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Pleural fluid neopterin levels in tuberculous pleurisy
Clinical Biochemistry 40 (2007) 876 – 880
Pleural fluid neopterin levels in tuberculous pleurisy Gursel Cok a,⁎, Zuhal Parildar b , Gunes Basol b , Ceyda Kabaroglu b , Ulku Bayindir a , Sara Habif b , Oya Bayindir b b
a Department of Chest Disease, School of Medicine, Ege University, Bornova, 35100, Izmir, Turkey Department of Clinical Biochemistry, School of Medicine, Ege University, Bornova, 35100, Izmir, Turkey
Received 28 November 2006; received in revised form 14 April 2007; accepted 18 April 2007 Available online 27 April 2007
Abstract Objectives: Neopterin is produced by stimulated macrophages under the influence of gamma interferon of lymphocyte origin. It is regarded as a biochemical marker of cell-mediated immune response. This study was designed to assess the diagnostic value of pleural fluid neopterin levels in tuberculous pleurisy in comparison with adenosine deaminase activity. Design and methods: Pleural fluid adenosine deaminase (ADA) activity and neopterin levels were measured in 16 patients with tuberculous pleurisy (TP) and 19 patients with malignant pleurisy (MP). ADA activity was determined by a colorimetric method, whereas neopterin levels were determined by a reversed-phase liquid chromatography technique. All values were given as median (min–max). Results: The mean age was 45.43 ± 20.39 years in the TP group and 60.42 ± 11.02 years in the MP group (p = 0.026). The median pleural fluid ADA activity was 51.75 U/L (3.50–62.40 U/L) in the TP group and was 2.30 U/L (1–8.20 U/L) in the MP group. The difference was statistically significant (p b 0.001). The median pleural fluid neopterin levels were 13.15 nmol/L (1.86–59.50 nmol/L) and 2.44 nmol/L (0.92–27.60 nmol/L) in the TP group and the MP group, respectively (p = 0.021). In order to evaluate the diagnostic value of pleural fluid neopterin concentrations, receiver-operating-characteristic curve analysis was performed. Conclusion: Pleural fluid neopterin concentration is significantly higher in TP when compared to MP, however when compared, its clinical use as a diagnostic marker is not valuable as ADA. © 2007 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved. Keywords: Neopterin; Tuberculous pleurisy; Malignant pleurisy
Introduction Important developments in the diagnosis and treatment of tuberculosis have been achieved, but there still remain some difficulties in the diagnosis of tuberculosis forms such as tuberculous pleurisy (TP). In TP, the possibility of showing Mycobacterium tuberculosis bacilli in pleural fluid with staining for acid–fast bacilli is about 5% in TP and 10–20% of the patients cannot be diagnosed despite the histopathologic examination and culture of the pleural biopsy material [1]. For this reason, efforts to find an alternative diagnosis method still continue.
⁎ Corresponding author. Fax: +90 232 388 71 92. E-mail address: [email protected] (G. Cok).
Neopterin is a compound derived from pteridine being produced during guanosine triphosphate (GTP) metabolism [2,3]. Neopterin was isolated for the first time in 1967 [2,3]. Macrophages secrete neopterin, as a result of the stimulus provided by interferon gamma derived from T lymphocytes. Since the neopterin source is monocytes and macrophages, it is evaluated as the biological marker of cellular immunity [4–8]. Macrophages, but especially T lymphocytes play an important role in tuberculosis. In this sense, studies have been conducted on the role of neopterin in tuberculosis activity and increased neopterin levels in various body fluids due to tuberculosis activity and immune response were reported [9–14]. It is known that neopterin may increase in various body fluids in infectious diseases such as sepsis, hepatitis C, human immunodeficiency virus and in non-infectious cases such as neoplasms and autoimmune diseases [5,10].
0009-9120/$ - see front matter © 2007 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved. doi:10.1016/j.clinbiochem.2007.04.009
G. Cok et al. / Clinical Biochemistry 40 (2007) 876–880
While there are studies revealing that neopterin increases in pleural fluid in TP, they do not provide a strong ground to allow routine use [9,11,14]. There is only one study which has been conducted to determine the cut-off value, which may be used for the diagnosis of TP [9]. This study was designed as a prospective study for the purpose of determining the role of pleural fluid neopterin levels in the TP diagnosis and a possible cut-off value. Methods The patients with TP constituted the study group, while the patients with malignant pleurisy (MP) constituted the control group. For both groups, having diabetes mellitus, chronic liver disease or chronic renal failure was used as exclusion criteria. Nineteen patients with MP and 18 patients with TP participated in the study. Two patients with TP were excluded as they were diagnosed with chronic renal failure and thus the findings of remaining 16 patients were evaluated. These sixteen patients did not receive any antituberculosis treatment before. The golden standard for the diagnosis of tuberculosis is either the identification of mycobacterium tuberculosis bacilli in pleural fluid or biopsy samples, or demonstration of caseating granulomatous inflammation in pleural biopsies [15]. In 12 of the patients, caseating granulomatous inflammation in the pleural biopsy was detected, where as remaining four were diagnosed according to the following clinical data: 1. Age less than 35 years old 2. Positive tuberculin skin test (N15 mm) 3. High pleural fluid adenosine deaminase (ADA) activity (N50 U/L) 4. Presence of lymphocytic pleural fluid cytologically (lymphocyte ratio N 50%) 5. Good response to anti-tuberculous treatment and after exclusion of other diagnosis. Malignancy was excluded due to benign description of fluid cytology and biopsy materials, empyema was excluded with regard to biochemical and macroscopic findings, effusions due to rheumatological disease were excluded by patients' clinical symptoms. Concerning the possible effects of chemotherapy on neopterin levels patients who did not receive any therapy were chosen for the MP group. The primary diagnosis of patients with MP were as follows: 9 squamous cell lung carcinoma, 3 adenocarcinoma, 5 non-small-cell lung carcinoma (could not be differentiated) and 2 primary breast cancer. Pleural fluid and serum samples were collected concurrently from each patient. Lactate dehydrogenase (LDH) activity, protein and glucose concentrations in serum and pleural fluid were determined by standard laboratory methods on an autoanalyzer. Pleural fluid and serum adenosine deaminase activity was determined by the colorimetric method described by Giusti and Galanti [16]. Pleural fluids, stored at −80 °C and protected from light, were thawed to measure neopterin levels by a reversed-phase chromatography technique described by Werner et al. [17].
877
SSPS 13.0 statistical program was used for statistical analysis. All values were given as median (min–max), unless stated otherwise. Mann–Whitney U and logistic regression analysis were conducted when appropriate. The value p b 0.05 was accepted as statistically significant. The diagnostic values of pleural fluid neopterin levels were calculated as sensitivity, specificity, negative predictive value (NPV) and positive predictive value (PPV). To illustrate the diagnostic performance of neopterin, a receiver-operating-characteristic curve was reported and the area under curve was calculated. Spearman's correlation analysis was performed to demonstrate a possible correlation between ADA activity and neopterin values. Results Thirteen (81.3%) male and three (18.7%) female patients were in the TP group. Out of the 19 patients in the MP group, 12 patients (63.2%) were male and 7 patients (36.8%) were female. The mean age in the TP and the MP groups were 45.43 ± 20.39 years and 60.42 ± 11.02 years, respectively (p = 0.026). Table 1 summarizes the measured results of the investigated parameters in the TP and MP groups. The median pleural fluid ADA activity was 51.75 U/L (3.50–62.40 U/L) in the TP group and was 2.30 U/L (1–8.20 U/L) in the MP group (p b 0.001). For ADA activity, at 40 U/L cut-off value, sensitivity was calculated as 75%, specificity as 100%, PPV as 100%, NPV as 82.6%. When the pleural fluid neopterin levels were considered, the TP group displayed significantly higher values (p = 0.021) (Fig. 1). After correcting for age, due to the significant age difference between the groups, the observed difference was still significant. In order to define a possible cut-off value for pleural fluid neopterin concentrations in the diagnosis of TP, four different concentrations were selected to calculate the diagnostic performance parameters. These results are summarized in Table 2. For pleural fluid neopterin levels, at 20 nmol/L concentration, sensitivity was 43.75%, specificity was 84.21%, positive predictive value was 70% and negative predictive value was 64%. Using 25 nmol/L as the cut-off value, the sensitivity was calculated as 31.25%, specificity was 94.73%, positive predictive value was 83.33% and negative predictive value was 62.06%.
Table 1 The results of the investigated parameters in the pleural fluid and serum
P/LDH (U/L) S/LDH (U/L) P/Protein (g/dL) S/Protein (g/dL) P/Glucose (mg/dL) S/Glucose (mg/dL) P/ADA (U/L) P/Neopterin (nmol/L)
TP (n = 16)
MP (n = 19)
p
627.50 (232–4495) 445.50 (283–1687) 4.95 (3.20–6.30) 6.90 (5.30–7.90) 79.50 (48–121) 95 (81–215) 51.75 (3.50–62.40) 13.15 (1.86–59.50)
596 (275–5600) 496 (238–873) 4.10 (3.10–5.90) 6.70 (3.80–7.70) 93 (10–151) 112 (80–179) 2.30 (1–8.20) 2.40 (0.92–27.60)
0.947 0.934 0.131 0.630 0.136 0.064 b0.001 0.021
Data are expressed as median (min–max). P: pleural fluid, S: serum, LDH: lactate dehydrogenase, ADA: adenosine deaminase, TP: tuberculous pleurisy, MP: malignant pleurisy.
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Fig. 1. The distribution of neopterin levels in pleural fluid in TP and MP groups. The median (min–max) neopterin concentration was 13.15 nmol/L (1.86–59.50 nmol/L) and 2.40 nmol/L (0.92–27.60 nmol/L), respectively. The difference was significant (p = 0.021). TP: tuberculous pleurisy, MP: malignant pleurisy.
In the TP group, there was one patient with an extremely high plasma neopterin value, 59.5 nmol/L. He was 36 years old with pleural effusion on the right side. In his pleural biopsy, the typical caseating granulomatous inflammation was shown. The following results were obtained in his pleural fluid sample; LDH activity was 368 U/L, glucose was 96 mg/dL, total protein was 4.8 g/dL and ADA activity was 62.30 U/L. The measured serum values were as follows; LDH activity was 463 U/L, glucose was 108 mg/dL, total protein was 6.3 g/dL. Receiver-operating-characteristic curve (ROC) analysis for neopterin levels and ADA activities is given in Fig. 2. Spearman's correlation analysis revealed a significant correlation between ADA activity and neopterin values (rs = 0.348, p = 0.04). Discussion The World Health Organization reported the occurrence of approximately 8 million new tuberculosis cases around the world in 1998 [18]. Tuberculous pleurisy, which is one of the important forms of extra-pulmonary tuberculosis, constitutes a
Table 2 The diagnostic performances of pleural fluid neopterin levels at different selected cut-off values Cut-off value (nmol/L)
Sensitivity (%)
Specificity (%)
PPV (%)
NPV (%)
15 20 25 30
50.00 43.75 31.25 12.50
73.68 84.21 94.73 100
61.53 70.00 83.33 100
63.63 64.00 62.06 57.57
PPV: positive predictive value, NPV: negative predictive value.
Fig. 2. Receiver-operating-characteristic curve analysis for pleural fluid neopterin and ADA levels. The calculated areas under the curves are 0.729 (S.E. = 0.085, 95% CI, p = 0.021) for neopterin and 0.980 (S.E. = 0.021, 95% CI, p b 0.001) for ADA.
major cause for pleural effusions. However, its incidence rate varies from country to country. A study conducted in Spain revealed that tuberculous pleurisy is the cause of 25% of pleural effusions [19] and reported that this rate increases to 80–90% in Africa [20,21]. It is known that this rate is below 5% in developed countries [22]. A study, which was conducted in Turkey and included 5480 patients with tuberculosis, reported that the rate of TP to the total number of tuberculosis cases is 6.7% [23]. Today, despite the high prevalence of the disease, difficulties are experienced in diagnosing the patients who carry other causes of exudative pleural effusions such as malignancies in the elderly. Absolute diagnosis of TP can be made by showing the reproduction of M. tuberculosis bacilli in the culture of the pleural fluid or pleural tissue samples or by showing caseous necrotic granulomas in the pleural biopsy. However, histological examination of needle biopsy material of pleura is nondiagnostic in 20–40% of patients with TP, and when the pleural biopsy is negative; mycobacteria can be cultured in pleural specimens in less than 10% of patients [15,24,25]. For this reason, studies on interferon gamma, ADA, antimicrobial antibodies and recently neopterin have been intensified for more accurate diagnosis [11,14,24,26]. Neopterin is produced by activated macrophages in response to interferon gamma of lymphocyte origin. Thus, it is believed that neopterin can be used as marker of cellular immunity [4–8]. It was reported that neopterin increases in various body fluids in patients with tuberculosis where cellular immunity plays an important role [9–14]. In our study, pleural fluid neopterin levels in the TP group were higher than the MP group. Baganha et al. was the first to compare 10 TP patients and 15 MP patients and reported that neopterin levels were higher both in the pleural fluid and the sera in TP group and thus it can be used in determining the pleural fluid aetiology and cellular immunity [11]. The correlation between serum and pleural fluid neopterin is well established, therefore in the
G. Cok et al. / Clinical Biochemistry 40 (2007) 876–880
current study assessment was made only in pleural fluid to save up from the cost [9,11,27]. Chiang et al. reported that pleural neopterin is lower in patients with MP than it is in patients with TP and that pleural neopterin level is below 25 nmol/L in 84% of the patients with MP [14]. Similarly, the neopterin level was found to be below 25 nmol/L in 94.73% of the patients with MP, in our study. After it was shown that neopterin can play a role in the TP diagnosis, Tozkoparan et al. studied 34 patients with TP and 29 patients with pleurisy of malignancy, parapneumonic effusion, congestive heart failure, empyema, pulmonary thromboembolism and liver cirrhosis, for the purpose of determining the cut-off point [9]. They decided on 30 nmol/L as the cut-off value and found the sensitivity as 85%, specificity as 93%, PPV as 94% and NPV as 84% [9]. In our study, at the same cut-off value, the sensitivity was found as 12.5%, specificity as 100%, PPV as 100% and NPV as 57.57%. The difference between our results and theirs may be attributed to their heterogenic group of 29 patients. We repeated the same calculations at cut-off values of 15, 20 and 25 nmol/L. Although it was found that cut-off values of 20 and 25 nmol/L were especially better in terms of sensitivity, when the performance characteristics were considered as a whole, the results were not sufficient. To evaluate the diagnostic accuracy at different cutoff levels, we performed ROC analysis for pleural fluid ADA activity and neopterin levels. The results of ROC analysis revealed 0.98 and 0.72 as areas under curve for ADA and neopterin, respectively. The significant correlation between ADA activity and neopterin values was expected since both molecules are secreted from macrophages under immune stimulation. The measurement of ADA activity is still the most valuable biochemical marker present in various body fluids in the diagnosis or differential diagnosis of tuberculosis [1,14,24,26,28– 35]. Determination of pleural fluid neopterin levels does not provide additional information in the differential diagnosis of TP and MP. Besides, with regard to neopterin, measuring ADA activity is more cost-effective and convenient for the routine practice. References [1] Valdes L, Pose A, San Jose E, Martinez Vazquez JM. Tuberculous pleural effusions. Eur J Intern Med 2003;14:77–88. [2] Sakurai A, Goto M. Neopterin: isolation from human urine. J Biochem 1967;61:142–5. [3] Anwaar I, GottsaE'ter A, Hedblad B, Palmqvist B, Mattiasson I, LindgaE'rde F. Endothelial derived vasoactive factors and leukocyte derived inflammatory mediators in subjects with asymptomatic atherosclerosis. Angiology 1998;49:957–66. [4] Hamerlinck FF, van Gool T, Faber WR, Kager PA. Serum neopterin concentrations during treatment of leishmaniasis: useful as test of cure? FEMS Immunol Med Microbiol 2000;27:31–4. [5] Berdowska A, Zwirska-Korczala K. Neopterin measurement in clinical diagnosis. J Clin Pharm Ther 2001;26:319–29. [6] Hoffmann G, Wirleitner B, Fuchs D. Potential role of immune system activation-associated production of neopterin derivatives in humans. Inflamm Res 2003;52:313–21.
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[7] Murr C, Widner B, Wirleitner B, Fuchs D. Neopterin as a marker for immune system activation. Curr Drug Metab 2002;3:175–87. [8] Fuchs D, Weiss G, Wachter H. Neopterin, biochemistry and clinical use as a marker for cellular immune reactions. Int Arch Allergy Immunol 1993; 101:1–6. [9] Tozkoparan E, Deniz O, Cakir E, et al. The diagnostic values of serum, pleural fluid and urine neopterin measurements in tuberculous pleurisy. Int J Tuberc Lung Dis 2005;9:1040–5. [10] Mohamed KH, Mobasher AA, Yousef AR, et al. BAL neopterin: a novel marker for cell mediated immunity in patients with pulmonary tuberculosis and lung cancer. Chest 2001;119:776–80. [11] Baganha MF, Mota Pinto A, Pego MA, Marques MA, Rosa MA, Cordeiro AJ. Neopterin in tuberculous and neoplastic pleural fluids. Lung 1992;170:155–61. [12] Horak E, Gassner I, Solder B, Wachter H, Fuchs D. Neopterin levels and pulmonary tuberculosis in infants. Lung 1998;176:337–44. [13] Yuksekol I, Ozkan M, Akgul O, et al. Urinary neopterin measurement as a noninvasive diagnostic method in pulmonary tuberculosis. Int J Tuberc Lung Dis 2003;7:771–6. [14] Chiang CS, Chiang CD, Lin JW, Huang PL, Chu JJ. Neopterin, soluble interleukin-2 receptor and adenosine deaminase levels in pleural effusions. Respiration 1994;61:150–4. [15] Ferrer J. Series ‘the pleura’. Pleural tuberculosis. Eur Respir J 1997;10: 942–7. [16] Giusti G, Galanti B. Colorimetric method. In: Bergmeyer HU, editor. Methods of Enzymatic Analysis. Veinheim: Verlag Chemie; 1984. p. 315–23. [17] Werner ER, Bichler A, Daxenblchier G, et al. Determination of neopterin in serum and urine. Clin Chem 1987;33:62–6. [18] World Health Organization. Global Tuberculosis Control 2000. [19] Valdés L, Alvarez D, Valle JM, Pose A, SanJosé E. The etiology of pleural effusions in an area with high incidence of tuberculosis. Chest 1996;109: 158–62. [20] Batungwanayo J, Taelman H, Allen S, Bogaerts J, Kagame A, Van de Perre P. Pleural effusion, tuberculosis and HIV-1 infection in Kigali, Rwanda. AIDS 1993;7:73–9. [21] Richter C, Perenboom R, Mtoni I, et al. Clinical features of HIVseropositive and HIV-seronegative patients with tuberculous pleural effusion in Dar es Salaam, Tanzania. Chest 1994;106:1471–6. [22] Marel M, Zrustová M, Stasny B, Light RW. The incidence of pleural effusion in a well-defined region: epidemiologic study in Central Bohemia. Chest 1993;104:1486–9. [23] Aktogu S, Yorgancioglu A, Çirak K, Köse T, Dereli SM. Clinical spectrum of pulmonary and pleural tuberculosis: a report of 5480 cases. Eur Respir J 1996;9:2031–5. [24] Diacon AH, Van de Wal WB, Wyser C, et al. Diagnostic tools in tuberculous pleurisy: a direct comparative study. Eur Respir J 2003;22:589–91. [25] Escudero Bueno C, Garcia Clemente M, Cuesta Castro B, et al. Cytologic and bacteriologic analysis of fluid and pleural biopsy specimens with Cope's needle. Study of 414 patients. Arch Intern Med 1990;150: 1190–4. [26] Valdes L, San Jose E, Alvarez D, et al. Diagnosis of tuberculous pleurisy using the biologic parameters adenosine deaminase, lysozyme, and interferon gamma. Chest 1993;103:458–65. [27] Zheng B, Cao KY, Chan CPY, et al. Serum neopterin for early assessment of severity of severe acute respiratory syndrome. Clin Immunol 2005;116:18–26. [28] Valdes L, Alvarez D, San Jose E, et al. Tuberculous pleurisy: a study of 254 patients. Arch Intern Med 1998;158:2017–21. [29] Villena V, Navarro-Gonzalvez JA, Garcia-Benayas C, et al. Rapid automated determination of adenosine deaminase and lysozyme for differentiating tuberculous and nontuberculous pleural effusions. Clin Chem 1996;42:218–21. [30] Burgess LJ, Maritz FJ, Le Roux I, et al. Use of adenosine deaminase as a diagnostic tool for tuberculous pleurisy. Thorax 1995;50:672–4. [31] Banales JL, Pineda PR, Fitzgerald JM, et al. Adenosine deaminase in the diagnosis of tuberculous pleural effusions: a report of 218 patients and review of the literature. Chest 1991;99:355–7.
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Pleural fluid neopterin levels in tuberculous pleurisy Gursel Cok a,⁎, Zuhal Parildar b , Gunes Basol b , Ceyda Kabaroglu b , Ulku Bayindir a , Sara Habif b , Oya Bayindir b b
a Department of Chest Disease, School of Medicine, Ege University, Bornova, 35100, Izmir, Turkey Department of Clinical Biochemistry, School of Medicine, Ege University, Bornova, 35100, Izmir, Turkey
Received 28 November 2006; received in revised form 14 April 2007; accepted 18 April 2007 Available online 27 April 2007
Abstract Objectives: Neopterin is produced by stimulated macrophages under the influence of gamma interferon of lymphocyte origin. It is regarded as a biochemical marker of cell-mediated immune response. This study was designed to assess the diagnostic value of pleural fluid neopterin levels in tuberculous pleurisy in comparison with adenosine deaminase activity. Design and methods: Pleural fluid adenosine deaminase (ADA) activity and neopterin levels were measured in 16 patients with tuberculous pleurisy (TP) and 19 patients with malignant pleurisy (MP). ADA activity was determined by a colorimetric method, whereas neopterin levels were determined by a reversed-phase liquid chromatography technique. All values were given as median (min–max). Results: The mean age was 45.43 ± 20.39 years in the TP group and 60.42 ± 11.02 years in the MP group (p = 0.026). The median pleural fluid ADA activity was 51.75 U/L (3.50–62.40 U/L) in the TP group and was 2.30 U/L (1–8.20 U/L) in the MP group. The difference was statistically significant (p b 0.001). The median pleural fluid neopterin levels were 13.15 nmol/L (1.86–59.50 nmol/L) and 2.44 nmol/L (0.92–27.60 nmol/L) in the TP group and the MP group, respectively (p = 0.021). In order to evaluate the diagnostic value of pleural fluid neopterin concentrations, receiver-operating-characteristic curve analysis was performed. Conclusion: Pleural fluid neopterin concentration is significantly higher in TP when compared to MP, however when compared, its clinical use as a diagnostic marker is not valuable as ADA. © 2007 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved. Keywords: Neopterin; Tuberculous pleurisy; Malignant pleurisy
Introduction Important developments in the diagnosis and treatment of tuberculosis have been achieved, but there still remain some difficulties in the diagnosis of tuberculosis forms such as tuberculous pleurisy (TP). In TP, the possibility of showing Mycobacterium tuberculosis bacilli in pleural fluid with staining for acid–fast bacilli is about 5% in TP and 10–20% of the patients cannot be diagnosed despite the histopathologic examination and culture of the pleural biopsy material [1]. For this reason, efforts to find an alternative diagnosis method still continue.
⁎ Corresponding author. Fax: +90 232 388 71 92. E-mail address: [email protected] (G. Cok).
Neopterin is a compound derived from pteridine being produced during guanosine triphosphate (GTP) metabolism [2,3]. Neopterin was isolated for the first time in 1967 [2,3]. Macrophages secrete neopterin, as a result of the stimulus provided by interferon gamma derived from T lymphocytes. Since the neopterin source is monocytes and macrophages, it is evaluated as the biological marker of cellular immunity [4–8]. Macrophages, but especially T lymphocytes play an important role in tuberculosis. In this sense, studies have been conducted on the role of neopterin in tuberculosis activity and increased neopterin levels in various body fluids due to tuberculosis activity and immune response were reported [9–14]. It is known that neopterin may increase in various body fluids in infectious diseases such as sepsis, hepatitis C, human immunodeficiency virus and in non-infectious cases such as neoplasms and autoimmune diseases [5,10].
0009-9120/$ - see front matter © 2007 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved. doi:10.1016/j.clinbiochem.2007.04.009
G. Cok et al. / Clinical Biochemistry 40 (2007) 876–880
While there are studies revealing that neopterin increases in pleural fluid in TP, they do not provide a strong ground to allow routine use [9,11,14]. There is only one study which has been conducted to determine the cut-off value, which may be used for the diagnosis of TP [9]. This study was designed as a prospective study for the purpose of determining the role of pleural fluid neopterin levels in the TP diagnosis and a possible cut-off value. Methods The patients with TP constituted the study group, while the patients with malignant pleurisy (MP) constituted the control group. For both groups, having diabetes mellitus, chronic liver disease or chronic renal failure was used as exclusion criteria. Nineteen patients with MP and 18 patients with TP participated in the study. Two patients with TP were excluded as they were diagnosed with chronic renal failure and thus the findings of remaining 16 patients were evaluated. These sixteen patients did not receive any antituberculosis treatment before. The golden standard for the diagnosis of tuberculosis is either the identification of mycobacterium tuberculosis bacilli in pleural fluid or biopsy samples, or demonstration of caseating granulomatous inflammation in pleural biopsies [15]. In 12 of the patients, caseating granulomatous inflammation in the pleural biopsy was detected, where as remaining four were diagnosed according to the following clinical data: 1. Age less than 35 years old 2. Positive tuberculin skin test (N15 mm) 3. High pleural fluid adenosine deaminase (ADA) activity (N50 U/L) 4. Presence of lymphocytic pleural fluid cytologically (lymphocyte ratio N 50%) 5. Good response to anti-tuberculous treatment and after exclusion of other diagnosis. Malignancy was excluded due to benign description of fluid cytology and biopsy materials, empyema was excluded with regard to biochemical and macroscopic findings, effusions due to rheumatological disease were excluded by patients' clinical symptoms. Concerning the possible effects of chemotherapy on neopterin levels patients who did not receive any therapy were chosen for the MP group. The primary diagnosis of patients with MP were as follows: 9 squamous cell lung carcinoma, 3 adenocarcinoma, 5 non-small-cell lung carcinoma (could not be differentiated) and 2 primary breast cancer. Pleural fluid and serum samples were collected concurrently from each patient. Lactate dehydrogenase (LDH) activity, protein and glucose concentrations in serum and pleural fluid were determined by standard laboratory methods on an autoanalyzer. Pleural fluid and serum adenosine deaminase activity was determined by the colorimetric method described by Giusti and Galanti [16]. Pleural fluids, stored at −80 °C and protected from light, were thawed to measure neopterin levels by a reversed-phase chromatography technique described by Werner et al. [17].
877
SSPS 13.0 statistical program was used for statistical analysis. All values were given as median (min–max), unless stated otherwise. Mann–Whitney U and logistic regression analysis were conducted when appropriate. The value p b 0.05 was accepted as statistically significant. The diagnostic values of pleural fluid neopterin levels were calculated as sensitivity, specificity, negative predictive value (NPV) and positive predictive value (PPV). To illustrate the diagnostic performance of neopterin, a receiver-operating-characteristic curve was reported and the area under curve was calculated. Spearman's correlation analysis was performed to demonstrate a possible correlation between ADA activity and neopterin values. Results Thirteen (81.3%) male and three (18.7%) female patients were in the TP group. Out of the 19 patients in the MP group, 12 patients (63.2%) were male and 7 patients (36.8%) were female. The mean age in the TP and the MP groups were 45.43 ± 20.39 years and 60.42 ± 11.02 years, respectively (p = 0.026). Table 1 summarizes the measured results of the investigated parameters in the TP and MP groups. The median pleural fluid ADA activity was 51.75 U/L (3.50–62.40 U/L) in the TP group and was 2.30 U/L (1–8.20 U/L) in the MP group (p b 0.001). For ADA activity, at 40 U/L cut-off value, sensitivity was calculated as 75%, specificity as 100%, PPV as 100%, NPV as 82.6%. When the pleural fluid neopterin levels were considered, the TP group displayed significantly higher values (p = 0.021) (Fig. 1). After correcting for age, due to the significant age difference between the groups, the observed difference was still significant. In order to define a possible cut-off value for pleural fluid neopterin concentrations in the diagnosis of TP, four different concentrations were selected to calculate the diagnostic performance parameters. These results are summarized in Table 2. For pleural fluid neopterin levels, at 20 nmol/L concentration, sensitivity was 43.75%, specificity was 84.21%, positive predictive value was 70% and negative predictive value was 64%. Using 25 nmol/L as the cut-off value, the sensitivity was calculated as 31.25%, specificity was 94.73%, positive predictive value was 83.33% and negative predictive value was 62.06%.
Table 1 The results of the investigated parameters in the pleural fluid and serum
P/LDH (U/L) S/LDH (U/L) P/Protein (g/dL) S/Protein (g/dL) P/Glucose (mg/dL) S/Glucose (mg/dL) P/ADA (U/L) P/Neopterin (nmol/L)
TP (n = 16)
MP (n = 19)
p
627.50 (232–4495) 445.50 (283–1687) 4.95 (3.20–6.30) 6.90 (5.30–7.90) 79.50 (48–121) 95 (81–215) 51.75 (3.50–62.40) 13.15 (1.86–59.50)
596 (275–5600) 496 (238–873) 4.10 (3.10–5.90) 6.70 (3.80–7.70) 93 (10–151) 112 (80–179) 2.30 (1–8.20) 2.40 (0.92–27.60)
0.947 0.934 0.131 0.630 0.136 0.064 b0.001 0.021
Data are expressed as median (min–max). P: pleural fluid, S: serum, LDH: lactate dehydrogenase, ADA: adenosine deaminase, TP: tuberculous pleurisy, MP: malignant pleurisy.
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Fig. 1. The distribution of neopterin levels in pleural fluid in TP and MP groups. The median (min–max) neopterin concentration was 13.15 nmol/L (1.86–59.50 nmol/L) and 2.40 nmol/L (0.92–27.60 nmol/L), respectively. The difference was significant (p = 0.021). TP: tuberculous pleurisy, MP: malignant pleurisy.
In the TP group, there was one patient with an extremely high plasma neopterin value, 59.5 nmol/L. He was 36 years old with pleural effusion on the right side. In his pleural biopsy, the typical caseating granulomatous inflammation was shown. The following results were obtained in his pleural fluid sample; LDH activity was 368 U/L, glucose was 96 mg/dL, total protein was 4.8 g/dL and ADA activity was 62.30 U/L. The measured serum values were as follows; LDH activity was 463 U/L, glucose was 108 mg/dL, total protein was 6.3 g/dL. Receiver-operating-characteristic curve (ROC) analysis for neopterin levels and ADA activities is given in Fig. 2. Spearman's correlation analysis revealed a significant correlation between ADA activity and neopterin values (rs = 0.348, p = 0.04). Discussion The World Health Organization reported the occurrence of approximately 8 million new tuberculosis cases around the world in 1998 [18]. Tuberculous pleurisy, which is one of the important forms of extra-pulmonary tuberculosis, constitutes a
Table 2 The diagnostic performances of pleural fluid neopterin levels at different selected cut-off values Cut-off value (nmol/L)
Sensitivity (%)
Specificity (%)
PPV (%)
NPV (%)
15 20 25 30
50.00 43.75 31.25 12.50
73.68 84.21 94.73 100
61.53 70.00 83.33 100
63.63 64.00 62.06 57.57
PPV: positive predictive value, NPV: negative predictive value.
Fig. 2. Receiver-operating-characteristic curve analysis for pleural fluid neopterin and ADA levels. The calculated areas under the curves are 0.729 (S.E. = 0.085, 95% CI, p = 0.021) for neopterin and 0.980 (S.E. = 0.021, 95% CI, p b 0.001) for ADA.
major cause for pleural effusions. However, its incidence rate varies from country to country. A study conducted in Spain revealed that tuberculous pleurisy is the cause of 25% of pleural effusions [19] and reported that this rate increases to 80–90% in Africa [20,21]. It is known that this rate is below 5% in developed countries [22]. A study, which was conducted in Turkey and included 5480 patients with tuberculosis, reported that the rate of TP to the total number of tuberculosis cases is 6.7% [23]. Today, despite the high prevalence of the disease, difficulties are experienced in diagnosing the patients who carry other causes of exudative pleural effusions such as malignancies in the elderly. Absolute diagnosis of TP can be made by showing the reproduction of M. tuberculosis bacilli in the culture of the pleural fluid or pleural tissue samples or by showing caseous necrotic granulomas in the pleural biopsy. However, histological examination of needle biopsy material of pleura is nondiagnostic in 20–40% of patients with TP, and when the pleural biopsy is negative; mycobacteria can be cultured in pleural specimens in less than 10% of patients [15,24,25]. For this reason, studies on interferon gamma, ADA, antimicrobial antibodies and recently neopterin have been intensified for more accurate diagnosis [11,14,24,26]. Neopterin is produced by activated macrophages in response to interferon gamma of lymphocyte origin. Thus, it is believed that neopterin can be used as marker of cellular immunity [4–8]. It was reported that neopterin increases in various body fluids in patients with tuberculosis where cellular immunity plays an important role [9–14]. In our study, pleural fluid neopterin levels in the TP group were higher than the MP group. Baganha et al. was the first to compare 10 TP patients and 15 MP patients and reported that neopterin levels were higher both in the pleural fluid and the sera in TP group and thus it can be used in determining the pleural fluid aetiology and cellular immunity [11]. The correlation between serum and pleural fluid neopterin is well established, therefore in the
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current study assessment was made only in pleural fluid to save up from the cost [9,11,27]. Chiang et al. reported that pleural neopterin is lower in patients with MP than it is in patients with TP and that pleural neopterin level is below 25 nmol/L in 84% of the patients with MP [14]. Similarly, the neopterin level was found to be below 25 nmol/L in 94.73% of the patients with MP, in our study. After it was shown that neopterin can play a role in the TP diagnosis, Tozkoparan et al. studied 34 patients with TP and 29 patients with pleurisy of malignancy, parapneumonic effusion, congestive heart failure, empyema, pulmonary thromboembolism and liver cirrhosis, for the purpose of determining the cut-off point [9]. They decided on 30 nmol/L as the cut-off value and found the sensitivity as 85%, specificity as 93%, PPV as 94% and NPV as 84% [9]. In our study, at the same cut-off value, the sensitivity was found as 12.5%, specificity as 100%, PPV as 100% and NPV as 57.57%. The difference between our results and theirs may be attributed to their heterogenic group of 29 patients. We repeated the same calculations at cut-off values of 15, 20 and 25 nmol/L. Although it was found that cut-off values of 20 and 25 nmol/L were especially better in terms of sensitivity, when the performance characteristics were considered as a whole, the results were not sufficient. To evaluate the diagnostic accuracy at different cutoff levels, we performed ROC analysis for pleural fluid ADA activity and neopterin levels. The results of ROC analysis revealed 0.98 and 0.72 as areas under curve for ADA and neopterin, respectively. The significant correlation between ADA activity and neopterin values was expected since both molecules are secreted from macrophages under immune stimulation. The measurement of ADA activity is still the most valuable biochemical marker present in various body fluids in the diagnosis or differential diagnosis of tuberculosis [1,14,24,26,28– 35]. Determination of pleural fluid neopterin levels does not provide additional information in the differential diagnosis of TP and MP. Besides, with regard to neopterin, measuring ADA activity is more cost-effective and convenient for the routine practice. References [1] Valdes L, Pose A, San Jose E, Martinez Vazquez JM. Tuberculous pleural effusions. Eur J Intern Med 2003;14:77–88. [2] Sakurai A, Goto M. Neopterin: isolation from human urine. J Biochem 1967;61:142–5. [3] Anwaar I, GottsaE'ter A, Hedblad B, Palmqvist B, Mattiasson I, LindgaE'rde F. Endothelial derived vasoactive factors and leukocyte derived inflammatory mediators in subjects with asymptomatic atherosclerosis. Angiology 1998;49:957–66. [4] Hamerlinck FF, van Gool T, Faber WR, Kager PA. Serum neopterin concentrations during treatment of leishmaniasis: useful as test of cure? FEMS Immunol Med Microbiol 2000;27:31–4. [5] Berdowska A, Zwirska-Korczala K. Neopterin measurement in clinical diagnosis. J Clin Pharm Ther 2001;26:319–29. [6] Hoffmann G, Wirleitner B, Fuchs D. Potential role of immune system activation-associated production of neopterin derivatives in humans. Inflamm Res 2003;52:313–21.
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