Abstract
Objectives: A steadily growing diversity of bacteria is reported in foreign body infections, and culture-independent methods have been shown to supplement established culture methods. Therefore, sampling and preservation of specimens have become an important issue. We report here experience from a prospective clinical study enrolling patients with joint prosthesis-related problems (www.joint-prosthesis-infection-pain.dk). From the patients a range of diagnostic and peroperative specimen types were obtained. Here we report primarily on the utility of different specimen types for culture -independent analytical methods.
Methods: Sampling for both culture-dependent and -independent analyses were done over a period of two years. Specimens were transferred directly to the lab, and cultures of tissue biopsies, joint fluid, sonication fluid from the prosthesis components, and eSwab™ (Copan, Italy) were performed within 24 h after sampling. The corresponding specimens for culture-independent methods were stored at -80°C until analyzed in batchs. Specimens for fluorescence in situ hybridization (FISH) analysis were stored for app. one year at -80°C in CyMol® (Copan, Italy), an alcohol based media, before the analyses were conducted. For direct visualization of microorganisms we used both FISH and peptide nucleic acid- fluorescence in situ hybridization (PNA-FISH®, AdvanDx, USA). Both FISH and PNA-FISH® were conducted according to previous publications, with broad range probes. An initial filtration step was performed for some samples in order to concentrate bacteria.
Results: We were able to perform FISH and PNA-FISH® on specimens stored for more than one year without further optimization of the hybridization protocol. The broad range probes demonstrated bacteria with a bright signal and a morphology comparable to the isolates obtained by culturing at the time of sampling. The detection limit for both FISH and PNA-FISH® were >10^3 bacteria/mL. With the eSwab™ system we were able to detect a broad range of bacteria including Staphylococcus spp., Streptococcus spp., Enterococcus spp., and Corynebacterium spp. by culture and 16S rRNA gene amplicon sequencing.
Conclusion: It is possible to preserve samples for FISH and PNA-FISH® for long-term storage by using CyMol® with an effective detection limit in the order of >10^3 bacteria/mL. Both the morphology and intensity of staining with nucleic acid and PNA probes were distinct. The eSwab™ was a convenient system for documenting a broad range of bacterial pathogens associated with foreign body infections.
Methods: Sampling for both culture-dependent and -independent analyses were done over a period of two years. Specimens were transferred directly to the lab, and cultures of tissue biopsies, joint fluid, sonication fluid from the prosthesis components, and eSwab™ (Copan, Italy) were performed within 24 h after sampling. The corresponding specimens for culture-independent methods were stored at -80°C until analyzed in batchs. Specimens for fluorescence in situ hybridization (FISH) analysis were stored for app. one year at -80°C in CyMol® (Copan, Italy), an alcohol based media, before the analyses were conducted. For direct visualization of microorganisms we used both FISH and peptide nucleic acid- fluorescence in situ hybridization (PNA-FISH®, AdvanDx, USA). Both FISH and PNA-FISH® were conducted according to previous publications, with broad range probes. An initial filtration step was performed for some samples in order to concentrate bacteria.
Results: We were able to perform FISH and PNA-FISH® on specimens stored for more than one year without further optimization of the hybridization protocol. The broad range probes demonstrated bacteria with a bright signal and a morphology comparable to the isolates obtained by culturing at the time of sampling. The detection limit for both FISH and PNA-FISH® were >10^3 bacteria/mL. With the eSwab™ system we were able to detect a broad range of bacteria including Staphylococcus spp., Streptococcus spp., Enterococcus spp., and Corynebacterium spp. by culture and 16S rRNA gene amplicon sequencing.
Conclusion: It is possible to preserve samples for FISH and PNA-FISH® for long-term storage by using CyMol® with an effective detection limit in the order of >10^3 bacteria/mL. Both the morphology and intensity of staining with nucleic acid and PNA probes were distinct. The eSwab™ was a convenient system for documenting a broad range of bacterial pathogens associated with foreign body infections.
| Original language | English |
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| Publication date | May 2014 |
| Number of pages | 1 |
| Publication status | Published - May 2014 |
| Event | 24th European Congress of Clinical Microbiology and Infectious Diseases, ECCMID 2014 - Barcelona, Spain Duration: 10 May 2014 → 13 May 2014 Conference number: 14 |
Conference
| Conference | 24th European Congress of Clinical Microbiology and Infectious Diseases, ECCMID 2014 |
|---|---|
| Number | 14 |
| Country/Territory | Spain |
| City | Barcelona |
| Period | 10/05/2014 → 13/05/2014 |