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 Table of Contents  
ORIGINAL ARTICLE
Year : 2021  |  Volume : 7  |  Issue : 1  |  Page : 43-47

Rapid detection of Mycobacterium tuberculosis in pus samples of suspected cases of extrapulmonary tuberculosis by GeneXpert MTB/RIF assay and its comparison with conventional methods


1 Department of Microbiology, IGIMS, Patna, Bihar, India
2 Department of General Surgery, IGIMS, Patna, Bihar, India

Date of Submission11-Jan-2021
Date of Decision20-Jan-2021
Date of Acceptance10-Mar-2021
Date of Web Publication28-Jun-2021

Correspondence Address:
Sweta Muni
Department of Microbiology, Indira Gandhi Institute of Medical Sciences, Patna - 800 014, Bihar
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jigims.jigims_11_21

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  Abstract 


Introduction: GeneXpert MTB/RIF or cartridge-based nucleic acid amplification test is a rapid diagnostic tool used for the detection of Mycobacterium tuberculosis and its resistance to rifampicin. It can be offered as a first-line diagnostic modality in suspected cases of extrapulmonary tuberculosis (TB). This study was done to evaluate the diagnostic accuracy of the Xpert MTB/RIF assay for the detection of M. tuberculosis in extrapulmonary specimens and its resistance to rifampicin.
Materials and Methods: This study was done in the Department of Microbiology at Indira Gandhi Institute of Medical Sciences, Patna, Bihar, over a period 18 months from January 2018 to June 2019 in 508 patients. The analysis and interpretation of the data were performed using Microsoft excel. The quantitative data obtained were expressed as percentage in tabular form.
Results: Out of a total of 508 pus samples in suspected extrapulmonary TB patients, the percentage of males was more than females with male-to-female ratio being 1.6:1. Among the testing of Mycobacterium tuberculosis by various methods, maximum detection was done by Xpert MTB/RIF assay in 37% of cases. The least number of cases were detected by Ziehl–Neelsen staining (8.1%). Rifampicin resistance was detected in 14.9% of cases (28/188) while its resistance was not detected in 85.1% of cases (160/188) among cases of M. tuberculosis detected. Many of the negative samples on fluorescent and Ziehl–Neelsen staining came to be positive with GeneXpert testing assuring the sensitivity and specificity of Xpert MTB/RIF assay.
Conclusion: In today's era when we are going for TB elimination, not only rapid TB case detection but also the early determination of multidrug resistance status is of prime value. This will not only assist in the identification of the patient with disease and drug resistance but also initiate prompt early intervention and treatment and moving a step closer in achieving the goal of TB elimination.

Keywords: Extrapulmonary, GeneXpert MTB/RIF, tuberculosis, ZN smear


How to cite this article:
Kumar R, Muni S, Pankaj D, Kumar R, Saurabh K, Kumar S, Kumari N, Shahi SK. Rapid detection of Mycobacterium tuberculosis in pus samples of suspected cases of extrapulmonary tuberculosis by GeneXpert MTB/RIF assay and its comparison with conventional methods. J Indira Gandhi Inst Med Sci 2021;7:43-7

How to cite this URL:
Kumar R, Muni S, Pankaj D, Kumar R, Saurabh K, Kumar S, Kumari N, Shahi SK. Rapid detection of Mycobacterium tuberculosis in pus samples of suspected cases of extrapulmonary tuberculosis by GeneXpert MTB/RIF assay and its comparison with conventional methods. J Indira Gandhi Inst Med Sci [serial online] 2021 [cited 2021 Sep 25];7:43-7. Available from: http://www.jigims.co.in/text.asp?2021/7/1/43/318918




  Introduction Top


The significance of Mycobacterium tuberculosis cannot be ruled out as an important infectious agent in causing death. The global incidence of tuberculosis (TB) is on the rise and India remains the highest-burden country accounting for one-fifth of global incidence. According to the Global TB Report 2018, India has the highest TB and multi-drug resistant TB burden in the world.[1] The estimated incidence rate of TB in India is 2.84 million and the cases of multidrug-resistant TB is increasing with around 1.3 lakhs cases emerging annually as per the World Health Organization (WHO).[2] Therefore, there is a need for more and more sensitive and rapid diagnostic techniques for TB identification and curb this disease which will go a long way in achieving its elimination. One such test for coeval quick TB diagnosis and rapid antibiotic sensitivity test is the Xpert MTB/RIF that can identify M. tuberculosis DNA and resistance to rifampicin. It is also known as the cartridge-based nucleic acid amplification test (NAAT). The WHO endorsed the use of this Xpert MTB/RIF test in TB endemic countries in 2010.[3] Many a times, traditional test for TB overlooks the disease and the role of GeneXpert MTB test becomes important in enabling diagnosis.[4],[5] Extrapulmonary TB also remains a major cause of concern as their steady rise may jeopardize TB elimination and there is a need for their identification, diagnosis, notification, and treatment of these patients to achieve this goal. The TB bacteria causing extrapulmonary TB is mostly a drug-resistant bacterium and these forms are mainly responsible for the extrapulmonary cases. Extrapulmonary TB is infections of part of the body other than the lungs which most commonly affects lymph nodes of the body. The difficulty lies in the fact that their detection becomes demanding due to low bacterial count and also there has to be proper collection of specimen by aspiration, excision, incision and drainage or other invasive/surgical means. An exact diagnosis of the mycobacterium TB depends on the identification of these bacteria in extrapulmonary samples.[6] The present study was done to identify the presence of Mycobacterial TB in pus samples obtained from various parts of the body by GeneXpert MTB assay. This test detects DNA sequences specific for M. tuberculosis and finding out multi-drug resistant TB by showing rifampicin resistance by polymerase chain reaction (PCR). This will not only identify cases of TB and rifampicin resistance but also instituting proper treatment by the treating doctor and ensues a step further in eliminating the disease. The objective of the present study was to detect the prevalence of TB and drug resistance in pus samples of extrapulmonary TB. Furthermore, this study was aimed to assess the efficacy of Xpert MTB/RIF assay for the diagnosis of TB as compared with conventional techniques.


  Materials and Methods Top


This study was done in the Department of Microbiology at Indira Gandhi Institute of Medical Sciences, Patna, Bihar, from January 2018 to June 2019. It was a single-centric, clinical microbiology hospital-based analytical study in 508 samples of pus collected over a period of 18 months. Patients having clinical features of extrapulmonary TB with evidence of pus confirmed from clinical examination and imaging techniques were included in the study. Patients having features suggestive of pyogenic pus, evidence of HIV infection and patients on antitubercular therapy were excluded from this study. Complete medical and surgical history including past history, family history, immunization history, and history of contact of TB was taken. Clinical specimen of pus was collected from microbiology and outpatients of various departments from various sites of the body (breast abscess, cold abscess, areas of lymph node and psoas abscess, etc.). All samples were processed for fluorescent smear microscopy and Ziehl–Neelsen[7] smear microscopy and also sent for GeneXpert assay with drug resistance testing. Routine investigations such as Hb%, TLC, DLC, tuberculin test, and HIV were done along with any special investigations if required. Chest X-ray and computed tomography scan where indicated was also done. For microscopic examination, smears were decontaminated and concentrated, then examined according to the Ziehl–Neelsen and Fluorochrome (auramine O) staining techniques as per the WHO recommendation. The 2 cm length of the smear or 300 fields with light microscope using ×1000, were scanned. The entire length was also used while using ×200 in case of an auramine slide with a fluorescent microscope. A negative smear was reported where no microorganisms were seen. The standard operating procedure for doing Xpert MTB/RIF test at our institute was followed. Around 2–5 ml of the pus sample was taken into a falcon tube and double volume of Xpert MTB/RIF sample reagent was added. This was vigorously shaken 10–20 times or vortexed for at least 10 s. Now, this was incubated for 10 min at room temperature, and again shaken vigorously 10–20 times or vortexed for at least 10 s. The sample was then incubated at room temperature for an additional 5 min. Now, using a fresh transfer pipette, 2 ml of the processed sample was transferred to the Xpert MTB/RIF cartridge. The cartridge was loaded into the GeneXpert instrument as per the manufacturer's instructions. The result was interpreted as following (a) MTB + VE and rifampicin resistance was not detected.(b) MTB + VE and Rifampicin resistance was detected.(c) MTB − VE (d) MTB + VE and rifampicin indeterminate [Figure 1]. The analysis and interpretation of the data were performed using Microsoft excel. The quantitative data obtained were expressed as percentage in tabular form.
Figure 1: Data with graphical interpretation of different test results (Real-time polymerase chain reaction array) on GeneXpert MTB/RIF assay (a) MTB-positive result with Rifampicin resistance not detected (b) MTB-positive result with rifampicin resistance detected (c) MTB-negative result

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  Results Top


A total number of 508 pus samples were included in the study. Among those 62% (n = 315) were male and 38% (n = 193) were female with male-to-female ratio being 1.6:1 [Table 1]. Among the tests for MTB by various methods, maximum detection was done by Xpert MTB/RIF assay in 37% of cases followed by fluorescence microscopy in 10.4% of cases. The least number of cases were detected by ZN staining (8.1%) [Table 2].
Table 1: Sex distribution in the present study

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Table 2: Positivity of clinical specimen of pus by three methods

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Among the 37% (188/508) cases of MTB detected by GeneXpert assay, rifampicin resistance was not detected in 85.1% of cases (160/188) while resistance was detected in 14.9% of cases (28/188) [Table 3].
Table 3: Results of drug resistance to rifampicin

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Among the results obtained, we could see that none of the fluorescent microscopy positive results, as well as ZN smear positive, gave negative results by GeneXpert. Whereas many of the negative samples on fluorescent and ZN staining came to be positive with GeneXpert testing. Among 455 negative results for MTB on fluorescence staining, 135 results came out to be positive on GeneXpert while among 467 negative results for MTB on ZN staining, 147 results came out to be positive on GeneXpert for MTB [Table 4] and [Table 5]. The result of the study revealed a higher positivity rate by the GeneXpert technique when compared to conventional methods.
Table 4: Comparison of GeneXpert Mycobacterium tuberculosis/rifampicin assay with fluorescence microscopy

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Table 5: Comparison of GeneXpert Mycobacterium tuberculosis/rifampicin assay with Ziehl–Neelson staining

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  Discussion Top


The Xpert MTB/RIF has been a revolution in TB control since its inception by contributing to the rapid diagnosis of TB disease and resistance to its drug. The test not only simultaneously detects M. tuberculosis complex but also resistance to rifampin in <2 h. As far as standard cultures are concerned, they take from 2 to 6 weeks for M. tuberculosis complex to grow and conventional drug resistance tests takes more than 3 weeks.[8] DNA sequences specific for M. tuberculosis is identified and rifampicin resistance is detected by PCR forming the basis of this test.[9] It is based on the Cepheid GeneXpert system, a rapid, simple-to-use NAAT. The test is a highly specific one as it uses 3 specific primers and 5 unique molecular probes to target rpoB gene of MTB, no cross-reaction have been observed with other bacterial species tested, thereby excluding nontubercular mycobacterium.[10] Studies on GeneXpert all over the globe prove the impact this technique has created worldwide and the importance of TB control. The range of MTB detected in suspected cases of extrapulmonary TB can vary between 22%–60% as per the study done by various researchers [Table 6].[11],[12],[13],[14],[15] The present study has 37% cases detected by GeneXpert technique which is quite similar to figures obtained by other Indian studies and express the increasing load of TB in our country.[12],[13],[14]
Table 6: Comparison of GeneXpert Mycobacterium tuberculosis/rifampicin assay done by various researchers

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The detection of rifampicin resistance of this study is also comparable to other findings.[12],[13] Fluorescent staining technique is little more sensitive in our study than Ziehl–Neelson staining, but the diagnostic yield of both these staining techniques in terms of M. tuberculosis detection is far less when compared to the CB-NAAT testing. The sensitivity of microscopy is compromised to a larger extent when bacterial load is <10,000 organisms per ml in cases where sputum is processed as sample and even more compromised in cases of extrapulmonary TB. Hence, the policy for samples collection in terms of time and days of reporting has to be more revised from time to time and has to be stringent.[16] Increasing drug resistance possess a great hindrance in TB treatment, control and prevention and the newer techniques of M. tuberculosis testing should be incorporated in health-care setting where facilities are suboptimal.[17] Promising results are being continually obtained since the start of this real-time PCR amplification test from past to present studies among patients with suspected extrapulmonary TB.[18] The findings of the present study corroborate that the Xpert MTB/RIF assay is an important advancement in the diagnosing extrapulmonary TB and favors the guidelines issued by WHO.[19] The rapidity with which results are obtained is an added advantage so its integration into a first-line diagnostic protocol as a routine must be considered. The high volume of TB cases and lack of simple, accurate, and time saving diagnostic test is a major hindrance in TB control at many centers in developing countries where the mainstay for diagnosis still remains sputum smear microscopy and chest X-ray.


  Conclusion Top


The GeneXpert MTB test is a magnificent tool for rapid detection of extra pulmonary TB. Not only the presence of TB infection but also GeneXpert machine detects drug resistance to rifampicin. In a developing country like India, drug-resistant TB is fast growing and TB control programs along with health education are needed to reduce the disease burden. In places where health-care facilities are limited, the CB-NAAT machine and its implementation at those centers will be an important step toward achieving TB eradication by 2025, a dream project of Government of India.

Limitations

In spite of the fact that MTB/RIF test is an important tool for rapid identification of RIF-resistant M. tuberculosis, especially in smear-positive clinical samples, we should always confirm the test results by culture and drug sensitivity testing.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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