Fluorescent in situ hybridization and Gram‑stained smears of whole blood as complementary screening tools in the diagnosis of sepsis

Zrodlowski, Tomasz; Flis, Agnieszka; Ziętkiewicz, Mirosław; Drwiła, Rafał

doi:10.20452/pamw.3949

Cited by 4 publications

(7 citation statements)

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“…Non-specific binding of Gram dyes or probes to non-bacterial targets might occur, but it is easily distinguished from the shape of bacterial cells. Owing to the blood sample purification method that was applied by our team, it was possible to obtain a high-quality microscopic image, which allowed us to directly visualize the Gram stained bacterial cells, but also was an opportunity to detect genetic material inside bacterial cells, which confirms our previous observations [20][21][22].…”

Section: Discussionsupporting

confidence: 83%

“…There are few studies that confirm the possibility of bacterial detection in negative blood culture fluids, with the use of an additional culture on enriched media or using Gram stain, however, due to the lack of a procedure for concentrating or purifying the samples or the use of FISH, the authors obtained lower percentages of bacterial detection compared to our results [37,38]. In the literature, there are no reports on the detection of bacteria directly in the blood using FISH, apart from the studies by our team, so we cannot compare the obtained results with other studies [13,20]. With the use of FISH, a high percentage of genetic material of Gram-negative bacteria in the blood samples of healthy individuals was detected, which would argue for the translocation of bacteria into the bloodstream and, generally, for the existence of bacterial DNAemia phenomenon, in completely healthy people [39][40][41].…”

Section: Discussioncontrasting

confidence: 66%

“…Therefore, screening methods could be very beneficial as they would facilitate the detection of bacteria (bacteremia) and fungi (candidemia) in the blood or, allow a higher efficacy of microorganism detection especially in standard blood culture that failed to show growth [16,17]. Among the screening methods, Gram staining and FISH (fluorescence in-situ hybridization) are useful as they allow the visualization of microbial cells in the direct smear [13,[18][19][20].…”

Section: Introductionmentioning

confidence: 99%

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Classical Microbiological Diagnostics of Bacteremia: Are the Negative Results Really Negative? What is the Laboratory Result Telling Us About the “Gold Standard”?

et al. 2020

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Standard blood cultures require at least 24–120 h to be reported as preliminary positive. The objective of this study was to compare the reliability of Gram staining and fluorescent in-situ hybridization (FISH) for detecting bacteria in otherwise negative blood culture bottles. Ninety-six sets were taken from patients with a diagnosis of sepsis. Six incomplete blood culture sets and eight blood cultures sets demonstrating positive growth were excluded. We performed Gram stain and FISH on 82 sets taken from post-operative septic patients: 82 negative aerobic blood cultures, 82 anaerobic blood cultures, and 82 blood samples, as well as 57 blood samples taken from healthy volunteers. From the eighty-two blood sets analyzed from the septic patients, Gram stain visualized bacteria in 62.2% of blood samples, 35.4% of the negative aerobic bottles, and in 31.7% of the negative anaerobic bottles. Utilizing FISH, we detected bacteria in 75.6%, 56.1%, and 64.6% respectively. Among the blood samples from healthy volunteers, FISH detected bacteria in 64.9%, while Gram stain detected bacteria in only 38.6%. The time needed to obtain the study results using Gram stain was 1 h, for FISH 4 h, and for the culture method, considering the duration of growth, 5 days. Gram stain and FISH allow quick detection of bacteria in the blood taken directly from a patient. Finding phagocytosed bacteria, which were also detected among healthy individuals, confirms the hypothesis that blood microbiome exists.

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Section: Discussionsupporting

confidence: 83%

Section: Discussioncontrasting

confidence: 66%

Section: Introductionmentioning

confidence: 99%

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Classical Microbiological Diagnostics of Bacteremia: Are the Negative Results Really Negative? What is the Laboratory Result Telling Us About the “Gold Standard”?

et al. 2020

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“…Unfortunately, we did not obtained consent of the bioethical commission, especially since the study also involved newborns. Our previous research showed that bacterial DNA could be detected by amplification method in the blood of healthy adult people but its taxonomic composition is completely different from the one seen in septic patients (Gosiewski et al 2017). Also, these results are comparable to our previous studies (Gosiewski et al 2005;Źródłowski et al 2017).…”

supporting

confidence: 89%

Comparison of PCR, Fluorescent in Situ Hybridization and Blood Cultures for Detection of Bacteremia in Children and Adolescents During Antibiotic Therapy

Zrodlowski

Jurkiewicz-Badacz

Sroka-Oleksiak

et al. 2018

Polish Journal of Microbiology

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A b s t r a c tThe gold standard in microbiological diagnostics of bacteremia is a blood culture in automated systems. This method may take several days and has low sensitivity. New screening methods that could quickly reveal the presence of bacteria would be extremely useful. The objective of this study was to estimate the effectiveness of these methods with respect to blood cultures in the context of antibiotic therapy. Blood samples from 92 children with sepsis were analyzed. Blood cultures were carried out in standard automated systems. Subsequently, FISH (Fluorescent In-Situ Hybridization) and nested multiplex-real-time-PCR (PCR) were performed. Blood cultures, FISH and PCR yielded positive results in 18%, 39.1%, and 71.7% of samples, respectively. Significant differences were found between the results obtained through culture before and after induction of antibiotherapy: 25.5% vs. 9.7%. There was no significant difference in FISH and PCR results in relation to antibiotics. The three methods employed demonstrated significant differences in detecting bacteria effectively. Time to obtain test results for FISH and PCR averaged 4-5 hours. FISH and PCR allow to detect bacteria in blood without prior culture. These methods had high sensitivity for the detection of bacteremia regardless of antibiotherapy. They provide more timely results as compared to automated blood culture, and may be useful as rapid screening tests in sepsis. K e y w o r d s: antibiotic therapy, FISH, PCR, sepsis Źródłowski T.W. et al.

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“…[11][12][13]. Within the last decade, insufficient identifications were switched to culture and eventually relied on other high accuracy methods for microbe identification in blood cultures such as matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF/MS) [14], molecular biology [15][16][17], multiplex PCR assays, specific hybridization assays, or microarrays [18][19][20][21]. Nevertheless, none of these methods, even though some of them are more costly, present a turn-around time that allows for rapid preliminary identification from the blood culture in the way Gram staining does.…”

Section: Introductionmentioning

confidence: 99%

Scanning Electron Microscope: A New Potential Tool to Replace Gram Staining for Microbe Identification in Blood Cultures

et al. 2021

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Blood culture is currently the most commonly used method for diagnosing sepsis and bloodstream infections. However, the long turn-around-time to achieve microbe identification remains a major concern for clinical microbiology laboratories. Gram staining for preliminary identification remains the gold standard. We developed a new rapid strategy using a tabletop scanning electron microscope (SEM) and compared its performance with Gram staining for the detection of micro-organisms and preliminary identification directly from blood cultures. We first optimised the sample preparation for twelve samples simultaneously, saving time on imaging. In this work, SEM proved its ability to identify bacteria and yeasts in morphotypes up to the genus level in some cases. We blindly tested 1075 blood cultures and compared our results to the Gram staining preliminary identification, with MALDI-TOF/MS as a reference. This method presents major advantages such as a fast microbe identification, within an hour of the blood culture being detected positive, low preparation costs, and data traceability. This SEM identification strategy can be developed into an automated assay from the sample preparation, micrograph acquisition, and identification process. This strategy could revolutionise urgent microbiological diagnosis of infectious diseases.

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Fluorescent in situ hybridization and Gram‑stained smears of whole blood as complementary screening tools in the diagnosis of sepsis

Cited by 4 publications

References 0 publications

Classical Microbiological Diagnostics of Bacteremia: Are the Negative Results Really Negative? What is the Laboratory Result Telling Us About the “Gold Standard”?

Classical Microbiological Diagnostics of Bacteremia: Are the Negative Results Really Negative? What is the Laboratory Result Telling Us About the “Gold Standard”?

Comparison of PCR, Fluorescent in Situ Hybridization and Blood Cultures for Detection of Bacteremia in Children and Adolescents During Antibiotic Therapy

Scanning Electron Microscope: A New Potential Tool to Replace Gram Staining for Microbe Identification in Blood Cultures

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