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Nontuberculous mycobacteria in clinical samples with negative acid-fast bacilli

Background: There is a progressive increase in nontuberculous mycobacteria (NTM) in pulmonary and extrapulmonary infections that might cause confusion with the Mycobacterium tuberculosis complex. To determine the frequency of finding NTM in clinical samples from patients diagnosed with active tuberculosis, with negative acid-alcohol-resistant bacilli (acid-fast bacillus [AFB]) in a third-level specialty hospital's mycobacterial laboratory between January 2013 and December 2014. Methods: This is a prospective, descriptive study where isolated strains of biological material were studied in Lowenstein–Jensen and BACTEC MGIT 960 cultures. Results: Clinical samples of 120 patients were studied, with pulmonary samples of 99/120 (82%) and extrapulmonary samples of 21/120 (18%). We identified NTM in 37/120 samples (30.8%), of which 16 in pulmonary, 13 in genitourinary, 3 in bone marrow, and 5 in various specimens. Mycobacterium avium was isolated in 20 samples, Mycobacterium intracellulare in seven samples, and various other species of NTM in the other 10 samples. Conclusion: To establish adequate treatment, we point out the importance of identifying the presence of NTM in the clinical samples of active tuberculosis patients with negative AFB, as possibly becoming confused with M. tuberculosis and which is essential in deciding which treatment is the most adequate.

the most susceptible and frequently present disseminated types. [15][16][17] These mycobacteria also affect the immunocompetent, usually localizing in the lung and in other organs, such as the lymph nodes, kidneys, pleura, meninges, and others. [18,19] NTM do not respond to primary treatment with antituberculous drugs and must be treated with other medication. Their presence is suspected when patients do not improve and requiring their identification to establish their specific therapy. [20] NTM cases are no longer considered clinical "curiosities" [21] and have been classified as causing emerging infections in countries where tuberculosis has decreased. [22,23] The pathogenesis of the infections produced by NTM has not been demonstrated clearly, and they are not transmitted from person-to-person and occur more frequently in women older than 40 of age with tall, thin phenotype and pectus excavatum or bronchiectasis. [24] The diagnosis and precise identification of the species of NTM require special molecular techniques, which are necessary for establishing treatment.
The objective of this study is to investigate the frequency in one third-level specialty hospital of NTM in samples taken from patients with clinical diagnosis of active tuberculosis, negative acid-fast bacillus (AFB), carried out in the hospital's mycobacteria laboratory between January 2013 and December 2014.

methods
The prospective study of clinical samples of consecutive cases was carried out to study the frequency of NTM, as causal agent of disease in patients diagnosed clinically with tuberculosis in a third-level hospital in Mexico City, Mexico.
Specimens were taken from patients over the age of 18, with HIV negative, with diagnosis of active tuberculosis and negative AFB, without previous treatment with antituberculosis drugs, and without evidence of pulmonary or extrapulmonary lesions.
Bronchial lavage and extrapulmonary biological material were obtained from each patient and cultivated in Lowenstein-Jensen (L-J) and BACTEC MGIT 960 medium. Each sample was stained with Ziehl-Neelsen (Z-N) for AFB, and polymerase chain reaction (PCR) sequencing was conducted and directed to gene hsp65 that codes for the thermic shock protein, in patients negative for the M. tuberculosis complex.
Bacteriologic studies were carried out in the samples previously homogenized in isotonic saline solution, phosphate buffer, and decontaminated with a solution of sodium hydroxide at 4%. After neutralization and centrifugal spinning, each of the homogenized specimens was suspended again in distilled water. For the cultures, 0.2 mL of this was inoculated in bottles containing BACTEC MGIT 960 liquid fluorescent medium (Mycobacterium Growth Indicator Tube, Becton Dickinson, San José, CA, USA) and in solid L-J medium. The cultures were incubated at 37°C for 8 weeks and inspected weekly to evaluate mycobacterial growth.
DNA was extracted by cell disruption with heat shock. PCR was carried out with Tb11 y Tb12 primers of hsp65 fragment gene, as described by Telenti, [25] and PCR products of 440 pb were obtained. Subsequent sequencing was achieved using the ABI PRISM_3130xl Genetic Analyzer (Applied Biosystems, Foster City, CA, USA). Phylogenetic analysis was performed with MEGAv. 6.0 software (distance Neighbor Joining method) using the data base for NTM species reported by Escamilla-Escobar, [10] including the GenBank sequences with ID as follows: HM056121, AY337278, KF432467, HM454220 and sequences from Table 1.

Results
Of the 120 clinical samples of HIV negative patients with diagnosis of tuberculosis, 82 (68%) were male and 38 (32%) were female, with an average age of 40, ranging between 18 and 76 years. Of these, 99 (82%) samples were taken by bronchial lavage and 21 (18%) were taken from extrapulmonary specimens.
Of the 120 samples studied, 51 (42.5%) were negative for M. tuberculosis complex in L-J culture and PCR. Thirty two (25.5%) with positive cultures were classified as confirmed cases of TB, and NTM were identified in 37 (30.8%) by culture and PCR sequencing, of which, 26 were male and 11 female from the following: NTM were found in 16 samples of bronchial lavage, 13 from urine, and three from bone marrow aspirate. There was one from each in gastric lavage, ascites liquid, cerebrospinal fluid, pleural liquid, and lymph node biopsy. The most frequent species were found M. avium and Mycobacterium intracellulare isolated [ Figure 1].
Of this group, four patients presented with >1 infected site by NTM as follows: (1) female patient, age 28, with peritoneal and pulmonary localization, and M. avium was isolated; (2) male patient, age 26, and had miliary (M. simie) and urogenital (M. avium) TB; (3) male patient, age 32, and with pulmonary and meningeal with M. avium; and (4) male patient, age 31, with pulmonary, and urogenital with M. avium.

dIscussIon
The M. tuberculosis complex causes the greatest number of tuberculous infections. However, NTM are increasingly being considered more frequent in both immunocompetent and immunocompromised subjects. The infection by NTM is suspected when there are risk factors such as advanced age, bronchiectasis, and poor response to specific and supervised antituberculosis treatment (directly observed treatment). In such cases, the possibility of pathogenic or pharmacologically resistant NTM infection should be considered. [7,15,26,27] Patients in our clinical samples might have NTM infection in spite of negative Z-N for AFB, which means that cultures and PCR are essential for a definitive diagnosis of the species of pathogenic mycobacteria involved and necessary for ruling out possible colonization. [28] Negative AFB does not imply absence of tuberculosis because its sensitivity is 50%-60%. Bronchial lavage specimens provide an alternative for obtaining useful specimens for bacteriologic diagnoses in cases of pulmonary affectations. [29,30] In Mexico, the increase in the frequency and characterization of nontuberculous disease-causing mycobacterial species is unknown. Studies in >30 countries worldwide found that M. avium complex is predominant, which is consistent with the findings in our research. Pulmonary infections and the increase in their extrapulmonary presentations are reported to be a growing problem in many countries, especially in the developing world where uncontrolled tuberculosis is a major issue. Although difficult, it is important to determine the sources and modes of transmission of NTM strains to implement effective prophylactic measures. [30] A delay in accurate diagnosis of NTM leads to an increased mortality, especially in immunocompromised patients. The medication employed may have significant side effects that can lead to treatment withdrawal and an increased resistance to the first-line drugs, and less than 50% of these drugs are successful, and relapses are estimated at >50% within 3 years. [31] The frequency of NTM in this study was 30.8% in our samples. Males were more affected, different from that informed by other authors [32,33] that report predominance in females. Systematic detection of NTM in samples from cases suspected of active tuberculosis with negative AFB is important to implement treatment according to sensitivity to antituberculosis drugs, with long-term follow-up and for the evaluation of results. [34,35] Increasing hospital costs for inpatients and outpatients are currently a priority for the most countries. The high costs affect the public hospitals. Diseases caused by NTM require a prolonged treatment for success, and the total cost involves many other expenses that are both financial and human, such as lost wages. Knowledge of the presence of NTM as a cause of illness in our region will enable the improvement of current prevention guidelines as well as treatment strategies. [36] conclusIon

Study limitations
This study confirms the existence of NTM in Mexico with patients diagnosed with active tuberculosis. However, since our sample is relatively small, our findings establish the need for further controlled studies to determine the actual correct frequency of these infections in the country.

Financial support and sponsorship
Nil. . using distance neighbor-joining algorithm, and nucleotide substitutions were corrected by Tamura-Nei model. Sequences obtained in this study were labeled with sample number/year of isolation followed by "HGM" (Hospital General de Mexico). Bootstrap values from 1000 replicates are indicated. Black triangles represent collapsed groups of nontuberculous mycobacteria species without genetic relationship to described HGM sequences