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Invest Clin 62(1): 28 - 36, 2021 https://doi.org/10.22209/IC.v62n1a03
The value of GeneXpert MTB/RIF in bronchoalveolar lavage fluid in the diagnosis of smear-negative pulmonary tuberculosis.
Yue-Ying Zhou, Ji-Chan Shi, Ning Pan, Zheng-Xing Wu, Ai-Qiong Cheng, Yan-Hong Mei, Lian-Peng Wu and Xian-Gao Jiang
Department of Infectious Diseases, Wenzhou Central Hospital, Wenzhou, Zhejiang, China.
culture; rifampicin resistance.
Abstract. This study aims to evaluate the diagnostic value of Xpert MTB/ RIF assay in bronchoalveolar lavage fluid (BALF) in subjects with smear-nega- tive pulmonary tuberculosis. From January 2019 to December 2019, 197 pa- tients with suspected pulmonary tuberculosis were recruited, and bronchoal- veolar lavage fluid was collected for acid-fast staining smear, liquid culture of Mycobacterium combined drug sensitivity and Xpert MTB/RIF detection. The sensitivity, specificity, positive predictive value and negative predictive value of Xpert MTB/RIF in bronchoalveolar lavage fluid (BALF) were calculated with smear-negative pulmonary tuberculosis as the reference standard. The consis- tency of xpert MTB/RIF in the diagnosis of rifampicin resistance was evaluated, with the results of Mycobacterium liquid culture drug sensitivity test and drug sensitivity test as the gold standards. The results showed that among 197 sus- pected tuberculosis patients, 55 patients were not diagnosed with tuberculosis and 142 patients were diagnosed with smear-negative pulmonary tuberculosis. One hundred and twenty three cases (86.62%) were positive for Xpert MTB/ RIF in bronchoalveolar lavage fluid, 15 cases (10.56%) were positive by acid-fast staining smear method, and 88 cases (61.97%) were positive by the liquid cul- ture method. The positive rate of Xpert MTB / RIF was 93.18% (82 / 88), which was higher than that of 75.93% (41 / 54) of the negative BALF mycobacterium culture (χ 2 = 8.598, P<0.01). The sensitivity and specificity of Xpert MTB/RIF for rifampicin resistance were 100.00% and 97.30%, respectively. Therefore, the diagnostic value of Xpert MTB/RIF in bronchoalveolar lavage fluid for bacterial- negative pulmonary tuberculosis is superior to the acid-fast staining smear of lavage fluid and the mycobacterium culture method.
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Valor de GeneXpert MTB / RIF en líquido de lavado broncoalveolar en el diagnóstico de tuberculosis pulmonar con frotis negativo
Invest Clin 2021; 62 (1): 28-36
Palabras clave: líquido de lavado broncoalveolar; Xpert MTB / RIF; tuberculosis; cultivo de Mycobacterium; resistencia a la rifampicina.
/ RIF en líquido de lavado broncoalveolar (BALF) en el diagnóstico de tubercu- losis pulmonar con frotis negativo. Se reclutaron 197 pacientes con sospecha de tuberculosis pulmonar desde enero de 2019 hasta diciembre de 2019 y se recogió líquido de lavado broncoalveolar para frotis de tinción acido-resistente, cultivo en medio líquido de sensibilidad a fármacos combinados de Mycobac- terium y detección de Xpert MTB/RIF. Se calcularon la sensibilidad, especifi- cidad, valor predictivo positivo y valor predictivo negativo de Xpert MTB/RIF en el líquido de lavado broncoalveolar (BALF), con tuberculosis pulmonar con frotis negativo como estándar de referencia. La consistencia de Xpert MTB/RIF en el diagnóstico de resistencia a la rifampicina se evaluó con los resultados de la prueba de sensibilidad al fármaco en cultivo en medio líquido de Mycobac- terium y la prueba de sensibilidad al fármaco como patrón oro. Los resultados mostraron que entre 197 pacientes sospechosos de tuberculosis, 55 no fueron diagnosticados con tuberculosis y 142 fueron diagnosticados con tuberculosis pulmonar con frotis negativo; 123 casos (86,62%) fueron positivos para Xpert MTB / RIF en el líquido de lavado broncoalveolar, 15 casos (10,56%) fueron po- sitivos por el método de frotis de tinción ácido-resistente y 88 casos (61,97%) fueron positivos por el método de cultivo en medio líquido. La tasa positiva de Xpert MTB/RIF fue del 93,18% (82/88), que fue superior a la del 75,93% (41/54) del cultivo de micobacterias BALF negativo (χ2 = 8,598, P <0,01). La sensibilidad y especificidad de Xpert MTB / RIF para la resistencia a la ri- fampicina fueron 100,00% y 97,30%, respectivamente. Por lo tanto, el valor de Xpert MTB/RIF en el líquido de lavado broncoalveolar en el diagnóstico de la tuberculosis pulmonar con frotis negativo, es superior al frotis de tinción acido- resistente del líquido de lavado y al método de cultivo de micobacterias.
Received: 14-08-2020 Accepted: 04-01-2021
The World Health Organization (WHO) reported that more than six million people contracted tuberculosis in 2018 (1). At pres- ent, China continues to be one of the highest TB burden countries in the world. In recent years, the prevalence rate of active pulmo-
nary tuberculosis in China has decreased slowly (2), with smear-negative tuberculosis patients accounting for about 70% of ac- tive pulmonary tuberculosis patients. Thus, smear-negative tuberculosis patients are a group that can hardly be ignored. The diag- nosis of tuberculosis mainly relies on the ac- id-fast staining of sputum smear, albeit with
low sensitivity. The sensitivity of positive Mycobacterium tuberculosis (MTB) culture specimens is only 44% - 80% (3,4), which makes it easy to miss. Fiber optic bronchos- copy is an important auxiliary examination and diagnosis method for smear-negative pulmonary tuberculosis (5). It can improve the disease diagnosis by collecting speci- mens through bronchoscopy, brush biopsy, bronchoalveolar lavage fluid (BALF), biopsy and other methods.
Xpert MTB/RIF designs primers and probes for the rifampicin resistance deter- mination region in the rpoB gene and 81bp gene of MTB to detect the presence of a mu- tation. It is also used to determine whether the patient had TB disease (6-8). Since most rifampicin-resistant strains are resistant to isoniazid, the resistance to rifampicin can be used as a monitoring indicator of suspected MDR-TB to a certain extent [9]. Numerous studies have found that this method has high sensitivity and specificity in detecting MTB by sputum samples (9-11), although the collection and results of sputum samples are affected by many factors. Using BALF as specimen not only solves the problem of no sputum, but it also comes directly from the segmental bronchus where the lesion is lo- cated. The bacterial content is relatively high, and the positive rate of detection is significantly improved. It can achieve the rapid early diagnosis of smear-negative pul- monary tuberculosis, which is of great sig- nificance for the early and rapid diagnosis of tuberculosis.
This study reports the value of Xpert MTB/RIF applied to BALF in the diagnosis of smear-negative pulmonary tuberculosis.
Fiber optic bronchoscopy was performed on 197 suspected pulmonary tuberculosis patients admitted to Wenzhou Central Hos- pital (Nanbaixiang District) from January 2019 to December 2019. The diagnostic cri- teria for tuberculosis adhered to the Chinese
Pulmonary Diagnosis Criteria WS288-2017
(12) (Mycobacterium tuberculosis was de- tected in bronchial etiology). This study was approved by the medical ethics committee of our hospital (approval number: L2020-01- 001). The laboratory procedures were under external quality control programs.
Inclusion criteria: (1) clinical mani- festations of pulmonary tuberculosis (e.g. cough, expectoration, hemoptysis and fever) and/or chest imaging manifestations (e.g. pneumonia and cavity); (2) three negative sputum acid-fast staining smears; (3) pa- tients’ informed consent; (4) subjects older than 14 years of age; (5) there was no gender limitation.
Exclusion criteria: (1) patients with contraindications to fiber optic bronchos- copy; (2) patients with cancer; (3) patients whose clinical data was incomplete.
Under the nasal catheter for oxygen inhalation, ECG, respiration and blood oxy- gen saturation monitoring, 80 mL of sterile
saline solution (37℃) were infused twice
into bronchoalveolar through the Olympus
BF-1TQ170 fibre bronchoscope using a dis- posable lavage device; it was then recovered with negative pressure. The negative pres- sure was 40–60 mmHg (1 mmHg = 1.333 kPa), and the recovery rate was more than 40% for a successful recovery. Each patient’s BALF was divided evenly into three parts and sent immediately for examination.
Five mL of BALF were added to a 10-mL disposable sterile test tube, and centrifuged at 3000× g for 10 min. Then, the superna- tant was gently aspirates and discarded it. Next, the bottom sediment was taken for smear, with the coating area being 10 × 20 mm and dried at a constant temperature using a drying machine. Afterwards, a car- bolic acid red dyeing solution was added, followed by a 10 min-decolorization with 3% of hydrochloric acid, alcohol for 1 min,
and then re-died with methylene blue for 30 s. Subsequently, washed and dried and then immersion oil was added on it. A bin- ocular microscope with a 10×-eyepiece and a 100×-objective was used for observation. It was considered positive if more than one acid-fast bacillus was detected per 300 fields examined.
The Bectec MGIT 960 system has been proved capable of measuring the sensitivity of second-line drugs. It is a fully automatic, radiation-free and non-invasive system for the identification and drug sensitivity of my- cobacteria (13,14). In this study, after BALF was fully mixed, 5 mL were added to a 50- mL disposable centrifuge tube. The volume of NALC-NaOH employed for culture was the same as the BALF volume. After 15 min, phosphate was added to 45 mL for mixing. Then, centrifuged at 3000 g for 15 min. the supernatant was discarded, and then a small amount of phosphate was added for resus- pension. After that, 0.5 mL of suspension was added to the MGIT tube for culture. The positive culture tube was confirmed by acid- fast staining, and the bacteria type of the acid-fast staining positive culture medium was identified. After the identification, if the result was MTB, the drug sensitivity test was carried out according to Modern Tuberculo- sis Diagnosis Technology.
The digestion solution was added to BALF to obtain attain a ratio of digestion liquid and BALF of 1:1 (V/V). The cover of the test tube was tightened and oscillated with a vortex oscillator for 30 s. the mixture was placed at room temperature for 15 min until there was no visible granular substance in the mixture. Then, 2 mL of the mixture was added to the cartridge, and then the car- tridge inserted into the Gene Xpert instru- ment for automatic detection. The system automatically displayed the test results after
about two hours. The method employed to identify M. tuberculosis from culture posi- tive tubes was an etiological culture. In this study, we employed a composite reference standard that combined bacteriological and clinical results. Additionally, we analyzed the results using the culture results as reference standard.
SPSS 17.0 software for the statistical analysis of data was used. The count data were expressed by rate, and the comparison was performed by the χ2 test. The difference of p < 0.05 was considered statistically sig- nificant.
Among the 197 suspected tubercu- losis patients, 55 patients were not diag- nosed with tuberculosis, and 142 patients were diagnosed with smear-negative pul- monary tuberculosis, including 95 males and 47 females, aged from 15 to 82-years old, with an average age of 37.54 ± 15.11. Among them, 129 cases were positive for etiological evidence (BALF acid-fast staining smears, Xpert MTB/RIF, mycobacterial cul- ture). Xpert diagnosed 13 MTB cases that were not diagnosed by other methods and 55 cases were non-tuberculosis respiratory diseases, including 33 males and 22 females, aged 17–67 years, with an average of 46.33
± 13.11 years old. Of these 55 cases , 35 had pneumonia, six cases had a chronic ob- structive pulmonary disease, six cases had bronchiectasis with hemoptysis, four cases had lung cancer, three cases had an atypi- cal mycobacterial lung disease, and one case had a pulmonary fungal disease. There were no significant differences in age and gender between the two groups (p > 0.05).
In smear-negative pulmonary tubercu- losis patients, 123 cases were positive for BALF Xpert MTB/RIF, 15 cases were positive in BALF acid-fast staining smear, and 88 cas-
es were positive in the BALF mycobacterium culture. Among the patients with non-tu- berculosis respiratory diseases, the BALF of three patients was cultured with nontuber- culous mycobacteria. BALF acid-fast stain- ing was negative in BALF Xpert.
The sensitivity, specificity, positive and negative predictive values of BALF Xpert MTB/RIF, BALF acid-fast staining smear and the BALF mycobacterium culture were 86.62%, 100.00%, 100.00%, 74.32%,
10.56%, 94.55%, 83.33%, 29.05%, 61.97%,
94.55%, 96.70% and 49.06%, respectively (see Table I). The ROC curve analysis showed that Xpert MTB/RIF and smear and myco- bacterium culture areas were 0.933, 0.526 and 0.783, respectively. The accuracy of BALF Xpert MTB/RIF was higher than that
of BALF acid-fast staining smear and BALF mycobacterium culture (Fig. 1).
The positive result of the BALF culture was used as the gold standard for diagnosis. Among the positive BALF mycobacterial cul- tures, 82 cases were positive, and six cases were negative in Xpert MTB/RIF. Among the nega- tive BALF mycobacterial culture, 41 cases were positive, and 13 cases were negative in Xpert MTB/RIF. The positive detection rate of BALF Xpert MTB/RIF was 93.18% (82/88), which was higher than 75.93% (41/54) of the negative BALF mycobacterium culture. The chi-square test was performed on BALF Xpert MTB/RIF and the BALF phenotypic drug susceptibility testing, χ2 = 8.598, p < 0.01. The results were statistically significant by these two kinds of detection methods.
Positive | 123 | 0 | 15 | 3 | 88 | 3 |
Negative | 19 | 55 | 127 | 52 | 54 | 52 |
Total | 142 | 55 | 142 | 55 | 142 | 55 |
COMPARISON OF THREE DETECTION METHODS IN THE DIAGNOSIS OF SMEAR NEGATIVE PULMONARY TUBERCULOSIS BY BALF.
Test method | Xpert MTB/RIF Lavage fluid smear Tuberculous culture in lavage fluid |
Pulmonary Non Pulmonary Non Pulmonary Non Tuberculosis tuberculosis Tuberculosis tuberculosis Tuberculosis tuberculosis |
Sensitivity (%) | 86.62 | 10.56 | 61.97 |
(123/142) | (15/142) | (88/142) | |
Specificity (%) | 100.00 | 94.55 | 94.55 |
(55/55) | (52/55) | (52/55) | |
Positive predictive | 100.00 | 83.33 | 96.70 |
value (%) | (123/123) | ( 15/18) | (88/91) |
Negative | 74.32 | 29.05 | 49.06 |
predictive value (%) | (55/74) | (52/179) | (52/106) |
Among 82 positive cultures, Xpert were
10 resistant and 72 sensitive. Taking the drug susceptibility test after the positive BALF mycobacteria culture as the gold stan- dard, the sensitivity and specificity of Xpert MTB/RIF were 8/8 and 72/74, respectively.
Tuberculosis is still a global public health problem endangering human health, with China suffering the most. Early diagno- sis and treatment are key to improving the prognosis of tuberculosis patients and also, controlling the spread of tuberculosis. At present, many tuberculosis patients do not have specific clinical manifestations, and they lack typical imaging features. Efficient and rapid diagnosis of patients with early infection plays a key role in controlling the spread of the tuberculosis epidemic. Accord- ing to the traditional sputum search for ac- id-fast bacilli and MTB culture methods, the
detection rate is low and time-consuming. It is very difficult to make a clear diagnosis of clinical patients with little or no sputum. Detection under bronchoscopy can hardly reveal the scope of the disease. In addition, alveolar lavage can more effectively collect specimens, thereby increasing the diagnos- tic rate. The Xpert MTB/RIF assay fully in- tegrates the three main steps (nucleic acid extraction, amplification and detection) re- quired for the traditional nucleic acid ampli- fication detection. After the pre-processed sample is put into the cartridge, the system will automatically proceed with nucleic acid extraction, nucleic acid amplification and product detection. Then, the results will be automatically given after the reaction is over. The whole method is straightforward and convenient; it requires low experimen- tal conditions, and it is suitable for primary hospitals.
Since the publication of the first re- port on Xpert MTB/RIF in 2010 (15), coun- tries with a high (16-18) and low (19) rate of tuberculosis worldwide have carried out
confirmatory evaluations of this method. The results show that the sensitivity of Xpert MTB/RIF is 76.9%–100.0%, although the sensitivity of the sputum Xpert MTB/ RIF test for the diagnosis of smear-negative tuberculosis is only 48.6%–77.7% (20, 21). This study showed that the sensitivity, spec- ificity, and positive and negative predictive values of BALF Xpert MTB/RIF in the diag- nosis of smear-negative pulmonary tuber- culosis are 86.62%, 100.00%, 100.00% and 74.32%, which are similar to those reported by Gao Chunjing et al. (22). The ROC curve analysis of these three detection methods showed that the accuracy of BALF Xpert MTB/RIF is higher than that of BALF acid- fast staining smear and the BALF mycobac- terium culture.
According to the recommendation of WHO, Xpert MTB/RIF was used as a prelimi- nary screening and diagnostic test for sus- pected MDR-TB or HIV-related tuberculosis among adults (23). The results showed that the sensitivity and specificity of Xpert MTB/ RIF for rifampicin resistance are 100.00% (8/8) and 97.30% (72/74), respectively, which are consistent with the previous re- ports indicating that the sensitivity and spec- ificity of Xpert MTB/RIF for rifampicin resis- tance are 92.9%–100.0% and 98.6%–100.0% (24-27), respectively. This study found that two samples were rifampicin resistant in BALF Xpert MTB/RIF, while they were sensi- tive on the phenotypic drug sensitivity test. The false resistance on Xpert MTB/RIF may be related to the silent mutation or misin- terpretation of the Xpert MTB/RIF detec- tion system (28,29).
Recently, the prevalence rate of active pulmonary tuberculosis in China has de- creased slowly, with smear-negative tubercu- losis patients accounting for about 70% of ac- tive pulmonary tuberculosis patients. Thus, smear-negative tuberculosis patients are a group that can hardly be ignored. Meng et al. (24) conducted a meta-analysis and found that Xpert Ultra exhibits a viable alternative
in sensitivities in both pulmonary TB and extrapulmonary TB, which was proved to be higher than Xpert in the comparative analy- sis, and shows a good performance in the de- tection of RIF’s resistance. This study evalu- ates the value of Xpert MTB/RIF applied to BALF in the diagnosis of smear-negative pulmonary tuberculosis. However, we did not conduct an ROC curve about the comparison of the three detection methods considering smear-negative pulmonary tuberculosis as the standard in this study, which should be further investigated in the future. Although the commercial molecular test, evaluated in this study was first recommended by WHO in 2010, this study had provided confirmatory evidence on the value of the assay to detect TB and rifampin resistant TB by using BALF clinical specimens.
Limitations. Firstly, two RIF-R resistant strains by X-Pert/MTB/RIF and sensitive by the phenotypic method used, still need fur- ther investigation. Secondly, as a preliminary study, we did not detected the additional yield of the Xpert over culture, which should be object of further research.
To sum up, Xpert MTB/RIF is a simple, rapid and accurate detection method. The BALF Xpert MTB/RIF detection has high specificity and sensitivity in the diagnosis of smear-negative pulmonary tuberculosis, and its sensitivity is significantly higher than that of BALF acid-fast bacillus and the BALF mycobacterium culture method. Moreover, detection is fast and can determine the pres- ence of rifampicin resistance. With the pop- ularity of bronchoscopy, BALF Xpert MTB/ RIF has a good application value in the rapid diagnosis of smear-negative pulmonary tu- berculosis.
This work was founded by Zhejiang Basic Public Welfare Research Project (LG- F18H010003) and Wenzhou Science and Technology Bureau Project (Y20180599).
Williamson DA, Basu I, Bower J. An eva- luation of the Xpert MTB/RIF assay and detection of false-positive rifampicin resis- tance in Mycobacterium tuberculosis. Diagn Microbiol Infect Dis 2012; 74(2):207-209.
Park SW, Klotzsche M, Wilson DJ, Boshoff HI, Eoh H, Manjunatha U, Blumenthal A, Rhee K, Barry CE, Aldrich CC. Evaluating the sensitivity of Mycobacterium tubercu- losis to biotin dprivation using regulated gene expression. Plos Pathogens 2011; 7(9):e1002264.
Vanna-Basil M, El Hajj H, Colangeli R. Rapid detection of rifampin resistance in Mycobacterium tuberculosis isolates from India and Mexico by a molecular beacon assay. J Clin Microbio1 2004; 42(12):5512
5516.
Canan Gürsoy N, Yakupoullar Y, Tekereko- lu MS, Otlu B. Evaluation of the diagnostic performance of Xpert MTB/RIF test for the detection of Mycobacterium tuberculosis and rifampin resistance in clinical samples. Mikrobiyol Bul 2016; 50(2):196-204.
sis. Chinese Tropical Medicine 2017; 17(3):
266-269.
Sivaramakrishnan G, Subramanyam B, Prem Kumar M, Golla R, Mondal R. Vali- dation of bedaquiline drug-susceptibility testing by BACTEC MGIT 960 system for Mycobacterium tuberculosis. Int J Myco- bacteriol 2019; 8(4):329-332.
www. who.int/ mediacentre/news/relea- ses/2010/tb_test_20101208/zh/.
Xpert MTB/RIF assay and Bactec MGIT 960 culture system for detection of rifampin-re- sistant Mycobacterium tuberculosis isolates in a country with a low tuberculosis (TB) in- cidence. J Clin Microbiol 2015;53(4):1351- 1354.
tion study. Lancet, 2011; 377(9776):1495-
1505.
He GQ, Li T, Shi JC. Evaluation of clinical application of rifampicin resistant Myco- bacterium tuberculosis in 214 patients with pulmonary tuberculosis. Chinese J Infect Dis 2016; 34(6):349-353.
pid detection of multidrug resistant MTB and Xpert MTB/RIF by gene B. Chinese J Nosocomial Infect 2017; 27(19):4340-
4343.
J. Evaluation of the Cepheid Xpert MTB/RIF assay for direct detection of Mycobacterium tuberculosis complex in respiratory speci- mens. J Clin Microbiol 2011;49(4):1621- 1623.