EBUS-GS with the GeneXpert MTB/RIF assay for diagnosis of Mycobacterium tuberculosis infection of isolated pulmonary nodules

Tuberculosis (TB) is a significant global public health problem. The 2021 Global Tuberculosis Report of the World Health Organization (WHO) concluded that about 9.9 million people were suffering from TB and 1.3 million died from TB during 2020. China had 841,500 newly diagnosed TB patients in 2020, accounting for 8.5% of the global total, second only to India [1]. Early diagnosis and treatment are keys for reducing the burden of TB [2]. The positive detection of pathogens in sputum (molecular testing, culture) is considered the gold standard for diagnosing tuberculosis [3]. However, a 2019 study reported that smear-negative pulmonary TB accounted for approximately 70% of all cases of pulmonary TB in China, and this was the main reason for the slow decline in the prevalence of pulmonary TB [4].

Pulmonary tuberculoma is a special type of secondary pulmonary TB that has no characteristic clinical manifestations, but has imaging manifestations of atypical non-calcified solitary pulmonary nodules [5]. Pulmonary tuberculoma can be easily confused with peripheral lung cancer, pulmonary sarcoidosis, benign lung tumor, and other conditions [6‐8]. Patients with pulmonary tuberculoma have extremely low rates of smear-positive and culture-positive sputum [9]. Samples obtained by minimally invasive examinations may improve the diagnostic efficiency based on pathological and etiological detection [10]. Clinicians commonly perform percutaneous pulmonary puncture for biopsy because this method is simple, minimally invasive, and inexpensive. However, this method is also less precise when the lesion is tiny or located at the inner one-third of lung, and can also lead to pneumothorax and bleeding [11]. It is often difficult for a clinician using a common bronchoscope to reach peripheral pulmonary lesions, so this method often fails to obtain a sufficient amount of tissue for the confirmation of bacteriologically-negative TB.

Endobronchial ultrasonography (EBUS) is an efficient and minimally invasive interventional bronchoscopic technique that is increasingly used because of its good performance in the diagnosis of pulmonary diseases, such as tumors [12‐14]. EBUS with a guide-sheath (EBUS-GS) uses a small-diameter, annular ultrasound probe that is inserted into a guide sheath. Clinicians can use EBUS-GS to enter a grade 8 to 9 bronchus, and then use the GS to establish a channel for biopsy under ultrasound guidance [14, 15]. There are few studies on EBUS-GS in the diagnosis of isolated pulmonary nodules caused by Mycobacterium tuberculosis (MTB) infection [14]. Due to the limited tissue obtained by EBUS-GS, it is still difficult to diagnose TB by pathological examination alone. To improve diagnostic efficiency, it is necessary to combine EBUS-GS with a rapid and effective pathogen detection method, such as GeneXpert MTB/RIF (Xpert; Cepheid, Sunnyvale, CA, USA), a cartridge-based, automated hemi-nested real time PCR system that uses five overlapping molecular beacon probes (A–E) which target the rifampicin resistance determining region (RRDR) of the rpoB gene [16]. This method thus allows detection of Mycobacterium tuberculosis complex (MTBC) and mutations conferring rifampicin resistance within 2 h [17]. The Xpert system is now widely used for the clinical diagnosis of pulmonary TB [18]. In this study, we analyzed the diagnostic value of EBUS-GS combined with Xpert for identification of MTB infections in isolated pulmonary nodules.

This study examined lesions of suspected pulmonary tuberculoma based on the characteristics of isolated nodules. Our major result was that the diagnostic sensitivity of EBUS-GS was significantly higher than that of BALF when used with Xpert, MGIT 960 culture, and acid-fast staining. Our results indicated that EBUS-GS with Xpert had the highest sensitivity and specificity for the diagnosis of pulmonary tuberculoma among all tested methods. This method therefore has significant value for use in clinical practice.

Previous research reported that the etiological diagnosis of smear-negative pulmonary TB was difficult [22]. Khan et al. found that the positive rate of acid-fast staining in BALF was only 25.9%, although the positive rate of BALF with culture reached 63% [23]. Zheng et al. performed a retrospective analysis and reported the sensitivity of BALF with Xpert was 68.8% [3]. Compared with smear-negative pulmonary TB, the diagnosis of pulmonary tuberculoma with isolated nodules is more difficult, resulting in a lower detection rate. Thus, previous studies found that 57.1% to 92% of TB patients in areas with endemic TB were misdiagnosed as having primary lung cancer [24]. It is common for some patients with pulmonary tuberculoma who were misdiagnosed as lung cancer and then received surgery in clinical practice.Therefore, a more accurate and minimally invasive examination is needed to obtain specimens and improve the diagnosis of pulmonary tuberculoma from isolated nodules.

Bronchoendoscopy techniques have developed rapidly during recent years. Ordinary bronchoendoscopy has limited value in the diagnosis of isolated pulmonary nodules [25]. The advantages of EBUS-GS are that it uses an ultra-fine bronchoscope and an annular ultrasound probe. As a minimally invasive diagnostic technology, EBUS-GS has greater flexibility, it leads to fewer complications, it can reach lesions around the central airway, and it can be used to guide entry into peripheral pulmonary lesions. The ultrasonic probe in EBUS-GS can help clinicians to avoid blood vessels in the surrounding regions, and thus improve safety and accuracy [26]. EBUS-GS has been used in clinical practice and in the differential diagnosis of peripheral pulmonary lesions [27], especially in the diagnosis of lung cancer. Zhu et al. found that the diagnostic rate of EBUS-GS for peripheral lung cancer was 64% in 150 patients, and that the postoperative complications from EBUS-GS were significantly less than those from CT-guided transthoracic needle aspiration [28]. Similarly, Bo et al. studied solitary pulmonary nodules (SPNs) and reported that EBUS-GS significantly increased the diagnostic rate of to 72.3% [15]. However, little is known about the use of EBUS-GS for the diagnosis of isolated pulmonary nodules caused by MTB. The present single-center prospective study demonstrated that the sensitivity when EBUS-GS was used with acid-fast staining, MGIT 960 culture, and Xpert were significantly higher when BALF was used with these assays. In other words, compared with bronchoalveolar lavage using a bronchoscope, EBUS-GS is better in locating the lesion and obtains more valuable specimens based on puncture and biopsy. Thus, the EBUS-GS greatly improved the diagnostic accuracy of pulmonary TB.

Xpert is an automated real-time nucleic acid amplification technology that provides rapid and simultaneous detection of tuberculosis and rifampicin resistance. Because Xpert MTB/RIF is more efficient and rapid than traditional methods, it is now widely used for the diagnosis of TB [29, 30], and the WHO recommends this method as a first-line rapid test for the diagnosis of pulmonary TB [31]. A previous study reported the sensitivity of BALF with Xpert among smear-negative or sputum-scarce patients with pulmonary TB was 58.9% [32], higher than in our study (34.78%). However, our sensitivity was 57.81% and specificity was 100% when EBUS-GS was used with Xpert. In addition, our AUC for EBUS-GS with Xpert was 0.789, significantly higher than the other methods. Although the sensitivity of EBUS-GS with Xpert in our study was still not ideal, it greatly improved the etiological diagnosis of isolated pulmonary nodules caused by MTB. The combined use of QFT, a PPD skin test, and other examinations makes it much easier to make a correct clinical diagnosis. We also found that EBUS-GS with Xpert and with MIGT 960 culture or pathological examination slightly increased the sensitivity. In the study by Lan Yao et al., ultrasound bronchoscopy combined with Xpert Ultra was used for the diagnosis of smear-negative pulmonary tuberculosis [33]. Xpert Ultra is a new diagnostic technology recommended by the World Health Organization (WHO). It integrates two different multicopy amplification targets, IS6110 and IS1081, and includes a larger DNA amplification chamber. The study found that Xpert Ultra had a higher sensitivity compared to Xpert (78.1% vs 64.6%) [33]. Therefore, combining EBUS-GS with Xpert Ultra technology may further improve the diagnostic sensitivity for isolated pulmonary tuberculosis nodules.

Pathological examination is crucial for the diagnosis of isolated pulmonary tuberculoma, and EBUS-GS can obtain pathological specimens from isolated pulmonary nodules. However, other diseases, such as nontuberculous mycobacterial pulmonary disease (NTM-PD) and granulomatous diseases of the lung, have clinical and pathological characteristics that are similar to pulmonary TB [34, 35]. In this study, the sensitivity of EBUS-GS with pathological examination (46.88%) was unacceptable, although the specificity was 95.77%; there were also 3 patients with false positive results, 1 with sarcoidosis and 2 with NTM-PD. In addition, drug resistance cannot be determined using a pathological examination. However, use of EBUS-GS with Xpert can quickly diagnose TB and confirm the presence of rifampicin resistance, suggesting that this method is more useful in clinical practice.

In general, compared with conventional invasive examinations, EBUS-GS is characterized by smaller wounds and fewer complications, and Xpert has a high diagnostic rate and is relatively rapid. The combined use of these two methods led to good diagnostic efficacy in isolated pulmonary nodules caused by MTB, and was also useful in the differential diagnosis of non-tuberculous pulmonary diseases.

There were some limitations in this study. Firstly, it was a single-center prospective study with a small sample size. Thus, the diagnostic value of EBUS-GS with Xpert for isolated pulmonary nodules caused by MTB needs verification by large multi-center studies. Secondly, the operators of EBUS-GS must have a relatively high level of technical expertise, and considering the angle of the bronchoscope, some lesions may not be reached or puncture failure might occur.

EBUS-GS combined with Xpert has great clinical value in the diagnosis of isolated pulmonary tuberculoma based on its moderate sensitivity and high specificity. The sensitivity of EBUS-GS is greater when used with Xpert. Thus, use of this method in clinical practice may help to provide early diagnosis pulmonary tuberculoma so that these patients can avoid misdiagnosis and receive prompt treatment.