MYCOBACTERIAL ANALYSIS

Brush biopsies (Flowgen Bioscience, UK) from ulcerated and non-ulcerated oral mucosa and unstimulated saliva samples were investigated in the mycobacterial analysis.

2.2.3.2.1 SAMPLE DECONTAMINATION

Saliva samples were decontaminated using the NaOH-NALC [2% sodium hydroxide (NaOH), 1.45% trisodium citrate and 0.5% N-acetyl-L-cysteine (NALC)] decontamination method as follows: 6 ml of the prepared NaOH-NALC were added to each salivary sample in a 50 ml Falcon tube and mixed by briefly vortexing and inverting the tube several times. The sample was allowed to be incubated with the NaOH-NALC for 30 minutes with

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periodic gentle vortexing. Subsequently, each sample was diluted (1:40 dilution) with phosphate buffer (pH 6.8) followed by centrifugation at 3000 xg for 30 minutes. The supernatant was discarded and 1.5 ml of sterile phosphate buffer was added to re-suspend the pellet.

Brush biopsy samples were treated with the 4% sulphuric acid decontamination method as follows: 2 ml of the acid was mixed with the brush biopsy sample by vortexing and then incubated for 30 minutes with periodic gentle mixing. Subsequently, each sample was diluted (1:40 dilution) with phosphate buffer and processed as mentioned above for the salivary samples.

2.2.3.2.2 CULTURING TECHNIQUES

0.25 ml of each decontaminated sample was cultured on Lowenstein-Jensen (LJ) slopes (Figure 2.18) and 0.5 ml was inoculated into mycobacterial growth indicator tubes (MGIT) which contained 4 ml of Middlebrook 7H9 broth base with a fluorescent indicator (Figures 2.14 and 2.15) (Becton Dickinson Diagnostic Instrument Systems, USA). MGIT bottles were incubated on the MGIT instrument which reads each tube hourly and triggers an alarm when growth is detected (Figure 2.16) (Becton Dickinson Diagnostic Instrument Systems, USA). All samples were allowed to be incubated for 6 weeks.

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Figure 2.14: LJ culture media [source: The National Mycobacterium Reference Laboratory].

Figure 2.15: MGIT bottles (Becton Dickinson Diagnostic Instrument Systems, USA) [source: The National Mycobacterium Reference Laboratory].

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Figure 2.16: MGIT instrument (Becton Dickinson Diagnostic Instrument Systems, USA) [source: http://www.bd.com/ds].

2.2.3.2.3 MYCOBACTERIAL IDENTIFICATION

The isolated mycobacterium was identified by the PCR and DNA-DNA hybridization technique using the GenoType Mycobacterium Common Mycobacterial (CM) kit, following the manufacturer’s instructions (Hain Lifescience, Germany).

The above-mentioned kit allows the identification of the following Mycobacterial species:

M. avium, M. chelonae, M. abscessus, M. fortuitum, M. gordonae, M. intercellulare, M. scrofulaceum, M. interjectum, M. kansasii, M. malmoense, M. peregrinum, M. marinum, M. ulcerans, M. tuberculosis complex and M. xenopi.

The identification procedure was divided into three stages: DNA isolation, multiplex amplification with biotinylated primes, and reverse hybridization.

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2.2.3.2.3.1 DNA ISOLATION

DNA was isolated from bacteria grown on LJ slopes or MGIT tubes. Bacterial colonies that had grown on LJ slopes were diluted directly with 300 µl of sterile deionised water. In the case of Mycobacteria grown on MGIT tubes, pellets were created by centrifuging 1 ml of the medium for 15 minutes at 10000 xg prior to the dilution step. Diluted samples were incubated for 20 minutes at 95 °C then for 15 minutes in an ultrasonic bath. Subsequently, the samples were centrifuged for 5 minutes at full speed and 5 µl of the supernatant were used in the PCR reaction.

2.2.3.2.3.2 DNA AMPLIFICATION

To amplify the isolated DNA, 45 µl of master mix were prepared for each DNA sample, as shown in table 2.10, then 5 µl of DNA sample were added. The amplification was performed in the thermal cycler using the thermal cycler conditions shown in table 2.11.

Table 2.10: Master mix for mycobacterial DNA amplification.

COMPONENT VOLUME REACTION PER

SAMPLE

PNM 35 µl

10 x polymerase incubation buffer 5 µl

MgCl2 1 µl (1.5 mM final concentration)

Thermostable DNA polymerase 1 µl

Nuclease-free H2O 3 µl

MgCl2: magnesium chloride; PNM: primer-nucleotide mix

Table 2.11: Thermal cycler conditions for mycobacterial DNA amplification.

STEP 1 1 CYCLE STEP 2 10 CYCLES STEP 3 20 CYCLES STEP 4 1 CYCLE Temperature 95 °C 95 °C 58 °C 95 °C 53 °C 70 °C 70 °C

Time 5 min 30 sec 2 min 25 sec 40 sec 40 sec 8 min

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2.2.3.2.3.3 HYBRIDIZATION

The hybridization stage included the following steps: chemical denaturation of the amplified product, hybridization of the single-stranded, biotin-labelled amplicons to membrane-bound probes, stringent washing, streptavidin conjugated alkaline phosphatise (AP) conjugation and AP mediated staining reaction.

2.2.3.2.3.3.1 CHEMICAL DENATURATION STEP

The chemical denaturation was created by mixing 20 µl of the amplified sample with 20 µl of the denaturation solution then incubating the mixture at room temperature for 5 minutes. 1 ml of warm hybridization buffer (45 °C) was added to each denatured sample and shaken gently until a homogeneous colour was created.

2.2.3.2.3.3.2 HYBRIDIZATION OF THE SINGLE-STRANDED, BIOTIN-

LABELLED AMPLICONS TO MEMBRANE-BOUND PROBES STEP

A GenoType Mycobacterium CM DNA strip was added to each sample and allowed to be completely covered with the solution. The strips were incubated for 30 minutes at 45 °C in a shaking water bath.

2.2.3.2.3.3.3 STRINGENT WASHING STEP

At the end of the incubation period, all the hybridization solution was aspirated and 1 ml of the stringent wash solution (STR) was incubated with the DNA strip for 15 minutes at 45 °C. The DNA strip was then removed and rinsed with 1 ml of rinse solution for 1 minute at room temperature.

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2.2.3.2.3.3.4 AP CONJUGATION STEP

1 ml of the diluted conjugate containing AP was added to each DNA strip and incubated for 30 minutes at room temperature on a shaking platform. The DNA strip was then washed twice for 1 minute each time with 1 ml of deionised water on a shaking platform at room temperature.

2.2.3.2.3.3.5 AP MEDIATED STAINING STEP

1 ml of the diluted substrate containing dimethyl sulfoxide was added to each DNA strip and incubated at room temperature in the dark for 20 minutes. The reaction was then stopped by rinsing the DNA strip twice with deionised water.

Each DNA strip contains 16 DNA probes, conjugate control (CC) and marker (Mr) (Figure 2.17). The first strip is the CC followed by the universal control (UC), which indicates the presence of bacteria, then the genus control (GC) that indicates the presence of mycobacteria in the tested sample. The other 14 bands are specific DNA probes used to identify the isolated mycobacterium. The hybridized strips were interpreted according to the manufacturer’s instructions, as illustrated in Figure 2.18.

CC UC GC Mr

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Figure 2.18. GenoType Mycobacterium CM kit (source: Hain Lifescience, Germany).