Article

Comparison of LED and Conventional Fluorescence Microscopy for Detection of Acid Fast Bacilli in a Low-Incidence Setting

Department of Epidemiology, Biostatistics and Occupational Health, McGill University, Montreal, Canada.
PLoS ONE (Impact Factor: 3.23). 07/2011; 6(7):e22495. DOI: 10.1371/journal.pone.0022495
Source: PubMed
ABSTRACT
Light emitting diode fluorescence microscopes have many practical advantages over conventional mercury vapour fluorescence microscopes, which would make them the preferred choice for laboratories in both low- and high-resource settings, provided performance is equivalent.
In a nested case-control study, we compared diagnostic accuracy and time required to read slides with the Zeiss PrimoStar iLED, LW Scientific Lumin, and a conventional fluorescence microscope (Leica DMLS). Mycobacterial culture was used as the reference standard, and subgroup analysis by specimen source and organism isolated were performed.
There was no difference in sensitivity or specificity between the three microscopes, and agreement was high for all comparisons and subgroups. The Lumin and the conventional fluorescence microscope were equivalent with respect to time required to read smears, but the Zeiss iLED was significantly time saving compared to both.
Light emitting diode microscopy should be considered by all tuberculosis diagnostic laboratories, including those in high income countries, as a replacement for conventional fluorescence microscopes. Our findings provide support to the recent World Health Organization policy recommending that conventional fluorescence microscopy be replaced by light emitting diode microscopy using auramine staining in all settings where fluorescence microscopy is currently used.

Full-text

Available from: Christina Greenaway
Comparison of LED and Conventional Fluo rescence
Microscopy for Detection of Acid Fast Bacilli in a Low-
Incidence Setting
Jessica Minion
1,2,3
, Madhukar Pai
1,2
*, Andrew Ramsay
4
, Dick Menzies
1,2
, Christina Greenaway
5
1 Department of Epidemiology, Biostatistics and Occupational Health, McGill University, Montreal, Canada, 2 Respiratory Epidemiology and Clinical Research Unit,
Montreal, Canada, 3 Department o f Medical Microbiology and Immunology, University of Alberta, Edmonton, Canada, 4 UNICEF/UNDP/World Bank/WHO Special
Programme for Research and Training in Tropical Diseases, World Health Organization, Geneva, Switzerland, 5 Department of Diagnostic Medicine, Division of Infectious
Diseases, SMBD-Jewish General Hospital, McGill University, Montreal, Canada
Abstract
Introduction:
Light emitting diode fluorescence microscopes have many practical advantages over conventional mercury
vapour fluorescence microscopes, which would make them the preferred choice for laboratories in both low- and high-
resource settings, provided performance is equivalent.
Methods:
In a nested case-control study, we compared diagnostic accuracy and time required to read slides with the Zeiss
PrimoStar iLED, LW Scientific Lumin, and a conventional fluorescence microscope (Leica DMLS). Mycobacterial culture was
used as the reference standard, and subgroup analysis by specimen source and organism isolated were performed.
Results:
There was no difference in sensitivity or specificity between the three microscopes, and agreement was high for all
comparisons and subgroups. The Lumin and the conventional fluorescence microscope were equivalent with respect to
time required to read smears, but the Zeiss iLED was significantly time saving compared to both.
Conclusions:
Light emitting diode microscopy should be considered by all tuberculosis diagnostic laboratories, including
those in high income countries, as a replacement for conventional fluorescence microscopes. Our findings provide support
to the recent World Health Organization policy recommending that conventional fluorescence microscopy be replaced by
light emitting diode microscopy using auramine staining in all settings where fluorescence microscopy is currently used.
Citation: Minion J, Pai M, Ramsay A, Menzies D, Greenaway C (2011) Comparison of LED and Conventional Fluorescence Microscopy for Detection of Acid Fast
Bacilli in a Low-Incidence Setting. PLoS ONE 6(7): e22495. doi:10.1371/journal.pone.0022495
Editor: Delia Goletti, National Institute for Infectious Diseases (L. Spallanzani), Italy
Received May 19, 2011; Accepted June 24, 2011; Published July 21, 2011
Copyright: ß 2011 Minion et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This project was supported in part by grants from the Canadian Institutes for Health Research (CIHR MOP-89918), the European Commission (EU-FP7;
TBSusgent), and the EDCTP (TB-NEAT). MP is supported by a CIHR New Investigator Award, DM by a Chercheur-national award, CG by a Chercheur Boursier
Clinicen award from the Fonds de Recherche en Sante
´
de Quebec, and JM by a Quebec Respiratory Health Training Fellowship. The funders had no role in study
design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: madhukar.pai@mcgill.ca
Introduction
Tuberculosis (TB) continues to be one of the world’s most
important infectious causes of morbidity and mortality. An
estimated 9.4 million people develop TB disease each year and
approximately 1.7 million die from the disease [1]. While the
preponderance of TB burden is borne by nations in Asia and
Africa, TB remains an important public health concern in high-
income as well as low- and middle-income countries globally [2].
One of the key steps in TB control is case detection. Although
advances in diagnostics are leading to the introduction of new tests
[3,4], the backbone of TB diagnosis worldwide continues to be
smear microscopy. Thus, increasing the sensitivity of smear
microscopy could have a large impact on global TB case detection
rates. As a result there have been several initiatives to optimise
smear microscopy including changes in specimen collection
procedures, specimen processing, and microscopy techniques
[5,6,7].
For microscopic detection of acid fast bacilli (AFB), fluorescence
microscopy (FM) using auramine staining has been shown to have
10% higher sensitivity compared to routine light microscopy used
with Ziehl-Neelsen (ZN) staining, without compromising specific-
ity [8]. FM is also more time efficient, with one large study
reporting FM to take only 25% of the time required for ZN
examinations [9]. In most high-income countries, FM has now
been widely adopted and is used routinely.
Light emitting diode (LED) microscopy is a novel diagnostic tool
developed primarily to allow resource-poor parts of the world
access to the benefits of FM [10,11,12]. Compared to conventional
mercury vapour fluorescence microscopes, LED microscopes are
less expensive and have lower maintenance requirements. The
diodes are very durable, do not require warm-up time, and do not
contain toxic products. Importantly, they are reported to perform
equally well without a darkroom. These qualities make them
attractive for use in low- and middle-income countries, and they
have performed well in evaluations in these settings
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[13,14,15,16,17,18,19,20]. Many of the benefits of LED technol-
ogy would also be appealing to laboratories in high-income
countries if LED microscopy is equivalent in performance to
conventional FM. Indeed, the World Health Organization (WHO)
recently recommended that conventional FM be replaced by
LED-FM in all settings where fluorescence microscopy is currently
used, and that LED FM be phased in as an alternative to
conventional ZN microscopy in all settings [21]. Despite this
recommendation, this is the first evaluation of LED-FM for TB
diagnosis based in a low-burden, high-resource setting.
The obje ctives of this study wer e to compare the sensitivi ty
and specificity of fl uorescence smea r microscopy in the detection
of AFB using two different LED micr oscopes: the Lumin
Por table Fl uroescence Kit (LW Scientific , Atlanta, Georgia,
USA) and the PrimoStar iLED (Carl Zeiss MicroI maging, Jena,
Ger many), with a conventional mercury vapour fluorescence
microscope, using mycobacterial culture as a reference standard,
in a low-incidence setting. Additionally, we compa red the time
required to read auramine-stained smears with the t hree FM
devices and collected feedba ck from microscopists regarding
user-im portant c haracteristics.
Methods
Study Setting
This study was conducted in Montreal, Quebec, Canada using
specimens submitted routinely for mycobacterial culture from
university and tertiary care centres, from April through September
2009. These included both diagnostic and follow-up specimens. In
2009 the province of Quebec reported 195 new and retreatment
cases, with an incidence rate of 2.5/100,000 [2]. The prevalence
of HIV infection in Canada is approximately 0.2% [22]. Overall
culture positivity in specimens submitted from patients with
suspected mycobacterial disease was approximately 2% with about
40% of these consisting of non-tuberculous mycobacteria (NTM).
Specimen Selection
Given the low culture positivity rate in our setting, we elected to
use a nested case control design so as to include all culture positive
specimens and an equal number of culture negative specimens. All
consecutive specimens submitted for mycobacterial culture had an
additional smear prepared and heat fixed. These unstained smears
were stored in dry, dark smear boxes. When culture results
became available, all smears originating from culture positive
specimens were selected for study inclusion. An equal number of
smears originating from culture negative specimens were random-
ly selected (using random number generators).
Fluorescence Microscopy Comparison
The Zeiss PrimoStar iLED microscope (Carl Zeiss MicroIma-
ging GmbH, Jena, Germany) was developed in collaboration with
FIND (Foundation for Innovative New Diagnostics) and is a stand-
alone microscope that can be used in bright-field or fluorescence
LED modes [23]. The Lumin Portable Fluorescent Kit (LW
Scientific, Atlanta, Georgia, USA) is a portable objective lens
attachment that is used with an existing light microscope (in this
study it was used with the Zeiss iLED) [24]. We used both the 20x
and 40x lenses, the latter for screening and the former for AFB
confirmation. We chose to evaluate these two devices upon
consideration of their suitability to high-resource settings (Zeiss),
their unique benefits compared to conventional fluorescent
microscopes (Lumin), and their current popularity in global
evaluations (Zeiss and Lumin).
All smear examinations were done by one of two technologists
with expertise in mycobacteriology and fluorescence microscopy,
who were blinded to the culture results and any patient details.
Smears were examined three times by the same microscopist using
the LW Scientific Lumin LED attachment, the Zeiss Primo Star
iLED, and a conventional mercury vapour microscope (Leica
DMLS). Between readings slides were randomized (with the aid of
random number lists) to maintain technologist blinding. Staining
of slides and readings with all three microscopes were done on the
same day to avoid possible fading of the fluorescent stain [25].
Slides were reported as doubtful, 1+,2+,3+,4+ or negative at
400x magnification [26]. Negative smears had 300 fields examined
before being declared negative. The time required to read slides
was estimated by logging the time at the start and at the end of
reading a group of slides. This time was then averaged for the
number of slides read to calculate a time per slide estimate, which
included mounting slides and recording results.
Specimen Processing
Respiratory specimens (including sputum, bronchioalveolar
lavage (BAL), bronchial wash (BW), lung aspirates) underwent
digestion/decontamination with NALC-NaOH and concentration
using centrifugation before slide preparation and culture inocula-
tion. Extrapulmonary specimens were processed using standard
procedures [26].
Each specimen was inoculated onto Lowenstein-Jensen (LJ)
media and into a MGIT tube (Becton Dickenson, Sparks, MD,
USA) for up to 8 weeks. Positive growth was confirmed by
Kinyoun staining, and mycobacterial isolates were sent to the
Quebec Provincial Laboratory for speciation using 16S ribosomal
sequencing.
Smears were heat-fixed before storage. Staining was performed
immediately before the first smear examination. Smears were
flooded with auramine O for 15 minutes, then rinsed with sterile
water; decolourized with acid-alcohol for 2 minutes, then rinsed
with sterile water; counterstained with potassium permanganate for
2-4 minutes, then rinsed with sterile water and allowed to air dry.
Analysis
Sensitivity, specificity, positive and negative predictive values,
and likelihood ratios were calculated for the LW Scientific Lumin
LED attachment, the Zeiss Primo Star iLED and conventional
mercury vapour (Leica DMLS) microscope using mycobacterial
culture as the reference standard. Confidence intervals were
constructed using exact methods for proportions [27]. Yield was
calculated using a second reference standard where any slide read
as positive using any microscope was considered positive.
Agreement between the three microscopy readings was estimated
using un-weighted kappa statistics (with results dichotomized as
positive or negative), as well as weighted kappa statistics with linear
weighting of 5 categories: negative, 1+,2+,3+, and 4+. Kappa
statistics were used to measure the agreement between readings
made using two microscopes, with the same reader evaluating the
same mycobacterial smears, while taking into account the
agreement occurring by chance [28].
Subgroup analysis was done by specimen type (sputum, non-
sputum respiratory, extrapulmonary) and mycobacterial species
isolated (M. tuberculosis complex, non-tuberculous mycobacteria
and acid fast non-mycobacteria).
Results
A total of 200 culture positive specimens were included in the
study, with 200 randomly selected culture-negative controls. 296
LED Microscopy in Low TB Incidence Settings
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specimens were submitted as sputum (74.0%), 64 originated from
the respiratory system but not classified as sputum (16.0%;
includes specimens such as BAL fluid, BW and lung biopsies), and
40 specimens were categorized as extrapulmonary (10.0%).
Using mycobacterial culture as a reference standard, the
accuracy of the 3 microscopes is shown in Table 1. Zeiss achieved
the highest sensitivity with 40.5% (95% CI: 33.6, 47.7), followed
by Lumin with 37.5% (95% CI: 30.8, 44.6) and conventional
fluorescence microscope with 36.5% (95% CI: 29.8, 43.6). None of
the differences in sensitivity were significantly different based on
overlapping confidence intervals. Specificity was very similar
between all 3 microscopes (conventional fluorescence microscope
and Zeiss were equal: 99.0% [95% CI: 96.4, 99.9]; and Lumin:
99.5% [95% CI: 97.2, 100]).
There were 87 specimens which were read as smear positive by
at least 1 of the 3 microscopes. The conventional fluorescence
microscope identified 75 specimens as smear positive, the Zeiss
identified 83 and the Lumin identified 76. Using a reference
standard where any positive microscopic reading was considered a
true positive, this resulted in sensitivities of 86.2% (95% CI: 77.1,
92.7), 95.4% (95% CI: 88.6, 98.7), and 87.4% (95% CI: 78.5,
93.5) for conventional fluorescence microscope, Zeiss and Lumin
respectively (Table 2).
Agreement was measured with the kappa statistic using
dichotomized results (where 1+,2+,3+ and 4+ were pooled as
positive). Agreement was high between all three microscopes:
unweighted kappa = 0.91 (95% CI: 0.85, 0.96) between
conventional fluorescence microscope and Zeiss; 0.89 (95% CI:
0.84, 0.95) between conventional fluorescence microscope and
Lumin; and 0.91 (95% CI: 0.86, 0.96) between Zeiss and Lumin.
Kappa values remained high if linear weights for categories of
smear positivity (negative, +1, +2, +3, +4) were used: weighted
kappa = 0.92 (95% CI: 0.89, 0.96) between conventional
fluorescence microscope and Zeiss; 0.92 (95% CI: 0.88, 0.96)
between conventional fluorescence microscope and Lumin; and
0.93 (95% CI: 0.90, 0.97) between Zeiss and Lumin. The
distribution of all positive smear readings is displayed in Figure 1.
Accuracy was also calculated depending on the category of
specimens examined and the species isolated. Table 3 shows
sensitivity and specificity of all 3 microscopes stratified by sputum
specimens, non-sputum respiratory specimens, and extra-pulmo-
nary specimens. There were no significant differences between the
microscopes for any of these subgroups, based on non-overlapping
confidence intervals.
Of the 200 culture positive specimens, MTB Complex
organisms were isolated from 115 (106 M. tuberculosis,9M.
africanum). The remaining 85 culture positive specimens isolated a
wide range of NTM as well as acid-fast organisms capable of
surviving mycobacterial decontamination and growing in myco-
bacterial growth media (2 Streptomyces species, 1 Nocardia puris,1
Tsukamurella tyrosinosolvens). These were considered true positives
since organisms from Streptomyces, Nocardia and Tsukamurella
genera are considered acid fast. The sensitivity of all 3 microscopes
was higher in detecting MTB Complex organisms compared to
NTM or other acid fast organisms; however, there was no
difference between the 3 devices (Table 4).
On average, reading slides using the conventional fluorescence
microscope took 1.51 mins/slide (95% CI: 1.47, 1.55). This was
Table 1. Accuracy Using a Culture Reference Standard.
TP/Cx
+
Sensitivity (95% CI) TN/Cx- Specificity (95% CI)
conventional FM 73/200 36.5% (29.8, 43.6 ) 198/200 99.0% (96.4, 99.9)
Zeiss 81/200 40.5% (33.6, 47.7) 198/200 99.0% (96.4, 99.9)
Lumin 75/200 37.5% (30.8, 44.6) 199/200 99.5% (97.2, 100)
PPV* NPV* LR
+
LR-
conventional FM 0.97 (0.91, 1.00) 0.61 (0.55, 0.66) 36.5 (9.1, 146.7) 0.6 (0.6, 0.7)
Zeiss 0.98 (0.92, 1.00) 0.62 (0.57, 0.68) 40.5 (10.1, 162.5) 0.6 (0.5, 0.7)
Lumin 0.99 (0.93, 1.00) 0.61 (0.56, 0.67) 75.0 (10.5, 534.2) 0.6 (0.6, 0.7)
TP = true positive, TN = true negative, Cx + = culture positive, Cx 2 = culture negative, PPV = positive predictive value, NPV = negative predictive value, LR+ =
positive likelihood ratio, LR- = negative likelihood ratio.
*PPV and NPV calculated for fixed prevalence of 50% due to case-control study design.
doi:10.1371/journal.pone.0022495.t001
Table 2. Sensitivity Using a Microscopic Reference Standard.
Smear
+
/ ‘‘Any
Smear Positive’’
Sensitivity
(95% CI)
conventional FM 75/87 86.2% (77.1, 92.7)
Zeiss 83/87 95.4% (88.6, 98.7)
Lumin 76/87 87.4% (78.5, 93.5)
doi:10.1371/journal.pone.0022495.t002
Figure 1. Distribution of positive smear readings. *No doubtful/
scanty results were reported.
doi:10.1371/journal.pone.0022495.g001
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identical to the time required using the Lumin (1.51 mins/slide;
[95% CI: 1.48, 1.54]), but longer than the time required using the
Zeiss (1.12 mins/slide; [95% CI: 1.09, 1.15]). The time savings
using the Zeiss microscope was statistically significant compared to
the other 2 microscopes, based on non-overlapping CIs.
Discussion
The benefits of improved sensitivity and reading efficiency of
FM compared to ZN microscopy have long been realized in high-
income countries through the use of conventional mercury vapour
fluorescence microscopes. The operational benefits of LED
microscopes over conventional fluorescence microscopes would
certainly be of interest for laboratories and technologists in high-
income as well as low- and middle-income settings. Working
without the need for a dark room using LED microscopes could
significantly improve workflow and maximize space utilization in
the lab, in addition to the benefits seen in tropical climates relating
to the absence of climate control in enclosed spaces. Lower
purchase price and maintenance costs, longer diode life, absence
of toxic components, and the lack of warm up time required
between turning on a conventional fluorescence microscope and
its use are all factors that would influence laboratories in high-
income countries to switch from conventional to LED FM.
However, these benefits would not be sufficient to adopt LED
FM in high-income settings unless the sensitivity and reading
efficiency associated with conventional FM were maintained with
LED FM. Additionally, laboratory technologists in high-income
countries are generally familiar with FM already and have ample
expertise using conventional fluorescence microscopes. While this
obviates the need for extensive training when introducing LED
FM, it sets high expectations of device quality and usability.
This is the first study evaluating LED microscopy for AFB
detection in a high-income, low-incidence setting, as a practical
improvement over existing conventional FM. Just as it is
unadvisable to extrapolate results from studies performed in
Table 3. Accuracy by Specimen Type.
a) Sputum
TP/Cx
+
Sensitivity (95% CI) TN/Cx- Specificity (95% CI)
conventional FM 63/169 37.3% (30.0, 45.0) 125/127 98.4% (94.4, 99.8)
Zeiss 68/169 40.2% (32.8, 48.0) 125/127 98.4% (94.4, 99.8)
Lumin 64/169 37.9% (30.5, 45.6) 126/127 99.2% (95.7, 100)
b) Other Respiratory*
TP/Cx
+
Sensitivity (95% CI) TN/Cx- Specificity (95% CI)
conventional FM 5/17 29.4% (10.3, 56.0) 47/47 100% (92.5, 100)
Zeiss 6/17 35.3% (14.2, 61.7) 47/47 100% (92.5, 100)
Lumin 6/17 35.3% (14.2, 61.7) 47/47 100% (92.5, 100)
c) Extrapulmonary
TP/Cx
+
Sensitivity (95% CI) TN/Cx- Specificity (95% CI)
conventional FM 5/14 35.7% (12.8, 64.9) 26/26 100% (86.8, 100)
Zeiss 7/14 50.0% (23.0, 77.0) 26/26 100% (86.8, 100)
Lumin 5/14 35.7% (12.8, 64.9) 26/26 100% (86.8, 100)
*includes specimens from respiratory system other than sputum (e.g. BAL, lung biopsy) .
TP = true positive, TN = true negative, Cx + = culture positive, Cx - = culture negative.
doi:10.1371/journal.pone.0022495.t003
Table 4. Sensitivity by Species Isolated.
MTB Complex
1
NTM
2
& others
3
TP/Cx
+
Sensitivity (95% CI) TP/Cx
+
Sensitivity (95% CI)
conventional FM 57/115 49.6% (40.1, 59.0) 16/85 18.8% (11.2, 28.8)
Zeiss 61/115 53.0% (43.5, 62.4) 20/85 23.5% (15.0, 34.0)
Lumin 58/115 50.4% (41.0, 59.9) 17/85 20.0% (12.1, 30.1)
1
includes 106 M. tuberculosis,9M. africanum.
2
NTM includes 26 M. avium,16M. gordonae,7M. kansasii,7M. chimaera,6M. intracellulaire,3M. conceptionense,2M. abscessus,2M. xenopi,2M. porcinum,2M. simiae
grp,1M. fortuitum,1M. shimoidei,1M. terrae,1M. celatum,1M. lentifalvum,3Mycobacterium spp (undetermined).
3
others include 2 Streptomyces spp., 1 Norcardia puris,1Tsukamurella tyrosinosolvens.
TP = true positive.
Cx+ = culture positive.
doi:10.1371/journal.pone.0022495.t004
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high-income, low-incidence settings to low-income, high-incidence
settings, it would be equally inappropriate to make inferences in
the opposite direction. Factors such as TB- and HIV-prevalence,
disease severity, proportion of non-tuberculous mycobacteria, and
type of specimens received will affect the external validity of any
TB diagnostic evaluation.
An illustration of this is seen in the diagnostic sensitivity we
report for all 3 FM modalities, which is lower than the pooled
estimates of sensitivity found in a recent systematic review and
meta-analysis (84% sensitivity compared to culture as a reference
standard) [29]. This emphasizes a common difference between a
high-income, low-incidence setting such as ours and the majority
of settings represented in the review. The fact that we have a much
higher proportion of smear negative, culture positive specimens
will lead to all types of microscopic TB diagnostics appearing to
underperform when compared to culture. While the same is often
seen with high incidences of TB and HIV co-infection, in this case
it is likely a combination of a relatively higher proportion of extra-
pulmonary TB and non-tuberculous mycobacterial infections,
which are more often smear-negative, as well as health system
practices such as the submission of specimens for follow-up of
incidental chest x-ray finding, symptom-free immigration screen-
ing, active case finding among TB contacts, and generally less
advanced disease among those diagnosed.
In this LED evaluation, we found no difference in diagnostic
accuracy between the Zeiss Primo Star iLED, the LW Scientific
Lumin and the conventional fluorescence microscope (Leica
DMSL). The agreement was high for all three microscopes
assessed (kappa .0.88 for all comparisons). When smears were
stratified by their specimen type or organism isolated, their
diagnostic accuracy remained equivalent. However, our evalua-
tion was limited by the small number of culture-positive and
smear-positive specimens available for inclusion, resulting in wide
confidence intervals around estimates of diagnostic accuracy. The
analysis was also performed by specimen and not by patient. While
this is consistent with most other studies in this field, we recognize
that the lack of independence between specimens arising from the
same patient may overestimate the precision of accuracy estimates.
For laboratory managers considering the implementation of
LED microscopy in either a low- or high-income setting, the
choice of LED device is important. There are several commercial
manufacturers now marketing LED microscopes, but few studies
comparing their head-to-head performance [15,30]. Given the
wide variety of devices available, each with different benefits
claimed and potential roles, it is important to compare them with
respect to a specific setting or situation. For instance, in high-
income, low-incidence laboratories, portability and the ability to
withstand power fluctuations and dusty environments are less
important considerations. In comparison, technologist acceptance,
speed of reading, and confirmation that readings can be made
without a darkroom continue to be important, specifically when
compared to currently available high quality mercury vapour
fluorescence microscopes.
The time required to examine slides was identical for the
conventional fluorescence microscope and the Lumin LED
attachment. However, the average time spent examining slides
with the Zeiss Primo Star iLED was significantly less than with the
other two microscopes. Subjective reports from our technologists
confirmed that the Zeiss was the easiest of the 3 to use, provided
the most convenient focusing and brightest viewing fields when
screening slides. Importantly, the technologists confirmed that the
Zeiss microscope was easily used without a darkroom. This was
similar to the user -reviews reported by Albert et al. [15].
The spectrum of light produced by LED devices is narrower
than that provided by mercury vapour conventional fluorescence
microscopes and its wavelength is produced to match specifically
the peak absorbance of auramine stains [10]. This likely
contributes to the increased brightness produced by LED
microscopes and explains why they can be used without a
darkroom. However, the Lumin attachment did not demonstrate
the same superior reading efficiency as the Zeiss. Our technologists
reported that the Lumin was more difficult to focus, and the
resulting fluorescence of the auramine-stained bacilli was dim and
they would not recommend its use without a darkroom. The
manufacturers of the Lumin have since recognized that the
objective light source was too dim, and newer models have been
improved in this regard. Another practical characteristic of the
Lumin (and other similar objective lens attachments) is the fact
that the light source needs to be plugged in directly to the objective
lens being used. Not only can this create an obstruction while the
technologist is working, but it also makes it inconvenient to switch
between different objective lenses and thus different viewing
magnifications. This assumes you have more than one Lumin
objective lens (as we did in this study); otherwise you are strictly
limited to one magnification. While the Lumin has received both
positive and negative reviews in other studies [13,15,18,30], many
of its benefits (including low upfront cost and portability) are less
important in most high-income settings.
Conclusions
The LED fluorescent microscopes (Zeiss Primo Star iLED and
LW Scientific Lumin) had nearly identical accuracy compared to a
conventional fluorescent microscope (Leica DMLS) for the
detection of AFB in patient specimens. The Zeiss required
significantly less time for smear examination compared to either
the conventional fluorescence microscope or the Lumin. Given the
practical benefits of LED microscopes for TB diagnosis, and
comparable accuracy to the current standard of a conventional
fluorescence microscope, we conclude that LED microscopy
should be considered by all TB diagnostic laboratories, including
those in high-income settings, as a replacement for conventional
FM. Our findings provide support for the recent WHO policy
which recommended that conventional FM be replaced by LED
FM using auramine staining in all settings where FM is currently
used [21].
Acknowledgments
The authors would like to thank laboratory technologists Kathy
Kyriazopoulos and Jennifer Glasgow, as well as the staff in the
microbiology laboratories of the Jewish General Hospital and Royal
Victoria Hospital, Montreal, Quebec.
Author Contributions
Conceived and designed the experiments: JM CG. Performed the
experiments: JM. Analyzed the data: JM MP CG. Contributed reagents/
materials/analysis tools: CG. Wrote the paper: JM MP AR DM CG.
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LED Microscopy in Low TB Incidence Settings
PLoS ONE | www.plosone.org 6 July 2011 | Volume 6 | Issue 7 | e22495
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  • Source
    • "To determine if the method can utilize inexpensive fluorescence microscopy equipment, we compared fluorescent intensity values obtained by using the battery-powered LED of Lumin fluorescence microscope to those obtained by using the more elaborate and expensive Olympus BX-41 unit. The development of light emitting diodes (LED) with fluorescent capabilities and low power requirements has led to the recent introduction of robust, inexpensive, battery-powered fluorescent microscopes such as the Lumin instrument [23,24]. Given that microtip visualization requires modest magnification (20X objective ) and uses a well-defined focal plane, we hypothesized that a low-cost instrument could function as well as a high-end one. "
    [Show abstract] [Hide abstract] ABSTRACT: An occupationally safe (biosafe) sputum liquefaction protocol was developed for use with a semi-automated antibody-based microtip immunofluorescence sensor. The protocol effectively liquefied sputum and inactivated microorganisms including Mycobacterium tuberculosis, while preserving the antibody-binding activity of Mycobacterium cell surface antigens. Sputum was treated with a synergistic chemical-thermal protocol that included moderate concentrations of NaOH and detergent at 60°C for 5 to 10 min. Samples spiked with M. tuberculosis complex cells showed approximately 10(6)-fold inactivation of the pathogen after treatment. Antibody binding was retained post-treatment, as determined by analysis with a microtip immunosensor. The sensor correctly distinguished between Mycobacterium species and other cell types naturally present in biosafe-treated sputum, with a detection limit of 100 CFU/mL for M. tuberculosis, in a 30-minute sample-to-result process. The microtip device was also semi-automated and shown to be compatible with low-cost, LED-powered fluorescence microscopy. The device and biosafe sputum liquefaction method opens the door to rapid detection of tuberculosis in settings with limited laboratory infrastructure.
    Full-text · Article · Jan 2014 · PLoS ONE
  • Source
    • "The iLED was preferred for overall handling and features especially with regard to the homogeneity of illumination. Other side-by-side comparisons found Fraen superior to Lumin [10], with no difference observed between Lumin and iLED in a high income, low incidence experienced laboratory setting [11]. We sought to compare the operational performance of the three LED FM methods in a cohort of HIV-positive TB suspects at a busy urban clinic in Kampala, Uganda, a high burden TB country. "
    [Show abstract] [Hide abstract] ABSTRACT: Light emitting diode (LED) fluorescence microscopy (FM) is an affordable, technology targeted for use in resource-limited settings and recommended for widespread roll-out by the World Health Organization (WHO). We sought to compare the operational performance of three LED FM methods compared to light microscopy in a cohort of HIV-positive tuberculosis (TB) suspects at an urban clinic in a high TB burden country. Two spot specimens collected from TB suspects were included in the study. Smears were stained using auramine O method and read after blinding by three LED-based FM methods by trained laboratory technicians in the Infectious Diseases Institutelaboratory. Leftover portions of the refrigerated sputum specimens were transported to the FIND Tuberculosis Research Laboratory for Ziehl Neelsen (ZN) smear preparation and reading by experienced technologist as well as liquid and solid culture. 174 of 627 (27.8%) specimens collected yielded one or more positive mycobacterial cultures. 94.3% (164/174) were M. tuberculosis complex. LED FM was between 7.3-11.0% more sensitive compared to ZN microscopy. Of the 592 specimens examined by all microscopy methods, there was no significant difference in sensitivity between the three LED FM methods. The specificity of the LED FM methods was between 6.1% and 7.7% lower than ZN microscopy (P<0.001), although exclusion of the single poor reader resulted in over 98% specificity for all FM methods. Laboratory technicians in routine settings can be trained to use FM which is more sensitive than ZN microscopy. Despite rigorous proficiency testing, there were operator-dependent accuracy issues which highlight the critical need for intensive quality assurance procedures during LED FM implementation. The low sensitivity of FM for HIV-positive individuals particularly those with low CD4 T cell counts, will limit the number of additional patients found by LED FM in countries with high rates of HIV co-infection.
    Full-text · Article · Sep 2013 · PLoS ONE
  • Source
    • "Advances in physics led to the development of light emitting diodes (LED), with appropriate fluorescent light output coupled with low power consumption, creating cheaper robust LED FM microscopes, requiring minimal mains or battery power. The WHO has recommended rolling out LED microscopes in lower income settings where they offer the throughput and sensitivity of more expensive fluorescent microscopes and are, therefore, of benefit in high HIV prevalence environments where sputum samples may carry a lower bacterial load; they can also be used successfully in middle or higher income set- tings [9,10]. For example, a multicenter study assessing the ease and effectiveness of LED-based fluorescence microscopy for TB detection (using PrimoStar iLED (Karl Zeiss, Oberkochen, Germany) was conducted in the Samara Region, Russia in 2008 and 2009 including two sites with no prior experience in fluorescence microscopy (unpublished data). "
    [Show abstract] [Hide abstract] ABSTRACT: In this article, we give an overview of new technologies for the diagnosis of tuberculosis (TB) and drug resistance, consider their advantages over existing methodologies, broad issues of cost, cost-effectiveness and programmatic implementation, and their clinical as well as public health impact, focusing on the industrialized world. Molecular nucleic-acid amplification diagnostic systems have high specificity for TB diagnosis (and rifampicin resistance) but sensitivity for TB detection is more variable. Nevertheless, it is possible to diagnose TB and rifampicin resistance within a day and commercial automated systems make this possible with minimal training. Although studies are limited, these systems appear to be cost-effective. Most of these tools are of value clinically and for public health use. For example, whole genome sequencing of Mycobacterium tuberculosis offers a powerful new approach to the identification of drug resistance and to map transmission at a community and population level.
    Full-text · Article · Aug 2013 · BMC Medicine
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