Comparison of CBNAAT, AFB Culture and Histopathology of Pleural Biopsy Specimens in Suspected Tuberculous Pleural Effusions Undergoing Pleural Biopsy – Case Series

Original Article

Comparison of CBNAAT, AFB Culture and Histopathology of Pleural Biopsy Specimens in Suspected Tuberculous Pleural Effusions Undergoing Pleural Biopsy – Case Series

Sridhar.R ¹ , Avinash Peddi ¹ , Sundararajan.L, ²  Narasimhan.R ².

¹Post Graduate, Department of Respiratory Medicine, Apollo Main Hospital, Chennai.
²Senior Consultant, Department of Respiratory Medicine, Apollo Main Hospital, Chennai.

Corresponding Author: Dr. Sridhar.R, Department of Respiratory Medicine. Email: dr.sridhar.ily@gmail.com

Tuberculous pleural effusion is the second most common extrapulmonary tuberculosis 1 . Diagnosis of pleural TB relies on the examination of pleural fluid or biopsy specimens using acid-fast microscopic examination, culture, polymerase

chain reaction, on evaluation of pleural fluid characteristics or histopathological examinations.
Due to its paucibacillary nature, it is difficult to demonstrate tubercle bacilli in pleural fluid by a standard AFB staining and culture, thus leading to a large number of cases being undiagnosed or misdiagnosed 2 . Pleural fluid is a suboptimal sample for bacterial confirmation of pleural TB, using any method 3 . Pleural biopsy is the preferred sample. Thoracoscopic biopsy has a higher diagnostic yield than closed pleural biopsy ⁵.

The CBNAAT (cartridge based nucleic acid amplification test) assay was endorsed by WHO in December 2010 12 . XPERT MTB/RIF is a commercially available diagnostic test for Mycobacterium tuberculosis complex, which uses polymerase chain reaction (PCR) to test specimens for genetic material specific to MTB(Mycobacterium tuberculosis), and simultaneously detects a gene which confers resistance to rifampicin, rpoB. Rapid diagnosis, mean time of detection is 30 days for solid culture. Rifampicin resistance was detected in < 1day in CBNAAT versus an average of 75 days for phenotypic drug susceptibility testing (DST) ⁶.

Since CBNAAT was specifically developed and optimized for testing sputum samples and initial large-scale evaluations were in patients with pulmonary TB 3 . However, evaluations of the assay have extended to a variety of non respiratory clinical samples from patients with extrapulmonary tuberculosis. WHO has endorsed standard operating procedures for the use CBNAAT MTB/RIF for non-respiratory specimens 6, 12 . According to WHO, CBNAAT MTB/RIF may be used as a replacement test for usual practice (including conventional microscopy, culture, and/or histopathology) for testing of specific non- respiratory specimens (lymph nodes and other tissues) from patients presumed to have extrapulmonary TB 7 . According to RNTCP, the sensitivity of CBNAAT compared to a liquid culture which is the gold standard test is high in biopsy specimens ⁴.

However, the preferred specimen for the diagnosing a pleural TB is a pleural tissue 3, 8 . To date, the studies on evaluation of CBNAAT performed on pleural tissue are very few.

To compare the yield of CBNAAT, AFB culture and histopathology of pleural tissue in suspected tuberculous pleural effusions undergoing pleural biopsy:

A case series comprising 20 of our patients with presumptive pleural tuberculosis with significant pleural effusion on imaging underwent thoracoscopy guided pleural biopsy in department of respiratory medicine. Presumptive pleural Tuberculosis was defined as presence of cough, chest pain or shortness of breath, with or without fever and weight loss, with evidence of a pleural effusion on examination or CXR 3. Specimens were sent for CBNAAT, AFB culture and for histopathological examination. Comparative analysis of these reports were carried out.

• Patients having clinical feature of tubercular pleural effusion.
• Pleural effusion an chest X- ray and ultrasonography.
• Patients who were more than 14 years of age.
• Patients giving consent for the study.

• Patients with well documented chronic history of heart failure, nephritic syndrome, liver cirrhosis.
• Cases with empyema thoracis.
• Contraindications to thoracocentesis like patient on mechanical ventilation, uncooperative patients, bleeding diathesis, patient on anticoagulation therapy.

Out of 20 cases, 11 were microbiologically diagnosed as tuberculous pleural effusion. Mycobacterium tuberculosis was detected by CBNAAT in 10 cases, culture showed AFB growth in 7 cases. Histopathological examination of pleural biopsy showed granulomatous inflammation in 13 cases. In CBNAAT positive 10 cases, AFB culture showed growth in 6 out of 10 cases and HPE showed granulomatous inflammation in all cases.

Table 1: CBNAAT, HPE and AFB culture results of all cases

Table 2:

Table 3:

Results

Statistic	Formula	Value	95% CI
Sensitivity		85.71%	42.13% to 99.64%
Specificity		69.23%	38.57% to 90.91%
Positive Likelihood Ratio		2.79	1.17 to 6.65
Negative Likelihood Ratio		0.21	0.03 to 1.31
Disease prevalence		35.00% (*)	15.39% to 59.22%
Positive Predictive Value		60.00% (*)	38.60% to 78.16%
Negative Predictive Value		90.00% (*)	58.58% to 98.28%
Accuracy		75.00% (*)	50.90% to 91.34%

Results

Statistic	Formula	Value	95% CI
Sensitivity		100.00%	59.04% to 100.00%
Specificity		53.85%	25.13% to 80.78%
Positive Likelihood Ratio		2.79	1.20 to 3.90
Negative Likelihood Ratio		0.00
Disease prevalence		35.00% (*)	15.39% to 59.22%
Positive Predictive Value		53.85% (*)	39.34% to 67.73%
Negative Predictive Value		100.00% (*)
Accuracy		70.00% (*)	45.72% to 88.11%

Seven of 20 cases met the diagnostic standardof pleural TB, defined as pleural biopsy specimens that had positive culture results. Compared with gold standard AFB culture results, the sensitivity and specificity of CBNAAT MTB/RIF were 85.7% and 69.2%.

Due to its paucibacillary nature, it is difficult to obtain microbiologically confirmed diagnosis in a tuberculous pleural effusion 2 . In many centres pleural fluid analysis was the primary investigation for pleural Tuberculosis. Pleural fluid cultures are positive for Mycobacterium tuberculosis in less than 40% and smears are virtually always negative 16 . Pleural fluid biochemical and cell count analysis had high number of false negatives and false positives, significant concern is that this would lead to wrong diagnosis and empirical treatment. Cases undergoing pleural fluid analysis alone, chance of missing other fatal disease like malignancy are more, it leads to worst outcomes. Nowadays the diagnosis of tuberculous pleural effusion has been most commonly made with biopsy of the pleura³ .

Pleural biopsies were sent often for histopathological examinations rather than AFB smear and culture due to very low sensitivity due to paucibacillary nature and delay in reports respectively. For an AFB smear to be positive there should be >10 ⁵ bacilli per ml in specimen, for an AFB culture to be positive >100 bacilli per ml should be in sample ¹⁵ , the new PCR diagnostic tool CBNAAT able to detect less than 10 bacilli per ml in different biological specimens ¹⁸. Due to advent of this newer diagnostic tool CBNNAT in many tertiary centres, pleural biopsy samples are being sent for CBNAAT to get microbiologically confirmed diagnosis. If MTB detected in CBNAAT, diagnosed as microbiologically confirmed tuberculosis ⁴.

According to WHO, CBNAAT had pooled crude sensitivity of 92.5% and pooled specificity of 98% for TB detection. rifampicin sensitivity and specificity were 98% and 99% in respiratory samples ⁶ . Data on utility of CBNAAT MTB/RIF in extra-specimens are still limited. A large study that when testing a range of non respiratory sample types from both adult and children suspected of having extrapulmonary tuberculosis, CBNATT MTB/RIF had a sensitivity of 81.3% and specificity of 99.8% ⁷. In a study of Inderpaul Singh Sehgal et al. found that the pooled sensitivity and specificity of CBNAAT MTB/RIF compared to those of pleural fluid mycobacterial culture were 51.4% and 98.6% respectively ¹⁰. Studies in CBNAAT MTB/RIF of pleural biopsies in pleural TB are very few. In a study of Jinghui Du et al. stated that the overall sensitivity, specificity, PPV and NPV of CBNAAT assay using biopsy specimens for pleural TB diagnosis were 85.5%, 97.2%, 95.9% and 89.6%, respectively and the overall sensitivity, specificity, PPV and NPV of CBNAAT assay using pleural fluid specimens for pleural TB diagnosis were 43.6%, 98.6%, 96.0% and 69.3%, respectively ¹¹.

In our study CBNAAT detected MTB in 10 cases, among these culture showed growth in 6 cases. And in one case culture showed AFB growth but CBNAAT negative. Compared with gold standard AFB culture results, the sensitivity, specificity, PPV and NPV of CBNAAT MTB/RIF were 85.7%, 69.2%, 53.8% and 100.0% respectively.

The diagnosis of tuberculous pleural effusion has been most commonly made with histopathological examination of the pleural biopsy. The demonstration of granuloma in the parietal pleura suggests tuberculous pleural effusion even caseous necrosis and AFB not demonstrated. Even though many physicians are treating pleural tuberculosis based on granuloma in histopathological examinations, reliance on histopathological examination of pleural biopsy to diagnose tuberculous effusion is debatable. Because it mimics other diseases, which might also produce granulomatous pleuritis such as fungal diseases, sarcoidosis, tularemia and rheumatoid pleuritis ¹⁷, still more than 95% of patients with granulomatous pleuritis have TB. Even granuloma is not demonstrated in pleural biopsy, the biopsy specimen should be examined for AFB because organisms are occasionally demonstrated when granuloma is not seen in biopsy ⁹.

In our study, granulomatous inflammation was present in 13 cases, out of these, 7 cases were confirmed as TB by gold standard AFB culture test. CBNATT MTB was detected in 10 cases. In our study the sensitivity and specificity of HPE were 100% and 53.85%.

It was also observed that the CBNAAT test detected MTB in 20% of our study of presumptive pleural TB cases whose AFB cultures were negative but who had positive radiological tests and positive histology report. In one case AFB culture showed growth in which CBNAAT was negative in Presumptive pleural TB with positive histology. CBNAAT is more sensitive than AFB smear and culture and it is more rapid test, mean time of detection 3.

Finally, not only M. tuberculosis detection but also rapidly determining the patient’s MDR status
is of prime importance in bringing to an end the spread of MDR-TB and decreasing mortality. Faster
methods that allow MDR regimens to be started early are urgently needed. Rifampicin resistance was detected in < 1day in CBNAAT versus an average of 75 days for phenotypic drug susceptibility testing (DST) ⁶. Recent studies have highlighted the problems with false-positive results of tests for rifampicin resistance ¹³. WHO recommends further confirmatory tests following detection of rifampicin-resistant M. tuberculosis strains ¹⁴. In our study CBNAAT detected rifampicin resistance in one case who had no risk factor for MDR. AFB culture had shown resistance to streptomycin in one case and to pyrazinamide in another case. We limited our study only to detection of MTB. The data on role of CBNAAT in MDR pleural TB are very few.

CBNAAT is likely to play an important role in providing rapid molecular diagnostic assessment of presumptive pleural TB. It is most rapid, highly sensitive test compared to AFB culture and more confirmatory to diagnose microbiologically confirmed tuberculosis compared to HPE in presumptive pleural TB patients.

Due to the advantage of newer PCR based test CBNAAT in pleural tissues rather than pleural fluid, there is increase in the yield of confirmed diagnosis of TB and reduces the missed or wrong diagnosis and empirical treatment and worst outcomes.

Limitation in our study is small sample size. Further studies should be done to evaluate the clinical impact of CBNNAT on pleural biopsies, including evaluation of the outcomes and effect on clinical practice decisions, management outcome, the cost-effectiveness and feasibility of implementing the assay.

We thank our post graduate friends and consultants in our department of respiratory medicine for their valuable inputs and suggestions for this article. We acknowledge the department of microbiology and pathology and staff in the bronchoscopy room for their support in data collection.

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