QBC ParaLens <sup>TM </sup> LED fluorescent microscope attachment with QBC F.A.S.T.<sup>TM </sup> AFB staining system

doi:10.4236/jtr.2013.13008

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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: [email protected]

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

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

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%

K. N’Guessan et al. / Journal of Tuberculosis Researc h 1 (2013) 40-43

direct examination were found to be negative 95% of the

time [9].

OPEN ACCESS

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.