|Year : 2022 | Volume
| Issue : 5 | Page : 17-21
Retrospective observational study to compare Xpert MTB/RIF assay with other diagnostic tests in lymph node tuberculosis
Nithya Haridas1, Asmita Mehta2, Akhilesh Kunoor2, Sadia Khan3
1 Associate Professor, Amrita Institute of Medical Sciences, Amrita Vishwa Vidya Peetham, Kochi, Kerala, India
2 Professor, Department of Respiratory Medicine, Amrita Institute of Medical Sciences, Amrita Vishwa Vidya Peetham, Kochi, Kerala, India
3 Associate Professor, Department of Microbiology, Amrita Institute of Medical Sciences, Amrita Vishwa Vidya Peetham, Kochi, Kerala, India
|Date of Submission||11-Aug-2022|
|Date of Decision||17-Aug-2022|
|Date of Acceptance||19-Aug-2022|
|Date of Web Publication||11-Nov-2022|
Guddy's, Chittayil House, Vaduthala, Kochi, Kerala,
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Background: Lymph node tuberculosis (TB) is the most common form of extrapulmonary TB in India. Standards for TB care in India recommend microscopy/culture/CBNAAT/molecular test/histopathology examination and drug sensitivity testing on appropriate specimens from the presumed sites of involvement for all patients with presumptive extrapulmonary TB. Objectives: To analyze the utility of Xpert MTB/Rif assay in lymph node TB. Methods: All patients who underwent lymph node sampling between July 2014 and June 2017 and for whom Xpert MTB/Rif assay was done were included. Demographic profile, Xpert MTB/Rif assay result, histopathology/cytology findings, smear acid-fast bacillus (AFB), and AFB culture results were noted. A composite reference score (CRS) was made. Results: Xpert MTB/Rif assay was positive in 63 of the 81 patients. Xpert had a sensitivity of 82.14% and specificity of 86.18%when compared against AFB culture and 75.61% and 98.97% when compared against CRS. Conclusion: Xpert MTB/Rif assay is a valuable test for rapid diagnosis of lymph node TB.
Keywords: Lymph node, polymerase chain reaction, tuberculosis
|How to cite this article:|
Haridas N, Mehta A, Kunoor A, Khan S. Retrospective observational study to compare Xpert MTB/RIF assay with other diagnostic tests in lymph node tuberculosis. Indian J Public Health 2022;66, Suppl S1:17-21
|How to cite this URL:|
Haridas N, Mehta A, Kunoor A, Khan S. Retrospective observational study to compare Xpert MTB/RIF assay with other diagnostic tests in lymph node tuberculosis. Indian J Public Health [serial online] 2022 [cited 2022 Dec 1];66, Suppl S1:17-21. Available from: https://www.ijph.in/text.asp?2022/66/5/17/360648
| Introduction|| |
Tuberculosis (TB) is the leading killer of infectious disease, ranking ninth among the overall causes of death worldwide. The global incidence of TB was 9.9 million in 2020. Globally, 14% of the incident cases represents extrapulmonary TB. According to the study by Gaur et al., 45.6% of the patients diagnosed with TB had extrapulmonary TB. The extrapulmonary TB burden ranges from 15% to 20% of all TB cases seen in HIV-negative patients, while in HIV-positive patients, it accounts for 40%–50% of the new TB cases.
The WHO TB statistics for India for 2020 provides an estimated incidence figure of 2.59 million, of which 29% are extrapulmonary.,
Lymph node TB is the most common form of extrapulmonary TB in India. A definitive diagnosis of tuberculous lymphadenitis requires demonstration of the organism Mycobacterium tuberculosis in the biopsy specimen or growth of the organism in a culture of the specimen. Most cases of tuberculous lymphadenopathy are paucibacillary; hence, acid-fast bacilli are not detected in most lymph node specimens. Culture for M. tuberculosis takes considerable time, with liquid cultures (the fastest) taking an average of 10–14 days to turn positive. Treatment of lymph node TB is frequently started based on histopathological demonstration of granuloma in excision biopsy specimens without definitive microbiological evidence of TB.
Molecular diagnostic tests like polymerase chain reaction (PCR), which detects the mycobacterial DNA, help in an early microbiological confirmation before instituting the treatment. Xpert MTB/Rif assay or Xpert MTB/Rif assay TB is a PCR-based rapid diagnostic test with high sensitivity and specificity in smear-positive TB cases. Standards for TB care in India recommend microscopy/culture/CBNAAT/molecular test/histopathology examination and drug sensitivity testing on appropriate specimens from the presumed sites of involvement for all patients with presumptive extrapulmonary TB.
Lymph node TB has been traditionally diagnosed by histopathological examination and other corroborative evidence such as positive Mantoux test and elevated ESR. It is time to ponder whether Xpert MTB/Rif assay can serve as a replacement for the conventional tests in a high TB incident country like India. There is a looming danger of initial drug resistance in pulmonary and extrapulmonary TB. Early identification of drug resistance will help in the early institution of appropriate treatment.
This study aims to assess the role of the Xpert MTB/Rif assay in the diagnosis of lymph node TB and determine the sensitivity, specificity, positive predictive value, and negative predictive value of the Xpert MTB/Rif assay in lymph node TB which was the primary objective.
A secondary objective was to study the role of Xpert MTB/Rif assay in assessing rifampicin resistance in lymph node TB.
| Materials and Methods|| |
This retrospective observational study was conducted at a tertiary care center in South India. The study period was between July 2014 and June 2017. The institutional ethics committee approved the study. Patients with suspected tuberculous lymph node enlargement whose samples were analyzed with Xpert MTB/Rif assay were included in the study.
Xpert MTB/Rif assay
Samples were processed according to the standard protocol recommended by the WHO. The samples were mixed with a double volume of Xpert MTB/Rif sample reagent and vortexed for 10 s, followed by 5 min of incubation at room temperature. A sample volume of 2 ml was transferred to Xpert MTB/Rif cartridge and loaded into the machine. The sample combines with the sample processing control (SPC) in the cartridge. A filter captures the sample and SPC. Ultrasonically, lysed cells release the DNA from bacterial cells if present. Eluted DNA combined with dried-down bead reagents in the cartridge. PCR and detection occur in real-time, and results are ready to be viewed and printed in 1 h and 52 min on average. Medical records of the included patients were traced using the medical records number. Demographic profile, reports of Xpert MTB/Rif assay, histopathology report, smear acid-fast bacillus (AFB) report, and AFB culture report of the patient were noted.
Composite reference score
A diagnosis of TB lymphadenitis was established based on a composite reference score (CRS) if any one of the criteria was met:
- Bacteriological confirmation of the presence of M. tuberculosis (direct smear or culture or histological finding)
- Histopathology/cytology finding of caseating granulomas; or epithelioid cell aggregate with or without Langerhans giant cells and necrosis
Plus, clinician diagnosis of tuberculous lymphadenitis with clinical response to anti-TB therapy.
Minimum number of positive cases was calculated to be 40 based on study by Bankar et al. (15% allowable error and 95% confidence). Xpert MTB/Rif assay results were compared against a CRS and culture results in this study. The demonstration of a granuloma has been conventionally considered surrogate evidence of TB. We analyzed the sensitivity and specificity of this finding by comparing it with the AFB culture results. AFB smear and Xpert MTB/Rif assay were also compared with AFB culture results. Standard equations were used for computing the sensitivity, specificity, positive predictive value, negative predictive value, and accuracy.
| Results|| |
The study included 212 patients with lymphadenopathy whose lymph node samples were subjected to Xpert MTB/Rif assay. AFB smear examination and Xpert MTB/Rif assay were done in all patients. AFB culture was done in 183 patients, and histopathology examination of lymph node specimens was done in 209 patients. The Xpert MTB/Rif assay was invalid in one patient. 179 patients whose samples were subjected to AFB smear, AFB culture, histopathological examination, and Xpert MTB/Rif assay were included in the study. The average age of the study population was 41.47 years. [Table 1] shows the demographic and lymph node characteristics of the study population.
[Figure 1] shows the results of the microbiological investigations. Histopathology suggestive of TB was seen in 60.89% (109/179) of the patients.
Based on the CRS, 81 cases were diagnosed as tuberculous lymphadenitis. Among the 81 tuberculous lymphadenitis cases, 62 showed a positive Xpert MTB/Rif assay report, while 19 patients had a negative Xpert MTB/Rif assay result. Xpert MTB/Rif assay has a sensitivity was 71.83% (59.9%–81.87%) and a specificity of 98.97% (94.39%–99.97%) smear-negative samples when compared to CRS
[Figure 2] shows the diagnostic yield of various tests against the AFB culture as the gold standard.
|Figure 2: Diagnostic value of Xpert MTB/Rif Assay, smear AFB, Histopathology against AFB culture. AFB: Acid-fast bacillus|
Click here to view
[Table 2] shows the diagnostic yield of Xpert MTB/Rif assay against culture and CRS. Xpert MTB/Rif assay has a sensitivity of 75.61%, specificity of 98.97%, a positive predictive value of 98.41%, and a negative predictive value of 82.76% against CRS. It has a high accuracy of 88.27%.
|Table 2: Diagnostic value of Xpert Mycobacterium tuberculosis/rifampicin assay against cultures and composite reference score|
Click here to view
Xpert MTb-Rif assay did not detect any rifampicin-resistant case in this study. One patient had indeterminate rifampicin resistance, which was sensitive to all first-line drugs in drug susceptibility tests. First-line drug sensitivity was done in 24 of the 56 culture-positive specimens. Fourteen patients showed M. tuberculosis sensitive to all first-line drugs. Mono-resistance to rifampicin, ethambutol, and streptomycin was seen in one patient each. Two patients had pyrazinamide resistance. While one case of multidrug resistance was seen (rifampicin, isoniazid, and streptomycin), various combinations of resistance were seen to first-line drugs in four patients - isoniazid and pyrazinamide, rifampicin and ethambutol and isoniazid and streptomycin.
| Discussion|| |
Microbiological confirmation of extrapulmonary TB remains a challenge due to the paucibacillary nature of the disease. Direct smear microscopy has sensitivity ranging from 27% to 50%,, in lymph node TB. Although LJ culture has a high specificity of 100% for the diagnosis of lymph node TB, its sensitivity is around 75%. Histopathology has a high sensitivity of about 96% for the diagnosis of tuberculous lymphadenitis with a compromised specificity. In this context, the WHO recommended rapid diagnostic PCR-based techniques.
Semi-automated radiometric culture BACTEC 460TB system is widely accepted as a reference standard in diagnosis of tuberculosis. However, the yield of culture may be less in lymph node TB due to the paucibacillary nature of the disease. Molecular tests which can detect the DNA even from nonviable bacilli may be positive even in culture-negative cases. CRS has been used in various studies to overcome such issues. Hence, Xpert MTB/Rif assay results were compared against a CRS and culture results in this study.
This study attempted to find whether Xpert MTB/RIF assay has enough diagnostic utility to serve as a replacement for the routine tests in lymph node TB.
Tuberculous lymphadenitis was seen in 46.66% of the study population. Xpert MTB/Rif assay could detect 76.5% of these cases. In the study by Tadesse et al., 64.3% of the patients were diagnosed as having tuberculous lymphadenitis. In the study mentioned above, 60.1% of the patients were diagnosed by Xpert MTB/RIF assay.
Xpert MTB/Rif assay was falsely positive in one patient whose culture, histopathology, and clinical follow-up did not reveal evidence of TB. Xpert MTB/Rif assay was positive in 46 of the 56 cultures growing M. tuberculosis. Ten patients had negative Xpert results despite a positive culture. This false negativity could have been due to sampling errors, inefficient extraction of DNA or the presence of PCR inhibitors in specimens. This could also be due to the absence of the IS6110 in these M. tuberculosis strains, reported in some studies.
Xpert MTB/Rif assay was positive in 13 patients whose culture was negative. These patients were already on anti-TB treatment, and the lymph node biopsy was done because of a delayed response to treatment. Xpert Mtb/Rif assay positivity indicates the presence of M. tuberculosis DNA in the specimen in the absence of viable bacilli.
Mediastinal lymph node was sampled using endobronchial ultrasound in 20 cases, in which 12 cases were diagnosed as tuberculous lymphadenitis. Xpert MTB/Rif assay was positive in 11 of these patients. Often, endoscopic ultrasound-guided lymph node biopsy specimens are too small for a proper histological examination. It should be noted that Xpert MTB/Rif assay could detect TB in these specimens.
Figure 2 shows that Xpert MTB/Rif assay has a sensitivity of 82.14% (95% confidence interval [CI] 69.60%–91.09%) and specificity of 86.18% (95% CI 78.80%–91.74%) when culture is taken as the gold standard. In the meta-analysis by Denkinger et al., the pooled sensitivity of 83.1% (95% CI 71.4%–90.7%) pooled specificity of 93.6% (95% CI 87.9%–96.8%). Our results are comparable with this meta-analysis. Histopathology had a better sensitivity at the cost of a lower specificity when compared to Xpert MTB/Rif assay when culture is taken as the gold standard. This would point to a more significant number of false positives if histopathology were taken as the single test for diagnosis of tuberculous lymphadenitis. In comparison, Xpert MTB/Rif assay provides a more accurate result.
Xpert MTB/RIF assay had 100% sensitivity in smear-positive samples while 71.83% in smear-negative samples. The study by Pandey et al. had shown a sensitivity of 100% and 77%, respectively, in smear-positive and smear-negative samples. Our results are comparable to that study.
58.33% of the patients in whom drug sensitivity was done showed sensitivity to all first-line drugs. Xpert MTB/RIF assay could correctly identify all these specimens as rifampicin sensitive. It failed to detect two cases which later proved to be rifampicin-resistant in first-line culture. Detailed analysis of the sensitivity and specificity of Xpert MTB/Rif assay in detecting rifampicin resistance was not attempted in this study, as drug sensitivity was done for <50% of the culture-positive cases. The survey by Chaudhary et al. reports a sensitivity of 91% and a specificity of 100% for detecting rifampicin resistance by Xpert MTB/RIF assay.
The advantages of the study include a large sample size of nearly 179 patients compared to other studies.,, The retrospective study design allows the pooling of cases from various specialties but may have limited the initial clinical assessment of cases. A prospective analysis would also have helped in better streamlining of samples for culture and drug sensitivity.
| Conclusion|| |
The present study supports Xpert MTB/Rif assay as a good test for the rapid diagnosis of lymph node TB. It helps in the rapid detection of rifampicin resistance as well.
Renjitha Bhaskar, Lecturer, Department of Biostatistics, Amrita Institute of Medical Sciences, Amrita Vishwa Vidya Peetham.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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