Molecular identification of non-tuberculous mycobacterial species isolated from extrapulmonary samples using real-time PCR and rpoB sequence analysis

Sampling

In total 198 EP samples suspected to NTM infection including lymph node biopsy, urine, skin legion aspiration, pleural fluid, and bone biopsy, were collected from patients admitted to referral hospitals in Ahvaz city, Southwest Iran, from the beginning to the end of year 2022. The initial proposal of the work was approved in the University high research and ethics combined committee and necessary permission for sample collection was granted.

Phenotypic identification

All samples were subjected to phenotypic identification. For all samples smear was prepared and Ziehl–Neelsen staining (ZNS) was performed for the presence of acid fast bacilli (AFB). For cultivation, the decontamination of all samples was done as described by Kent (1985). In brief, this was done by using 4% N-Acetyl-L cysteine-sodium hydroxide with subsequent centrifugation at 3000 g for 15 min and re-suspensin of decontaminated sample in phosphate buffer. About half a milliliters aliquot of decontaminated samples was inoculated onto Lowenstein Jenson (LJ) media (Biomerieux, F-69,280 Marcy l’Etoile, France), incubated at 37 °C for 8 weeks, and examined weekly for growth. Mycobacterial isolates were identified by conventional phenotypic and biochemical tests including colony morphology, growth at 25, 37, and 42 °C, pigment production, semi-quantitative catalase test, Tween 80 hydrolysis, arylsulfatase test, heat-stable catalase (pH 7, 68 °C), urease, and nitrate reduction test (Kent 1985). Out of the total 198 suspected samples, 74 (37.3%) were identified as Mycobacterium tuberculosis and excluded from the study. The rest 124 (62.6%) suspected samples to NTM infection, were included in the study for definitive identification.

DNA extraction

The mycobacterial isolates grown on LJ medium were used for extraction of genomic DNA using DNA extraction QIAamp Mini Kit (Qiagen, Hilden, Germany), according to the manufacturer’s instructions. The obtained DNA was diluted 10 fold using distilled water and the concentration was determined using a Nanodrop instrument (Thermo Fisher Scientific, Waltham, MA, USA), which was then used as a template DNA for molecular assays.

Identification of NTM species by PCR sequencing

For NTM molecular identification, a 750-bp fragment of the rpoB gene was amplified using MycoF and MycoR primers (Adékambi et al. 2003). The PCR mixture was prepared in a final volume of 25 µl comprising 10X PCR buffer, 1.5 mmol of MgCl2, 0.2 mmol of each dNTP, 1 U/µl of Taq DNA Polymerase, 1 µmol of each primer, 5 µl genomic DNA (50 ng), and 18 µl sterile deionized water. The PCR products were visualized by electrophoresis on 1% agarose and the results were recorded using a gel documentation system (Protein Simple, San Jose, California, USA), after staining with DNA safe stain (Yektatajhiz, Iran). A 100 bp molecular marker was used to determine the size of produced fragments.

Nucleotide sequencing

The amplified PCR products for each isolate were purified with the Gene JETTM Gel Extraction Kit (Fermentas, Lithuania), according to manufacturer’s guidelines. An ABI PRISM 7500 Sequence Detection System (Applied Biosystems, Foster City, CA, United States) was used to determine the sequences of the products. The sequences of the rpoB gene for each isolate were examined using BLAST separately, and multiple sequence alignment (MSA) was carried out on sequences and the available pertinent sequences of NTM recovered from the GenBank database, using the MEGA7 program (Saitou and Nei 1987).

The real-time PCR method for the detection of NTM

All samples were investigated by Seegene NTM PCR test (Seegene, Soul, South Korea). Overall, 5 µl of an aliquot of the supernatant was mixed with 15 µl of master mix, which contains 10 µl×2 Anyplex PCR master mix, 2 µl 10×MTB/NTM oligonucleotide mix, and 3 µl 8- methoxypsoralen. CFX96 Touch RT-PCR Detection System (Bio-Rad Laboratories Inc., USA) was used for amplification and identification of NTM, which detects the 16 S rRNA gene. For quality control, the kit contains in-house extraction controls, positive and negative amplification, and internal control in the master mix, and we processed them in each run. The interpretation of data was done automatically by CFX Maestro software that showed the results to threshold and cutoff values. Positive and negative controls were used for quality control in each run.

Nucleotide sequence accession numbers

The sequences for each detected NTM isolate were aligned separately and compared with all existing relevant sequences of mycobacteria recovered from GenBank database, and the sequences were deposited in GenBank under accession numbers OQ466451-OQ466527.

Statistical analysis

Statistical Package for Social Sciences (SPSS), version 22 (IBM Inc., Armonk, New York, USA) was used to analyze the data. Data were presented as mean ± SD, frequencies, and percentages. LJ culture was used as a gold standard test, whereas ZNS and NTM RT-PCR as screening test. Specificity, sensitivity, positive predictive, and negative predictive values were measured as recommended by Standards for Reporting of Diagnostic Accuracy Studies.

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