In vitro assessment of a rapid colorimetric method to detect ex-tracellular DNA in cerebrospinal fluid: a platform for rapid diagnosis of bacterial meningitis
In vitro assessment of a rapid colorimetric method to detect ex-tracellular DNA in cerebrospinal fluid: a platform for rapid diagnosis of bacterial meningitis
Roya Darbani,1,*Amir razmezani,2Lars Eng,3Gunnel Svenstäter,4Annette Theodorsson,5Fariba Nayeri,6
1. The Institute for Protein Environmental Affinity Surveys (PEAS Institute) 2. Division of Neuro inflammation Science, Departmant of clinical and experimental medicine, linköping university 3. The institute for protein Enviromental Affinity Surveys( Peas Institut) 4. Departemant of Oral Biology, Faculity of Odontology, Malmö University 5. Division of Neuro inflammation Science, Departmant of clinical and experimental medicine, linköping university 6. MD, PhD, PEAS Institut, BeryllFoundation for Clinical and Experimental Science
Introduction: Rapid and organ-specific diagnosis of acute infections saves lives and resources. As part of the innate immune system, neutrophils trap and kill bacteria by way of neutrophil extracellular traps, which are composed of extracellular DNA released into bodily fluids during infection. A combination of sulfated glycan and aniline dye (test solution /strip test) was recently used to develop the rapid colorimetric extracellular DNA detector, which showed a positive result spe-cifically in the infected organ. In the present work we used stimulation of healthy neutrophils by bacteria and assessed experimentally the colorimetric reaction compared to CSF from patients
Methods: Fresh cerebrospinal fluid (CSF) samples were obtained from patients admitted to the neurosur-gical intensive care unit (n=55) and analyzed by rapid test. Neutrophils from healthy subjects (28 and 32 years of age, one female) were stimulated with bacteria and incubated in the test solution. The results were studied by spectrophotometer.
Results: A shift in absorption from the γ-peak (521 nm, pink, negative) to the α-peak (630 nm, blue, posi-tive), which was inhibited by pre-incubation with DNase I or antibiotics effective against the bacteria was observed through several controlled attempts on healthy neutrophils. Light mi-croscopy revealed that neutrophils in test-positive CSF samples (n=26) differed morphologically from test-negative samples (n=29), resembling the bacterial-stimulated healthy neutrophils. The absorption shift occurred in test-positive samples after addition of DNase.
Conclusion: A diagnostic method based on detection of extracellular DNA in CSF can be employed at point-of-care to distinguish meningitis at an early stage even in the damaged brain tissue.