DSpace Repository

Optimising fluorescein diacetate sputum smear microscopy for assessing patients with pulmonary tuberculosis

Show simple item record

dc.contributor.author Datta, S.
dc.contributor.author Alvarado, K.
dc.contributor.author Gilman, Robert Hugh
dc.contributor.author Valencia, T.
dc.contributor.author Aparicio, C.
dc.contributor.author Ramos, E.S.
dc.contributor.author Montoya, R.
dc.contributor.author Evans, Carlton Anthony William
dc.date.accessioned 2019-12-06T21:04:41Z
dc.date.available 2019-12-06T21:04:41Z
dc.date.issued 2019
dc.identifier.uri https://hdl.handle.net/20.500.12866/7567
dc.description.abstract Background: Assessing Mycobacterium tuberculosis (TB) viability by fluorescein diacetate (FDA) microscopy can predict TB culture results, treatment response and infectiousness. However, diverse methods have been published. We aimed to optimise FDA microscopy, minimising sputum processing, biohazard and complexity for use in resource-constrained settings. Methods and results Optimization: Patients with smear-positive pulmonary TB before treatment and healthy control participants provided sputa. These were divided into equal aliquots that were tested directly or after NaOH centrifuge-decontamination. Each aliquot was cultured and used to prepare slides (n = 80). FDA microscopy used: 1 or 3 drops of sputum; with/out acid-alcohol wash; with/out phenol sterilization; with 0/30/60 seconds KMnO4 quenching. Control samples all had negative culture and microscopy results. FDA microscopy had higher sensitivity when performed directly (without centrifuge-decontamination) on 1 drop of sputum (P<0.001), because 3 drops obscured microscopy. Acid-alcohol wash and KMnO4 quenching made bacilli easier to identity (P = 0.005). Phenol sterilization did not impair microscopy (P>0.1). Validation: The 2 protocols that performed best in the optimization experiments were reassessed operationally by comparing duplicate slides (n = 412) stained with KMnO4 quenching for 30 versus 60 seconds. FDA microscopy results were similar (P = 0.4) and highly reproducible, with 97% of counts agreeing within +/-1 logarithm. Storage: Smear microscopy slides and aliquots of the sputum from which they were made were stored for 4 weeks. Twice-weekly, paired slides (n = 80) were stained with freshly prepared versus stored FDA and read quantitatively. Storing sputum, microscopy slides or FDA solution at 4C or room temperature had no effect on FDA microscopy results (all P>0.2). Cost: Material costs for each slide tested by FDA microscopy using reagents purchased locally were USD $0.05 and required the same equipment, time and skills as auramine acid-fast microscopy. Conclusions: We recommend a simple, bio-secure protocol for FDA microscopy that provides sensitive and repeatable results without requiring centrifugation. en_US
dc.language.iso eng
dc.publisher Public Library of Science
dc.relation.ispartofseries PLoS ONE
dc.rights info:eu-repo/semantics/restrictedAccess
dc.rights.uri https://creativecommons.org/licenses/by-nc-nd/4.0/deed.es
dc.subject Article en_US
dc.subject centrifugation en_US
dc.subject clinical protocol en_US
dc.subject controlled study en_US
dc.subject false negative result en_US
dc.subject false positive result en_US
dc.subject fluorescein diacetate en_US
dc.subject human en_US
dc.subject lung tuberculosis en_US
dc.subject microscopy en_US
dc.subject process optimization en_US
dc.subject reproducibility en_US
dc.subject respiratory tract disease assessment en_US
dc.subject sensitivity analysis en_US
dc.subject sputum smear en_US
dc.subject storage temperature en_US
dc.title Optimising fluorescein diacetate sputum smear microscopy for assessing patients with pulmonary tuberculosis en_US
dc.type info:eu-repo/semantics/article
dc.identifier.doi https://doi.org/10.1371/journal.pone.0214131
dc.subject.ocde https://purl.org/pe-repo/ocde/ford#1.06.01
dc.subject.ocde https://purl.org/pe-repo/ocde/ford#3.02.07
dc.relation.issn 1932-6203


Files in this item

Files Size Format View

There are no files associated with this item.

This item appears in the following Collection(s)

Show simple item record

info:eu-repo/semantics/restrictedAccess Except where otherwise noted, this item's license is described as info:eu-repo/semantics/restrictedAccess

Search DSpace


Browse

My Account

Statistics