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 Table of Contents  
ORIGINAL ARTICLE
Year : 2017  |  Volume : 12  |  Issue : 2  |  Page : 93-98

Study of serum interferon gamma in tubercular pleural effusions


Department of Respiratory Medicine, Jawaharlal Nehru Medical College, Wardha, Maharashtra, India

Date of Web Publication8-Sep-2017

Correspondence Address:
Saood Ali
Department of Respiratory Medicine, Jawaharlal Nehru Medical College, Sawangi Meghe, Wardha - 442 001, Maharashtra
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jdmimsu.jdmimsu_53_17

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  Abstract 

Background: Pleural effusions (PEs) are a common reason for admission in Acharya Vinoba Bhave Rural Hospital, and in developing countries like India, tuberculosis (TB) is the most common cause of exudative PE. The diagnosis of tuberculous PE with standard analysis of pleural fluid is often inaccurate. Pleural tissue histopathology and culture for Mycobacterium tuberculosis are considered the gold standard, but the procedure is invasive, requires skilled clinicians, and does not provide immediate results. To address these limitations, we evaluated whether serum interferon gamma (IFN-γ) can be used as a diagnostic aid for tubercular PEs and to compare its efficacy with adenosine deaminase (ADA) and cytology of pleural fluid. Materials and Methods: The sample size was 86 patients aged 16 years and above and who were diagnosed having tubercular PE either on the basis of ADA or cytology or by both methods, and serum IFN-γ levels were evaluated in these patients. Results: Serum IFN-γ was significantly raised in patients with tubercular PE. Conclusion: Pleural fluid ADA and IFN-γ are almost equally sensitive in diagnosing tubercular effusion; however, in cases where obtaining pleural fluid or other diagnostic material is difficult, serum IFN-γ can be considered as aid in diagnosing TB.

Keywords: Adenosine deaminase, cytology, interferon gamma, pleural effusion, tuberculosis


How to cite this article:
Ali S, Ghewade B, Jadhav U, Cladius S. Study of serum interferon gamma in tubercular pleural effusions. J Datta Meghe Inst Med Sci Univ 2017;12:93-8

How to cite this URL:
Ali S, Ghewade B, Jadhav U, Cladius S. Study of serum interferon gamma in tubercular pleural effusions. J Datta Meghe Inst Med Sci Univ [serial online] 2017 [cited 2017 Sep 26];12:93-8. Available from: http://www.journaldmims.com/text.asp?2017/12/2/93/214191


  Introduction Top


A pleural effusion (PE) is an excessive accumulation of fluid in the pleural space, which indicates an imbalance between pleural fluid formation and removal. The normal pleural space contains a relatively small amount of fluid, 0.1–0.2 mL/kg of body weight on each side.[1]

PE can be of two types: transudative and exudative. Transudative PEs are caused by fluid leaking into the pleural space and increased hydrostatic pressure or decreased oncotic pressure. Exudative effusions are caused due to increased capillary permeability, which may be due to blocked blood vessels, inflammation, lung injury, or drug reactions, which cause damage or disruption of pleural membranes or vasculature.

Each year, approximately 1 million patients develop PE. PEs may occur with many different infections or as a complication of pulmonary disease. In addition, PEs frequently complicate malignant disease, heart disease, liver disease, gastrointestinal disease, kidney disease, and collagen vascular disease.[2]

Tuberculosis (TB), the single most frequent infectious cause of death worldwide, also is a major cause of PE, where the fluid has lymphocytic and exudative characteristics. Differential diagnosis between TB and nontuberculous PE can be sometimes difficult, representing a critically important clinical problem.[3] The diagnosis of tuberculous pleuritis should be considered in any patient with an exudative PE. Tuberculous PE is the second most common extrapulmonary manifestation of active Mycobacterium tuberculosis (MTB) infection after lymph node TB, accounting for up to 23% of TB cases and 30% of PEs in Western Europe.[4],[5] PE occurs in approximately 5% of patients with TB. TB may account for 14% of all pleural exudates and 25% of all PEs in areas with high incidence of TB.

Pleural biopsy for culture of MTB and histopathological detection of caseating granulomas are regarded as the gold standard for the diagnosis of tuberculous PE, with sensitivities of 39%–80% and 50%–97%, respectively. Pleural biopsy is performed because the chances of obtaining a diagnosis are much greater than examination of pleural fluid alone, but it is more invasive, requires greater expertise, and is subject to sampling errors.[6]

The laboratory tests on pleural fluid which help in etiological diagnosis of PE include cell count (total and differential count) and cytology, biochemical tests (protein, lactate dehydrogenase [LDH], adenosine deaminase [ADA]), and microbiological tests (Ziehl–Neelsen [ZN] stain, culture for aerobic bacteria, fungi, and Mycobacteria).

Though easier to collect, examination of the pleural fluid by cytology requires an experienced pathologist and culture or ZN stain has poor sensitivity for tuberculous PE.

Pleural fluid ADA levels are very useful in diagnosing pleural TB. It was first proposed as a diagnostic test in 1978,[7] and since then, it is frequently used in diagnosis of tubercular effusions. However, ADA is also raised in other conditions such as empyema and rheumatoid arthritis [8] and in small percentages in Q fever,  Brucellosis More Details, some neoplasms, and legionnaires disease; hence, making the diagnosis of tubercular effusion on ADA alone is difficult.

Therefore, diagnosis of tuberculous PE is typically made based on a compatible clinical presentation and the presence of suggestive pleural fluid characteristics including elevated protein levels and lymphocytosis.

More recently, MTB-specific T-cell interferon (IFN)-γ release assays (TIGRAs) have been developed for the diagnosis of MTB infection from the peripheral blood. While these tests are unable to distinguish between active and latent TB when performed on blood samples, recent studies report high sensitivity and specificity for tuberculous PE when TIGRAs are performed on the pleural fluid.[9] This study was designed to determine whether serum IFN-γ can be reliably sampled from tuberculous PE suspects admitted to Acharya Vinoba Bhave Rural Hospital (AVBRH), and if so, whether they provide data regarding the sensitivity and specificity of the assay in a TB-endemic setting.


  Materials and Methods Top


The cross-sectional study was conducted at AVBRH, Sawangi (Meghe), in indoor patients of tubercular PE diagnosed with the help of ADA and cytology or both. The study included 86 patients who were admitted (inpatient department [IPD]) in AVBRH.

A structured standard pro forma was prepared and used for recording the history, symptoms, side of effusion, hemogram and erythrocyte sedimentation rate, pleural fluid findings, which included pH, sugar, proteins, LDH, total leukocyte count (TLC) and differential leukocyte count (DLC), and ADA. Serum IFN-γ was also recorded.

The study was conducted from July 2014 to June 2016.

Inclusion criteria

Patients with diagnosed tubercular effusion by ADA, cytology or both, who were admitted (IPD) at AVBRH and were of age 16 years and above were included.

Exclusion criteria

Patient unwilling for consent were excluded.

These patients were assessed on the basis of the following clinical symptoms:

  1. Dry cough
  2. Chest pain
  3. Shortness of breath or dyspnea
  4. Fever
  5. Hemoptysis
  6. Weight loss
  7. Loss of appetite
  8. Any other.


Chest X-ray

Every patient enrolled in this study underwent chest X-ray posteroanterior view.

Features that were noted on chest X-ray included side of PE, hydropneumothorax if present, and cavitation.

Pleural fluid aspiration and cytology

Pleural aspiration was done and the fluid collected was sent for biochemical and cytological testing, which help in the further diagnosis of tubercular effusion.

Pleural fluid investigations

Aspirated pleural fluid was sent for routine biochemical analysis which included pH, sugars, proteins, LDH, TLC, and DLC, which included polymorphs and lymphocytes. Fluid collected was sent for ADA and cytological examination. Pleural fluid was analyzed using lights criteria which suggested a pleural fluid protein/serum protein >0.5 as an exudative effusion.

A cutoff of 36 IU/L for ADA was taken in diagnosing tubercular effusion.

Serum interferon gamma

Patients in whom either ADA or cytology was suggestive of tubercular effusion were selected to evaluate for serum IFN-γ levels. Venous blood was collected in plain bulbs, which was centrifuged to obtain serum, and then samples were deep frozen at −20°C. Similar methods were done until a slot of 36 samples was achieved which was the minimum sample requirement to procure IFN-γ test.

Out of 96 tests which could be performed by IFN-γ-EASIA Kit, ten were used as a control and standard and the rest 86 samples were evaluated by the kit.

Method for calculating interferon gamma levels by ELISA method

DIA source IFN-γ-EASIA Kit with catalog number KAP1231 was used for the analysis of serum interferon levels. Each kit could perform 96 tests. It was manufactured by DIA source ImmunoAssays S. A. Rue du Bosquet, 2, B-1348 Louvain-la-Neuve, Belgium.


  Observations and Results Top


[Table 1] shows age-wise distribution of patients in the study population. Maximum number of patients belonged to the age group 46–55 years (21 patients), followed by 26–35 years (20 patients). Patients with older age group were the least age group, i.e., 66–75 years, which included only six patients.
Table 1: Age-wise distribution of study populations

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[Table 2] shows gender-wise distribution of patient in this study population. Maximum number of patients, i.e. 62 (72.09%) of 86, were males and females were only 24 (27.90%).
Table 2: Gender-wise distribution of study populations

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[Table 3] shows the distribution of patients diagnosed with tubercular effusion by cytology, ADA, or both. Eighty patients (93.03%) had cytology positive for TB, out of which 58 were males and 22 were females. Eighty-four patients (97.67%) had a positive ADA >36 IU/L which was taken as cutoff for diagnosing TB. Out of these 84 patients, 61 were males and 23 were females. Seventy-eight patients (90.69%) had both cytology and ADA positive, which included 57 males and 21 females.
Table 3: Distribution of patients according to diagnostic modalities

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Majority of patients in this study were from age 26–55 years. The mean age group with interferon levels <0.28 was 45.06 ± 18.14, and mean age group with interferon levels >0.28 was 41.08 ± 14.42. This relation was not statistically significant [Table 4].
Table 4: Age-wise distribution of patients in relation with interferon gamma

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Dry cough and dyspnea (79.07%) were the predominant chief complaints in this study, followed by fever in 68.60% patients and chest pain in 56.98% patients. Hemoptysis was the least common symptom occurring in only 8.14% patients [Table 5].
Table 5: Distribution of patients according to complaints in relation with interferon gamma

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Pleural fluid pH, sugar, protein, polymorph, and lymphocyte showed no major difference in both groups, and their relation was statistically not significant. LDH and TLC showed a raised value in group with a low INF-γ, and LDH had a mean of 1615.86 ± 3022.20 in group with low INF-γ as compared to 775.08 ± 685.02 in group with positive INF-γ. TLC was also raised in group with a low INF-γ with a mean of 2195.53 ± 3131.25 as compared to 639.52 ± 742.56 in group with a positive and raised INF-γ [Table 6].
Table 6: Comparison of pleural fluid parameters in two groups

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Cytology was tubercular in 80 (93.02%) cases, out of which 65 (81.25%) cases had a raised serum INF-γ. Six cases had a positive or raised serum IFN-γ, and in those, cytology was not tubercular. Hence, such cases should be evaluated further and only cytology should not be considered for diagnosing TB [Table 7].
Table 7: Association of cytological findings with interferon gamma

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  • Sensitivity = 100% (95% confidence interval [CI] = 78.20%–100%)
  • Specificity = 8.45% (3.16%–17.49%)
  • Positive predictive value = 18.75% (95% CI = 10.89%–29.03%)
  • Negative predictive value = 100% (54.07%–100%)
  • Accuracy = 24.41%.


[Table 8] shows that out of total 86 cases, 84 (97.67%) had a positive and raised ADA. Out of these 84 cases, 71 cases (84.52%) showed both a positive ADA and INF-γ. Two cases were ADA and INF-γ negative. This relation was statistically significant.
Table 8: Association of adenosine deaminase with interferon gamma

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  • Sensitivity = 86.67% (95% CI = 59.54%–98.34%)
  • Specificity = 0%
  • Positive predictive value = 15.48% (95% CI = 8.50%–25.01%)
  • Negative predictive value = 0%
  • Accuracy = 15.11%.


The mean value of ADA was 59.83 with a standard deviation (SD) of 12.39. The mean value of INF-γ was 9.10 with an SD of 9.27 [Table 9].
Table 9: Correlation between adenosine deaminase level and interferon gamma

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[Graph 1] shows a linear progression between ADA and INF-γ. As ADA increases, IFN-γ also increases, and as ADA decreases, IFN-γ also decreases.




  Discussion Top


In the present study, maximum number of patients was from age 26–55 years. The mean age group with interferon levels <0.28 was 45.06 ± 18.14, and mean age group with interferon levels >0.28 was 41.08 ± 14.42, and in this study, 72.09% were males and 27.91% were females hence showing a male predominance; these findings were consistent with a study done by Das on 127 patients of malignant and tuberculous PE and he found that tuberculous effusion is a disease of younger people mostly below 40 years of age and majority of patients were male.[10]

Similarly, Liam et al. did a retrospective study on 186 patients with exudative PE, and they found a mean age (±SD) of 51.2 (±19.2) years with exudative PEs; 131 (70.4%) were males and 55 (29.6%) were females.[11] In a study done recently by Baumann et al., the mean age of 14,000 patients of tuberculous pleuritis reported to the Communicable Disease Center in the United States between 1993 and 2003 was 49.9 years.[12] In another series study done in Qatar by Ibrahim et al., the mean age of 100 patients with tuberculous pleuritis was 31.5 years.[13] Probable cause for male predominance in this study was that most of the males were alcoholic and smokers, which are the predisposing factors for TB.

Dry cough and dyspnea (79.07%) were the predominant chief complaints in this study, followed by fever in 68.60% and chest pain in 56.98% patients. Hemoptysis was the least common symptom occurring in only 8.14% patients. In studies done by Moudgil et al., Berger and Mejia, Richter et al., pleuritic chest pain was present in almost 75% cases and the second most common symptom was cough present in 70%.[14],[15],[16] In this study, dyspnea was the predominant complaint which could be because majority of patients had a large PE, leading to compressive symptoms such as dyspnea.

Pleural fluid pH, sugar, protein, polymorph, and lymphocyte showed no major difference in both groups and their relation was statistically not significant. LDH and TLC showed a raised value in group with a low INF-γ, and LDH had a mean of 1615.86 ± 3022.20 in group with low INF-γ as compared to 775.08 ± 685.02 in group with positive INF-γ. TLC was also raised in group with a low INF-γ with a mean of 2195.53 ± 3131.25 as compared to 639.52 ± 742.56 in group with a positive and raised INF-γ. Similarly, in a study done by Valdés et al. on 254 patients with tuberculous pleuritis, only 17 had fewer than 50% lymphocytes in the pleural fluid.[17] In another series study done by Bielsa et al. on 214 patients, only 11% had more than 50% pleural fluid polymorphonuclear leukocytes.[18] Ruan et al. did a study on 382 patients in Taiwan with tuberculous pleuritis and found that the median lymphocyte percentage of total cells in pleural fluids was 84% and 17% of cases had a lymphocyte percentage of <50%.[19]

Out of 86 cases, 84 (97.67%) had a positive and raised ADA. Out of these 84 cases, 71 cases (84.52%) showed both a positive ADA and INF-γ. Two cases were ADA and INF-γ negative. This relation was statistically significant. Ocaña et al. measured pleural fluid ADA levels in 221 PEs. All patients with a pleural fluid ADA level >70 U/L had TB, whereas no patient with a pleural fluid ADA level <40 U/L had tuberculous pleuritis.[20] Porcel et al. studied ADA levels in 2104 PEs which included 221 tuberculous pleuritic; they demonstrated a sensitivity of 93%, specificity of 90%, positive likelihood ratio of 10.05, and negative likelihood ratio of 0.07.[21] Liang et al. performed a meta-analysis of 63 articles evaluating the diagnostic usefulness of ADA that included 2796 patients with tuberculous pleuritis and 5297 patients with other diseases. They reported mean sensitivity of 0.92, mean specificity 0.90, mean positive likelihood ratio 9.03, and mean negative likelihood ratio 0.10.[22] Sharma et al. studied the ADA and pleural fluid IFN-γ in tubercular effusions. Krenke et al. compared the efficacy of ADA and IFN-γ levels in diagnosing tuberculous pleuritis. They studied 94 patients (50 males and 44 females, mean age 60 ± 18 years, range 18–95 years) with PE. Tubercular PE was diagnosed in patients with (i) positive pleural fluid or pleural biopsy culture or (ii) granulomas in the pleural biopsy specimen, after exclusion of other granulomatous diseases. Pleural fluid ADA activity was measured with the colorimetric method of Giusti, while IFN-γ level was measured with ELISA. Tubercular PE was diagnosed in 28 patients. The nontubercular PE group consisted of 35 patients with malignant PE, twenty patients with parapneumonic effusion/pleural empyema, five with pleural transudate, and six with miscellaneous PE. The ADA activity and IFN-γ concentration were significantly higher in tubercular PE than in nontubercular PEs (614.1 ± 324.5 vs. 15.1 ± 36.0 pg/mL, P < 0.0001 and 75.1 ± 39.1 vs. 11.0 ± 16.6 U/L, respectively, P < 0.0001). The diagnostic sensitivity and specificity of IFN-γ measurement (cutoff value of 75.0 pg/mL) were 100% and 98.5%, respectively, and were similar to those of ADA (100% and 93.9% at the cutoff value of 40.3 U/L).[23]

Katti did a study on 72 patients which included 33 untreated TB patients, 19 contacts of TB patients, and twenty healthy individuals. Interleukin-1 (IL-1), IL-2, and IFN-γ were determined in serum samples of all these patients; he found that an increased level of IFN-γ in patients with untreated TB.[24] Similarly, Hasan et al. did a study on serum levels of IFN-γ, IL-10, CXCL9, CCL2 in pulmonary and extrapulmonary patients. They found that IFN-γ is consistently raised in both pulmonary and extrapulmonary patients and levels of serum IFN-γ depend on the severity of disease.[25]


  Conclusion Top


We conclude that pleural fluid ADA and IFN-γ are almost equally sensitive in diagnosing tubercular effusion; however, in cases where obtaining pleural fluid or other diagnostic material is difficult, serum IFN-γ can be considered as diagnostic aid.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
  References Top

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Valdés L, Pose A, San José E, Martínez Vázquez JM. Tuberculous pleural effusions. Eur J Intern Med 2003;14:77-88.  Back to cited text no. 5
    
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Ocaña I, Ribera E, Martinez-Vázquez JM, Ruiz I, Bejarano E, Pigrau C, et al. Adenosine deaminase activity in rheumatoid pleural effusion. Ann Rheum Dis 1988;47:394-7.  Back to cited text no. 8
    
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Richeldi L. Rapid identification of Mycobacterium tuberculosis infection. Clin Microbiol Infect 2006;12 Suppl 9:34-6.  Back to cited text no. 9
    
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Das DK. Age and sex distribution in malignant and tuberculous serous effusions: A study of 127 patients and review of the literature. Geriatr Gerontol Int 2015;15:1143-50.  Back to cited text no. 10
    
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Liam CK, Lim KH, Wong CM. Causes of pleural exudates in a region with a high incidence of tuberculosis. Respirology 2000;5:33-8.  Back to cited text no. 11
    
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Baumann MH, Nolan R, Petrini M, Lee YC, Light RW, Schneider E, et al. Pleural tuberculosis in the united states: Incidence and drug resistance. Chest 2007;131:1125-32.  Back to cited text no. 12
    
13.
Ibrahim WH, Ghadban W, Khinji A, Yasin R, Soub H, Al-Khal AL, et al. Does pleural tuberculosis disease pattern differ among developed and developing countries. Respir Med 2005;99:1038-45.  Back to cited text no. 13
    
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Moudgil H, Sridhar G, Leitch AG. Reactivation disease: The commonest form of tuberculous pleural effusion in Edinburgh, 1980-1991. Respir Med 1994;88:301-4.  Back to cited text no. 14
    
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Richter C, Perenboom R, Mtoni I, Kitinya J, Chande H, Swai AB, et al. Clinical features of HIV-seropositive and HIV-seronegative patients with tuberculous pleural effusion in Dar es Salaam, Tanzania. Chest 1994;106:1471-5.  Back to cited text no. 16
    
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Valdés L, Alvarez D, San José E, Penela P, Valle JM, García-Pazos JM, et al. Tuberculous pleurisy: A study of 254 patients. Arch Intern Med 1998;158:2017-21.  Back to cited text no. 17
    
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Bielsa S, Palma R, Pardina M, Esquerda A, Light RW, Porcel JM, et al. Comparison of polymorphonuclear- and lymphocyte-rich tuberculous pleural effusions. Int J Tuberc Lung Dis 2013;17:85-9.  Back to cited text no. 18
    
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Ruan SY, Chuang YC, Wang JY, Lin JW, Chien JY, Huang CT, et al. Revisiting tuberculous pleurisy: Pleural fluid characteristics and diagnostic yield of mycobacterial culture in an endemic area. Thorax 2012;67:822-7.  Back to cited text no. 19
    
20.
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21.
Porcel JM, Esquerda A, Bielsa S. Diagnostic performance of adenosine deaminase activity in pleural fluid: A single-center experience with over 2100 consecutive patients. Eur J Intern Med 2010;21:419-23.  Back to cited text no. 21
    
22.
Liang QL, Shi HZ, Wang K, Qin SM, Qin XJ. Diagnostic accuracy of adenosine deaminase in tuberculous pleurisy: A meta-analysis. Respir Med 2008;102:744-54.  Back to cited text no. 22
    
23.
Krenke R, Safianowska A, Paplinska M, Nasilowski J, Dmowska-Sobstyl B, Bogacka-Zatorska E, et al. Pleural fluid adenosine deaminase and interferon gamma as diagnostic tools in tuberculosis pleurisy. J Physiol Pharmacol 2008;59 Suppl 6:349-60.  Back to cited text no. 23
    
24.
Katti MK. Assessment of serum IL-1, IL-2 and IFN-γ levels in untreated pulmonary tuberculosis patients: Role in pathogenesis. Arch Med Res 2011;42:199-201.  Back to cited text no. 24
    
25.
Hasan Z, Jamil B, Khan J, Ali R, Khan MA, Nasir N, et al. Relationship between circulating levels of IFN-gamma, IL-10, CXCL9 and CCL2 in pulmonary and extrapulmonary tuberculosis is dependent on disease severity. Scand J Immunol 2009;69:259-67.  Back to cited text no. 25
    



 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6], [Table 7], [Table 8], [Table 9]



 

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