Cartridge-based nucleic acid amplification test: a novel rapid diagnostic tool to study the burden of tuberculosis from a tertiary care hospital

Abstract

Despite efforts to limit the morbidity and mortality from tuberculosis (TB), it continues to be an important cause of death. There is an urgent need for a diagnostic test that accurately and quickly diagnoses TB, especially if it is also a near-point-of-care test. The GeneXpert polymerase chain reaction test (known in India as CBNAAT [cartridge-based nucleic acid amplification test] and is capable of diagnosing TB and rifampicin resistance within 2 h) is a promising tool.

The duration of our study was two years and was carried out in the DOTS centre of a tertiary care hospital in India. A total of 5449 samples were processed using CBNAAT. Of the total samples tested, 2068 were extra-pulmonary. The following information was collected: number of extra-pulmonary samples processed; number of Mycobacterium tuberculosis (M. tuberculosis)-positive samples; patterns of rifampicin sensitivity; number of people living with HIV (PLHIV); and number of children.

Of the samples, 62.1% were from suspected pulmonary TB patients. Out of the total samples tested using CBNAAT, 21.8% were positive for M. tuberculosis. Rifampicin resistance was seen in 9.2%, 8.5% and 10.3% of the total, pulmonary and extra-pulmonary samples, respectively, in M. tuberculosis-positive samples. Overall, 36.9% samples were from the paediatric population and 5.7% belonged to PLHIV. Rifampicin resistance was seen in 8.8% and 8.3% of the M. tuberculosis-positive paediatric and PLHIV samples, respectively.

Keywords

CBNAAT Xpert MTB/RIF tuberculosis paediatric tuberculosis PLHIV tuberculosis

Introduction

Tuberculosis (TB) still constitutes one of the top 10 causes of mortality and is the most important cause of death due to infection even surpassing HIV/AIDS. In 2017, TB was responsible for the ill-health of approximately 10 million individuals, of whom one million were children, and 9% were associated with HIV. Of the 6.4 million incident cases notified in 2017, 14% were extra-pulmonary.¹ The World Health Organization (WHO) recently announced three separate lists of high burden countries for TB, TB/HIV and multidrug-resistant TB (MDR-TB) for 2016–2020 based on recent available estimates. India is one of the 14 countries present in all three lists and was the largest contributor of TB cases in 2017. India, along with China, Indonesia and the Philippines, contributed 50% of the global total.^1,2

Of the estimated 558000 incident MDR/RR-TB cases, 82% were also MDR-TB. India alone contributed almost one-quarter of the world's total load of MDR/rifampicin-resistant TB (RR-TB) cases (MDR-TB or RR-TB but isoniazid-susceptible TB). Multidrug resistance is defined as resistance to at least both isoniazid and rifampicin, whereas rifampicin resistance is defined as any resistance to rifampicin (mono-resistance, poly-resistance, MDR or extensively drug-resistant [XDR]), with or without resistance to other anti-TB drugs.¹

Pulmonary TB may present with similar clinical and radiologic features to those of community-acquired pneumonia (CAP).³ Physicians usually prescribe two-week trial antibiotic courses for lower respiratory tract infections in presumptive TB cases. The failure of response to antibiotic treatment often guides the physicians to reach to the correct diagnosis of TB.^4,5 This may further accentuate the problem of antimicrobial drug resistance. Meanwhile these cases may continue to transmit and spread TB in the community. Sputum microscopy, being a simple and rapid method, has remained the primary tool for pulmonary TB diagnosis, followed by conventional sputum culture. The sensitivity of smear microscopy is poor and a conventional solid culture technique suffers the limitation of a long turnaround time extending up to several weeks.⁶ Even liquid culture techniques have a mean turnaround time of 21 days.⁷ Therefore, despite culture being the ‘gold standard’ for the diagnosis of TB, culture is not a suitable diagnostic test in curbing TB transmission. Furthermore, the positivity of smear and culture is low for extra-pulmonary TB due to its paucibacillary nature and irregular distribution of tubercular bacilli.⁸ Detection of pulmonary TB in the paediatric population is a challenging task as it is difficult to obtain good quality sputum samples from children. Moreover, the alternative samples are difficult to obtain and the quantity of the available sample is often insufficient. The paucibacillary nature of paediatric TB further makes the diagnosis difficult.^9,10 TB is the most frequently encountered opportunistic infection among people living with HIV/AIDS (PLHIV). These cases of HIV-TB co-infection have a risk of mortality.¹¹ Diagnosis of TB using conventional methods is often difficult in PLHIV due to the production of scanty sputum and the paucibacillary nature of disease.¹²

In view of the challenges encountered in an accurate and timely diagnosis of TB, particularly in resource-limited settings with high TB prevalence, there was an urgent need for a diagnostic test that could fulfil these requirements along with being valuable as a point-of-care test. The development of CBNAAT (cartridge-based nucleic acid amplification test)/Xpert MTB/ RIF is a significant milestone in TB diagnostics. CBNAAT is the only rapid test currently recommended by the WHO for rapid TB diagnosis. CBNAAT is an automated, heminested real-time polymerase chain reaction (PCR) that simultaneously detects Mycobacterium tuberculosis (M. tuberculosis) and rifampicin resistance as a surrogate marker of MDR-TB using molecular beacons.¹³ This test is performed directly on clinical specimens and results become available in < 2 h, which is in sharp contrast to the turnaround time of 8–10 weeks taken by conventional TB drug-sensitivity testing. CBNAAT has no particular prerequisites and needs limited technical training for its performance. Moreover, the biosafety risks associated with CBNAAT are minimal as M. tuberculosis bacilli are inactivated in vitro, which further emphasises its utility as a rapid point-of-care TB diagnostic test.¹⁴

The WHO initially approved the use of CBNAAT as a rapid TB diagnostic test in December 2010. Since 2013, the WHO endorsed CBNAAT for use in paediatric and specific forms of extra-pulmonary TB.¹ Therefore, CBNAAT can serve as a promising tool to aid in the achievement of global objective of early detection of TB cases and improved TB care. Early diagnosis of TB can consequently reduce the lag time in the initiation of effective TB treatment, henceforth reducing the risk of TB transmission to contacts and progression of disease within the patient.¹⁵

Methods

This prospective study was carried out in the Department of Microbiology and DOTS center of a tertiary care hospital, in New Delhi. The study extended over a period of two years from August 2016 to August 2018. CBNAAT was performed on the samples from the suspected TB cases as per the RNTCP guidelines.¹⁶ All patients with any suggestive symptoms and signs of TB inclusive of cough > 2 weeks, persistent fever > 2 weeks, haemoptysis, significant weight loss or abnormal chest radiograph were defined as suspected pulmonary TB cases. The cases with constitutional symptoms of significant weight loss, fever > 2 weeks, night sweats and/or organ-specific symptoms and signs such as swollen lymph nodes, joint pains and swelling, stiffness of neck, disorientation, etc. were considered suspected extrapulmonary TB cases.¹⁷

CBNAAT was performed using Xpert MTB/RIF assay (Cepheid Inc., Sunnyvale, CA, USA) following the manufacturer's instructions. Xpert MTB/RIF assay is CBNAAT for the simultaneous detection of M. tuberculosis and rifampicin resistance. It is based on the principle of Hemi-nested PCR. The information regarding tests performed using CBNAAT, number of extrapulmonary samples processed, number of M. tuberculosis-positive samples and patterns of rifampicin sensitivity, number of PLHIV and paediatric population among M. tuberculosis-positive and rifampicin-sensitive or resistance samples was collected.

Results

During the study duration, 5449 samples were processed using CBNAAT. Of them, 2068 (38%) of the total samples tested were extrapulmonary samples. Table 1 demonstrates the six-monthly distribution of total and extrapulmonary samples tested using CBNAAT and their M. tuberculosis positivity. Out of the total samples tested using CBNAAT, 4024 (73.8%) were M. tuberculosis-negative. Overall, 1189 (21.8%) were positive for M. tuberculosis by CBNAAT. Rifampicin resistance was seen in 109 (9.2%) of the total M. tuberculosis-positive samples. Figure 1 and Table 2 show the pattern of rifampicin sensitivity among the M. tuberculosis-positive samples. Overall, 3381 (62.1%) of the samples were from suspected pulmonary TB patients. Of these pulmonary samples, 751 (22.2%) were positive for M. tuberculosis by CBNAAT. In 64 (8.5%) of these M. tuberculosis-positive pulmonary samples, rifampicin resistance was seen. Among the M. tuberculosis-positive samples, 12 (1.6%) were rifampicin indeterminate. Distribution of extrapulmonary samples among the overall M. tuberculosis-positive samples is shown in Figure 2. Table 3 depicts the distribution of CBNAAT-positive extrapulmonary TB samples. The most common extrapulmonary sample was pus (59.8%), followed by cerebrospinal fluid (24%), lymph node (7.5%), biopsy (3.2%), pleural fluid and ascitic/peritoneal fluid (2.3%) and synovial fluid (0.9%). Figure 3 further demonstrates the rifampicin sensitivity patterns among M. tuberculosis-positive extrapulmonary TB samples using CBNAAT. In our study, 10.3% of the M. tuberculosis-positive extrapulmonary samples were rifampicin-resistant.

Figure 1.

Distribution of rifampicin sensitivity patterns among M. tuberculosis-positive samples using CBNAAT (n = 1189).

Figure 2.

Distribution of extrapulmonary samples among M. tuberculosis-positive samples tested using CBNAAT (n = 1189).

Figure 3.

Distribution of M. tuberculosis positivity and rifampicin sensitivity patterns among EPTB samples using CBNAAT (n = 438).

Table 1.

Distribution of M. tuberculosis positivity in CBNAAT-tested samples (n = 5449).

Duration	Total tests performed using CBNAAT	Total MTB positive samples (n (%))	Total extrapulmonary TB samples processed (n)	Total extrapulmonary TB positive samples (n (%))
Aug 2016–Jan 2017	444	122 (27.5)	151	49 (32.5)
Feb 2017–Jul 2017	1393	297 (21.3)	536	143 (26.7)
Aug 2017–Feb 2018	1437	249 (17.3)	538	105 (19.5)
Mar 2018–Aug 2018	2175	521 (24)	843	141 (16.7)
Total	5449	1189	2068	438

Table 2.

Distribution of rifampicin sensitivity patterns among M. tuberculosis-positive samples using CBNAAT.

	Duration
	Aug 2016– Jan 2017	Feb 2017– Jul 2017	Aug 2017– Feb 2018	Mar 2018– Aug 2018
Total MTB + samples (n)	122	297	249	521
Total MTB + and RIF-sensitive (total MTB+/RIF−) samples (n (%))	103 (84.4)	263 (88.6)	225 (90.4)	477 (91.6)
Total MTB + and RIF-resistant (total MTB+/RIF+) samples (n (%))	18 (14.8)	33 (11.1)	20 (8.0)	38 (7.3)
Total MTB + and RIF-indeterminate (total MTB+/RIF-indeterminate) samples (n (%))	1 (0.8)	1 (0.3)	4 (1.6)	6 (1.2)
Extrapulmonary TB MTB + samples (n)	49	143	105	141
Extrapulmonary TB MTB + and RIF-sensitive (total MTB+/RIF−) samples (n (%))	41 (83.7)	128 (89.5)	97 (92.4)	127 (90.1)
Extrapulmonary TB MTB + and RIF-resistant (total MTB+/RIF+) samples (n (%))	8 (16.3)	15 (10.5)	8 (7.6)	14 (9.9)

Table 3.

Distribution of CBNAAT-positive extrapulmonary TB samples based on site involved (n = 438).

	Duration
	Aug 2016– Jan 2017	Feb 2017– Jul 2017	Aug 2017– Feb 2018	Mar 2018– Aug 2018
Pus	23 (46.9)	88 (61.5)	59 (56.2)	92 (65.2)
Lymph node	10 (20.4)	7 (4.9)	11 (10.5)	5 (3.5)
Cerebrospinal fluid	12 (24.5)	34 (23.8)	28 (26.7)	31 (22.0)
Biopsy	2 (4.1)	5 (3.5)	3 (2.9)	4 (2.8)
Pleural fluid	1 (2.0)	4 (2.8)	1 (1.0)	4 (2.8)
Synovial fluid/joint aspirate	0 (0)	1 (0.7)	2 (1.9)	1 (0.7)
Ascitic/peritoneal fluid	1 (2.0)	4 (2.8)	1 (1.0)	4 (2.8)
Total (n)	49	143	105	141

Values are given as n (%) unless otherwise specified.

Out of the total samples, 2010 (36.9%) were from the paediatric population and 313 (5.7%) belonged to PLHIV. Figure 4 depicts the distribution of M. tuberculosis-positive samples belonging to PLHIV and the paediatric population among the CBNAAT-tested samples. Table 4 shows the distribution of CBNAAT-positive PLHIV and paediatric samples on the basis of site involved. In CBNAAT-tested M. tuberculosis-positive PLHIV cases, 85.4% had pulmonary TB and 14.6% had extrapulmonary TB.

Figure 4.

Distribution of PLHIV and the paediatric population among the CBNAAT-tested rifampicin-sensitive M. tuberculosis-positive samples (n = 1068).

Table 4.

Distribution of CBNAAT-positive PLHIV and paediatric samples based on site involved.

Duration	PLHIV samples			Paediatric samples
Duration	Total (n)	Pulmonary (n (%))	Extrapulmonary TB (n (%))	Total (n)	Pulmonary (n (%))	Extrapulmonary TB (n (%))
Aug 2016–Jan 2017	13	13 (100)	0 (0)	11	6 (54.5)	5 (45.5)
Feb 2017–Jul 2017	22	19 (86.4)	3 (13.6)	59	38 (64.4)	21 (35.6)
Aug 2017–Feb 2018	9	6 (66.7)	3 (33.3)	27	22 (81.5)	5 (18.5)
Mar 2018–Aug 2018	4	3 (75)	1 (25)	63	47 (74.6)	16 (25.4)
Total	48	41 (85.4)	7 (14.6)	160	113 (70.6)	47 (29.4)

Among CBNAAT-tested M. tuberculosis-positive samples from PLHIV, 44 (91.7%) were rifampicin-sensitive. In these samples from PLHIV, rifampicin resistance was seen in 4 (8.3%) samples. In CBNAAT-tested M. tuberculosis-positive paediatric cases, 70.6% had pulmonary TB and 29.4% had extrapulmonary TB. Among paediatric samples, 14 (8.8%) out of 160 M. tuberculosis-positive samples were rifampicin-resistant.

Discussion

In an endemic country such as India, early diagnosis of TB along with accurate detection of drug resistance are critical for prevention of TB transmission and development of resistant cases. In resource-limited settings, the adoption of newer techniques remains challenging due to the involved start-up and training costs. However, CBNAAT has been reported to be cost-effective even in these settings.^18,19 CBNAAT can be performed directly on the clinical specimens, including the ones from extrapulmonary sites. Xpert MTB/RIF is capable of detecting both live and dead bacteria.²⁰ Xpert MTB/RIF can be useful as an initial investigation for the diagnosis of TB or can be used as an adjunct in smear-negative TB suspects. Xpert MTB/RIF has a better sensitivity and specificity than smear microscopy and culture.²¹ The chances of contamination are lower and this assay does not impose a biosafety concern. The conventional techniques for the detection of drug resistance are slow, more prone to contamination and labour-intensive. This may lead to an inadvertent delay in the initiation of effective and appropriate antitubercular treatment (ATT) which may further enhance the risk of disease transmission and development of resistance.²²

In the present study, 21.8% of the all samples tested by CBNAAT were positive for M. tuberculosis. In accordance with our study, Al-Ateah et al. also reported 24.6% positivity by Xpert® assay.²³ However, Iram et al., in their study, reported a higher positivity of 45.3% in TB suspects by Xpert MTB\RIF assay.²⁴ In our study, 22.2% of the pulmonary samples were positive for M. tuberculosis. Sharma et al. reported 31.3% of the Xpert MTB/RIF assayed pulmonary samples positive for M. tuberculosis.¹⁴ The present study has reported rifampicin resistance in an overall 9.2% of the total and 8.5% of the pulmonary M. tuberculosis-positive samples. Sharma et al. documented a higher rifampicin resistance of 25.4% in pulmonary M. tuberculosis-positive samples.¹⁴ However, Sowjanya et al. observed a lower (2.8%) rifampicin resistance in M. tuberculosis-positive sputum samples.²⁵ In the present study, 21.2% of the extrapulmonary samples were positive for M. tuberculosis. Another study reported that 22.5% of the extrapulmonary samples were positive for M. tuberculosis.²⁴ Similarly, Iram et al., in their study, reported 22.5% of the extrapulmonary samples positive for M. tuberculosis.²⁴ The previous study did not document any cases of rifampicin resistance in extrapulmonary samples from TB suspects.²⁴

In the current study, more than one-third of samples processed belonged to the paediatric population. Positivity for M. tuberculosis was seen in 8% of these samples by CBNAAT. Another study involving the paediatric tuberculosis suspects reported a positivity for M. tuberculosis in 26.4% of the samples by CBNAAT.²⁶ In our study, rifampicin resistance was reported in 8.8% of the M. tuberculosis-positive paediatric samples. Kumar et al. have documented 5.7% resistance for rifampicin in paediatric samples positive for M. tuberculosis.²⁶ In developing countries, which very often have logistic constraints, coexistent TB in PLHIV makes the imparting of adequate antiretroviral therapy (ART) a challenging task. The odds are made worse by the presence of MDR-TB in the same patients. Therefore, there is an urgent need for a rapid and effective screening technique such as CBNAAT for TB that can aid in the early detection of drug resistance in PLHIV. Furthermore, it has been shown that the sensitivity of CBNAAT performance is not significantly altered in HIV-TB co-infection as seen with sputum microscopy.²⁷ In our study, we reported 15.3% of the samples from PLHIV being CBNAAT-positive for M. tuberculosis. Rifampicin resistance was seen in 8.3% of these M. tuberculosis-positive samples. Dewan et al. have reported a higher positivity of 40% for M. tuberculosis in the sputum samples from PLHIV pulmonary TB suspects by CBNAAT. Furthermore, the same study also reported a higher rifampicin resistance of 25% in these M. tuberculosis-positive samples from PLHIV.¹²

CBNAAT, despite being an extremely useful investigation for the diagnosis of TB, has a few limitations. Xpert cannot detect cases with isoniazid mono-resistance. CBNAAT is capable of detecting rifampicin resistance only; therefore, it is not useful in differentiating MDR-TB from XDR-TB cases. Moreover, it is not considered suitable in monitoring response to treatment.²⁶

Conclusion

The rapid availability of reliable test results makes CBNAAT an appealing tool for the TB diagnosis. It may play a critical role in the timely detection and appropriate treatment of paucibacillary TB such as smear-negative pulmonary, extrapulmonary, paediatric TB and TB co-existent with HIV. Its ability to detect resistance to rifampicin speedily gives it an edge over conventional diagnostic methods.

Footnotes

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

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