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Laboratoire de Microbiologie
Departement de Pediatrie, Hpital Necker-Enfants Malades, Assistance Publique— Hpitaux de Paris (AP-HP), 75015 Paris
Laboratoire de Microbiologie
Departement de Pediatrie, Hpital Armand Trousseau, AP-HP, 75012 Paris
Laboratoire de Microbiologie
Departement de Pediatrie, Hpital Robert Debre, AP-HP, 75019 Paris
Centre de Ressources en Biostatistiques, Epidemiologie et Pharmacoepidemiologie
Centre National de Reference des Mycobacteries, Institut Pasteur, 75015 Paris
Laboratoire de Microbiologie, Hpital Raymond Poincare, AP-HP, 92380 Garches, France
ABSTRACT
We studied the prevalence and species distribution of nontuberculous mycobacteria (NTM) in relation to age in 385 patients with cystic fibrosis (CF) (mean age ± standard deviation , 12.0 ± 6.1 [1 to 24] years; sex ratio, 0.53) attending three Parisian centers. The overall prevalence of NTM in sputum was 8.1% (31 out of 385). The following NTM were isolated (n = 33): Mycobacterium abscessus (n = 13, 39.4%), Mycobacterium avium complex (MAC) (n = 7, 21.2%), Mycobacterium gordonae (n = 6, 18.2%), and other (n = 7, 21.2%). Sixteen patients met the American Thoracic Society microbiological criteria for NTM infection, including 11 patients positive for M. abscessus, 4 for MAC, and 1 for MAC and Mycobacterium kansasii. The overall prevalence of NTM was significantly lower in patients under 15 years old than for patients equal to or more than 15 years old (4.8 versus 14.9%, respectively; P = 0.001). M. abscessus was isolated at all ages, while MAC was not recovered before 15 years (prevalence of 0.0 and 5.2% in patients aged 1 to 14 and 15 to 24, respectively; P = 0.001).
TEXT
Since 1990, an increasing number of studies have reported the recovery of nontuberculous mycobacteria (NTM) from the respiratory tract of patients with cystic fibrosis (CF) (1, 12, 13, 16). In a recent multicenter study carried out in the United States using standardized bacteriological methods, the overall prevalence of NTM in sputum (percent of patients with at least one positive NTM culture) was 13%, ranging from 7 to 24% depending on the center (16). Mycobacterium avium complex (MAC) (72%) and Mycobacterium abscessus (16%) were the most common species. Other NTM (Mycobacterium gordonae, Mycobacterium kansasii, Mycobacterium lentiflavum, Mycobacterium peregrinum) were much rarer. About 20% of the culture-positive subjects met the American Thoracic Society (ATS) microbiological criteria for NTM pulmonary disease (2); most of these patients were positive for MAC or M. abscessus.
Paradoxically, although we know that CF children are vulnerable to bacterial infections from birth, most previous studies on NTM in CF have been conducted in populations mostly or exclusively composed of adults (1, 13, 16). For example, only patients who were 10 years or older were recruited in the multicenter United States study cited above, and the mean age ± standard deviation (SD) was 21 ± 9 years (16). Very few case series involving children with CF have been carried out, and those that have been done mainly used inadequate culture and species identification techniques. However, although imperfect, these studies suggest some differences in the epidemiology of NTM between pediatric and adult CF populations. One of the most intriguing differences is the apparent propensity of CF children to be infected with rapidly growing mycobacteria (RGM). In 1980, Boxerbaum reported that 8 out of 430 CF children were infected with RGM: 6 with organisms referred to as Mycobacterium chelonei (now the M. chelonae-abscessus group) and two with M. fortuitum (4). M. chelonei infection was believed to be the cause of death in one of the six patients. In a prospective study carried out at a French pediatric CF center, 4 out of 106 patients had at least one sputum sample positive for M. fortuitum complex organisms, most likely M. abscessus, but no patients were positive for MAC (8).
To study possible age-related differences in the overall epidemiology of NTM in CF, we conducted prospectively a large multicenter prevalence study including children, teenagers, and young adults. We show that both the overall prevalence of NTM and the distribution of NTM species in CF patients vary in relation with age, suggesting major differences in the epidemiology of the various NTM species encountered in CF.
The Parisian area (France) possesses three CF centers located in teaching hospitals (Armand Trousseau, Necker-Enfants Malades, and Robert Debre hospitals). These centers are pediatric centers, but they continue to follow up patients until the age of 20 to 25 years to ensure a progressive and less psychologically difficult transfer to adult centers. In 2000, at least three sputum samples from each of the CF patients attending these three centers were tested for NTM. All sputum samples studied were collected as part of a routine search for common CF pathogens. The French National Ethics Committee states that approval and informed consent are not required for this type of research. Sputum samples were processed at each of the three centers using the same protocol. Samples were decontaminated with NALC-NaOH-oxalic acid (0.25% N-acetyl-L-cysteine-1% sodium hydroxide-5% oxalic acid) (20). Acid-fast bacilli (AFB) smears were stained with auramine-rhodamine and scored as previously described (21). Two Lwenstein-Jensen slants were inoculated per specimen; one was incubated at 37°C and the other at 30°C. The slants were examined twice weekly for 2 weeks and then weekly for a further 10 weeks. RGM were identified by standard techniques (15) and hsp65 sequencing (17). MAC was identified by the Accuprobe technique (Gen Probe). All strains were sent to the reference laboratory (Centre National de Reference des Mycobacteries, Institut Pasteur, Paris, France) for confirmation of identification. The Fisher exact test and chi-square test were used for statistical analysis. Statistical significance was accepted for P < 0.05.
A total of 385 CF patients were screened for NTM during the study period (total number of sputum samples, 1,567; mean [SD] number of sputum samples per patient, 4.1 [1.8]; total number of sputum samples with bacterial overgrowth, 133 [8.5%]). The age of these 385 patients at inclusion ranged from 1 to 24 years (mean [SD], 12.0 [6.1] years); the sex ratio was 0.53 (203 males/182 females). The demographic characteristics of the included patients were similar in the three centers and consistent to those of the overall CF population followed in France (3). Of the 385 patients, 31 (8.1%; ranging from 6.0 to 11.7% according to center) provided at least one sample positive for NTM during the study period. These 31 patients were 16 males and 15 females (sex ratio, 0.52). Their mean age at the time of the first culture positive for NTM was 14.6 years, with a range of 1 to 22 years. Thirteen patients provided at least one sample with positive AFB smear. These 13 patients were also culture positive.
Thirty-three mycobacterial strains were isolated from the 31 NTM-positive subjects; 29 subjects had a single species and two had two species (MAC and M. kansasii in one subject, M. abscessus and M. gordonae in one subject). The most common organisms were M. abscessus (13 out of 33, 39.4%), MAC (7 out of 33, 21.2%), and M. gordonae (6 out of 33, 18.2%); other organisms (7 out of 33, 21.2%) included M. kansasii (n = 2), M. chelonae (n = 1), M. lentiflavum (n = 1), M. scrofulaceum (n = 1), M. szulgai (n = 1), and M. xenopi (n = 1) (Fig. 1). Of the 31 culture-positive subjects, 16 (51.6% of culture-positive subjects, 4.2% of all study subjects) met the ATS bacteriological criteria for NTM pulmonary disease (at least three positive sputum cultures with negative AFB smears or two positive sputum cultures and one positive AFB smear) (2): 11 were positive for M. abscessus, 4 for MAC, and 1 for MAC and M. kansasii (Fig. 1). Thirteen of these patients had positive AFB smears (M. abscessus, 9 patients; MAC, 3 patients; MAC and M. kansasii, 1 patient).
We analyzed the prevalence and the distribution of the NTM species within each of the following age brackets: 1 to 4, 5 to 9, 10 to 14, 15 to 19, and 20 to 24 years. The overall prevalence of NTM was relatively stable at around 5% between 1 and 14 years (Table 1). It then increased sharply, reaching about 15% at older ages (prevalence of 4.8 and 14.9% in patients aged 1 to 14 and 15 to 24 years, respectively; P = 0.001). However, there were marked differences according to the NTM species. M. abscessus was isolated at all ages up to 22 years (mean [SD] age, 14.1 [5.5] years), without significant differences between the age groups. Conversely, MAC was not found before 15 years (mean [SD] age, 17.3 [2.0] years), with prevalence values of 0.0 and 5.2% in patients aged 1 to 14 and 15 to 24 years, respectively (P = 0.001); its prevalence in patients aged 15 years and over was similar to that of M. abscessus (Table 1). M. gordonae was mostly isolated before 15 years (mean [SD] age, 9.2 [5.3] years), with prevalence values of 2.0 and 0.7% in patients aged 1 to 14 and 15 to 24 years, respectively (the P value was not significant). Finally, the other NTM species, such as M. kansasii, tended to behave like MAC (mean [SD] age, 17.3 [4.3] years), with prevalence values of 0.4 and 4.4% in patients aged 1 to 14 and 15 to 24 years, respectively (P < 0.01). These differences were reflected by the distribution of NTM species as a function of age (Fig. 2): M. abscessus and M. gordonae predominated between 1 and 14 years, accounting for 90% of NTM recovered in this age bracket; MAC and M. abscessus predominated at older ages, each accounting for one-third of NTM, far ahead of NTM species such as M. kansasii (9%).
This is the first time the prevalence of NTM species has been studied in relation with age in a large CF population including patients of all ages, from very young children to adults. The age pyramid of our population reflects the current demography of CF patients in France (3). We did not, however, include patients aged over 24 years. Thus, our study does not provide any information about older adults, who currently account for about 17% of CF patients in France (3). Our results show that the overall prevalence of NTM is relatively steady until about 14 or 15 years of age (about 5%). It increases considerably thereafter, reaching about 15%. Similar data have been published previously, based on studies on either children or adults (1, 4, 8, 12, 13, 16, 18). However, their relevance can be questioned given the major methodological differences between studies. Our study is not subject to this type of bias, because we included patients with the same demographic characteristics from three centers and because all the patients were screened using the same microbiological techniques (decontamination, culture, and identification). The increase in the prevalence of NTM observed in adolescence is probably multifactorial. Most NTM are probably opportunistic pathogens that can develop only on preexisting lesions, such as bronchiectases (11). It is also possible that some NTM, due to the fact that they are resistant to most antibiotics classically used to treat CF, are progressively selected by the repeated antibiotic treatments received by patients, as has been shown for other CF pathogens, such as Stenotrophomonas maltophilia and Alcaligenes xylosoxidans (9, 14). Finally, there is probably a cumulative effect linked to the fact that some infections caused by NTM such as M. abscessus start in childhood and persist indefinitely in most cases (6, 18, 19).
The most striking finding of our study is that the various NTM species recovered were associated with different age classes. The difference was particularly striking between the two most common NTM, M. abscessus and MAC: whereas M. abscessus was isolated at all ages, including from very young children, MAC was only found from adolescence onwards. It is possible that this is due to differences in the sources or modes of contamination. MAC and the M. chelonae-abscessus group are common in soils and water (5, 7). However, M. abscessus has never been found in the environment of CF patients. A second explanation, which is equally plausible and does not exclude the first hypothesis, is that M. abscessus, as was recently suggested by Griffith, is more closely related to a true respiratory pathogen than to an opportunistic pathogen unlike MAC (10). The requirement for underlying pulmonary lesions in the establishment of durable infection may thus be less strict for M. abscessus than for MAC, explaining the greater capacity of M. abscessus to develop in young subjects.
ACKNOWLEDGMENTS
We thank Gil Bellis (Institut National d'Etudes Demographiques, Paris) for demographic information about CF in France and Claire Bernede for help with statistical analysis.
This work was supported by a grant from the Association "Vaincre la Mucoviscidose."
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