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Year : 2022  |  Volume : 10  |  Issue : 1  |  Page : 3-5

Role of analysis of threshold cycle (Ct) values in cartridge-based nucleic acid amplification test (CBNAAT) result: Impact on public health in relation to tuberculosis control and drug resistance

Intermediate Reference Laboratory (IRL), Kolkata, State Training & Demonstration Centre (STDC); Department of Health & Family Welfare, Govt.of West Bengal, India

Date of Submission30-Aug-2021
Date of Acceptance04-Jun-2022
Date of Web Publication12-Nov-2022

Correspondence Address:
Dr. Indranath Roy
Indranath Roy, Intermediate Reference Laboratory (IRL), Kolkata, State Training & Demonstration Centre (STDC), West Bengal. Dept. of Health & Family Welfare, Govt.of West Bengal
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jpsic.jpsic_21_21

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How to cite this article:
Roy I. Role of analysis of threshold cycle (Ct) values in cartridge-based nucleic acid amplification test (CBNAAT) result: Impact on public health in relation to tuberculosis control and drug resistance. J Patient Saf Infect Control 2022;10:3-5

How to cite this URL:
Roy I. Role of analysis of threshold cycle (Ct) values in cartridge-based nucleic acid amplification test (CBNAAT) result: Impact on public health in relation to tuberculosis control and drug resistance. J Patient Saf Infect Control [serial online] 2022 [cited 2023 Mar 30];10:3-5. Available from: https://www.jpsiconline.com/text.asp?2022/10/1/3/361003

Recent global estimates indicate that about a half million new Rifampicin-resistant tuberculosis (RR-TB) cases occurred in 2019, with 78% of them having confirmed multidrug-resistant TB (MDR-TB). The estimated number of MDR/RR-TB cases in India is 124 000 (9.1/100,000 population). The first National Anti-tuberculosis Drug Resistance Survey has shown that about 28% of TB patients were resistant to any drugs (22% amongst new and 36.82% amongst previously treated) and nearly 6.19% had MDR-TB (2.84% amongst new and 11.62% amongst previously treated).

Long before, in December 2010, the WHO recommended the use of the cartridge-based nucleic acid amplification test (CBNAAT) assay for early detection of TB along with prompt initiation of antitubercular drugs. This has been the main objective to curb the transmission of TB with early detection and initiation of treatment. The WHO's policy statement was issued in early 2011 and supported by a rapid implementation document, which provided the technical 'how-to' and operational considerations for rolling out the use of the CBNAAT assay.[1] An unprecedented uptake of this new technology followed the release of the WHO's policy. By the end of December 2013, more than 2000 CBNAAT instruments and more than 5 million cartridges had been procured in the public sector in 98 countries eligible for concessional prices.

The development of the CBNAAT assay is considered an important breakthrough in the fight against TB. CBNAAT provides a robust and promising role towards 'End TB strategy'.[2]

For universal drug susceptibility testing and upfront nucleic acid amplification test (NAAT), CBNAAT has been widely used in Low Middle Income Countries (LMICs), like India. Hence, in the recently published 'Programmatic Management of Drug-Resistant TB' guidelines, 2021, by the National Tuberculosis Elimination Programme, India, CBNAAT has been considered a pivotal role for TB diagnostic algorithm. Nearly all peripheral healthcare centres in India are almost equipped with the service of NAAT platform including CBNAAT. The laboratory technologists (LTs)/CBNAAT operators were trained and are being sensitised for easy specimen processing and reporting. The point of concern has been that the training of LTs working with CBNAAT focused mainly towards specimen processing, running the specimens, reporting and maintenance of the machine.[3]

Before elaborating the point of concern, it is very pertinent to discuss the ways in which CBNAAT machine actually works and interprets the result. It provides rapid identification of Mycobacterium tuberculosis complex (MTBc) and Rifampicin resistance from direct specimens within 2 h. CBNAAT machine is equipped with five[4] probes, namely Probe A, B, C, D and E, and specimen processing control (SPC).

In this semi-nested real-time polymerase chain reaction assay, Mycobacterium tuberculosis Complex (MTBc) is detected by five overlapping molecular probes complementary to the entire 81-bp rpoB core region. M. tuberculosis is detected when at least two of the five probes give positive signals with a cycle threshold (Ct) of ≤38 cycles. A semi-quantitative estimate of the concentration of bacilli can be defined by Ct range (>28 = very low, 22–28 = low, 16–22 = medium and <16 = high). Rifampicin resistance is reported when the difference between the first (early Ct) and the last (late Ct) M. tuberculosis-specific beacons is >4 cycles and was reported sensitive if ≤4 cycles.[5] In simple words, we can say that Rifampicin resistant detected occurs when the difference between the highest and the lowest Ct values differ by more than 4(ΔCt max>4).

Now, it has been observed that if there is one mutation detected by any probe (usually in probe E), it will give a Ct value of 0. In this case, the Ct difference between the lowest and the highest Ct values usually will give a difference of more than 4, which is interpreted by the software of CBNAAT platform as Rifampicin resistant detected. However, sometimes, if the bacillary load is very low in the specimen, the CBNAAT platform would continue to run and there could be a possibility that, to achieve the Ct values above threshold level, the CBNAAT platform would run for a considerable number of cycles. In most of these circumstances, there would be no probe showing a Ct value of 0. In this scenario, the difference between probes with highest and lowest Ct values would give a difference more than 4, which would be falsely interpreted by the machine as Rifampicin resistance detected.[4] Hence, in the practical field, we usually observe the result of MTBc detected with very low level and Rifampicin resistance. In most of the cases, if the results were analysed by checking the individual Ct value of the probes, the results could establish that most of the Rifampicin resistant were false Rifampicin resistant shown by the machine.

Hence, it is always recommended that while reporting the CBNAAT result, the LTs or CBNAAT operators located in peripheral laboratories or CBNAAT sites should check the probe status along with SPC value. They need to check the Ct values of all the probes. If there is a true mutation, there shall be Ct value of a particular probe zero.[6]

LTs or CBNAAT operators should re-run the test either with a fresh specimen if possible or with the same specimen if it is not readily available. There is every possibility that patients are often diagnosed and notified as false Rifampicin resistance and overtreated. In apex body laboratory, the same specimen might show MTBc negative/invalid/uninterpretable in Line probe Assay(LPA) method and can create discrepant result between CBNAAT and LPA methods of MTBc detection especially with the status of Rifampicin resistance.

So, it is always a good approach that if the LTs be trained and sensitized to check the Ct values of five probes while taking out the report from the software attached to CBNAAT machine so that they can analyze the result and make a corrective action afterwards. They should be encouraged not to check the presence of MTBc and Rifampicin status only but go for further checking of the Ct values of the concerned probes.

Another point of concern has been the detection of silent mutation after analysing the zero Ct value of any particular probe. In India, the most common mutation occurs in mutation of probe E followed by D. Mutation in probe B is often considered silent so to report it as Rifampicin resistant; we need to consider the epidemiological background of the patient.

After providing a hands on training and sensitization programme, Laboratory technologists (LT)/CBNAAT operators at peripheral CBNAAT sites are expected to exercise the analysis of CBNAAT results with the followings points:

  1. Checking SPC
  2. Ct values of five probes
  3. Checking mutation of probe which is supposed to be zero (If Rifampicin resistance is detected)
  4. The type of probe which shows mutation.(For epidemiological study)

This information can be forwarded to apex body laboratory to analyze the data and cross check any discrepancy. Atleast LTs/CBNAAT operators should be encouraged to check the M. tuberculosis detection(If MTB is detected) along with the checking of semi quantitative value of MTB detection (very low level, low level, moderate, high ) and the difference between the highest and lowest probe Ct values. This information will enable us to check the bacillary load, and we can minimize the false Rifampicin resistant reporting.

To summarise, the LTs or operators at CBNAAT centres in the periphery should check the CBNAAT report in details particularly for those reports with Rifampicin-resistant cases to avoid any over/misdiagnosis. They should check the probe Ct values and be sure that the results being generated are valid and quality assured. If they need refresher training or sensitisation, the higher authority should recognise and address the issues. Ultimately, the LTs/CBNAAT operators reporting from CBNAAT sites are playing a pivotal role in TB diagnostic and treatment regimen algorithm, and we need to be with them with all possible theoretical and technical support. Another thing to address is that we can also minimise the overuse of anti-TB drugs and can prevent the unnecessary emergence of drug resistance by reducing antibiotic selective pressure in the community.[7]

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.

  References Top

World Health Organization. Meeting Report of the WHO Expert Consultation on the Definition of Extensively Drug-Resistant Tuberculosis; 2020. Available from: https://www.who.int/publications/i/item/meeting-report-of-the-who-expert- consultation-on-the-definitionof-extensively-drug-resistant-tuberculosis. [Last accessed on 2021 Mar 16].  Back to cited text no. 1
Central TB Division, Government of India. Technical and Operational Guidelines for TB Control in India 2016. New Delhi: Central TB Division, Directorate General of Health Services, Ministry of Health and Family Welfare; 2016.  Back to cited text no. 2
WHO. WHO consolidated guidelines on tuberculosis Module 3: Diagnosis – Rapid diagnostics for tuberculosis detection [Internet]. 2020. p. 108.  Back to cited text no. 3
Prakash AK, Datta B, Tripathy JP, Kumar N, Chatterjee P, Jaiswal A. The clinical utility of cycle of threshold value of GeneXpert MTB/RIF (CBNAAT) and its diagnostic accuracy in pulmonary and extra-pulmonary samples at a tertiary care center in India. Indian J Tuberc 2018;65:296-302.  Back to cited text no. 4
Lawn SD, Nicol MP. Xpert® MTB/RIF assay: Development, evaluation and implementation of a new rapid molecular diagnostic for tuberculosis and rifampicin resistance. Future Microbiol 2011;6:1067-82.  Back to cited text no. 5
Barry P, Lin G. Experts on Xpert: A Laboratorian and a Clinician Discuss Interpretation of Xpert MTB/RIF Results. Curry International Tuberculosis Center. 2018 Webinar.URL: https://www.currytbcenter.ucsf.edu/trainings/experts-xpert-laboratorian-and-clinician-discuss-interpretation-xpert-mtbrif-results/materials. [Last accsssed on 2021 Feb 20].  Back to cited text no. 6
World Health Organization. Integrated Surveillance on Antimicrobial Resistance: Guidance from a WHO Advisory Group. Geneva: World Health Organization; 2013.  Back to cited text no. 7


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