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Journal of Bronchology & Interventional Pulmonology:
doi: 10.1097/LBR.0000000000000063
Original Investigations

Value of the Polymerase Chain Reaction Method for Detecting Tuberculosis in the Bronchial Tissue Involved by Anthracosis

Mirsadraee, Majid MD*; Shafahie, Ahmad MD; Reza Khakzad, Mohammad MSc‡,§; Sankian, Mojtaba PhD§

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Author Information

*Department of Internal Medicine, 22 Bahman Hospital, Islamic Azad University—Mashhad Branch

Department of Immunology and Zakaria Research Center, Medical School of Islamic Azad University—Mashhad Branch

§Immunology Research Center, Buali Research Center, Mashhad University of Medical Sciences, Mashhad

Kerman University of Medical Sciences, Kerman, Iran

M.M.: designed the research/study, collected data, analyzed data, and wrote the paper; A.S.: collected data and wrote the paper; M.R.K.: performed the research/study and contributed important reagents; M.S.: performed the research/study and contributed important reagents.

This research was conducted in Zakaria Research Center under the supervision and financial support of the deputy of research of the Islamic Azad University, Mashhad branch.

Disclosure: There is no conflict of interest or other disclosures.

Reprints: Majid Mirsadraee, MD, No 80, 15th Kosar, Kosar Ave., Vakilabad Blvd, Mashhad, 91786, Iran (e-mails: majidmirsadraee@yahoo.com; majidmirsadraee@mshdiau.ac.ir).

Received October 23, 2013

Accepted February 18, 2014

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Abstract

Background: Anthracofibrosis is the black discoloration of the bronchial mucosa with deformity and obstruction. Association of this disease with tuberculosis (TB) was approved. The objective of this study was to find the additional benefit of assessment of TB by the polymerase chain reaction (PCR) method.

Methods: Bronchoscopy was performed on 103 subjects (54 anthracofibrosis and 49 control subjects) who required bronchoscopy for their pulmonary problems. According to bronchoscopic findings, participants were classified to anthracofibrosis and nonanthracotic groups. They were examined for TB with traditional methods such as direct smear (Ziehl-Neelsen staining), Löwenstein-Jensen culture, and histopathology and the new method “PCR” for Mycobacterium tuberculosis genome (IS6110).

Results: Age, sex, smoking, and clinical findings were not significantly different in the TB and the non-TB groups. Acid-fast bacilli could be detected by a direct smear in 12 (25%) of the anthracofibrosis subjects, and adding the results of culture and histopathology traditional tests indicated TB in 27 (31%) of the cases. Mycobacterium tuberculosis was diagnosed by PCR in 18 (33%) patients, but the difference was not significant. Detection of acid-fast bacilli in control nonanthracosis subjects was significantly lower (3, 6%), but PCR (20, 40%) and accumulation of results from all traditional methods (22, 44%) showed a nonsignificant difference.

Conclusions: The PCR method showed a result equal to traditional methods including accumulation of smear, culture, and histopathology.

Bronchial anthracofibrosis (BAF) of the lung is the black discoloration of the bronchial mucosa that causes bronchial obstruction and chronic lung disease.1 The association between tuberculosis (TB) and BAF was reported in one fifth2 to more than half3 of BAF subjects, which is significantly higher than patients who required bronchoscopy. These findings usually force the pulmonologist to obtain enough samples for evaluation of TB when BAF is detected during bronchoscopy. Direct smear for acid-fast bacilli (AFB), Mycobacterium tuberculosis cultures, and histopathology are commonly used for diagnosing TB. The polymerase chain reaction (PCR) has been used as a faster and more sensitive method for the diagnosis of Mycobacterium tuberculosis; however, this method has been used in only 1 study of BAF subjects.4

The objective of this study was to determine the additional benefits of the PCR method for diagnosing TB in patients who demonstrate BAF with a prospective controlled study design.

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MATERIALS AND METHODS

One hundred three subjects requiring flexible bronchoscopy for various indications in 2 university hospitals during the years 2006 to 2008 were enrolled in this prospective case-control study. Demographic data, history of exposure to smoke, and important clinical findings were recorded. During bronchoscopy, subjects with black discoloration that causes deformity or stenosis of the bronchus were classified as BAF. The control group consisted of the subjects mentioned above, but without any black discoloration in their bronchial tree. TB was detected by bronchoscopy with different methods. Bronchial lavage was collected in all subjects for Ziehl-Neelsen staining and Löwenstein-Jensen culture. Two samples of bronchial tissue from BAF patients were sent for PCR analysis. A histopathology examination was performed when infiltration into the bronchial surface was observed. The accuracy of different methods was compared between subjects detected to suffer from TB in the BAF and the control groups.

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PCR METHOD

Two samples of bronchial tissue obtained by bronchial biopsy were used for PCR evaluation. Bronchial washing was not used due to possible contamination of the bronchial washing with the remaining material in the bronchoscope and oropharyngeal secretions. The lung tissue was homogenized completely by a tissue homogenizer, and proteinase K was added. DNA was extracted by the PrimePrep genomic DNA isolation kit (Genet Bio Company). The CinnaGen kit was used to amplify the IS6110 target using PCR amplification primers and conditions. The presence of 163-bp fragments compared with DNA size markers indicates a positive test (Fig. 1).

Figure 1
Figure 1
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The Ethical Committee of the Medical School of Islamic Azad University—Mashhad Branch approved this study, and all patients gave their written consent.

The sample size was calculated according to the prevalence of anthracofibrosis in our region (11%).1 A positive culture of bronchial lavage and or a histopathology consistent with TB was the gold standard for the diagnosis of TB. The χ2 test and 95% confidence level were used to evaluate the differential significance between groups. EPI INFO 2003 and SPSS 16 software were used for statistical analysis. Significance was accepted at P<0.05.

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RESULTS

The mean age of BAF subjects (70.5±10.4 y) was significantly higher than that of the control group (t=6.64, P=0.0001). The comparison of demographic data (Table 1) including the sex ratio, occupation, and bread baking in a rustic household oven did not show a significant difference between the BAF and the control groups and TB and non-TB subjects, but the frequency of cigarette smoking in the BAF group (18%) was significantly lower than that in the control group (45%) (χ2=8.3; P=0.004; odd ratio=0.26; 95% confidence level, 0.1-0.67). Coughing (74%) and dyspnea (66%) were the most frequent clinical findings in BAF and TB subjects, which did not show a significant difference from the control group (Table 1). Upper lobe localization was also more frequent in both BAF and TB groups, but the differences were not significant (Table 1).

Table 1
Table 1
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According to the results of culture, histopathology, and PCR, TB was detected in 49 subjects, 27 of whom suffered from BAF (55%), and 22 (45%) belonged to the control group (Table 2).

Table 2
Table 2
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Traditional laboratory examinations of TB in BAF subjects showed positive results for AFB in 25% (12) of the patients, 11% (10) for culture, and 32% (6) for histopathology (Table 3). Accumulating these results of traditional methods confirmed the diagnosis of TB in 31% of the patients, which was not significantly different from that determined by the PCR method (33%).

Table 3
Table 3
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Although evaluating the accuracy of the diagnostic method, the PCR method showed the highest sensitivity in BAF subjects (66%), followed by the histopathology method. However, in evaluating the accuracy by the likelihood ratio method, the highest result was shown with an accumulation of traditional methods (Table 2), although the comparison of PCR and the accumulation of traditional methods was not significant (18 positive subjects in comparison with 20, respectively).

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DISCUSSION

In this prospective study, the result of the PCR method for diagnosing TB was compared with traditional TB tests. PCR was able to diagnosis TB better than traditional tests, but the difference between PCR and the accumulation of traditional methods was not significant. PCR, as the most sensitive test in this study, could increase the yield of TB diagnosis in the BAF group, but the benefit of this test was more significant in the control group (from 4% to 20%), and it showed that TB was more frequent in the control group than in BAF subjects, although the difference was not significant. Therefore, a complete profile of traditional TB tests including AFB, culture, and histopathology had results comparable to those of PCR in BAF subjects.

The benefit of the PCR method compared with the traditional method could be explained in 2 ways: (1) it is less operator dependent than traditional methods (compared with the direct smear test), and (2) it is less time consuming (compared with the culture) with minimum tissue obtained. Obtaining a biopsy for histopathology is also difficult as it may cause severe bleeding; therefore, this may limit the number of specimens that is needed for detecting granoluma.3 Nevertheless, the result of this study showed that traditional methods are able to detect TB as well as the PCR method and without additional cost. In this regard, choosing the PCR method should be individualized according to the expertise of the laboratory staff in traditional methods.

Na et al4 reported the frequency of positive PCR for TB in anthracofibrosis patients to be at 37%, which is higher than that evaluated by the traditional method in that study (30%).

The most important methods for detecting mycobacterium TB in BAF patients were different in previous studies. Towhidi et al5 detected TB by the direct smear for AFB in 96% of patients and Törün et al6 detected TB by culture in 100% of patients. In contrast, in some studies, histopathologic evaluation was the mainstay for the diagnosis of TB.7,8 Ghanei et al9 reported cavitary lesion, a positive PPD test, and a high sedimentation rate as the most helpful ancillary paraclinical tests for the diagnosis of TB in anthracosis patients. In the present study, a fluctuation in the frequency of TB over time was seen. It seems that this frequency has been affected by the TB frequency in their community.

In conclusion, PCR could replace traditional methods if the experience of the staff with traditional methods showed unsatisfactory results or due to bleeding if the tissue obtained for histopathologic evaluation is not sufficient. Otherwise, traditional methods still are reliable methods for detecting TB.

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REFERENCES

1. Mirsadraee M, Saaedi P.Anthracosis of lung; evaluation of potential causes.J Bronchol.2005;12:84–87.

2. Mirsadraee M, Asnashari A, Attaran D.Tuberculosis in patients with anthracosis of lung; underlying mechanism or superimposed disease.Iran Red Crescent Med J.2011;13:670–673.

3. Chung MP, Kyung SL, Joungho H.Bronchial stenosis due to anthracofibrosis.Chest.1998;113:344–350.

4. Na JO, Lim CM, Lee SD, et al..Detection of Mycobacterium tuberculosis in bronchial specimens using a polymerase chain reaction in patients with bronchial anthracofibrosis.Tuberc Respir Dis.2002;53:161–172.

5. Towhidi M, Keshmiri M, Attaran D, et al..Tuberculous bronchostenosis presenting as anthracofibrosis.Med J Mashhad Univ Med Sci.2003;45:73–76.

6. Törün T, Güngör G, Özmen I, et al..Bronchial anthracostenosis in patients exposed to biomass smoke.Turkish Respir J.2007;8:048–051.

7. Hemmati SH, Shahriar M, Molaei NA.What causes anthracofibrosis? Either tuberculosis or smoke.Pak J Med Aci.2008;24:395–398.

8. Kim HY, Im JG, Goo JM, et al..Bronchial anthracofibrosis (inflammatory bronchial stenosis with anthracotic pigmentation).AJR.2000;174:523–527.

9. Ghanei M, Aslani J, Peyman M, et al..Bronchial anthracosis: a potent clue for diagnosis of pulmonary tuberculosis.Oman Med J.2011;26:19–22.

Keywords:

anthracosis; anthracofibrosis; tuberculosis; polymerized chain reaction; acid-fast bacilli

© 2014 by Lippincott Williams & Wilkins.

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