Evaluation of flow cytometry for the detection of bacteria in biological fluids

Abstract

Objectives Conventional microbiological procedures for the isolation of bacteria from biological fluids consist of culture on solid media and enrichment broth. However, these methods can delay the microbiological identification for up to 4 days. The aim of this study was to evaluate the analytical performance of Sysmex UF500i (Sysmex, Kobe, Japan) as a screening method for the detection of bacteria in different biological fluids in comparison with direct Gram staining and the conventional culture on solid media and enrichment broth. Methods A total of 479 biological fluid samples were included in the study (180 ascitic, 131 amniotic, 56 synovial, 40 cerebrospinal, 36 pleural, 24 peritoneal, 9 bile and 3 pericardial fluids). All samples were processed by conventional culture methods and analyzed by flow cytometry. Direct Gram staining was performed in 339 samples. The amount of growth on culture was recorded for positive samples. Results Bacterial and white blood cell count by flow cytometry was significantly higher among culture positive samples and samples with a positive direct Gram stain compared to culture negative samples. Bacterial count directly correlated with the amount of growth on culture (Kruskall-Wallis H χ 2 (3) = 11.577, p = 0.009). The best specificity (95%) for bacterial count to predict culture positivity was achieved applying a cut-off value of 240 bacteria/μL. Conclusions Bacterial and white blood cell counts obtained with flow cytometry correlate with culture results in biological fluids. Bacterial count can be used as a complementary method along with the direct Gram stain to promptly detect positive samples and perform other diagnostic techniques in order to accelerate the bacterial detection and identification.

Evaluation of flow cytometry for cell count and detection of bacteria in biological fluids

“…Also, samples with culture-positive results had significantly higher bacterial and WBC counts than samples with culture-negative results. Our results are in agreement with previous studies which have demonstrated that bacterial count by FCM showed a good correlation with culture results 6,8,10,11 . We established optimal cut-off points for bacterial count by FCM in order to obtain DGS and culture-positive results.…”

“…Regarding the differential leukocyte counts, UF4000 (Sysmex, Kobe, Japan) showed excellent performance for body fluids which did not contain abnormal cells. Our results are in agreement with those of other studies, having demonstrated that the FCM allows to correctly classify and enumerate WBCs in the CSF, PRF and PDF [5][6][7][8][9][10] . Due to its characteristics, the XN10 (Sysmex, Kobe, Japan) is suitable for the analysis of samples containing a large quantity of RBC and WBC (> 10 3 / µL).…”

Evaluation of Flow Cytometry for Cell Count and Detection of Bacteria in Biological Fluids

“…Regarding the differential leukocyte counts, UF4000 (Sysmex, Kobe, Japan) showed excellent performance for body fluids which did not contain abnormal cells. Our results agree with those of other studies, having demonstrated that the FCM allows to correctly classify and enumerate WBCs in the CSF, PRF and PDF ( 5 – 10 ). Because of its characteristics, the XN10 (Sysmex, Kobe, Japan) is suitable for the analysis of samples containing a large quantity of RBC and WBC (>10 3 /μL).…”

Pseudomonas aeruginosa Biofilm Lung Infection in Cystic Fibrosis: The Challenge of Persisters

“…Another useful technique capable of providing direct bacterial quantification is flow cytometry. Although it has mostly been employed to investigate bacterial physiology and metabolic responses [82], efforts to optimize its quantification ability have made it suitable for some diagnostic applications [83]. In particular, flow cytometry analysis and imaging now enable detection and enumeration of non-culturable and intracellular P. aeruginosa cells [84,85].…”