Egyptian Journal of Chest Diseases and Tuberculosis (2012) 61, 139–144

The Egyptian Society of Chest Diseases and Tuberculosis

Egyptian Journal of Chest Diseases and Tuberculosis

www.elsevier.com/locate/ejcdtwww.sciencedirect.com

ORIGINAL ARTICLE

ESAT-6-ELISpot and interferon c in the diagnosis

of pleural tuberculosis

Waheed Shouman a,*, Mohamed El-Gammal a, Amany Shaker a,

Ashraf El-Shoura a, Ayman Marei b, Mohammed El-Ahmady b, Ghada Boghdadi b

a Department of Chest, Zagazig University, Egyptb Department of Microbiology and Immunology, Zagazig University, Egypt

Received 15 April 2012; accepted 25 April 2012Available online 30 January 2013

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KEYWORDS

ESAT6;

IFN-c;Tuberculosis;

Pleural effusion

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Abstract Background: Appropriate diagnostic methods for tuberculous pleural effusion are vital.

The IFN-c tests using specific Mycobacterium Tuberculos is antigens in samples from the site of

infection may be promising in diagnosis of tuberculosis. Objective we examined the ability of

ELISpot test using circulating peripheral blood mononuclear cells (PBMC) and compartmentalized

pleural fluid mononuclear cells (PFMC) for diagnosis of active TB infection in patients with tuber-

culous pleural effusion. Methods PBMC and PFMC-based ELISpot test for IFN-c test using

specific M. tuberculosis antigen: Early Secretory Antigen Target-6 protein (ESAT-6) was used for

diagnosis of active TB infection. Thirty-five patients with clinically suspected tuberculous pleural

effusion were enrolled over a 12-month period. Results 11 patients out of 35 were positive by culture

and PCR (31.4%). Incubation of PBMC with ESAT-6 for 8 h showed sensitivity and specificity of

82% and 92%, respectively, for the PBMC–ELISpot as compared to PFMC–ELISpot that was

54% and 96% respectively. With 24 h incubation of ESAT-6 there was around 2.5 fold increase

in the median number of spot forming cells (SFCs) in PFMC from 30 to 74, whereas there was min-

imal increase of median number of SFCs in PBMC from 55 to 60. Conclusion ESAT-6 – ELISpot

using PBMC and PFMC is useful as a tool for diagnosis of TB effusion. PFMC needs longer period

of incubation for processing of ESAT-6 than PBMC. Moreover, IFN-c in pleural effusion (PE) is

another useful way for diagnosis of TB pleurisy which is sensitive, simple and cheap.ª 2012 The Egyptian Society of Chest Diseases and Tuberculosis. Production and hosting by Elsevier B.V.

All rights reserved.

(W. Shouman).

e Egyptian Society of Chest

g by Elsevier

of Chest Diseases and Tuberculos

.012

Introduction

Tuberculosis (TB) is a major public health problem and it isestimated by the World Health Organization that there areapproximately 9.4 million incident cases, and 1.3 million

deaths among HIV-negative people [1]. The disease is espe-cially prevalent in developing countries, where it accounts for

is. Production and hosting by Elsevier B.V. All rights reserved.

140 W. Shouman et al.

more than a quarter of all preventable adult deaths [2]. Thediagnosis of tuberculous pleural effusion remains a seriousclinical problem. Routine analysis of pleural fluid, signs and

symptoms and radiological finding are often inadequate forstarting of empirical therapy.

Currently, the only sure criterion for definite diagnosis of

TB is the demonstration of the presence of tubercle bacilli inclinical specimens. This is based on traditional methods: theZiehl-Neelsen (ZN) acid fast stain and laboratory culture of

Mycobacterium tuberculosis on Lowenstein Jensen media (LJ)medium. However, ZN stains lacks specificity and sensitivity,whilst confirmation by culture requires several weeks [3].Although, new rapid diagnostic culture methods have been

investigated that are based on either bacteriophage detectionmethods [4] or liquid culture technique such as BACTEC [5],they require specialized personnel and equipment. In addition

their expenses have limited their use in many routine diagnos-tic laboratories.

Several newly developed diagnostic assays for TB based on

M. tuberculosis – specific antigens encoded by genes in the re-gion of difference 1 (RD1 region) gave promising results fordiagnosis of M. tuberculosis infection. These specific antigens

such as early secreted antigenic target-6 (ESAT-6) and culturefiltrate protein-10 (CFP-10) are proved to be accurate markerof M. tuberculosis infection and to distinguish M. tuberculosisinfection from the response to BCG vaccination [6].

ESAT-6 and CFP-10 forms 1:1 complex when co expressedin an engineered BCG strains which found to be difficult to beprocessed and presented to T Cells. Consequently, single anti-

gen is preferred to be used rather than ESAT-6 and CFP-10form [7]. ESAT-6 or CFP-10 have been frequently used in En-zyme-linked immunospot (ELISpot) [8–10], Flow-cytometry

[7,11–14] and Quantiferon assays [15,16]. The usefulness ofthese assays for diagnosis of tuberculous pleural effusion in ac-tual clinical practice is limited especially in endemic area. It has

been shown that mononuclear cells (MC) compartmentalizedin the infected sites such as pleural fluid [17], bronchoalveolarlavage fluid [18] and CSF [12,19] secrets higher IFN-c in in-fected sites than peripheral blood mononuclear cells.

In this study we aimed to evaluate the ability of ELISpottest using circulating peripheral blood mononuclear cells(PBMC) and compartmentalized pleural fluid mononuclear

cells (PFMC) for diagnosis of active TB infection in patientswith tuberculous pleural effusion.

Figure 1 IFN-c producing T-cell responses (A) (Negative

control), (B) (Positive Control) and (C) (ESAT-6).

Materials and methods

This study was done at Chest and Microbiology & Immunol-ogy Departments, Zagazig University Hospitals between

November 2009 and October 2010. Written informed consentswere taken from all subjects.

Thirty-five patients with suspected TB pleural effusion(ages 37 ± 11.2 years, male/female = 24/11) were included.

The following were done:

(1) Full clinical examination.

(2) Plain chest X ray and chest CT(3) Pleural fluid tapping was performed according to the

standards procedures (20). Cytological, microbiological

ZN stain, culture, and pleural fluid PCR were processedusing standard techniques [8,12].

(4) Thoracoscopy and/or Abram’s pleural biopsy was per-

formed to cases in which final diagnosis was not reachedby the previous procedures.

(5) A total of nineteen patients with non-tuberculous pleu-

ral effusion were enrolled as a control in this study (ages32 ± 7 years, male/female = 15/4).

Antigen

ESAT-6 was produced as recombinant proteins in Escherichiacoli (13).

Table 1 Clinical features of patients enrolled (n= 35).

Number

Features:

Male 24 (68.5%)

Female 11 (32.2%)

Age 37 ± 11.2

Chest X-ray infiltrates 4 (11.4%)

Clinical Symptoms:

Cough 14 (40%)

Expectoration 10 (28.5%)

Haemoptysis 3 (8.5%)

Breathlesnes 27 (77.1%)

Chest pain 15 (20%)

Fever 9 (25.7%)

Weight loss 17 (48.5%)

ESAT-6-ELISpot and interferon c in the diagnosis of pleural 141

ELISpot assay

MTB-specific assay was performed using test–plates from R &D systems (EL285-Human IFN-c ELISpot Kit). PBMC andPFMC were prepared by Ficoll-Hypaque gradient centrifuga-

tions from 5-ml heparinised blood and 50 ml to 75 ml pleuralfluid respectively. Briefly, 250,000 PBMC or 250,000 PFMCwere plated on two plates pre-coated with anti-human IFN-cantibody after stimulation with ESAT-6 peptide (5 lg/ml final

concentration). One plate was incubated for 8 h. and the otherone was incubated for 24 h. at 37 �C in a humid atmosphere of5% CO2. Spots-counting and analysis were performed accord-

ing to manual’s instruction. For each sample, negative controlsample (unstimulated) and a positive control (stimulated withphorbol myrisate acetate (P-1585, Sigma) at a final concentra-

tion of 20 ng/ ml and Ionomycin (I-0634, Sigma) at 1 lM wereincluded Figure 1. The test was scored positive if the averagenumber of SFCs was higher than the average number of SFCs

in controls using ROC curve test. Background number of spotsin negative control was always less than 10 and positive controlmore than 50.

PE – IFN-c

IFN-c in pleural effusion (PE) was measured using ELISA kit(Biosource Europe, Belgium).

Tuberculin Skin test (TST)

The TST is performed by injecting 0.1 ml of tuberculin purified

protein derivative (PPD) into the inner surface of the forearmintradermally. The skin test reaction read between 48 and 72 hafter administration.

Statistical analysis

Statistical analysis was performed by SPSS (SPSS Inc., Chicago,II, USA). TheMann-Whitney andROC curve test were used for

comparison in the patient group between different tests.

Results

Thirty-five patients with suspected tuberculous pleural effusionwere prospectively enrolled in this study. Eleven patients haddefinite diagnosis of tuberculous pleural effusion either by cul-

ture or PCR or presence of caseating granuloma in the pleuralbiopsy taken by Abram’s blind needle biopsy or thoracoscopy.The other 24 patients were considered to have presumptive

tuberculosis. Clinical characteristics of the patients’ groupare shown in Table 1. Nineteen controls with non – tubercu-lous pleural effusion; 12 parapeumonic, 6 malignant and 1

with hepatic hydrothorax were included.Among all of the 11 patients with confirmed tuberculous

pleural effusion, the ELISpot TB were positive in (9/11)81.8% in PBMCs (median number of SFCs was 60 out of

250,000 T cells) and (6/11) 54.5% in PFMCs (median numberof SFCswas 40 out of 250,000T cells)Figure 2. In the 24 patientswith presumptive diagnosis of TBPE, the ELISpot TB were po-

sitive in 3/24 (12.5%) in PBMC and 1/24 (4%) in PFMC.More-

over, the median number of SFCs was 20 out of 250,000 T cellsfor both PBMCs and PFMCs. Themedian number of SFCs was

20 and 18 for PBMCs and PFMCs respectively in the controlgroup. The number of SCFs in PBMCs and PFMCs was statis-tically different between controls and patients with confirmed

tuberculosis (p < 0.001). The sensitivity and specificity ofESAT-6-ELISpot for the diagnosis of TBPE was 82% 92%,respectively, for PBMCs whereas those for PFMC were 54%

and 96% respectively (Table 2).In the 11 confirmed cases, the PBMC ELISpot results did

not correlate with PFMC in five cases: four cases were positiveby PBMC (one of them gave borderline results and the other

three were negative by PFMC), the fifth case gave positive re-sult by PFMC and was negative by PBMC. This case was alsopositive by PCR.

To demonstrate the effect of the time of incubation in activa-tion of primed T cells. The separated PBMCandPFMC from10patients were cultured for 24 h in the presence of ESAT-6 and

IFN-c production was assessed by ELISpot test. The mediannumber of SFCs increased from 55 to 60 in PBMCs whereasthere was a significant increase in the median number of SFCsin PFMCs from 30 to 74 after the addition of ESAT-6 for 24 h

Figure 3. With 24 h incubation, the sensitivity and specificityof both PBMC and PFMC were 100% and 60% respectively.In comparison, the 8 h culture PBMC had sensitivity and spec-

ificity of 100% and 60% whereas, these of PFMCs were 40%and 80% respectively for the same 10 patients (Table 3).

The size of indurations of TST did not match with the

intensity of SFC. Moreover, TST had a sensitivity of 70%which is lower than that of PBMC–ELISpot (82%) and higherthan that of PFMC (54%). However, the specificity of TST

(88%) was lower than both PBMC–ELISpot (92%) andPFMC (96%).

Four patients out of 35 gave intermediate ELISpot result(around 30 spots) by both PBMC and PFMC: The culture

was negative for those four patients but there was only one pa-tient had positive tuberculin and PCR results.

Pleural fluid IFN-c level in 11 patients with confirmed po-

sitive cases and presumptive TBPE were 5.5 and 1.0 pg/ml,respectively, whereas this for non tuberculous effusion was0.45. There was significant difference between the three groups

(p 6 0.01). Interestingly, there was one case which was positiveby TST, PBMC and PFMC and PE – IFN-c and was negativeby both culture and PCR.

Figure 2 Concentration of ESAT-6 – IFN-c producing SFCs after 8 h incubation in PBMCs or PFMCs of 35 patients with suspected

pleural tuberculosis.

Table 2 Sensitivity and Specificity of different tests among 35

cases with suspected TBPE after 8hrs incubation of ESAT-6.

Test No of

positive cases

Sensitivity

(%)

Specificity

(%)

ESAT-6 PBMC (8 h) 12 (35) 82 92

ESAT-6 PFMC (8 h) 7 (35) 54 96

IFN–c in PE 10 (35) 85.7 95.6

Table 3 Comparison of 8 h and 24 h. incubations of ESAT-6

among 10 random cases with suspected tuberculous pleural

effusion.

Test No of

positive cases

Sensitivity

(%)

Specificity

(%)

ESAT-6 PBMC (8 hrs) 7(10) 100 60

ESAT-6 PFMC (8 h) 3(10) 40 80

ESAT-6 PBMC (24 h) 7 (10) 100 60

ESAT-6 PFMC (24 h) 7(10) 100 60

142 W. Shouman et al.

PBMCs or PFMCs of random 10 patients with suspectedpleural tuberculosis.

Discussion

IFN-c based assays have been studied in the diagnosis of extra-

pulmonary TB [17,19–22]. In tuberculous pleural effusion anti-gen specific memory T-lymphocytes migrate from blood to thesite of infection and release T-helper cell cytokines up on con-

tact with antigens. Diagnosis of TB from antigen specific T-cells cytokines release have been extensively studied usingRD-1 encoded antigens such as ESAT-6 and CFP-10. More-

over, it is showed that the sensitivity and specificity ofESAT-6 antigen alone is higher than CFP-10 and ESAT-6+CFP10. While antigen-specific–T-cells cytokines release assaysin blood have better sensitivity and specificity than other

Figure 3 Concentration of ESAT-6 – IFN-c producing SFCs after 2

suspected pleural tuberculosis.

assays such as tuberculin skin test (TST), they cannot distin-guish between active and latent TB. Furthermore, it has been

demonstrated that using antigen-specific T-cells cytokines re-lease assays directly on mononuclear cells from the site ofinfection has better performance in the diagnostic ability in

discrimination between active and latent infection (12).However, there are no detailed studies in our area on the

diagnostic ability of the ESAT-6-specific T-cells cytokines re-

lease assays in discrimination between active and latent infec-tion. Also, there is no adequate evidence on effect ofincubation time and processing of antigens on production ofthe antigen-specific-cells cytokines.

4 h incubation in PBMCs or PFMCs of random 10 patients with

ESAT-6-ELISpot and interferon c in the diagnosis of pleural 143

In the present study, diagnosis of tuberculous pleural effu-sion by enumerating ESAT-6 – specific T-cells from the periph-eral blood and pleural fluid was evaluated. In confirmed TB

positive patients the mean numbers of ESAT-6–SFCs inPBMC ELISpot was higher than that of PFMC ELISpot by1.5 fold. With 8 h of incubation, our result is similar to that

of Baba and his colleagues [16], who found that QFT–TB inblood was higher than that of pleural fluid with sensitivity of71% and 44% respectively. However, this result is not in

agreement with study done by Losi and his colleagues [8],who found that PFMC has a high sensitivity of 95% whichis 7.5% higher than PBMC suggesting a compartmentalizationof antigen-specific T-cells in TBPE. The possible explanation

may be explained by the different ethnic background of pa-tients and different kits (T Spot.TB Kit) in Losi’s study.

The ESAT-6-ELISpot specificity in our study was 82% and

96% for both PBMC and PFMC respectively. Three out of 35patients had a false positive result. One of these patients wasfalse positive by PBMC and PFMC and positive by TST as

well had a history of treatment of tuberculosis. One patientwas positive by PFMC and PCR and negative by PBMC–ELI-Spot assay. This patient was cured by antituberculous drugs.

This specificity test was higher than that of Losi and colleagueswho reported 76%.

The SFC increased by about 2.5 fold in PFMC when incu-bation time increased from 8 h to 24 h However, there was

slight increase in the no of SFC in PBMC. The results showthat increase time of incubation allows longer time for anti-gen processing by monocytes in PFMC for digestion of pep-

tide. Moreover, increase time of incubation may havedifferential expression of cytokines profile between PBMCand PFMC.

Measurement of whole IFN-c in the pleural fluid has beenused as diagnostic markers for tuberculous pleural effusion[23]. In this study, IFN-c in pleural fluid has sensitivity of

85.7 and specificity of 95.6 which is comparable to that ofHiraki [23]. The sensitivity of IFN-c was the same as that ofELISpot PBMC (85.75) and higher than that of PFMC(71.4). The specificity of IFN-c in pleural fluid was higher than

that of PBMC–ELISpot (85.7 vs (82%) & similar to that ofPFMC-ELISpot (96%).

Conclusion

ESAT-6-ELISpot using PBMC and PFMC is useful adjuncttool for diagnosis of TB effusion. However, PFMC needs long-

er period of incubation for processing of ESAT-6 than PFMC.Moreover, IFN-c in pleural fluid is another useful way fordiagnosis of TB pleurisy which is sensitive, simple and cheap.

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