well, sequencing failed to identify Cutibacterium acnes in most culture- and PCR-positive samples. These challenges limited characterization of bacteria in presumed aseptic implants, but genera were identified for further investigation. In all, we provide further support for the hypothesis that there is likely a microbiota present in clinically uninfected joint implants, and we show that methods other than 16S rRNA gene sequencing may be ideal for its characterization. This work has illuminated the importance of further study of microbiota of clinically uninfected joint implants with novel molecular and computational tools to further eliminate contaminants and artifacts that arise in low bacterial abundance samples.

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Section: Discussionmentioning

confidence: 99%

Deciphering the low abundance microbiota of presumed aseptic hip and knee implants

et al. 2021

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“…The same authors using an in vitro biofilm model, have shown that PMA treatment enhanced the observed differences after chlorhexidine rinse as compared to non‐PMA treated samples (Exterkate et al, 2014 ). In the field of orthopedic surgery, when PMA in combination to PCR was compared to culture and standard PCR techniques for the detection of residual periprosthetic joint infection, an enhancement of the specificity and sensitivity of 89% and 79% respectively, was reported in PMA‐treated samples (Askar et al, 2019 ).…”

Section: Discussionmentioning

confidence: 99%

Microbiological testing of clinical samples before and after periodontal treatment. A comparative methodological study between real‐time PCR and real‐time‐PCR associated to propidium monoazide

Sereti

Zekeridou

Cancela

et al. 2021

Clinical & Exp Dental Res

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Objectives: The aim of the present methodological study was to evaluate the discrepancies in the detection of a number of periodontally involved pathogenic bacteria obtained from clinical samples by two methods: the quantitative Polymerase Chain Reaction (qPCR) and the qPCR combined with pre-treatment by Propidium Monoazide (PMA).Material and methods: Plaque and saliva samples were obtained from 30 subjects: 20 subjects with chronic or aggressive periodontitis in need of periodontal therapy with or without antibiotics and 10 subjects in Supportive Periodontal Treatment (SPT). The clinical samples taken before treatment (BL) and 1 month later (M1), were divided in two aliquots: one was immediately treated with PMA while the other was left untreated. All samples were further analyzed with qPCR after DNA extraction, for the detection of Aggregatibacter actinomycetemcomitans (Aa), Porphyromonas gingivalis (Pg), Tannerella forsythia (Tf), Treponema denticola (Td), Parvimonas micra (Pm), and Prevotella intermedia (Pi).Results: Large inter-individual variations were observed in the concentration of the studied bacteria. At both instances (BL and M1) and for the three groups, significantly lower counts of bacteria were depicted when plaque and saliva samples were pretreated with PMA as compared to those without treatment. Treatment resulted in significant decreases in the number of bacteria, mainly in the plaque samples. However, these changes were almost similar in the three groups independently of the method of detection used (PMA-qPCR vs. q-PCR). Conclusion:Removal of DNA from non-viable cells with PMA treatment is an easily applied step added to the classical qPCR that could give accurate information on the presence of viable bacterial load and evaluate the response to periodontal treatment.

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“…Among these dyes, PMA is the most widely used dye for the preselection of the viable microorganisms and it has been reported in some studies that PMA showed higher sensitivity than EMA in the detection of viable microbial cells ( a strong covalent nitrogen-carbon bond. This modification results in the structural changes and insolubility of the DNA, which leads to the loss of the DNA during the extraction procedure and inaccessibility of the DNA for elongation by DNA polymerase (Askar et al, 2019;Nocker et al, 2006). Therefore, the DNA template in dead bacteria pretreated with PMA fails to amplify during PCR procedure (Figure 4).…”

Section: Nucleic Acid Amplification-based Detection Methodsmentioning

confidence: 99%

The diagnostic tools for viable but nonculturable pathogens in the food industry: Current status and future prospects

Rui

Liao

Zhao

et al. 2021

Comp Rev Food Sci Food Safe

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Viable but nonculturable (VBNC) microorganisms have been recognized as pathogenic contaminants in foods and environments. The failure of VBNC cells to form the visible colonies hinders the ability to use conventional media for their detection. Efficient and rapid detection of pathogens in the VBNC state is a prerequisite to ensure the food safety and public health. Despite their nonculturability, VBNC cells have distinct characteristics, such as morphology, metabolism, chemical composition, and gene and protein expression, that have been used as the basis for the development of abundant diagnostic tools. This review covers the current status and advances in various approaches for examining microorganisms in the VBNC state, including but not limited to the methodological aspects, advantages, and drawbacks of each technique. Existing methods, such as direct viable count, SYTO/PI dual staining, and propidium monoazide quantitative polymerase chain reaction (PCR), as well as some techniques with potential to be applied in the future, such as digital PCR, enhanced‐surface Raman spectroscopy, and impedance‐based techniques, are summarized in depth. Finally, future prospects for the one‐step detection of VBNC bacteria are proposed and discussed. We believe that this review can provide more optional methods for researchers and promote the development of rapid, accurate detecting methods, and for inspectors, the diagnostic tools can provide data to undertake risk analysis of VBNC cells.

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Propidium monoazide–polymerase chain reaction for detection of residual periprosthetic joint infection in two-stage revision

Cited by 12 publications

References 33 publications

Deciphering the low abundance microbiota of presumed aseptic hip and knee implants

Deciphering the low abundance microbiota of presumed aseptic hip and knee implants

Microbiological testing of clinical samples before and after periodontal treatment. A comparative methodological study between real‐time PCR and real‐time‐PCR associated to propidium monoazide

The diagnostic tools for viable but nonculturable pathogens in the food industry: Current status and future prospects

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