Evaluation of microbiological variants of sputum processing and concentration of mycobacteria to optimize the microscopic and imaging diagnosis of tuberculosis

Jorge E. Coronel, Christian C. Del Carpio, Erwin J. Dianderas, Edgar A. Florentini, Guillermo L. Kemper, Patricia Sheen, Mirko Zimic

Research output: Contribution to journalArticle

Abstract

Background: Direct sputum smear is still the first-choice tool for screening of tuberculosis worldwide. Variants of this technique, to improve the sensitivity are desired. Methods: Two microbiological variants of the standard sputum smear ('pellet' and 'diluted-pellet') for both Ziehl-Neelsen (ZN) and auramine fluorescence (AF) staining were evaluated. In addition, two methods for concentration of mycobacteria in sputum, using positive and negative pressure filtration, were tested and compared. The evaluation of the microbiological variants was performed on 98 culture positive sputum samples from different TB patients. The diagnostics sensitivity and the level of detritus in the processed sputum were determined. Bacilli load in the smear variants was determined by microscopic observation and by manual inspection of microscopic digital images. The comparison of the mycobacteria filtration methods was performed on 76 smear positive sputum samples. Filters retaining the concentrated mycobacteria were stained with AF and compared with the direct smear. Bacilli load, detritus level, filtered volume, filtration time and background noise level, were determined. Results: The sensitivity of microscopy with the microbiological variants was 7.1% and 2% higher in ZN and AF respectively, compared to direct smear. The sensitivity of AF in diluted pellet was significantly higher than all ZN variants (P < 0.05). Detritus level observed in slides was significantly lower in the diluted pellet than the pellet and direct smear in ZN and AF (P < 0.001). A significant increase in the bacilli load in microscopic observation and digital images analysis was observed in pellet and diluted pellet than the direct method (P <0.0001). The concentration of mycobacteria using positive-pressure filtration showed a trend to produce a higher bacilli load compared to the negative-pressure filtration and direct smear, although it was not significant. Detritus levels were significantly higher in both variants of filtration (P < 0.0001). Filtered volumes were higher in positive-pressure compared to negative-pressure filtration. Filtration times were significantly higher in negative-pressure compared to positive-pressure filtration (P < 0.0001). Conclusion: The proposed variants improved the performance of the standard sputum smear, making it an important test for settings with high rates of smear-negative TB cases.

Original languageEnglish (US)
Pages (from-to)75-82
Number of pages8
JournalInternational Journal of Mycobacteriology
Volume8
Issue number1
DOIs
StatePublished - Jan 1 2019
Externally publishedYes

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Mycobacterium
Sputum
Benzophenoneidum
Tuberculosis
Pressure
Fluorescence
Bacillus
Noise
Microscopy
Staining and Labeling

Keywords

  • Concentration
  • filtration
  • sensitivity
  • smear
  • sputum
  • tuberculosis

ASJC Scopus subject areas

  • Microbiology (medical)
  • Infectious Diseases

Cite this

Evaluation of microbiological variants of sputum processing and concentration of mycobacteria to optimize the microscopic and imaging diagnosis of tuberculosis. / Coronel, Jorge E.; Del Carpio, Christian C.; Dianderas, Erwin J.; Florentini, Edgar A.; Kemper, Guillermo L.; Sheen, Patricia; Zimic, Mirko.

In: International Journal of Mycobacteriology, Vol. 8, No. 1, 01.01.2019, p. 75-82.

Research output: Contribution to journalArticle

Coronel, Jorge E. ; Del Carpio, Christian C. ; Dianderas, Erwin J. ; Florentini, Edgar A. ; Kemper, Guillermo L. ; Sheen, Patricia ; Zimic, Mirko. / Evaluation of microbiological variants of sputum processing and concentration of mycobacteria to optimize the microscopic and imaging diagnosis of tuberculosis. In: International Journal of Mycobacteriology. 2019 ; Vol. 8, No. 1. pp. 75-82.
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abstract = "Background: Direct sputum smear is still the first-choice tool for screening of tuberculosis worldwide. Variants of this technique, to improve the sensitivity are desired. Methods: Two microbiological variants of the standard sputum smear ('pellet' and 'diluted-pellet') for both Ziehl-Neelsen (ZN) and auramine fluorescence (AF) staining were evaluated. In addition, two methods for concentration of mycobacteria in sputum, using positive and negative pressure filtration, were tested and compared. The evaluation of the microbiological variants was performed on 98 culture positive sputum samples from different TB patients. The diagnostics sensitivity and the level of detritus in the processed sputum were determined. Bacilli load in the smear variants was determined by microscopic observation and by manual inspection of microscopic digital images. The comparison of the mycobacteria filtration methods was performed on 76 smear positive sputum samples. Filters retaining the concentrated mycobacteria were stained with AF and compared with the direct smear. Bacilli load, detritus level, filtered volume, filtration time and background noise level, were determined. Results: The sensitivity of microscopy with the microbiological variants was 7.1{\%} and 2{\%} higher in ZN and AF respectively, compared to direct smear. The sensitivity of AF in diluted pellet was significantly higher than all ZN variants (P < 0.05). Detritus level observed in slides was significantly lower in the diluted pellet than the pellet and direct smear in ZN and AF (P < 0.001). A significant increase in the bacilli load in microscopic observation and digital images analysis was observed in pellet and diluted pellet than the direct method (P <0.0001). The concentration of mycobacteria using positive-pressure filtration showed a trend to produce a higher bacilli load compared to the negative-pressure filtration and direct smear, although it was not significant. Detritus levels were significantly higher in both variants of filtration (P < 0.0001). Filtered volumes were higher in positive-pressure compared to negative-pressure filtration. Filtration times were significantly higher in negative-pressure compared to positive-pressure filtration (P < 0.0001). Conclusion: The proposed variants improved the performance of the standard sputum smear, making it an important test for settings with high rates of smear-negative TB cases.",
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AU - Del Carpio, Christian C.

AU - Dianderas, Erwin J.

AU - Florentini, Edgar A.

AU - Kemper, Guillermo L.

AU - Sheen, Patricia

AU - Zimic, Mirko

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N2 - Background: Direct sputum smear is still the first-choice tool for screening of tuberculosis worldwide. Variants of this technique, to improve the sensitivity are desired. Methods: Two microbiological variants of the standard sputum smear ('pellet' and 'diluted-pellet') for both Ziehl-Neelsen (ZN) and auramine fluorescence (AF) staining were evaluated. In addition, two methods for concentration of mycobacteria in sputum, using positive and negative pressure filtration, were tested and compared. The evaluation of the microbiological variants was performed on 98 culture positive sputum samples from different TB patients. The diagnostics sensitivity and the level of detritus in the processed sputum were determined. Bacilli load in the smear variants was determined by microscopic observation and by manual inspection of microscopic digital images. The comparison of the mycobacteria filtration methods was performed on 76 smear positive sputum samples. Filters retaining the concentrated mycobacteria were stained with AF and compared with the direct smear. Bacilli load, detritus level, filtered volume, filtration time and background noise level, were determined. Results: The sensitivity of microscopy with the microbiological variants was 7.1% and 2% higher in ZN and AF respectively, compared to direct smear. The sensitivity of AF in diluted pellet was significantly higher than all ZN variants (P < 0.05). Detritus level observed in slides was significantly lower in the diluted pellet than the pellet and direct smear in ZN and AF (P < 0.001). A significant increase in the bacilli load in microscopic observation and digital images analysis was observed in pellet and diluted pellet than the direct method (P <0.0001). The concentration of mycobacteria using positive-pressure filtration showed a trend to produce a higher bacilli load compared to the negative-pressure filtration and direct smear, although it was not significant. Detritus levels were significantly higher in both variants of filtration (P < 0.0001). Filtered volumes were higher in positive-pressure compared to negative-pressure filtration. Filtration times were significantly higher in negative-pressure compared to positive-pressure filtration (P < 0.0001). Conclusion: The proposed variants improved the performance of the standard sputum smear, making it an important test for settings with high rates of smear-negative TB cases.

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