Identification and Susceptibility Testing on Samples of Mycobacterium spp

Background: Tuberculosis is still a major global health problem. Human tuberculosis is caused by species of bacteria belonging to the Mycobacterium genus. In this study we determined mycobacterial species affecting patients from Botucatu, Brazil, and tested M. tuberculosis sensitivity to different drugs. Methods: Data were obtained from Clinical Laboratory Analysis records at Botucatu Medical School University Hospital, UNESP. All samples were processed according to standard isolation procedures from the 2008 Brazil Ministry of Health Mycobacteria Manual, which consist of staining smears by the Ziehl-Neelsen technique and seeding cultures in the Löwenstein-Jensen medium. Results: Samples were isolated from sputum (80.5%), bronchoalveolar lavage (13.8%), pleural fluid (4.6%), and cerebrospinal liquor (1.1%). Smears were evaluated in 87 cases and a total of 59 patients showed positive smears; 55 from 70 sputum samples and 4 from 12 bronchoalveolar lavage samples. No pleural fluid (4) or cerebrospinal liquor (1) samples showed positive smears. The most commonly identified strain was M. tuberculosis (61 cases); followed by M. avium and M. gordonae 2 cases each, and M. peregrinum and M. abscessus 1 case each. Mycobacteria were not identified in 20 patients. Only two strains of M. tuberculosis were multidrug resistant; one was resistant to isoniazid, rifampicin, and pyrazinamide. These two patients evolved to cure. Conclusion: This study highlights a small but troubling percentage of multidrug resistant samples and reveals the occurrence of nontuberculous mycobacteria, emphasizing the importance of correctly identifying species and testing sensitivity to antibacilar drugs to assure an adequate therapy.


INTRODUCTION
Despite being one of the oldest infectious diseases, tuberculosis is still a major global health problem. Contributory factors include: social inequality, insufficient research aimed at developing new treatments and vaccines, population migration, disabled health systems, the high prevalence of multi-drug resistant cases, and an association with human immunodeficiency virus (HIV) infection [1].
Currently, tuberculosis affects approximately one-third of humanity; in 2012, there were an estimated 8.6 million new cases. Most cases (95%) occurred in middle to low-income countries. Each year, at least 1.3 million people die from tuberculosis; 12% of these cases are associated with the Aids epidemic [2,3].
Brazil has the largest number of cases in Latin America and is one of 22 [8][9][10]. The combination of HIV infection and multidrug resistant M. tuberculosis (MDR-TB), which are, at a minimum resistant to isoniazid and rifampicin, represent a potential public health threat in many countries, particularly in those where AIDS is at epidemic levels and tuberculosis incidence is high. It is therefore necessary to know the sensitivity pattern of M. tuberculosis lineages so that adequate treatment can be provided [11].
The aim of this study was to determine mycobacterial species affecting patients from the micro-region of Botucatu, SP, Brazil, and to evaluate the frequency of atypical Mycobacteria infection and their sensitivity to different drugs.

MATERIALS AND METHODS
Laboratory data were obtained from Clinical Laboratory Analysis records at Botucatu Medical School University Hospital, UNESP, between January 2008 and December 2010. All procedures were approved by the Research Ethics Committee of Botucatu Medical School. The following parameters were evaluated: number of patients with clinically suspected tuberculosis; type of biological sample; patient age, gender, and geographical origin; HIV serology; mortality; and result of species identification and sensitivity to drug treatments. One or more samples from a total of 87 patients were studied. All samples were processed according to standard isolation procedures from the 2008 Ministry of Health Mycobacteria Manual, which consist of staining smears by the Ziehl-Neelsen technique and seeding cultures in the Löwenstein-Jensen medium [12].

Samples Procedures
Samples were placed in 15 mL tapered tubes with an equal volume of NaOH 4%, agitated, and rested for 15 min. After adding the same volume of sterile distilled water, samples were centrifugedat 3,000 g for 15 min. The supernatant was discarded and Red phenol 0.4% was added to the sediment, followed by 1 N HCl for neutralization. Then, 0.2 ml of sediment was seeded on Löwenstein-Jensen tubes, which were incubated up to 60 days at 37ºC.
Positive cultures were sent to the Adolfo Lutz Institute (IAL) in Sorocaba, Brazil, for species identification and drug susceptibility testing, using an automated Bactec MGIT 960 and modified Middlebrook 7H9 broth. When Mycobacteria identification was not possible using this technique, strains were sent to IAL in Sao Paulo, Brazil, for identification by polymerase chain reaction (PCR) using specific primers for M. tuberculosis. MNT identification was performed by PCR-restriction enzyme analysis (PRA)-hsp65 as previously described [13].  [15]. In a study by IAL, Sao Jose do Rio Preto, Brazil, between 1996 to 2005, there was an increased NTM rate corresponding to 34.3%. The increase in NTM frequency may in part have been due to the use of an automated liquid culture method which is more sensitive than solid media [16].

RESULTS AND DISCUSSION
It should be pointed out that we use the traditional solid medium based methodology for Mycobacteria isolation. Additionally, concordance between positive smears and cultures was 67.8%, showing that culture is still the gold standard for laboratory diagnosis of Mycobacterium spp. However, recent studies have shown that PCR method is a valuable, costeffective and alternative tool for quick diagnosis of active tuberculosis in different clinical specimens [17]. Our data were essential for corroborating a new protocol on biological sample processing. Due to the low percentage of positive pleural fluid and bronchoalveolar lavage smears (25%), cultures have been performed to eliminate false negative results, even when not requested by a patient's physician.
The isolated NTM species were M. avium, M. gordonae, M. abscessus, and M. peregrinum. Pedro et al. [16] observed a high incidence of M. avium complex, almost 50% of all isolated NTM samples; M. gordonae corresponded to 10% of all NTM. According to Fontana et al. [18], these NTMs are present in nature, and can be isolated from river water, the ground, house dust, and vegetation. Although NTMs are usually saprophytic, there are reports of them causing disease. Several authors have described the importance of identifying Mycobacteria to establish adequate therapy, as they have different drug resistance patterns and treatment periods may be long, ranging from 18 to 24 weeks [18,19].
Patient demographics revealed a 2:1 men-towomen ratio, which agrees with studies carried out in other regions of Brazil [20]. The age range with the highest frequency of tuberculosis was 35-45 years old, which accounted for 41% of all patients; this has also been observed by other authors [18,19,21]. Mortality and positive HIV serology rates were 16.6% and 10.1%, respectively. Taking these two variables together, from the 10 patients who died, three (30%) had HIV+ serology. As there were 6 HIV+ patients in all and three died, their death rate was high (50%). Several authors have considered multidrug resistance as a result of human error involved in prescribing or distributing patients' medicines [22]. According to the World Health Organization [23], drug resistance is a man-made amplification of the natural phenomenon of spontaneous mutations in M. tuberculosis genes. Another factor associated with drug resistance is nonadherence to therapy, especially in drug addicts, which is the most difficult to control. Furthermore, in a recent European research, it was observed that previous treatment for tuberculosis was the strongest risk factor for MDR-TB [24].

CONCLUSION
Our study highlights a small but troubling percentage of multidrug resistant samples in the Botucatu region of Sao Paulo State, Brazil. It also revealed the occurrence of nontuberculous Mycobacteria, emphasizing the importance of correctly identifying species and testing sensitivity to antibacilar drugs to assure an adequate therapy.

CONSENT
It is not applicable.