Rapid and accurate detection of RMP- and INH- resistant Mycobacterium tuberculosisin spinal tuberculosis specimens by CapitalBio™ DNA microarray: A prospective validation study

From March 2009 through December 2011, we performed this prospective validation study in Southwest Hospital, Chongqing, China. One hundred and fifty-three consecutive patients with clinically and pathologically diagnosed spinal tuberculosis were enrolled into this study. Clinical specimens (e.g., pus, caseous or granulation tissue) were collected during surgery and were divided into two aliquots for tuberculosis detection; one of the aliquots was subjected to species identification and detection of gene mutations by the CapitalBio™ DNA microarray (CapitalBio Corp., Beijing, China), and the other aliquot was processed for culture and DST. The study was approved by the Ethics Committee of Southwest Hospital. Written informed consent was obtained from all of the patients.

Diagnosis of spinal tuberculosis was made with reference to clinical and radiological findings and was verified histopathologically after debridement in all patients. Clinical and laboratorial presentation: Fever, night sweat and weight loss, back pain, neurological signs, angular deformity, positive response to anti-tuberculosis treatment, relative lymphocytosis, a low level of haemoglobin and a raised ESR. Radiology: X-ray, CT and MRI revealed bone destruction, vertebral collapse, kyphosis, tubercular lesions and paravertebral abscess. Histopathology: Epitheloid granulomas, a granular necrotic background and lymphocytic infiltration.

Oligonucleotide probes were printed onto OPAldehydeslide aldehyde-activated slides at a concentration of 10 μmol/L in 50% dimethyl sulfoxide using a SmartArrayer-48 microarrayer (both from CapitalBio Corp., Beijing, China) and were covalently immobilized on the slides via an amino group at their 5’ ends (spotting pattern shown in Figure 1). In each array, sixteen oligonucleotide probes were designed to detect mutations of rpoB (codons 531, 526, 513, 516, 511, 533), inhA (nucleotide-15 within the promoter) and katG (codon 315) (Figure 1 a, b, c). In addition, seventeen oligonucleotide probes were chosen in several species-specific sequence regions of the 16S rRNA gene for identification of different Mycobacterium species (Figure 2).

Spinal tuberculosis specimens were subjected to DNA microarray analysis at the Laboratory Department of Southwest Hospital. Investigators and laboratory staff were blinded to the culture and DST results of the clinical specimens. The CapitalBio™ DNA microarray was performed according to the manufacturer’s instructions [11, 12]. Briefly, 1–2 ml of the spinal tuberculosis specimen was inactivated for 30 min at 95°C followed by sonication in an ultrasonic water bath for 25 min. Next, the inactivated specimen was liquefied with an equal volume of liquefying solution (containing 0.5% N-acetylcysteine, 1.45% trisodium citrate and 2% NaOH) for 30 min at 37°C. After inactivation and liquefaction, 1 ml of the specimen was centrifuged at 12000 rpm for 5 min to pellet the bacteria. Following centrifugation, the supernatant was discarded, and the pellet was resuspended in 1 ml of 0.9% (w/v) saline and centrifuged at 12000 rpm for 5 min. This supernatant was subsequently discarded, and the pellet was resuspended in 50 μl of 10 mM Tris-EDTA buffer and then transferred to an extraction tube. The total DNA was isolated from the sample by vortexing the tube at maximum speed in an Extractor™ 36 (CapitalBio) for 5 min. Next, the extraction tube was incubated at 95°C for 5 min, centrifuged briefly and the supernatant was stored at −20°C until use. Next, asymmetric PCR was performed on the samples by a Peltier PTC225 thermal cycler (MJ Research, Watertown, MA) for two amplification rounds according to the manufacturer’s instructions. Following PCR amplification, chip hybridization was performed on the samples in a three-dimensional tilting agitator BioMixer II hybridization; an automated SlideWasher-8 (both from CapitalBio) was then used to wash and dry the hybridized slides. The fluorescent signal on the slides that was emitted by the microarrays was detected using a GeneArray scanner (LuxScan™-10K confocal laser scanner, CapitalBio, China), and the fluorescence intensities were quantified by the tailor-made software developed by CapitalBio. The whole protocol took less than 9 h to perform, with up to 4 specimens being analyzed for M. tuberculosis identification and 2 specimens being analyzed for drug resistance detection in parallel.

The quality-assured culture and DST was conducted using the BACT/MGIT 960 system and the absolute concentration method on L-J medium at the Department of Clinical Laboratory, Infectious Disease Medical Center in Chongqing. The specimens were processed according to standard methodologies [13]. The following critical concentrations were used respectively: 1 and 10 μg/ml for INH, and 50 and 250 μg/ml for RMP.

We enrolled a total of 153 patients with spinal tuberculosis at Southwest Hospital during the study period. Among the 153 patients, 76 were female and 77 were male. The mean age was 36.4 years (range, 4 to 76 years). A total of 77.1% (118/153) of the patients were initial treatment cases, and the remaining 22.9% (35/153) were retreatment cases who had received previous chemotherapy for a mean period of 7.1 months (range, 3 to 74 months) at the time of enrollment. None of the patients was HIV-seropositive.

Among the 153 specimens that were cultured, a total of 60.1% of the specimens (93/153) produced a positive culture. Following culture testing, the positive specimens were subjected to DST. The results of the DST using the absolute concentration method on L-J medium showed that a total of 15/93 (16.13%) isolates were resistant to INH, 18/93 (19.35%) were resistant to RMP, and 11/93 (11.83%) of these isolates were resistant to both INH and RMP (MDR-TB). The mean turnaround time of culture and DST was 56.8 days (range, 49 to 77 days).

Among the 153 spinal tuberculosis specimens, 114 samples were identified to contain M. tuberculosis strains by the DNA microarray, and the positive detection rate was 74.51%. Among the 93 patients with culture-positive tuberculosis, the overall sensitivity of the M. tuberculosis identification by the DNA microarray was 93.55% (87/93). The sensitivity was 97.4% for smear- and culture-positive cases and 73.3% for smear-negative, culture-positive cases (Table 1). The hybridization pattern of the M. tuberculosis identification was shown in Figure 3a.