Slides were then incubated for 30 minutes at 37°C. In low-income and middle-income countries, direct (unconcentrated) sputum smear microscopy is the primary method for diagnosing pulmonary tuberculosis. To increase operational relevance, we measured liquids including sputum as drops from disposable transfer pipettes, which we found to have an average volume of 40 μl. Supervision, 6. National Center for Biotechnology Information, Unable to load your collection due to an error, Unable to load your delegates due to an error. 2020 Jun 26;36:134. doi: 10.11604/pamj.2020.36.134.16652. All of these procedures were performed in duplicate: 1 slide from each pair of slides was stained using FDA working solution that was prepared daily as described above; the other duplicate from each pair of slides was stained using FDA working solution that had been prepared on the first day of the experiment and stored at room temperature, protected from direct light. In patient samples, 13 slides had false-negative results, of which 11 were stained with protocol A-C. Bacilli/ml results in protocols A-C were lower than other protocols (Fig 2), and were also significantly less than the CFU/ml in the corresponding direct sputum culture (all P≤0.05, Fig 3B). We present a method for the automated identification of Mycobacterium tuberculosis in images of Ziehl‐Neelsen‐stained sputum smears obtained using a bright‐field microscope. Fluorescein diacetate (FDA) is a vital stain that generally causes viable cells to fluoresce because non-specific esterase in the cytoplasm of metabolically-active cells must be present to hydrolyse the stain to a fluorescent form [4]. Classification of Note. Zamora J, Muriel A, Abraira V. Meta-DiSc for windows: a software package for the meta-analysis of diagnostic tests. USA.gov. Table demonstrating the factors that improved the quality of slides if fluorescein diacetate (FDA) microscopy was positive (n = 122). Specifically, 46% (13/28, 95% CI = 28–66%) of bacilli/ml results in FDA protocols D-G were within +/- 1 logarithm of the corresponding CFU/ml in unprocessed sputa, and 89% (25/28, 95% CI = 72–98%) within +/- 2 logarithms. The pellet was re-suspended in phosphate buffered saline at pH 6.8 to a final volume of 2 ml. https://doi.org/10.1371/journal.pone.0214131.g006. Writing – original draft, When there were duplicate results, geometric means were calculated. Please check your email for instructions on resetting your password. No, Is the Subject Area "Distillation" applicable to this article? Data Availability: Data are uploaded into a publicly available repository (http://www.ifhad.org/data-repository/). However, diverse methods have been published. As shown in Table 1, this protocol has been evaluated previously [12]. Writing – review & editing. The method is fast, inexpensive, and specific for Mycobacterium tuberculosis in high incidence areas. Biomedical Signal and Image Processing in Patient Care. The full text of this article hosted at iucr.org is unavailable due to technical difficulties. Click through the PLOS taxonomy to find articles in your field. For the protocols using thick smears from direct sputum, identifying bacilli was difficult because they were often obscured by cells and other sputum contents. Computer Methods and Programs in Biomedicine. Resources, https://doi.org/10.1371/journal.pone.0214131.g003. Automated object and image level classification of TB images using support vector neural network classifier. Working off-campus? All sputa and slides were stored protected from light for up to 4 weeks. The results of this review provide a point We did not specifically test the specificity of FDA microscopy on non-mycobacteria microorganisms, but we assume that the acid-fast wash in our protocol would generally prevent them from staining, as is the case for other acid-fast stains. A stock solution of 5 mg/ml FDA (Sigma-Aldrich, MO, USA) in acetone was stored at -20°C. By searching many sources, we identified 83 studies. Labour costs and equipment such as microscope, glassware, Bunsen burners, drying racks and distilled water were not included in the cost analysis because these costs would have been purchased by a laboratory that was already performing TB fluorescent microscopy with auramine staining, as recommended by the World Health Organization [16]. 6. IFHAD: Innovation For Health And Development, Laboratory of research and development, Universidad Peruana Cayetano Heredia, Lima, Peru, Geometric transformation invariant features are extracted for implementation of the second stage, namely one‐class object classification. Lab Med. https://doi.org/10.1371/journal.pone.0214131.g004. https://doi.org/10.1371/journal.pone.0214131.t002. Data curation, In high-income countries, fluorescence microscopy rather than conventional Centrifuge-decontamination may also selectively kill a specific phenotype of M. tuberculosis, distorting findings important to monitoring treatment response in both quantitative culture and FDA microscopy [23]. NIH Investigation, However, there were only 6 blood-stained sputum samples in this study (Table 3). XI Cochrane Colloquium. Slides were then rinsed with distilled water. 5. Note, NS = not specified, mins = minutes, CPC = cetylpyridinium chloride, and NAOH-NALC = sodium hydroxide and N-acetyl cysteine. Furthermore, PCR usually does not identify resistance to other drugs, and cannot differentiate between live and dead M. tuberculosis, so cannot reliably assess early treatment response [42]. Store at -20°C. 11. There is insufficient evidence to determine the value of fluorescence In culture the number of colony-forming units (CFU) in each serial dilution was recorded. Phenol at 5% concentration was applied to slides and left for 10 minutes. 1. Project administration, Affiliations Get the latest research from NIH: https://www.nih.gov/coronavirus. The quality of slides for patient samples were high, with median score 300 (IQR = 245–320) out of 400 and this did not change with storage time. This hypothesis is supported by our finding that all slides prepared from sputum from healthy control participants were consistently negative with the chosen FDA microscopy protocol. Sputum smear microscopy has been the primary method for diagnosis of pulmonary tuberculosis in low and middle income countries 3, which is where nearly 95 … Sputum microscopy is not only reliable; it is also very simple and cost effective. NIH Direct microscopy versus sputum cytology analysis and bleach sedimentation for diagnosis of tuberculosis: a prospective diagnostic study. 5. Examination of smears for tubercle bacilli by fluorescence microscopy. The first comprises a one‐class pixel classifier for object segmentation. Operational studies are needed to determine whether the increased sensitivity provided by processing methods is sufficient to offset their increased cost, complexity, and potential biohazards, and to examine their feasibility. ScienceDirect ® is a registered trademark of Elsevier B.V. ScienceDirect ® is a registered trademark of Elsevier B.V. Sputum processing methods to improve the sensitivity of smear microscopy for tuberculosis: a systematic review. Minimum variance-embedded deep kernel regularized least squares method for one-class classification and its applications to biomedical data. Cybernetic microbial detection system using transfer learning. 2014 Fall;45(4):e140-1. There were 9 acid-fast microscopy positive patients and 2 healthy controls who provided samples that were used to make 80 slides and 264 culture wells in this study. Dusthackeer A, Balasubramanian M, Shanmugam G, Priya S, Nirmal CR, Sam Ebenezer R, Balasubramanian A, Mondal RK, Thiruvenkadam K, Hemanth Kumar AK, Ramachandran G, Subbian S. Front Microbiol. However, this potential limitation was reduced by taking photos of the microscopy views, which were reviewed by other team members. 5. World Health Organisation. and you may need to create a new Wiley Online Library account. Thin sputum smears with the use of 30 seconds of potassium permanganate helped quench fluorescence from background material sufficiently to allow the mycobacteria to be optimally visualized. No, Is the Subject Area "Mycobacterium tuberculosis" applicable to this article? Public health mycobacteriology: a guide for the level III laboratory. Data curation, Automated Method of Analysing Sputum Smear Tuberculosis Images Using Multifractal Approach. Fluorescence microscopy is credited However, there is no published evidence to guide which of these protocols should be used. As microscopists gain more experience this slide reading time may reduce. A fluorescent staining procedure for determining the viability of Mycobacterial cells. By continuing you agree to the use of cookies. here. Cultures were incubated in un-supplemented air at 37°C. -, Medzon EL, Brady ML. Conceptualization, Thus, there is a need to identify methods to improve the sensitivity of microscopy. 1.2 μl/mm2, as previously used for FDA microscopy, see Table 1 [13,14,17]. Characterization of antibody response against 16kD and 38kD of. J Bacteriol. Yes The corresponding author had full access to all study data and was responsible for the decision to publish. Bar graphs comparing the quantitative sensitivity of fluorescein diacetate (FDA) microscopy protocols A-G: (A) to identify acid-fast bacilli (AFB), shown as the difference in logarithmic base 10 counts between FDA bacilli/ml of sample versus AFB/ml in acid-fast (auramine) (B) to identify colony-forming units (CFU), shown as the difference in logarithmic base 10 counts between FDA bacilli/ml of sample versus CFU/ml in quantitative culture.

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