Nontuberculous Mycobacteria Isolation from Clinical and Environmental Samples in Iran: Twenty Years of Surveillance

Nontuberculous mycobacteria (NTM) are opportunistic pathogens that are widely distributed in the environment. There is a lack of data on species distribution of these organisms from Iran. This study consists of a review of NTM articles published in Iran between the years 1992 and 2014. In this review, 20 articles and 14 case reports were identified. Among the 20 articles, 13 (65%) studies focused on NTM isolates from clinical specimens, 6 (30%) studies examined NTM isolates from environmental samples, and one (5%) article included both clinical and environmental isolates. M. fortuitum (229/997; 23%) was recorded as the most prevalent and rapid growing mycobacteria (RGM) species in both clinical (28%) and environmental (19%) isolated samples (P < 0.05). Among slow growing mycobacteria (SGM), M. simiae (103/494; 21%) demonstrated a higher frequency in clinical samples whereas in environmental samples it was M. flavescens (44/503; 9%). These data represent information from 14 provinces out of 31 provinces of Iran. No information is available in current published data on clinical or environmental NTM from the remaining 17 provinces in Iran. These results emphasize the potential importance of NTM as well as the underestimation of NTM frequency in Iran. NTM is an important clinical problem associated with significant morbidity and mortality in Iran. Continued research is needed from both clinical and environmental sources to help clinicians and researchers better understand and address NTM treatment and prevention.


Introduction
In 1996, the Working Group of the Bacteriology and Immunology Section of the International Union against Tuberculosis and Lung Disease contacted 50 laboratories in several countries, including Iran, in order to collect and analyze epidemiological data for nontuberculous mycobacteria (NTM) or mycobacteria other than tuberculosis (MOTT). At this time, the Iranian reference laboratory provided data from 98 patients (1980)(1981)(1982)(1983), of which M. fortuitum and M. kansasii were identified as the most dominant NTM in clinical samples [1]. In the following years, many researchers attempted to determine the prevalence of NTM and its importance in Iran [2][3][4]. Unfortunately, these studies failed to capture a comprehensive measure of NTM in Iran. The majority of NTM in Iran consist of small samples or data confined to small geographical areas that cannot be generalized. As a result, no clear data on the epidemiology of NTM is available on the national scale.
Iran is an intermediate tuberculosis-(TB-) burden country where TB remains a major public health problem. The incidence of TB in Iran is 21 per 100,000 people.
The significant number of multi-drug resistant (MDR), extensive drug resistant (XDR), and totally drug resistant (TDR) tuberculosis underline the possibility of NTM infection among tuberculosis suspected cases [5,6]. In most cases, patients with positive sputum smear microscopy are treated with first line pulmonary tuberculosis therapy. Clinical failures prompt the transfer of TB samples to central laboratories for further identification of isolates and in case of NTM infection. Therefore, the diagnosis and treatment of resistant TB begin with considerable delay [7]. So far, limited investigation on NTM infections is reported from TB endemic countries with limited laboratory resources. Instead, NTM infection is more documented in developed countries including geographical variability [8][9][10]. For example, Mycobacterium avium complex (MAC) followed by M. gordonae and M. xenopi is considered the most predominant NTM in the United States and Europe [11,12]. Given the complex treatment challenges particularly in low resource countries, understanding geographical diversity of NTM within the country is particularly important. With this background, we aim to retrospectively analyze and compare the NTM data published in the last 20 years. In addition, we evaluated the long-term trends of NTM isolation from clinical and environmental specimens.

Methods
A literature search was performed in PubMed, Scopus, SID and Google Scholar, Embase, and the Cochrane Library on nontuberculous mycobacteria in Iran. The search keywords were "atypical Mycobacteria, " "nontuberculous mycobacteria, " and "Iran, " Original articles, case reports, and reviews published on nontuberculous mycobacteria in Iran in peerreviewed journals including Persian and English journals were considered [2][3][4]. Congress abstracts were excluded. The following data were abstracted for the purpose of review: the name of the city, research methods, and individual NTM species as well as sample source. The statistical significance of observed trends of NTM in the last 20 years was tested using Poisson log-linear regression. All analyses were performed using the statistical software packages SPSS version 21 (IBM SPSS, Inc., Chicago, IL).
As shown in Table 1, the primary method of NTM detection was based on culture using Löwenstein-Jensen media. Identification was performed by conventional methods in 38% (8/20) and molecular methods in 15% (3/20) of articles. In 9 (45%) studies, both molecular and conventional methods were applied.  (Table 3).

NTM in Clinical
In the case report articles, M. marinum (4/14; 28.5%) had higher detection rate, most frequently isolated from nodules or lesions of the hand ( Table 2).  Figure 2, the frequency of NTM among pulmonary TB cases was studied in only 8 studies. In 1995, 18 (8%) of 225 respiratory samples were recorded as NTM as compared to 2013 when 55 (18%) were recorded as NTM from 291 samples. This trend shows a significant increase in NTM detection rates during the study period ( < 0.05).

Discussion
To the best of our knowledge this is the first study in which trends in clinical and environmental NTM species have been investigated over the past twenty years. Overall, from 34 published reports (original and case reports), 997 NTM strains were identified (494 isolated from clinical samples and 503 from environmental samples). The majority of clinical (86.2%) and environmental (65.2%) NTM species were isolated from Tehran ( = 261) and Isfahan ( = 53), respectively. These data are incomplete considering that Iran consists of 31 provinces. Here, we showed that NTM was isolated only from 14 (46%) provinces during the past years ( Figure 1). In the remaining 17 provinces, there is no data available on prevalence of environmental and clinical NTM. These numbers suggest that NTM is a neglected disease in Iran, which is likely true for other neighboring countries in the region, where the incidence of TB is higher, such as Afghanistan, Iraq, and Pakistan [41][42][43][44][45][46][47][48]. In Pakistan, three clinical NTM reports were published in 1984, 2011, and 2013 with total sample size of 4, 62, and 104 subjects, respectively [43,44]. M. fortuitum was identified as the most prevalent NTM in Pakistan (13.5%). In Iraq, few studies recently reported the frequency of NTM in dairy products and environmental samples such as milk powder and fresh milk, drinking water, and fecal samples from horses [46][47][48]. The most prevalent NTM species in Iraq was M. chelonae (18.2%) [46,48].
In this study we also aimed to identify NTM distribution and trends within Iran. Variable techniques were used in different laboratories. From 1992 to 2006, most laboratories used traditional methods for identification of NTM in both clinical and environmental samples. However, from 2009 to 2014, advances in laboratory technique allowed combinations  Mycobacterium lentiflavum [38] 2 0 1 0 T e h r a n S p u t u m Mycobacterium marinum [39] 2008 Sari Nodules and bulls on the back of the right hand Mycobacterium thermoresistibile [40] 2006 Tehran Cervical lymph node of traditional and molecular methods to be used (Table 1) resulting in the detection of more species of NTM in clinical and environmental samples. Reports from high-tech laboratories proposed the use of commercial line probe assay supplemented with sequencing for identification [49,50]. Ideally, the use of the commercial method may support standardization and it facilitates the comparison of results within different settings. Our data demonstrate PRA (PCR restriction analysis) methods with either hsp65 or 16s-23s rRNA, rpoB genes as the optional molecular test [18,51,52]. This highlights the need for standardized methods and guidelines for NTM identification in Iran. We also showed that the majority of NTM were collected from respiratory samples. The results underline the importance of identifying NTM from suspected pulmonary TB patients. Molecular and phenotypic identification revealed a geographical distribution of NTM in Iran. From 494 clinical NTM isolates, 28.3% and 21.4% were recorded to be M. fortuitum and M. simiae, respectively. Analyzing the previous studies [15][16][17][18][19] [20,53,54]. In three other regional settings (Isfahan, Sistan-Baluchestan, and Kerman), M. simiae was not isolated. The clinical importance of M. simiae in various geographical regions of Asia, including Turkey and Japan, has been already documented [55,56]. The frequency of M. simiae wasreported to be from 1.5% to 10% across studies [53,54]. For environmental SGM, the frequency of M. flavescens (44/503; 9%), M. thermoresistibile (24/503; 5%), and M. terrae (21/503; 5%) was higher than other species (Table 4). In contrast to RGM group, the distribution and frequency of slow growing mycobacteria in clinical and environmental samples were different.
The current study found a considerable number of environmental NTM (157/503; 31.2%) that remained unidentifiable (Table 3). This highlights the importance of the implementation of new techniques in order to improve NTM identification. At present, 8 regional and one national reference TB laboratories are functioning in Iran. Recently, due to global fund, they have been equipped with molecular diagnostic testing capabilities. As a result, it is our expectation that NTM detection will increase within the next few years.
Drug susceptibility tests (DST) for NTM were not performed in the majority of published studies in Iran. In developed countries, a variety of susceptibility testing methods such as the -test, TREK, and microbroth dilution are used to carry out DST [57]. Given the well-described resistance patterns emerging in developed countries with low incidence of NTM, susceptibility testing is a particularly important clinical tool for countries such as Iran.
In conclusions, the trends of isolation and identification of NTM have been increased in Iran in the last 20 years. This increasing trend is attributable to the implementation of enhanced molecular techniques that have improved the detection coupled with the enhanced awareness of NTM in the clinical setting. However, further research is needed to address this important public health threat including enhancing the epidemiology of NTM throughout Iran, standardizing laboratory techniques for detection and drug susceptibility testing, and improving clinicians knowledge on NTM diagnosis and treatment in Iran.