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

Abstract

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.

Bandemia as an Early Predictive Marker of Bacteremia: A Retrospective Cohort Study

“…The advantage of bandemia is that the presence or absence of findings can be determined inexpensively, using labor-saving and labor-free machine counts. In recent years, new techniques such as an antibody microarray coupled to a Surface Plasmon Resonance imager (SPRi), PCR methods and Fluorescent in situ hybridization (FISH), were developed [ 32 , 33 , 34 , 35 ]. These techniques are labor-intensive and costly at the moment; future developments may advance diagnostic strategies for bacteremia.…”

Mass Spectrometry Proteotyping-Based Detection and Identification of Staphylococcus aureus, Escherichia coli, and Candida albicans in Blood

“…Drawbacks exist in performing the cultivation step, including the time required for the culturing itself, as well as the fact that many blood cultures are inconclusive, in the sense that the bacteria or fungi from patient samples may not grow in the culture or cannot be recovered, i.e. false negative results ( Murray and Masur, 2012 ; Skvarc et al., 2013 ; Sinha et al., 2018 ; Hazwani et al., 2020 ; Źródłowski et al., 2020 ). However, false negative blood cultures may result from the presence of antibiotics in the culture, originating from the patient blood, from infections caused by opportunistic microorganisms that grow poorly in standardized, automated, blood culture systems or that only few viable cells of the pathogen have been recovered from patient blood samples ( Sinha et al., 2018 ).…”

“…Recently, methods for detection of genetic material ( Mancini et al., 2010 ; Skvarc et al., 2013 ; Gosiewski et al., 2014 ; Liesenfeld et al., 2014 ; van de Groep et al., 2018 ), as well as DNA-sequencing-based methods ( Grumaz et al., 2016 ; Gosiewski et al., 2017 ; Watanabe et al., 2018 ; Grumaz et al., 2020 ), have been used for the detection of pathogens in blood. Serological methods, including detection of lipopolysaccharides for Gram-negative bacteria or galactomannan for fungi ( Opal, 2010 ; Dickson and Lehmann, 2019 ), but also methods based on Gram-staining and fluorescence in-situ hybridization (FISH) have been successfully applied for direct detection of pathogens in blood ( Gosiewski et al., 2014 ; Źródłowski et al., 2020 ). An advantage of these methods is that they do not rely on isolates from blood cultures and can be used on samples where antibiotic treatment has been initiated ( Źródłowski et al., 2018 ; Źródłowski et al., 2020 ).…”

“…Serological methods, including detection of lipopolysaccharides for Gram-negative bacteria or galactomannan for fungi ( Opal, 2010 ; Dickson and Lehmann, 2019 ), but also methods based on Gram-staining and fluorescence in-situ hybridization (FISH) have been successfully applied for direct detection of pathogens in blood ( Gosiewski et al., 2014 ; Źródłowski et al., 2020 ). An advantage of these methods is that they do not rely on isolates from blood cultures and can be used on samples where antibiotic treatment has been initiated ( Źródłowski et al., 2018 ; Źródłowski et al., 2020 ). These procedures are efficient, although some of them are relatively expensive and not suitable for the routine in clinical laboratories with large numbers of samples ( Mancini et al., 2010 ; Skvarc et al., 2013 ; Opota et al., 2015 ; Źródłowski et al., 2018 ; Briggs et al., 2021 ).…”

Healthcare-Associated Laboratory-Confirmed Bloodstream Infections—Species Diversity and Resistance Mechanisms, a Four-Year Retrospective Laboratory-Based Study in the South of Poland

“…Yet, many blood cultures are negative and the empirical treatment of culture-negative BSIs is of great interest [ 3 ]. Molecular tests based on polymerase chain reaction (PCR) and fluorescent in situ hybridization (FISH) can help increase the sensitivity of detecting bacteria in the blood and also speed up this stage, but they are still not routine diagnostics in Poland [ 4 ].…”