Specific and Rapid Detection by Fluorescent In Situ Hybridization of Bacteria in Clinical Samples Obtained from Cystic Fibrosis Patients

Abstract

We report on the rapid and specific detection of bacteria commonly isolated from clinical specimens from cystic fibrosis (CF) patients by fluorescent in situ hybridization (FISH). On the basis of comparative sequence analysis, we designed oligonucleotide probes complementary to species-specific 16S rRNA regions of these microorganisms and demonstrated the specificities of the probes by hybridization of different remotely related as well as closely related reference strains. Furthermore, in a pilot project we investigated 75 sputum samples and 10 throat swab specimens from CF patients by FISH and detected Pseudomonas aeruginosa, Burkholderia cepacia, Stenotrophomonas maltophilia,Haemophilus influenzae, and Staphylococcus aureus within these specimens. The specificity of FISH was 100% in comparison to the results of conventional microbial culture. In contrast, the sensitivity of standard laboratory cultivation was moderately higher, since the limit for microscopic detection of bacteria within sputum samples by FISH was approximately 4 × 105 CFU/ml of sputum (resulting in a 90% sensitivity for FISH). Moreover, we demonstrated that FISH will be useful for the rapid detection of bacteria that cause acute pulmonary exacerbations in CF patients, as demonstrated in patients with H. influenzae, S. aureus, andP. aeruginosa exacerbations. Therefore, FISH is a valuable additional method for the rapid and specific detection of bacteria in clinical samples from CF patients, in particular, patients with pulmonary exacerbations.

Molecular diversity of bacterial endosymbionts associated with dagger nematodes of the genus Xiphinema (Nematoda: Longidoridae) reveals a high degree of phylogenetic congruence with their host

“…The FISH probe 5 0 -/5ATTO633N/TGC CAC CCG TAG GTG T-3 0 (Brown et al, 2015) was used to detect Candidatus Xiphinematobacter rivesi (in green), and the FISH probe Burkho_1 5 0 -5ATTO550/RCC CTC TGT ACC GAC CAT-3 0 was designed to target the 16S rRNA of our Burkholderiaceae endosymbiont (in red) in Xiphinema nematodes was based on the probe Burkho designed for Burkholderia spp. (Hogardt et al 2000). After hybridization, specimens were washed twice at 46°C for 30 min in 1 mL of prewarmed hybridization wash buffer (20 mM Tris-HCl, 0.02% wt/vol SDS, 0.008 M NaCl, 5 mM EDTA).…”

Rapid differentiation of Candida albicans from non-C. albicans directly in a variety of clinical specimens using fluorescent in situ hybridisation

“…This study is subject to a limitation of the minimum detection limit (FISH 10 3 microorganisms ml )1 vs. 10 5 microorganisms ml )1 for Gram stain). 6 Only the samples that showed yeast by Gram staining were evaluated by a follow-up FISH assay. Some specimens with a low number of yeast cells could be missed when performing the Gram stain screening process, which would affect the sensitivity analysis of the FISH assay in this study.…”

“…Recently, fluorescent in situ hybridisation (FISH) of whole cells with a fluorescently-labelled, 18S rRNAtargeted, oligonucleotide probe has been used for the sensitive detection of microorganisms. [3][4][5][6] Several authors have reported the identification of Candida species from blood cultures using FISH, 7-9 but only a few studies have investigated the identification of Candida species in other types of clinical samples. 10,11 In this study, we developed a modified FISH protocol for differentiation of C. albicans from non-C. albicans species in clinical specimens without culturing (except for blood cultures), evaluated the sensitivity and specificity of this protocol compared with traditional identification methods, and described the protocol detail for the application of the FISH protocol on blood culture, bronchoalveolar lavage (BAL) fluid, abscess ⁄ puncture liquid and urine samples.…”

Rapid identification of Leishmania spp. in formalin‐fixed, paraffin‐embedded tissue samples by fluorescence in situ hybridization

“…Fluorescence in situ hybridization (FISH) is a rapid diagnostic staining procedure based on the specific binding of fluorescently labelled probes to ribosomal target RNA. It has been broadly evaluated for the identification of various microorganisms in clinical samples by fluorescence microscopy (Hogardt et al 2000;Kempf et al 2000;Poppert et al 2002;Poppert et al 2005Poppert et al , 2010aWellinghausen et al 2005). FISH-based differentiation of pathogens is even possible from more difficult specimens such as blood culture materials (Hogardt et al 2000;Kempf et al 2000;Poppert et al 2010a,b) or formalinfixed, paraffin-embedded tissue samples (Rü ssmann et al 2003;Yilmaz et al 2007;Hagen et al 2011).…”

“…We developed and evaluated a rapid FISH-based microscopic screening procedure for Leishmania spp. in tissue samples under standard conditions (Hogardt et al 2000;Kempf et al 2000) as a useful tool for endemic areas and travel medicine clinics. It is based upon newly designed probes to optimize screening and to facilitate diagnosis.…”