Rapid Identification of Bacterial Species by Fluorescence Spectroscopy and Classification Through Principal Components Analysis

Giana, Héctor Enrique; Silveira, Landulfo; Zângaro, Renato Amaro; Pacheco, Marcos Tadeu Tavares

doi:10.1023/b:jofl.0000008059.74052.3c

Cited by 101 publications

(63 citation statements)

References 11 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…Very few studies on the use of autofluorescence for the detection of bacteria have been reported (2,14). Some bacteria exhibit spontaneous emission spectra following excitation at specific wavelengths.…”

Section: Discussionmentioning

confidence: 99%

Autofluorescence of Mycobacteria as a Tool for Detection of Mycobacterium tuberculosis

Patino¹,

Alamo²,

Cimino³

et al. 2008

Journal of Clinical Microbiology

View full text Add to dashboard Cite

The diagnosis of tuberculosis in developing countries still relies on direct sputum examination by light microscopy, a method that is easy to perform and that is widely applied. However, because of its poor sensitivity and requirement for significant labor and training, light microscopy examination detects the bacilli in only 45 to 60% of all people whose specimens are culture positive for Mycobacterium tuberculosis. Therefore, new diagnostic methods that would enable the detection of the undiagnosed infected population and allow the early commencement of antituberculosis treatment are needed. In this work, the potential use of mycobacterial cyan autofluorescence for the detection of Mycobacterium tuberculosis was explored. The tubercle bacilli were easily visualized as brilliant fluorescent bacilli by microscopy and were easily tracked ex vivo during macrophage infection. Assays with seeded sputum and a 96-well microplate reader fluorimeter indicated that <10 6 bacilli ml ؊1 of sputum could be detected. Moreover, the use of microplates allowed the examination of only 200 l of sputum per sample without a loss of sensitivity. Treatment with heat or decontaminating chemical agents did not interfere with the autofluorescence assay; on the contrary, they improved the level of bacterial detection. Autofluorescence for the detection of bacilli is rapid and easy to perform compared to other methodologies and can be performed with minimal training, making this method suitable for implementation in developing countries.The diagnosis of tuberculosis (TB) in low-and middle-income countries, where more than 90% of TB cases occur, is mainly performed by microscopy examination of stained sputum smears for acid-fast bacilli (25). The International Standards for Tuberculosis Care considers the microscopic analysis of two or three specimens per patient to be a cornerstone for the diagnosis of TB (13). Although light microscopy is inexpensive, easy to perform, and highly specific in areas where there is a high prevalence of TB, it is relatively insensitive, requiring Ն10 4 bacilli ml Ϫ1 of sputum to achieve a positive result (15,35). In addition, the procedure requires the observation of from 100 to 300 fields in order to obtain accurate results (31).The sensitivity of microscopy is influenced by numerous factors, such as the prevalence and severity of disease, the quality of specimen collection, the type of specimen, the method of processing (direct or concentrated, centrifugation, liquefaction), staining, and finally, the quality of the examination (20,23,26,29). In well-trained hands, the test detects tubercle bacilli in 75% of all people who have active pulmonary TB, but the sensitivity may fall to 45 to 60%, depending on the training, eye, and motivation of the laboratory technician (1). The basis of conventional light microscopy was developed more than 100 years ago, and, at present, conventional light microscopy uses carbolfuchsin Ziehl-Neelsen or Kinyoun acid-fast stains. Fluorescence microscopy with fluorochrome dyes suc...

show abstract

Section: Discussionmentioning

confidence: 99%

Autofluorescence of Mycobacteria as a Tool for Detection of Mycobacterium tuberculosis

Patino¹,

Alamo²,

Cimino³

et al. 2008

Journal of Clinical Microbiology

View full text Add to dashboard Cite

show abstract

“…In order to realize rapid detection of bacteria, some spectroscopy techniques, such as fluorescence [14][15][16], infrared [17][18][19], and Raman [20][21][22][23], have also been used for the determination of characteristic parameters of bacteria. The disadvantages associated with those spectroscopy techniques include the high performance requirements for instrumentation and the inability to provide detailed information of the structural and compositional characteristics of the bacteria cells.…”

Section: Introductionmentioning

confidence: 99%

Analytic Method on Characteristic Parameters of Bacteria in Water by Multiwavelength Transmission Spectroscopy

Yu-xia

Zhao

Gan

et al. 2017

Journal of Spectroscopy

View full text Add to dashboard Cite

An analytic method together with the Mie scattering theory and Beer-Lambert law is proposed for the characteristic parameter determination of bacterial cells (Escherichia coli 10389) from multiwavelength transmission spectroscopy measurements. We calculate the structural parameters of E. coli cells, and compared with the microscopy, the relative error of cell volume is 7.90%, the cell number is compared with those obtained by plate counting, the relative error is l.02%, and the nucleic content and protein content of single E. coli cells are consistent with the data reported elsewhere. The proposed method can obtain characteristic parameters of bacteria as an excellent candidate for the rapid detection and identification of bacteria in the water.

show abstract

“…A variety of label-free methods have been implemented to identify bacteria including laser-induced breakdown spectroscopy [16][17][18], Fourier transform infrared spectroscopy [19,20], Raman spectroscopy [1,21], and autofluorescence [22,23].…”

Section: Introductionmentioning

confidence: 99%

The use of microfluidics and dielectrophoresis for separation, concentration, and identification of bacteria

et al. 2016

View full text Add to dashboard Cite

Traditional bacterial analyses take one to two days under favorable conditions where the bulk of the time is spent waiting for bacteria to divide and grow until visual colonies can be observed for identification. In the case of bacteria with slow doubling times, this process can take weeks. This delay in analysis is unacceptable, especially in cases of life threatening diseases or emergencies. It is clear that in order to decrease the analysis time of the bacteria, the culturing and growth step must be circumvented. The goal of this research is to design, build, and test a device that could decrease the analysis time of bacteria using label-free methods of dielectrophoresis and Raman spectroscopy.Testing for device design was performed with clinical samples in mind, which consist of bacteria grown in a variety of environmental conditions (i.e. available food sources, growth stage, temperature, etc.) and accompanied by sample debris. Raman spectra of bacteria grown in varying media and metabolic stages were collected and analyzed. Results indicate that growth phase and media have an impact on Raman spectra iv and is distinguishable by linear discriminant analysis (LDA). Despite these spectral differences, it was found that LDA classification of closely related bacteria remains fairly high (90%) regardless of growth phase. Sample debris were also considered in device design and accommodated for by dielectrophoresis. Devices were built with the goal to isolate bacteria from a mixed sample and simultaneously acquire Raman spectra for identification.For this dissertation, a device was designed, built, and tested that incorporates dielectrophoresis for particle isolation and Raman spectroscopy for identification. The device was modeled in COMSOL to ensure that an appropriate electrical field gradient could be obtained to isolate bacteria from 5 µm diameter polystyrene spheres. The device was built and successfully trapped bacteria away from polystyrene spheres and Raman spectra of the bacteria were collected while trapped. These results indicate a clear potential for contactless dielectrophoresis-Raman devices to isolate and identify bacteria from sample debris, and thereby decrease the analysis time of bacteria. Typical bacterial analysis involves culturing and visualizing colonies on an array of agar plates. The growth patterns and colors among the array are used to identify the bacteria. For fast growing bacteria such as Escherichia coli, analysis will take one to two days. However, slow growing bacteria such as mycobacteria can take weeks to identify.In addition, there are some species of bacteria that are viable but nonculturable. This lengthy analysis time is unacceptable for life-threatening infections and emergency situations. It is clear that to decrease the analysis of the bacteria, the culturing and growth steps must be avoided. The goal of this research is to design, build, and test a device that could decrease the analysis time of bacteria.

show abstract

Rapid Identification of Bacterial Species by Fluorescence Spectroscopy and Classification Through Principal Components Analysis

Cited by 101 publications

References 11 publications

Autofluorescence of Mycobacteria as a Tool for Detection of Mycobacterium tuberculosis

Autofluorescence of Mycobacteria as a Tool for Detection of Mycobacterium tuberculosis

Analytic Method on Characteristic Parameters of Bacteria in Water by Multiwavelength Transmission Spectroscopy

The use of microfluidics and dielectrophoresis for separation, concentration, and identification of bacteria

Contact Info

Product

Resources

About