Vol.1, No.3, 40-43 (2013) Journal of Tubercul osis Resear ch
http://dx.doi.org/10.4236/jtr.2013.13008
QBC ParaLens™ LED fluorescent microscope
attachment with QBC F.A.S.T.™ AFB
staining system*
Kouassi N’Guessan1, Brian Joseph Hnatkovich2#, N’Guetta Aka1, Armand Achy-Brou3,
Bakary Coulibaly1, Jean Marc Assande1, Philomène Mohoudi1, Mireille Dosso1
1Département de Bactériologie-Virologie, Institut Pasteur de Côte d’Ivoire, Abidjan, Côte d’Ivoire
2QBC Diagnostics, Inc., Port Matilda, USA; #Corresponding Author:
3Département d’Epidémiologie, Institut Pasteur de Côte d’Ivoire, Abidjan, Côte d’Ivoire
Received 4 September 2013; revised 22 October 2013; accepted 1 November 2013
Copyright © 2013 Kouassi N’Guessan et al. This is an open access article distributed under the Creative Commons Attribution Li-
cense, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
ABSTRACT
This study was designed to evaluate the QBC
ParaLens™ LED fluorescent microscope at-
tachment and the QBC F.A.S.T.™ AFB staining
system for the detection of Acid Fast Bacilli in
pathological samples in Abidjan, Cote d’Ivoire. A
total of 50 patients were tested using direct
smear specimens with both Ziehl-Neelsen (ZN)
light microscopy and LED fluorescence mi-
croscopy with QBC F.A.S.T. AFB stain. The sam-
ples were also cultured and tested using an im-
munochromatograpic test for detection of anti-
gen MPT 64 and the results were compared to
direct examination. ZN light microscopy detect-
ed 20 positive cases and LED fluorescent mi-
croscopy with QBC F.A.S.T. AFB stain detected
21. The sensitivity and specificity of ZN light
microscopy was determined to be 84.2% and
87.1% respectively. The sensitivity and specific-
ity of LED fluorescent microscopy with QBC
F.A.S.T. AFB stain was determined to be 94.7%
and 90.3% respectively. Compared to ZN light
microscopy, LED fluorescent microscopy with
QBC F.A.S.T. AFB stain increased the sensitivity
of direct examination without concentration by
10.5%.
Keyw ords: Tuberculosis; AFB; Ziehl-Neelsen; LED
Fluorescence; QBC ParaLens; QB C F.A.S.T.
1. INTRODUCTION
Tuberculosis remains a global scourge throughout the
developing world because of its endemoepidemic char-
acter and person to person transmission. The TB control
strategy is focused on the identification of cases that
transmit infection and on implementation of therapeutic
regimes. In poorer countries, the diagnostic and thera-
peutic decisions on patients with suspected TB are often
based on direct examination of Ziehl-Neelsen stained
slides using light microscopy, a technique whose sensi-
tivity is variable [1]. Detection of AFB in stained smears
may provide the initial evidence of the presence of my-
cobacterium in suspected TB patients, however, smear
examination provides only a presumptive diagnosis of
TB as the AFB may be acid-fast organisms other than M.
tuberculosis.
A systematic review conducted by Steingart et al. has
shown that the sensitivity of direct examination for the
detection of Acid-Fast Bacilli (AFB) from pathological
samples is better with fluorescent microscopy than that
with conventional light microscopy [2]. The routine use
of fluorescent microscopy for the diagnosis of TB has
been limited for various reasons including the short life-
time of the lamp and the overall cost of implementation
[3]. Technological advances have allowed for the de-
velop- ment of light-emitting diode (LED) fluorescent
micro- scopes. LED fluorescence microscopy is more
economic than conventional fluorescent microscopes that
utilize halogen or mercury bulbs [2 ,3].
The QBC ParaLens LED fluorescent microscope at-
tachment transforms a light microscope into a fluorescent
microscope. The QBC ParaLens is easier to use than
more conventional fluorescent microscopes [3,4] and
does not requir e change to existing infrastructure.
*Conflicts of Interest: B. J. Hnatkovich is employed by QBC Diagnos-
tics, Inc. and assisted in the preparation of this manuscript for publica-
tion. Other authors in this study have no conflict of interest or financial
relationship with the manufacturer or distributor of the QBC ParaLens,
QBC F.A.S.T. AFB stain or QBC Diagnostics, Inc.

K. N’Guessan et al. / Journal of Tuberculosis Researc h 1 (2013) 40-43 41
Previous work has shown that there is a good correla-
tion between the results obtained with the QBC ParaLens
and those results obtained with classical fluorescent mi-
croscopy [5]. In some cases, the performance of LED
FM is better than conventional fluorescent microscopy
methods [5,6].
This study was designed to evaluate the QBC Para
Lens LED fluorescent microscopy attachment and the
QBC F.A.S.T. AFB staining system for the detection of
AFB in pathological specimens of suspected TB patients
in Abidjan, Côte d’Ivo ire in 20 10.
2. METHODS
2.1. Sample Collection
Patient samples were collected at the CHU Services of
Cocody, Centre Antituberculeux de Treichville and Ad-
jamé. Samples were collected from 50 patients with
symptoms suggestive of tuberculosis. The 50 samples
included 31 spontaneous sputum samples, 16 gastric as-
pirates sample, 1 bronchial aspiration sample, and 2
pleural liquid samples Table 1. Samples were then tran s-
ported at 4˚C to the Tubercu losis Laboratory at the Insti-
tute Pasteur of Cote d’Ivoire. The samples were exam-
ined for non-conformance per internal quality control
procedures and accepted for use in the study.
2.2. Direct Examination
Direct examination without concentration was per-
formed on each of the samples. Two smears were pre-
pared according to internal protocols. The first smear
was made on a common use microscope slide. The sec-
ond smear was made on a QBC F.A.S.T. SureFo cus slide.
Both smears were air-dried and fixed using flame. The
first smear was stained according to accepted procedures
with a 0.5% Carbol Fuschsin solution prepared in the
laboratory. The second smear was stained using the QBC
F.A.S.T. AFB Stain kit according to the manufacturer’s
instructions for use.
Direct examination was conducted using an Olympus
CX21 microscope. To facilitate fluorescent examination,
the QBC ParaLens fluorescent microscope attachment
with 60× objective was mounted to the nosepiece of the
same microscope.
Ziehl-Neelsen (ZN) stained smears were reviewed us-
ing a 100× objective under oil. QBC F.A.S.T. AFB fluo-
rescent stained smears were reviewed using the QBC
ParaLens with 60× objective under oil. Two technicians
conducted blinded microscope examination according to
accepted procedures for both the ZN and QBC F.A.S.T.
AFB examination techniques. Discordant results were
reviewed by a third, more experienced technician. Direct
examination results were quantified using the WHO
scale [7].
2.3. Culture
Each of the samples was also cultured according to
accepted procedures. Samples were decontaminated us-
ing a 2% N-Acetyl-L-Cysteine (NALC) solution and
incubating for 15 minutes. After incubation, the samples
were centrifuged at 3000 × g for 20 minutes. The super-
natant was removed and the pellet re-suspended in a
phosphate suspension buffer (pH 7). The solution was
homogenized by aspiration. Each sample was used to
inoculate three cultures of Lowenstein-Jensen (LJ) media.
The inoculants were incubated at 37˚C for eight weeks.
After incubation, all positive LJ cultures were tested us-
ing an Immunochromatographic (SD BioLine) test for
detection of antigen MPT 64. Data was collected and
analyzed using Epi-Info 6.04 (CDC, Atlanta, GA, USA).
3. RESULTS
Direct examination of ZN stained specimens resulted
in 40%; 95% C.I. [0.26 - 0.54] % (20 of 5 0 samples) posi-
tive incidence rate. When LED fluorescent microscopy
was used to examine the specimens stained with the
QBC F.A.S.T. AFB stain, a 42%; 95% C.I. [0.28 - 0.56]%
(21 of 50 samples) positive incident rate was seen (Table
2).
Of the samples examined, positive results were ob-
served using ZN stain in 16 spontaneous sputum sam-
ples, 3 gastric aspirates and 1 liquid bronchial aspiration.
LED fluorescent microscopy with QBC F.A.S.T. AFB
stain resulted in positive samples being observed in 17
spontaneous sputum samples, 3 gastric aspirates and 1
liquid bronchial aspiration. The MPT 64 antigen detec-
tion method identified 18 positive cases and 1 strongly
suggestive suspected case.
Compared to the MPT 64 antigen, the sensitivity,
specificity, positive and negative predictive value of ZN
were determined to be 84.2%; C.I. 95% [0.74 - 0.91]%,
87.1%; C.I. 95% [0.78 - 0.96]%, 80% and 90% respec-
tively (Table 3).
Compared to the MPT 64 antigen, the sensitivity,
specificity, positive and negative predictive value of LED
fluorescent microscopy with QBC F.A.S.T. AFB stain
were determined to be 94.7%; C.I. 95% [0.88 - 1.00]%,
90.3%; C.I. 95% [0.82 - 0.99]%, 85.7% and 90.6% re-
spectively (Table 4).
ZN resulted in 5 discordant results; 3 false negatives
and 2 false positives compared to LED fluorescent mi-
croscopy with QBC F.A.S.T. AFB stain. Of the 3 false
negative findings, 2 were found to be positive for MPT
64 and 1 was found to be negative. The results of the
smear examination of the 2 false positives were recorded
as “scanty” and in both cases the sample was negative
for MPT 64.
LED fluorescent microscopy with QBC F.A.S.T. AFB

K. N’Guessan et al. / Journal of Tuberculosis Researc h 1 (2013) 40-43
42
Table 1. Distribution of samples according to origin.
Origin Sputum
Gastric
Aspiration Pleural Fluid Bronchial Aspiration Total
CHU Cocody 20 16 1 2 39
CAT Adjamé 3 0 0 0 3
CAT
Treichville 8 0 0 0 8
Total 31 16 1 2 50
Table 2. Distribution of the results according to the method
used.
Method Negative Scanty 1+ 2+ 3+Total
ZN 30 4 2 5 950
LED 29 4 4 8 550
*WHO/IUATLD Scale.
Table 3. Performance of ZN light microscopy compared to
antigen MPT 64 test on cultured samples.
Direct Examination MPT 64 (+) MPT 64 (−) Total
ZN (+) 16 4 20
ZN (−) 3 27 30
Total 19 31 50
Table 4. Performance of LED Fluorescent Microscopy with
QBC F.A.S.T. AFB Stain compared to antigen MPT 64 Test on
cultured samples.
Direct Examination MPT 64 (+) MPT 64 (−) Total
LED (+) 18 3 21
LED (−) 1 28 29
Total 19 31 50
stain also resulted in 5 disco rdan t results compared to ZN;
2 false negative and 3 false positives. Of the 2 false
negative findings, both were determined to be negative
for MPT 64. Of the results of the smear examination of
the 3 false positives, 2 were recorded as “scanty” and 1
was recorded as “1+”. When cultured and tested, 1
“scanty” sample and 1 “1+” sample were positive for
MPT 64
The sensitivity of direct examination, without concen-
tration, of AFB increased from 84.2% with ZN to 94.7%
with LED fluorescent microscopy with QBC F.A.S.T.
AFB stain.
4. DISCUSSION
Despite its low sensitivity compared to culture, the
diagnostic decisions and treatment of tuberculosis in
low-income countries are based on the direct examina-
tion of ZN stained pathological specimens. The current
strategy in the fight against TB is centered on the detec-
tion of infected individuals and requires detection meth-
ods with a higher sensitivity and specificity compared to
conventional ZN light microscopy to identify a larger
number of positive patients that present with symptoms
of TB. With a sensitivity of 94.7%, LED fluorescent mi-
croscopy with the QBC ParaLens and QBC F.A.S.T. AFB
stain is a credible alternative to ZN light microscopy for
the detection of TB.
Previous work comparing conventional ZN light mi-
croscopy to LED fluorescent microscopy concluded that
the sensitivity of the LED method was superior to ZN
light microscopy [2,3,6]. In the study presented here, 50
patients from TB centers were used to compare ZN light
microscopy to LED fluorescent microscopy. As a diag-
nostic tool, LED fluorescent microscopy with QBC
F.A.S.T. AFB stain identified 2 false negative samples
compared to ZN light microscopy. When cultured and
tested, these samples were determined to be positive
MPT 64 antigen.
While showing superior sensitivity to ZN light mi-
croscopy, the specificity of the two methods were rela-
tively close; ZN light microscopy resulted in a specificity
of 87.1% and LED fluorescent microscopy with QBC
F.A.S.T. AFB stain resulted in a specificity of 90.3%.
Table 5.
Compared to previous studies that have compared
LED fluorescent microscopy to ZN light microscopy, a
sample size of 50 specimens is high [3,6]. The sample
size, to a certain extent, contributed to the performance
of LED fluorescent microscopy presented herein. Marsh,
et al., with 221 sputum samples, found LED fluorescent
microscopy to have a sens itivity of 84.7% and a sp ecific-
ity of 98.9% for the routine detection of AFB [6]. It can
also be mentioned that 8% of the samples with results
classified as “scanty” by direct examination are more
likely to be false positives [8] both with ZN light mi-
croscopy and LED fluorescent microscopy. In some
studies, cultures from samples identified as “scanty” by
Table 5. Comparison of the direct examination methods.
Performance ZN
LED FM with
QBC
F.A.S.T. AFB Stain
Difference
Sensitivity (95% C.I.)84.2%
(0.74 - 0.94) 94.7% (0.88 - 1.01) +10.5%
Specificity
(95% C.I.)
87.1%
(0.78 - 0.96) 90.3% (0.82 - 0.99) +3.2%
Positive Predictive
Value (PPV) 80.0% 85.7% +5.7%
Negative Predictive
Value (NPV) 90.0% 90.6% +0.6%

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43
direct examination were found to be negative 95% of the
time [9].
In conclusion, we believe that LED fluorescent mi-
croscopy with QBC F.A.S.T. AFB stain to be a credible
alternative to ZN light microscopy for routine TB
screening in countries endemic for pulmonary tuberculo-
sis.
5. ACKNOWLEDGEMENTS
We extend our sincere thanks to Mr. Alain Noudofinin for the finan-
cial support he brought to this work and the equipment, reagents and
consumables used in this study.
REFERENCES
[1] Salfinger, M. and Pfyffer, G.E. (1994) The new diagnostic
mycobacteriology laboratory. European Journal of Cli-
nical Microbiology & Infectious Diseases, 13, 961-979.
http://dx.doi.org/10.1007/BF02111498
[2] Steingart, K.R., Henry, M., Ng, V., Hopewell, P.C., Ram-
say, A., Cunningham, J., Urbanczik, R., Perkins, M., Aziz,
M.A. and Pai, M. (2006) Fluorescence versus conven-
tional sputum smear microscopy for tuberculosis: A sys-
tematic review. The Lancet Infectious Diseases, 6, 570-
581. http://dx.doi.org/10.1016/S1473-3099(06)70578-3
[3] Affolabi, D., Torrea, G., Odoun, M., Senou, N., Ali, L.M.,
Anagonou, S. and Van Deun, A. (2010) Comparison of
two LED fluorescence microscopy build-on modules for
acid-fast-bacilli smear microscopy. The International
Journal of Tuberculosis and Lung Disease, 14,160-164.
[4] Kuhn, W., Armstrong, D., Atteberry, S., Dewbrey, E.,
Smith, D. and Hooper, N. (2010) Usefulness of the Pa-
ralens fluorescent microscope adaptor for the identifica-
tion of mycobacteria in both field and laboratory settings.
The Open Microbiology Journal, 4, 30-33.
http://dx.doi.org/10.2174/1874285801004010030
[5] Nguyen, V.H., Dinh, N.S., Anthony, R.M., Cobelens, F.G.
and van Soolingen, D. (2007) Fluorescence microscopy
for tuberculosis diagnosis. The Lancet Infectious Disea-
ses, 7, 238-239.
http://dx.doi.org/10.1016/S1473-3099(07)70059-2
[6] Marais, B.J., Brittle, W., Painczyk, K., Hesseling, A.C.,
Beyers, N., Wasserman, E., van Soolingen, D. and Warren,
R.M. (2008) Use of light-emitting diode fluorescence mi-
croscopy to detect acid fast bacilli in sputum. Clini cal In-
fectious Diseases, 47, 203-207.
http://dx.doi.org/10.1086/589248
[7] World Health Organization (1998) Laboratory services in
tuberculosis control. Part II: Microscopy. WHO/TB/
98.258. WHO, Geneva.
[8] Van Deun, A., Salim, A.H., Cooreman, F., Daru, P., Das,
A.P., Anug, K.J.M. and Reider, H.L. (2004) Scanty AFB
smears: What’s in a name? The International Journal of
Tuberculosis and Lung Disease, 8, 816-823.
[9] Lawson, L., Yassin, M.A., Ramsay, A., Emenyonu, N.E.,
Squire, S.B. and Cuevas, L.E. (2005) Comparison of
scanty AFB smears against culture in an area with high
HIV prevalence. The International Journal of Tubercu-
losis and Lung Disease, 9, 933-935.