Due to increasing incidence of Mycobacterium infection, rapid diagnosis and differentiation of the Mycobacterium species is crucial for successful treatment. Traditional methods usually take about 4-10 weeks (or more) to give the results. Therefore, molecular methods can be an appropriate alternative for rapid diagnosis and differentiation of Mycobacterium species. Although, Mycobacterium genome is more than 4000 bp in length, a few genes can be the candidate target for molecular diagnosis of these bacteria. Some of the most prevalent NTM species lack a unique sequence within the candidate genome region to be differentiated from each other, due to the high genomic similarity among the Mycobacterium species .
Most of the PCR assays which are used for detecting mycobacteria, identify a single or limited number of Mycobacteriums species . Therefore, these methods seem to be inefficient for identifying Mycobacteria to the species level. Restriction fragment length polymorphism (RFLP) is known as a reliable method for differentiation of mycobacteria, but interpretation of its results is actually quite difficult, because of low distance between band sizes in some species and the emergence of new patterns that have not been reported before . Furthermore, RFLP method is unreliable in detection of mixed cultures and is not applicable to clinical samples. Real time PCR technique is also too costly and requires considerable expertise. Although, sequencing is known as the gold standard method in detection of mycobacteria, it is unable to identify mixed culture samples or co-infection with more than one NTM species in a patient as it requires a high quality and pure PCR product. Generally, the advantages and disadvantages of different molecular techniques are summarized in table 4.
Due to disadvantages of these diagnostic techniques, use of a molecular method based on reverse hybridization can be useful in detecting and differentiating of Mycobacterium species. The PCR-LPA is one of the reverse hybridization techniques which is able to differentiate a large number of Mycobacterium species from each other. LPA is faster than traditional methods, more accurate than chromatographic tests, and less expensive than sequencing for diagnosis of mycobacteria . However, LPA assay is a PCR-based method. The low amount of DNA and high amount of inhibitors in the clinical specimen can lead to false-negative results, especially in smear-negative specimens .
Therefore, cultivation of bacteria from clinical specimens and testing on colonies is the most appropriate way for NTM identification. However, a major drawback with mycobacterial culture is the overgrowth of one NTM species that may cause to miss the growth of another one in the cases of co-infection with more than one NTM species.
In the present study, ITS showed a strong potential for discriminating closely related Mycobacterium species. The sequence analysis of ITS in different Mycobacterium species revealed that the 16S-23S spacer sizes of slowly growing Mycobacteria are shorter than those of rapidly growing species (Figure1). Moreover, the differences in the ITS length of various Mycobacterium species have made it easy to detect the mixed culture or co-infections by electrophoresis of PCR products. We found the presence of more than one species in 2 samples according to the electrophoresis and sequencing results. The rest of the samples (1,2,3,4,5,6,8) were showed only one species (Figure3). Therefore, this advantage of ITS target can be very useful for diagnostic purposes.
To date, several lung infections have been reported with more than one species of NTM or more than one genotype of a specific species. However, information on such infections is very limited. The highest incidence of simultaneous infection with two NTM species is related to M. avium complex and M. abscessus co-infection , but in this study, we found a co-infection with M. simiae and M. fortuitum, and a mixed infection with different M. fortuitum genotypes.
However, PCR-LPA analysis indicated that the discrimination between M. marinum and M. ulcerans which have a high degree of sequence homology is impossible. Interestingly, PCR-LPA was able to discriminate between M. chelonae and M. abscessus with two nucleotide differences between their ITS sequences, and between M. avium and M. intracellulare which cannot be distinguished by chromatographic methods .
In our pilot study on nine clinical samples and their cultured isolates, M. fortuitum was the most common mycobacterial species in the sample collection region (Tehran, Iran). In consistent with our results, Irandoost and colleagues reported the same species as the most prevalent one in the same region . Our finding showed that this method is capable of detecting mycobacterium species in clinical samples as accurate as culture or sequencing methods (Figure 4). Although, the results of LPA performed on clinical samples showed band intensity weaker than those of cultured isolates, we had no problem for interpretation of the results.
In the present study, we described a kind of PCR-LPA which is readily applicable for respiratory specimens in clinical laboratories. This is the first study which compared the results of PCR-LPA in clinical and cultivated samples from the same patients. Our PCR-LPA based on the ITS target can differentiate 12 mycobacterial species and genotype by the use of 15 probes. The speed of the assay is similar to commercial kits with high specificity and required no radioactive material. Moreover, the cost of this test is very low compared to the similar available kits. Each test only has a cost of 6-7€ (Table 4). We routinely performed the amplification by PCR in less than 3 h, followed by a line probe assay in 4 h.