Fluorescencein situhybridization (FISH) in the microbiological diagnostic routine laboratory: a review

Abstract

Early identification of microbial pathogens is essential for rational and conservative antibiotic use especially in the case of known regional resistance patterns. Here, we describe fluorescence in situ hybridization (FISH) as one of the rapid methods for easy identification of microbial pathogens, and its advantages and disadvantages for the diagnosis of pathogens in human infections in the laboratory diagnostic routine. Binding of short fluorescence-labeled DNA or nucleic acid-mimicking PNA probes to ribosomes of infectious agents with consecutive analysis by fluorescence microscopy allows identification of bacterial and eukaryotic pathogens at genus or species level. FISH analysis leads to immediate differentiation of infectious agents without delay due to the need for microbial culture. As a microscopic technique, FISH has the unique potential to provide information about spatial resolution, morphology and identification of key pathogens in mixed species samples. On-going automation and commercialization of the FISH procedure has led to significant shortening of the time-to-result and increased test reliability. FISH is a useful tool for the rapid initial identification of microbial pathogens, even from primary materials. Among the rapidly developing alternative techniques, FISH serves as a bridging technology between microscopy, microbial culture, biochemical identification and molecular diagnostic procedures.

Open Source 3D‐printed focussing mechanism for cellphone‐based cellular microscopy

“…The increasing sophistication of lab-on-chipbased diagnostics at the micrometre scale (St John & Price, 2014) has resulted in shrinking the size of laboratory equipment and made cellphone-based microscopy relevant to not just resource-poor settings, but also diagnostics in developed countries. Although continued and dramatic developments in molecular and genetic diagnostics have made DNA-and protein-based tests routine diagnostic tools, parallel improvements in dyes and conjugated sensors have improved the precision of phenotypic tests (Frickmann et al, 2017;Lee et al, 2017;Zainol Abidin et al, 2017). Indeed, the relevance of phenotypic assays in clinics and biology teaching laboratories has been revived by these developments in molecular probes and microscopy.…”

Fluorescence In Situ Hybridization for Diagnosis of Whipple’s Disease in Formalin-Fixed Paraffin-Embedded Tissue

“…In our study, we could show in 12 patients from routine practice that FISH has the potential to diagnose active WD in FFPE specimens and enables the direct visualization and detection of individual vital bacteria in the infected tissue (5, 6, 34, 38, 49, 50). While sensitivity of FISH is not yet ideal, the commercially available FISH represents a valuable addition to the diagnostic kit and may be improved by the deglycolisation method proposed by Audoly et al (41).…”

“…The permeability of the cellular wall of bacteria for the oligonucleotide probes (with a molecular weight of about 6,500 Da) in formalin-fixed specimen is not fully resolved; however, FISH analysis with oligonucleotide probes was successfully used in several previous studies including smears and tissue preparations from human and animals but also whole cell hybridization of bacteria (34, 35, 38, 45, 46). The T. whipplei species-specific probe was analyzed, was also successful used in three studies before (5, 15, 41).…”

“…Bacterial species have previously been detected in situ by hybridizing fluorescent oligonucleotide probes (of 15–30 base pairs in length) targeted against specific sequences in either the 16S or 23S ribosomal RNA (rRNA) of bacteria (34–38). Fluorescence in situ hybridization (FISH) can be applied on cytological smears of fluids and formalin-fixed paraffin-embedded (FFPE) specimens alike.…”

Detection of Hepatitis B Virus M204I Mutation by Quantum Dot-Labeled DNA Probe

“…Real-time PCR technology has proven its usefulness in different research areas, especially in the quantification and genotyping of pathogens [14,15,16]. Fluorescence in situ hybridization (FISH) [17] is one of these rapid methods for the easy identification of microbial pathogens. As a microscopic technique, FISH has the unique potential to provide information about spatial resolution, morphology and identify key pathogens in mixed species samples.…”