Introduction: Among the first line drugs used for treatment of Drug Sensitive Tuberculosis, Rifampicin plays a crucial role. Hence it is important to know the resistance of Rifampicin using molecular technologies like CBNAAT and line probe Assay. However, a diagnostic dilemma occurs when there is discordance in result of rifampicin resistance at periphery and in higher laboratories like Intermediate Reference Laboratory (IRL).

Aims and objectives: To study various patterns of Gene for rifampicin that is demonstrated in probes of Line Probe Assao for First line –Line Probe Assay, which showed discordance with CBNAAT at periphery.

Methodology: A retrospective observational study was conducted from 1^st January 2021 to 31^st December 2021 for all those samples which showed rifampicin discordance.

Result: A total of 203 results were found discordant in Rifampicin. The resolution for rifampicin discordance is resolved using the NAAT facility at (IRL). Most of the resolution occurred in concurrence with result of Rifampicin in field. Eight of them gave inconclusive results (MTB not detected)

Conclusion: It is very important to understand the gene patterns in LPA for providing appropriate regimen to the patients.

Keywords: Rifampicin, discordance, gene patterns, NAAT, LPA

Tuberculosis (TB), caused by Mycobacterium tuberculosis is disease of major concern as India accounts to 27% of Global burden. Diagnosis of drug resistant TB and their management remained challenge to National TB program because of poorer outcomes. The important first line drugs Isoniazid, Rifampicin, Pyrazinamide and Ethambutol remained powerful weapons to combat TB disease when used in correct doses and duration.

National drug resistant surveys in 2016 mentioned that Rifampicin resistance is observed in 2-3 % in new cases and 11-12% in retreatment cases, overall amounting to 6% of all TB Cases. The tests to detect Rifampicin status have been increasing at district level and sub district level. The molecular tests like CBNAAT (Cartridge Based Nucleic Acid Amplification Test) and FLLPA (First Line –Line Probe Assay). First line Probe Assay gives information of resistance about two drugs i.e. Isoniazid and Rifampicin. The genes that are detect for Isoniazid are Kat G and Inh A whereas rpoB is the gene detected for Rifampicin.

Rifampicin was discovered in 1965, marketed in Italy in 1968, and approved in the United States in 1971. Rifampicin inhibits bacterial DNA-dependent RNA synthesis by inhibiting bacterial DNA-dependent RNA polymerase. Crystal structure data and biochemical data suggest that rifampicin binds to the pocket of the RNA polymerase β subunit within the DNA/RNA channel, but away from the active site. The inhibitor prevents RNA synthesis by physically blocking elongation, and thus preventing synthesis of host bacterial proteins. By this "steric-occlusion" mechanism, rifampicin blocks synthesis of the second or third phosphodiester bond  between the nucleotides in the RNA backbone, preventing elongation of the 5' end of the RNA transcript past more than 2 or 3 nucleotides.

Rifampicin is the drug used in combination for treatment of Mycobacterium tuberculosis, Mycobacterium leprae, A typical mycobacterium, Zoonotic infections like Brucella species.

Resistance to rifampicin arises from mutations that alter residues of the rifampicin binding site on RNA polymerase, resulting in decreased affinity for rifampicin. Resistance mutations map to the rpoB gene, encoding the beta subunit of RNA polymerase. An alternative mechanism of resistance is through Arr-catalyzed ADP-ribosylation of rifampicin. With the assistance of the enzyme Arr produced by the pathogen Mycobacterium smegmatis, ADP-ribose is added to rifampicin at one of its ansa chain hydroxy groups, thereby inactivating the drug.

Rational of study

Rifampicin resistance is surrogate marker for Multi drug resistance (MDR). The resistance for rifampicin prompts program managers to tailor the suitable treatment regimen.

It is very important to know the status of rifampicin in diagnosis and management of DRTB. The status of rifampicin determines the MDR status. CBNAAT /Tru NAAT are molecular tests which will not only detect TB infection but also determine the resistance to Rifampicin. The CBNAAT (cartridge based Nucleic acid amplification test) at district level and sub district level are made available as expansion of diagnostic services for DRTB.

As part of diagnostic DRTB (Drug resistant Tuberculosis) algorithm 2021 in National TB Elimination Program, India, and all the samples which detected Rif resistance should undergo both first line and second line Probe Assay along with liquid Culture. The First line LPA not only reconfirms Rif’s resistance but also gives information about genes related to Isoniazid (Inh A & Kat G). Similarly, the samples where Gene Xpert showed no resistance to rifampicin, are subjected to FLLPA. At times there is an observation that the discordance in the both molecular tests leads management of such TB patient in cross roads.

Research question

1. What are reasons for rifampicin discordance?
2. What are suitable solution to resolve to design appropriate DRTB regimen?

Aim of the study

To study various patterns of Gene that are demonstrated in probes of Line Probe Assay for First line –Line Probe Assay which were discordant from the results in CBNAAT.

Objectives

1. To understand the patterns of rifampicin resistance detected and rifampicin resistance inferred which showed rifampicin resistance detected in CBNAAT at district and sub district level.
2. To resolve discordance by offering CBNAAT at Culture and drug susceptibility laboratory (C&DST) which prompts the appropriate decision on Rifampicin Status.

All the consecutive samples will be taken from January 1^st 2021 to December 31^th 2021, which showed discordance in Rifampicin.

Study period: January 2021- December 2021

Place of Study: State TB training and demonstration Center, Hyderabad.

Samples size: All consecutive samples which showed rifampicin discordance during the study period.

Type of Study: retrospective Observational study

Inclusion criterion

1. All samples which are Rif resistant at district level and rif resistance not detected in FLLPA at C&DST laboratory
2. All samples which were Rif’s resistance not detected and Rif Resistance detected in FLLPA at C&DST laboratory.

Exclusion criterion

There was no exclusion criterion for this study.

All samples which showed discordance in rifampicin resistance are recorded. The samples showing discordance are resolved by performing Gene Xpert again at C&DST laboratory. Two out of three molecular test performed is taken as the final result of Rifampicin.

The entire data was collected in Excel sheet and will be updated according.

Training was being given to assigned person on updating of the data. Appropriate statistics was applied along with demographics.

A total of 40 TruNAAT and 38 CBNAAT were available in public sector in the state of Telangana in the year 2021. First line LPA was offered in Public sector at Intermediate Reference laboratory and is collocated at State TB training and Demonstration Center (STDC), Hyderabad, Telangana, India.

A total of 203 were found to be rifampicin discordant when tested in NAAT at districts and LPA at Reference laboratory in the year 2021.

The more number of samples were shown in districts of Hyderabad, Sangareddy, Rangareddy and Nalgonda.

Kumurambheem Asifabad was only district which did not show any discordance in Rifampicin when tested in CBNAAT and TruNAAT.

Only one discordant result was seen in Mahabubnagar, Nagarkurnool, Rajanna Siricilla and Yadadri bhongir districts.

All the districts had TruNAAT facility at the time of study. Only one district of Narayanpet did not had CBNAAT facility at the time of study.

Having NAAT facility in the districts was very important in conducting this study as this had impact and the diagnosis of Rifampicin resistance. District wise distribution is shown in Table 1.

As a policy and protocol of CDST laboratories, all samples (irrespective of Rif’s status) reaching CDST laboratories would be subjected to smear microscopy. All those which read smear negative would be inoculated in liquid culture for reading in Line probe Assay (LPA).All those which are positive will be directed to LPA

Smear results of those 203 samples which showed discordance were recorded (Table 2).

The second sample according to algorithm are directed to CDST laboratory (Table 3).

Type of samples received (Table 4).

In FLLPA tests all the Samples read PCR, TUB band Rpo B gene and following gene types were studied (Tables 5–7).

Rifampicin, as the most effective first-line antituberculosis drug, also develops resistance due to the mutation on Mycobacterium tuberculosis (Mtb) RNA polymerase. Multidrug-resistant tuberculosis (MDR-TB) is defined as disease due to Mycobacterium tuberculosis that is resistant to isoniazid (H) and rifampicin (R) with or without resistance to other drugs. Rifampicin-resistant TB (RR-TB) defined as resistance to rifampicin detected using genotypic or phenotypic methods with or without resistance to other first-line anti-TB drugs. MDR-TB/RR-TB has been an area of growing concern to human health worldwide and posing a threat to the control of TB. The Global TB Report 2016 estimated that of 3.9% newly diagnosed and 21% of previously treated TB cases had MDR-TB. It has been estimated that 580,000 cases of TB resistant to at least rifampicin (RR-TB) globally in 2015, of whom, 480,000 were having resistant to both rifampicin and isoniazid (MDR-TB), and 250,000 deaths occurred due to MDR-TB/RR-TB in 2015 globally. Out of estimated 580,000 MDR-TB/RR-TB cases, only 132,120 (23%) were detected, and even fewer 124,990 (20%) started treatment, and only 52% of them were treated successfully.¹

Rifampicin resistance status was made more available after decentralizing the TB services in form of TruNAAT and CBNAAT (Cartridge Based Nucleic Acid Amplification test) at block level. According Programmatic management of Drug resistance TB (PMDT) guidelines 2019 and 2021, The samples which detected rifampicin resistance in the field would go through the cascade of FLLPA (First line -Line Probe Assay), SLLPA (Second line-line probe Assay) and LC-DST (liquid Culture Drug susceptibility testing).At times the discordance in the result of rifampicin is encountered which is amounting to 1.7%(i.e. 203 out of 11839 samples tested in FLLPA).

As per PMDT guidelines 2019 and 2021, discordance in RR results between NAAT & FL-LPA to be resolved with a repeat NAAT at C&DST lab and microbiologists will provide the final decision.²

A total of 203 results showed discordance in CBNAAT and LPA. Among the geographical distribution, Hyderabad holds 37 (majority of cases), amounting to more than 15% of total cases.

This could be attributed to reasons like more number of NAAT machines doing more number of tests. All the 37 tests which showed rifampicin discordance were resolved.

In our study the samples which detected Rifampicin resistance in field NAAT, showing sensitive in FLLPA were 88. Out of 88, when discordance was resolved, 34 out of 88 showed resistance, 53 out of 88 showed resistance not detected. Only one case showed MTB not detected.

The samples which detected Rifampicin Sensitive in field NAAT, showing resistance in FLLPA were 115.Out of 115, when discordance was resolved, 53 out of 115 showed resistance, 55 out of 115 showed resistance not detected. Seven case showed MTB not detected.

Whenever the result of MTB is not detected, the treating physician had to take a decision on whether to treat the patient as drug sensitive TB or Drug resistant TB. Molecular test like CBNAAT and LPA failed to demonstrate the status even after subjecting to NAAT third time.

The line probe Assay which is read manually should be understood in depth on presence of TUB band, expression of wild types and mutations. The following table helps to understand the interpretation’s in LPA (Table 8).

Resistance to rifampicin is predominantly (95% of cases) caused by genetic variants in the rifampicin resistance-determining region (RRDR) of the RNA polymerase β subunit (rpoB) gene.³ Discordant results occurred mostly (20/22) in specimens with very low bacterial load.⁴ This can be known in present study by smear results of negative, scanty and 1+ are more in number approximately 70% of cases.

Few studies have investigated the mechanism causing discordances between two molecular tests. In a recent article, Hofmann-Thiel et al. hypothesized that several mechanisms can cause discordances, but they did not provide evidence for or the relative importance of these mechanisms in routine conditions.⁵ The most frequent cause of discordance observed was false-negative results by MTBDRplus in the presence of ‘disputed’ mutations, defined as rpoB mutations associated with low-level rifampicin resistance that is missed by rapid phenotypic drug susceptibility tests.⁶

Discordant results between two molecular tests (especially Xpert and MTBDRplus) can have important implications for clinical care and public health as they can result in additional tests requested and can pose challenges to patient management. Patients wrongly diagnosed with rifampicin-resistant TB may receive a prolonged period of unnecessarily toxic and poorly effective drugs, while those misclassified as having drug-susceptible TB after an initial diagnosis of rifampicin-resistant TB may receive ineffective first-line treatment. Our observation that three in four patients with discordant results can be resolved by a follow-up Xpert assay support the recent guidelines that recommend a confirmatory Xpert assay in patients at ‘low rifampicin-resistant TB risk’ and/or a MTBDRplus assay to assess for MDR TB following a rifampicin-resistant TB diagnosis.⁷

Molecular assays have become the main method for detection and confirmation of rifampicin resistance in M. tuberculosis, but discordances between test results have important implications for the laboratory, clinician and patient. Our findings of a high (1.7%) prevalence of discordant results between CBNAAT and LPA under routine conditions and an array of underlying causes highlight both the importance and complexity of this issue. Future research should assess the value of a repeat CBNAAT in the case of a discrepant result to guide healthcare workers in their treatment decisions.

Laboratory staff and NTEP, Telangana, India for providing the data.

The author has no conflicts of interest to declare.

TB Association of India, Telangana Chapter who funded to conduct the research.

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