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 Table of Contents  
ORIGINAL ARTICLE
Year : 2022  |  Volume : 17  |  Issue : 3  |  Page : 557-562

Cartridge-based nucleic acid amplification test: An early tool for the diagnosis of sputum smear-negative pulmonary tuberculosis


1 Department of Respiratory Medicine, DMIMS and JNMC Sawangi, Wardha, Maharashtra, India
2 Department of Respiratory Medicine, BYL and Nair Hospital, Mumbai, Maharashtra, India

Date of Submission16-Feb-2022
Date of Decision08-Jul-2022
Date of Acceptance14-Aug-2022
Date of Web Publication2-Nov-2022

Correspondence Address:
Dr. Pankaj Wagh
Department of Respiratory Medicine, DMIMS and JNMC Sawangi, Wardha, Maharashtra
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jdmimsu.jdmimsu_62_22

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  Abstract 


Introduction: Tuberculosis (TB) of the lung is an infectious disease caused by Mycobacterium tuberculosis, which is an aerobic, acid-fast, Gram-positive bacillus. India has the world's highest number of TB cases, accounting for one-fourth of all TB cases. For efficient treatment of pulmonary TB, microbiological diagnosis is the mainstay. Acid-fast bacillus (AFB) smear-negative patients could account for 31% of new cases. After multiple negative induced-sputum smear tests, we conducted this study to observe whether cartridge-based nucleic acid amplification test (CBNAAT) provides an additional and early diagnostic yield for undiagnosed cases of pulmonary TB. Objectives and Goals: The purpose of this study was to observe if CBNAAT could be used to diagnose pulmonary TB early in patients who had TB clinically and radiologically but negative for sputum direct smear. Materials and Methods: At our institute, 82 patients were involved in an observational study for assessing the utility of CBNAAT in the early confirmation of TB in individuals who had an X-ray chest that is indicative of PTB, but a negative sputum smear for AFB. Our hospital's department of respiratory medicine was the site of the current clinical study. Results: The patients were mostly between the ages of 31 and 40 years, with a male majority. Cough was the most common symptom in 72 (88%) patients. In 30 patients, the sputum CBNAAT test was positive (36.5%). All 82 instances were given a bronchoalveolar lavage (BAL). In 46 (56%) patients, BAL CBNAAT was positive. In 12 (15%) cases, bronchial brushings tested positive for AFB on smear examination. Caseating epithelioid granuloma was seen in 4 (14%) patients after a transbronchial lung biopsy. Conclusion: In individuals with strong indication of pulmonary TB, sputum CBNAAT and BAL CBNAAT provide a higher bacteriological confirmation of diagnosis.

Keywords: Acid-fast bacillus, bronchoalveolar lavage, cartridge-based nucleic acid amplification test, Mycobacterium tuberculosis


How to cite this article:
Wagh P, Rajpurohit R, Ali S, Adwani S, Kadukar J, Aurangabadkar G. Cartridge-based nucleic acid amplification test: An early tool for the diagnosis of sputum smear-negative pulmonary tuberculosis. J Datta Meghe Inst Med Sci Univ 2022;17:557-62

How to cite this URL:
Wagh P, Rajpurohit R, Ali S, Adwani S, Kadukar J, Aurangabadkar G. Cartridge-based nucleic acid amplification test: An early tool for the diagnosis of sputum smear-negative pulmonary tuberculosis. J Datta Meghe Inst Med Sci Univ [serial online] 2022 [cited 2023 Feb 1];17:557-62. Available from: http://www.journaldmims.com/text.asp?2022/17/3/557/360230




  Introduction Top


Mycobacterium tuberculosis (MTB), an aerobic, acid-fast, Gram-positive bacillus, causes tuberculosis (TB). In India, TB is a major public health issue. Although India is the world's second most populous country after China, It has the most TB cases, having approximately one-quarter of all TB cases worldwide. In 2013, India was expected to have 2.1 million cases of TB, out of a global annual incidence of 9 million cases.[1]

TB is a global public health epidemic of epic proportions, with developing countries bearing the brunt of the TB burden. For the diagnosis of pulmonary TB culture and microscopic examination of conventional sputum, IS, gastric washings, and fiberoptic bronchoscopy are used. Each of these tests' sensitivity, specificity, and diagnostic yield varies greatly between investigations.[2],[3],[4],[5],[6],[7],[8] Microscopic examination of sputum and culture is reasonably simple to do, despite the fact that in a significant number of individuals with pulmonary TB, the test might be negative with sensitivity levels being observed to range from 25% to 45%.[2],[9],[10] Microbiological diagnosis is the mainstay for efficient treatment of pulmonary TB. Although the proper sputum is obtained, the bacillary load must be 10,000/ml at least in order for the report to be positive for TB bacilli.[11] In addition, it is contingent on the preceding therapy, default behavior, and cough effectiveness. Smear negative for acid-fast bacillus (AFB) might be found in 31% of new cases.[12] Induced sputum using hypertonic saline necessitates additional resources and training, but it has been proven in multiple trials to improve the diagnostic yield of sputum testing.[13],[14],[15] Few research studies have established that sputum and BAL cartridge-based nucleic acid amplification test (CBNAAT) has a greater diagnostic yield than sputum and BAL direct smear for AFB in the confirmation of lung TB. When a patient with active pulmonary TB receives numerous negative IS findings, clinicians are frequently faced with a difficult decision. They can either start empiric therapy for pulmonary TB or have an invasive test like a bronchoscopic BAL CBNAAT and sputum CBNAAT to reach the diagnosis. The query then becomes whether bronchoscopic BAL CBNAAT and sputum CBNAAT can help individuals with active pulmonary TB who have had several negative induced-sputum outcomes. Depending on the research design and patient demographic analyzed, the available literature on this issue indicates variable diagnostic yields for bronchoscopic BAL and sputum CBNAAT.[16],[17] Patients having bronchoscopic BAL CBNAAT have higher diagnostic yields because they are unable to expectorate any sputum. Patients who had bronchoscopic BAL after a sufficient sputum sample was judged to be smear negative did not have this problem.[18],[19],[20] Other studies, on the other hand, have found that bronchoscopic BAL yields no more than sputum induction.[21],[22]

We conducted this research to determine if bronchoscopic BAL CBNAAT and sputum CBNAAT give extra and early diagnostic yield following numerous negative IS smear examination. As a result, the current research was conducted at our tertiary care center to find early diagnostic value of bronchoscopic BAL CBNAAT and sputum CBNAAT in suspected pulmonary TB patients.


  Aims and Objectives Top


To know the early diagnostic utility of CBNAAT in direct sputum smear-negative lung TB.


  Materials and Methods Top


The relevance of BAL and sputum CBNAAT in the confirmation of PTB in patients with X-ray chest indicative but not diagnosed due to a negative sputum smear for AFB was evaluated in an institutional-based observational research with 82 individuals over a period of one and half year. The current clinical study was conducted in a tertiary care center's department of respiratory medicine. Patients who were agreed to participate in the trial, who were more than 18 years old, PTB patients suspected (as per RNTCP guidelines), patients with X-ray that suggests TB, but sputum that was negative for AFB, and patients with pneumonia that does not resolve were included in the study. Patients with HIV, bleeding diathesis, arrhythmias, a history of myocardial infarction, X-ray that suggests something other than TB were excluded from the research.

Methodology

The study included all patients who visited our tertiary care center's outpatient department and inpatient department and fulfilled the inclusion and exclusion criteria. All patients were evaluated for investigations such as Hb%, TLC, DLC, erythrocyte sedimentation rate, BT, computed tomography, platelet count, urine examination, RBS, chest X-ray, electrocardiography, HIV, and HBSAg. Sputum for AFB and CBNAAT-2 samples were taken, 1 spot sample and 1 sample in early morning. Ziehl–Neelsen was the technique for staining. Those patients who were negative for sputum AFB direct smear were investigated for sputum CBNAAT. Fiberoptic bronchoscopy was utilized to assess the bronchial tree and retrieve bronchoalveolar lavage (BAL) fluid for CBNAAT. Transbronchial lung biopsy and bronchial brushings (for 2 slides) were also collected where feasible. All of the participants had at least two sputum smear tests that were negative for acid-fast bacilli. Under local anesthetic, two bronchoscopists conducted a transnasal bronchoscopy with an Olympus bronchoscope. The nasal cavity and throat were sprayed with lignocaine 10%, and the vocal cords, trachea, and bronchi were sprayed with lignocaine 2% solution. Bronchial trees were anesthetized with lignocaine doses ranging from 40 to 120 mg. All inpatients received premedication with pethidine 50–75 mg and atropine 0.6 mg intramascularly half an hour before the surgery, while outpatients did not. Bronchoalveolar lavage (BAL), bronchial brushing, and transbronchial lung biopsy (TBLB) were taken after a comprehensive evaluation of the bronchial tree. The collected samples were mounted on slides for Ziehl–Neelsen staining and were investigated for CBNAAT.


  Ethical clearance Top


The Institutional Ethics Committee of DMIMSDU has approved the Research work proposed to be carried out at Jawaharlal Nehru Medical College, Sawangi(M), Wardha. Date : 1st July 2019 with Reference no DMIMS(DU)/IEC/2019-20/91.


  Observations and Results Top


The advantage of CBNAAT in the early diagnosis of PTB was investigated in an institutionally based observational study with 82 patients. The most of the individuals (44%) were in the age range of 31–40 years [Table 1]. Male patients made up 55% of the study samples, while female patients made up 45% [Graph 1]. Cough and fever were present in 72 (88%) and 58 (71%) patients, respectively. Shortness of breath 42 (51%), lack of appetite 36 (44%), chest discomfort 34 (41%), hemoptysis 28 (34%), and weight of loss 13 (16%) were the other symptoms [Graph 2]. The upper zone was more typically engaged, with infiltration occurring more frequently in the right UZ (12), left UZ (6), and bilaterally (4). Right UZ consolidation (5), left UZ consolidation (3), bilateral (2), cavity in left UZ (1), bilateral military mottling (4), and fibrosis in right UZ (3) [Graph 3]. In 33 (40%) of the patients, bronchoscopy revealed no pathological lesion. Out of 49 patients with bronchoscopy revealing pathology, 25 (31%) had bronchus discharge, 16 (20%) participants had an abnormal mucosa/granuloma, 6 (7%) had oozing of blood from bronchus, and only 2 (2%) had growth evident [Graph 4]. In all 82 cases, CBNAAT of the sputum was performed. CBNAAT was found to be positive in 30 (36.5%) patients, including 18 males and 12 females. There was no significant relationship between sputum CBNAAT and patient gender (P = 0.478) [Table 2]. In all 82 cases, CBNAAT of BAL fluid was performed. CBNAAT was found to be positive in 46 (56%) patients, including 23 males and 23 females [Table 2]. All 30 patients that were tested positive on sputum CBNAAT were positive on the BAL CBNAAT as well. There was no significant relationship between CBNAAT of BAL fluid and patient gender (P = 0.316). In all 82 instances, bronchial brushings were obtained from the diseased section. In 12 (15%) patients, smear tests for AFB were positive in bronchial brushings. Nine out of the 12 positive patients also tested positive for sputum and BAL CBNAAT, whereas 3 instances tested positive only for bronchial brushing smear. Bronchial brushing smear for acid-fast bacilli had no significant relationship with patient gender (P = 0.794). Only 29 out of 82 individuals had bronchial biopsies that were possible. In 4 (14%) patients, TBLB revealed caseating epithelioid granuloma, with 2 being positive in all three tests and 2 being positive only in TBLB. Malignancy was found in 4 (14%) individuals, while nonspecific inflammation was seen in 21 (72%) patients [Graph 5]. 30 (36.5%) cases were diagnosed with the help of sputum CBNAAT. 46 (56%) patients were confirmed for TB by BAL fluid CBNAAT and sputum CBNAAT. Bronchial brushing smear and TBLB helped further diagnosis of pulmonary TB. Diagnosis of 51 cases out of 82 (62%) were done by combining all investigations [Table 3]. Sputum CBNAAT and BAL CBNAAT diagnosed 56% of patients. BAL CBNAAT was the most successful approach with a 90.2% (46/51) sensitivity. Sputum CBNAAT and bronchial brushing smear had a sensitivity of 58.8% (30/51) and 23.5% (12/51), respectively. 4 out of 51 positive cases were diagnosed by TBLB, with a sensitivity of 7.8% (4/51) [Table 4]. The greatest yield was found in BAL CBNAAT (46/82) with a 56% yield, followed by sputum CBNAAT (21/82) with a 36.5% yield. Bronchial brushing smear and TBLB exhibited low diagnostic yields, with diagnostic yields of just 12/82 (15%) and 4/29 (14%), respectively [Table 5].
Table 1: Distribution of patients according to age and gender

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Table 2: Cartridge-based nucleic acid amplification test of bronchoalveolar lavage fluid and sputum

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Table 3: Role of bronchoscopy in diagnosis of pulmonary tuberculosis

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Table 4: Sensitivity of various diagnostic methods

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Table 5: Total yield of various diagnostic methods

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  Discussion Top


The relevance of CBNAAT in the early diagnosis of PTB suspects was evaluated in an institutionally based observational research with 82 individuals. India accounts for 2.2 million of the 8.6 million new TB cases identified every year worldwide, more than double the number of any other country.[23]

The yardstick for pulmonary TB diagnosis is the recognization of acid-fast bacilli in sputum. Microscopic examination of sputum is an important part of the WHO's directly observed treatment short-course approach. Chronic symptomatic patients who do not have acid-fast bacilli should be closely observed and AKT should only be started after bacteriological confirmation of diagnosis. Most of the patients in this study (44%) were in the age range of 31–40 years, with male patients constituting 55% of the study sample and females 45%. The average age of the participants in the research by Bhaglani et al.[24] was 41.18 years. Males were 42.35 years old on an average, whereas females were 38.50 years old. In a research by Kumar et al.,[25] most patients were from 20–29-year-old age group (33.33%), followed by 40–49-year-old with 22 (66.66%) males and 11 (33.33%) females. In a research by Prakash et al.,[26] 33 (66%) of the 50 cases were male and 17 (34%) were female. In a study by Patel and Kamdar,[27] males accounted for 36 (68%) of the 53 patients, whereas females accounted for 17 (32%) with more prevalent age group being 20 to 49 years.

Cough and fever were reported by 88% and 71% of the participants in our research, respectively, followed by breathlessness (51%), lack of appetite (42%), chest discomfort (41%), hemoptysis (34%), and weight loss (16%). In a research by Bhaglani et al.,[24] the most common finding was cough (93.93%), followed by dyspnea (86.36%), chest pain (50%), fever (45.45%), and hemoptysis (28.78%). In a study by Patel and Kamdar,[27] the cardinal signs of TB were fever (83%), cough (74%), chest discomfort (64%), and weight loss (60%).

The present study's radiological findings revealed that the UZ was the most typically implicated, with infiltration occurring most frequently in the right UZ (12), left UZ (6), and bilaterally (4). Right UZ consolidation (5), left UZ consolidation (3), bilateral (2), cavity in left UZ (1), bilateral military mottling (4), and right UZ fibrosis (3). The most prevalent radiological features were consolidation (53%), cavitary lesion (26%), and mediastinal lymphadenopathy, according to Patel and Kamdar.[27]

In our study, 33 (40%) patients had no pathological lesion on bronchoscopy. 49 patients on bronchoscopy had pathology, out of that 25 (31%) had bronchus discharge, 16 (20%) had abnormal mucosa/granuloma, 6 (7%) had oozing of blood from bronchus, and only 2 (2%) had growth evident. Kumar et al.'s[25] study found no abnormalities in 9 (27.27%) and obvious abnormalities in 24 (72.72%) patients. Hyperemic and edematous mucosa in 16 (48.48%). Four (12.12%) patients had a distinct granular surface, mucopurulent secretion in 13 (39.39%), bleeding in 6 (18.18%), atrophy of mucosa and stenosis of bronchus in 4 (12.12%), growth in 3 (9.09%). In Prakash et al.'s[26] study, no pathological lesion in 21 (42%) patients and 29 (58%) patients with bronchoscopy revealing pathology with 14 (28%) had bronchus discharge, 11 (22%) had abnormal mucosa/granuloma, 5 (10%) had compression, and another 5 (10%) had oozing of blood from bronchus, whereas 2 cases (4%) cases had growth.

All 82 patients in our research had sputum CBNAAT. Sputum CBNAAT was positive in 30 patients (36.5%), including 18 males and 12 females. There was no significant relationship between sputum CBNAAT and patient gender (P = 0.478). Mohanty T et al.'s[28] study on 100 cases with 74 men and 26 women had BAL CBNAAT yield in 38/71 (54%), sputum CBNAAT positive in 23 (32%), BAL culture reference standard yield in 42/71 (59%).

All 82 patients in our research had BAL fluid CBNAAT. CBNAAT was positive in 46 patients (56%), including 23 males and 23 females. There was no significant relationship between CBNAAT of BAL fluid and patient gender (P = 0.316). Mohanty et al.'s[28] study on 100 cases with 74 men and 26 women had BAL CBNAAT yield in 38/71 (54%), SI CBNAAT positive in 23 (32%), and BAL culture reference standard yield in 42/71 (59%).

In our study, bronchial brushings were collected from the diseased section in 82 instances. In 12 (15%) of the patients, smear tests for AFB were positive in bronchial brushings. Bronchial brushing smear examination for acid-fast bacilli had no significant relationship with patient gender (P = 0.794). In Kumar et al.'s[25] study, bronchial brushing was performed on 16 of the 33 patients. AFB was found in 4 of the 25 individuals who had a bronchial brushing smear. In Bhaglani et al.'s[24] study in three instances, bronchial brushings were obtained; however, only one bronchial brushing generated a positive Ziehl–Neelsen staining smear result.

Bronchial biopsies were performed in only 29 of the 82 participants in the current research when biopsy was possible. A TBLB revealed caseating epithelioid granuloma in 4 (14%), malignancy in 4 (14%), and nonspecific inflammation in 21 (72%) patients. Kumar et al.'s[25] study reported that 2 (15.38%) of the 13 patients had caseating epithelioid granuloma, 3 (23.07%) had malignancy, and 8 (61.54%) had nonspecific inflammation. In a research by Prakash P et al.,[26] 3 out of 12 instances (25%) of TBLB revealed caseating epithelioid granuloma, with AFB in 2 of them (16.66%).

The BAL fluid CBNAAT and sputum CBNAAT were used to diagnose 46 (56%) patients in this investigation. The use of a bronchial brushing smear and TBLB for confirmation of the lung TB was helpful. By integrating all diagnostic procedures, we were able to identify 51 instances out of 82 (62%).

BAL CBNAAT was the most successful approach, with a 90.2% (46/51) sensitivity, followed by sputum CBNAAT and bronchial brushing smear with a sensitivity of 58.8% (30/51) and 23.5% (12/51), respectively. 4 out of 51 positive cases were diagnosed by TBLB, with a sensitivity of 7.8% (4/51). In a 2017 observational research, Mohanty et al.[28] evaluated multiple diagnostic methods in sputum smear-negative patients, with a focus on CBNAAT. The researchers discovered 100 cases, 74 men and 26 women. With a sensitivity, specificity, PPV, and NPV of 88.09%, 96.5%, 97.36%, and 84.84%, respectively, BAL CBNAAT yielded 38/71 (54%). In a research by Jacomelli et al.,[29] BAL CBNAAT had a sensitivity and specificity of 60% and 100%, respectively, for the diagnosis of TB.

BAL CBNAAT yielded the greatest yield (46/82) of 56% in our study, followed by sputum CBNAAT (30/82) of 36.5%. Bronchial brushing smear and TBLB exhibited low diagnostic yields, with diagnostic yields of just (12/82) 15% and (4/29) 14%, respectively. In Prakash et al.'s[26] study, BAL CBNAAT was the most successful approach with a sensitivity of 87.5% in 21 cases (21/24), followed by brushings with a sensitivity of 54.17% in 13 instances (13/24).

By combining all bronchoscopic-assisted procedures, 51 (62.2%) of the 82 patients in our research were identified as pulmonary TB. The most successful approach for diagnosing TB was BAL CBNAAT, which was used in 46 of the 51 individuals and had a sensitivity of 90.2%. Sputum CBNAAT, which was used in 30 instances, had a sensitivity of 58.8%. The bronchial brushing smear was done in 12 individuals and had a sensitivity of 23.5, whereas TBLB was successful in diagnosing 4 cases and had sensitivity of 7.8%. Other remaining 31 (37.8%) were diagnosed as other diseases out of which 27 were diagnosed as a bacterial pneumonia and 4 as a malignancy. The study's limitations were only temporary. It is carried out through our health-care system and was not involved entire population of Maharashtra. Because no culture for MTB was performed, the positive and negative predictive values of sputum CBNAAT and BAL CBNAAT could not be computed.


  Conclusion Top


India is one of the 22 high-burden nations for PTB, and because of the disease's high contagiousness, early detection is critical. The findings of this study imply that BAL CBNAAT and sputum CBNAAT can be used to diagnose SSN-PTB patients. In SSN-PTB patients, BAL CBNAAT and sputum CBNAAT demonstrate a high rate of bacteriological confirmation of diagnosis. In sputum-negative individuals, BAL CBNAAT and sputum CBNAAT along with different bronchoscopic methods can offer a prompt and conclusive diagnosis of PTB.

In sputum smear-negative individuals, CBNAAT is a valuable method for early identification of pulmonary TB. CBNAAT offers more bacteriological confirmation of diagnosis in individuals with considerable clinical and radiographic signs of lung illness. Individuals with sputum smear-negative lung TB who have a high degree of clinicoradiological suspicion should have BAL CBNAAT and sputum CBNAAT, as well as additional bronchoscopic-aided techniques.

Acknowledgment

We acknowledge Dr. Panjabrao Deshmukh Memorial Medical College, Amravati.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5]



 

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