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ORIGINAL ARTICLE
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Role of cartridge-based nucleic acid amplification test in diagnosing extrapulmonary tuberculosis


 Department of Microbiology, Dr. D. Y. Patil Medical College, Hospital and Research Centre, Dr. D. Y. Patil Vidyapeeth, Pune, Maharashtra, India

Date of Submission30-Nov-2020
Date of Decision20-Dec-2021
Date of Acceptance22-Dec-2021

Correspondence Address:
Shahzad Mirza,
Department of Microbiology, Dr. D. Y. Patil Medical College, Hospital and Research Centre, Dr. D. Y. Patil Vidyapeeth, Pune - 411 018, Maharashtra
India
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/mjdrdypu.mjdrdypu_678_20

  Abstract 


Background: Tuberculosis (TB) is a major health concern globally. Extrapulmonary tuberculosis (EPTB) accounts for around 20% of the total (EPTB) cases which are difficult to diagnose due to the lack of availability of diagnostic tools. Although pulmonary TB is most common presentation, EPTB is also an important problem clinically. Cartridge-based nucleic acid amplification (CBNAAT) test has a well-documented role in diagnosing pulmonary tuberculosis. The potency of CBNAAT in the diagnosis of EPTB cases is still debated. Aim: To establish the importance and role of diagnosing EPTB by CBNAAT. Settings: Conducted in Dr. D Y Patil Medical College Hospital and Research Centre, Dr. D Y Patil Vidyapeeth, Pimpri, Pune-411018. Design: Retrospective cross-sectional study of suspected EPTB patients in a tertiary care center area from April 2020 to September 2020. Materials and Methods: Data of suspected EPTB patients were retrieved. Potency of CBNAAT in diagnosing EPTB was assessed. Statistical Analysis: All the statistical analyses were carried out using the Statistical Package for the Social Sciences (SPSS) version 20. Results: Samples collected from 556 suspected EPTB patients were subjected to CBNAAT. The collected samples included pus, pleural fluid, cerebrospinal fluid, gastric aspirate, peritoneal fluid, tissue biopsy, pericardial fluid, ascitic fluid, synovial fluid, drain fluid, para-spinal fluid. In CBNAAT results, 67 cases were positive for TB bacilli, rifampicin resistance detected in 4 samples. Conclusion: Xpert®MTB/RIF assay is a rapid and effective method for early detection of EPTB and can be used in conjunction with routine culture methods for detection of the same.

Keywords: Cartridge-based nucleic acid amplification test, extrapulmonary tuberculosis, genexpert, rifampacin



How to cite this URL:
Kannuri S, Mirza S, Misra RN, Vyawahare R C, Das NK, Gandham NR, Khan S, Bhaumik S, Mukhida SS. Role of cartridge-based nucleic acid amplification test in diagnosing extrapulmonary tuberculosis. Med J DY Patil Vidyapeeth [Epub ahead of print] [cited 2022 Dec 6]. Available from: https://www.mjdrdypv.org/preprintarticle.asp?id=339947




  Introduction Top


Tuberculosis (TB) even now is considered a major health concern for the world with a third of the total population being infected with Mycobacterium tuberculosis.

According to the World Health Organization (WHO), India tops the list for the highest TB burden countries in the world with 26% of the world's cases.[1] In 2019, a record number of ~24lakh cases were reported in India which was an increase of ~12% compared to the previous year, this translates to an incidence rate of 159 cases/lakh population. Among the reported cases, ~28% were reported from private sector alone. Of all the states in the country, these five states, namely Uttar Pradesh (20%), Maharashtra (9%), Madhya Pradesh (8%), Rajasthan (7%), Bihar (7%), contribute majorly to the number of cases.[2],[3]

Pulmonary TB is the most common presentation but extrapulmonary tuberculosis (EPTB) is also as important from a clinical perspective. Statistically EPTB accounts for ~16%[1] of the total reported cases. India being among the top five countries with high TB burden, to curb this situation, the government has implemented the National Tuberculosis Elimination Program; formerly Revised National TB Control Program.[2] TB primarily is a disease affecting the lung parenchyma but it is also known to cause extrapulmonary complications, like TB meningitis, TB osteomyelitis, etc. The many diagnostic techniques available at the clinicians' disposal are more attuned for pulmonary origin of TB, for detecting Extra-pulmonary TB(EPTB) is still a bit of a challenge either because of the paucibacillary nature of the sample or lack of diagnostic tools. Among the many diagnostic modalities, CBNAAT is one of the faster techniques available to a treating clinician or a microbiologist for reporting of TB. An added advantage of this testing technique apart from being fast is the high specificity and sensitivity ranging from 93.4-97.2% and 98.9-99.8% respectively. CBNAAT was approved for direct diagnosis of TB by WHO and the indian government, also the important role of this diagnostic method was discussed in many studies done prior. This study aims to emphasize the role of Cartridge based Nucleic Acid Amplification Test(CBNAAT) in diagnosing extra pulmonary tuberculosis.

Materials and Methods

This study is a retrospective cross-sectional study conducted at a tertiary care center. The study used data from April 2020 to September 2020.

All patients suspected to have EPTB and whose samples were sent to Microbiology department for processing were included in the study.

All the samples suspected of EPTB received at the Microbiology department were processed using Xpert®MTB/RIF (Cepheid) assay. This biomedical equipment is a closed system consisting of disposable closed cartridges hence preventing cross contamination. This system works on the principle of nucleic acid amplification to detect the presence of M. tuberculosis specific genes and also it detects resistance towards rifampicin by checking for mutations in rpoB gene. Using this device is simple and does not warrant any special training, and the turn-around time is ~2 h making it a rapid diagnostic test compared to other conventional diagnostic methods.

Received samples were added with master mix in the ratio of 1:2(specimen:reagent) and vortex for 10min then let it rest, repeat the vortex and allow it rest for another 10min, later add 2mL of the supernatant into the cartridge, load into the equipment.

The processed samples included pleural fluid, gastric aspirate, lymph node aspirate, pericardial fluid, synovial fluid, cerebrospinal fluid (CSF), peritoneal fluid, ascitic fluid, and other miscellaneous samples. Study has been submitted for ethical clearance from University Ethics Committee of Dr. D. Y. Patil Vidyapeeth, Pune.

Statistical analysis

All the statistical analyses were carried out using the Statistical Package for the Social Sciences (SPSS) version 20 (IBM, USA, New York).

Results

A total number of 556 (n) extrapulmonary samples were processed using Xpert®MTB/RIF assay (Cepheid).

Out of the 556, 328 (58.99%) samples were from male patients and 228 (41.01%) samples were from female patients [Figure 1]. Out of the 556 received samples, 67 were positive; positivity rate of 12% [Figure 2] and 6 were invalid. Among the positive samples, 35 (52.23%) samples received from female patients and 32 (47.76%) samples received from male patients [Figure 3]. Four samples (5.97%) were detected with rifampicin resistance, and apart from rifampicin resistance, another 4 patient samples (5.97%) that were indeterminate for rifampicin resistance [Figure 4].
Figure 1: Gender distribution

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Figure 2: Results of all samples run

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Figure 3: Gender distribution in positive tests

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Figure 4: Rifampicin resistance distribution

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Most common sample received for processing was pleural fluid accounting for 25.89% (144 samples) of the total samples followed by CSF accounting for 16.54% (92 samples), ascitic fluid accounting to 10.97% (61 samples), and lastly, gastric aspirate amounting for 7.55% (41 samples) with other miscellaneous samples making up the other 39.05% (217 samples) [Figure 5].
Figure 5: Distribution of extra pulmonary samples

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Out of 67 MTB detected samples, 17 samples obtained from lymph node (25.37%), followed by 12 pleural fluid (17.91%), 8 CSF (11.94%), and 3 gastric aspirate specimens (4.47%) and 1 ascitic fluid (1.49%) were included [Figure 6].
Figure 6: Distribution of samples in CBNAAT positive tests

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We noted that majority of the samples we received were belonging to 21–30 age group, 108 (19.42%) out of 556 samples received for testing. One third (22 samples) part of positivity was shared by these young age group patients. 68 samples (12.23%) received from paediatric age group for MTB detection testing by CBNAAT where 5 (7.35%) paediatric samples tested positive for MTB out of those 5 samples 3 samples were gastric aspirate


  Discussion Top


EPTB accounts around 15%–20% of all TB cases in our country. Although it is a known fact that there are more cases of TB among males than in females but the statistics for EPTB states otherwise, it is noted that there are more cases of EPTB among females than males which is shown in my study and it corroborates with other studies.[4],[5],[6]

In current study, we processed 58.99% male patient samples and 41.01% female patient samples for EPTB for detection by CBNAAT method, additionally, in this study, 52.23% specimens from female patients and 47.76% from males tested positive for EPTB contrary to conventional notion that TB is more prevalent in males.[4],[5],[6]

In our study, we noted that majority of the positive cases (n = 67) were rifampicin sensitive (88.05%) with four (5.97%) being rifampicin resistant and four (5.97%) being rifampicin indeterminate. This trend in rifampicin resistance has also been noted in another study by Reddy R et al.,[7] and also, the number of samples with rifampicin resistance and rifampicin indeterminate were higher in comparison to the reference study,[7] this difference in number can be attributed to lower sample size and shorter study duration.

In the current study, we observed that among the MTB detected specimen, majority of the samples were that of lymph node (25.37%) and pleural fluid (17.91%) which were predominant followed by CSF (11.94%), gastric aspirate (4.47%), and ascitic fluid (1.49%) were seen among the extrapulmonary samples like some studies Prakasha SR et al., Alwani H et al., Shrivastava AK et al., Rai DK.[8],[9],[10],[11] Differences in results for CSF, gastric aspirate, and ascitic fluid with that of other studies[8],[9],[10],[11] can be due the low volume of the respective samples received for processing.

In the current study, it was noted that majority of the samples processed and positivity rate (32.84%) was from 21 to 30 age group as mentioned in other study.[11] Furthermore, among the 68 samples that were received and processed 3 gastric aspirate samples were positive for MTB, this finding in our study was a bit higher compared to other study like Aygun et al.,[12] where lymph node samples were in higher number this variation may be due patient unwilling for invasive procedure or the doctor unwilling to subject the patient for the same.

Advantages of CBNAAT are many to mention such as economical, time saving, i.e., it has a very short turn-around time of about 2 h like mentioned in some studies like Yadhav et al., Kasat et al.,[13],[14] and also it detects for rifampicin resistance and according to Global TB Report-2020-78% of rifampicin-resistant TB are multidrug resistant[1] and also the learning curve to learn toperform this diagnostic test is shorter. Disadvantages of this equipment: can only detect mono resistance, needs electricity to run so in very rural places with lack of power this diagnostic is not of much use.

Many limitations can immediately be noted in the study starting with not having a gold standard test to determine the positivity rate of the assay and also the study period being only 6 months limits the association factors among the various elements in the study.


  Conclusion Top


Xpert®MTB/RIF assay is a very useful, rapid and economical tool for diagnosing EPTB, especially in a resource-limited setup which provides the clinicians a certain level of surety when diagnosing EPTB.

Limitations

study samples were not correlated with biopsy, culture or clinic-radiological reports.

Acknowledgment

The authors would like to thank Department of Microbiology, Dr. D. Y. Patil Medical College, Hospital and Research Centre, Dr. D. Y. Patil Vidyapeeth, Pune - 411 018, Maharashtra, India.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
Global Tuberculosis Report 2020. Geneva: World Health Organization; 2020. Licence: CC BY-NC-SA 3.0 IGO. Available at https://www.who.int/publications-detail-redirect/9789240013131. [Last accessed on 2022 Mar 5].  Back to cited text no. 1
    
2.
India TB Report; 2020. Available at https://tbcindia.gov.in/showfile.php?lid=3538. [Last accessed on 2022 Mar 5].  Back to cited text no. 2
    
3.
Maurya AK, Kant S, Nag VL, Kushwaha RA, Dhole TN. Trends of anti-tuberculosis drug resistance pattern in new cases and previously treated cases of extrapulmonary tuberculosis cases in referral hospitals in northern India. J Postgrad Med 2012;58:185-9.  Back to cited text no. 3
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4.
Pitre A. A Rapid Gender Assessment of Tuberculosis in India; 2018. Available at http://stoptb.org/assets/documents/communities/CRG/TB%20Gender%20Assessment%20India.pdf. [Last accessed on 2022 Mar 5].  Back to cited text no. 4
    
5.
Sarpal SS, Goel NK, Kumar D, Janmeja AK. Gender disparities in retreatment patients of tuberculosis: A north Indian study. J Nat Sci Biol Med 2015;6:63-6.  Back to cited text no. 5
    
6.
Mehraj J, Khan Z, Saeed D, Shakoor S, Hasan R. Extrapulmonary tuberculosis among females in South Asia – Gap analysis. Int J Mycobacteriol 2016;5:392-9.  Back to cited text no. 6
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7.
Reddy R, Alvarez-Uria G. Molecular epidemiology of rifampicin resistance in Mycobacterium tuberculosis using the GeneXpert MTB/RIF assay from a rural setting in India. J Pathog 2017;2017:6738095.  Back to cited text no. 7
    
8.
Prakasha SR, Suresh G, D'sa IP, Shetty SS, Kumar SG. Mapping the pattern and trends of extrapulmonary tuberculosis. J Glob Infect Dis 2013;5:54-9.  Back to cited text no. 8
    
9.
Alwani H, Subhankar S, Rao CM, Dash DP. Role of CBNAAT in extrapulmonary tuberculosis – An ongoing pilot study. Eur Respir J 2019;54 Suppl 63:PA3007.  Back to cited text no. 9
    
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Shrivastava AK, Brahmachari S, Pathak P, Ratan K, Sainia T, Patel U, et al. Clinico-epidemiological profile of extra-pulmonary tuberculosis in central India. Int J Med Res Rev 2015;3:223-30.  Back to cited text no. 10
    
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Rai DK, Pandey S. A hospital-based cross-sectional study on clinico-demographic characteristic of extrapulmonary tuberculosis cases coming to a tertiary hospital of Bihar. Indian J Community Med 2018;43:122-3.  Back to cited text no. 11
[PUBMED]  [Full text]  
12.
Aygun D, Akcakaya N, Cokugras H, Camcıoglu Y. Evaluation of clinical and laboratory characteristics of children with pulmonary and extrapulmonary tuberculosis. Medicina (Kaunas) 2019;55:428.  Back to cited text no. 12
    
13.
Yadhav K, Veena M. Role of GeneXpert in rapid molecular detection of extrapulmonary tuberculosis in tertiary care hospital. Int J Med Res Rev 2018;6:271-6.  Back to cited text no. 13
    
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Kasat S, Biradar M, Deshmukh A, Jadhav S, Deshmukh H. Effectiveness of CBNAAT in the diagnosis of extrapulmonary tuberculosis. Int J Res Med Sci 2018;6:3925-8.  Back to cited text no. 14
    


    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]



 

 
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