Sputum or gargle specimens were divided into 2 equal parts: half was decontaminated with N-acetyl-l-cysteine–NaOH and half was decontaminated by using 6% sulfuric acid to compare these decontamination procedures for culture of mycobacteria (9). Specimens were cultured in Mycobacteria Growth Indicator Tubes (Becton Dickinson Microbiology Systems, Cockeysville, MD, USA) according to the manufacturer’s instructions and guidelines reported by Master (10). Mycobacterium isolates were identified by using the Accuprobe culture confirmation test for the M. tuberculosis complex (Accuprobe; bioMérieux, Marcy l’Etoile, France) or by 16S rRNA gene sequencing (11). Serologic testing for HIV was performed by using a qualitative immunoassay (Determine HIV-1/2; Abbott Laboratories, Abbott Park, IL, USA) and the Vidas HIV DUO assay (bioMérieux).

Data were entered into SPSS version 6 software (SPSS Inc., Chicago, IL, USA) and analyzed by using STATA version 8.0 (StataCorp., College Station, TX, USA). Student t-tests were used to assess different means between groups; proportions were compared by using χ² tests. Univariate odds ratios with 95% confidence intervals were calculated to assess associations of potential risk factors for NTM positivity. A stepwise backward regression approach was used for multivariate analysis.

Body mass index (BMI) was calculated as weight in kilograms divided by squared height in meters. Underweight was defined as a BMI <18.

NTM lung disease was diagnosed if patients had respiratory symptoms, abnormal chest radiographic results suggestive of Mycobacterium infection, and 2 positive sputum cultures with the same NTM. Patients or controls with positive NTM cultures who did not meet these criteria were considered colonized.

Of 180 patients, 60 (33%) had only M. tuberculosis isolates in their sputum samples, 12 (7%) had M. tuberculosis and NTM, and 19 (11%) had only NTM (Table 1). Microscopic results of Ziehl-Neelsen–stained smears of sputum samples were positive in 46 (67%) of 69 patients with M. tuberculosis isolates and in 1 patient with an NTM-positive culture.

Of the 31 NTM-positive patients, 29 had 2 consecutive sputum samples subjected to culture. In 22 of the 29 patients, only 1 of 2 cultures was positive. Four NTM-positive patients had 2 positive NTM cultures; 1 of these patients had pulmonary disease. M. intracellulare was isolated twice from 2 of these 4 patients, M. avium was isolated from both samples from 1 patient, and NTM of 2 species and M. tuberculosis was isolated from 1 patient. One of 2 sputum cultures from 3 other NTM-positive patients contained mycobacteria that could not be identified. Two of these 3 patients had M. intracellulare in 1 sputum sample; M. porcinum was isolated from 1 patient.

Characteristics of the 4 patients with NTM isolates in both sputum samples and of the 3 NTM-positive patients from whom 1 of the 2 sputum samples contained mycobacteria that could not be identified are shown in Table 2. For 3 patients (1, 2, and 4), NTM-associated disease was suspected because of the combination of symptoms, positive cultures, and pathologic changes consistent with TB seen on chest radiographs. However, only 1 patient (patient 4) fulfilled the ATS criteria for NTM lung disease. M. intracellulare was isolated from all 3 of these patients.

The first patient (patient 4) was a 32-year-old HIV-positive man who reported having had a productive cough with hemoptysis for 17 weeks. He also was vomiting and had diarrhea. His BMI was 15. He had been treated for TB. Results of a chest radiograph were consistent with TB and showed alveolar infiltration and interstitial pathologic changes. His condition did improve after treatment with antimicrobial drugs (chloramphenicol and tetracycline), and he was again given treatment for TB. Mycobacteria were cultured from 2 sputum samples and identified as M. intracellulare. The patient died 5 weeks later.

The second patient (patient 1), who had M. intracellulare pulmonary disease, was a 55-year-old HIV-positive man. He was admitted because of a productive cough with hemoptysis for 17 weeks. Diarrhea was also reported; his BMI was 17. He had been receiving treatment for TB for 4 months. Sputum obtained before treatment was AFB negative. Radiographic investigation of the chest showed cavities and alveolar consolidation. No improvement was seen after he was treated with antimicrobial drugs (chloramphenicol, amoxicillin, gentamicin, and metronidazole). Sputum was examined again and was smear positive for AFB. The first sputum culture showed M. intracellulare. In the second sputum culture, the isolated mycobacteria could not be identified because of logistic reasons. The patient died 3 weeks later.

The third patient (patient 2), who had M. intracellulare pulmonary disease, was a 45-year-old HIV-positive woman who had had respiratory signs and symptoms for >1 year. She was known to have asthma. Physical examination found enlarged submandibular, supraclavicular, and axillary lymph nodes. Her BMI was 20. Alveolar infiltration was seen on a chest radiograph. Treatment with chloramphenicol was started. Culture of the first sputum sample showed mycobacteria that could not be identified; the second sputum showed M. intracellulare. Three days after admission, the patient was taken home by her family and was lost to follow-up.

M. tuberculosis was cultured from sputum or gargle specimens from 3 (0.8%) of the 385 controls; in 1 of these 3 controls, NTM and M. tuberculosis were isolated (Table 1). In 61 (16%) controls, only NTM were isolated; this number was comparable with the proportion of patients among whom only NTM were isolated (11%; p = 0.2). In 61 of 62 NTM-positive controls, only 1 sputum or gargle specimen was culture positive for NTM. In 1 control, who was HIV negative, 2 mycobacteria, M. porcinum and an unknown Mycobacterium sp., were isolated. No chest radiographs suggestive of TB were observed for any of the controls.

From 383 controls, 2 sputum or gargle samples were cultured for Mycobacterium spp. Significantly fewer controls (1/383) than patients (4/154) had 2 sputum or gargle cultures positive for NTM (p<0.05).

To compare the influence of the decontamination method on the yield of mycobacteria, we divided sputum or gargle samples from all patients and controls into 2 equal parts before decontamination (9). A total of 635 sputum samples were cultured from 180 patients, and 1,532 sputum or gargle samples were cultured from 385 controls. The results of the cultures are shown in Table 3. The number of NTM (72) isolated from 635 sputum samples of patients was significantly higher than the number of NTM (99) isolated from 1,532 sputum or gargle samples from controls (11% and 6%, respectively, (p<0.001).

Mycobacteria were isolated from 273 (43%) of 635 sputum samples from patients; M. tuberculosis isolates were found in 201 (74%) of the 273 positive sputum specimens, and NTM were found in 72 (26%). The most frequently isolated NTM was M. intracellulare, which was found in 12 specimens.

Mycobacteria were isolated from 104 (6.8%) of 1,532 sputum or gargle samples cultured from the controls. M. tuberculosis was found in 5 (4.8%) of the 104 positive cultures, and NTM were found in 99 (95%). The predominant NTM isolated from the controls were M. avium (5 specimens), M. goodii (4 specimens), and M. peregrinum (4 specimens).

A total of 55 (32%) of 171 NTM isolated from patients and controls were not identified because their 16S rDNA sequences were absent in the BLAST (National Center for Biotechnology Information, Bethesda, MD, USA, www.ncbi.nlm.nih.gov) database. These 55 NTM species were closely related to various Mycobacterium spp. such as M. intracellulare, M. malmoense, M. fortuitum, M. smegmatis, and M. terrae.

The 93 patients and controls with NTM-positive cultures had different clinical and radiographic features than the 472 patients and controls without NTM in sputum samples (Table 4). These persons were more likely to report vomiting and diarrhea, were more often underweight (BMI<18), more often had general malaise, and their chest radiographs more frequently showed changes consistent with TB, such as consolidation and interstitial changes. HIV status and presence of M. tuberculosis in the sputum culture did not differ between the NTM-positive and NTM-negative groups. There were no significant differences between these groups in terms of age (mean 36.7 years vs. 34.8 years; p = 0.2), sex, smoking habits, alcohol use, and previous treatment for TB (Table 5). The percentages of farmers and of persons in both groups who used unboiled milk were comparable. Moreover, NTM-positive persons used tap water more often than NTM-negative persons (p = 0.004). A subgroup analysis, restricted to patients with NTM and patients without NTM in sputum, yielded similar results (data not shown).

Independent risk factors for NTM culture–positive sputum were determined by using multivariate analysis. Two factors, underweight (BMI<18) and use of tap water, were independently associated with having an NTM-positive sputum culture (Table 6).