Direct detection of resistance to fluoroquinolones/SLIDs in sputum specimen by GenoType MTBDRsl v.2.0 assay A study from Eastern Uttar Pradesh, India

According to World Health Organization (WHO), drug-resistant tuberculosis (DR-TB) is a major contributor to antimicrobial resistance globally and continues to be a public health threat. Annually, about half a million people fall ill with DR-TB globally. The gradual increase in resistance to fluoroquinolones (FQs) and second-line injectable drugs (SLIDs), poses a serious threat to effective TB control and adequate patient management. Therefore, WHO suggests the use of GenoType MTBDRsl v.2.0 assay for detection of multiple mutations associated with FQs and SLIDs. Hence, the study was conducted to determine the prevalence of resistance to FQs and SLIDs by comparing direct GenoType MTBDRsl v.2.0 assay with phenotypic drug susceptibility testing (DST). The study was conducted on 1320 smear positive sputum samples from a total of 2536 RR-TB, confirmed by GeneXpert MTB/RIF. The smear positive specimens were decontaminated, and DNA extraction was performed. Furthermore, the extracted DNA was used for GenoType MTBDRsl v.2.0 assay. While 20% of the decontaminated specimens were inoculated in Mycobacterium growth indicator tube (MGIT) for drug susceptibility testing (DST). Out of 1320 smear positive sputum samples, 1178 were identified as Mycobacterium tuberculosis complex (MTBC) and remaining were negative by GenoType MTBDRsl v.2.0 assay. Of the 1178 MTBC positive, 26.6% were sensitive to both FQs and SLIDs, whereas 57.3% were only FQs resistant and 15.9% were resistant to both FQs and SLIDs. Further DST of 225 isolates by liquid culture showed that 17% were sensitive to both FQs and SLIDs, 61.3% were only FQs resistant and 21.3% were resistant to both. The specificity for FQs and SLIDs was 92.31% and 100% whereas sensitivity was 100% respectively by GenoType MTBDRsl v.2.0 assay in direct sputum samples. Our study clearly suggests that GenoType MTBDRsl v.2.0 assay is a reliable test for the rapid detection of resistance to second-line drugs after confirmation by GeneXpert MTB/RIF assay for RR-TB. Though, high rate FQ (ofloxacin) resistance was seen in our setting, moxifloxacin could be used as treatment option owing to very low resistance.


Background
Drug-resistant tuberculosis (DR-TB) has emerged as an enormous global challenge for TB control. It develops either because of an infection with a resistant strain or as a result of inadequate treatment. Multidrug-resistant TB (MDR-TB) is the form of TB that is resistant to rifampicin and isoniazid, the two most powerful anti-TB drugs [1]. It is of grave concern to public health, with higher mortality rates than drug-susceptible TB [2]. These TB patients require treatment with second-line drugs that are costlier and have increased toxicity; and remain infectious longer than patients infected with drug-susceptible strains [3]. Extensively drug-resistant TB (XDR-TB) is defined as MDR-TB plus resistance to fluoroquinolones (FQs) and one of the second line injectable drugs (SLIDs) utilized in MDR-TB treatment regimens [4].
With the increasing use of the GeneXpert MTB/RIF assay for simultaneous detection of TB and resistance to rifampicin (without further testing for isoniazid resistance), an increasing number of RR-TB cases are being detected and notified [5]. Rifampicin resistant TB (RR-TB) is considered as a surrogate marker for MDR-TB [6].
In recent years several molecular methods have been developed for the diagnosis of tuberculosis and detection of drug resistance, including line probe assays [8]. Most of the species of Mycobacteria grow slowly with a generation time of 18 to 24 h; resulting in delay of TB diagnosis by conventional methods [9]. Molecular techniques reducing turnaround time (TAT) are able to detect and identify Mycobacterium tuberculosis complex (MTBC) within 1-2 days instead of several weeks with the traditional methods [10]. It is essential to detect drug resistance to second-line drugs before initiating treatment for better management of MDR-TB.
Line probe assays (LPAs) detect mutations associated with drug resistance. The Genotype MTBDRplus assay (Hain Lifescience Nehren Germany) identifies MTBC and detects resistance to rifampicin and isoniazid. Gen-oType MTBDRsl v.1.0 assay detects resistance to FQs, SLIDs and ethambutol (EMB) [11]. However, in recent years several novel mutations were identified that also confer resistance. Thus, WHO recommended the use of GenoType MTBDRsl v.2.0 assay (Hain Lifescience Nehren German) which detects multiple mutations which are associated with resistance to FQs and SLIDs. Mutations in gyrA and gyrB are detected for resistance to FQs, while resistance to SLIDs are detected through mutations in rrs and eis gene [12]. In 2018, Revised National Tuberculosis Control Program (RNTCP) recommended GenoType MTBDRsl v.2.0 as the initial test, instead of phenotypic drug susceptibility testing (DST) [13]. Uttar Pradesh is a region where highest number of TB cases are reported annually in India (https:// tbfac ts. org/ tb-stati stics-india/). With this in mind, the study was designed to compare the direct GenoType MTBDRsl v.2.0 assay with phenotypic drug susceptibility testing and determine the prevalence of resistance to FQs and SLIDs.

Location and samples
The study was undertaken in TB Culture & DST lab, Department of Microbiology, Institute of Medical Sciences, Banaras Hindu University, Varanasi, India, over the period of January 2019 to December 2019. Specimen processing, inoculation, and DST were performed in RNTCP certified laboratory with well-equipped BSL II & III facilities under standard biosafety precautions. All presumptive TB samples were directly detected by GeneXpert MTB/RIF assay, which identified TB along with rifampicin resistance (RR). Since RR is a surrogate marker for MDR-TB, the diagnosis of MDR-TB was based on this data. Over a period of one year, a total of 2536 RR-TB sputum specimens were collected, among which 1320 samples were smear positive and 1216 were smear negative. The smear positive samples were included in this study and subjected to GenoType MTB-DRsl v.2.0 assay while phenotypic DST was performed for 20% of the samples. Every fifth sample was systematically selected for culture.

Specimen processing and Genomic DNA extraction
The collected specimens were decontaminated as described elsewhere [6,14]. Briefly, specimens were decontaminated using N-acetyl-L-cysteine and sodium hydroxide (NALC-NaOH) method. The decontaminated and concentrated sediments were used for Ziehl-Neelsen staining (ZN staining) as well as for DNA extraction and inoculation into BACTEC MGIT 960 (BD, USA) automated liquid culture system for DST.
DNA extraction was performed by using GenoLyse kit (Hain Lifescience, Nehren, Germany) according to manufacturer's instructions. In brief, 1000 μl of the decontaminated sample was centrifuged at 10,000×g for 15 min. Supernatant was discarded and the pellet was re-suspended into 100 μl lysis buffer followed by vortexing for 30 s. This suspension was incubated in water bath at 95 •C for 5 min. Following this 100 μl neutralization buffer was added and vortexed for 30 s, then it was centrifuged at 13,000×g for 5 min. The supernatant containing DNA was transferred in a separate tube for further use.

GenoType MTBDRsl v.2.0 assay
The extracted genomic DNA was subjected to GenoType MTBDRsl v.2.0 assay for the detection of MTBC and resistance to FQs and SLIDs, according to the manufacturer's instructions (Hain Lifescience, Nehren, Germany).

Drug susceptibility testing
For 264 samples a portion of the decontaminated samples (0.5 ml) was inoculated into a mycobacterium growth indicator tube (MGIT) and incubated in an automated BACTEC MGIT-960 system for a maximum of 42 days, with constant monitoring. ZN microscopy was performed from each positive-flagged MGIT tube to check for the presence of acid-fast bacilli (AFB). Simultaneously, a loopful of liquid culture was inoculated in the brain heart infusion (BHI) agar and incubated for 24 h to check the sterility of the positive culture tube [15]. To confirm MTBC, immunochromatography-based MPT64 rapid antigen detection kit (SD BIOLINE TB Ag, South Korea) was used [16]. The confirmed MTBC isolates were further subjected to DST using MGIT 960 system for ofloxacin (2ug/ml), kanamycin (2.5ug/ml), capreomycin (2.5ug/ml) and moxifloxacin (0.25ug/ml). The H37Rv strain was used as a quality control testing in DST.

Statistical analysis
The sensitivity and specificity of GenoType MTB-DRsl v.2.0 assay was analyzed with MedCalc statistical software taking the phenotypic DST as gold standard.  Table 1.

Concordance between GenoType MTBDRsl v.2.0 assay and Phenotypic DST
For assessing the performance of GenoType MTBDRsl v.2.0 assay, phenotypic DST was taken as gold standard. GenoType MTBDRsl v.2.0 assay accurately identified all strains except one, as shown in Table 2, thus the overall concordance between GenoType MTBDRsl v.2.0 assay and phenotypic DST was 224/225.

Discussion
The present study highlights the rapid and accurate diagnosis of drug resistant-TB directly from sputum specimens by using GenoType MTBDRsl v.2.0 assay, whereas, previous studies have shown that it could be performed on DNA extracted from culture isolates [17][18][19][20]. According to WHO, an effective treatment regimen depends on optimal susceptibility testing of M. tuberculosis to anti-TB drugs [21]. Under TB control programs DR-TB portends a prominent threat globally. Efficient treatment of MDR-TB and XDR-TB is very expensive, which is especially an issue in low-income countries. Therefore, a sensitive and specific diagnostic tool is essential for effective diagnosis and subsequent treatment.
In our study, the specificity for FQs and SLIDs was 92.31% and 100% whereas sensitivity was 100% respectively by GenoType MTBDRsl v.2.0 assay in direct sputum samples when compared against phenotypic DST. A previous study by Tagliani et al. [21] demonstrated a sensitivity and specificity of GenoType MTBDRsl v.2.0 assay for FQs was respectively 93% and 98.3% and for SLIDs 88.9% and 91.7%, from culture positive isolates when compared with phenotypic DST.

Conclusions
From this study we can clearly suggest that the sputum specimen with the clinical diagnosis of MTB could be directly subjected to GenoType MTBDRsl v.2.0 assay, after confirmation by GeneXpert MTB/RIF for rifampicin resistance (RR) and ZN staining. This bypasses the long incubation period required for liquid culture and DST and may help for the rapid detection of second line drug resistance for FQs and SLIDs in order to initiate treatment among the critical patients. The findings showed alarming level of FQ (ofloxacin) resistance from the geographical region (Uttar Pradesh) of India which contributes the highest cases of TB. However, another drug moxifloxacin could be used as treatment option owing to very low resistance.