To the Editors:
Nontuberculous mycobacteria (NTM) are opportunistic pathogens that are widely present in our environment, i.e. in the soil and in natural and processed water. This group of organisms, with varying biological properties and clinical relevance, has gained notoriety over the past two decades due to their capability to cause severe disease in patients with immunodeficiency and/or chronic lung disease [1]. Despite their rising isolation frequency and growing clinical importance, NTM infections receive little scientific or public health attention. This partly results from the dogma that these NTM infections are not transmitted from male-to-male [1].
During our previous research of Mycobacterium malmoense infection in the Netherlands [2], we studied two patients with M. malmoense pulmonary infection with a strong epidemiological link. Human transmission was suspected because an acid-fast bacilli (AFB) smear-positive patient was in close contact with an immunocompromised patient. Herein, we present details of the cases and the results of the molecular diagnostics that were performed. The patients gave informed consent; ethical approval was not required for this retrospective study.
A 38-yr-old male with a medical history of chronic obstructive pulmonary disease and spontaneous pneumothorax reported to the respiratory physician with progressive dyspnoea, productive cough, fever, night sweats, malaise and weight loss. A chest radiograph revealed infiltrates with cavities in the upper lobes. Laboratory diagnostics showed a raised erythrocyte sedimentation rate (90 mm·h−1) and leukocytosis (white blood cell count 22.4 cells·mL−1); serological tests for HIV were negative. Three sputum smears were negative for AFB on direct microscopy. Bronchoalveolar lavage was performed, which was positive for AFB on direct microscopy. A regimen of isoniazid, rifampicin, ethambutol and pyrazinamide was initiated, based on a presumptive diagnosis of pulmonary tuberculosis. Later, sputum appeared to be positive for AFB. All corresponding cultures yielded M. malmoense, identified by Inno-LiPA Mycobacteria v2 reverse line blot assay (Innogenetics, Ghent, Belgium). Since the patient met the American Thoracic Society diagnostic criteria for clinical disease [1] the regimen was changed to 24 months of rifampicin, ethambutol and clarithromycin and the patient slowly improved.
11 months after the diagnosis was made in patient one, a second patient was diagnosed with M. malmoense disease in the same hospital. This patient, a 59-yr-old male, had a history of kidney transplantation and subsequent immunosuppressive treatment with cyclosporine and prednisone. He presented with productive cough, malaise, fever and weight loss. Erythrocyte sedimentation rate was 110 mm·h−1 and the white blood cell count was 8.5 cells·mL−1. A chest computed tomography scan revealed an infiltrate with cavitation in the left upper lobe and mediastinal lymphadenopathy. A transbronchial biopsy showed granulomatous inflammation with central necrosis and visible AFB on histological examination. Sputum samples revealed AFB on direct microscopy and corresponding cultures, and two simultaneously obtained blood cultures yielded M. malmoense. A regimen of rifampicin, ethambutol and clarithromycin was initiated; owing to clarithromycin intolerance, this drug was changed to ciprofloxacin. After initial progression of disease, including sepsis, progressive pulmonary infiltrates and spontaneous rupture of a nectrotising mediastinal lymph node to the oesophagus, the patient recovered slowly. Both patients completed 24 months of drug therapy and have remained culture negative during 2 yrs of follow-up. Patient two died after the 2-yr follow-up period due to a Grawitz tumour.
Epidemiological investigation revealed that the two patients were acquainted and had intensive contact because they met very regularly in the same bar. The first patient worked in a flower bulb processing factory. Isolates of both patients, as well as unrelated patients from the same region and bordering regions, were subjected to genotyping by repetitive element (rep)-PCR typing. The results were confirmed using amplified fragment length polymorphism (AFLP) typing as described previously [3, 4]. Rep-PCR typing revealed a 97.4% similarity between isolates of both patients, suggesting they were possibly the same single strain. However, this same (>97% similarity) pattern was also observed from the M. malmoense isolate of an elderly female with pulmonary M. malmoense disease living in a nearby city; this patient was not epidemiologically related. Other strains from nearby and more distant regions, as well as from the same region, although at a later period, revealed distinct patterns by rep-PCR typing (fig. 1a). However, AFLP typing showed that the isolates of both patients had only 90% similarity. AFLP included the strain of a patient with pulmonary M. malmoense disease from the neighbouring region (05–1477) in the cluster with isolates of both our patients (fig. 1b).We subsequently sequenced the 16S rDNA gene, 16S–23S internal transcribed spacer (ITS), as well as partial hsp65 and rpoB genes as previously described [5]. The isolates of both patients had identical 16S, ITS and rpoB sequences; the 441bp Telenti-fragment of the hsp65 gene showed two base-pairs difference between both isolates [6].
Gene sequencing results of the patient’s isolates including related strains isolated from other patients. 04–1708 is the strain isolated from patient one and 05–1634 was isolated from patient two. a) Typing results obtained by repetitive element (rep)-PCR typing (DiversiLab; BioMerieux, Marcy l'Etoile, France). 97.4% similarity was calculated between the two patient isolates using rep-PCR. b) Amplified fragment length polymorphism (AFLP) typing results. 90% similarity was calculated between the two patient isolates using AFLP. Strain 05–1681 was isolated from an elderly female with pulmonary Mycobacterium malmoense disease in a nearby city; 04–817 was isolated from a child with lymphadenitis in the same region; and 05–1477 was isolated from a patient with pulmonary M. malmoense disease in a neighbouring district. More distantly related 09–142 and 05–1401 were isolated in the same region but 4 yrs later and in a region in another part of the country, respectively. Strain 05–1473 is a Mycobacterium fortuitum isolate used as an out group. Mycobacterium smegmatis mc2–155 and Mycobacterium marinum 2000–01053 are control strains.
Although there is a strong epidemiological link between both patients with a perfect scenario for human transmission (an AFB positive and coughing source in close contact with an immunocompromised receiver), the results of AFLP and the hsp65 gene sequences, showing two base-pairs difference between the strains of both patients, render human transmission in this case unlikely. Cases of NTM human transmission have been suggested previously, as reviewed by Wolinsky [7]. One setting showed some similarities to ours with two brothers who developed pulmonary Mycobacterium kansasii disease, with an interval of 1 yr [8]. Still, human transmission of NTM has, however, never been proven.
The interpretation of genotyping results in order to make definite conclusions about human NTM transmission is complicated: even if both strains had been fully identical by the methods described, this cannot rule out the possibility of infection from an identical environmental reservoir. The similarity with other strains from the same region (fig. 1) suggests the presence of a local ecotype. If local ecotypes exist, as our typing data suggests, the environmental source need not even have been identical, they might just have been infected in the same region. In conclusion, we couldn't establish human transmission in this case of an AFB positive and coughing source in close contact with an immunocompromised receiver.
Footnotes
Statement of Interest
None declared.
- ©ERS 2012
