Pathogen Detection Using Frequency Domain Fluorescent Lifetime Measurements.

OBJECTIVE

Inflammation of the meninges is a source of severe morbidity and therefore is an important health concerns worldwide. The conventional clinical microbiology approaches used today to identify pathogens suffer from several drawbacks and frequently provide false results. This research describes a fast method to detect the presence of pathogens using the frequency domain (FD) fluorescence lifetime (FLT) imaging microscopy (FLIM) system.

METHODS

The study included 43 individuals divided into 4 groups: 9 diagnosed with different types of bacteria; 16 diagnosed with different types of viruses; 5 healthy samples served as a control; and 12 samples were negative to any pathogen, although presenting related symptoms. All samples contained leukocytes that were extracted from the cerebrospinal fluid (CSF) and were subjected to nuclear staining by 4', 6-diamidino-2-phenylindole (DAPI) and FLT analyses based on phase and amplitude crossing point (CRPO).

RESULTS

Using notched boxplots, we found differences in 95% probability between the first three groups through different notch ranges (NR). Pathogen samples presented a longer median FLT (3.28 ns with NR of 3.24-3.32 ns in bacteria and 3.18 ns with NR of 3.16-3.21 ns in viruses) compared to the control median FLT (2.65 ns with NR of 2.63-2.67 ns). Furthermore, we found that the undetected forth group was divided into two types: a relatively normal median FLT (2.72 ns with NR of 2.68-2.76 ns) and a prolonged FLT (3.22 ns with NR of 3.17-3.27 ns).

CONCLUSION

FLT measurements can differentiate between control and pathogen by the CRPO method.

SIGNIFICANCE

The FD-FLIM system can provide a high throughput diagnostic technique that does not require a physician.