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.
The aim of the study was to evaluate particular polymerase chain reaction primers targeting selected representative genes and the influence of a preincubation step in a selective broth on the sensitivity of group B Streptococcus (GBS) detection by nucleic acid amplification techniques (NAAT). Research samples were vaginal and rectal swabs collected in duplicate from 97 pregnant women. They were used for enrichment broth culture-based diagnostics, bacterial DNA isolation, and amplification, using primers based on species-specific 16S rRNA, atr and cfb genes. To assess the sensitivity of GBS detection, additional isolation of samples preincubated in Todd-Hewitt broth with colistin and nalidixic acid was performed and then subjected to amplification again. The introduction of the preincubation step increased the sensitivity of GBS detection by about 33–63%. Moreover, NAAT made it possible to identify GBS DNA in an additional six samples that were negative in culture. The highest number of true positive results compared to the culture was obtained with the atr gene primers, as compared to cfb and 16S rRNA primers. Isolation of bacterial DNA after preincubation in enrichment broth significantly increases the sensitivity of NAAT-based methods applied for the detection of GBS from vaginal and rectal swabs. In the case of the cfb gene, the use of an additional gene to ensure the appropriate results should be considered.
The goal of the study was to assess if screening methods of Gram staining and Fluorescent Hybridization In Situ (FISH) can increase the rate of detection of bacteria in blood of patients with negative blood culture results.METHODS: Blood cultures and 2 ml-blood samples of patients fulfilling the criteria of sepsis were analyzed. Blood cultures underwent standard testing in automated systems. Blood samples of patients with negative blood culture results were subjected to erythrocytes lysis (creating a strong background) according to the methodology described by Gosiewski et al. [patent: US9879311 (B2)]. The remaining sediment was used to prepare microscope slides for Gram staining and FISH. The preparations were analyzed using the light microscope and the BX51 (Olympus) fluorescence microscope respectively. RESULTS:In total, 143 samples were analyzed. Blood cultures demonstrated growth in 15.38% (n¼22).Remaining 121 blood samples obtained from patients with negative blood cultures were tested with the use of Gram staining, and FISH. Gram staining visualized bacteria in 51.24% (n¼62), and FISH detected bacteria in 61.98% (n¼75). The time required to obtain test results was 1 hour for Gram staining and 4 hours for FISH. CONCLUSIONS:The implemented methodology allows the detection of bacteria directly in blood samples collected from septic patients with negative blood culture results with the use of Gram staining and FISH.The study confirms the effectiveness of both methods in microbiological testing of whole blood, but FISH demonstrates greater sensitivity when compared to Gram staining. Gram staining and FISH are inexpensive, easily available methods that provide results in a timely manner. They could be used as quick screening methods in diagnostics of bacteremia. CLINICAL IMPLICATIONS:The microbiological diagnostics of bacteremia consists of blood culture. Although culture-positive bacteremia is found in about 30% of patients in septic shock, its presence is associated with higher mortality rates. The advantage of blood culture is its simplicity and cost effectiveness. The disadvantage is that this method is time-consuming and has low sensitivity. Time needed to initiate a targeted therapy has a direct impact on patient's survival. Molecular methods based on the genetic analysis are costly and are currently only available in a select number of research centers. Therefore, new screening methods that could quickly reveal the presence of bacteria would be extremely useful.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.