Mycobacterium avium Complex Infections: Detailed Phenotypic and Functional Immunological Work-Up Is Required despite Genetic Analyses.

INTRODUCTION: Cervical scrofulous lymphadenitis due to Mycobacterium avium complex (MAC) in immunocompetent adults is a rare disease. The presence of MAC infections demands meticulous clinical evaluation of patients along with detailed phenotypic and functional evaluation of their immune system including next-generation sequencing (NGS) analyses of target genes. METHODS: Exact clinical histories of the index patients both suffering from retromandibular/cervical scrofulous lymphadenitis were obtained along with phenotypic and functional immunological evaluations of leukocyte populations followed by targeted NGS-based sequencing of candidate genes. RESULTS: Immunological investigations showed normal serum immunoglobulin and complement levels, but lymphopenia, which was caused by significantly reduced CD3+CD4+CD45RO+ memory T-cell and CD19+ B-cell numbers. Despite normal T-cell proliferation to a number of accessory cell-dependent and -independent stimuli, the PBMC of both patients elaborated clearly reduced levels of a number of cytokines, including IFN-γ, IL-10, IL-12p70, IL-1α, IL-1ß, and TNF-α upon TCR-dependent T-cell stimulation with CD3-coated beads but also superantigens. The IFN-γ production deficiency was confirmed for CD3+CD4+ helper and CD4+CD8+ cytotoxic T cells on the single-cell level by multiparametric flow cytometry irrespective of whether PMA/ionomycin-stimulated whole blood cells or gradient-purified PBMC was analyzed. In the female patient L1, targeted NGS-based sequencing revealed a homozygous c.110T>C mutation in the interferon-γ receptor type 1 (IFNGR1) leading to significantly reduced receptor expression on both CD14+ monocytes and CD3+ T cells. Patient S2 presented with normal IFNGR1 expression on CD14+ monocytes but significantly reduced IFNGR1 expression on CD3+ T cells, despite the absence of detectable homozygous mutations in the IFNGR1 itself or disease-related target genes. Exogenous addition of increasing doses of IFN-γ resulted in proper upregulation of high-affinity FcγRI (CD64) on monocytes from patient S2, whereas monocytes from patient L1 showed only partial induction of CD64 expression after incubation with high doses of IFN-γ. CONCLUSION: A detailed phenotypic and functional immunological examination is urgently required to determine the cause of a clinically relevant immunodeficiency, despite detailed genetic analyses.

N-acetyl-cysteine mediates protection against Mycobacterium avium through induction of human ß-defensin-2 in a mouse lung infection model.

Mycobacterium avium complex is a causative organism for refractory diseases. In this study, we examined the effects of N-acetyl-cysteine on M. avium infection in vitro and in vivo. N-acetyl-cysteine treatment suppressed the growth of M. avium in A549 cells in a concentration-dependent manner. This effect was related to the induction of the antibacterial peptide human ß-defensin-2. In a mouse model, N-acetyl-cysteine treatment significantly reduced the number of bacteria in the lungs and induced murine ß-defensin-3. In interleukin-17-deficient mice, the effects of N-acetyl-cysteine disappeared, indicating that these mechanisms may be mediated by interleukin-17. Moreover, an additional reduction in bacterial load was observed in mice administered N-acetyl-cysteine in combination with clarithromycin. Our findings demonstrate the potent antimycobacterial effects of N-acetyl-cysteine against M. avium by inducing antimicrobial peptide, suggesting that N-acetyl-cysteine may have applications as an alternative to classical treatment regimens.

The rough colony morphotype of Mycobacterium avium exhibits high virulence in human macrophages and mice.

Introduction. The incidence of Mycobacterium avium complex (MAC) pulmonary disease (MAC PD), a refractory chronic respiratory tract infection, is increasing worldwide. MAC has three predominant colony morphotypes: smooth opaque (SmO), smooth transparent (SmT) and rough (Rg).Aim. To determine whether colony morphotypes can predict the prognosis of MAC PD, we evaluated the virulence of SmO, SmT and Rg in mice and in human macrophages.Methodology. We compared the characteristics of mice and human macrophages infected with the SmO, SmT, or Rg morphotypes of M. avium subsp. hominissuis 104. C57BL/6 mice and human macrophages derived from peripheral mononuclear cells were used in these experiments.Results. In comparison to SmO- or SmT-infected mice, Rg-infected mice revealed severe pathologically confirmed pneumonia, increased lung weight and increased lung bacterial burden. Rg-infected macrophages revealed significant cytotoxicity, increased bacterial burden, secretion of proinflammatory cytokines (TNF-α and IL-6) and chemokines (CCL5 and CCL3), and formation of cell clusters. Rg formed larger bacterial aggregates than SmO and SmT. Cytotoxicity, bacterial burden and secretion of IL-6, CCL5 and CCL3 were induced strongly by Rg infection, and were decreased by disaggregation of the bacteria.Conclusion. M. avium Rg, which is associated with bacterial aggregation, has the highest virulence among the predominant colony morphotypes.

Genetic Variation/Evolution and Differential Host Responses Resulting from In-Patient Adaptation of Mycobacterium avium.

Members of the Mycobacterium avium complex (MAC) are characterized as nontuberculosis mycobacteria and are pathogenic mainly in immunocompromised individuals. MAC strains show a wide genetic variability, and there is growing evidence suggesting that genetic differences may contribute to a varied immune response that may impact the infection outcome. The current study aimed to characterize the genomic changes within M.avium isolates collected from single patients over time and test the host immune responses to these clinical isolates. Pulsed-field gel electrophoresis and whole-genome sequencing were performed on 40 MAC isolates isolated from 15 patients at the Department of Medical Microbiology at St. Olavs Hospital in Trondheim, Norway. Isolates from patients (patients 4, 9, and 13) for whom more than two isolates were available were selected for further analysis. These isolates exhibited extensive sequence variation in the form of single-nucleotide polymorphisms (SNPs), suggesting that M. avium accumulates mutations at higher rates during persistent infections than other mycobacteria. Infection of murine macrophages and mice with sequential isolates from patients showed a tendency toward increased persistence and the downregulation of inflammatory cytokines by host-adapted M. avium strains. The study revealed the rapid genetic evolution of M. avium in chronically infected patients, accompanied by changes in the virulence properties of the sequential mycobacterial isolates.

Epidemiology and Predictors of NTM Pulmonary Infection in Taiwan - a Retrospective, Five-Year Multicenter Study.

Multicenter, longitudinal studies on nontuberculous mycobacteria (NTM) pulmonary infection (PI) are lacking. This study provides a 5-year epidemiological overview of NTM-PI in Taiwan and investigated its predictors. The clinical relevance of each respiratory NTM isolate in six hospitals between 2008 and 2014 was determined according to current guidelines. Recurrent episodes were judged by serial bacteriological results. New episodes of NTM-PI and pulmonary colonization (PC) occurring since 2010 were analyzed. Logistic regression analysis was performed to identify the predictors of NTM-PI. Between 2010 and 2014, the incidence rate of NTM-PI was 46.0 episodes per 100,000 hospital-based patient-years. Mycobacterium avium intracellulare complex (MAC) was predominant in Northern Taiwan, whereas MAC and M. abscessus were copredominant in Southern Taiwan. Multiple episodes occurred in 9.5% of NTM-PI patients. No female predominance was observed, except for MAC-PI. Previous pulmonary tuberculosis and chronic obstructive pulmonary disease (COPD) were the most common pulmonary comorbidities and independent risk factors for NTM-PI. Other risk factors included M. kansasii, M. abscessus, and southern Taiwan. Geographical variation of NTM-PI exists in Taiwan. Clinicians should keep a high suspicion on NTM-PI in the risk population. In endemic area of tuberculosis and COPD, there may be no female predominance in NTM-PI.

Health-related quality of life is inversely correlated with C-reactive protein and age in Mycobacterium avium complex lung disease: a cross-sectional analysis of 235 patients.

BACKGROUND: Mycobacterium avium complex (MAC) lung diseases generally cause chronic disease in immunocompetent hosts. Although a few studies have examined health-related quality of life (HRQL) in patients with MAC lung disease, there have been no large studies. This study aimed to evaluate HRQL and its correlation with clinical outcomes in MAC lung disease. METHODS: A cross-sectional study was conducted at Keio University Hospital to investigate the factors associated with HRQL in pulmonary nontuberculous mycobacterial diseases. MAC lung diseases were diagnosed according to the 2007 ATS/IDSA guidelines for nontuberculous mycobacterial diseases. The 36-item short form health survey (SF-36) was administered to assess clinical outcomes. Clinical variables included treatment status, latest haematological data, and bacterial smear and culture results. RESULTS: The SF-36 scores for the 235 patients (median age, 69 years; 45 men and 190 women) with MAC lung disease, except for the bodily pain and mental health subscale scores, were significantly lower than the Japanese population norms. In the multivariable analyses, current treatment for MAC and a positive sputum smear or culture within the past year were significantly associated with lower SF-36 scores. C-reactive protein (CRP) and age showed stronger inverse correlations with SF-36 scores. CONCLUSIONS: HRQL, especially the physical component, was impaired in patients with MAC lung diseases; this appears to be related with current treatment status, positive sputum smear or culture within the previous year, and particularly CRP and age. Further studies including qualitative assessments are needed to investigate the efficacy of CRP as a marker for progression or treatment response in MAC lung disease. TRIAL REGISTRATION: Clinical trial registered with UMIN ( UMIN000007964 ).

Role of IL-6 in Mycobacterium avium--associated immune reconstitution inflammatory syndrome.

Immune reconstitution inflammatory syndrome (IRIS) is a major adverse event of antiretroviral therapy in HIV infection, and paradoxically occurs as HIV viremia is suppressed and CD4 T cell numbers recover. IRIS reflects pathogenic immune responses against opportunistic infections acquired during the period of immunodeficiency, but little is understood about the mechanisms of inflammatory pathology. In this study, we show that IL-6 and C-reactive protein levels transiently rise at the time of the IRIS event in HIV-infected patients, unmasking Mycobacterium avium complex infection after starting antiretroviral therapy. To directly test the role of IL-6 in IRIS pathology, we used a model of experimentally inducible IRIS in which M. avium-infected T cell-deficient mice undergo a fatal inflammatory disease after reconstitution with CD4 T cells. We find that IL-6 neutralization reduces C-reactive protein levels, alleviates wasting disease, and extends host survival during experimental IRIS. Moreover, we show that combined blockade of IL-6 and IFN-γ further reduces IRIS pathology, even after the onset of wasting disease. The combination of these clinical and experimental-model data show that the IL-6 pathway is not only a biomarker of mycobacterial IRIS but also a major mediator of pathology distinct from IFN-γ and may be a useful target for therapeutic intervention.

Distribution of mycobacterial antigen based on differences of histological characteristics in pulmonary Mycobacterium avium infectious diseases--consideration of the extent of surgical resection from the pathological standpoint.

Quantitative assessment of mycobacterial antigens is very important to determine the surgical indication, as well as the area of resection for pulmonary Mycobacterium avium complex (MAC) infectious disease. However, at present, pathological assessment is only possible as a postoperative examination. We performed quantitative evaluation of mycobacterial antigens using lung tissues with MAC pulmonary infection obtained from surgical resection. The distribution of mycobacterial antigens was evaluated by immunohistochemical staining with monoclonal antibody for mycobacteria. In exudative reactions, many monocyte-lineage cells containing mycobacterial antigens were observed in alveoli, whereas the quantity of mycobacterial antigens was extremely decreased in proliferative reactions. Epithelioid cells or multinucleated giant cells contained mycobacterial antigens in necrotic granulomas. In solitary nodules with central necrosis, mycobacterial antigens were frequently observed, whereas they were rarely observed in solitary nodules without caseous necrosis. Mycobacterial antigens were not observed in the epithelial layer of bronchioles in any cases, although proliferative granulomas were notably observed in the developed lymphoid follicles in subepithelial lesions of bronchiole. Thus, exudative reactions or nodules with caseous necrosis indicate the possibility of numerous mycobacteria remaining in the pulmonary focus. Therefore, intraoperative histological assessment may help in the determination of the area of surgical resection. This is the first study to quantitatively evaluate mycobacterial antigens according to histological characteristics in MAC pulmonary disease.

IL-32 expression in the airway epithelial cells of patients with Mycobacterium avium complex lung disease.

Lung disease due to Mycobacterium avium complex (MAC) organisms is increasing. A greater understanding of the host immune response to MAC organisms will provide a foundation to develop novel therapies for these recalcitrant infections. IL-32 is a newly described pro-inflammatory cytokine that enhances host immunity against various microbial pathogens. Cytokines that induce IL-32 such as interferon-gamma, IL-18, IL-12 and tumor necrosis factor-alpha are of considerable importance to mycobacterial immunity. We performed immunohistochemistry and morphometric analysis to quantify IL-32 expression in the lungs of 11 patients with MAC lung disease and 10 controls with normal lung tissues. After normalizing for basement membrane length, there was a profound increase in IL-32 expression in the airway epithelial cells of the MAC-infected lungs compared with controls. Following normalization for alveolar surface area, there was a trend toward increased IL-32 expression in type II alveolar cells and alveolar macrophages in the lungs of MAC patients. Human airway epithelial cells (BEAS-2B) infected with M. avium produced IL-32 by a nuclear factor-kappa B-dependent mechanism. In both BEAS-2B cells and human monocyte-derived macrophages, exogenous IL-32γ significantly reduced the growth of intracellular M. avium. This finding was corroborated by an increase in the number of intracellular M. avium recovered from THP-1 monocytes silenced for endogenous IL-32 expression. The anti-mycobacterial effect of IL-32 may be due, in part, to increased apoptosis of infected cells. These findings indicate that IL-32 facilitates host defense against MAC organisms but may also contribute to the airway inflammation associated with MAC pulmonary disease.

Structure and host recognition of serotype 13 glycopeptidolipid from Mycobacterium intracellulare.

The Mycobacterium avium-M. intracellulare complex (MAIC) is divided into 28 serotypes by a species-specific glycopeptidolipid (GPL). Previously, we clarified the structures of serotype 7 GPL and two methyltransferase genes (orfA and orfB) in serotype 12 GPL. This study elucidated the chemical structure, biosynthesis gene, and host innate immune response of serotype 13 GPL. The oligosaccharide (OSE) structure of serotype 13 GPL was determined to be 4-2'-hydroxypropanoyl-amido-4,6-dideoxy-ß-hexose-(1 → 3)-4-O-methyl-α-L-rhamnose-(1 → 3)-α-L-rhamnose-(1 → 3)-α-L-rhamnose-(1 → 2)-α-L-6-deoxy-talose by using chromatography, mass spectrometry, and nuclear magnetic resonance (NMR) analyses. The structure of the serotype 13 GPL was different from those of serotype 7 and 12 GPLs only in O-methylations. We found a relationship between the structure and biosynthesis gene cluster. M. intracellulare serotypes 12 and 13 have a 1.95-kb orfA-orfB gene responsible for 3-O-methylation at the terminal hexose, orfB, and 4-O-methylation at the rhamnose next to the terminal hexose, orfA. The serotype 13 orfB had a nonfunctional one-base missense mutation that modifies serotype 12 GPL to serotype 13 GPL. Moreover, the native serotype 13 GPL was multiacetylated and recognized via Toll-like receptor 2. The findings presented here imply that serotypes 7, 12, and 13 are phylogenetically related and confirm that acetylation of the GPL is necessary for host recognition. This study will promote better understanding of the structure-function relationships of GPLs and may open a new avenue for the prevention of MAIC infections.

Preparation and characterization of spray dried inhalable powders containing chitosan nanoparticles for pulmonary delivery of isoniazid.

The aim of this study was to prepare spray dried inhalable powders containing isoniazid-loaded chitosan/tripolyphosphate (TPP) nanoparticles for sustained delivery of the drug to the lung. Nanoparticles were prepared by ionic gelation method. In-vitro drug release study indicated that the rate of drug release from nanoparticles was decreased by increasing the amount of chitosan. Entrapment of isoniazid into chitosan/TPP nanoparticles decreased minimum inhibitory concentrations (MIC) of the drug against mycobacterium avium intracellulare. Nanoparticles were spray dried using excipients such as lactose, mannitol and maltodextrin alone or with leucine. Results showed that the obtained powders had different aerosolization property. It was observed that by adding leucine, the particle size of microparticles deceased and the process yield and fine particle fraction (FPF) increased significantly. The in-vitro deposition data indicated that spray drying of isoniazid-loaded nanoparticles with lactose in the presence of leucine resulted in the production of inhalable powders with the highest FPF (45%).

Immunological markers of lung disease due to non-tuberculous mycobacteria.

Lung disease due to non-tuberculous mycobacteria (NTM) is a poorly understood condition that is difficult to treat. Treatment remains problematic as few tools are available to help clinicians monitor disease progression or predict treatment outcome. In this study, plasma levels of several inflammatory molecules and the frequency of circulating T cell subsets were measured in patients with NTM lung disease and known treatment status, and compared with their adult offspring and with unrelated healthy controls. Plasma levels of the chemokine CXCL10 and IL-18 were assessed for associations with treatment efficacy. CXCL10 was higher in patients than adult offspring (p< 0.001) and unrelated controls (p< 0.001). Plasma CXCL10 was also lower in patients who responded well to therapy or who controlled their infection without requiring therapy, when compared to patients who did not respond to therapy (p=0.03). Frequencies of activated (HLA-DR(+)) CD4(+) T cells were higher in patients than adult offspring (p<0.001) and unrelated controls (p<0.05), with the highest frequencies in individuals who had completed at least 6 months of treatment. Frequencies of activated (CD38(+)) CD8(+) T cells in most treatment responders were similar to unrelated controls. Low plasma levels of CXCL10 may reflect successful control of NTM lung disease with or without therapy. Compared with responders, patients who responded poorly to treatment generally had higher plasma levels of CXCL10 and IL-18, and higher frequencies of activated CD8(+) T cells.

Control of Mycobacterium fortuitum and Mycobacterium intracellulare infections with respect to distinct granuloma formations in livers of BALB/c mice.

Mycobacterium fortuitum is a rapidly growing nontuberculous Mycobacterium that can cause a range of diseases in humans. Complications from M. fortuitum infection have been associated with numerous surgical procedures. A protective immune response against pathogenic mycobacterial infections is dependent on the granuloma formation. Within the granuloma, the macrophage effector response can inhibit bacterial replication and mediate the intracellular killing of bacteria. The granulomatous responses of BALB/c mice to rapidly and slowly growing mycobacteria were assessed in vivo and the bacterial loads in spleens and livers from M. fortuitum and Mycobacterium intracellulare-infected mice, as well as the number and size of granulomas in liver sections, were quantified. Bacterial loads were found to be approximately two times lower in M. fortuitum-infected mice than in M. intracellulare-infected mice and M. fortuitum-infected mice presented fewer granulomas compared to M. intracellulare-infected mice. These granulomas were characterized by the presence of Mac-1+ and CD4+ cells. Additionally, IFN-γmRNA expression was higher in the livers of M. fortuitum-infected mice than in those of M. intracellulare-infected mice. These data clearly show that mice are more capable of controlling an infection with M. fortuitum than M. intracellulare. This capacity is likely related to distinct granuloma formations in mice infected with M. fortuitum but not with M. intracellulare.

Moxifloxacin pharmacokinetics/pharmacodynamics and optimal dose and susceptibility breakpoint identification for treatment of disseminated Mycobacterium avium infection.

Organisms of the Mycobacterium avium-intracellulare complex (MAC) have been demonstrated to be susceptible to moxifloxacin. However, clinical data on how to utilize moxifloxacin to treat disseminated MAC are scanty. In addition, there have been no moxifloxacin pharmacokinetic-pharmacodynamic (PK/PD) studies performed for MAC infection. We utilized an in vitro PK/PD model of intracellular MAC to study moxifloxacin PK/PD for disseminated disease. Moxifloxacin doses, based on a serum half-life of 12 h, were administered, and the 0- to 24-h area under the concentration-time curve (AUC(0-24)) to MIC ratios associated with 1.0 log(10) CFU/ml per week kill and 90% of maximal kill (EC(90)) were identified. The AUC(0-24)/MIC ratio associated with 1.0 log(10) CFU/ml kill was 17.12, and that with EC(90) was 391.56 (r(2) = 0.97). Next, the moxifloxacin MIC distribution in 102 clinical isolates of MAC was identified. The median MIC was 1 to 2 mg/liter. Monte Carlo simulations of 10,000 patients with disseminated MAC were performed to determine the probability that daily moxifloxacin doses of 400 and 800 mg/day would achieve or exceed 1.0 log(10) CFU/ml per week kill or EC(90). Doses of 400 and 800 mg/day achieved the AUC(0-24)/MIC ratio of 17.12 in 64% and 92% of patients, respectively. The critical concentration of moxifloxacin against MAC was identified as 0.25 mg/liter in Middlebrook media. The proposed susceptibility breakpoint means that a larger proportion of clinical isolates is resistant to moxifloxacin prior to therapy. For patients infected with susceptible isolates, however, 800 mg a day should be examined for safety and efficacy for disseminated M. avium disease.

Concentrations of clarithromycin and active metabolite in the epithelial lining fluid of patients with Mycobacterium avium complex pulmonary disease.

OBJECTIVES: Clarithromycin (CAM) is widely accepted for the treatment of Mycobacterium avium complex (MAC) pulmonary diseases. This study measured (a) the concentrations of CAM and its active metabolite (14OH-CAM) in bronchial epithelial lining fluid (ELF) obtained by bronchoscopic microsampling (BMS), and (b) the minimal inhibitory concentrations (MIC) of CAM for each MAC isolate. METHODS: We studied eight patients with MAC pulmonary disease treated with oral CAM, 400 (n=4) or 800 (n=4) mg/day. BMS was performed 3h after the last CAM dose, and the concentrations of CAM and 14OH-CAM were measured in ELF collected from the diseased and normal contralateral pulmonary segments, and in serum. RESULTS: The mean+/-SEM ELF concentrations of CAM (23.85+/-7.64 microg/ml) and CAM+14OH-CAM (28.71+/-8.37 microg/ml) in the 800 mg/day treatment group were significantly higher than in the 400mg/day group (7.48+/-2.58 microg/ml and 9.63+/-2.99 microg/ml, respectively; both p<0.05), while the serum concentrations were similar in both groups. In both treatment groups, the mean ELF concentrations sampled from diseased and normal segments were higher than the MIC against MAC isolates. The mean ELF concentration of CAM in the 400mg/day treatment group was <8 mircog/ml (the breakpoint concentration of CAM against M. avium recommended by the Clinical Laboratories Standards Institute), while the mean concentration in the 800 mg/day treatment group was >8 microg/ml. CONCLUSION: These observations suggest that CAM, 800 mg/day, is an appropriate dose to treat MAC pulmonary disease, and prevent its spread from diseased to non-diseased lung segments.

Bronchoscopic microsampling to analyze the epithelial lining fluid of patients with pulmonary Mycobacterium avium complex disease.

BACKGROUND: A new bronchoscopic microsampling (BMS) probe has recently been developed, which allows the minimally invasivecollection of epithelial lining fluid (ELF) simultaneously from multiple sites. While the number of immunocompetent patients with pulmonary Mycobacterium avium complex(MAC) disease is growing, the precise pathogenesis of the disease remains to be clarified. OBJECTIVES: This study aimed to measure biochemical markers in the ELF of patients with pulmonary MAC disease using BMS. METHODS: We studied 8 healthy volunteers (controls) and 8 patients with pulmonary MAC disease. In the patients, ELF was collected from visibly abnormal (involved) and normal (uninvolved) sites using high-resolution computed tomography (HRCT). RESULTS: The concentration of IFN-gamma (means +/- SEM) was significantly higher in patient ELF sampled from involved (26.62 +/- 9.66 pg/ml) than from uninvolved sites (5.33 +/- 1.44 pg/ml; p < 0.05) or from normal controls (<1.56 pg/ml; p < 0.05). In most patients, there was no difference in IFN-gamma concentrations between the involved and uninvolved sites. The concentration of KL-6 in ELF was significantly higher in the involved (793 +/- 108 U/ml) than in the uninvolved sites (192 +/- 43 U/ml;p < 0.05) or in normal controls (186 +/- 38 U/ml;p < 0.05). CONCLUSIONS: The specific inflammatory reaction caused by MAC infection might not be limited to sites that were visibly abnormal on HRCT. Although further improvement has to be considered, BMS may help to increase the understanding of the pathogenesis of various respiratory disorders.

Pulmonary Mycobacterium avium complex infection: association with NRAMP1 polymorphisms.

The present study aimed to elucidate risk factors for nonimmunocompromised pulmonary Mycobacterium avium complex (MAC) infection. Epidemiological data and variations of candidate genes for mycobacterial diseases were analysed in 111 patients with pulmonary MAC infection. Four polymorphisms of the human natural resistance-associated macrophage protein (NRAMP)1 gene, the 5'(GT)n, 469+14 G/C, D543N and the 3'untranslated region (3'TGTG) insertion/deletion, were genotyped using PCR-based methods. Fok I and Taq I polymorphisms of the vitamin D receptor gene and -221 X/Y and codon 54 A/B polymorphisms of the mannose binding lectin gene were also evaluated. Females were more susceptible to MAC infection mainly affecting the right middle lobe or lingular segment of the lung. Patients' residence at the onset of the disease was distributed evenly irrespective of a waterfront or city water supply system. As compared with homozygotes for major alleles of the D543N and TGTG insertion/deletion polymorphism of the NRAMP1 gene, heterozygotes containing minor alleles were less often observed in MAC cases than in controls. This genetic effect was more significant in patients without comorbidity but not in patients with comorbidity. Other polymorphisms did not show any association with the MAC infection. The human natural resistance-associated macrophage protein 1 gene might be involved in susceptibility to pulmonary Mycobacterium avium complex infection.