article: Incidences of nontuberculous mycobacteria (NTM) causing pulmonary and extrapulmonary diseases are reportedly increasing globally and the current epidemiologic situation in Saudi Arabia remains unclear. To study such trend, we carried out a nationwide systematic epidemiological study focusing on NTM diseases for the first time in the country. A nationwide collection of NTM isolates with clinical and demographical data was conducted for a period of 24 months. Primary species identification was carried out by line probe assays followed by sequencing of 16S rRNA, 16S-23S ITS region, rpoB and hsp65 genes. The laboratory findings were comprehensively analysed against demographical and clinical data. A total of 527 isolates were enrolled with a higher proportion of Saudi citizens (76. 5%), elderly (>60 years) patients (34. 2%), and male gender (65. 3%) respectively. Overall, 75. 1% isolates were pulmonary origin with a proven clinical significance of 44. 7%. In total, 34 NTM species including 17 rare species were identified, in addition to 8 ‘undefined’ isolates. M. simiae (22. 6%), M. fortuitum (18. 1%) and M. abscessus (17. 8%) were predominant species. Interestingly, 27 new cases of clinically relevant M. riyadhense were also noticed (Primary data on emergence of rare NTM species and M. riyadhense has been recently reported). Results showed, rare clinical events such as mycobacteremia, cecum abscess, peritonitis and ascites caused by M. wolinskyi, M. holsaticum, M. duvalii and M. monacence respectively. Diabetes mellitus (P value-0. 04) and previous history of tuberculosis (P value- 0. 001) were identified as independent risk factors associated with NTM diseases. NTM disease spectrum and pathogen diversity is an emerging challenge to any nation, including Saudi Arabia. Therefore, more priorities will be given to NTM’s with an immediate initiative to develop diagnostic infrastructures and disease management plans.
  Globally, pulmonary and extrapulmonary human infections caused by various non-tuberculous mycobacteria (NTM) were reportedly increased [1–5]. Although, immunosuppressive illnesses and therapies are regarded as major predisposing factors, there was recent increase of NTM incidences among immunocompetent patients. Despite this rise, NTM diseases are not yet considered as serious public health threat and these diseases remain largely neglected in most parts of the world. Unlike tuberculosis, NTM causing diseases are not notifiable conditions in most of the countries, except in regions like Australia [6]. Scarcity of evidences to support human-to-human transmission and the facultative nature of NTM entities have contributed remarkably towards such negligence. To date, almost 150 species of NTM’s are officially recognized as per the List of Prokaryotic Names with Standing in Nomenclature (http: //www. bacterio. net/mycobacterium. html). The pathogenic potential of most of the species is still not fully defined and hardly few dozen are only familiar to clinicians and microbiologist [7]. Species spectrum of NTM’s are mostly associated with geographical distribution, thus the diversity varies from region to region [8]. Approximately, 75–90% of NTM’s are isolated from pulmonary samples, however majority remains as colonizers [9]. Extrapulmonary involvement had been commonly observed in lymphnode, skin, soft tissues, bone and less frequently in central nervous, urogenital and gastro intestinal systems [10]. Regardless of the broad disease spectrum, data on definitive NTM incidences are scarce in most part of the world, particularly from developing countries. To date, no nationwide systematic epidemiological study on clinical relevance of NTM’s was conducted in Saudi Arabia. However, recently Varghese et al. showed, considerable threats from several NTM species causing pulmonary and extrapulmonary diseases [11,12]. Therefore, for the first time a prospective study has been carried out to analyse the species spectrum, clinical relevance, underlying risk factors, possible mortality and geographical adaptation of NTM’s in the country.
  This study has been reviewed and approved by the Office of Research Affairs of King Faisal Specialist Hospital and Research Centre, Riyadh. During April-2014 to March-2016,527 non repetitive NTM isolates were collected from different provincial laboratories. The sample volume has been calculated following a previously published national data on NTM isolation [13]. Clinical and demographical data including data on previous NTM isolations have been archived from medical and laboratory records. The study had two major case groups; pulmonary (396 isolates) and extrapulmonary (131 isolates). Clinical relevance of pulmonary group was defined according to the American Thoracic Society (ATS) guidelines [9]. Briefly, clinical relevance was confirmed if cases meet any of the following criteria, culture positivity detected in a minimum of two separate sputum or tracheal aspirates; one lung tissue with histopathological relevance; or bronchio-alveolar lavage. All the extrapulmonary cases were considered as clinically relevant. Mortality data has been collected after referring to the laboratory records at the end of sample collection period (month 22–24) from each study sites. Isolates were primarily identified by using line probe assays (Mycobacterium CM and AS -Hain Life Science, Germany). Isolates which identified only up to Mycobacterium species level were subjected to sequencing of four highly conserved genes, 16S rRNA, rpoB, 16S-23S ITS region and hsp65 using the BigDye Terminator cycle sequencing chemistry (Applied Biosystems, USA). Primary sequencing was targeted on 16S rRNA (645-655bp hyper variable region) and a 342bp region of rpoB genes, followed by hsp65 (439bp) and 16S-23S ITS region (480bp) genes [14–17].
  A total of 527 isolates from different geographical region were collected with a demographical predominance of male gender (65. 3%), age group elderly (34. 2%) and Saudi nationals (76. 5%). Geographical case distribution showed more incidences in West (33. 8%) followed by Central (30%) and East (22. 2%) (Table 1). Pulmonary isolates were dominant (75. 1%) and 82. 6% of pulmonary NTM isolation was from sputum samples. Following ATS guidelines, a clinical relevance of 44. 7% and 55. 3% colonization were observed. Lymphnode (29. 8%) and skin (22. 9%) were the most affected extrapulmonary sites. Interestingly, 13 (9. 9%) cases of mycobacteremia and 17. 6% of various other infections also reported (Fig 1). Only, 426 (80. 8%) cases were tested for HIV and reported with 14 (3. 3%) positive cases. Overall, 178 (33. 8%) cases received treatment and most among them had extrapulmonary diseases (129/131; 98. 5%) (Table 1). A total of 34 different species including 17 rarely reported species were identified. The slow growing species (56. 7%) were predominant with an increased isolation of M. simiae (22. 6%). M. fortuitum (18. 1%), M. abscessus (17. 8%) and M. gordonae (7. 6%) were the other major species isolated. Overall, 29 (5. 5%) isolates were identified as ‘rare’ species, which mainly included M. monacence and M. cosmeticum. M. riyadhense was isolated from 27 patients and 8 (1. 5%) isolates were identified only up to Mycobacterium species (Fig 2). Published primary findings of the study revealed existence of 12 M. riyadhense and 27 ‘rare’ NTM cases [11,18]. M. avium, M. riyadhense and M. gordonae were frequently isolated from pulmonary samples. However, most of the identified species (20/34 species) were involved in different extrapulmonary anatomical sites. M. fortuitum (22. 1%), M. abscesuss (21. 4%) and M. simiae (19. 1%) were the predominant in extrapulmonary infections. Interestingly, 75 (57. 2%) isolates were rapid growers, including 6 rare species (M. holsaticum, M. duvalii, M. monacense, M. wolinskyi, M. peregrinum, and M. cosmeticum). The analysis showed rare clinical events such as mycobacteremia, cecum abscess, peritonitis and ascites caused by M. wolinskyi, M. holsaticum, M. duvalii and M. monacence respectively (Table 2). We analysed geographical distribution of 8 common species (M. simiae, M. kansasii M. intracellulare, M. riyadhense, M. gordonae, M. abscessus, M. fortuitum, and M. avium). M. abscessus and M. fortuitum were commonly isolated from the West, South, and East regions. In Central province domination of M. simiae (48%) was evident with consistent isolation of M. riyadhense. Pulmonary cases were high in Central region (81. 6%) while extrapulmonary in North (60%) and West (31. 5%). Clinical relevance of pulmonary cases was observed high (43. 4%) in Central province (Fig 3). The Proportion of pulmonary and extrapulmonary infection in each region is also depicted. Clinical relevance and colonization of pulmonary NTM infections after consulting the American Thoracic Society guidelines also presented. Overall 29 different types of underlying conditions among 376 (71. 3%) of cases were noticed including 39. 4% cases with multiple immunosuppressive conditions. Majority of the confounding factors were recorded among Saudi population (80. 5%). Diabetes mellitus (21. 8%) was the most common among Saudi nationals. Smoking and hypertension were the other two factors found among both Saudi and non-Saudi nationals. There were five infectious diseases other than tuberculosis and HIV observed as underlying conditions (Table 3). Among the demographical factors, the findings showed a clear statistical association of patients in age groups 16–29 (p-value, 0. 02), 30–45 (p-value, 0. 003) and >60 years (p-value, 0. 007). Diabetes mellitus (p-value, 0. 04) and previous history of tuberculosis disease (p-value, 0. 001) were also statistically significant (Table 3). Although detailed clinical follow ups were limited under the scope of the study, we analyzed the NTM mortality using laboratory records. Overall, 12 (2. 3%) patients died in the cohort due to pulmonary (58. 3%) or extrapulmonary (41. 7%) diseases (Table 1). Analysis of causative organism showed, presence of M. simiae (66. 7%) in most cases associated with extra pulmonary diseases including mycobacteremia. M. abscessus was found in other 4 (33. 3%) died patients with pulmonary disease (Table 1, Table 2).
  In conclusion, Saudi Arabia currently faces several challenges from NTM diseases and they are generally neglected. The emergence of new pathogens such as M. riyadhense or difficult to treat species like M. simiae, should be addressed immediately. Presence of rare species of NTM’s which cause both pulmonary and extrapulmonary diseases to the Saudi population may show the potential adaptability of them in the country. Higher prevalence of Saudi population with NTM diseases must be investigated thoroughly to rule out the possibilities of NTM disease vulnerability. Finally, detailed management plan for mycobacterial disease control must be developed with urgent priorities.
summary: Non-tuberculous mycobacteria (NTM) are opportunistic pathogens and saprophytes found largely in wet soil, marshland, streams, rivers and estuaries. Pulmonary and extrapulmonary Infections caused by NTM’s increased globally in immune-suppressed and immune-competent individuals without an exemption to Saudi Arabia. However, recent developments in diagnostic platforms improved the identification of several new and rare species of NTM’s. Interestingly, recent studies linked the direct impact of species spectrum on clinical outcome and therapies. To date, in Saudi Arabia, no systematic nationwide surveillance study was conducted on the challenges of NTM causing diseases. Therefore, for the first time, a nationwide inventory study on the spectrum of clinically relevant pulmonary and extra pulmonary diseases with an attempt to explore epidemiological data has been carried out. The study results showed a huge species spectrum of NTM’s causing highly diverse clinical manifestations. Clinical relevance pulmonary infection was relatively high and several very rare extrapulmonary infections also were observed. The study reported the emergence of a rare pathogen, Mycobacterium riyadhense and predominance of Mycobacterium simiae as the most common species. As the first large scale study from the GCC region, the findings cements the growing potential of various NTM species to cause significant clinical implications in the country and warrants an immediate change on disease management priorities.
keywords: sequencing techniques
  medicine and health sciences
  pathology and laboratory medicine
  elderly
  population dynamics
  pathogens
  geographical locations
  microbiology
  species delimitation
  retroviruses
  viruses
  immunodeficiency viruses
  age groups
  saudi arabia
  gene sequencing
  rna viruses
  speciation
  molecular biology techniques
  population biology
  bacteria
  research and analysis methods
  lentivirus
  medical microbiology
  hiv
  microbial pathogens
  actinobacteria
  molecular biology
  people and places
  nontuberculous mycobacteria
  asia
  viral pathogens
  biology and life sciences
  population groupings
  geriatrics
  evolutionary biology
  dna sequencing
  evolutionary processes
  organisms
  geographic distribution
title: The first Saudi Arabian national inventory study revealed the upcoming challenges of highly diverse non-tuberculous mycobacterial diseases