Journal of Epidemiology and Global Health

Volume 10, Issue 2, June 2020, Pages 153 - 156

Utility of Xpert MTB/RIF Assay for Diagnosis of Pediatric Tuberculosis Under Programmatic Conditions in India

Authors
Rakesh Yadav1, #, Pankaj Vaidya2, #, Joseph L Mathew2, Sanjay Verma2, Rajiv Khaneja3, Priyanka Agarwal4, Pankaj Kumar5, Meenu Singh2, Sunil Sethi1, *
1Department of Medical Microbiology, Postgraduate Institute of Medical Education and Research, Chandigarh, India
2Pediatric Medicine, Advanced Pediatric Center, Postgraduate Institute of Medical Education and Research, Chandigarh, India
3State TB Cell, Sector 34, Chandigarh, India
4WHO Country Office for India, New Delhi, India
5Department of Paediatrics, Govt Medical College, Chandigarh, India
#

First two authors are joint first author.

*Corresponding author. Email: sunilsethi10@hotmail.com
Corresponding Author
Sunil Sethi
Received 7 June 2019, Accepted 7 December 2019, Available Online 10 January 2020.
DOI
10.2991/jegh.k.191215.002How to use a DOI?
Keywords
Xpert MTB/RIF; pediatric tuberculosis; diagnosis; India
Abstract

Tuberculosis (TB) diagnosis in children still remains a challenge in developing countries. We analyze the performance of Xpert MTB/RIF assay for the diagnosis of pediatric TB under programmatic conditions. We retrospectively analyzed the performance of Xpert MTB/RIF assay from February 2016 to March 2018. A total 2678 samples from TB suspects below 14 years were received in the laboratory and were frontline tested by Xpert MTB/RIF assay according to the manufacturer’s instructions. If sample was sufficient, the smear microscopy and culture were performed as per standard World Health Organization’s guidelines. The smears and cultures were performed in 2178 and 588 samples, respectively. Among 2678 samples, 68 were rejected, Xpert MTB/RIF assay was positive in 357/2610 (13.6%) cases, while the smear was positive in 81/2178 (3.3%) cases. The sensitivity of smear and Xpert MTB/RIF when compared with culture was 24.6% (14.1–37.8%) and 81% (68.6–90.1%), respectively. The diagnostic accuracy of Xpert MTB/RIF and smear was 97.1% and 92.2%, respectively. Thirty samples (8.5%) were detected as rifampicin resistance by Xpert MTB/RIF assay. The Xpert MTB/RIF increased the detection rate up to fourfold when compared with smear microscopy. Xpert MTB/RIF assay is the most rapid, sensitive, and specific method for microbiological confirmation and rifampicin resistance detection in pediatric tuberculosis.

Copyright
© 2020 Atlantis Press International B.V.
Open Access
This is an open access article distributed under the CC BY-NC 4.0 license (http://creativecommons.org/licenses/by-nc/4.0/).

1. INTRODUCTION

Childhood tuberculosis (TB) constitutes a major but underappreciated burden of disease in endemic countries as the national TB programs mainly focus on adult TB [1,2]. According to the global TB report, 1.04 million children were diagnosed with TB and 0.2 million deaths were estimated in 2016 [3]. In India, pediatric TB accounts for 6% of the total TB burden [4]. For better treatment outcome, timely treatment initiation is required with the help of rapid diagnostics. However, especially in children, the diagnosis of pediatric TB has become more complex because of limited World Health Organization (WHO) endorsed tests. Each test has its own limitations, for example, smear microscopy has a low sensitivity due to paucibacillary nature of TB in children and lacks reproducibility [5]. It is unable to differentiate the disease caused by other mycobacterial species. The gold standard for TB diagnosis is Mycobacterium tuberculosis culture, which is laborious and time consuming [6]. The sensitivity of culture for the diagnosis of pediatric TB, as comparison to clinical standard, ranges from 25 to 75% depending upon the specimen’s type, quality, and also the severity of disease [6].

Nucleic acid amplification test (NAAT) for TB diagnosis, especially in adults, has a very high sensitivity and specificity [79]. However, for the diagnosis of pediatric TB, the sensitivity and specificity of different NAATs are lower than in adults, taking culture as a gold standard. For diagnosis of adult TB and rifampicin resistance, Xpert MTB/RIF assay (a hemi-nested real-time polymerase chain reaction) showed very high sensitivity and specificity in smear-positive TB. The assay provides results in 90 min and also offers a promising solution in addressing the challenges in the diagnosis of pediatric pulmonary tuberculosis (PTB). Several studies have been conducted for determination of accuracy of Xpert MTB/RIF assay in pediatric TB. Among smear-positive and smear-negative samples, the sensitivity of Xpert MTB/RIF assay was 95−96% and 55–62%, respectively, using culture as a reference standard [10]. After a meta-analysis, WHO recommended that the Xpert MTB/RIF assay can be used as frontline test rather than conventional microscopy and culture in all children suspected of TB. In India, under Revised National Tuberculosis Control Program (RNTCP), the Xpert MTB RIF assay was also recommended to be used as a first choice for the diagnosis of pediatric TB at different levels. This study was thus conducted to retrospectively analyze the utility of Xpert MTB/RIF assay under routine programmatic conditions in a tertiary care center.

2. MATERIALS AND METHODS

2.1. Study Design

A total of 2678 samples were received from pediatric population under RNTCP from February 2016 to March 2018. All the samples, respiratory and nonrespiratory, were received from presumptive TB patients as defined by RNTCP, India. These samples were received in the RNTCP center, Mycobacteriology laboratory of the Department of Medical Microbiology, Post Graduate Institute of Medical Education & Research (PGIMER), Chandigarh, for routine diagnosis of pediatric TB. The Mycobacteriology laboratory is certified by the International Organization for Standardization 15189:2007 and also a DST-approved center by India’s RNTCP. The data were analyzed retrospectively and patient information including age, sex, type of sample, HIV status, previous history, and contact history were collected from the RNTCP request form received in the laboratory. These samples were first processed for Xpert MTB/RIF assay, and if the sample was sufficient, then the other methods like smear microscopy and culture were performed. As this is a retrospective analysis of laboratory data, approval was taken from Intramural Institutional Ethics Committee, PGIMER, Chandigarh.

2.2. Xpert MTB/RIF Assay

The Xpert MTB/RIF assay was used as frontline test for diagnosis of TB. About 1 ml sample was mixed with double the amount of reagent buffer and incubated and transferred into the cartridge. The cartridge was then placed into the instrument. The Xpert MTB/RIF were interpreted as M. tuberculosis complex detected or not detected and rifampicin resistance detected and not detected [11].

2.3. Routine Microbiological Assays

The direct smear was prepared and reported as per the RNTCP guideline, India. The remaining sample was processed/decontaminated by NALC–NaOH method [12]. A total of 500 µl of the processed sample was transferred in MGIT tube (Becton Dickinson, USA). The MGIT tube was incubated at 37°C for 42 days in the MGIT 960 instrument as per the manufacturer’s instructions. The positive tubes given by MGIT 960 instrument were confirmed by using SD BIOLINE TB Ag MPT64 Rapid (Standard Diagnostics, Inc., Republic of Korea) for M. tuberculosis complex.

2.4. Statistical Analysis

All the statistical parameters were calculated using M. tuberculosis culture as a reference standard. The parameters like sensitivity and specificity were calculated using online calculator (https://www.medcalc.org/calc/diagnostictest.php). The positive predictive value (PPV), negative predictive value (NPV), and concordance were also calculated.

3. RESULTS

3.1. Study Population

A total of 2678 samples were received from presumptive pediatric TB patients from February 2016 to March 2018. Sixty-eight samples were rejected due to unavailability of complete information (n = 66) and invalid test results (n = 2) by Xpert MTB/RIF assay. Therefore, 2610 samples were analyzed for this study, of which 1551 (59.4%) were male and 1059 (40.6%) were females, with age ranging from 1 month to 14 years with median age of 7 years. Among the 2610 samples, 1626 (62.1%) were respiratory samples including induced sputum and sputum (584, 22.4%), gastric aspirate/gastric lavage (740, 28.4%), bronchoalveolar lavage (252, 9.6%), endotracheal (ET) aspirate (50, 1.9%). The nonrespiratory samples were 984 (37.9%) including ascitic fluid (33, 1.3%), bone marrow aspirates (24, 0.9%), tissue biopsies (24, 0.9%), cerebrospinal fluid (CSF) (384, 14.7%), endobronchial ultrasound-guided transbronchial needle aspirate (EBUS-TBNA) (3, 0.1%), fine needle aspiration cytology (FNAC) (142, 5.6%), lymph node aspirate (50, 1.9%), pleural fluid (183, 7%), pus (120, 4.6%), synovial fluid (8, 0.3%), and other extra-pulmonary tuberculosis (EPTB) samples (13, 0.5%) (Table 1). Among 2610 samples, the smear and culture were performed for 2178 and 588 samples, respectively. The smear was positive in 81/2178 (3.3%) cases and the culture for M. tuberculosis complex was positive for 58/588 (9.9%) cases (Figure 1). The smear and culture positive were found in 14/567 (9.9%) cases, smear negative and culture positive were 43/567 (7.6%), and there was 1 (0.2%) smear positive and culture negative case.

Type of sample Total no Xpert MTB/RIF positive (%)
Bronchoalveolar lavage 252 24 (9.5)
ET aspirate 50 10 (20)
Gastric aspirate/gastric lavage 740 56 (7.6)
Induced sputum/sputum 584 160 (27.4)
Ascitic fluid 33 3 (9.1)
Aspirate 24 2 (8.3)
Biopsy 24 0
CSF 384 29 (7.5)
EBUS–TBNA 3 2 (66.7)
Fine-needle aspiration 142 28 (19.7)
Lymph node aspirate 50 9 (18)
Pleural fluid 183 7 (3.8)
Pus 120 24 (20)
Synovial fluid 8 1 (12.5)
Other EPTB 13 2 (15.4)
Table 1

Xpert MTB/RIF positivity among the respiratory and nonrespiratory samples

Figure 1

Participant flowchart in the study. AFP, acid-fast Bacilli.

A total of 357 (13.6%) samples were diagnosed as TB by Xpert MTB/RIF assay, including 250 (70.02%) respiratory samples and 107 (29.9%) nonrespiratory samples. Among positive respiratory samples, 24/252 (9.5%) were bronchoalveolar lavage (BAL), 10/45 (22.2%) ET aspirates, 56/740 (7.6%) gastric aspirate (GA)/gastric lavage (GL), and 160/584 (27.4%) were induced sputum or sputum. Among positive nonrespiratory samples, there were 3/33 (9.1%) ascitic fluid, 2/24 (8.3%) aspirates, 29/384 (7.6%) CSF, 2/3 (66.7%) EBUS-TBNA, 28/142 (19.7%) FNAC, 9/50 (18%) lymph node aspirate, 7/183 (3.8%) pleural fluid, 24/120 (20%) pus, 1/8 (12.5%) synovial fluid, and 2/13 (15.4%) other EPTB samples. Thirty (8.5%) samples were found rifampicin resistant, of which there were 20 (2 BAL, 2 ET aspirate, 5 GL, and 11 sputum) respiratory samples and 10 (3 CSF, 2 FNAC, 1 LN aspirate, 1 pleural fluid, and 3 pus) were nonrespiratory samples.

3.2. Sensitivity and Specificity of Xpert MTB/RIF

Among the 588 TB suspects, whose request was also received for culture and processed for liquid culture, smear was positive in 15 cases and Xpert MTB/RIF was positive in 71 samples. Among 71 Xpert MTB/RIF-positive samples, 47 and 24 were culture positive and culture negative, respectively. The sensitivity and specificity of smear were 24.6% (14.1–37.7%) and 99.8% (98.9–100%), respectively. The overall sensitivity of Xpert MTB/RIF was 81.03% (68.6–90.1%) and specificity was 95.5% (93.3–97.1%) (Table 2). The PPV and NPV of Xpert MTB/RIF was 66.2% (56.5–74.7) and 97.8% (96.4–98.8%), respectively. The diagnostic accuracy of smear and Xpert MTB/RIF assay was 92.2% (89.7–94.3%) and 94% (91.8–95.8%), respectively. In respiratory samples, the Xpert MTB/RIF was positive in 53 cases including 49 in culture-positive samples. The overall sensitivity of Xpert MTB/RIF in respiratory samples was 81.2% (67.3–91.5%), while the specificity of Xpert MTB/RIF was 96.6% (94.3–98.1%). The PPV and NPV of Xpert MTB/RIF were 73.6% (62.1–82.6%) and 97.8% (96–98.7%), respectively. In nonrespiratory samples, the Xpert MTB/RIF was positive in 12 cases including 9 in culture-positive samples. The overall sensitivity of Xpert MTB/RIF in nonrespiratory samples was 80% (44.4–97.5%), while the specificity of Xpert MTB/RIF was 94.6% (90.2–97.4%). The PPV and NPV of Xpert MTB/RIF were 44.4% (28.9–61.2%) and 98.8% (96.2–99.7%), respectively. A κ-value and proportion of agreement between two diagnostic tests, that is, smear microscopy and Xpert MTB/RIF, were detected as 0.35 and 0.89, respectively (Table 3).

Type of diagnostic test Sensitivity in % (95% CI) Specificity in % (95% CI) PPV (95% CI) NPV (95% CI)
Smear 24.6 (14.1–37.7) 99.8 (98.9–100) 93.3 (65.2–99.1) 92.2 (91–93.2)
Xpert MTB/RIF—overall 81 (68.6–90.1) 95.5 (93.3–97.1) 66.2 (56.5–74.7) 97.8 (96.4–98.7)
Xpert MTB/RIF—respiratory samples 81.2 (67.4–91.5) 96.6 (94.3–98.1) 73.6 (62.1–82.6) 97.7 (96–98.7)
Xpert MTB/RIF—nonrespiratory samples 80 (44.4–97.5) 94.6 (90.2–97.4) 44.4 (28.9–61.2) 98.8 (96.2–99.7)
Table 2

Performance of diagnostic tests for detection of M. tuberculosis

Smear positive Smear negative k-value Proportion of agreement
Xpert MTB/RIF—positive 77 233 0.3555 0.89 (0.88–0.90)
Xpert MTB/RIF—negative 5 1923
Table 3

Concordance between smear and Xpert MTB/RIF assay

4. DISCUSSION

The accurate diagnosis of pediatric TB is still a difficult task. In this study, we retrospectively analyzed the data of routinely used Xpert MTB/RIF from February 2016 to March 2018 in a tertiary care hospital of North India under programmatic conditions. Overall, the bacteriological confirmation by Xpert MTB/RIF was demonstrated in 13.6% cases, which was much higher than the smear microscopy that had 3.3%. The previous Indian studies have also shown two- to threefold increase in positivity rate by using Xpert MTB/RIF as a frontline test in pediatric TB suspects [13]. Other studies from South Africa and Uganda have also shown the proportion of Xpert MTB/RIF-positive results ranging from 13 to 14% in pediatric TB [14,15]. Overall, the smears were positive in 3.3% cases and the culture was positive in 9.9% cases. There were low positivity of smear and culture in our study because both types of samples, respiratory and nonrespiratory, were included. In a meta-analysis by Detjen et al. [16], the sensitivity of smear in respiratory samples ranges from 0 to 60%. In our study also, the Xpert MTB/RIF had fourfold higher positivity than the microscopy. In the nonrespiratory samples, the microbiological confirmation was 10.9%, which was substantially higher than that of Gupta et al. [17] who reported 4% positivity.

The sensitivity of Xpert MTB/RIF when culture was taken as a reference standard was 79.5%. One study from Uganda has also shown similar sensitivity of 81.3% in pediatric TB while another from Germany has shown a pooled sensitivity of 54.7% [15]. The specificity was 95.5% and comparable to the meta-analysis done by Detjen et al. [16], in which the specificity ranged from 86 to 100% in respiratory samples. In respiratory samples, Xpert MTB/RIF’s sensitivity was 81.2% in our study while other studies have shown a range of 25–100%. In nonrespiratory samples, Xpert MTB/RIF’s sensitivity was 80%, which was comparable to the respiratory samples.

Only a very few studies report resistance in pediatric TB. In our study, Xpert MTB/RIF assay detected 8.5% (30) cases of rifampicin resistance in respiratory and nonrespiratory samples. In India, Raizada et al. [13] also have shown 17.4% rifampicin resistance cases among the bacteriological confirmed cases. There is one limitation of this study that culture was not performed on all samples due to less amount of the samples.

5. CONCLUSION

The Xpert MTB/RIF assay was found to have a pooled sensitivity of 81.2% and a specificity of 95.5% for rifampicin resistance detection, which was found in line with WHO’s recommendation for the use of Xpert MTB/RIF assay as a frontline test for the diagnosis of pediatric TB.

CONFLICTS OF INTEREST

The authors declare they have no conflicts of interest.

AUTHORS’ CONTRIBUTION

RY and SS designed the study; RY wrote the manuscript; JM, PV, MS, SV, PK, and RK helped in sample collection; RY and SS did the statistics; JM, PV, MS, SS, and PA modified the manuscript; and SS supervised the project.

FUNDING

This was a retrospective analysis of the routine testing under RNTCP program and the consumables provided by Central TB Division, India.

ACKNOWLEDGMENTS

The financial, administrative, and technical support of Department of Medical Microbiology, PGIMER, Chandigarh and RNTCP is acknowledged.

REFERENCES

[3]World Health Organization (WHO), Global Tuberculosis Report 2017, World Health Organization, Geneva, 2017. Available from: http://www.who.int/tb/publications/global_report/gtbr2017_main_text.pdf (last accessed November 3, 2017).
[10]World Health Organization (WHO), Automated real-time nucleic acid amplification technology for rapid and simultaneous detection of tuberculosis and rifampicin resistance: Xpert MTB/RIF assay for the diagnosis of pulmonary and extra pulmonary TB in adults and children, World Health Organization, Geneva, 2011. Available from: http://apps.who.int/iris/bitstream/10665/112472/1/9789241506335_eng.pdf?ua=1
Journal
Journal of Epidemiology and Global Health
Volume-Issue
10 - 2
Pages
153 - 156
Publication Date
2020/01/10
ISSN (Online)
2210-6014
ISSN (Print)
2210-6006
DOI
10.2991/jegh.k.191215.002How to use a DOI?
Copyright
© 2020 Atlantis Press International B.V.
Open Access
This is an open access article distributed under the CC BY-NC 4.0 license (http://creativecommons.org/licenses/by-nc/4.0/).

Cite this article

TY  - JOUR
AU  - Rakesh Yadav
AU  - Pankaj Vaidya
AU  - Joseph L Mathew
AU  - Sanjay Verma
AU  - Rajiv Khaneja
AU  - Priyanka Agarwal
AU  - Pankaj Kumar
AU  - Meenu Singh
AU  - Sunil Sethi
PY  - 2020
DA  - 2020/01/10
TI  - Utility of Xpert MTB/RIF Assay for Diagnosis of Pediatric Tuberculosis Under Programmatic Conditions in India
JO  - Journal of Epidemiology and Global Health
SP  - 153
EP  - 156
VL  - 10
IS  - 2
SN  - 2210-6014
UR  - https://doi.org/10.2991/jegh.k.191215.002
DO  - 10.2991/jegh.k.191215.002
ID  - Yadav2020
ER  -