Evaluate “Rifampicin Resistance” as Surrogate Marker for Rapid Detection of MDR-TB Using Real-Time PCR Directly on FNAC Samples of Tuberculous Lymphadenitis

Background: India has the dubious distinction of having second largest burden of MDR-TB cases in the world. According to WHO, MDR-TB is defined as resistance to isoniazid and rifampicin, the two most important drugs for treatment of TB. “Rifampicin resistance” is recommended as surrogate marker for MDR-TB by WHO, as at least, 90% of all rifampicin-resistant clinical isolates found resistant to isoniazid. Localization of genetic alterations in the 81-bp “Rifampicin Resistance-Determining Region” of rpoB gene in 96% of rifampicin resistant strains make PCR. Results: Eighteen samples were identified as MDR-TB cases by DST. Real-Time PCR picked up mutated ropB gene in 17 cases out of these 18 MDR-TB cases. Conclusion: “ Rifampicin resistance” is an efficient surrogate marker for timely detection of MDR-TB using rapid, accurate and sensitive molecular technique like Real-Time PCR.


INTRODUCTION
The spread of isoniazid and rifampicin resistant M. tuberculosis known as Multi Drug Resistant [MDR] is a major setback to tuberculosis [TB] control programs worldwide [1]. India is home to the second largest number of MDR-TB cases in the world [2]. Rifampicin resistance is recommended by WHO as "surrogate marker" for detecting MDR-TB because mono-resistance to rifampicin in M. tuberculosis is rare [1]. At least 90% of all rifampicin resistant clinical isolates are also resistant to isoniazid [1]. Genetic mutations are mainly responsible for resistance to rifampicin in M. tuberculosis [3]. Ninety six percent of rifampicin resistant M. tuberculosis strains acquire genetic alterations within 81-bp "Rifampicin Resistance-Determining Region" [RRDR] corresponding to codons 507 to 533 in the rpoB gene [4]. This region acts as an ideal target for molecular tests for rifampicin resistance. Real-Time Polymerase Chain Reaction (PCR) is one such accurate, sensitive and rapid molecular method that can yield virtually unlimited copies of specific DNA fragments from minute quantities of the source DNA material in very short time.
The key to prevent the rapid rise and spread of MDR-TB is early detection and appropriate case management.
Detection of "rifampicin resistance", the surrogate marker of MDR-TB, using rapid molecular technique like Real-Time PCR could save precious time and lives. This may also help in averting the catastrophic effect of drug resistance on TB control programs extensively relying on rifampicin as the first line drug of Anti-Tubercular Treatment.
In the present study, we evaluated "rifampicin resistance" as surrogate marker for rapid detection of MDR-TB using Real

MATERIALS AND METHODS
The prospective study was conducted on eighty clinico-cytologically confirmed cases of TBLN who had taken 5 months or more of standardized ATT (DOTS regimen) and showed persistence of initial LN or appearance of new

Culture
Before culture, the specimens were purified by digestion and decontamination by the N-acetyl-L-cysteine [NALC]-NaOH to prevent overgrowth by non-mycobacterial microorganisms in the culture. M. tuberculosis was cultured in the conventional LJ medium at 37ºC in 5% CO 2 for 1 week, at 37ºC without CO 2 for another 7 weeks and thereafter were observed once a week for M. tuberculosis growth.

DNA Extraction
Mycobacterial genomic DNA was extracted from the FNAC samples as described by Van Sooligen et al.
[8] with minor modifications. The quality and quantity of DNA was assessed by measuring absorbance ratio at 260/280 nm. An absorbance ratio within 1.8 to 2.0 suggested good quality extraction.

ZN Staining
Sixty three percent [51/80] cases were AFB [Acid Fast Bacilli] positive. None of these cases were gram stain positive. None of the 10 negative controls were AFB positive, 3 control samples from pyogenic abscess were gram stain positive for cocci.

DST Results
The results of DST were divided into following categories: i) 18 out of 30 cases were resistant to both isoniazid & rifampicin, i.e. MDR strains. ii) 11 cases were sensitive to both drugs iii) One case was sensitive to rifampicin but resistant to isoniazid. iv) 19 cases were resistant to isoniazid.

DISSCUSSION
MDR-TB is defined as resistance to isoniazid and rifampicin with or without resistance to other drugs [1]. Rifampicin is the most effective bactericidal drug that helps in shortening the duration of ATT [10]. It inhibits transcription and elongation of bacterial RNA by binding with the bacterial DNA-dependent RNA polymerase [11,12]. This RNA polymerase has 4 subunits but mutations in the rpoB gene coded sub-unit play a significant role in development of resistance. Mutations in this sub-unit prevent rifampicin from binding the bacterial DNAdependent RNA polymerase [11] making the bacilli resistant to the drug. Random, single step spontaneous genetic mutations are responsible for resistance to rifampicin in M. tuberculosis. High degree of resistance to rifampicin has been reported due to mis-sense mutations at codons 526 to 531of rpoB [3]. The reported frequency of mutation at codon 531 is 29-74%, at 526 is 0-43% and at 516 is 0-38% [13,14].
Importance of rifampicin resistance in MDR-TB is highlighted by the fact that WHO has recommended it as surrogate marker for detecting MDR-TB [1]. Rifampicin resistance has been linked with treatment failure and outbreaks, further adding to its clinical and public health relevance [1] . Also rifampicin resistance is more amenable for genotypic DST because 95-98% rifampicin resistance occurs via genetic mutations in 81 bp region of rpoB gene [3,4]. As compared to rifampicin resistance, molecular resistance to other anti-TB drugs like isoniazid is more complex and requires assessment of mutations in multiple genes for better correlation with phenotypic DST. Accurate genotypic DST is technically more challenging for the other firstline and second-line anti-TB drugs whereas molecular tests for rifampicin resistance are much more developed [15].
With the aim to evaluate "rifampicin resistance" as surrogate marker, the versatile rapid molecular technique of Real-Time PCR was used directly on 30 FNAC samples. Conventional DST using the proportion method [5] was also performed as it is considered "gold standard". By DST, 18 samples were found resistant to both isoniazid and rifampicin i.e. MDR-TB, 11 cases were susceptible to both the drugs and 1 case was sensitive to rifampicin but resistant to isoniazid. Thus 18 cases were found to be rifampicin resistant by the conventional DST. On analysis by Real-Time PCR using rpoB probe that covered RRDR [codons 531 and 526] of rpoB gene we could detect 17 of 18 [94%] rifampicin resistant isolates. Thus by both the methods, none of the rifampicin resistant cases were found to be sensitive to isoniazid. The study corroborates the fact that" rifampicin resistance" is an excellent surrogate marker for MDR-TB.
In the study, none of the cases found to be "rifampicin sensitive" by DST were revealed as "rifampicin resistant" by Real-Time PCR. Thus no false positives were observed with Real-Time PCR. Although the study cohort was limited, yet it brings out the high sensitivity of Real-Time PCR for detection of drug resistance. Sensitivity of Real-Time PCR for detecting rifampicin resistance has been reported at 82.8% by Mokrousov et al. [16] and 97.5% by Ruíz et al. [17].
In this study the sensitivity of Real-Time PCR was found to be 94.4% and the positive predictive value was one.
Although in recent years, several molecular techniques have been applied to detect mutations associated with anti-tubercular drug resistance, but most of these techniques have been evaluated in cultures. Very few studies in the literature have been published for direct detection of resistance in clinical samples [16,17,18,19,20,21]. The study also supports the fact that Real-Time PCR can be successfully applied on clinical samples directly (without the need for culture), as we were able to pick up 94% rifampicin resistant cases from FNA samples of TBLN.

Fig. 1. Real time PCR results showing two distinct peaks (Tm) for wild & mutant rpoB Gene. (Melt data for Melt A. Green)
In 3-5% of rifampicin resistant isolates, additional mechanisms like rifampicin permeability and novel mutations in alternate sub units of RNA polymerase may confer resistant phenotype [3,22]. In our study also Real-Time PCR could not detect mutation in the single sample that was resistant to rifampicin by DST. The probable reason could be that novel mutations in alternate sub units of RNA polymerase may have been missed by the probe which detected mutation in the "hot spot" region of the rpoB gene. Using probes with higher resolving power may further improve the specificity. However, our study corroborates findings from earlier studies from South India which have also found that codon 531 and codon 526 are most frequently involved in mutations [23,24].
Culture is the most specific method for diagnosis but is not very sensitive [<50%] and is also time consuming [results available only after 4-8 weeks] [25]. In the study too, culture based results were available after 6-8 weeks, but Real-Time PCR results were available within hours of DNA extraction. Culture requires viable organisms [10 to 100 viable organisms per sample]. This is often a problem in partially treated patients and pauci-bacillary cases [26]. Detection of M. tuberculosis DNA in clinical samples of such cases by Real-Time PCR is a promising approach for rapid diagnosis of the disease as this technique is independent of the viability of organism. Extra-pulmonary TB is even more difficult to diagnose as samples are frequently obtained using invasive or semiinvasive methods and may not have adequate number of bacilli [25].
Most of the studies [27,28] have reported culture positivity for FNAC samples of TBLN varying from 17-55.5%. Thus culture may not be available in many cases where DST needs to be performed before initiating appropriate treatment. In our study culture was positive in only 37% (30/80) cases, which limited the DST to only 30 out of the total 80 cases. Fifty cases could not be evaluated further. Reasons for low culture positivity in the study could be presence of scant bacilli and bactericidal substances as majority of the patients were receiving ATT and use of harsh decontamination procedure [29]. PCR positivity in culture negative samples highlights the importance of this technique in detection of drug resistance cases where culture based methods may not be available. Thus the main advantages of Real-Time PCR are rapid detection [1.5-2.0 hours after DNA extraction], quantitative analysis, ability to pick up very minute quantity of DNA independent of the viability of organism and a lower risk of contamination.
In a study by Payanandan et al. [30] the incidence of MDR-TB in Thailand was found to be same as that of rifampicin resistance, establishing that rifampicin resistance can be effectively used as surrogate marker for MDR-TB. In this study rifampicin resistance in 17 out of 18 MDR-TB cases was detected by rpoB probe targeting codons 531 and 526 indicating that rifampicin resistance could be effectively used as a proxy for MDR-TB, averting cost of analyzing the status of isoniazid resistance in suspected cases.
The alarming rise in MDR-TB is threatening to weaken TB control programs worldwide. There is an emergent need for rapid diagnosis of MDR-TB for early initiation of treatment, improved prognosis and interrupting transmission of MDR strains which is not possible with the conventional DST due to longer turn-aroundtime. Rifampicin resistance, surrogate marker for MDR TB, can be easily detected by targeting the 81-bp RRDR of rpoB gene using molecular methods like Real-Time PCR [31,32,33] that can yield rapid results for timely initiation of treatment of MDR-TB particularly in countries like India with high prevalence.

LIMITATIONS OF THE STUDY
In developing country like ours with high case burden of MDR TB, relatively faster, reliable and readily usable technique like Real-Time PCR could be of more value for timely & accurately detecting MDR-TB. We tried to assess the possibility of utilizing Rifampicin resistance (rpoB gene) as surrogate marker for MDR-TB using Real-Time PCR directly on FNA samples. It also needs to be mentioned that sequencing of rpoB gene was not a part of our study. The only limitation we feel regarding use of Real-Time PCR based methods are requirement of appropriate equipments and reagents, standardization of techniques, and availability of trained human resources.

CONCLUSION
The study underlines importance and usefulness of "rifampicin resistance" as surrogate marker for MDR TB using Real-Time PCR directly on FNAC samples to screen both naïve as well as treated TB cases to save valuable time.