Clinical specimens
A total of 241 baseline sputum specimens collected as part of a standard patient care were randomly selected from clinical specimens sent to our TB laboratory between October 2012-February 2013.
Ethics
The study protocol was reviewed and approved by the Institutional Review Board (IRB) at Joint Clinical Research Centre (JCRC). Individual informed consent was not sought because the study was conducted on routine samples only and it did not involve any intervention, additional samples or change in patient management. A patient consent waiver was approved by the IRB of JCRC.
Processing of sputum specimens
All specimens were decontaminated according to the available laboratory protocol [14]. In brief, specimens were 1:1 mixed with N-acetyl-l-cysteine (NALC)-NaOH (final concentrations 1.5% NaOH, 0.7% NaCitrate, 0.25% N-acetyl-cysteine) and put on plat form shaker (Thermo scientific Inc. USA) at 60 rpm for 20 min. After neutralization with 0.5 M phosphate buffer (pH 6.8) and centrifugation (3000 × g for 20 min) in order to concentrate the mycobacteria, the sediment was re-suspended with 2 ml phosphate buffer.
Of the sediment, 500 μl were inoculated into Mycobacteria Growth Indicator Tubes (MGIT™) (Becton-Dickenson, Heidelberg, Germany) already supplemented with 800 μl final concentration of 12.5 U/ml polymyxin B, 1.25 mg/ml amphotericin B, 5 mg/ml nalidixic acid, 1.25 mg/ml trimethoprim and 1.25 mg/ml azlocillin (PANTA) and incubated in the Bactec™ MGIT 960 system (Becton, Dickinson and Company, Franklin Lakes, NJ) according to the manual of the manufacturer. The leftover suspension (500 to 1000 μl) was kept at 2–8°C until further processing in the frame of the present study. The tubes were automatically and continuously monitored for growth and remained in the instrument until it signaled positive for growth or negative at the end of the 42-day incubation. All liquid cultures that turned positive, were screened for acid fastness using Ziehl Neelsen microscopy, inoculated on blood agar plates to detect contaminants and then M. tubeculosis confirmed using MPB64-protein based immuno-chromatographic assay (capilia TB, TAUNS, Japan) following manufacturers’ guidelines. All the Mycobacteria other than tuberculosis (MOTTs) cultures were screened to confirm the presence of M. avium and M. kansasii using DNA line probe assay (GenoType Mycobacterium CM, Hain Lifesciences, Nehren, Germany) (see Figure 1).
Isolation of genomic DNA
In brief, 500 μl of the decontaminated sputum sample was spun and pellet re-suspended in 20 μl of nuclease free water; heat killed in a heat block set 95°C for 1 hour to lyse the bacteria and later sonicated at 37 kHz (Elma S30, Gottlieb-Daimler-Str. Singen, Germany) for 15 minutes. The resultant genomic DNA in the supernatant was recovered by centrifugation at 8000 g for 3 min for eventual use in the Real time PCR assay.
Quality control
Reagents were aliquoted and each aliquot was used only once. Sterile microfuge tubes and 96 PCR well plates for Real time PCR assay use. Reagent preparation, DNA extraction, DNA amplification and detection were performed in separate rooms to avoid cross contamination of amplicons.
A positive control (Mycobacterium tuberculosis H37Rv, M. avium and M. kansasii) was included in each test and distilled water was included as a negative test control. Uracil-N-glycosylase (UNG) was used in the amplification process to avoid post PCR DNA contamination.
Mycobacterium real time PCR
Real time PCR was performed on a LightCycler® 480 II (Roche diagnostics, Mannheim, Germany) according to the manufacturer’s instructions using light cycler mycobacterium detection assay. This assay comprised of two major steps: (i) Amplification of internal control sequences and genomic target sequences (ii) melting curve detection by florescence measurement at 640 nm. A 20 μl reaction mixture contained 4 μl of sample lysate (or 4 μl of positive/negative control), 11 μl detection mix (primers and probes), 4 μl of PCR master mix (Taq polymerase, DNTPs, Mg2+), 0.75 μl internal control and 0.25 μl uracil-DNA gylcosylase. Thermal cycling was as follows: 10 min at 95°C, then 45 cycles of 10 sec 95°C, 10 sec 50°C and final extension of 20 sec at 72°C. Melting curve detection to determine the melting temperature (Tm) values for the target sequences was set as follows: 1 min at 95°C, 2 min at 40°C, 75°C continuous and then cooling at 10 sec at 40°C.
Detection
The LightCycler® 480 II analyzed the samples in 2 steps: (i) PCR amplification of the target region where the target amplicon for each sample were detected between the annealing and elongation as sigmoid curves at 640 nm (see Figure 2) (ii) Tm calling using the LightCycler 480®software to determine the melting temperature (Tm) specific for each subtype in the samples (see Figure 3). Range Tm included according to the manufacturer instructions include; 54.4 to 57.4°C for M. tuberculosis, 47.5 to 50.5°C for M. avium and 57.5 to 60.5°C for M, kansasii.
Analysis of data
The time that elapsed from MGIT sample inoculation and incubation to MGIT culture positivity or negativity was registered as the turnaround time (TAT) for liquid culture. The time difference between the start and stop time for each real time PCR run was used as the turnaround time for Real time PCR assay. The unit sample cost between Real time PCR and liquid culture use was achieved by comparing the requirements and their costs to test 241 clinical specimens for identification of M. tuberculosis, M. avium and M. kansasii.
Statistical data were entered and analyzed using Epi™ StatCal info version 7 software (CDC, Atlanta). The values got were validated using statistical diagnostic software MedCalc version 15.2.2 (MedCalc Software bvba, Belgium).