PCR-reverse blot hybridization assay in respiratory specimens for rapid detection and differentiation of mycobacteria in HIV-negative population

The genus Mycobacterium contains a number of acid-fast bacilli (AFB), including Mycobacterium tuberculosis complex (MTBC), Mycobacterium leprae, and non-tuberculosis mycobacteria (NTM) [1]. NTM are ubiquitous environmental organisms that incidentally cause opportunistic NTM pulmonary disease (NTM-PD) and extrapulmonary infections in immunocompromised individuals [2]. With the prevalence of acquired immunodeficiency syndrome (AIDS), the recognition of clinical importance of NTM is also growing [3]. Recently, many countries have reported a dramatically increased incidence of NTM-PD. [4‐7] NTM infections constitutes 0.5–35% of all human mycobacterial infections [8]. For patients with risk factors, such as bronchiectasis, cystic fibrosis, and immune deficiency, the figure was higher still, over 50% [9]. However, a high percentage of patients have no risk factors [10]. Infections of most NTM species have been traditionally considered from environmental sources rather than person-to-person transmission like MTBC [11]. In addition, many NTM strains are insensitive to most anti-tuberculosis drugs [12, 13]. Therefore, quality and timely identification of mycobacterium species is necessary for better patient management and appropriate chemotherapy. However, most clinical features (cough, sputum production, fatigue, hemoptysis and fever) and abnormalities on chest radiography (centrilobular nodules, tree-in-bud opacity and cavitation) generally associated with NTM-PD are atypical, which may lead to false diagnoses as PTB, and hence to overtreatment of patients who do not have TB [14, 15]. The traditional methods used in mycobacterial laboratories require cultivated isolates and the results are only obtained after weeks to months of incubation. Therefore, these culture-based methods are time-consuming and labor-intensive [16]. Moreover, the traditional methods could not discriminate NTM to the species level, or detect mixed infections in which two or more mycobacterial species were simultaneously detected in the same specimen [17]. All NTM isolates from respiratory samples should be identified at least to species level, since that optimal therapeutic regimen differs according to different species, especially between slow-growing and rapid-growing species. Therefore, current clinical methods are not conducive to guide clinical decisions in a timely manner. Clinicians sometimes refer only to interferon-γ release assays (IGRAs). IGRAs are based on M. tuberculosis specific antigens. These antigens are not present in NTM strains other than M. marinum, M. kansasii, M. gordonae and M. szulgai [18]. However, it is worth mentioning that a number of TB patients are also negative in IGRAs [19]. So, IGRAs could not make a reliably distinction between NTM and MTBC.

Advances in molecular assays have accelerated the diagnosis of mycobacteria.

infections over recent years. Among these advances, nucleic acid amplification (NAA)-based techniques permit for identifying mycobacteria at the species level [20‐23]. Sequencing of 16S rRNA has been recommended as the gold standard method for definitive identification and discrimination of mycobacterial species [24]. Unfortunately, although fast, some of these techniques still require cultured strains and expensive equipment such as Hain line-probe and minion [22]. A commercial kit based on 16S rRNA sequencing and nucleic acid probes and reverse blot hybridization (REBA), PCR-REBA Myco-ID (Yaneng BioSciences, Shenzhen), was developed for simultaneous genotyping of 22 clinical important mycobacterial species including MTB, M. smegmatis, M. Intracellulare, M. kansasii, M. chelonae, M. marinum, M. fortuitum, M. terrae, M. nonchromogenicum, M. avium, M. scrofulaceum, M. abscessus, M. xenopi, M. gilvum, M. phlei, M. triviale, M. gordonae, M. gastri, M. vaccae, M. szulgai, M. diernhoferi, M. simiae. Moreover, as a major advantage, PCR-REBA Myco-ID can be performed with clinical specimens. Despite the fact that this technology has been used as a rapid, low cost and easy-to-use method in mycobacterial research works, it has not been incorporated into workflow of many mycobacterial laboratories. Therefore, the purpose of this study was to evaluate clinical applicability of PCR-REBA Myco-ID assay for prompt and accurate identification of NTM at species level directly from 696 clinical specimens in China.

In current retrospective study, we evaluated the clinical applicability of PCR-REBA Myco-ID assay for rapid detection and differentiation of mycobacterial species directly from respiratory samples (sputum and BALF) in clinically suspected NTM-PD patients. Our results indicate that PCR-REBA Myco-ID assay has higher sensitivity and PPV than MGIT 960-TBc ID, which is consistent with the results of previous study by Lee et al. [28]. Because NTM are ubiquitous in the environment, a single isolated NTM strain from sputum without repeated isolation on culture, is not clinically significant, in general. Therefore, in the investigation of clinically suspected NTM-PD patients, sputum samples should be collected at least two other days for mycobacterium culture. Differentiation of Mycobacteria at the species level by phenotypic and biochemical testing is time-consuming because of the slow growth rate of mycobacteria. However, the major advantage of some direct molecular detection is to rapidly differentiate MTB from NTM in clinical samples. PCR-REBA Myco-ID assay has a short turnaround time of about 4 h. Timely intervention is also the key to the success of NTM treatment. Another limitation of MGIT 960-TBc ID test is that the accuracy is heavily affected by mycobacterial species and the quality of specimen. Relatively high contamination rate was reported [29]. Our results showed that the overall contamination rate was 1.7% (12/696) in this study. In addition, there were two false positive cultures in the non-TB group including one MTBC and one NTM isolate. Culture of NTM can be difficult even in mycobacterial reference laboratories. For example, while many NTM species grow best at 28 °C or 45 °C, conventional cultures are performed only at 35 °C. Other NTM species require prolonged incubation or special medium. Some NTM cultivars require supplementary media or extended culture cells. In our current study, six strains of NTM were not cultured, but were detected by PCR-REBA Myco-ID assay and confirmed by PCR-HRM analysis. Routinely culture also cannot discriminate NTM species. At present, more than 160 distinct Mycobacterium species have been validly published (http://​www.​bacterio.​net/​mycobacterium.​html). The clinical relevance of isolated NTM differs strongly by species, from pathogenic species (e.g., Mycobacterium kansasii and Mycobacterium malmoense) to typical saprophytes (Mycobacterium gordonae and Mycobacterium phlei) [30]. In practice, treatment recommendations presented for NTM are based on the NTM species. NTM isolates species should be subspeciated. For example, the prognosis of diseases caused by M. intracellulare is better than that caused by M. avium, and the prognosis of diseases caused by M. abscessus is worse than that caused by M. massiliense [31, 32]. However, PCR-REBA Myco-ID assay cannot further classify M. abscessus as M. massiliense, M. bolletii and M. abscessus subspecies. If person-to-person transmission of M. abscessus is suspected, it is recommended to use whole genome sequencing (WGS), which has been developed in recent years [33]. Several methods based on WGS achieve subspecies-level resolution [34]. However, this technique is more sophisticated but not cost-effective and requires expensive equipment. It is too expensive to be used routinely in mycobacterial laboratories especially in source-limited countries. A limit of this study is that our method was not evaluated using metagenomic data. However, it should be noted that although we investigated 22 clinical important mycobacterial species in clinical samples, PCR-REBA Myco-ID assay appears to include the potential to analyze more NTM species in future studies.

Currently, mass spectrometry (MS) analysis is popular in large hospitals. For its low running costs. However, it was reported that closely related species could not be distinguished correctly [35]. Another limit of MS analysis is that this method still requires cultured NTM cells similar to Hain Geno Type MTBC test [36]. Polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) analysis (PRA) of some house-keeping genes does not need cultured NTM cells and special expensive equipment. However, PRA cannot discriminate different mycobacterial species in mixed infections [37]. In this study, 22 specimens with M. intracellulare and M. abscessus mixed infection and 10 specimens with M. avium and M. abscessus mixed infection were identified by PCR-REBA Myco-ID assay.

Our finding showed that PCR-REBA Myco-ID assay is an efficient method and has higher specificity and rapidity than conventional methods. Furthermore, it does not require expensive specialized equipment. It should be incorporated into workflow of mycobacterial laboratories especially in source-limited countries.