Enhancing the Accuracy of Peripheral Pulmonary Tuberculosis Diagnosis: A Comparative Evaluation of CapitalBio^TM Mycobacterium Nucleic Acid Detection Test and Mycobacterium tuberculosis Isothermal RNA Amplification Assay Using Endobronchial Ultrasonography with Guide Sheath

Introduction

Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis (MTB) that continues to pose a significant risk to human health.¹ TB is classified into two types: pulmonary TB (PTB) and extrapulmonary TB (EPTB), depending on the part of the body affected by the MTB infection.² PTB is responsible for almost 80% of all TB cases, and patients with PTB are the main source of TB transmission, thus, it is essential to diagnose and treat patients as early as possible.

However, in the very early stages of tuberculosis bacterial infection, if the infection is located in the peripheral zones of the lungs, which we call peripheral pulmonary tuberculosis (PTB), diagnosis is often difficult. Diagnosing peripheral PTB is particularly challenging compared with central lesions.³

Generally, test samples can be acquired through bronchoscopy or lung puncture biopsy. Bronchoscopy is the least invasive of the two methods and has a lower incidence of adverse reactions. However, lesions located in the periphery of the lungs have a significantly lower positive diagnostic rate because of the subpleural bronchioles’ slenderness and the difficulty of bronchial penetration.⁴ The use of radial endobronchial ultrasound has substantially improved the precision of bronchoscopy for peripheral pulmonary lesions, resulting in an overall diagnostic yield of 70.6%.^5–7 Endobronchial ultrasonography with guide sheath (EBUS-GS) is a tool that consists of a small circular ultrasound probe placed inside a guiding sheath. Medical professionals use it to reach Grade 8–9 bronchi. Once reached, the GS can establish pathways for ultrasound-guided biopsies, allowing direct access to lavage and biopsy samples.⁸

Advancements in nucleic acid testing have significantly improved timely and swift detection of TB, overcoming limitations of traditional diagnostic methods⁹ that could impede the diagnostic process and impact the overall prognosis.^10,11 Several molecular detection methods are currently being studied for diagnosing TB, including the CapitalBio^TM Mycobacterium nucleic acid detection test (CapitalBio MTB test) and the Mycobacterium tuberculosis isothermal RNA amplification test (MTB-RNA), are widely used in TB hospitals in China to diagnose TB rapidly.^12,13 The CapitalBio MTB test is a commercial test that uses real-time fluorescent polymerase chain reaction (PCR) and TaqMan probe technology. In contrast, MTB-RNA thermostatic amplification real-time detection technology uses thermostatic amplification of 16S rRNA as a target to generate a fluorescent signal to detect the presence of viable bacteria. Up to now, there are limited reports on certain methods for diagnosing peripheral PTB, particularly in obtaining bronchoalveolar lavage fluid (BALF) samples. It is unclear whether combining EBUS-GS with these methods is effective for evaluating peripheral PTB. Therefore, this study aims to determine the accuracy of these two methods in detecting nucleic acids in EBUS-GS BALF samples, providing healthcare professionals with further insights into the diagnosis of peripheral PTB.

Materials and Methods

Study Participants and Design

A retrospective analysis was conducted at the TB Diagnosis and Treatment Center at Zhejiang Chinese and Western Medicine Integrated Hospital. The clinical data of patients who underwent EBUS-GS operations and bronchial lavages between June 1, 2020 and November 30, 2023 were reviewed. All patients demonstrated a solitary nodular lesion measuring <3 cm in diameter, located in the outer region of the lungs. Because of its location, traditional bronchoscopy could not access the distant bronchial areas, making it necessary to use EBUS-GS to determine whether the patient had PTB. The doctor was fully informed the patients about the procedure and possible risks and signed written consent form about the EBUS-GS and bronchial lavage. This was a retrospective study, the study did not involve patient privacy or harm to patients, informed consent from participants was not required, and the Ethics Committee of Zhejiang Chinese and Western Medicine Integrated Hospital agreed to waive informed consent for patients (2024-YS-067).

We based our diagnosis on the People’s Republic of China Health Industry Standard for PTB Diagnosis (WS 288–2017) (National Health Commission of the People’s Republic of China. 2018). We used the composite reference standard (CRS) as the final diagnosis. PTB was considered confirmed when there was a positive result of MTB culture; or molecular biology test (such as CapitalBio MTB, MTB-RNA, Xpert MTB/RIF) positive and imaging results are consistent with PTB; or confirmation of pathological tissue changes consistent with TB. Probable PTB is indicated by a positive blood immunological test, characteristic PTB changes on chest CT images, and a positive response to anti-tuberculosis treatment. Non-PTB was diagnosed when there was no evidence of MTB infection and lung lesions resolved without anti-tuberculosis treatment. We considered both confirmed and probable PTB as peripheral PTB in this study.

Specimen Collection and Handling

BALF was collected from the peripheral lesion site in the lung using the EBUS-GS procedure. Fresh BALF samples were evenly distributed for AFB smear, MTB culture, CapitalBio MTB test, and MTB-RNA detection.

AFB Smear

A 1-mL BALF sample was dropped on a dry and clean glass slide. It was allowed to dry naturally and stained and rinsed with successive drops of carbolic acid and methylene blue. Finally, 300 different fields on the slide were observed using a microscope at 100× magnification. If more than one antacid bacillus was found, the result was reported as positive.

MTB Culture

We added 5 mL NaOH solution to an equal volume of BALF, mixed it thoroughly, and then centrifuged. The supernatant was discarded, and phosphate-buffered saline solution was added to the sediment. Subsequently, 0.5 mL of the mixture was transferred to a 960 liquid culture tube and incubated. The BACTEC MGIT 960 Mycobacteria Culture System automatically reports positive results based on the fluorescence signal.

CapitalBio^TM Mycobacterium Nucleic Acid Detection Test (CapitalBio MTB Test)

An equal amount of NaOH solution was added to 1 mL of BALF sample, centrifuged, and the supernatant was discarded. The nucleic acid extract was added to the precipitate, placed in a water bath at 95°C and incubated for 10 min, centrifuged again, and the supernatant was discarded. Nucleic acid DNA within the precipitate was extracted according to the manufacturer’s instructions. RT-PCR was performed using a fluorescence quantification RT-PCR instrument (SLAN-96S Real-Time PCR System ZEESAN Xiamen CN) to amplify and detect IS6110 elements for MTB. A sample was considered positive when the amplification produced an S-curve and the Ct value was <40.

Mycobacterium Tuberculosis Isothermal RNA Amplification Assay (MTB-RNA)

One milliliter of the lavage specimen was added to an equal amount of 4% NaOH solution in a sterile tube and allowed to stand for 20 min before centrifugation. The supernatant was removed, and 1 mL of saline was added to the precipitate for washing, followed by another centrifugation. Subsequently, 50 μL of dilution solution was added to complete the sample preparation. The test samples and positive and negative control solutions were subjected to ultrasonication at 300W for 15 min and then added to the amplification detection solution. The kit was then placed in a fluorescence detection instrument with a constant temperature, where PCR amplification detection was performed using FAM as the fluorescein channel (RenDu Biotechnology Company, Shanghai, China). A positive result was indicated when the dt value was ≤35.

Statistical Analysis

Data were analyzed using SPSS, version 20.0 (IBM Corp., Armonk, NY, USA). Continuous variables that followed a normal distribution were presented as means with standard deviations, whereas categorical data were analyzed using either the chi-squared test or Fisher’s exact test. MedCalc Statistical (v15.2.2, MedCalc Software bvba, Ostend, Belgium) was used to determine the sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and area under the curve (AUC), with a 95% confidence interval for the tests being evaluated. The results were compared with that of the final clinical diagnosis. McNemar’s test compared variances in paired data, and the Z-test compared different AUCs. The receiver operating characteristic (ROC) curve was drawn for each test, and a Venn diagram was created using an interactive tool (http://www.bioinformatics.com.cn/static/others/jvenn/example.html). A p-value <0.05 was considered statistically significant between the two groups.

Results

Participants’ Clinical Characteristics

A retrospective study was conducted on 315 patients suspected of peripheral PTB. However, 28 patients without complete data were excluded, and finally, 287 patients and their clinical information and examination results were analyzed from June 1, 2020, to November 30, 2023. Each patient provided a sample of EBUS-GS operated BALF, and AFB smear, culture, CapitalBio MTB test, and MTB-RNA test were completed. All patients were HIV-negative, the mean patient age was 51.14 ± 16.89 years, and 65.16% were male.

Based on the CRS, 70 specimens were confirmed PTB, 54 of which were CapitalBio MTB test positive and 40 were MTB-RNA positive. Probable PTB was identified in 104 specimens. Although 25 were CapitalBio MTB test positive, none were MTB-RNA positive. Further, 113 specimens were considered confirmed non-PTB, and no one was positive for the CapitalBio MTB test or MTB-RNA (Figure 1).

Figure 1 Diagnostic categorization of the participants in the research.

Among the 113 patients with non-PTB, 62 were finally diagnosed with bacterial pneumonia, 9 with fungal pneumonia, 1 with mycoplasma pneumonia, 9 with nontuberculous mycobacteria pulmonary disease, 17 with lung cancer, and 15 with other noninfectious lung lesions.

Diagnostic Accuracy of the AFB Smear, MTB Culture, CapitalBio MTB Test, MTB-RNA, and Parallel Tests

When the CRS was used as the reference standard, the sensitivity, specificity, PPV, NPV, and AUC of AFB smear for peripheral PTB were 9.20% (5.35–14.50%), 97.35% (92.44–99.45%), 84.21% (60.42–96.62%), 41.04% (35.10–47.19%), and 0.53 (0.47–0.59). Those of MTB culture were 31.03% (24.25–38.48%), 100.00% (96.79–100.00%), 100.00% (93.40–100.00%), 48.50% (41.92–55.11%), and 0.69 (0.62–0.76). Those of the CapitalBio MTB test were 44.83% (37.30–52.54%), 100.00% (96.79–100.00%), 100.00% (95.38–100.00%), 54.07% (47.06–60.96%), 0.72 (0.67–0.78). Those of the MTB-RNA test were 22.99% (16.96–29.96%), 100.00% (96.79–100.00%), 100.00% (91.91–100.00%), 45.75% (39.42–52.18%), 0.61 (0.56–0.67). Lastly, those of the parallel test (CapitalBio MTB test or MTB-RNA) were 46.55% (38.97–54.96%), 100.00% (96.79–100.00%), 100.00% (95.55–100.00%), 54.85% (47.79–61.78%), 0.73 (0.68–0.78). The detailed results are presented in Table 1.

Table 1 Diagnostic Accuracy of AFB Smear, MTB Culture, CapitalBio MTB Test, MTB RNA and Parallel Test (CapitalBio MTB Test or MTB-RNA) for Rapid Diagnose Peripheral PTB Compared with a Composite Reference Standard

The CapitalBio MTB test and MTB-RNA demonstrated higher precision than the AFB smear and MTB culture detection assays, with statistical significance (P < 0.001). Table 2 outlines the differences in diagnostic precision among the AFB smear, MTB culture, CapitalBio MTB test, and MTB-RNA. Figure 2 shows the ROC curves for the four tests, whereas Figure 3 presents a Venn diagram depicting the positive results from each test.

Table 2 Compare the AFB Smear, MTB Culture, CapitalBio MTB Test, MTB-RNA and Parallel Test (CapitalBio MTB Test or MTB-RNA) for Rapid Diagnose Peripheral PTB

Figure 2 ROC curves for AFB smear, MTB culture, CapitalBio MTB test, and MTB-RNA tests.

Figure 3 Venn diagram of the positive results from the four tests.

Evaluation of the Precision of the CapitalBio MTB Test, MTB-RNA, and Parallel Test (CapitalBio MTB test or MTB-RNA)

The results showed that the parallel test was significantly better than the MTB-RNA detection assay (P < 0.001, as shown in Table 2). Using the parallel and individual tests can improve the accuracy of diagnosis and significantly increase the diagnostic efficiency compared to the MTB-RNA test alone (P < 0.05). However, the difference between the CapitalBio MTB and other tests was insignificant (P > 0.05; Table 2). Figure 4 shows the ROC curves for the CapitalBio MTB test, MTB-RNA and parallel test.

Figure 4 ROC curves of the CapitalBio MTB test, MTB-RNA tests, and parallel test.

Discussion

Recently, the diagnosis and treatment of PTB have improved tremendously, and many patients are now able to actively seek medical treatment in the early stages of the disease, get a clear diagnosis of the disease as soon as possible, and initiate antituberculosis treatment (ATT), which is socially important for our country to actively and effectively control the spread of tuberculosis in the population. However, in patients with lesions present only in the peripheral lung bands or subpleura, it is difficult for conventional bronchoscopy to deeply penetrate into the distal bronchioles, leading to a significant decrease in the diagnostic rate. Therefore, we used EBUS-GS as an alternative approach to reach the distal end of the tracheal bundle to more efficiently diagnose peripheral PTB. This enables us to perform the lavage and biopsy with greater precision, resulting in optimal diagnostic performance with minimal patient trauma.⁸

Clinicians frequently use EBUS-GS because of its safety, effectiveness, and low bleeding risk, especially in the early diagnosis of lung cancer.^14,15 Yet, the effectiveness of EBUS-GS in evaluating solitary nodules resulting from MTB has not been thoroughly assessed. Sun et al discovered that the percentage of AFB smear positivity in BALF was just 35.2%, while the positivity rates for BALF culture and Xpert were 84.9% and 87.2%, respectively.¹⁶ In our study, the sensitivity of AFB smear was only 9.2%, while that of culture was 31.03%. These lower values could be due to the small number of lung lesions in our patient sample, with many cases detected through physical examination screening in the early stages of the disease, whereas other studies were conducted in the middle or late stages. Additionally, after a few weeks, three false-positive samples of AFB smear were later confirmed to be nontuberculous mycobacteria by culture. Thus, conventional techniques for TB diagnosis, such as AFB smears and MTB culture are inadequate to meet clinical diagnostic needs.

Nucleic acid molecular testing offers a cost-effective, rapid, and accurate diagnostic method for identifying MTB. Cao et al reviewed past data and found that the accuracy of EBUS-GS combined with Xpert was notably better than BALF combined with Xpert in detecting isolated lung nodules.⁵ A previous study showed that the CapitalBio MTB test had an 83.0% sensitivity for detecting TB, similar to the diagnostic precision of Xpert MTB/RIF (84.4%), costing only 1/9th as much as Xpert.¹⁷ Bai et al found that the sensitivity and specificity of the tuberculosis bacillus DNA for BALF were 67.7% and 98.9%, respectively, higher than our study findings of 44.73% sensitivity and 100.00% specificity.¹⁸ However, Bai’s research did not include imaging data, whereas our study focused on lesions in the peripheral lung bands, which are challenging to access with routine bronchoscopy.

The CapitalBio MTB test uses DNA nucleic acid testing techniques that allow the DNA nucleic acid to remain intact even after the bacteria have died and can even survive for several years. Thus, a positive test result does not necessarily indicate active PTB. In contrast, RNA is less stable than DNA, degrading within approximately one week, and exists only in living bacteria. Previous studies found that the MTB-RNA test had a sensitivity of 49.6% and 63.9% for sputum and BALF samples, respectively, and greatly increased positive rates of molecular diagnosis of PTB.¹⁹ In this study, the MTB-RNA assay demonstrated a sensitivity of 22.99%, a specificity of 100.00%, and an AUC value of 0.61. The reason for this analysis is that the lung lesions in our study population were mild; therefore, it was not easy to obtain positive results. MTB-RNA has half the sensitivity of the CapitalBio MTB test, indicating a significant difference in diagnostic efficacy (P < 0.001). In our study, the diagnostic efficacy of the CapitalBio MTB test was not significantly different from the parallel test (P > 0.05). In clinical practice, the CapitalBio MTB test is preferred for the diagnosis of PTB, but in patients who have had PTB within the past 2–3 years and have been treated regularly, a combination of MTB-RNA test is required to determine the presence of viable MTB in order to decide whether or not to return to ATT.

This study has some limitations. First, patient selection may have been biased due to the constraints of a single retrospective study design. Second, the study was conducted in an area with a high prevalence of TB, with our department serving as a TB diagnostic facility in Zhejiang Province, where TB cases are concentrated. This may limit the generalizability of the results. Third, EBUS-GS requires a high level of technical skill, and lesions close to the subpleura may not be accessible or could result in puncture failure.

Conclusion

EBUS-GS is a method known for its precise localization and low complication rate. It is beneficial for diagnosing single nodular lesions in the outer bands of the lungs. When PTB is suspected in peripheral nodular lesions in the outer bands of the lungs, the CapitalBio MTB test is recommended in conjunction with this technique for the early diagnosis of PTB.