Evaluating Diagnostic Utility of geneXpert Ultra (Mycobacterium
tuberculosis/Rifampicin), Microscopy and Liquid Culture to Isolate
Mycobacterium tuberculosis and
Nontuberculous Mycobacteria among Pulmonary Tuberculosis Suspects
Apoorva Gota, Vishnu Prasad Shenoy, Asha Kamath1
Abstract
CONTEXT: Nontuberculous mycobacteria (NTM) are posing major public health problems and have recently gained importance as they are being isolated from patients with pulmonary disorders. Most of the time, NTM are misinterpreted as tuberculosis (TB) and this misdiagnosis leads to treatment failure. Hence, this study aimed to identify NTMs isolated from suspected cases of pulmonary TB cases and was aimed to distinguish Mycobacterium TB (MTB) from NTM infections.
AIMS: Our study aimed to compare microscopy grading of respiratory samples with the Ct values of GeneXpert Ultra (MTB/rifampicin [RIF]), to assess the diagnostic accuracy of microscopy, GeneXpert ultra (MTB/RIF), liquid culture, to compare GeneXpert ultra (MTB/RIF) results with microscopy with culture and clinical implications, importance of performing a rapid test for pulmonary TB and pulmonary NTM infections in suspected cases of adults.
SETTINGS AND DESIGN: The study was conducted in the Mycobacteriology laboratory, Department of Microbiology, in Kasturba Hospital, Manipal. The study was a prospective, cross‑sectional study, single center.
SUBJECTS AND METHODS: A total of 452 respiratory samples obtained from April to July 2023 were included in the study. Smearpositive pulmonary TB cases were identified and subjected to amplification by GeneXpert ultra (MTB/RIF) and 84 samples were GeneXpert ultra (MTB/RIF) positive and RIF sensitive. Liquid culture by mycobacteria growth indicator tube (MGIT) 960 was performed and for positive tubes, TB Ag MPT64 was performed. The results were compared and analyzed with patients’ demographic information. Clinical and radiological data from the requisition forms were assessed.
STATISTICAL ANALYSIS USED: IBM SPSS statistics software version 22 was used. Correlations between GeneXpert ultra (MTB/RIF) Ct values and smear status were calculated by polychoric correlation. Extended McNemar’s test was used to find the association between the variables.
RESULTS: GeneXpert ultra (MTB/RIF) yielded a positivity rate of 22.2% compared to smear microscopy 17.2%. Liquid culture yielded a positivity rate of 20.3%. Ct value and smear grading yielded a positive correlation (ρ = 0.8681; P < 0.05). Undetected cases of TB by GeneXpert ultra (MTB/RIF) Department of
Microbiology, Kasturba Medical College, Manipal Academy of Higher Education, 1Department of Data Science, Prasanna School of Public Health, Manipal Academy of Higher Education, Manipal, Karnataka, India Access this article online
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DOI:10.4103/jpdtsm.jpdtsm_133_23
How to cite this article: Gota A, Shenoy VP, Kamath A. Evaluating diagnostic utility of geneXpert ultra (Mycobacterium tuberculosis/rifampicin), microscopy and liquid culture to isolate Mycobacterium tuberculosis and nontuberculous mycobacteria among pulmonary tuberculosis suspects. J Prev Diagn Treat Strat Med 2023;2:236-42.
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correspondence:
Dr. Vishnu Prasad Shenoy, Department of Microbiology, Kasturba Medical College, Manipal Academy of Higher Education, Manipal - 576 104, Karnataka, India.
E-mail: vishnu.shenoy@
manipal.edu Submitted: 20-Aug-2023 Revised: 19-Oct-2023 Accepted: 02-Dec-2023 Published: 22-Dec-2023
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Introduction
M
ycobacteria encompass 4 groups based on the growth rate, capability of causing tuberculosis (TB), and nonpathogenic mycobacteria.Group 1 includes Mycobacterium TB (MTB) complex which includes organisms causing TB. Group 2 includes Mycobacterium leprae, the organism responsible for causing leprosy. Group 3 includes nontuberculous mycobacteria (NTM) or atypical mycobacteria, which are ubiquitous, diverse group of mycobacteria isolated from animal, soil, and water originating from natural environments and cause infection in both immunocompetent and immunocompromised individuals.[1] About 170 species of mycobacteria are comprised in NTM. Pulmonary diseases in humans are mostly caused by Mycobacterium avium complex, Mycobacterium kansasii, and Mycobacterium abscessus (M. abscessus) species.[2] Recently, NTMs are gaining more importance as there is an increase in comorbid conditions and they cause infections in HIV/AIDS, diabetes, renal failure, transplant recipients and immunosuppressive therapy, and lung disorders individuals.[3] NTMs can cause infections in pulmonary and extrapulmonary regions ranging from skin and soft tissue infections which occurs following surgery, trauma, device‑associated infections, central line‑associated bloodstream infection, infections caused due to catheters. However, the most common site of infection is the lungs.[4]
Symptoms such as night sweats, loss of appetite, fever, weight loss, and chest pain overlap with the symptoms of TB. NTMs are often misdiagnosed, and misinterpreted as tubercule bacilli by physicians based on their appearance as acid‑fast bacilli (AFB) under microscopy in sputum smears or pulmonary specimens. For these reasons, the diagnosis of NTM infections is confusing and misleading.
NTMs are also difficult to treat as they are resistant to most of the anti‑tubercular drugs. Therefore, it leads to prolong treatment of the anti‑tubercular drugs and they are labeled as drug‑resistant TB and lead to treatment failure. With so many existing challenges, NTM infections have gained importance and therefore, it is crucial to understand their biology, epidemiology, diagnosis, and management of infections and has caused to be undergone research globally.[5,6]
Even though microscopy is considered as gold standard, it cannot differentiate between MTB and NTM as they appear morphologically similar. For NTM infections, specimen can be collected from the site involved, for pulmonary NTM infections, sputum samples, bronchial brush, induced sputum, endotracheal (ET) aspirates, Bronchoalveolar lavage fluid (BAL) are collected, but contamination must be avoided from tap water as environmental mycobacteria are present.
The ideal staining technique for both MTB and NTM is the fluorescent staining method. Culture methods for isolation of NTM and MTB include mycobacteria growth indicator tube (MGIT) 960 which contains a growth supplement and antibiotic mixture, fluorescent indicator.
Culture by Lowenstein–Jensen medium is also used which is considered gold standard. Biphasic media, such as the Septi‑Chek System (Becton Dickinson), provide enhanced recovery of most NTM in one system. 28°C–37°C is considered the optimal temperature for incubation of NTM culture. To identify the real pulmonary NTM disease, the American Thoracic Society and Infectious Diseases Society of America have provided some set of criteria. The criteria require radiological and clinical finding of the lung/chest, evidence by microbiological methods, and requires the identification of strain, to confirm NTM disease.[2,7] The test to distinguish MTB and NTM is a rapid, immunochromatographic test Standard Diagnostics (SD) Bioline TB Ag Mycobacterium tuberculosis protein (MPT) 64 RAPID has been used routinely. This kit detects a protein that is specific for MTB, MPT 64 using a mouse monoclonal antibody. This assay is rapid and is highly efficient.
GeneXpert ultra (MTB/RIF) is a cartridge‑based nucleic acid amplification test which detects target sequences based on semi‑quantitative, nested real‑time polymerase chain reaction (PCR), and melt curve analysis. GeneXpert ultra (MTB/RIF) includes the two specific insertion sequences IS1081 and IS6110 of MTB, and rpoB gene and it detects the disease in smear‑negative cases, with increased sensitivity comparted to microscopy. It detects “trace” amounts of MTB DNA in the sample along with “high,” “medium,”
“low,” and “very low”[8‑10] and also detects the disease in cases of smear negativity, which aids in improving diagnostic accuracy and sensitivity.[11]
were identified as positive under liquid culture by MGIT 960. TB Ag MPT64 was performed and yielded NTM and speciated as Mycobacterium abscessus complex by line probe assay.
CONCLUSIONS: Our study states the need for identification and differentiation of MTB from NTM.
Keywords:
Auramine O phenol staining technique, Ct value, fluorescence microscopy, GeneXpert ultra (Mycobacterium tuberculosis/
rifampicin), liquid culture, Mycobacterium tuberculosis, nontuberculous mycobacteria, TB Ag MPT64
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Thus, microscopy alone is not sufficient to classify NTM from MTB. Therefore, an accurate method to differentiate and distinguish MTB from NTM is essential to prevent the wrong diagnosis of the cases. In this study, we isolated and differentiated pulmonary MTB from NTMs by microscopy by Auramine O phenol staining technique and GeneXpert ultra (MTB/RIF) followed by liquid culture by MGIT960 and TB Ag MPT64. The diagnostic efficacy of each test was calculated. M. abscessus complex was isolated and speciated by line probe assay (LPA).
Our findings suggest that NTMs play a vital role in causing pulmonary infections and it is important to differentiate MTB from NTM.
Materials and Methods
Ethical statementThe study was performed after the approval from institutional ethics committee. This was single‑center, cross‑sectional study. The study was performed in the Mycobacteriology Laboratory, Department of Microbiology, Kasturba Hospital, Manipal, Karnataka, India, from April 2023 to July 2023.
Study design
The microscopy was performed for all the samples and grading was done. The grading results of respiratory samples were compared with the Ct values of GeneXpert ultra (MTB/RIF) and these results were simultaneously compared with liquid culture by MGIT 960 from adult patients with suspected cases of pulmonary TB and the MTB and NTMs were differentiated based on radiographic, clinical findings and by performing a rapid immunochromatographic test.
During the study, smear microscopy, GeneXpert ultra (MTB/RIF), and liquid culture were systematically performed on the respiratory samples suspected of pulmonary TB; however, GeneXpert ultra (MTB/RIF) and liquid culture were performed only if they were selectively requested by the physicians for the patients with a high suspicion of PTB. Only the requisition forms sent to the mycobacteriology laboratory were analyzed and routine tests for diagnosis of TB were done. Clinical, radiological, and microbiological data were recorded from the requisition forms only.
Study samples
Samples were collected in suspected cases of pulmonary TB.
Inclusion criteria
All the cases of pulmonary TB in adults were included.
The samples sent to the mycobacteriology laboratory for routine diagnosis of TB were taken into the study.
Exclusion criteria
Extrapulmonary TB cases and pediatric cases were excluded from the study.
Microscopy results (grading) and GeneXpert ultra (MTB/
RIF) Ct values were recorded for each sample.
Type of sampling
The type of sampling method involved was stratified sampling. Population was divided into pulmonary and extrapulmonary cases. Only pulmonary samples were taken to be a part of this study. Extrapulmonary samples and extrapulmonary TB cases were not considered.
Bacteriological methods Microscopy
All the respiratory samples sputum, BAL, ET aspirates received at the laboratory were subjected to smear microscopy for AFB detection by fluorescence microscopy by Auramine O phenol staining technique to evaluate smear‑positives and smear‑negatives. Smears were graded according to NTEP guidelines.[8]
GeneXpert ultra (Mycobacterium tuberculosis/
rifampicin) (Cepheid, Sunnyvale, CA)
GeneXpert ultra (MTB/RIF) (Cepheid Inc., Sunnyvale, CA, USA) assay was performed on the requested pulmonary samples to evaluate and detect the presence of MTB and RIF resistance (RIF). Samples were received in sterile screw‑capped containers and were processed in Class IIA Biosafety cabinet. To 1 mL sample, 2 mL buffer was added and vortexed, and kept at room temperature for 10 min, and was vortexed again and kept at room temperature for 5 min. 2.5 mL of vortexed sample was added into the GeneXpert cartridge and the cartridge was loaded into the machine and results were displayed after 1 h 20 min along with drug resistance. Ct values were displayed if MTB was detected: Ct value <16 as high; Ct value 16–22 as medium; Ct value 22–28 as low; Ct value > 28 as very low; and trace. Ct values for each of the five probes were recorded. Ct value for (IS1081–IS6110) was selected for statistical analysis.
Liquid culture by mycobacteria growth indicator tube 960
Liquid culture was performed only on the requested samples to isolate positive results. MGIT 960 tubes were labeled and MGIT growth supplement/PANTA was added to each tube with a sterile pipette. Later processed sample (0.5 mL) was added to the tube and was mixed properly by inverting it several times. All the steps were performed in a biosafety cabinet to avoid contamination.
For every step, a new, sterile pipette was used. Caps were closed tightly and all tubes were scanned in the BACTEC MGIT 960 instrument. Inoculated MGIT tubes
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were incubated at 37°C, for 4 weeks and read manually under ultraviolet light for fluorescence. If there is no growth in the tubes, the tubes are declared negative by the instrument after 6 weeks. After the instrument signaled the tube as positive, the tubes were removed and observed for growth. For MTB, light turbidity or small granular appearance was seen in the medium, and growth was seen at the bottom. For some NTMs, light turbidity is seen. All the negative tubes were observed for growth manually before discarding.
For culture‑positive tubes, a smear was made and stained with Ziehl–Neelsen staining technique and an ICT was performed to differentiate MTB from NTM.
Tuberculosis Ag MPT64
MPT64, a rapid immunochromatographic test was performed for all the positive MGIT 960 tubes to differentiate MTB from NTM. 100 µL of sample (from MGIT 960 tube) was added with a sterile pipette into the sample well after a dense mixing from the colonies. Test results were interpreted after 15 min. If both the control band and the test band are visible, the test is considered positive. If only the control band is visible, the test is considered negative. If the control band is not visible, the test is considered invalid.
Statistical analysis
IBM SPSS statistics software version IBM SPSS statistics software version 22 for Windows was used for statistical analysis. The performance of Ct values for the detection of smear‑positive patients was evaluated using the extended McNemar test which was used to find the
association between the variables. The polychoric correlation coefficient was used to obtain the correlation.
The diagnostic accuracy for microscopy, GeneXpert ultra (MTB/RIF), and liquid culture was calculated using the rate of positivity formula.
Results
Demographic characteristics and clinical presentations of the patients
A total of 452 respiratory samples were included in the study. Of the 452 respiratory samples, 339 (74.7%) were sputum, 100 (22%) were BAL fluid, and 13 (2.9%) were ET aspirates. Among the 452 cases, 283 were male (62.3%), and 169 were female (37.2%). Majority of the patients belonged to the age group of 40–59 years (42.1%) who were 191, followed by the age group of 60–
79 years (37.4%) who were 170 as mentioned in the above Table 1.
Performances of the tests
Of the 452 samples received, 84 samples were GeneXpert ultra (MTB/RIF) positive and RIF sensitive and 77 cases were AFB positive. Among the 77 AFB positives, 5 cases were AFB scanty smear which on liquid culture by MGIT 960 were identified as NTM and speciated as M. abscessus complex by LPA Common Mycobacteria (CM) as GeneXpert ultra (MTB/RIF) could not detect MTB in these 5 cases as shown in Table 2. Figure 1 was followed for speciation of NTM into Mycobacterium abscessus.
A total of 72 AFB positives were isolated. In addition, GeneXpert ultra (MTB/RIF) could detect MTB in 15 smear‑negative cases, (14/84) trace MTB, and (1/84) very Table 1: Association of acid‑fast bacilli smear status by fluorescence microscopy by Auramine O phenol
staining technique and GeneXpert ultra (Mycobacterium tuberculosis/rifampicin) in patients with underlying conditions
Demography, clinical, radiological data, n (%) Smear positivity,
n (%)
Smear negativity,
n (%)
GeneXpert ultra (MTB/RIF) MTB detected (including
trace), n (%)
GeneXpert ultra (MTB/
RIF) MTB not detected, n (%)
AFB positivity and MTB detected,
n (%)
Total number of cases ‑ 452 77 (17) 375 (82.9) 84 (18.5) 368 (81.4) 69 (15)
Number of male patients ‑ 283 (62.3) 50 (17.6) 233 (82) 58 (20.4) 225 (79.5) 45 (15.9)
Number of female patients ‑ 169 (37.2) 24 (14.2) 145 (85.7) 21 (12.4) 148 (87.5) 17 (10)
Number of patients with RVD ‑ 17 (3.7) 3 (17.6) 14 (82.3) 4 (23.5) 13 (76.4) 4 (23.5)
Number of patients previously infected with PTB ‑ 36 (7.9) 11 (30.5) 25 (69.4) 14 (38.8) 22 (61.1) 10 (27.7) Number of patients with diabetes mellitus ‑ 65 (14.3) 16 (24.6) 49 (75.3) 18 (27.6) 47 (72.3) 11 (16.9)
Number of patients with COPD ‑ 33 (7.3) 8 (24.2) 25 (75.7) 7 (21.2) 26 (78.7) 12 (36.3)
Number of patients with abnormal renal function ‑ 22 (4.8) 6 (27.2) 16 (72.7) 5 (22.7) 17 (77.2) 9 (40.9)
Number of patients with hypertension ‑ 25 (5.5) 12 (48) 13 (52) 13 (52) 12 (48) 12 (48)
Number of patients with fever ‑ 77 (17) 13 (16.8) 64 (83.1) 12 (15.5) 65 (84.4) 25 (32.4) Number of patients with cough and expectoration ‑ 41 (9.7) 6 (14.6) 35 (85.3) 7 (17) 34 (82.9) 6 (14.6) Number of patients with shortness of breath ‑ 32 (7) 7 (21.8) 25 (78.1) 8 (25) 24 (75) 10 (31.2)
Number of patients with hemoptysis ‑ 16 (3.5) 4 (25) 12 (75) 5 (31.2) 11 (68.7) 8 (16)
Number of patients with abnormal CXR ‑ 105 (23.1) 48 (45.7) 57 (54.2) 50 (47.6) 55 (52.3) 51 (48.5) AFB: Acid‑fast bacilli, MTB: Mycobacterium tuberculosis, RIF: Rifampicin, PTB: Pulmonary tuberculosis, COPD: Chronic obstructive pulmonary disease, CXR: Chest X‑ray, RVD: Retroviral Disease
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Respiratory samples
Sputum BAL fluid
Respiratory samples subjected to microscopy for AFB detection by Auramine O phenol staining technique Scanty AFB detected in 3 Sputum, 2 BAL fluid samples= 5 Scanty AFB
Samples with Scanty AFB subjected for MTB detection by GeneXpert ultra (MTB/RIF) MTB not detected
Undetected MTB samples inoculated into liquid culture by MGIT 960 MGIT positive obtained
Ziehl-Neelsen staining performed for culture positive tubes Acid-fast bacilli seen
Rapid ICT test TB Ag MPT64 performed from MGIT 960 tubes Negative
Nontuberculous mycobacteria
Speciated as Mycobacterium abscessus complex
by LPA
Figure 1: Flow diagram of methodologies for identification and differentiation of Mycobacterium tuberculosis from nontuberculous mycobacteria for respiratory samples.
AFB: Acid‑fast bacilli, MTB: Mycobacterium tuberculosis, RIF: Rifampicin, MGIT: Mycobacteria growth indicator tube, BAL: Bronchoalveolar lavage, LPA: Line probe assay, ICT:
Immunochromatographic test
low case, which on subjecting to liquid culture by MGIT 960, MTB complex was not isolated. A total of 69 cases were identified AFB and GeneXpert ultra (MTB/RIF) positive. Among them, 44 were sputum, 22 were BAL fluid, and 3 were ET aspirates. Smear grading was done for AFB‑positive smears and overall, the results observed are given respectively scanty AFB in 19 cases (27.5%), 1+ in 23 cases (33.3%), 2+ in 11 cases (15.9%), and 3+ in 16 cases (23.1%) as shown in above Table 3.
Comparison and analysis of the test methods Smear positivity was significantly correlated with the Ct of IS1081‑IS6110 of the MTB‑detected cases. Polychoric correlation coefficient was used to obtain the correlation and it was obtained as 0.8681. A positive correlation was found between Ct value and AFB smear grading with polychoric correlation ρ of 0.8681 (P < 0.05). An extended
McNemar test was used to find the association between the variables.
Figure 2 was followed for identification and differentiation of NTM from MTB, which included detail testing.
Discussion
In this study, GeneXpert ultra (MTB/RIF) showed a positivity rate of 22.2% whereas microscopy (Auramine O phenol staining technique) showed a positivity rate of 17.2%, and liquid culture by MGIT 960 showed a positivity rate of 20.3%. A positive correlation of 0.8681 was obtained between smear microscopy (fluorescence microscopy by Auramine O phenol staining technique) and from this value, we can conclude that there is a high positive correlation between the variables and hence, Table 2: Association of pulmonary nontuberculous mycobacteria infections with clinical and radiological findings Type of sample Clinical presentations Previous history/comorbidities CXR findings Final diagnosis
BAL† fluid Fever, cough with expectoration Old case of tuberculosis Upper lobe fibrosis NTM (M. abscessus complex)
Sputum Fever, cough, headache Diabetes Bronchiectasis NTM (M. abscessus complex)
Sputum Fever, cough, shortness of breath Old case of tuberculosis Bilateral bronchiectasis NTM (M. abscessus complex) Sputum Left side body weakness, fever,
cough, headache Old case of tuberculosis Right lower lobe mass NTM (M. abscessus complex) BAL fluid Fever, cough with expectoration
shortness of breath Old case of tuberculosis Left lower lobe nodule
formation NTM (M. abscessus complex)
†Scanty. NTM: Nontuberculous mycobacteria, BAL: Bronchoalveolar lavage, M. abscessus: Mycobacterium abscessus, CXR: Chest X‑ray
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both microscopy and GeneXpert ultra (MTB/RIF) would give almost similar results and use of one test can be replaced by another.
GeneXpert ultra (MTB/RIF) detected MTB trace in 16.6% (14/84) of the respiratory samples and detected MTB very low in (1/84) when the smear status was negative. Out of 69 AFB and GeneXpert ultra (MTB/
RIF) positives, 21 cases (30.4%) were identified positive in liquid culture (23.8%) by MGIT 960, among these 21 cases, 5 (1.1%) were detected as NTM which showed smear positive scanty when MTB was not detected.[9]
Trace MTB results were correlated with liquid culture by MGIT 960, out of which MTB complex was not isolated. Based on these results, trace MTB detection can be reclassified as TB-negative as it was not obtained by liquid culture, but it must be confirmed on the basis of the patient’s previous history of TB or requesting a repeat sample.[10] This suggests that in some situations, GeneXpert ultra (MTB/RIF) can be more sensitive than the liquid culture to detect the presence of MTB. NTM’s were identified as M. abscessus complex. Therefore, most pulmonary NTM cases are misdiagnosed as TB based on staining by microscopy as microscopy cannot differentiate MTB and NTM. Therefore, microscopy alone is not sufficient to detect MTB and requires a series of tests to differentiate pulmonary MTB from pulmonary NTM infections such as microscopy, GeneXpert ultra (MTB/RIF), liquid culture, and TB Ag MPT64.[3,5]
Culture is useful to detect the presence of the viable bacilli, and has even lesser limit of detection, 10 CFU/
mL.[9,12] GeneXpert ultra (MTB/RIF) is important in these
situations to distinguish MTB from NTM as it amplifies only MTB‑specific insertion sequences; IS1081 and IS6110. In this study, GeneXpert ultra (MTB/RIF) had a significant role in detecting NTM infections. Therefore, GeneXpert ultra (MTB/RIF) must be performed while suspecting NTM infections as amplification of the MTB-specific insertion sequences does not occur.
Therefore, GeneXpert ultra (MTB/RIF), liquid culture, and TB Ag MPT64 must be performed while suspecting pulmonary NTM infections. Liquid culture detects is used for isolation of both MTB and NTM, whereas, a rapid immunochromatographic test like TB Ag MPT 64 confirms the presence of only MTB complex from the sample but not NTM.
In this study, all the 5 cases of pulmonary NTMs were isolated from the patients having clinical presentations such as fever, cough, shortness of breath, and abnormal CXR, i.e. patients with bronchiectasis, nodule formation, and with a previous history of pulmonary TB. It indicates that for the detection of pulmonary NTM, the presence of clinical symptoms followed by a radiological diagnosis of chest/lung, along with microbiological evidence is
Samples (pulmonary/extrapulmonary samples)
Perform staining technique for AFB detection by Auramine O phenol staining technique
AFB positive
GeneXpert ultra (MTB/RIF) performed for MTB detection
detectedMTB MTB not detected
Inoculated into MGIT 960 tubes for liquid culture
Culture positive
Perform Ziehl–Neelsen staining to confirm the presence of AFB
Perform TB Ag MPT64 test AFB seen
MPT64 positive
MPT64 negative Mycobacterium
tuberculosis complex
Non-tuberculous mycobacteria AFB positive, GeneXpert ultra
(MTB/RIF) positive, MGIT
960 positive= MTB complex AFB positive, GeneXpert ultra (MTB/RIF) negative, MGIT 960 positive= NTM.
Figure 2: Flow diagram for identification and differentiation of Mycobacterium tuberculosis from nontuberculous mycobacteria. AFB: Acid‑fast bacilli, MTB: Mycobacterium tuberculosis, RIF: Rifampicin, TB: Tuberculosis,
MGIT: Mycobacteria growth indicator tube
Table 3: Comparative results of GeneXpert ultra (Mycobacterium tuberculosis/rifampicin) with acid-fast bacilli smear grading results by fluorescence microscopy by Auramine O phenol staining technique
GeneXpert ultra (MTB/RIF) AFB smear grading results
MTB detection by
GeneXpert ultra (MTB/RIF)* Total positives,
n (%) Negative,
n (%) Positive
3+, n (%) 2+, n (%) 1+, n (%) Scanty, n (%)
High 14 (16.6) ‑ 13 (92.8) 1 (7.1) ‑ ‑
Medium 18 (21.4) ‑ 3 (16.6) 9 (50) 5 (27.7) 1 (5.5)
Low 26 (30.9) ‑ ‑ 1 (3.8) 18 (69.2) 7 (26.9)
Very low 12 (14.2) 1 (9) ‑ ‑ ‑ 11 (91.6)
Trace 14 (16.6) 14 (100) ‑ ‑ ‑ ‑
*Mycobacterial load defined by GeneXpert ultra (MTB/RIF) software: Ct value <16 as high; Ct value 16–22 as medium; Ct value 22–28 as low; Ct value >28 as very low; and trace. AFB: Acid‑fast bacilli, MTB: Mycobacterium tuberculosis, RIF: Rifampicin, Ct: Cyclic threshold
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necessary and the diagnosis was done according to the criteria. Similar results were seen in a study conducted by Otchere ID et al., 43 (2.5%) isolates were identified as NTMs after an unsuccessful PCR amplification of IS6110, but positive hsp65 gene amplification. In our study, NTMs were identified after unsuccessful PCR amplification by GeneXpert ultra (MTB/RIF) and a negative TB Ag MPT64. In this study, there was a significant association between NTMsinfected patients and clinical mycobacteria from presumptive TB cases.[3,4,6]
Although detection by AFB microscopy is useful in identifying the AFB, it cannot distinguish between MTB and NTM.
M. abscessus is a species of a fast‑growing NTM, responsible for causing pulmonary infections infamously in patients with underlying lung disorders and prior TB.[13] Therefore, for the correct diagnosis of NTM infections, guidelines given by the American Thoracic Society/Infectious Diseases Society of America must be followed. It is, therefore, important to declare pulmonary NTM infections by following the diagnostic criteria given by the American Thoracic Society and the Infectious Diseases Society of America.
Conclusion
M. abscessus complex comprises a group of rapidly growing, multidrug‑resistant, nontuberculous mycobacteria that are responsible for a wide spectrum of infections. Therefore, it is difficult to interpret results based on microscopy. Therefore, it is important to identify and differentiate MTB from NTM by further testing. GeneXpert ultra (MTB/RIF) is a valuable screening test in patients who are suspicious of having PTB and helps to differentiate MTB complex from NTM infections. However, when a clinical sample is detected MTB negative by GeneXpert ultra (MTB/RIF) but positive by smear microscopy, liquid culture has to be requested to rule out NTM infections.
Outcome of the study
GeneXpert ultra (MTB/RIF) is a valuable screening test in patients who are suspicious of having PTB and helps to differentiate MTB complex from NTM infections.
Rational of the study
To differentiate pulmonary MTB from NTMs by various technique and to calculate the diagnostic efficacy of each test.
Limitations of the study
The limitations of the study were as follows: the sample size was less, follow‑up with the patients in case of trace MTB detection was not done, and repeat sample was
not asked in case of trace MTB detection. Treatment strategies for NTM infections were not done in the study.
Follow‑up with patients with pulmonary NTM infections was not done.
Financial support and sponsorship Nil.
Conflicts of interest
There are no conflicts of interest.
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