Evaluation of newer drugs (Linezolid & Meropenem) and classical second line drugs (Amikacin & Levofloxacin) in clinical isolates of MDR-TB utilizing broth based Bactec MGIT 960 technique

Abstract

With the increase in MDR-TB strains around the globe, there has been an urgent need to carry out drug susceptibility to first and second line antituberculosis drugs. It is imperative that treatment of patients suffering from drug resistant TB should be carried out based on quick, reliable and quantitative measure of susceptibility testing. This endeavor is a cornerstone for prevention of resistance in treatment of tuberculosis and a way forward for optimal exploitation of the available antituberculosis drugs (Mukherjee et al., 2004). The increase in MDR-TB rates has lead to pressing demands for appropriate treatment with second line antituberculosis drugs and need to find newer compounds with potential in vitro activity against MDR-TB. A number 17 of antimicrobial compounds i.e. Linezolid, Levofloxacin, Moxifloxacin, Carbepenems and Amoxicillin/ clavulananic acid have been considered as potentially active agents against MDR- TB (Schectoret al., 2009; Wong et al., 1988; Bozeman et al., 2005). The reliable drug susceptibility testing method provides us with detailed knowledge on quantitative drug resistance pattern which ultimately paves the way for empirical treatment of drug resistant tuberculosis. During the last decade or so MGIT 960 system has been extensively studied and validated for susceptibility testing of first line antituberculosis drugs (Bemer et al., 2002). The multicentre laboratory validation of the BACTEC MGIT 960 technique for testing susceptibilities of M. tuberculosis to classical second line drugs and newer antimicrobials (Rusch-Gerdes et al., 2006) has provided us with a guideline for resource poor countries like Pakistan to endeavor testing such compounds against our local isolates. According to WHO global report 2013, tuberculosis culture facility in Pakistan is possible in only seven laboratories accounting to 0.2 laboratory per 5 million population while in whole country only four laboratories can perform drug susceptibilities accounting to only 0.1 laboratory per 5 million population. To add fuel to the fire, Pakistan also could not achieve the target of having at least one centre for carrying out smear microscopy under the WHO global plan to stop TB 2011-2015 (WHO, 2013 ). In the backdrop of such sorry state of affairs our laboratory was one of the very few in Pakistan with capacity to carry out DST to first and second line antituberculosis drugs (Ghafoor et al., 2014)). With the aim of finding the susceptibility pattern to classical second line and newer investigational drugs, it was challenge to embark upon journey on the guidelines provided by validation studies. Aims & Objectives The study has been undertaken keeping in view two objectives. 1. To evaluate the in vitro effectiveness of multiple concentrations of newer compounds (Linezolid and Meropenem) against MDR-TB isolates in Pakistani population. 2. To find out the susceptibilities of MDR-TB isolates against Amikacin and Levofloxacin to find out extent of XDR & pre XDR TB cases in our set up. Study Design It is a quantitative cross sectional research carried out at Armed Forces Institute of Pathology, Rawalpindi Pakistan from Sept, 2011 to Aug, 2013. Material and Methods The methodology of study comprised of two parts. The first part comprised of all the procedures leading to the determination of MDR-TB, while the second part consisted of evaluation of newer compounds (Linezolid & Meropenem) and two of the classical second line antituberculosis drugs (Amikacin & Levofloxacin). All MDR-TB isolates were subjected to susceptibility testing against two classical second line drugs Amikacin (AK) and Levofloxacin (LEVO). These drugs were obtained from chemically pure form from (Sigma, Taufbirchen, Germany). Amikacin disulfate salt 710 μg/mg cat.N. A1774 with storage at 2-8 ͦ C manufactured by Sigma and Levofloxacin > 98% HPLC cat.N. 28266 with storage at 2-8 ͦ C manufactured by Sigma were used. These drugs were dissolved in deionized water. The stock solutions of AMK (84μg/l and LEVO (84μg/ml) were prepared. Before subjecting the MDR-TB isolates to the test drugs full susceptible and quality control strain (ATCC 27294) was subjected to the critical concentration of drug used. The drugs panel consisted of three MGIT tubes, one for growth control and two for second line drugs. Each 7ml MGIT tube was checked for any contamination or turbidity and labelled properly. After mixing the growth supplement (OADC), 0.1 ml of each antibiotic stock solution was added in respective MGIT tubes. 0.5ml of culture proven MDR-TB sample was added to two MGIT tubes while 0.5ml of 1:100 diluted sample was added to control tube. After bar code scanning all the inoculated tubes were entered in the instrument and incubated at a temperature of 37oC. An un-inoculated MGIT tube was used as a negative control. All MDR-TB isolates were separately subjected to susceptibility testing against two newer investigational drugs Linezolid (LNZ) and Meropenem (MER). Linezolid was provided by Continental pharmaceuticals Karachi while Meropenem was provided by Adam & Musa Jee Karachi. Linezolid as pure substance ca.100% Cat.N. 165800-03-3 with storage recommendation at room temperature and manufactured by Pfizer was used. These drugs were dissolved in deionized water. The stock solutions of LNZ and MER (84 μg/ml) were prepared in sterile water as per instructions provided in leaflets of respective drugs. The three concentrations were used for both drugs for BACTEC MGIT 960 system. For Linezolid 0.5, 1.0 & 2.0 μg/ml and for Meropenem 4.0, 8.0 & 16μg/ml respectively (Rusch-Gerdes et al., 2006). Before subjecting the MDR-TB isolates to the test drugs full susceptible and quality control strain (ATCC 27294) was subjected to the three concentrations of drugs used. The drugs panel consisted of three MGIT tubes, one for growth control and two for each of the three concentrations of investigational drugs. Each 7ml MGIT tube was checked for any contamination or turbidity and labelled properly. Mixing of the growth supplement (OADC), and further processing with antibiotic stock solutions and addition of cculture proven MDR-TB sample was similar to Second line drugs. The MGIT 960 system flags the completion of a DST when the growth unit (GU) of the growth control reaches 400 and reports S for susceptible or R for resistant, as well as a GU value for each drug-containing MGIT tube on the printout. An isolate was interpreted to be susceptible when the GU of a drug-containing MGIT tube was equal to or less than 100 or as resistant when the GU was greater than 100. If an isolate was interpreted to be resistant, a smear was made and stained to prove the presence of acid-fast bacilli (AFB) with morphology compatible with that of MTBC and the absence of contaminants. So based on the multi centre validation studies to find out cut off concentrations of second line and newer drugs for testing with MGIT 960 method (Rodrigues et al., 2008; Sanders et al., 2004). Following concentrations were used for the interpretations of MDR-TB isolates being sensitive or resistant. 1. Amikacin 1.0 μg/ml 2. Levofloxacin 2.0 μg/ml 3. Linezolid 1.0 μg/ml 4. Meropenem 4.0 μg/ml Data management and analysis All data was analyzed and processed using statistical software (Statistical Package for Social Sciences; SPSS 19. Results were expressed as frequencies or as mean ± SD unless indicated otherwise. The Pearson chi-square test was used to determine the association between classical second line drugs as well as susceptibilities of MDR TB isolates to different concentrations of Linezolid and Meropenem. We considered p value less than 0.05 to be significant.