Fading of Auramine-Stained Mycobacterial Smears and Implications for External Quality Assurance

Abstract

Light-emitting diode fluorescence microscopy is being scaled up for tuberculosis control, but fading of auramine-stained slides could compromise external quality assurance. We stored auramine-stained slides and reexamined them over time. Slides stored in all environments faded quickly, with significant changes in the proportion of positive slides in as little as 1 week.Fluorescence microscopy (FM) can increase sensitivity and decrease the time required for sputum smear examination compared to Ziehl-Neelsen (ZN) staining and light microscopy (6). Low-cost, robust light-emitting diode (LED) microscopes have facilitated the expansion of FM into low-resource settings (2, 3), and in 2010, the World Health Organization (WHO) recommended that LED FM be phased in to replace ZN microscopy for tuberculosis (TB) diagnosis (9). As part of FM expansion, a system for external quality assurance (EQA) is needed and is currently being developed (9). While many EQA programs for ZN microscopy involve saving a sample of ZNstained smears for blinded rereading by a centralized reference laboratory (1, 8), fluorochrome-based stains fade over time (4). However, there is a wide spectrum of opinion regarding the rate of fading and its effect on the sensitivity of fluorochromestained smears. To generate evidence for the development of EQA strategies for LED FM, we examined the effect of storing auramine-stained slides at different temperatures and in different environments on the concordance of smear readings over time.All of the specimens submitted for mycobacterial diagnostics at Hinduja Hospital and Medical Research Centre, Mumbai, India, had an extra slide prepared and stained with auramine O-KMnO 4 (HiMedia, Mumbai, India). These smears were examined by two independent microscopists using a Lumin LED objective lens attachment (LW Scientific) in conjunction with an LED FM evaluation (5). A subset of slides was selected for storage and inclusion in this study. The sample was enriched with low-positive smears as follows: approximately 25% scanty, 25% 1ϩ, 25% 2ϩ, 15% 3ϩ, and 10% negative. All included smears had been identically scored by the two microscopists on initial reading. A total of 330 slides were allocated to one of four storage environments, darkness at room temperature (22°C), darkness in a humidified incubator (30°C), darkness in a refrigerator (4°C), and light at room temperature (22°C). Sixty slides in each of the 3 dark environments (closed slide boxes) were read monthly, 30 additional slides in all 4 environments were read weekly, and 30 slides were stored in the dark at room temperature and read once after 3 months. All slides were reread by both original microscopists during routine daily work and recorded as negative, scant, 1ϩ, 2ϩ, or 3ϩ. Blinding, reassortment, and restorage were performed by an unblinded researcher not involved in smear reading. Smear examination results were dichotomized as positive/negative, with smears read as scanty, 1ϩ, 2ϩ, or 3ϩ considered positive.In all of the storage environments, there wa...

LED fluorescence microscopy in the diagnosis of tuberculosis: Fading and restaining of smears for external quality assessment

“…Another subsets of slides (composed of 24 slides with 4 negative smears and a similar proportion of smears having different grades of positivity as previously described) were kept under the four previously described environments until the third month, when smears were examined for the first time; of 40 strong positive slides, 38 (95 %) remained positive whereas this proportion reached 72.50 %% (29/40) in those slides with ''less numerous AFB''; these proportions did not significantly differ from those obtained with slides read at monthly intervals (96.10 % and 72.66 % for ''numerous AFB'' and ''less numerous AFB'' slides, respectively, p < 0.05) ( Table 1). Moreover, we found that weakly positive slides began to fade during the first month of observation; however, according to Xia et al 11 and Radhakrishnan et al, 6 the proportion of fading of fluorescence-stained smears reported by us with weakly positive smears (10 %/month) was considerably lower than that described by Minion et al 3 , who found a fading speed of about 25 %/month. This difference could be attributed to the limitations of the device used by Minion et al, 2 who informed that the Lumin attachment was difficult to focus and showed lower excitation light intensity than other devices, which might limit the identification of those AFB that were slowly losing their intensity over time.…”

Improving acid-fast fluorescent staining for the detection of mycobacteria using a new nucleic acid staining approach

“…In all cases, SYBR Gold proved to be advantageous by producing stronger fluorescent intensities of stained MTB, and remarkably low background fluorescence. The SYBR Gold staining procedure required less time, used fewer reagents and strongly resisted fading; which has been identified as a problem with the current clinically used fluorescent Auramine-O stain [39].…”

“…D) Single bacillus dualstained with rhodamine (red) and SYBR Gold[39] with surface intensity plots obtained through Nikon NIS Elements software. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.…”

Implementation of LED Fluorescence Microscopy for Diagnosis of Pulmonary and HIV-Associated Tuberculosis in a Hospital Setting in Indonesia

“…However, the appropiate and feasible external quality assurance (EQA) procedures are needed for TB laboratories planning to implement LED-FM because the fluorochrome-based stained fade over time [25].…”