Detection of clinically important non tuberculous mycobacteria (NTM) from pulmonary samples through one-step multiplex PCR assay

The burden of non-tuberculous mycobacterial (NTM) disease is increasing worldwide but still its diagnosis is delayed and it is mistaken as multidrug-resistant tuberculosis (MDR-TB).The present study was performed to develop a multiplex PCR assay for detection and identification of clinically most common NTM to the species level from pulmonary samples. Out of 50 isolates, 26 were identified as Mycobacterium kansasii (MK), 20 were identified as Mycobacterium abscessus (MA) and 4 were identified as Mycobacterium avium complex (MAC) through multiplex PCR and further confirmed by sequencing. Our study showed that multiplex PCR assay is a simple, convenient, and reliable technique for detection and differential identification of major NTM species.


Background
Tuberculosis (TB), caused by Mycobacterium tuberculosis complex (MTBC), persists as the principal killer disease worldwide, notably in the developing countries and has been a major public health problem in spite of considerable progress in diagnosis and treatment [1]. The genus Mycobacterium comprises several species that are divided into three groups, the MTBC, Mycobacterium leprae and atypical or non-tuberculous mycobacteria (NTM) [2]. NTM, also known as environmental mycobacteria or mycobacteria other than tuberculosis (MOTT), are mycobacteria which are generally free-living organisms and found ubiquitously in the environment [3]. There has been approximately 200 NTM species identified to-date [4]. They can cause a wide range of infections, with pulmonary infections being the most frequent (65-90%) [5].
Nowadays NTM have become important human pathogens as the incidence and prevalence of disease caused by them continue to increase worldwide [6]. The disease causing agents among NTM differ geographically, but the most common species that are frequently isolated from patients with NTM infection are Mycobacterium avium complex (MAC) (Mycobacterium avium, Mycobacterium intracellulare and Mycobacterium chimaera), Mycobacterium abscessus complex (Mycobacterium abscessus subspecies bolletii, subspecies massiliense and Mycobacterium chelonae) and Mycobacterium kansasii [7,8]. The identification and differentiation of NTM from MTBC is of important diagnostic value as the pathogenesis and treatment regimens for these diseases are different [6,9,10].The varying pattern of susceptibility towards anti-TB drugs imposes need of different treatment strategies even among the NTM of same species complex [11][12][13]. Thus, rapid differentiation of MTBC from NTM and species-specific identification of NTM is crucial for proper treatment and appropriate patient management.
Usually, the prevalence of NTM infections has been notified from TB non-endemic countries and rarely from TB endemic countries because the chances of missing NTM infections are higher in TB endemic countries [14,15]. The current standard of care for diagnostic tests does not include bacterial characterization leading to smear positive NTM cases being misclassified as MTBC. So, majority of NTM infections remain either undetected or receive chemotherapy commonly used for tuberculosis causing evolution of drug resistant NTM strains. Besides cases of mixed infection have also been reported by a few workers [16,17].Mostly these NTM species are identified by phenotypic methods which are very cumbersome and time taking. Thus the study was designed to develop a multiplex PCR assay for detection and identification of clinically most common NTM to the species level from pulmonary samples.

Result
In this study, there were a total of 50 cultures which were smear positive but negative by capilia and Geno-Type MTBDR plus Assay. The multiplex PCR using four sets of primers for three unrelated mycobacterial species was successfully developed. At first multiplex PCR was tested with control strains of Mycobacterium avium complex (MAC), Mycobacterium kansasii (MK) and Mycobacterium abscessus (MA). In each strain of MAC, MK and MA we found two bands, one genus specific 688 bp and another band of 169 bp, 218 bp and 271 bp respectively for each species as shown in Fig. 1.In Mycobacterium tuberculosis(H37Rv), clinically confirmed MTBC and known strain of Mycobacterium fortuitum only one genus specific (688 bp) band was found as shown in Fig. 2. Out of 50 isolates, 26 were identified as Mycobacterium kansasii, 20 were identified as Mycobacterium abscessus and 4 were identified as Mycobacterium avium complex through multiplex PCR and further confirmed by sequencing. The sequencing data of control strains and other isolates were analyzed with the help of Basic Local Alignment Search Tool (BLAST) which finds regions of local similarity between the Internal transcribed spacer (ITS) region sequences of reference strains such as Mycobacterium avium complex: accession no.CP040255.1, Mycobacterium abscessus: accession no.CP030860.1, Mycobacterium kansasii: accession no.LR031424.1.

Discussion
In the current study, we identified the most common NTM species from pulmonary samples. Out of these NTM species 26 (52%) were identified as Mycobacterium kansasii, 20 (40%) Mycobacterium abscessus and 4(8%) as Mycobacterium avium complex by multiplex PCR and further confirmed by sequencing.

Conclusion
In conclusion, multiplex PCR is a simple, fast, convenient and reliable technique for identification of NTM species in the routine laboratory. This method can be used in developing countries for identification of most common NTM from pulmonary samples. To the best of our knowledge this is the first type of study conducted in India.

Study design and identification of isolates
This study was undertaken in the Department of Microbiology, Institute of Medical Sciences, Banaras Hindu University, at Varanasi. It is the extension of our previous work [32] where out of 60 positive cultures, 10 (16.7%) were found positive by both GenoType MTBDR plus assay (LPA) and PCR but remaining 50 which were liquid culture (MGIT 960) positive, but capilia and LPA negative were included in this study. Control strains of Mycobacterium avium complex (MAC), Mycobacterium kansasii (MK) and Mycobacterium abscessus (MA) were obtained from National Reference Laboratory, National Institute of Tuberculosis and Respiratory Diseases, New Delhi, India.

DNA extraction
DNA isolation from the positive MGIT cultures as well as solid culture was done by CTAB-chloroform method with some modifications in BSL-3 laboratory [33,34]. The quality and quantity of DNA were analyzed by a spectrophotometer (Thermo Scientific NanoDrop 2000).

Primer designing for the study
At first sequence of Mycobacterium tuberculosis, Mycobacterium avium complex, Mycobacterium kansasii and Mycobacterium abscessus was downloaded from National Center for Biotechnology Information (NCBI) data base. Then the genus specific primer was designed from 16S rRNA region which is specific for Mycobacterium genus. The species specific primer from the Internal transcribed spacer (ITS) region of Mycobacterium avium complex, Mycobacterium abscessus and Mycobacterium kansasii were designed. Annealing temperature and GC content were calculated for both forward and reverse complementary primer using Tm calculator Thermo Fisher Scientific software. The detail of prepared primers is enlisted in Table 1.  Final extension step at 72°C for 10 min.

Sequencing
The ITS region was amplified with the help of primers for MAC, MK and MA. Product size was confirmed by agarose (2%) gel electrophoresis. Four representative strains from each species were sent for sequencing.

Sequence data analysis
The sequencing data of control strains and other isolates were analyzed with the help of Basic Local Alignment Search Tool (BLAST). BLAST finds regions of local similarity between sequences [35].