|FULL LENGTH ARTICLE
|Year : 2016 | Volume
| Issue : 4 | Page : 437-445
Evaluation of different laboratory methods for rapid diagnosis of tuberculous pleurisy*
Safaa Amer1, Azza El Hefnawy1, Nashwa Abdel Wahab2, Hadir Okasha1, Amira Baz1
1 Medical Microbiology and Immunology Department, Faculty of Medicine Alexandria University, Alexandria, Egypt
2 Chest Disease Department, Faculty of Medicine Alexandria University, Alexandria, Egypt
|Date of Web Publication||14-Feb-2017|
Medical Microbiology and Immunology Department, Faculty of Medicine, Alexandria University, El Khartoum Square, Azarita
Source of Support: None, Conflict of Interest: None
Background/Objective: Tuberculous pleurisy is a diagnostic challenge due to its nonspecific clinical presentation, paucibacillary nature of the effusion together with the inefficiency of conventional laboratory methods motivating the evaluation of variable diagnostic strategies.
Methods: Using thoracoscopy, the pleural cavity of 50 patients with undiagnosed exudative pleural effusion were fully examined and biopsy specimens of affected parietal pleura were taken under direct vision. Pleural fluid and biopsy specimen were subjected to microscopic examination (direct and after cytocentrifugation), culture, PCR, and histopathological examination.
Results: The pleural biopsy specimens proved to have a higher detection rate of tubercle bacilli than pleural fluid. Also, cytocentrifugation improved the sensitivity of microscopic detection for both pleural fluid and biopsy specimens.
Conclusion: The combination of microbiological results and histopathology examination of the pleural biopsy specimens is essential for the diagnosis of tuberculous pleurisy, as microbiological examination of pleural biopsy specimens has proved to have a higher detection rate than pleural fluid examination.
Keywords: Biopsy, Mycobacterium, Pleural tuberculosis, Thoracoscopy, Tuberculosis, Nonspecific pleurisy
|How to cite this article:|
Amer S, El Hefnawy A, Wahab NA, Okasha H, Baz A. Evaluation of different laboratory methods for rapid diagnosis of tuberculous pleurisy*. Int J Mycobacteriol 2016;5:437-45
|How to cite this URL:|
Amer S, El Hefnawy A, Wahab NA, Okasha H, Baz A. Evaluation of different laboratory methods for rapid diagnosis of tuberculous pleurisy*. Int J Mycobacteriol [serial online] 2016 [cited 2019 Nov 20];5:437-45. Available from: http://www.ijmyco.org/text.asp?2016/5/4/437/200127
| Introduction|| |
According to the World Health Organization, the incidence of tuberculosis (TB) was 9.6 million in 2014 and it is estimated that deaths from TB will increase from 3 million a year to 5 million by the year 2050. Between 2002 and 2020, approximately 1 billion people will be newly infected, 200 million people will get sick, and 36 million will die of TB if proper control measures are not instituted ,.
Although the majority of patients with TB have pulmonary TB, extrapulmonary TB affecting mainly the lymph nodes and pleura constitutes the initial presentation in ˜25% of adults . It was mentioned that among all cases presenting with pleural effusion, 25% are unable to be attributed to a specific diagnosis, even after thoracentesis and closed pleural biopsy (PB) . As many as 50% of the patients in this undiagnosed group will eventually be diagnosed with a malignancy. Other diagnostic possibilities include TB, fungal disease, connective tissue disease-related pleuritis, pulmonary infarction, rib fractures, asbestos-related pleural effusion, and nonspecific pleuritis (NSP) .
Still, TB is the main cause of pleural effusions in some countries . It is important to consider the possibility of tuberculous pleurisy in all patients with an undiagnosed pleural effusion. A pleural effusion as an isolated manifestation of TB is self-limited and of little immediate concern, but may lead to serious disease many years later. Tuberculous pleurisy is thought to represent primarily a hypersensitivity reaction to tuberculous protein while the bacillary burden in the pleural space is low .
Extrapulmonary tuberculosis usually presents more of a diagnostic problem than pulmonary TB. The combination of small numbers of bacilli and inaccessible sites causes bacteriologic confirmation of diagnosis to be more difficult, and invasive procedures are frequently required to establish a diagnosis .
The aim of the current study was to evaluate the different diagnostic methods: (1) direct smears; (2) cytocentrifugation prepared smears; (3) cultures; and (4) polymerase chain reaction (PCR) for detection of Mycobacterium tuberculosis in pleural fluid (PF) and PB specimen obtained by thoracoscopy from patients with undiagnosed lymphocytic exudative pleural effusion.
| Patients and methods|| |
The present study was conducted on 50 patients who presented to the Chest Diseases Department in Alexandria Main University Hospital, Alexandria, Egypt, with exudative pleural effusion (according to Light's criteria), but of unclear etiology after biochemical, bacteriological, and cytological examination of the PF. All patients were subjected to full history taking including age, sex, smoking status, occupation, residence, and history of other diseases or previous malignancies, thorough clinical examination and routine laboratory investigations including hemoglobin, total and differential white blood cell count, renal and liver function tests, fasting blood glucose level, coagulation profile including prothrombin time and activity, international normalized ratio, and platelet count. Oxygen saturation was detected with a pulse oximeter and an electrocardiogram was performed on all patients. Radiological evaluation in form of a plain chest X-ray in the posteroanterior view to evaluate the amount of pleural effusion, position of mediastinum, and any other abnormality, and contrast enhanced computed tomography to view pleural thickening, pleural nodules, mediastinal pleural involvement, lung masses or consolidation, lymphadenopathy, and any other abnormality was performed on all patients. Also, thoracic ultra sound (Sonos 100 CF, Hewlett Packard, MS, USA) was done to assess loculations, adhesions, and apparent masses, and to determine the most appropriate site of entry. Thoracentesis was performed and PF analysis for total protein and albumin content, lactate dehydrogenase content, total and differential leucocytic count, Ziehl–Neelsen (ZN) stain for acid-fast bacilli (AFB), and cytological examination for malignant cells were performed.
Thoracoscopy was performed in the bronchoscopy suite, with the patient under conscious sedation and local anesthesia lying in the lateral decubitus position. An examination was carried out with a rigid thoracoscope. Patients were monitored regarding blood pressure and pulse rate, an electrocardiograph was attached, a pulse oximeter was used, and supplementary oxygen was provided to maintain oxygen saturation > 90%. Equipment used included a rigid thoracoscope (Karl Storz, Tuttlingen, Germany), a straight forward telescope 0 ° with an angled eyepiece, 10 mm in diameter, working length at 27 cm with a 6-mm working channel, a metallic trocar 11 mm in diameter, cold (xenon) light source, an endoscopic camera attached to the eyepiece, video monitor and recorder, and other accessories commonly available in a chest tube insertion tray. A single port of entry was required in all patients. The patient was positioned in the lateral decubitus position breathing spontaneously, with the normal lung in the dependent position and with the arm raised above the head. The involved side of the chest was disinfected: 15–30 mL of lidocaine 2% was injected at the point of entry, through all layers of chest wall as far as the pleura. Thoracentesis to confirm the presence of PF at the insertion site was performed. A single puncture, which involved a 1-cm incision in the midaxillary line between the fourth and seventh intercostal space of the chest wall was done, and a track was created by blunt dissection. A trocar was inserted and the pleural cavity was opened to atmospheric pressure. Any remaining PF was then aspirated . A full examination of the pleural cavity was then made and biopsy specimens of parietal pleura were taken as appropriate under direct vision. Multiple (5–7) biopsy samples were taken. At the end of the procedure, a chest tube was inserted and lung expansion was radiographically confirmed before removal of the tube. A chest radiograph was taken within 24 h and patients were put under close observation postthoracoscopy. Both PF samples and PBs were sent to the TB lab in the Department of Medical Microbiology and Immunology, Faculty of Medicine, University of Alexandria, as well as undergoing pathological examination. The specimen sent for microbiology was put separately in normal saline. The study was approved by the Alexandria Faculty of Medicine Ethical Committee and informed consent was obtained from patients before sampling.
Specimens were delivered aseptically to the TB lab in the Department of Medical Microbiology and Immunology, Faculty of Medicine, University of Alexandria.
With regards to the PF samples, they were divided into three parts: (1) one part (20 mL) was centrifuged at 1200×g for 15 min and the sediment was used for ZN smear and Lowenstein–Jensen (LJ) culture ; (2) another part (10 mL) of the sample was centrifuged at 1200×g for 15 min and the deposit was used to detect AFB after cytocentrifugation in cytospin ; (3) the last 10 mL of the samples were used for the detection of mycobacterial DNA by PCR .
The PB obtained by medical thoracoscopy was delivered in part in sterile normal saline, minced finely, and homogenized by shaking with 1-mm sterile glass beads, suspended in 4-mL sterile distilled water by vortex shaker, then the sample was divided to be used to perform a direct ZN smear, cytocentrifugation ZN smear, LJ culture, and PCR for mycobacterial DNA . A portion of the punch PB was also sent for histopathological examination.
Two types of smears were done from each processed PF and PB: (1) a direct smear from the centrifuged deposit was stained by ZN and examined microscopically for AFB ; (2) a cytocentrifugation smear was done for PF and PB by centrifugation at 1200×g for 15 min, then the deposit was decontaminated by mixing with an equal volume of 6% Na hypochlorite and was used to prepare a ZN stained smear after cytocentrifugation in cytospin (rottofix32A, Hettich, Kirchlengern, Germany) and examined microscopically for AFB.
The processed PF and PB specimens were decontaminated and homogenized according to Petroff's method. The sediment was then inoculated onto two slants of LJ medium. One tube was plain LJ and another tube containing p-Nitrobenzoic acid (500 mg/L) for the detection of atypical mycobacterium. Both slants were incubated at 37 °C and were examined using a hand lens 5–7 days after incubation and weekly thereafter for 6–8 weeks. Suspected mycobacterial colonies required > 7 days to grow and were further identified as M. Tuberculosis on the basis of colonial morphology on LJ medium, acid alcohol fast staining, preference of growth at 35–37 °C, lack of photo reactivity, and inability to grow on LJ medium containing p-Nitrobenzoic acid (500 mg/L) ,.
DNA extraction from PF and PB samples was done using QIAGENQIA amp DNA Mini and Blood Mini extraction kit (Qiagen Inc., Valencia, CA, USA) according to manufacturer instructions. The amplification of the extracted DNA was then carried out using the following primers: forward primer (P1), 5'-CCT GCG AGC GTA GGC GTC GG3' and reverse primer (P2), 5' CTC GTC CAG CGC CGC TTC GG3', to amplify a target fragment of 123 bp from the insertion of the M. Tuberculosis sequence element IS6110 . Each amplification reaction was carried out in a final volume of 25 μL containing 12 μL of maxima hot start PCR master mix (2×), 2- μL P1, 2- μL P2, 5- μL DNA extract from PF, or 7- μL DNA extract from PB. The volume was adjusted to 25 μL using sterile distilled water. The PCR amplification was done in a thermal cycler (Genius Techne, Cambridge, England) and was subjected to 40 cycles of denaturation at 94 °C for 2 min, annealing at 68 °C for 2 min, and extension at 72 °C for 1 min. The amplified products were separated on 2% agarose gel electrophoresis in 1×40 mM Tris–acetate plus 1 mM EDTA). Separated DNA segments of 123 bp were stained with ethidium bromide and visualized on a UV-light transilluminator ([Figure 1]).
|Figure 1: Polymerase chain reaction-based detection of the Mycobacterium tuberculosis complex targeting IS6110. Electrophoresis separation of the amplicon into 2% agarose gel is documented across Lanes 1–8. The presence of a 123-bp amplicon in Lanes 1 and 3–5 indicated the presence of the target while the absence of the amplicon in Lane 2 pointed towards the absence of the target. Note. L = ladder 100 bp; NC = negative control; PC = positive control.|
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Statistical analysis was carried out using SPSS statistics software (version 20.0; IBM Corp., Armonk, NY, USA). Categorical variables were described using frequencies and percentages. Chi-square, Fisher's exact, and Monte Carlo tests were used for testing the associations between categorical variables. Quantitative data were tested for normality using Kolmogorov–Smirnov test. Normally distributed variables were described using mean and standard deviation and in such cases the independent sample t test was used for comparing two groups. Agreement test, reported as a kappa statistic, was used as a quantitative measure of the strength of agreement between tests. It lies on a −1 to 1 scale where 1 is perfect agreement and 0 is expected to be due to chance. A p value of kappa was also reported. Statistical significance was accepted as p < .05. All applied statistical tests of significance were two-tailed.
LJ culture was considered the gold standard for each processed specimen when dealing with the other tests performed (microscopy and PCR) on same sample, to calculate sensitivity, specificity, accuracy, positive predictive value, and negative predictive value results for these tests.
| Results|| |
Of the 50 patients included in the study 24 (48%) were men and 26 (52%) were women with a mean age of 53.08±11.9 years for both sex and their age ranged from 27 years to 80 years.
Using the PB culture results, 24 (48%) patients were found to be tuberculous and their mean age was significantly lower (47.71±12.28 years) than the 26 (52%) patients with a negative PB culture (58.04±9.21 years; p = .001). With regards to sex distribution, a significant difference was found between both sexes regarding LJ positive cultures, with a female: male ratio of 2:1 (χ2 = 3.978, p = .046).
Among the studied patients, 16 patients (32%) were current or ex-smokers and 32 patients (64%) were passive smokers. Regarding previous medical history, 19 patients (38%) were known to be diabetics, and 10 patients (20%) had a history of previous malignancy. The main presenting symptoms were dyspnea (90%), chest pain (58%), significant weight loss (40%), fever (34%), cough (20%), night sweating (4%), and hemoptysis (2%) ([Table 1]).
|Table 1: Demographic characteristics and baseline clinical and radiological data of the studied patients.|
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The right side was involved in 28 (56%) patients and the left side in 21 (42%), while bilateral effusion was found in only one patient, with no significant difference found regarding the side of pleural involvement (MCp = .694). The amount of effusion was massive in 21 patients (42%), moderate in 27 (54%), and mild in two (4%) patients. Contrast enhanced computed tomography of the studied patients showed smooth pleural thickening in 22 patients (10 were diagnosed as tuberculous, 10 malignant, one empyema, and one idiopathic effusion) and irregular pleural thickening was present in 10 patients. All were finally diagnosed as malignant effusion. Pleural nodules were found in 11 patients and mediastinal pleura were involved in six patients (all diagnosed as malignant).
Thoracoscopic examination of the pleura revealed pleural nodules, hyperemia and increased vascularity, adhesions, masses, fibrinous pleural peel, pleural plaques, and micronodules ([Table 2] and [Figure 2]).
|Table 2: Thoracoscopic findings (macroscopic appearance) among the studied patients.|
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|Figure 2: Radiology and thoracoscopic view of a tuberculous pleurisy patient. (A) Chest X-ray showing right-sided pleural effusion; (B) chest computed tomography showing right encysted pleural effusion with pleural thickening (maximally reaching 7 mm); (C) thoracoscopic view showing micronodules; (D) Disruption of adhesions using rigid forceps. Histopathological findings showed nonspecific pleurisy with foreign body giant cells and direct smear staining of pleural biopsy for acid-fast bacilli showed positive results. Patient was diagnosed as tuberculous pleurisy.|
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Results of different microbiological methods for diagnosis of tropical pulmonary eosinophilia from both PF and PB specimens
Out of 50 PF specimens, two (4%) were positive by direct ZN smear, seven (14%) by cytocentrifugation ZN smear, 16 (32%) by LJ culture, and 17 (34%) by PCR. Out of the 50PB specimens, three (6%) showed positive results by direct ZN smear, 12 (24%) by cytocentrifugation ZN smear, 24 (48%) by LJ culture, and 26 (52%) by PCR ([Table 3]).
|Table 3: Results of different microbiological methods for diagnosis of tropical pulmonary eosinophilia.|
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Using LJ culture as the gold standard, PCR had the highest sensitivity in detecting Tuberculous Pleural Effusion (TPE) from PF and PB specimens, 93.75% and 100%, respectively, followed by cytocentrifugation ZN smear (37.50% and 50%), then direct smear with a sensitivity of 12.50% for both specimens. Regarding specificity, direct ZN smear showed the highest specificity (100%) for both specimens, followed by cytocentrifugation ZN smear (97.06% and 100%), then PCR (94.12% and 92.31%; [Table 4]) ([Table S1],[Table S2],[Table S3],[Table S4],[Table S5],[Table S6]).
|Table 4: Evaluation of direct smear, cytocentrifugation Ziehl–Neelsen (ZN) smear, and polymerase chain reaction (PCR) for the diagnosis of tuberculosis in pleural fluid (PF).|
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When comparing LJ culture results (gold standard) for PF and PB, [Table 5] shows that 24 (48%) had positive PB culture results and 16 (32%) had positive PF culture results. This difference was statistically significant (McNemar p = .021). Out of the 24 (48%) with positive PB culture results, 15 (30%) were also positive by PF culture and nine (18%) were negative. Out of 16 (32%) PF specimens with positive culture results, 15 (30%) were positive by PB culture and only one (2%) was negative. The kappa value was 0.594, which reflects the moderate agreement of LJ culture of PF with LJ culture of PB for the detection of AFB.
|Table 5: Comparison between Lowenstein–Jensen (LJ) culture results for pleural fluid (PF) and pleural biopsy (PB) specimens.|
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We also compared PCR results for PF and PB, where 26 (52%) had positive PB PCR results and 17 (34%) had positive PCR results from PF specimens. This difference was statistically significant. (McNemar p = .004). Out of the 26 (52%) PB specimens with positive PCR, 17 (34%) were also positive by PF and nine (18%) were negative, indicating that if PCR was used on PF only nine cases would have been missed. All the 17 (34%) PCR positive PF specimens were also positive by PB PCR. The kappa value was .645, which reflects good agreement of PCR results for PF with the PCR results for PB for detection of AFB ([Table 6]).
|Table 6: Results of pleural fluid (PF) and pleural biopsy (PB) polymerase chain reaction (PCR).|
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Twenty-four (48%) of the 50 studied patients were diagnosed as malignant effusion (4 mesothelioma and 20 metastatic pleural carcinomas) and 22/50 patients (44%) were diagnosed as nonspecific fibrinous pleurisy. Three patients (6%) showed caseating granulomas and one out of 50 patients (2%) showed septic pleurisy. Of the 24 patients with positive biopsy LJ culture results, three showed caseating granuloma, two showed malignancy, 18 showed NSP, and one showed septic pleurisy diagnosed as tuberculous empyema. All of the LJ culture positive biopsy specimens that showed NSP on histopathological examination were positive by PCR. Both TB and malignancy coexisted in two patients. Histopathological examination failed to significantly discriminate between positive and negative patients compared with PB LJ culture (MCp = .197) ([Table 7]).
|Table 7: Results of histopathological examination in comparison to Lowenstein–Jensen (LJ) culture for pleural biopsy (PB).|
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| Discussion|| |
In the present study we evaluated different laboratory diagnostic tests on PF and PB specimens taken from patients with undiagnosed exudative pleural effusion. Thoracoscopy was performed under local anesthesia to obtain PB as it allowed direct visualization of the pleura to determine the macroscopic lesions in addition to the feasibility of obtaining biopsy for both bacteriological and pathological examinations. Sugiyama et al. classified tuberculous pleurisy into four stages: Stage I where the parietal pleura is swollen, reddened, and show tiny white nodules; Stage II where the redness and swelling become more extensive and military white nodules extending diffusely and coalescing together; Stage III white fibrin deposits extend over the pleura in a cord or a membrane-like fashion; and Stage IV (the chronic stage) the fibrin deposits become fibrous. Part of the pleural effusion becomes encapsulated with a fibrin net, and the parietal pleura become white, thickened, firm, and difficult to biopsy. This thickened, firm pleura cannot be biopsied except under direct vision as in cases of rigid thoracoscopy in order to avoid injury to the visceral pleura and lung ,.
The mean age of TPE-proven cases in the current study was 47.71±12.28 years, with a female:male ratio of 2:1. Studies showed that women may have a higher rate of disease progression in their reproductive years, whereas men have higher rates at older ages which means that hormonal factors might be involved . Also, in a study conducted among African-born cases, women were at higher risk of extrapulmonary TB, those of the 25–59 years group were the most likely to present as extrapulmonary TB, with women at higher risk being in North Africa .
Our results revealed that ZN smear had the lowest detection rate for both PF and PB specimens followed by cytocentrifugation ZN smear, then LJ culture, and PCR. This was in agreement with other researchers evaluating microscopy and cultures on similar samples ,. Of importance in our study, ZN smear from PF had a sensitivity of 12.50%, which increased to 37.50% when applying cytocentrifugation prior to ZN staining. Specificity was 100% for direct ZN smear and 97.06% for cytocentrifugation ZN smear. This specificity result can be explained by the presence of one case in this study that showed positive cytocentrifugation ZN smear result with negative culture which was attributed to the presence of nonviable mycobacteria detected also by PCR. This patient has been on antituberculous therapy due to high clinical suspicion. The explanation of the low sensitivity value of ZN smears in the present study is due to the low bacillary load in PF, as the detection by microscopy depends on the presence of more than 104–5 bacilli/mL for microscopic positivity ,,,. However the use of cytocentrifugation method for preparing ZN increases sensitivity without a loss of specificity, compared to the direct smear method .
Regarding PB specimens ZN had a sensitivity of 12.5% which increased to 50% for ZN smear after cytocentrifugation, while specificity was 100% for both smears. Others examined pleural tissue for AFB by direct ZN, revealing variable rates of detection by smear. This difference may be explained by using different methods for collecting, processing, and homogenization of PB specimens. To our knowledge no studies were previously done for evaluation of ZN cytocentrifugation smears on PB after homogenization ,,,,.
The specificity of both direct and cytocentrifugation smear from PB was (100%) as processing and concentrating methods do not yield any gain in specificity in comparison to direct smear, yet the difference was reflected on the increased sensitivity of cytocentrifugation ZN smears over direct ZN smear in both PF and PB specimens . Revealing that cytocentrifugation smear has many advantages including: (1) rapid results; (2) increasing sensitivity of detection when small numbers of bacilli are present; (3) safety, because the bleached specimen has no viable organisms; and (4) cost-effectiveness and simplicity, enabling laboratories of all sizes to perform accurate AFB smears around the clock. The only limitation of this method is the inability to produce viable mycobacterial cultures from the bleached cytocentrifuged specimens .
The lower culture detection rate in PF samples when compared with that of PB samples can be explained by the presence of viable bacilli in the involved pleural tissue, while the pleural effusion is considered a manifestation of a hypersensitivity reaction to the mycobacterium .
Regarding IS6110 PCR performed on both PF and PB specimen, the higher sensitivity of PB specimens compared with PF specimens is explained by the fact that a biopsy specimen allows for a better quality of sample with sufficient amount of tissue containing mycobacterial DNA, and thus increases the sensitivity of the PCR. Also medical thoracoscopy allows for the direct inspection of the pleura, thus biopsies can be taken under direct vision, giving a diagnostic yield superior to that of blind closed PB. However, the relatively lower specificity of PCR in comparison with culture in this study is a reflection of two PF specimens with positive PCR and negative culture results. The first specimen showed positive cytocentrifugation ZN smear result with negative culture due to presence of nonviable mycobacteria which was detected also by PCR. This patient had been on antituberculous therapy for the previous 6 weeks. The second specimen had false positive PCR results which might be due to the presence of nonviable mycobacteria or cross contamination that could have occurred during specimen collection or processing or from latent infection with TB.
In similar studies which used thoracoscopy to improve diagnosis of patients with undiagnosed pleural effusion by thoracocentesis and closed PB, Helala et al. and Huang et al.  found that the most common diagnosis was malignancy which highlights the importance of thoracoscopy versus other methods of sampling the pleura. This is in contrast to others who reported that histopathologic examinations of the PB specimens showed prevalence of caseating granuloma in patients with exudative pleural effusion ,,.
In the current study the most common thoracoscopic finding was the presence of adhesions, loculations, fibrinous pleural peel, and nodules (sometimes also small micronodules). There was widespread affection of the pleura explaining the high yield of closed transthoracic PB in tuberculous patients. The sensitivity of thoracoscopic macroscopic appearance suggestive of tuberculous pleural involvement was 66.67% and the specificity was 92.31%; the positive predictive value and negative predictive value were 88.89% and 75% ([Table S7]), respectively. The macroscopic appearance at thoracoscopy proved reliable.
In the current study the most common pathological findings for PB specimens with positive LJ culture and PCR was Nonspecific pleurisy NSP. A study conducted by Amini et al.  in Iran, assessed the value of PCR IS6110 assay in tissue specimens of needle PB in patients suspicious of pleural TB, showed that out of 68 total patients, 15 positive PCR results belonged to patients with nonspecific biopsy reports, and out of 12 malignant patients only one had positive PCR for TB. Also Yum and Choi  reported three (25%) of 12 cases diagnosed as chronic or NSP were positive in PCR for M. Tuberculosis. Those 12 patients were diagnosed as TB clinically with therapeutic trial with antituberculous drugs and clinical follow up. Kim et al.  reported that out of 23 patients with NSP (that were followed up until a diagnosis was reached), 11 (48%) were found to have tuberculosis based on their response to therapy and neoplasms were detected in two (8.7%). In another study conducted by Oshita et al.  on 69 cases of NSP diagnosed with pleura core needle biopsy and then followed up for > 2 years, where a final diagnosis was established in 56 cases, the causes of the effusion were tuberculous in 29 cases and neoplastic in 13 cases (27 out of 29 cases [90%] with tuberculous pleuritis were therapeutically diagnosed).
The two PBs showing coexisting TB with malignancy is a finding supported by several studies as patients with malignancy have reduced immunocompetence due to their primary disease and/or the effects of anticancer treatment which predispose to TB ,,. Also, with the progression of lung cancer, old foci of TB would reactivate and dissemination of TB bacilli could occur .
Chawla et al.  compared PCR results with histopathological findings and stated that positive PCR/negative histopathological examination results for TB was due to disease still developing and well-developed granuloma had not yet formed, but the presence of mycobacterial DNA in tissues could still be easily detected by PCR at the early stage.
Similar results were found in another study where TB PCR results were also positive in 10 (36%) of 28 cases with chronic inflammation without definite granulomatous lesions. This result was explained by immunosuppression or the small size of biopsy specimens. In immunosuppressed patients, such as patients infected with the human immunodeficiency virus, the tissue reaction to TB includes a spectrum of changes that may not involve the presence of a granuloma, despite the presence of TB pleuritis. In addition, because the size of submitted histologic specimens is usually small, fully developed histopathologic features of TB, such as a well-formed granuloma with caseous necrosis, were infrequently seen .
In the current study histopathological examination showed that out of the 24 patients with positive biopsy LJ culture results, only three (12.5%) showed caseating granulomas which emphasize the value of LJ culture for the diagnosis of TB from PB specimens.
Hasaneen et al.  reported that histopathologic examination of the PB specimen had a sensitivity of 53.8% and was a rapid method for reaching the diagnosis of tuberculous pleural effusion; however, its sensitivity was low in comparison to that of PB culture (92.3%). These results were in agreement with the results reported by Katiyar et al.  showing that the culture of PB was more sensitive in diagnosing tuberculous pleural effusion when compared to the pathologic examination of PB specimens. Also, Sugiyama et al.  reported that the diagnostic rate of thoracoscopic PB was 90.1%.
The difference in detection rates in the histopathologic examinations in these studies could be attributed to the fact that repeated sampling is needed for histopathologic examinations to detect caseating lesions, while for cultures one sample seems to be sufficient. In addition to the need for multiple sampling, histopathology alone cannot distinguish between a disease caused by M. Tuberculosis from other causes of granuloma . Rosso et al.  found that the best sensitivity was achieved by combining the results of pleura tissue culture and histopathology 91.8%.
As a conclusion microbiological examination of PB specimens has proved to have a higher detection rate than PF examination which failed to diagnose eight tuberculous pleurisy patients out of the 24 patients diagnosed by PB LJ culture. Cytocentrifugation prepared ZN smear proved to be safe, cheap, applicable, and easy to be used as an effective screening method that can be efficiently applied to pleural punch biopsy samples. PCR provides a rapid diagnostic test for TPE but false positivity was a limitation, therefore the molecular methods cannot substitute culture (gold standard) but may successfully complement them. Also, a combination of the results from microbiological and histopathology examinations of the PB specimens is recommended and considering NSP histopathology reports from pleural punch biopsies in TPE clinically suspected cases as an alert to improve the clinical vision for early diagnosis and treatment of TPE.
| Conflicts of interest|| |
The authors declare no conflict of interest.
| Appendix A. Supplementary data|| |
Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ijmyco. 2016.07.001.
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[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6], [Table 7]
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