Primary Multidrug Resistant Tuberculosis and Utility of Line Probe Assay for Its Detection in Smear-Positive Sputum Samples in a Tertiary Care Hospital in South India

Primary Multidrug Resistant Tuberculosis and Utility of Line Probe Assay for Its Detection in Smear-Positive Sputum Samples in a Tertiary Care Hospital in South India Fahmiya Leena Yacoob, Beena Philomina Jose, Sarada Devi Karunakaran Lelitha, and Sreelatha Sreenivasan Department of Microbiology, Government Medical College, Kozhikode, Kerala 673008, India Received 16 October 2015; Revised 5 January 2016; Accepted 2 March 2016 Academic Editor: Abhineet S. Sheoran Copyright © 2016 Fahmiya Leena Yacoob et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract In a high tuberculosis burdened country like India, rapid, cost-effective, and reliable diagnostic tools for tuberculosis are an urgent need of the hour to prevent inappropriate treatment strategies and further spread of resistance. This study aimed to estimate the proportion of new smear-positive tuberculosis cases with primary resistance to rifampicin and/or isoniazid as well as identify the common mutations associated with it. Sputum of 200 newly diagnosed smear-positive cases of 1+ score and above was directly subjected to Line Probe Assay using the GenoType MTBDRplus assay kit. All samples were inoculated onto solid media and 61 samples were inoculated in automated liquid culture also. The Line Probe Assay gave hundred percent interpretable results with 2.5% of the study population showing resistant pattern. Only 1% of the cases were primary multidrug resistant tuberculosis and 1.5% showed isoniazid monoresistance. S531L and C15T were the most common genetic mutations seen for rifampicin and isoniazid resistance, respectively. 40% had absent rpoB wild type 8 band indicating probable silent mutation after clinical correlation. The average turnaround time for Line Probe Assay was far less (3.8 days) as compared to solid and liquid cultures (35.6 days and 13.5 days, resp.). 1. Introduction Tuberculosis (TB) earned the sobriquet “The Captain of All These Men of Death” during the 18th and 19th century for the morbidity and mortality it caused and now ranks alongside HIV as a leading cause of death worldwide [1]. India ranks first among the high TB burdened countries contributing to 24% of the estimated global incidence. It is also one of the three countries contributing to more than half of the global burden of multidrug resistant tuberculosis (MDR TB), the other two being China and the Russian Federation [2, 3]. Improved rapid diagnostic methods, vigilant monitoring of case reporting, and follow-up through national surveillance programs in these countries will have a global impact on the TB statistics. Although MDR TB is reported to be low with 2.2% among new cases and 15% among retreatment cases, due to the large population and high TB burden, it translates into a high number of actual cases (71,000 estimated MDR TB cases among notified pulmonary TB cases in 2014) [1]. Sputum smear microscopy remains the mainstay of diagnosis due to its simplicity, cost-effectiveness, and feasibility. However, molecular diagnostic techniques like GeneXpert, Polymerase Chain Reaction (PCR), and Line Probe Assays (LPAs) are being increasingly used recently. These newer techniques aid in the rapid diagnosis of drug resistance and thus prevent inappropriate treatment regimes, amplification, and spread of drug resistance in countries like India with high TB load. The World Health Organization (WHO) recommended a new policy to use LPAs for rapid screening of patients at risk of MDR TB in 2008 [4]. The LPA is a reverse hybridization technique wherein the patient’s sample, after undergoing DNA extraction and amplification, is subjected to hybridization with membrane strips coated with complementary probes targeted against specific genes. Highly specific binding of complementary DNA strands is ensured by stringent conditions which result from the combination of buffer composition and a certain temperature. During hybridization, streptavidin-conjugated alkaline phosphatase is added which binds to the amplicon’s biotin via the streptavidin moiety. Finally, the alkaline phosphatase transforms an added substrate into a dye which become visible on the membrane strips as a coloured precipitate. A template helps in the interpretation of the banding patterns obtained. GenoType MTBDRplus assay (Hain Lifesciences, GmbH, Nehren, Germany) targets the rpoB (coding for beta subunit of RNA polymerase), katG (coding for catalase peroxidase), and promoter region of inhA (coding for NADH enoyl ACP reductase) genes in both culture isolates and clinical samples. Thus, in addition to rifampicin resistance (rpoB gene), MTBDRplus assay aids in the detection of high level and low level resistance to isoniazid (INH) via the katG gene and inhA gene, respectively. In India, as per the Revised National TB Control Programme (RNTCP), the definition of presumptive drug resistant TB (DR TB) cases includes all retreatment cases at diagnosis, any smear-positive cases during follow-up, contacts of confirmed DR TB cases, and HIV associated TB cases at diagnosis [5]. For drug susceptibility testing (DST), rapid molecular tests like LPA and CB-NAAT (cartridge based-nucleic acid amplification tests), if available, are the preferred DST methods for first-line drugs as per RNTCP protocol [5]. This study aimed at finding the proportion of cases with primary drug resistance in the study population and to assess the utility of LPA in our set-up in comparison to solid and automated liquid culture systems. 2. Materials and Methods2.1. Specimen Collection and Storage Early morning expectorated sputum samples of 200 new smear-positive cases of grade 1+ and above were collected in sterile leakproof containers during the study period of one and a half years. All the samples were stored at a temperature of 2–8°C for not more than 4 days before decontamination. 2.2. Specimen Processing2.2.1. Direct Smears The specimens were stained by Ziehl-Neelsen technique. The smears were graded as “scanty” for 1–10 AFB/100 oil emulsion fields (OIF), “1+” for 10–99 AFB/100 OIF, “2+” for 1–9 AFB/OIF, and “3+” for >10 AFB/OIF. Smears graded “scanty” positive were excluded from this study. 2.2.2. Decontamination It was done by N-acetyl-L-cysteine- (NALC-) NaOH-sodium citrate method in which equal amounts of 4% NaOH and 2.9% sodium citrate were mixed with NALC powder (0.5 g NALC/100 mL of NaOH-sodium citrate solution). This mixture was used within 24 hours of preparation. In a 50 mL plastic sterile conical bottom graduated centrifuge tube, specimen ≤ 10 mL and NALC solution were added in equal volume. The capped tubes were vortexed (about 5–20 seconds/tube) and after allowing it to stand for 15–20 minutes, the tubes were filled to 50 mL with sterile phosphate buffer solution at pH 6.8 and swirled by hand to mix. The specimens we (...truncated)