The high morbidity and mortality associated with drug resistant tuberculosis (DR-TB) makes it a matter of grave concern [1]. The widespread availability of rapid molecular diagnostic techniques has improved the case detection rate for DR-TB [2]. World Health Organization (WHO) approved the first version of line probe assay (Genotype MTBDRplus Ver 1.0, Hain Lifescience, Nehren, Germany [LPA V.1]) in 2008. The utility was limited to higher grade of smear-positive and culture-positive samples [3]. An improved version of line probe assay (LPA) (Genotype MTBDRplus Ver 2.0, Hain Lifescience [LPA V.2]) was endorsed by WHO in 2011 [4] which has high sensitivity and specificity when compared to Xpert MTB/RIF [5,6].

DNA based molecular diagnostics are based on screening alterations in the nucleic acid sequences. The principle of LPA is based on rpoB gene, an 81 base-pair rifampicin (RIF) resistance-determining region which accounts about 95% of RIF resistance. Missing of wild-type (WT) bands with or without presence of mutation (MUT) bands is interpreted as resistant. RIF specific mutation, S531L, H26Y, H526D, and D516V are the most prevalent mutation found in rpoB gene in LPA [2,7]. Disputed mutations have also been observed at positions L511P, D516Y, H526L, and L533P in rpoB gene [8,9]. In LPA V.2, a strain having missing or light band of WT3 (codon 513 to 517) or WT8 (codon 530 to 533) without any MUT band, could be due to silent mutation [7] and failure of hybridization probe [10]. Non-interpretable (NI) results in LPA V.2., albeit a small proportion, are a diagnostic challenge. Our study evaluated the role of phenotypic drug susceptibility testing (pDST) and sequencing in NI results of LPA V.2.

Out of total 1,614 sputum specimens, 1,212 (75.1%) were smear-positive and 402 (24.9%) were found to be smear-negative using ZN staining and microscopy. Among smear-positive specimens, 1,166 of 1,212 (96.2%) were culture-positive, 22 of 1,212 (1.8%) were culture-negative and contamination was found in 24 of 1,212 (2%) specimens. Among smear-negative specimens, 152 of 402 (37.8%) were culture-positive, 228 of 402 (56.7%) were culture-negative and rest 22 of 402 (5.5%) were found to be contaminated in LC (Table 1). After satisfying the inclusion and exclusion criteria, 1,340 samples were included for the study. All smear-positive specimens were directly processed for the LPA while, in case of smear-negative specimens, LPA were done only when the culture turned positive in LC (indirect).

In last decade, the use of several molecular diagnostic techniques like Xpert MTB/RIF and LPA have increased the DR-TB case notification rate worldwide [12]. RIF-resistant (RR) associated rpoB gene hotspot (codon 507-533) is identified as the principle RR-TB defining region in all the nucleic acid amplification tests based molecular diagnostics [13].

We found that a low proportion of LPA V.2 results are NI (n=24, 1.8%). Silent mutations (WT3 [codon 514] and WT8 [codon 533]) and rare rpoB gene mutations [7,12] have been previously identified to cause NI results. In the recent version of LPA, mutation in WT8 band (codon 530-533) is a common cause of the NI result. In present study, rpoB associated WT8 band was completely missing without presence of any MUT bands in the strips (defined as NI) thereby leading to diagnostic dilemma. As per the programmatic guidelines in India, all RIF mono-resistant patients are eligible for treatment as MDR patient [1]. This limitation associated with LPA V.2 may lead to patients either misdiagnosed as RIF-sensitive TB resulting in high failure and relapse rate or RR-TB resulting in unnecessarily being treated as MDR-TB [14,15].

In this study, the most frequent mutation in rpoB coded gene was found at codon S531L in both RIF mono-resistant (n=7, 58.3%) as well as in MDR-TB isolates (n=157, 74.2%) which is consistent with the previously published studies [6,16,17]. For the detection of INH resistant, LPA is equipped with two genes katG and inhA, both of them showed similar frequency of mutation as published previously [6]. Apart from the predesigned mutation bands in rpoB coded gene, other mutations like L530P (n=2, 9.1%), S531F (n=6, 27.2%), S531W (n=10, 45.5%), A532V (n=2, 9.1%), and L533P (n=2, 9.1%) were observed in sequencing and these were also found resistant to RIF in pDST. In previous studies, disputed mutation at position L533P has been reported on both Lowenstein-Jensen and MGIT-960 culture medium [18,19]. Two specimens were sensitive to both sequencing and pDST. No MUT band was observed in LPA of these samples possibly due to failure of hybridization probe. The similar explanation has been quoted by Seifert et al. (2016) [10]. These patients’ isolates could have missed the proper resistant patterns to start the required treatment with single diagnostic test alone. This type of mutation frequencies was reported in ≥10% of total mutations associated with rpoB gene [19]. Mutation at codon 533 (L533P) is likely to be missed by Xpert MTB/RIF [20], but usually detected in LPA V.2, as seen in present study also (L533P [n=2, 9.1%]). LPA V.2 covers more coverage regions of rpoB gene (from codon 505 to 534) when compared to Xpert MTB/RIF (codon 509 to 533) [20]. However, some other deletions were also noted within and out of the hot spot region of rpoB gene which were associated with low level resistant in pDST [21,22]. Sequencing aided the identification of some other polymorphism in rpoB gene also. This finding of our study underscores that uncommon and/or silent mutation can cause NI results which can cause therapeutic dilemmas. Similarly, Nikam et al. [23] observed NI results in both RIF and INH without mentioning any specific loci of genes in MTBDRplus assay. Their study also highlighted this limitation of LPA which is especially of concern in TB endemic areas.

Therefore, this study highlights the limitations of LPA in detection of mutation patterns associated WT8 region (codon 530-533) [7]. To overcome this limitation, pDST and sequencing are recommended in select cases, such as NI results from LPAs. In the present study, we showed the usefulness of pDST and sequencing for elucidating the NI results from the Genotype MTBDRplus assay.

In most real-life scenario in high TB burden areas, sequencing may not be feasible. pDST can be a reasonable alternative in resource-limited settings. However, more prospective studies with clinical correlation can throw more light in this regard.

In the present study, we found that a small proportion of clinical samples were reported as NI in the Genotype MTBDRplus assay. Almost all of the strains with NI results (22/24, 91.7%) were RR in the pDST. Sequencing detected various rpoB mutations in these 22 cases. These results suggest that pDST and rpoB sequencing are useful in elucidating the cause and determining the clinical meaning of NI results from the Genotype MTBDRplus assay.