Long-term storage of clinical samples in CyMol® medium for PNA- FISH® and culturing from the eSwab™ system

Lone Heimann Larsen, Yijuan Xu, Malene Schibler Pedersen, Henrik Carl Schønheyder, Trine Rolighed Thomsen

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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.

Original languageEnglish
Publication dateMay 2014
Number of pages1
Publication statusPublished - May 2014
Event24th European Congress of Clinical Microbiology and Infectious Diseases, ECCMID 2014 - Barcelona, Spain
Duration: 10 May 201413 May 2014
Conference number: 14

Conference

Conference24th European Congress of Clinical Microbiology and Infectious Diseases, ECCMID 2014
Number14
CountrySpain
CityBarcelona
Period10/05/201413/05/2014

Bibliographical note

Poster no. P0606

Cite this

Larsen, L. H., Xu, Y., Pedersen, M. S., Schønheyder, H. C., & Thomsen, T. R. (2014). Long-term storage of clinical samples in CyMol® medium for PNA- FISH® and culturing from the eSwab™ system. Poster presented at 24th European Congress of Clinical Microbiology and Infectious Diseases, ECCMID 2014, Barcelona, Spain.