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Year : 2020  |  Volume : 69  |  Issue : 1  |  Page : 1-5

Role of bronchoalveolar lavage cartridge-based nuclear acid amplification test in the diagnosis of sputum smear-negative pulmonary tuberculosis

1 Department of Pulmonary Medicine, Kempegowda Institute of Medical Sciences (KIMS), Bengaluru, Karnataka, India
2 Department of Microbiology, Kempegowda Institute of Medical Sciences (KIMS), Bengaluru, Karnataka, India

Date of Submission21-Nov-2018
Date of Acceptance27-Jan-2019
Date of Web Publication31-Jan-2020

Correspondence Address:
MBBS, DNB, DTCD Archana B.
Department of Pulmonary Medicine, Kempegowda Institute of Medical Sciences (KIMS), Bengaluru 560004, Karnataka
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/ejcdt.ejcdt_185_18

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Background Approximately 40–60% of clinical and radiological suspected cases of pulmonary tuberculosis (PTB) will have sputum smear result negative for acid-fast bacilli (AFB). Establishing early diagnosis and starting treatment early can have significant effect on morbidity and mortality in these patients. Bronchoalveolar lavage (BAL) samples obtained by bronchoscopy can be tested with cartridge-based nuclear acid amplification test (CBNAAT) for rapid diagnosis of sputum smear-negative PTB cases.
Objectives To assess the role of BAL CBNAAT in the early diagnosis of sputum smear-negative PTB cases.
Patients and methods A total of 112 patients with clinicoradiological findings suggestive of PTB with two consecutive sputum smear-negative results for AFB were enrolled in the study. BAL samples were subjected to Ziehl–Neelsen staining, CBNAAT (Xpert MTB/Rif; Cepheid), and liquid culture by mycobacteria growth indicator tube method as standard reference. Sputum samples from the same patients were also cultured.
Results BAL samples were positive for AFB on Ziehl–Neelsen staining in 64.2% of smear-negative patients. CBNAAT was positive in 75.8%, whereas mycobacteria growth indicator tube was positive in 76.7% as compared with sputum culture positivity, which was positive in 19.6% of patients. Sensitivity and specificity of CBNAAT were 96.51 and 86.3%, respectively, when compared with culture results. Overall, 76.7% of patients were diagnosed as having culture-positive PTB.
Conclusion BAL CBNAAT is useful in the early diagnosis of sputum smear-negative PTB, thus reducing the morbidity and mortality among these patients.

Keywords: bronchoalveolar lavage, cartridge-based nuclear acid amplification test, fiberoptic bronchoscopy, smear-negative pulmonary tuberculosis

How to cite this article:
B. A, Singh A, N. H, Gopi A. Role of bronchoalveolar lavage cartridge-based nuclear acid amplification test in the diagnosis of sputum smear-negative pulmonary tuberculosis. Egypt J Chest Dis Tuberc 2020;69:1-5

How to cite this URL:
B. A, Singh A, N. H, Gopi A. Role of bronchoalveolar lavage cartridge-based nuclear acid amplification test in the diagnosis of sputum smear-negative pulmonary tuberculosis. Egypt J Chest Dis Tuberc [serial online] 2020 [cited 2020 Apr 3];69:1-5. Available from: http://www.ejcdt.eg.net/text.asp?2020/69/1/1/277294

  Introduction Top

Tuberculosis (TB) is a major global health problem causing significant mortality and morbidity worldwide. There were an estimated 10.4 million TB cases globally, with estimated 1.3 million TB deaths among HIV-negative people and an additional 374 000 deaths among HIV-positive people in 2016 [1]. Most of the estimated number of cases occurred in South-East Asia region (45%), African region (25%), and Western Pacific region (17%), and a smaller proportion of cases occurred in the WHO Eastern Mediterranean region (7%), the WHO European region (3%), and the WHO region of the Americas (3%) [1]. The top five countries with 56% of estimated cases were India, Indonesia, China, Philippines, and Pakistan [1].

Detecting patients with active pulmonary tuberculosis (PTB) is an important component of TB control, as early treatment renders them noninfectious and interrupts the chain of transmission. Smear microscopy is cornerstone for the diagnosis of the TB. Overall, 40–60% of clinically and radiologically suspected cases of PTB will have sputum smear-negative results for acid-fast bacilli (AFB) [2]. Studies have suggested that more than 50% of these patients would be needing chemotherapy by 12 months if left untreated [3]. Gold standard for diagnosis of TB is the detection of mycobacterium tuberculosis (MTB) by solid culture, but it requires 2–8 weeks. Fiberoptic bronchoscopy (FOB) with bronchoalveolar lavage (BAL) can be used for diagnosis of smear-negative PTB. BAL sample can be subjected to Ziehl–Neelsen (ZN) staining, microbial cultures, and molecular testing.

Many PCR-based molecular methods have been developed for early and rapid diagnosis of TB and drug resistance from clinical specimens [4],[5],[6],[7]. Xpert MTB/Rif (Cepheid) is a cartridge-based nuclear acid amplification test (CBNAAT), which is a fully automated real-time PCR that detects MTB along with rifampicin resistance within 2 h, and it is recommended by WHO on sputum samples [8]. Formal recommendations have not yet been established for FOB samples. Hence, further studies are required to assess its performance on FOB samples.

This study was conducted in an institution-based hospital to evaluate the role of BAL CBNAAT in the diagnosis of smear-negative PTB.

  Materials and methods Top

This is a prospective study conducted in the Department of Pulmonary Medicine, Kempegowda Institute of Medical Sciences, Bangalore, India, from January 2017 to March 2018. Consecutive patients with clinicoradiological findings suggestive of PTB with two consecutive sputum smear-negative results for AFB were screened and treated with 2 weeks of broad-spectrum antibiotics (amoxyclav). A total of 112 patients aged more than 18 years and ready to give written consent, who showed no improvement and in whom alternative diagnosis was ruled out, were enrolled in the study.

Patients with sputum smear-positive results, isolated extra-PTB, HIV-positive patients, and patients with contraindications for FOB, such as coagulation disorders, refractory hypoxia, severe thrombocytopenia, cardiovascular instability, and severe pulmonary hypertension were excluded from the study. Informed written consent was taken from the study participants, and the study was approved by the institutional ethical committee.

Bronchoscopy procedure

FOB was performed using Olympus BF type 1T150 (Bronchoscope Olympus corporation, Okaya, Nagano, Japan). For the collection of BAL specimens, 100–150 ml of normal saline, at room temperature, was instilled by 50-ml aliquots. The BAL fluid was sent for CBNAAT testing, ZN smear microscopy, and mycobacteria growth indicator tube (MGIT) culture.

Processing of samples for acid-fast staining

Samples were subjected for decontamination by modified Petroff’s technique and then centrifuged at 3000 rpm for 15 min. Supernatant was discarded, and the deposit was used for smear preparation.

Acid-fast bacilli culture

Samples were digested and decontaminated using N-acetyl-L-cysteine. Culture was done using BACTEC mycobacteria growth indicator tube method containing modified middle brook 7H9 broth. Cultures were incubated at 37°C and screened for any growth at regular interval once in 24 h. All tubes were checked for positivity till 42 days. Cultures found positive were further confirmed by ZN staining and fluorescence staining and also by immunochromatography assay for detection of MTB antigen MPT 64.

Cartridge-based nuclear acid amplification test assay

CBNAAT testing was performed under RNTCP guidelines. BAL sample was inactivated with 2 : 1 sample reagent; 2 ml of this material was transferred to the test cartridge and inserted into MTB/RIF (Cepheid, Sunnyvale, California, USA) test platform. Filter-captured organisms then underwent ultrasonic lysis to release DNA, which is then mixed with PCR reagent, from which real-time amplification and detection of the MTB and rifampicin resistance is possible in less than 2 h.

Statistical analysis

Data analysis was done using SPSS-16.0 for Windows version (IBM SPSS Statistics, IBM, USA). Sensitivity, specificity, positive predictive value, and negative predictive value were calculated, along with the corresponding 95% confidence intervals (CIs). BAL CBNAAT was compared with BAL culture using kappa statistics.

  Results Top

A total of 112 patients were enrolled in the study. Among 112 patients, 76 (67.8%) were males and 36 (32.1%) were females. Mean age of the patients was 41 years (22.5–60 years). The baseline clinical, demographic, clinical, and radiological characteristics of patients are shown in ([Table 1]). Of 112 BAL samples, ZN smear, CBNAAT, and culture were positive in 72 (64.2%), 85 (75.8%), and 86 (76.7%) patients, respectively. Sputum culture was positive in 22 (19.6%) patients only ([Table 2]). Of 112 patients, 86 (76.7%) were diagnosed as BAL culture-positive PTB as compared with 22 (19.6%) as sputum culture-positive PTB. Overall, 86 (76.7%) patients were diagnosed as having culture-positive PTB ([Table 3]). Four (3.57%) more patients were diagnosed as having TB based on histopathology of transbronchial lung biopsy specimen. A confirmed diagnosis of TB was made in 90 (80.35%) patients ([Figure 1]). Among the CBNAAT-positive and culture-positive patients, seven patients had rifampicin resistance.
Table 1 Baseline characteristics of patients with smear-negative pulmonary tuberculosis (N=112)

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Table 2 Smear/culture and cartridge-based nuclear acid amplification test results

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Table 3 Comparison of bronchoalveolar lavage cartridge-based nuclear acid amplification test and bronchoalveolar lavage Ziehl–Neelsen smear microscopy results with culture

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Figure 1 Flow diagram of patients included in the study. FOB, fiberoptic bronchoscopy; BAL, bronchoalveolar lavage; PTB, pulmonary tuberculosis; CBNAAT, cartridge-based nuclear acid amplification test; TBLB, transbronchial lung biopsy; ILD, interstitial lung disease, CA, carcinoma.

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When compared with culture as gold standard, sensitivity, specificity, positive predictive value, and negative predictive values for CBNAAT were 96.51% (95% CI, 90.14–99.27%), 92.31% (95% CI, 74.87–99.05%), 97.65% (95% CI, 91.63–99.37%), and 88.89% (95% CI, 72.36–96.07%), respectively. The corresponding value for BAL ZN smear microscopy were 76.67% (95% CI, 66.57–84.94%), 86.36% (95% CI, 65.09–97.09%), 95.83% (95% CI, 88.87–98.51%), and 47.50% (95% CI, 37.53–57.68%), respectively ([Table 4]). As per the kappa statistic (κ=0.87), BAL CBNAAT is in near-perfect agreement with the culture results.
Table 4 Performance value of the cartridge-based nuclear acid amplification test, Ziehl–Neelsen smear microscopy when compared with culture

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In our study, among the 24 CBNAAT- and culture-negative patients, 4 patients grew nontuberculous mycobacterium (NTM), and eight were diagnosed as having pneumonia, 4 as having interstitial lung disease, and two as having bronchogenic carcinoma. No definite diagnosis was made in two patients. No complications occurred among the patients undergoing FOB ([Figure 1]).

  Discussion Top

TB remains one of the deadliest diseases worldwide. Early diagnosis of TB is very important to break the chain of transmission. FOB plays a significant role in the diagnosis of patients who are suspected to have PTB based on radiological findings but have sputum smear result negative for AFB. FOB is considered as a safe diagnostic and interventional procedure even in young and premature infants [9].

Among the various bronchoscopic specimens such as BAL, bronchial washings, brushings, bronchial biopsy, and transbronchial biopsy, BAL is considered far superior for the diagnosis of TB [10]. This is owing to the fact that the topical anesthetics such as lidocaine used during bronchoscopy has been shown to inhibit the growth of mycobacterium [11] and the concentration of aspirated local anesthetic could be lower in BAL as compared with bronchial washings. Because of the higher sensitivity of tests using specimens obtained through bronchoscopy, some experts recommend that in regions with a high frequency of TB, bronchoscopy should be performed routinely for patients suspected of having TB [12],[13].

In our study, we evaluated the diagnostic yield of BAL CBNAAT in 112 sputum smear-negative patients. Only 22 (19.6%) patients could be able to diagnose as having TB based on sputum culture positivity. However, BAL CBNAAT yielded a positive result in 85 (75.8%) patients. Using culture as the reference standard, BAL CBNAAT showed a high sensitivity of 96.51% which was similar to previous studies (80.0–95.7%) [14],[15],[16],[17],[18]. This high sensitivity may be owing to the clinical and radiological evaluation before FOB, including a course of board spectrum antibiotic, which ensured a high pretest probability. Moreover, in our study, early diagnosis of TB was possible in these patients by BAL CBNAAT as compared with sputum smear microscopy.BAL CBNAAT showed a specificity of 92.3% compared with previous studies (91–100%) [14],[15],[16],[17],[18]. It is because four culture samples were positive for NTM, and CBNAAT only detects MTB. In other three CBNAAT-negative and culture-positive cases, it is possible that the bacterial load may have been too low for CBNAAT to detect the DNA from MTB complex. It shows that a patient with a negative CBNAAT result can still have TB with MTB or NTM [16],[18].

Two of our patients had culture-negative result and CBNAAT positivity. This could be owing to DNA amplification from dead bacilli leading to positive CBNAAT result, whereas culture remains negative [19]. This is in line with the study by Barnard et al. [18] who had raised caution in the interpretation of Xpert positive results, which were culture negative. In our study, BAL CBNAAT outperformed BAL smear microscopy (sensitivity 96.51% vs. 80.23%), and we could diagnose seven patients as having MDR. Moreover, FOB and BAL contributed to an alternative diagnosis in 18 patients, including four NTM cases.

Our study had few limitations. The sensitivity and specificity of MTB/RIF assay to detect rifampicin resistance was not evaluated in the present study and also the cost-effectiveness of CBNAAT was not evaluated.

  Conclusion Top

In sputum smear-negative PTB suspected cases, BAL CBNAAT has good sensitivity and specificity. Widespread use of CBNAAT can increase the case detection rates of PTB, thereby facilitating early treatment decisions and curbing transmission.


Authorship Statement: Dr Archana and Dr Amandeep Singh designed the study. Dr Archana and Dr Amandeep Singh performed the study. Dr Huliraj and Dr Anjana Gopi contributed important reagents. Dr Archana and Dr Amandeep Singh collected the data. Dr Archana, Dr Amandeep Singh, and Dr Huliraj analyzed the data. Dr Archana and Dr Amandeep Singh wrote the paper.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.

  References Top

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  [Figure 1]

  [Table 1], [Table 2], [Table 3], [Table 4]


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