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Evaluating the diagnostic accuracy of the Xpert MTB/RIF assay on bronchoalveolar lavage fluid: A retrospective study

  • Yanjun Lu
    Affiliations
    Department of Laboratory Medicine, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Jiefang Road 1095, Wuhan 430030, China
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  • Yaowu Zhu
    Affiliations
    Department of Laboratory Medicine, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Jiefang Road 1095, Wuhan 430030, China
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  • Na Shen
    Affiliations
    Department of Laboratory Medicine, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Jiefang Road 1095, Wuhan 430030, China
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  • Lei Tian
    Affiliations
    Department of Laboratory Medicine, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Jiefang Road 1095, Wuhan 430030, China
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  • Ziyong Sun
    Correspondence
    Corresponding author.
    Affiliations
    Department of Laboratory Medicine, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Jiefang Road 1095, Wuhan 430030, China
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Open AccessPublished:February 08, 2018DOI:https://doi.org/10.1016/j.ijid.2018.01.030

      Abstract

      Objective

      Limited data on the diagnostic accuracy of the Xpert MTB/RIF assay using bronchoalveolar lavage fluid from patients with suspected pulmonary tuberculosis (PTB) have been reported in China. Therefore, a retrospective study was designed to evaluate the diagnostic accuracy of this assay.

      Methods

      Clinical, radiological, and microbiological characteristics of 238 patients with suspected PTB were reviewed retrospectively. The sensitivity, specificity, positive predictive value, and negative predictive value for the diagnosis of active PTB were calculated for the Xpert MTB/RIF assay using TB culture or final diagnosis based on clinical and radiological evaluation as the reference standard.

      Results

      The sensitivity and specificity of the Xpert MTB/RIF assay were 84.5% and 98.9%, respectively, and those for smear microscopy were 36.2% and 100%, respectively, when compared to the culture method. However, compared with the sensitivity and specificity of final diagnosis based on clinical and radiological evaluation, the sensitivity and specificity of the assay were 72.9% and 98.7%, respectively, which were significantly higher than those for smear microscopy.

      Conclusions

      The Xpert MTB/RIF assay on bronchoalveolar lavage fluid could serve as an additional rapid diagnostic tool for PTB in a high TB-burden country and improve the time to TB treatment initiation in patients with PTB.

      Keywords

      Introduction

      Tuberculosis (TB) is a serious global health problem and is one of the leading causes of death worldwide. TB results in high morbidity and mortality worldwide: approximately 10.4 million new TB cases and 1.4 million deaths were reported in the World Health Organization (WHO) global TB report of 2016 (
      • World Health Organization
      Global tuberculosis report 2016.
      ).
      China has the world’s third largest TB epidemic, with more than 1.3 million new cases of TB each year (
      • Wang L.
      • Liu J.
      • Chin D.P.
      Progress in tuberculosis control and the evolving public-health system in China.
      ). Despite its life-threatening pathogenesis, TB can be cured when diagnosed correctly and treated effectively. The ‘gold standard’ for TB diagnosis is solid or liquid culture; however, this method typically requires 4–8 weeks (
      • Foulds J.
      • O’Brien R.
      New tools for the diagnosis of tuberculosis: the perspective of developing countries.
      ,
      • Pfyffer G.E.
      • Wittwer F.
      Incubation time of mycobacterial cultures: how long is long enough to issue a final negative report to the clinician?.
      ). Smear microscopy for acid-fast bacilli (AFB) is rapid and inexpensive, but its poor sensitivity and positive predictive value (PPV) limit its use (
      • Centers for Disease Control and Prevention (CDC)
      Updated guidelines for the use of nucleic acid amplification tests in the diagnosis of tuberculosis.
      ,
      • Moure R.
      • Muñoz L.
      • Torres M.
      • Santin M.
      • Martín R.
      • Alcaide F.
      Rapid detection of Mycobacterium tuberculosis complex and rifampicin resistance in smear-negative clinical samples by use of an integrated real-time PCR method.
      ). Thus, the development of new nucleic acid amplification diagnostic technologies is required to provide the potential to make an early diagnosis and thus improve patient outcomes.
      The WHO has recommended the Xpert MTB/RIF assay (Cepheid, USA) for the diagnosis of pulmonary TB (PTB) and detection of rifampicin resistance (
      • Geneva World Health Organization
      WHO guidelines approved by the guidelines review committee. Policy statement: automated real-time nucleic acid amplification technology for rapid and simultaneous detection of tuberculosis and rifampicin resistance: Xpert MTB/RIF system.
      ). The Xpert MTB/RIF assay is an automated, single cartridge-based nucleic acid amplification test that simultaneously detects Mycobacterium tuberculosis (MTB) and rifampicin resistance within 2–3 h. This test has a demonstrated sensitivity on sputum in the range of 79.7–100% for PTB when compared to culture methods (
      • Van Rie A.
      • Page-Shipp L.
      • Scott L.
      • Sanne I.
      • Stevens W.
      Xpert(®) MTB/RIF for point-of-care diagnosis of TB in high-HIV burden, resource-limited countries: hype or hope?.
      ,
      • Helb D.
      • Jones M.
      • Story E.
      • Boehme C.
      • Wallace E.
      • Ho K.
      • et al.
      Rapid detection of Mycobacterium tuberculosis and rifampicin resistance by use of on-demand, near-patient technology.
      ,
      • Boehme C.C.
      • Nabeta P.
      • Hillemann D.
      • Nicol M.P.
      • Shenai S.
      • Krapp F.
      • et al.
      Rapid molecular detection of tuberculosis and rifampicin resistance.
      ). However, only a few studies so far have focused on assessing the performance of the Xpert MTB/RIF assay on fiberoptic bronchoscopy samples for TB diagnosis (
      • Lee H.Y.
      • Seong M.W.
      • Park S.S.
      • Hwang S.S.
      • Lee J.
      • Park Y.S.
      • et al.
      Diagnostic accuracy of Xpert® MTB/RIF on bronchoscopy specimens in patients with suspected pulmonary tuberculosis.
      ,
      • Theron G.
      • Peter J.
      • Meldau R.
      • Khalfey H.
      • Gina P.
      • Matinyena B.
      • et al.
      Accuracy and impact of Xpert MTB/RIF for the diagnosis of smear-negative or sputum-scarce tuberculosis using bronchoalveolar lavage fluid.
      ,
      • Yin Q.Q.
      • Jiao W.W.
      • Han R.
      • Jiao A.X.
      • Sun L.
      • Tian J.L.
      • et al.
      Rapid diagnosis of childhood pulmonary tuberculosis by Xpert MTB/RIF assay using bronchoalveolar lavage fluid.
      ,
      • Lu Jie
      • Li Huimin
      • Dong Fang
      • Shi Jin
      • Yang Hui
      • Han Shujing
      • et al.
      The feasibility of Xpert MTB/RIF testing to detect rifampicin resistance among childhood tuberculosis for prevalence surveys in Northern China.
      ).
      The guidelines recommend bronchoscopy for patients with suspected TB who show negative results on AFB sputum smears (
      • Lewinsohn D.M.
      • Leonard M.K.
      • LoBue P.A.
      • Cohn D.L.
      • Daley C.L.
      • Desmond E.
      • et al.
      Official American Thoracic Society/Infectious Diseases Society of America/Centers for disease control and prevention clinical practice guidelines: diagnosis of tuberculosis in adults and children.
      ). Additionally, bronchoscopic examination could improve the diagnostic accuracy and aid in the rapid diagnosis following advance treatment for PTB in cases with minimal lesions or during the early stages of PTB (
      • Shin J.A.
      • Chang Y.S.
      • Kim T.H.
      • Kim H.J.
      • Ahn C.M.
      • Byun M.K.
      Fiberoptic bronchoscopy for the rapid diagnosis of smear-negative pulmonary tuberculosis.
      ,
      • Choudhary S.
      • Tayade B.
      • Kharbade S.
      • Sontakke A.
      • Khan S.
      Outcome of fiber optic bronchoscopy in sputum smear negative pulmonary tuberculosis.
      ). Therefore, testing respiratory specimens obtained from bronchoscopy (such as washings or lavage) using the Xpert MTB/RIF assay may help to improve the diagnosis and the time to TB treatment initiation.
      In China, the Xpert MTB/RIF assay using bronchoalveolar lavage fluid (BALF) has been used to assist in the diagnosis of childhood PTB (
      • Yin Q.Q.
      • Jiao W.W.
      • Han R.
      • Jiao A.X.
      • Sun L.
      • Tian J.L.
      • et al.
      Rapid diagnosis of childhood pulmonary tuberculosis by Xpert MTB/RIF assay using bronchoalveolar lavage fluid.
      ,
      • Lu Jie
      • Li Huimin
      • Dong Fang
      • Shi Jin
      • Yang Hui
      • Han Shujing
      • et al.
      The feasibility of Xpert MTB/RIF testing to detect rifampicin resistance among childhood tuberculosis for prevalence surveys in Northern China.
      ). However, data on the sensitivity of the Xpert MTB/RIF assay performed on BALF are limited, especially for adults. Therefore, the aim of this study was to evaluate the diagnostic efficiency of the Xpert MTB/RIF assay performed on BALF from patients with a clinical suspicion of PTB.

      Methods

      Study population

      The records of all patients with suspected PTB who underwent bronchoscopy at Tongji Hospital from August 2015 to August 2017 were reviewed retrospectively. Tongji Hospital is affiliated to Tongji Medical College, Huazhong University of Science and Technology, and is located in Wuhan in the middle of Hubei Province, China.
      A total of 258 patients suspected of having PTB were enrolled in the study. The clinical suspicion of PTB was based on clinical features (e.g., cough, hemoptysis, and fever) or imaging features (e.g., nodule, shadow, and cavitation) or a history of contact with TB (patients who had been in contact with a known TB patient). Clinical information was collected from the patients, including clinical symptoms, imaging features, age, and previous medical history. In the study hospital, laboratory testing for TB included smear microscopy, culture, and Xpert MTB/RIF assay on BALF. Based on the total clinical information and the clinicians’ analyses, the patients were classified as having confirmed PTB, a clinical diagnosis of TB, or as not having TB. Twenty patients were eventually excluded as a final clinical diagnosis could not be made. Thus, the performance of the MTB component of the MTB/RIF assay was evaluated for the remaining patients. Confirmed active PTB was diagnosed based on a positive culture. The clinical diagnosis of TB was based on symptoms and radiographic findings compatible with a response to anti-TB treatment within 2 months, or the results of histological examination showing lung tissue with caseating granuloma.
      This study was conducted according to the principles of the Declaration of Helsinki. The study was approved by the Ethics Committee of Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China. A waiver of consent was approved due to the retrospective nature of the study.

      AFB microscopy and culture

      Every specimen was divided equally into two parts. One part was used for smear microscopy and MTB culture, and the other part was used for the Xpert MTB/RIF assay. One part of the specimen was sent to the clinical microbiology laboratory division to be tested directly by AFB microscopy (Ziehl–Neelsen staining), followed by processing with N-acetyl-l-cysteine and sodium hydroxide (NALC NaOH). The resuspended pellet was then subjected to culture on both solid medium (Lowenstein–Jensen) and liquid medium (BACTEC MGIT 960 culture). A positive result in either solid culture or liquid culture indicated a culture-positive result.

      Xpert MTB/RIF assay

      The other part of the BALF specimen was used in the Xpert MTB/RIF assay. In accordance with the manufacturer’s instructions, a 2:1 volume of sample reagent buffer (SR) was added to the BALF, except for samples of <1 ml volume, which were processed directly by adding SR up to 2 ml. The mixture was vortexed for 20 s and left for 15 min. Next, 2 ml of the mixture was added to the cartridge containing washing buffer, reagents for lyophilized DNA extraction and PCR amplification, and fluorescent detection probes. The results of the Xpert MTB/RIF assay were generated automatically within 2 h and reported as MTB-negative or MTB-positive. As well as being designed to detect MTB, the Xpert MTB/RIF assay also detects mutations that confer rifampicin resistance using three specific primers and five unique molecular probes targeting the 81-bp core region of the bacterial RNA polymerase β subunit (rpoB) gene. The presence of any mutations in the rpoB gene was also investigated in this study.

      Statistical analysis

      The sensitivity, specificity, PPV, and negative predictive value (NPV) for the diagnosis of active PTB were calculated using the website http://vassarstats.net/clin1.html. The 95% confidence intervals (CIs) were estimated according to exact binomial distribution. Sensitivity and specificity values were compared using McNemar’s test. All CIs were two-sided. A p-value of <0.05 was considered statistically significant. SPSS statistics version 17.0 software (SPSS Inc., Chicago, IL, USA) was used for the statistical analysis.

      Results

      Patient characteristics

      During the study period, 258 patients with suspected TB underwent bronchoscopy. Twenty patients were excluded due to a lack of definitive diagnosis. The characteristics of the remaining patients are shown in Figure 1. Of the 238 study subjects, 125 were male and 113 were female. The patients ranged in age from 17 to 85 years (mean age 47.2 years). The principal clinical symptoms were cough (55.4%), fever (21.4%), hemoptysis (12.2%), and chest distress or chest pain (12.6%) (Table 1). The majority of chest images obtained by computed tomography (CT) scan showed patchy shadow (48.3%), nodules (16.8%), cavitation (4.6%), fiber streak foci (10.5%), and pleural effusion (5.0%) (Table 1). Based on the clinical information including clinical symptoms, chest images, and treatment effect, 85 patients were diagnosed with PTB. Fifty-eight of these patients had positive cultures, including nine who had positive cultures but showed a negative result in the Xpert MTB/RIF assay. With regard to the nine false-negative Xpert MTB/RIF assay results, two samples (2/9) were low volume (<1 ml) and four (4/9) were bloody. In the Xpert MTB/RIF assay true-positive specimens, one sample (1/49) was low volume (<1 ml) and no samples were bloody. Twenty-seven patients were diagnosed with PTB based on clinical and histological/radiological features. The final diagnoses for the other patients are detailed in Table 1.
      Figure 1
      Figure 1Flow diagram of patients included in the study.
      Table 1Demographic, clinical, and radiological characteristics of the 238 patients included in the study
      CharacteristicNumber (%)
      Median age, years (range)47.2 (17.0–85.0)
      Sex
       Male125 (52.5)
       Female113 (47.5)
      Symptoms
       Cough131 (55.4)
       Fever51 (21.4)
       Hemoptysis29 (12.2)
       Chest distress or chest pain30 (12.6)
       Physical examination18 (7.6)
       NA9 (3.8)
      Chest CT scan
       Patchy shadow115 (48.3)
       Nodules40 (16.8)
       Cavitation11 (4.6)
       Fiber streak foci25 (10.5)
       Pleural effusion12 (5.0)
       NA10 (4.2)
       Lack of imaging data25 (10.5)
      Final diagnosis
       Pulmonary tuberculosis75 (31.5)
       Pleural tuberculosis10 (4.2)
       NTM infection2 (0.8)
       Lung cancer17 (7.1)
       Bacterial pneumonia85 (35.7)
       Pulmonary nodules12 (5.0)
       Pulmonary fungal infection7 (2.9)
       Interstitial lung diseases6 (2.5)
       COPD5 (2.1)
       Old TB18 (7.5)
       Chronic granuloma1 (0.4)
      NA, not available; CT, computed tomography; NTM, non-tuberculous mycobacteria; COPD, chronic obstructive pulmonary disease, TB, tuberculosis.

      Diagnostic sensitivity and specificity of AFB smear, mycobacterial culture, and Xpert MTB/RIF assay

      With the culture method as the reference standard, the Xpert MTB/RIF assay with BALF specimens showed positive results for 49 of the 58 patients with culture-positive TB; the sensitivity of the Xpert MTB/RIF assay was 84.5% (95% CI 72.1–92.2%). Among the cases that showed a positive result on Xpert MTB/RIF, two were finally diagnosed with bacterial pneumonia; thus, the specificity was 98.9% (95% CI 95.6–99.8%) (Table 2). Although these two patients initially showed the clinical symptoms of cough and fever, based on which they were first suspected to have TB, antibiotic treatment eliminated these symptoms, resulting in TB being excluded by the physicians. All of the samples were also tested by smear microscopy, which had a sensitivity of 36.2% (95% CI 24.3–49.9) and a specificity of 100.0% (95% CI 97.4–100%). Further, a comparison was made between Xpert MTB/RIF and smear microscopy for the detection of TB, and a significantly higher sensitivity was found for Xpert MTB/RIF than for smear microscopy (p< 0.001) (Table 2).
      Table 2Sensitivity and specificity of the Xpert MTB/RIF assay, smear microscopy, and culture for the diagnosis of pulmonary tuberculosis.
      Performance relative to culturePerformance relative to final diagnosis
      Sensitivity %

      95% CI
      Specificity %

      95% CI
      Sensitivity %

      95% CI
      Specificity %

      95% CI
      Xpert MTB/RIF assay84.598.972.998.7
      72.1–92.295.6–99.862.0–81.794.9–99.8
      49/58178/18062/85151/153
      Smear microscopy36.210024.798.7
      24.3–49.997.4–100.016.3–35.594.9–99.8
      21/58180/18021/85151/153
      Culture68.298.7
      57.1–77.794.9–99.8
      58/85151/153
      p-Value0.000
      p-Value calculated using McNemar’s test.
      /0.000
      p-Value for Xpert MTB/RIF assay vs. smear microscopy.
      /
      0.523
      p-Value for Xpert MTB/RIF assay vs. culture.
      /
      0.000
      p-Value for culture vs. smear microscopy.
      /
      CI, confidence interval.
      a p-Value calculated using McNemar’s test.
      b p-Value for Xpert MTB/RIF assay vs. smear microscopy.
      c p-Value for Xpert MTB/RIF assay vs. culture.
      d p-Value for culture vs. smear microscopy.
      When pathologically and clinically diagnosed PTB in addition to culture-confirmed TB was used as a combined reference standard, the sensitivity and specificity of the Xpert MTB/RIF assay were 72.9% (95% CI 62.0–81.7%) and 98.7% (95% CI 94.9–99.8%), respectively; the sensitivity of the assay was significantly higher than that of smear microscopy (24.7%; 95% CI 16.3–35.5%;). However, there was no significant difference in the sensitivities between the Xpert MTB/RIF assay and culture (68.2%; 95% CI 57.1–77.7%; p = 0.523) (Table 2).
      With regard to the positive Xpert MTB/RIF results, three of the 62 cases of PTB showed rpoB gene mutations; thus the rpoB gene mutation rate was 4.8%. However, reference phenotypic susceptibility testing was not performed to confirm rifampicin resistance; hence the true rifampicin resistance rate may not be consistent with the rpoB gene mutation rate.

      PPV and NPV of smear microscopy, culture, and Xpert MTB/RIF

      With culture as the reference standard, the overall PPV and NPV for the Xpert MTB/RIF assay were 96.1% (95% CI 85.4–99.3%) and 95.2% (95% CI 90.8–97.6%), respectively. The corresponding values for smear microscopy were 100% (95% CI 80.8–100.0%) and 82.9% (95% CI 77.1–87.6%), respectively. When considered against the clinical reference standard, PPV and NPV were 100.0% (96.9% CI 88.2–99.5%) and 86.8% (95% CI 80.6–91.3%), respectively, for the Xpert MTB/RIF assay, while these values were measured as 91.3% (95% CI 70.5–98.5%) and 70.2% (95% CI 63.6–76.2%), respectively, for smear microscopy, and as 96.7% (95% CI 87.5–99.4%) and 84.8% (95% CI 78.5–89.6%), respectively, for culture (Table 3).
      Table 3Positive predictive value and negative predictive value of the Xpert MTB/RIF assay, smear microscopy, and culture for the diagnosis of pulmonary tuberculosis
      Performance relative to culturePerformance relative to final diagnosis
      PPV %

      (95% CI)
      NPV %

      (95% CI)
      PPV %

      (95% CI)
      NPV %

      (95% CI)
      Xpert MTB/RIF assay96.1 (85.4–99.3)95.2 (90.8–97.6)96.9 (88.2–99.5)86.8 (80.6–91.3)
      Smear microscopy100 (80.8–100)82.9 (77.1–87.6)91.3 (70.5–98.5)70.2 (63.6–76.2)
      Culture96.7 (87.5–99.4)84.8 (78.5–89.6)
      PPV, positive predictive value; NPV, negative predictive value; CI, confidence interval.

      Discussion

      To date, sputum smear microscopy remains the first microbial analysis for both the diagnosis of TB and the assessment of patient infectiousness, which guides airborne isolation measures (
      • Guenaoui K.
      • Harir N.
      • Ouardi A.
      • Zeggai S.
      • Sellam F.
      • Bekri F.
      • et al.
      Use of GeneXpert Mycobacterium tuberculosis/rifampicin for rapid detection of rifampicin resistant Mycobacterium tuberculosis strains of clinically suspected multi-drug resistance tuberculosis cases.
      ). However, the limited sensitivity of this method hinders its widespread application for TB diagnosis. In addition, smear microscopy shows limited specificity, because AFB staining cannot distinguish between MTB and non-tuberculosis mycobacteria (NTM). Although culture is the ‘gold standard’ for TB diagnosis, it is slow and may take up to 2–8 weeks to yield results. The Xpert MTB/RIF assay is a rapid, automated molecular test with good sensitivity for PTB on sputum samples. However, with regard to its utility on BALF samples, especially in smear-negative cases and in patients who find it difficult to cough up sputum, only a few studies have been conducted so far (
      • Lee H.Y.
      • Seong M.W.
      • Park S.S.
      • Hwang S.S.
      • Lee J.
      • Park Y.S.
      • et al.
      Diagnostic accuracy of Xpert® MTB/RIF on bronchoscopy specimens in patients with suspected pulmonary tuberculosis.
      ,
      • Theron G.
      • Peter J.
      • Meldau R.
      • Khalfey H.
      • Gina P.
      • Matinyena B.
      • et al.
      Accuracy and impact of Xpert MTB/RIF for the diagnosis of smear-negative or sputum-scarce tuberculosis using bronchoalveolar lavage fluid.
      ,
      • Mok Y.
      • Tan T.Y.
      • Tay T.R.
      • Wong H.S.
      • Tiew P.Y.
      • Kam J.W.
      • et al.
      Do we need transbronchial lung biopsy if we have bronchoalveolar lavage Xpert® MTB/RIF?.
      ,
      • Ullah I.
      • Javaid A.
      • Masud H.
      • Ali M.
      • Basit A.
      • Ahmad W.
      • et al.
      Rapid detection of Mycobacterium tuberculosis and rifampicin resistance in extrapulmonary tuberculosis and sputum smearnegative pulmonary suspects using Xpert MTB/RIF.
      ). It appears that only two studies have been conducted in China previously, both of which were aimed at assisting in the diagnosis of childhood PTB (
      • Yin Q.Q.
      • Jiao W.W.
      • Han R.
      • Jiao A.X.
      • Sun L.
      • Tian J.L.
      • et al.
      Rapid diagnosis of childhood pulmonary tuberculosis by Xpert MTB/RIF assay using bronchoalveolar lavage fluid.
      ,
      • Lu Jie
      • Li Huimin
      • Dong Fang
      • Shi Jin
      • Yang Hui
      • Han Shujing
      • et al.
      The feasibility of Xpert MTB/RIF testing to detect rifampicin resistance among childhood tuberculosis for prevalence surveys in Northern China.
      ).
      In this study, the Xpert MTB/RIF assay was found to have a sensitivity of 84.5% when compared to culture, which was higher than the sensitivity of smear microscopy (36.2%). Furthermore, the PPV for the assay was 96.1% and the NPV was 95.2%. The results for the sensitivity of the Xpert MTB/RIF assay are consistent with those of previous studies conducted in Korea by
      • Lee H.Y.
      • Seong M.W.
      • Park S.S.
      • Hwang S.S.
      • Lee J.
      • Park Y.S.
      • et al.
      Diagnostic accuracy of Xpert® MTB/RIF on bronchoscopy specimens in patients with suspected pulmonary tuberculosis.
      , who reported a value of 81.6%, and a study conducted in Pakistan by
      • Ullah I.
      • Javaid A.
      • Masud H.
      • Ali M.
      • Basit A.
      • Ahmad W.
      • et al.
      Rapid detection of Mycobacterium tuberculosis and rifampicin resistance in extrapulmonary tuberculosis and sputum smearnegative pulmonary suspects using Xpert MTB/RIF.
      , who reported a sensitivity of 80.0%. Two cases in the present study that showed positive results in the Xpert MTB/RIF assay were finally diagnosed with bacterial pneumonia; therefore, the specificity of the assay was finally concluded to be 98.9% (95% CI 95.6–99.8%). The false-positive reports were consistent with extremely high cycle threshold (Ct) values (>28). A previous study by
      • Barnard D.A.
      • Irusen E.M.
      • Bruwer J.W.
      • Plekker D.
      • Whitelaw A.C.
      • Deetlefs J.D.
      • et al.
      The utility of Xpert MTB/RIF performed on bronchial washings obtained in patients with suspected pulmonary tuberculosis in a high prevalence setting.
      showed some discordance between the results of the Xpert MTB/RIF assay and culture when the Ct values were very high for the detection of MTB. In this study as well, some cases were finally excluded from a diagnosis of TB. When the final diagnosis included clinical or radiological evaluation as a reference standard, the Xpert MTB/RIF assay showed a significantly higher sensitivity of 72.9% compared with smear microscopy (24.7%), but no significant difference was observed between the Xpert MTB/RIF assay and culture (68.2%) in terms of sensitivity. Moreover, the Xpert MTB/RIF assay also showed good PPV (96.1% and 96.9%) and NPV (95.2% and 86.8%) whether compared to culture or the final diagnosis of TB based on clinical and radiological/histological examination. These results further strengthen the evidence indicating the superiority of Xpert MTB/RIF over smear microscopy for the detection of TB when using BALF samples.
      Thirteen cases were identified to be Xpert MTB/RIF-positive but culture-negative, and these cases were finally diagnosed with PTB based on the improvements upon treatment with anti-TB agents. A similar situation has also been reported in previous studies.
      • Theron G.
      • Peter J.
      • Meldau R.
      • Khalfey H.
      • Gina P.
      • Matinyena B.
      • et al.
      Accuracy and impact of Xpert MTB/RIF for the diagnosis of smear-negative or sputum-scarce tuberculosis using bronchoalveolar lavage fluid.
      reported 11 patients who showed positive results for Xpert MTB/RIF but negative results for culture, and
      • Walters E.
      • Goussard P.
      • Bosch C.
      • Hesseling A.C.
      • Gie R.P.
      GeneXpert MTB/RIF on bronchoalveolar lavage samples in children with suspected complicated intrathoracic tuberculosis: a pilot study.
      found that the Xpert MTB/RIF assay had a 14% additional diagnostic yield because it confirmed PTB in two cases that showed negative results in culture. One explanation for these differences in the results between Xpert MTB/RIF and culture may be the nature of the PCR test. The Xpert MTB/RIF assay amplifies any DNA whether it originates from live or dead bacilli; therefore, it cannot be assumed that a positive result equates to active disease. Therefore, in the near future, it will be important to determine how to manage patients who show a positive result on Xpert MTB/RIF but a negative result on culture. In the present study, 13 of 15 Xpert MTB/RIF-positive and culture-negative cases showed improvements after anti-TB treatment, which finally supported a diagnosis of PTB. Although implementation of the Xpert MTB/RIF resulted in a more rapid diagnosis of TB in this study, further studies are needed to determine whether the Xpert MTB/RIF assay results in detectable improvements in the time to TB treatment initiation.
      In this study, there were also 11 cases that tested culture-positive and Xpert MTB/RIF-negative; two cases were finally confirmed to be NTM and the other nine were found to have MTB infection. A possible explanation for this discrepancy could be the presence of PCR inhibitors or insufficient nucleic acid material in some specimens. Previous studies have shown that the use of 5–10 ml of centrifuged urine from HIV-infected sputum-scarce individuals improves the performance of the Xpert MTB/RIF assay compared with uncentrifuged urine (
      • Peter J.G.
      • Theron G.
      • Muchinga T.E.
      • Govender U.
      • Dheda K.
      The diagnostic accuracy of urine-based Xpert MTB/RIF in HIV-infected hospitalized patients who are smear-negative or sputum scarce.
      ). This suggests that enrichment of the sample could improve the diagnostic sensitivity. However, in the present study, most of the samples for Xpert MTB/RIF testing only had a volume of 2 ml. Another problem was that four BALF samples in this study were contaminated with a little blood, which could have affected detection using the Xpert MTB/RIF assay. In support of this speculation, a previous study reported that the sensitivity of Xpert MTB/RIF on bloodstained sputum is lower because blood is a known inhibitor of DNA amplification (
      • Meyer A.J.
      • Atuheire C.
      • Worodria W.
      • Kizito S.
      • Katamba A.
      • Sanyu I.
      • et al.
      Sputum quality and diagnostic performance of GeneXpert MTB/RIF among smear-negative adults with presumed tuberculosis in Uganda.
      ).
      The Xpert MTB/RIF assay simultaneously identifies MTB and detects rifampicin resistance with molecular beacons to detect five overlapping 81-bp regions in the rpoB gene known as the rifampicin resistance determining region (
      • Ramaswamy S.
      • Musser J.M.
      Molecular genetic basis of antimicrobial agent resistance in Mycobacterium tuberculosis: 1998 update.
      ). It has been shown that 95% of all rifampicin-resistant MTB strains contain mutations in the region of the rpoB gene (
      • Lawn S.D.
      • Mwaba P.
      • Bates M.
      • Piatek A.
      • Alexander H.
      • Marais B.J.
      • et al.
      Advances in tuberculosis diagnostics: the Xpert MTB/RIF assay and future prospects for a point-of-care test.
      ). Given the importance of the prompt and accurate identification of rifampicin resistance, Xpert MTB/RIF has been recommended for confirming rifampicin resistance in all TB cases in the USA (
      • Centers for Disease C and Prevention
      Availability of an assay for detecting Mycobacterium tuberculosis, including rifampicin-resistant strains, and considerations for its use — United States, 2013.
      ). According to a national survey of drug-resistant TB in China performed in 2007, 6.7% of new TB cases and 29.4% of previously treated cases were resistant to rifampicin (
      • Zhao Y.
      • Xu S.
      • Wang L.
      • Chin D.P.
      • Wang S.
      • Jiang G.
      • et al.
      National survey of drug-resistant tuberculosis in China.
      ). In the present study, although three cases showed rpoB gene mutations (gene mutation rate of 4.8%), the true rifampicin resistance rate may not be consistent with the rpoB gene mutation rate due to the lack of reference phenotypic susceptibility testing for confirmation. So the rifampicin resistance results obtained using the Xpert MTB/RIF assay in this study should be interpreted with caution.
      There are some limitations to this study. First, the study was performed at a single site, which may limit generalization of the results. Second, the study was performed retrospectively, and studies with a retrospective design are generally prone to bias. It was also not possible to evaluate the impact of Xpert MTB/RIF with BALF samples on improvements in the time to TB treatment initiation.
      Nevertheless, the Xpert MTB/RIF assay was shown to be a useful tool for TB testing on BALF samples. It may be used as an add-on test to routine culture with these samples, as it enabled rapid diagnosis as well as the detection of drug-resistant TB infections in one case and added to the total diagnostic yield.

      Conflict of interest

      None.

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