|Year : 2017 | Volume
| Issue : 2 | Page : 122-126
Diagnostic performance of genoType® MTBDRplus line probe assay in bronchoalveolar lavage for pulmonary tuberculosis diagnosis in sputum scarce and smear-negative patients
Farah Idrees1, Muhammad Irfan1, Kauser Jabeen2, Joveria Farooqi2, Rumina Hasan2
1 Department of Medicine, Aga Khan University, Karachi, Pakistan
2 Department of Pathology and Laboratory Medicine, Aga Khan University, Karachi, Pakistan
|Date of Web Publication||19-May-2017|
Department of Medicine, Aga Khan University, Stadium Road, Karachi
Source of Support: None, Conflict of Interest: None
Background: MTBDRplus line probe assay (LiPA) has been endorsed by the World Health Organization for pulmonary tuberculosis (TB) diagnosis. However, its value for Mycobacterium tuberculosis (MTB) detection in bronchoalveolar lavage (BAL) needs exploration. This study determined the diagnostic performance of MTBDRplus in BAL for MTB complex detection and isoniazid/rifampicin resistance in smear-negative and sputum scarce patients. Materials and Methods: Retrospective evaluation of data (January–December 2013) from patients who underwent bronchoscopy was done. Of these, patients with high TB suspicion with available data on acid-fast bacilli (AFB) smear/culture and MTBDRplus were selected. Results of MTBDRplus were compared with AFB smear/culture and drug susceptibility. Sensitivity and specificity of MTBDRplus was determined using TB culture as gold standard. Results: Data on 383 patients who underwent bronchoscopy were collected. Of these, 154 previously untreated TB suspect patients that were either smear negative on sputum microscopy or sputum scarce were selected. Out of 154 patients, 11 were smear positive and 34 patients were AFB culture positive. MTBDRplus detected MTB in 23/34 cases, the sensitivity and specificity being 67.6% and 85% (P < 0.001) versus 32.4% and 100% (P < 0.001) compared to smear microscopy. All smear-positive cases (n = 11) were detected by MTBDRplus. There were no discrepancies between phenotypic drug susceptibility testing and LiPA for isoniazid and rifampicin resistance in patients. Two cases of multidrug-resistant TB were detected. Conclusion: MTBDRplus detected TB more rapidly and accurately than smear microscopy with significant accuracy for isoniazid and rifampicin resistance. Its use in clinical practice would lead to rapid detection and effective management.
Keywords: Bronchoalveolar lavage, MTBDRplus line probe assay, pulmonary tuberculosis
|How to cite this article:|
Idrees F, Irfan M, Jabeen K, Farooqi J, Hasan R. Diagnostic performance of genoType® MTBDRplus line probe assay in bronchoalveolar lavage for pulmonary tuberculosis diagnosis in sputum scarce and smear-negative patients. Int J Mycobacteriol 2017;6:122-6
|How to cite this URL:|
Idrees F, Irfan M, Jabeen K, Farooqi J, Hasan R. Diagnostic performance of genoType® MTBDRplus line probe assay in bronchoalveolar lavage for pulmonary tuberculosis diagnosis in sputum scarce and smear-negative patients. Int J Mycobacteriol [serial online] 2017 [cited 2017 May 24];6:122-6. Available from: http://www.ijmyco.org/text.asp?2017/6/2/122/206606
| Introduction|| |
Tuberculosis (TB) results in high morbidity and mortality worldwide with ~10.4 million new TB cases and 1.4 million deaths as reported in global TB report 2016. Among these deaths, ~190000 could be attributed to multidrug-resistant (MDR) TB. Pakistan has high TB and MDR-TB burden with ~323,856 cases reported in 2015. The rate of MDR-TB among untreated patients is estimated to be 4.2%.
With increasing TB drug resistance, effective management relies heavily on accurate and early diagnosis. The situation becomes even more challenging when a patient with a high TB suspicion is either smear negative or sputum scarce., In such patients, specimen collection using bronchoscopy has been recommended. Diagnostic yield of TB culture in bronchoalveolar lavage (BAL) of smear-negative cases has been reported from 15% to 24%.,
A number of rapid nucleic acid amplification-based TB diagnostic methods have been developed and validated.,,, The World Health Organization (WHO) has recommended Xpert MTB/RIF assay for pulmonary TB diagnosis and rifampicin resistance detection in both smear negative and positive patients. Studies evaluating Xpert MTB/RIF assay performance for BAL report sensitivity of 80%–93% in culture-positive patients and recommended this assay for TB diagnosis in BAL.,,,
Use of MTBDRplus line probe assay (LiPA) was recommended in 2008 by the WHO for pulmonary TB diagnosis. However, it was not recommended in smear-negative cases due to low sensitivity of earlier version of this assay in this patient population. A later study comparing the performance of MTBDRplus LiPA version 2 with Xpert MTB/RIF assay has shown equivalent performance in both smear-negative and smear-positive pulmonary TB patients. MTBDRplus has an advantage over Xpert MTB/RIF as it can detect both isoniazid and rifampicin resistance. So far, the performance of MTBDRplus has been validated mainly on sputum samples, and its role in diagnosing TB in sputum scarce patients requiring BAL has not been assessed.,,
This study was performed to evaluate the diagnostic performance of MTBDRplus in BAL using TB culture as a reference standard.
| Materials and Methods|| |
The study was conducted at Aga Khan University (AKU) Hospital, Karachi, Pakistan. Records were evaluated for all bronchoscopies performed in the hospital from January 2013 to December 2013 after taking approval from the Ethical Review Committee of AKU.
Identification of patients with suspected TB was done through file review. Only those cases were selected who were sputum smear negative (sputum microscopy negative twice) or sputum scarce and on whom BAL samples had been sent with a request for acid-fast bacilli (AFB) smear, culture, and MTBDRplus on the same sample. Patients who had received previous TB treatment or were on current treatment for 2 weeks or more were excluded from the study.
All bronchoscopy procedures were performed by faculty and staff in the pulmonology section of hospital. A flexible bronchoscope with a 5.9 mm diameter (Olympus Optical Co., Tokyo, Japan) was used. After inspecting bronchial trees, samples were collected from the segment or subsegment that showed abnormal lesions on chest X-ray or computed tomography scan. Bronchial washing fluid was obtained by instillation of 20 ml of normal saline, and for BAL, 50–100 ml of normal saline was instilled and aspirated from the lung segments involved. BAL was collected and sent to AKU laboratory in a sterile container. A number of patients also underwent bronchial biopsy along with BAL, and data on histopathology for those specimens were also collected.
AKU laboratory is a Supranational Reference Laboratory for TB diagnosis in the region. All three tests, microscopy, culture, and MTBDRplus were performed at this laboratory.
Acid-fast bacilli smear and isolation of Mycobacterium tuberculosis
BAL fluid samples submitted for AFB smear and culture underwent digestion and decontamination using 5% NaOH-NALC solution and centrifuged. The pellet was resuspended in phosphate buffer saline to inoculate culture media and smear preparation. Auramine-based fluorescent stain was used to report AFB smear results as recommended by Global Laboratory Initiative in Mycobacteriology Laboratory Manual (http://www.stoptb.org/wg/gli/assets/documents/gli_mycobacteriology_lab_manual_web.pdf. Last accessed July 9, 2016. Last updated April 2014). During the study period, Mycobacterium tuberculosis (MTB) culture was performed using the standard methods. Pulmonary specimens were inoculated on Lowenstein–Jensen and MGIT (Becton Dickinson). MTB identification was confirmed using the absence or presence of growth on Middlebrook 7H10 Agar containing 0.5 mg/ml p-nitrophenyl butyrate (Sigma-Aldrich, St. Louis, USA) prepared in-house.
Antimicrobial susceptibility test
Antituberculous drug susceptibility testing (DST) was performed according to CLSI M24-A42 guidelines  by agar proportion technique on enriched Middlebrook 7H10 medium (BBL). The concentration that was used was rifampicin, 1 μg/ml; isoniazid, 0.2 and 1 μg/ml; ethambutol, 5 and 10 μg/ml; and streptomycin, 2 and 10 μg/ml. Pyrazinamide resistance was detected at 100 μg/ml using the BD BACTEC ™ MGIT 960 PZA test Medium and Kit (Becton Dickinson, USA). For quality control of susceptibility testing, MTB H37Rv was tested.
Line probe assay
LiPA was performed using GenoType ® MTBDRplus version 2.0 (Hain Lifescience, Germany). About 0.5 ml of decontaminated specimen was centrifuged, heat inactivated, and DNA released by enzymatic lysis. Multiplex polymerase chain reaction was run on 5 μl of the supernatant (the extracted DNA) according to the instructions provided by manufacturers. Amplicons were hybridized to the DNA probe-labeled strip with conjugate; amplification and gene locus controls for MTB complex, rpoB, inhA, and katG; and wild type and mutation probes for inhA promoter, katG, and rpoB genes. An isolate was categorized as sensitive to both drugs if all wild-type probes tested positive and absence of hybridization with mutation detection probes. If at least one wild-type probe is absent or a mutation probe is present, resistance of the strain to the respective antibiotic was considered.
The data were obtained from the computerized records and file review. Descriptive analysis was done for demographic and clinical features. Data were entered and analyzed by Statistical Package for the Social Sciences (SPSS) software version 19 IBM Corp. Released 2010. IBM SPSS Statistics for Windows, Version 19.0. Armonk, NY: IBM Corp. Results were expressed as number (percentage) as all variables were categorical. Measures of diagnostic accuracy (sensitivity, specificity, positive predictive value [PPV], and negative predictive value) were calculated with 95% confidence interval taking culture as a gold standard. For further analysis, a composite standard was developed combining culture positivity, histology consistent with TB, and response to TB treatment.
| Results|| |
Characteristics of patients
Of the 383 patients who underwent bronchoscopy between January 1, 2013, and December 31, 2013, 160 TB suspects were selected in whom BAL was sent for all three tests: AFB smear, TB culture, and MTBDRplus. Six patients who had been on antitubercular therapy (ATT) were excluded from the study. Out of 154 study patients, 67.53% were male and 32.47% were female. About 96 (62.33%) were unable to produce sputum and 58 (37.66%) had previously been smear negative on two occasions. In addition, bronchial biopsy or cytology of bronchial brushings was performed on 34 patients [Figure 1]. The clinical details of 154 study patients are shown in [Table 1].
|Table 1: Demographic and clinical characteristics of 154 patients with suspected pulmonary tuberculosis|
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On clinical and radiological feature, 72 patients were diagnosed to have pulmonary TB and were started on ATT. Eight two patients were excluded as they have alternate diagnosis and not treated as TB [Table 1]. Cultures of BAL from 34 patients yielded growth of MTB, whereas BAL from 38 patients remained negative on culture. All 72 patients were started on anti-TB therapy. Of these, 64 successfully completed their treatment. However, eight patients were lost to follow-up between 2 and 4 months of therapy, despite having shown signs of improvement during the initial 2 months of treatment.
Diagnostic accuracy of acid-fast bacilli smear, tuberculosis culture, and MTBDRplus
MTB was detected with MTBDRplus in 23 (67.6%) of the 34 culture-positive patient samples. There were three cases of culture-proven MDR-TB, in which MTB was not detected by MTBDRplus. TB smear was positive in 11 of the 34 (32.4%) culture-positive patients. All smear-positive cases (11/11; 100%) were detected by MTBDRplus. Sensitivity, specificity, PPV, and PPV of TB smear and MTBDRplus in comparison to culture are shown in [Table 2] and [Table 3].
|Table 2: Frequency table for Mycobacterium tuberculosis detection by acid-fast bacilli smear and MTBDRplus among 72 samples included in the study. Mycobacterium tuberculosis culture was taken as gold standard|
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|Table 3: Diagnostic performance of MTBDRplus in bronchoalveolar lavage using culture as a reference standard (n=72)|
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The study included six MTBDRplus-positive and culture-negative cases. Two of these patients were expired during the 1st week of hospital stay with a final diagnosis of squamous cell carcinoma of lung in first patient and antineutrophil cytoplasmic antibodies positive granulomatosis with polyangiitis in second patient. Four patients were started on ATT. Of the 4 who were started on ATT, two patients showed chronic granulomatous inflammation on endobronchial biopsy and two were started on clinical grounds. Three patients had successfully completed ATT and were labeled cured and one was lost to follow-up. When these four cases were included, sensitivity and specificity of MTBDRplus increased to 79.41% and 94.74%, respectively.
The drug susceptibility pattern of 34 culture-positive samples is shown in [Table 4]. Two cases of MDR-TB were detected 4 weeks earlier with MTBDRplus than conventional phenotypic testing. Of the 23 culture positive and MTBDRplus cases on which the results of susceptibility testing were known, the agreement of rifampicin and isoniazid resistance detection between conventional DST and MTBDRplus was 100%. Two out of 23 isoniazid-resistant MTB strains and 2/23 rifampicin-resistant strains were accurately detected by MTBDRplus.
| Discussion|| |
Collection of respiratory secretions by bronchoscopy is performed for pulmonary TB diagnosis in patients who cannot produce sputum or those who are sputum AFB smear negative with clinical suspicion of TB. No previous study has evaluated the performance of MTBDRplus in BAL samples for TB diagnosis in a high-burden country.
In this study, MTBDRplus clearly outperformed smear, detecting 12 out of 23 smear-negative cases 4 weeks before the culture results. Furthermore, our data showing sensitivity and specificity of 79.41% and 94.74%, respectively, for MTBDRplus in BAL suggest that this test will be valuable for MTB detection.
The sensitivity and PPV of MTBDRplus for BAL have been found to be comparatively lower than Xpert MTB/RIF assay.,,, Published data report that diagnostic accuracy of MTBDRplus and Xpert MTB/RIF assay in sputum is similar., However, head-to-head comparison of these assays in BAL has not been reported yet.
An advantage of MTBDRplus is that it additionally detects isoniazid resistance along with rifampicin resistance. In our study, the agreement of rifampicin and isoniazid resistance detection between conventional DST and MTBDRplus was 100% making it a reliable modality to start treatment before the availability of culture results. In our study, there was no discrepancy found for rifampicin and isoniazid resistance with the phonotypical assay. This could be because of low number of samples, as it is well known that although the rifampicin has a good correlation with the phenotypic DST, the correlation decreases in isoniazid since there could be missense mutations or mutations in representative resistance genes (katG and inhA) that cannot be detected by the molecular assay. More MDR and non-MDR and smear-negative patients should be included in the future studies for an accurate evaluation of MTBDRplus.
One of the limitations of our study is the low sample size of culture-positive TB cases. The low numbers may have contributed to a higher sensitivity and specificity of resistance detection by MTBDRplus as compared to previous studies.,
MTBDRplus also detected six cases that were culture negative. This phenomenon has been observed in previous studies with both Xpert MTB/RIF assay and MTBDRplus , and may represent either cross-contamination (either during specimen collection or in the laboratory) or due to detection of DNA from nonviable bacteria in previously treated patients. Although treated patients were excluded, a transcription error in the patient file may have led to a failure to detect such cases. An earlier study has reported two such cases which were initially culture negative but became culture positive during treatment follow-up. In our study, out of these six patients, four were treated with ATT and three successfully completed the treatment. It is possible that these cases may have represented “partially treated” TB as in our setup, many antibiotics including fluoroquinolones have already been used by physicians before the patient is referred to a pulmonary specialist or a diagnosis of TB is considered.
| Conclusion|| |
Our study indicates that MTBDRplus detected TB more rapidly and accurately than smear microscopy with significant accuracy for isoniazid and rifampicin resistance. Its use in clinical practice would lead to rapid detection and effective management of TB in sputum scarce or smear-negative patients. In cases where the LiPA is positive and culture is negative, the management should be based on clinical decision-making and follow-up cultures.
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Conflicts of interest
There are no conflicts of interest.
| References|| |
Mohammad AB, Iliyasu G, Habib AG. Prevalence and genetic determinant of drug-resistant tuberculosis among patients completing intensive phase of treatment in a tertiary referral center in Nigeria. Int J Mycobacteriol 2017;6:47-51.
] [Full text]
Tadesse M, Aragaw D, Rigouts L, Abebe G. Increased detection of smear-negative pulmonary tuberculosis by GeneXpert MTB/RIF® assay after bleach concentration. Int J Mycobacteriol 2016;5:211-8. [Full text]
Getahun H, Harrington M, O'Brien R, Nunn P. Diagnosis of smear-negative pulmonary tuberculosis in people with HIV infection or AIDS in resource-constrained settings: Informing urgent policy changes. Lancet 2007;369:2042-9.
Brown M, Varia H, Bassett P, Davidson RN, Wall R, Pasvol G. Prospective study of sputum induction, gastric washing, and bronchoalveolar lavage for the diagnosis of pulmonary tuberculosis in patients who are unable to expectorate. Clin Infect Dis 2007;44:1415-20.
Charoenratanakul S, Dejsomritrutai W, Chaiprasert A. Diagnostic role of fiberoptic bronchoscopy in suspected smear negative pulmonary tuberculosis. Respir Med 1995;89:621-3.
Greco S, Girardi E, Navarra A, Saltini C. Current evidence on diagnostic accuracy of commercially based nucleic acid amplification tests for the diagnosis of pulmonary tuberculosis. Thorax 2006;61:783-90.
Ling DI, Flores LL, Riley LW, Pai M. Commercial nucleic-acid amplification tests for diagnosis of pulmonary tuberculosis in respiratory specimens: Meta-analysis and meta-regression. PLoS One 2008;3:e1536.
Pai M, Flores LL, Pai N, Hubbard A, Riley LW, Colford JM Jr. Diagnostic accuracy of nucleic acid amplification tests for tuberculous meningitis: A systematic review and meta-analysis. Lancet Infect Dis 2003;3:633-43.
Buyankhishig B, Oyuntuya T, Tserelmaa B, Sarantuya J, Lucero MG, Mitarai S. Rapid molecular testing for multi-resistant tuberculosis in Mongolia: A diagnostic accuracy study. Int J Mycobacteriol 2012;1:40-4. [Full text]
Lawn SD. Diagnosis of pulmonary tuberculosis. Curr Opin Pulm Med 2013;19:280-8.
Barnard DA, Irusen EM, Bruwer JW, Plekker D, Whitelaw AC, Deetlefs JD, et al.
The utility of Xpert MTB/RIF performed on bronchial washings obtained in patients with suspected pulmonary tuberculosis in a high prevalence setting. BMC Pulm Med 2015;15:103.
Lee HY, Seong MW, Park SS, Hwang SS, Lee J, Park YS, et al.
Diagnostic accuracy of Xpert® MTB/RIF on bronchoscopy specimens in patients with suspected pulmonary tuberculosis. Int J Tuberc Lung Dis 2013;17:917-21.
Le Palud P, Cattoir V, Malbruny B, Magnier R, Campbell K, Oulkhouir Y, et al.
Retrospective observational study of diagnostic accuracy of the Xpert ®
MTB/RIF assay on fiberoptic bronchoscopy sampling for early diagnosis of smear-negative or sputum-scarce patients with suspected tuberculosis. BMC Pulm Med 2014;14:137.
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. Thorax 2013;68:1043-51.
Causse M, Ruiz P, Gutierrez JB, Zerolo J, Casal M. Evaluation of new genotype MTBDRplus for detection of resistance in cultures and direct specimens of Mycobacterium tuberculosis
. Int J Tuberc Lung Dis 2008;12:1456-60.
Lacoma A, Garcia-Sierra N, Prat C, Ruiz-Manzano J, Haba L, Rosés S, et al.
Genotype MTBDRplus assay for molecular detection of rifampin and isoniazid resistance in Mycobacterium tuberculosis
strains and clinical samples. J Clin Microbiol 2008;46:3660-7.
Miotto P, Piana F, Cirillo DM, Migliori GB. Genotype MTBDRplus: A further step toward rapid identification of drug-resistant Mycobacterium tuberculosis
. J Clin Microbiol 2008;46:393-4.
Zhang L, Ye Y, Duo L, Wang T, Song X, Lu X, et al
. Application of genotype MTBDRplus in rapid detection of the Mycobacterium tuberculosis
complex as well as its resistance to isoniazid and rifampin in a high volume laboratory in Southern China. Mol Biol Rep 2011;38:2185-92.
Shakoor S, Ahsan T, Jabeen K, Raza M, Hasan R. Use of p-nitrobenzoic acid in 7H10 agar for identification of Mycobacterium tuberculosis
complex: A field study. Int J Tuberc Lung Dis 2010;14:1644-6.
Clinical Laboratory Standards Institute (CLSI). Susceptibility Testing of Mycobacteria, Nocardia
and Other Aerobic Actinomycetes; Approved Standards. 2nd
ed. CLSI Document M24 -A2. Wayne, Pennsylvania, USA: Clinical Laboratory Standards Institute; 2011.
Barnard M, Gey van Pittius NC, van Helden PD, Bosman M, Coetzee G, Warren RM. The diagnostic performance of the Genotype MTBDRplus version 2 line probe assay is equivalent to that of the Xpert MTB/RIF assay. J Clin Microbiol 2012;50:3712-6.
Bablishvili N, Tukvadze N, Avaliani Z, Blumberg HM, Kempker RR. A comparison of the Xpert ®
MTB/RIF and GenoType ®
MTBDRplus assays in Georgia. Int J Tuberc Lung Dis 2015;19:676-8.
Farooqi JQ, Khan E, Alam SM, Ali A, Hasan Z, Hasan R. Line probe assay for detection of rifampicin and isoniazid resistant tuberculosis in Pakistan. J Pak Med Assoc 2012;62:767-72.
Nathavitharana RR, Cudahy PG, Schumacher SG, Steingart KR, Pai M, Denkinger CM, et al.
Accuracy of line probe assays for the diagnosis of pulmonary and multidrug-resistant tuberculosis: A systematic review and meta-analysis. Eur Respir J 2017;49. pii: 1601075.
[Table 1], [Table 2], [Table 3], [Table 4]