Retrospective Analysis of 10 Cases of Disseminated Nontuberculous Mycobacterial Disease with Osteolytic Lesions

Introduction

Nontuberculous mycobacterial (NTM) infection can lead to disseminated NTM (DNTM) disease, which usually affects multiple organs including the lung, skin and soft tissues and lymph nodes, as well as and bone can also be affected.^1,2 Previously, NTM infection of bone leading to osteolysis has been rarely reported and is often overlooked by clinicians. Recent research revealed that 34% of anti-IFN-γ-autoantibody (AIGA)-associated NTM disease can involve the bone and joints,³ suggesting that NTM infection of bone is not rare in AIGA-positive NTM patients. However, the mechanism of NTM leading to osteolysis is unclear, and it may be related to AIGAs.^4,5 Osteolytic destruction due to NTM is frequently misdiagnosed as tuberculosis or other fungal infections,^6–8 indicating insufficient recognition of this condition by clinicians. Moreover, systemic research on the clinical and imaging characteristics of patients with NTM involving bone and leading to osteolytic lesions is lacking, leading to delays in correct diagnosis and treatment. Therefore, we retrospectively analyzed the clinical and imaging characteristics of 10 human immunodeficiency virus (HIV)-negative patients with DNTM disease with osteolytic lesions to provide clinical experience for the early diagnosis of this condition.

Patients and Methods

Study Population

The medical records of ten HIV-negative patients diagnosed with DNTM disease with osteolytic lesions between January 1, 2011, and December 31, 2020, at the First Affiliated Hospital of Guangxi Medical University and the Fourth People’s Hospital of Nanning City were retrospectively evaluated. Data extracted from the medical records included demographic information (sex, age and occupation), clinical characteristics, laboratory findings, imaging manifestations, and clinical outcomes.

This study was approved by the Signation Ethical Committee of the First Affiliated Hospital of Guangxi Medical University [2021[KY-E-118]]. All patients or patients’ parents provided written informed consent.

Methods Used to Diagnose NTM Infection

Patients were diagnosed with NTM infection according to the following criteria: 1) NTM culture: Clinical specimens (such as sputum, blood, skin lesion pus, bone marrow, and pathological tissues) were inoculated on solid and liquid medium and incubated at 35°C. Positive RGM cultures were characterized by colonies that were visible to the naked eye within 7 days, while positive SGM cultures by colonies formed within 2 to 3 weeks.^9,10 2) Identification of NTM species: Gene sequencing technology, such as direct homologous gene method or sequence alignment method (16S DNA, hsp65), is currently the “gold standard” for strain identification.^10,11 Previously, due to the limitation of the laboratory conditions, only species were identified, but no subspecies were identified. Patients who met one of the above criteria were diagnosed with NTM infection.

Diagnostic Criteria for DNTM Disease

The diagnostic criteria for DNTM disease were as follows: two or more nonadjacent tissues or organs were involved, positive NTM culture/positive molecular biology detection from the blood, and/or positive NTM culture/positive molecular biology detection from any of the following: respiratory secretions, skin lesion pus, bone marrow, and biopsy specimens from the liver, lymph nodes, and lung.⁹

Diagnostic Criteria for NTM with Osteolytic Lesions

Patients were diagnosed according to one of the following criteria: 1) NTM identified in bone and/or bone marrow biopsy samples using culture and metagenomics next-generation sequencing (mNGS); 2) disseminated NTM disease with osteolytic lesions diagnosed based on the presence of osteolytic lesions on imaging examination, clinical symptoms including ostealgia, improvement after receiving anti-NTM treatment alone, and exclusion of other diseases that cause osteolysis (tuberculosis, cancer, hematological diseases, and fungal infections such as African histoplasmosis, blastomycosis, cryptococcosis, Talaromycosis marneffei and coccidioidomycosis).

Anti-IFN-γ Autoantibody Assay

Serum samples were obtained under sterile conditions before the patient received antimicrobial therapy and during the active stage of the infection. Serum samples were retrieved from a serum bank and stored at −80°C. AIGAs antibody was detected in all participants. All serum samples were tested at the first thaw. The detection of AIGAs in the serum was performed using an enzyme-linked immunosorbent assay kit (Cloud-Clone Corp., Wuhan, China) according to the manufacturer’s protocols. The normal range for the AIGA concentration was defined as the 99th percentile for the healthy controls and was estimated using the log-normal distribution. Outlying concentrations were classified as positive for AIGAs.¹³

Results

Demographic Data, Clinical Characteristics, NTM Culture and Histopathology

During the 10-year study period, ten HIV-negative patients with DNTM disease with osteolytic lesions were evaluated. The study population included six males with a median age of 54.5 years (range 36.5–74.25 years). Their occupations included farmers (n = 7), retirees (n = 2) and workers (n = 1). The median time from symptom onset to diagnosis was 12 months (range: 8.5–18.5 months). Among the ten patients, five patients had 1–2 underlying diseases and/or a previous surgical history, and two (20%) had previous glucocorticoid therapy. Six patients had other bacterial and fungal coinfections. All patients showed disseminated infection of NTM, with the lung, lymph nodes, skin, bone, joint, and liver being the most commonly involved organs. Microbiological studies demonstrated M. abscessus in three patients, M. avium complex in two patients, and M. kansasii, M. fortuitum, M. chelonae, M. intracellulare, and M. intermedium in one patient each. NTM were isolated from sputum samples (6/10, 60%), pus from subcutaneous abscesses (5/10, 50%), pus from joints (1/10, 10%), pus from lymph nodes (1/10, 10%) and bronchoalveolar lavage fluid (1/10, 10%). In addition, histopathology or cytology of specimens obtained from five patients showed suppurative inflammation, including 2 with and 3 without granuloma formation. All patients were misdiagnosed with tuberculosis (including pulmonary tuberculosis, bone and joint tuberculosis, lymph node tuberculosis, and tuberculous pleurisy), and the duration of antituberculosis treatment varied from 4 to 48 months (Table 1).

Table 1 Demographic Data, Clinical Characteristics, NTM Culture and Histopathology of 10 Patients with Disseminated Nontuberculous Mycobacterial Disease with Osteolytic Lesions

Anemia (100%) and ostealgia (100%) were the most common symptoms and signs, followed by fever (70%), emaciation (70%), cough and expectoration (70%), subcutaneous abscesses (70%), anorexia (60%) and lymphadenopathy (60%). Joint pain (10%), joint dysfunction (10%), abdominal pain and distension (10%), diarrhea (10%) and bloody stool (10%) were also present (Supplemental Table 1).

Laboratory Examination

Complete blood count examinations revealed increased white blood cell and neutrophil counts in nine patients (90%) (median 14.65 × 10⁹/L, range 11.85–22.1× 10⁹/L; median 11.49 × 10⁹/L, range 8.64–18.7 × 10⁹/L, respectively) and normal absolute lymphocyte counts in ten patients. Ten patients showed decreased hemoglobin concentrations (median 83 g/L, range 68.65–96.25 g/L). Four of nine patients showed reductions in CD4+ T lymphocyte counts (median 511 cells/µL, range 182–614 cells/µL). Three of nine patients showed reductions in CD8+ T lymphocyte counts (median 331 cells/µL, range 122.5–429.5 cells/µL). Nine patients (90%) showed decreased albumin levels (median 29.4 g/L, range 24.5–33.6 g/L), four patients (40%) showed decreased globulin levels (median 39.5, range 35.2–46.2 g/L), and three patients (30%) showed liver dysfunction. C-reactive protein (CRP) concentrations and erythrocyte sedimentation rate (ESR) were significantly increased in all patients (median 101 mm/h, range 45.5–133 mm/h; median 134 mg/L, range 69.6–175.6 mg/L, respectively). Serum samples obtained from 10 patients were tested for AIGAs. Among the ten patients, seven patients were defined as AIGA-positive, and the other three were defined as AIGA-negative. The positivity rate of AIGAs was 70% (7/10), with a median titer of 31,220.2 ng/mL and a range of 4266.2–56,088.2 ng/mL (Supplemental Table 2).

Skeletal Localization

NTM could affect any bone of the body, presenting with multiple bone involvement. The bony structures involved were as follows: bones of the limbs (6/9, 66.67%), trunk (5/9, 55.56%), skull 2/9, 22.22%), and cartilage (1/9, 11.11%). The most common sites were the vertebrae, sternum, clavicle, and ribs (3/9, 33.33%). The femur and ilium were affected in two patients (2/9, 22.22%), and the humerus, scapula, metacarpal, phalanx, tibia, talus, frontal bone and mandible were affected in one patient (1/9,11.11%). Two patients (2/9, 22.22%) showed joint involvement, and seven patients showed surrounding abscess formation (7/9, 77.78%) (Table 1).

Image Features of Disseminated NTM Disease with Osteolytic Lesions

All patients showed multiple areas with osteolytic destruction (more than two sites). Radiographic and CT examinations were performed on nine patients and showed moth-eaten or irregular destruction of bone, bone defects, and single/multiple, well-circumscribed, rounded low-density areas with bone destruction (Figure 1). Pathological fracture was identified in two patients (Figure 1A), periosteal proliferation in two patients (Figure 1F), and surrounding abscess formation in seven patients (Figure 1B, D and E). A magnetic resonance imaging (MRI) scan was performed on one patient and showed high signals in the T2-weighted image and the fat-saturated T2-weighted image (Figure 1C). An ECT bone scan was performed in one patient and showed significantly increased uptake in multiple bones (Figure 1G). Positron emission tomography/CT (PET/CT) was performed in two patients and showed metabolic activity in multiple bones of the body, indicating systemic bone marrow involvement. Chest computed tomography (CT) revealed abnormal findings in eight patients, presenting as patchy exudation, fiber proliferation, consolidation, bronchiectasis, ground-glass opacity, cavities, atelectasis, nodules, mediastinal and/or hilar lymphadenopathy, pleural thickening, pleural effusion and pericardial effusion (Figure 2 and Table 2).

Table 2 Imaging Manifestations of 10 Patients with Disseminated Nontuberculous mycobacteria Disease with Osteolytic Lesions

Figure 1 X-ray radiography revealed a pathological fracture in the right clavicle (arrow) (A from patient 5). CT of the right wrist and palm revealed moth-eaten and irregular destruction of the bone in the right metacarpal and adjacent joints, narrowing of the joint space and swelling in the surrounding soft tissues (arrow) (B from patient 3). A sagittal magnetic resonance imaging (MRI) scan (fat-saturated T2-weighted image) revealed high signals within the L3 vertebral body (arrow) (C from patient 8). Computed tomography (CT) of the head and brain revealed irregular destruction of the bone in the inner plate of the left frontal bone with surrounding abscess formation (arrow) (D from patient 5). Pelvic CT revealed irregular destruction of the bone in the left acetabulum and femoral head, narrowing of the hip joint space, and surrounding abscess formation (arrow) (E from patient 3). Chest CT scan revealed an irregular bone defect in the manubrium of the sternum with a sclerotic edge (arrow) (F from patient 8). ECT bone scan revealed increased radioactive concentrations in the manubrium of the sternum, multiple ribs, L3 vertebral body, bilateral sacroiliac joints, left ilium, and upper end of the right femur (G from patient 8).

Figure 2 Chest computed tomography (CT) showing exudation and consolidation in the upper lobe of the right lung (A from patient 3); atelectasis in the middle lobe of the right lung and right pleural effusion (B from patient 7); consolidation in the lower lobe dorsal segment of the left lung with cavity formation and thick-walled cavities, nodules, patches, and fiber proliferation in the upper lobe of the right lung (C from patient 8); right thoracic collapse, large consolidation in the upper lobe of the right lung with air bronchogram signs, and multiple ground-glass opacities, patchy exudations and nodules in the upper lobe of the left lung (D from patient 1). Multiple patchy exudations, bronchiectasis and multiple thin-walled cavities of varied sizes in the lower lobe of the bilateral lung (E from patient 8). Multiple areas of bronchiectasis, consolidation and exudation in the middle lobe of the right and left lungs (F from patient 8).

Treatments and Outcomes

All patients received anti-nontuberculous therapy with a combination of 2–5 types of antibiotics dominated by clarithromycin after antimicrobial susceptibility testing (AST) of NTM Infections. Six patients received sensitive drugs for other bacterial and fungal coinfections. In addition to medical treatment, five patients underwent surgery (50%), consisting of debridement, incision for drainage and internal fixation. Seven patients improved, five of whom were cured after the initial treatment, but the other two were cured after changing the therapeutic regimen many times due to the aggravation of symptoms or recurrence. The duration of treatment ranged from 18 to 65 months. Patient 3, who had joint dysfunction that influenced activities of daily living, recovered completely after treatment. Two patients died of comorbidities during the treatment period. Patient 1, who had chronic obstructive pulmonary disease (COPD) was misdiagnosed with tuberculosis for one and a half years and finally died of respiratory and circulatory failure and septic shock after 27 days of treatment for DNTM disease. Patient 6, who had diabetes, died of hypoglycemic coma after the diagnosis of DNTM disease and treatment for 2 years. The remaining patient was lost to follow-up (Table 3 and Supplemental Table 3).

Table 3 Treatment and Outcomes of 10 Patients with Disseminated Nontuberculous Mycobacterial Disease with Osteolytic Lesions

Discussion

Nontuberculous mycobacteria are regarded as opportunistic pathogens and have been reported in immunosuppressed individuals.^12,13 In recent years, several studies demonstrated that AIGAs may be an important susceptibility factor for NTM infections in previously healthy individuals.^3,14 Hase et al³ found that 34% AIGA-positive patients with NTM disease have bone and joint involvement by reviewing 111 AIGA-positive patients with NTM disease. In this study, all ten patients had multiple osteolytic destructions and 70% of these patients with positive AIGAs, which were confirmed in the previous research. These phenomenon suggests that NTM infection of bone is not rare in AIGA-positive patients, and the mechanism of osteolysis may be related to AIGAs.^4,5 NTM infection and inflammation break the balance of osteoclast formation and bone destruction, resulting in a bias toward bone resorption.⁴ In addition, NTM infection of bone has been rarely reported and is often overlooked by clinicians.

¹⁷ Interferon-γ (IFN-γ) is important in the maintenance of the balance between osteoclasts and osteoblasts. Osteoclast formation and bone destruction were more pronounced in mice lacking a functional IFN-γ.^15,16 IFN-γ directly inhibit osteoclast formation from osteoclast precursors, and indirectly stimulate osteoclast formation by stimulating antigen-dependent T-cell activation and secretion of the osteoclastogenic factors RANKL and TNF-α.¹⁷ Recently, blockade effects of AIGAs present in NTM patients on IFN-γ have been described by several studies, suggesting that AIGAs can neutralize IFN-γ, affect the activation of the IFN-γ receptor (IFN-γR) and downregulate the production of its downstream factors (eg, TNF-α and IL-12), and inhibit IFN-γ-STAT-1 phosphorylation.¹⁸ Thus, osteolytic lesions in NTM patients with positive AIGAs may be due to the blockade and neutralizing effects of AIGAs on anti-IFN-γ, leading to a disruption in the balance between osteoclasts and osteoblasts, NTM infection and inflammation bias toward bone resorption.

In our study, all patients presented with fever, emaciation, anemia, ostealgia, subcutaneous abscess and markedly increased leukocytes counts, CRP concentrations and ESRs. Chest CT revealed extensive and multiple pulmonary parenchymal exudations, consolidation, cavities, and bronchiectasis, while radiography and bone CT revealed osteolytic lesions. Histopathological examination revealed suppurative granulomas in the lesions. These clinical and laboratory findings, variable imaging manifestations, and histopathological changes resemble those seen in osteomyelitis caused by tuberculosis. Thus, all patients were misdiagnosed with tuberculosis before being diagnosed with NTM infection. These results indicate insufficient recognition of DNTM disease with osteolysis by clinicians. Therefore, systemic research on NTM infection of bone that investigates clinical characteristics, especially radiographic presentations, should be considered a necessity for improving differential diagnosis.

According to our research, the clinical characteristics of disseminated NTM infection of bone leading to osteolytic lesions can be summarized as follows: (1) NTM infection is mainly characterized by chronic suppurative infection manifesting as significantly increased white blood cell and neutrophil counts, ESR concentrations and CRP levels, and wasting systemic symptoms such as anemia and hypoproteinemia may be obvious. In addition, NTM infection commonly presents as disseminated infection of multiple organs including the lungs, lymph nodes, skin, bone and joints, with multiple subcutaneous abscesses, increased purulent secretions in the airway and suppurative changes in lymph nodes with pain. (2) NTM can affect any bone of the body, often presenting as multiple bone involvement. The most commonly involved sites were the vertebrae, sternum, clavicle and ribs, followed by the femur and ilium. The humerus, scapula, metacarpal, phalanx, tibia, talus, frontal bone and mandible were also involved. Radiography and CT of bone revealed osteolytic lesions characterized by multiple lucent defects showing moth-eaten or irregular destruction of bone, bone defects, well-circumscribed, rounded low-density areas, pathological fractures, and periosteal proliferation. Bone ECT showed significantly increased uptake in multiple bones, while PET/CT showed metabolic activity in multiple bones of the body. When NTM disseminates to an osseous site, significant focal neutrophil aggregation and lysosome release leads to ostealgia, subcutaneous abscess and osteolytic destruction. (3) Histopathological examination of pathological tissue specimens obtained from patients with NTM infection demonstrated suppurative inflammation dominated by neutrophil infiltration, with or without granuloma formation. Compared with osteoarticular infections caused by NTM, infections caused by tuberculosis most commonly involve the thoracolumbar region of the spine and load-bearing joints such as the hips and knees.¹⁹ Imaging examinations revealed sequestrum in the bony destruction area, intervertebral disc destruction, paravertebral cold abscess and spinal deformity when tuberculosis bacteria infected bone.⁶ Histopathology of pathological tissue specimens obtained from patients with tuberculosis demonstrates granulomas dominated by lymphocyte infiltration, with a central area of caseating necrosis.⁶ Therefore, taking the typical clinical and imaging features and histopathological manifestations into consideration, osteolytic destruction caused by NTM infection can be differentiated from that caused by tuberculosis, especially when the effect of anti-tuberculosis therapy is poor. Thus, etiological culture remains the gold standard for the diagnosis of NTM infection, tuberculosis and other fungal infections. In recent years, the development of molecular biology techniques, such as follow-up PCR, NGS and gene sequencing, has provided great help in the identification of mycobacterium strains and drug resistance profiles, greatly improving the clinical diagnosis rate and prognosis.^20–22

According to guidelines,⁹ for the treatment of DNTM disease, long-term chemotherapy with a combination of 5–6 antimycobacterial agents should be administered after species identification and in vitro AST for at least one to two years. For the treatment of DNTM disease with osteolytic lesions, systemic antimycobacterial chemotherapy should be primarily administered, though surgery can also play an important role in some cases. Surgical treatments, such as debridement and incision for drainage, should be considered in cases with poor response to chemotherapy, extensive bone destruction and abscesses formation.^9,23 For patients with pathological fractures, internal fixation of the fracture should also be performed immediately. In addition, long-term follow-up of DNTM patients with osteolysis after discharge is crucial to patient rehabilitation. The course and dosage of oral antibiotics, imaging examination findings, the ESR, and CRP levels should be monitored to assess the progression of the disease.²³ In addition, It has been reported that in the cases of bone and joint NTM infections, the most common causative NTM species are SGM dominated by the M. avium-intracellulare complex, while RGMs such as M. fortuitum, M. abscessus and M. chelonae have also been reported.^2,23 A multicenter retrospective study consisting of 117 patients with extrapulmonary NTM infections suggests that bone and joint infections were predictors of SGM infections.²⁴ These findings may be helpful in early empirical antimicrobial regimen selection for bone NTM infection in basic hospitals that are unable to identify subspecies.

Limitations

There were some limitations of our study. First, although this was a retrospective study, the number of patients included was not large. Furthermore, NTM patients without bone involvement were not included as a control group to calculate the incidence of NTM involving bone. Second, as a retrospective study, the medical record data of some of the patients were incomplete. Third, elucidating the reason why AIGA-positive patients with NTM infection are more prone to osteolytic destruction will require large, multicenter research, which will be done in subsequent studies. In spite of these limitations, we systematically analyzed the clinical and imaging characteristics of patients with NTM infection involving bone leading to osteolytic lesions for the first time, which provides information about this condition to clinicians and lays the foundation for further research.

Conclusions

DNTM infection of bone leading to osteolysis usually occurs in patients with AIGA-positive antibodies and is often misdiagnosed as tuberculosis. When patients present with increased white blood cell and neutrophil counts, focal suppurative granulomas, multiple areas with worm-eaten appearance or irregular destruction of bone with increased radioactive concentrations, DNTM disease with osteolytic lesions should be suspected, especially when the effect of antituberculous therapy is poor. On the basis of species identification and in vitro antimicrobial susceptibility testing, early systemic anti-nontuberculous chemotherapy, combined with surgical treatment if necessary, can improve the clinical efficacy and reduce the recurrence, disability and mortality of patients with DNTM disease with osteolytic lesions.