Osteomyelitis with a Pathologic Fracture, Mortal Liver Abscess, and Cholelithiasis: Distinct Features in two Families with the Same p47-phox Mutation

Chronic granulomatous disease (CGD), first described in 1954 as the “fatal granulomatous disorder of childhood,”¹ is a primary immunodeficiency disorder that results from the absence or malfunction of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunits in phagocytic cells.² Nicotinamide adenine dinucleotide phosphate oxidase is composed of 2 membrane-spanning subunits, gp91-phox and p22-phox (encoded by CYBB and CYBA, respectively), and 3 cytoplasmic subunits, p40-phox, p47-phox, and p67-phox (encoded by NCF4, NCF1, and NCF2, respectively). A defect in 1 of 5 subunits and Rac2 gene defects abolish the activity of the oxidase and lead to CGD.³ The major clinical manifestations of CGD are pneumonia, lymphadenitis, liver abscess, pyoderma, inflammation of the gastrointestinal tract, and osteomyelitis.⁴ Serious recurrent life-threatening infections develop due to bacteria and fungi that produce catalase, such as Staphylococci, Burkholderia cepacia complex, Serratia marcescens, Nocardia spp., Aspergillus spp., Salmonella,Bacille Calmette-Guérin (BCG), Mycobacterium tuberculosis, and Candida are important infectious agents.^5,6 Here, we report 4 patients with CGD who suffer from the same variant but have distinct clinical presentations at different ages. Verbal and written informed consent was obtained from the patients and the patients' families who agreed to take part in the study.

Case Presentations

Patient 1

A 16-year-old female, the third child of consanguineous parents, was admitted with a 7-month history of painful right hand swelling following trauma. Her brother had died at 22, likely due to pulmonary tuberculosis. The patient underwent 2 abscess drainage and debridement procedures, with biopsy showing granulomatous inflammation. An X-ray revealed resorption of the distal fourth metacarpal and a fracture in the third metacarpal, along with osteopenia around the carpal bones (Figure 1A and B). Nine months later, bullous lesions developed on her hand. After 9 months, right-hand bullous lesions occurred and drained afterward. On examination, she had a tender 2 cm × 4 cm swelling and a chronic discharging sinus. Despite a year of anti-tuberculosis treatment, fistulized osteomyelitis persisted, leading to suspicion of Aspergillus spp. osteomyelitis. We administered prophylactic therapy with itraconazole and trimethoprim-sulfamethoxazole. Pyrazinamide, isoniazid, rifampicin, ciprofloxacin, streptomycin, and ethambutol were given for persistent lesions but with a poor response. Right-hand magnetic resonance imaging (MRI) revealed active osteomyelitis, and we added vancomycin to the treatment. She underwent debridement and biopsy for the right-hand radial dorsal site with a suspicion of Aspergillus spp. osteomyelitis.

Figure 1.

(A, B) Sixteen years and 9 months old patient. X-ray shows complete resorption of the distal part of the fourth metacarpal bone and a pathological fracture of the third metacarpal bone. Osteopenia around the carpal bones is prominent. (C) The X-ray below is the one at the age of 17 years. The lytic lesion in the fourth metacarpal bone healed partially and the fracture in the third metacarpal bone completely healed. (D) The X-ray below is when the patient was 22 years old years old patient. Bone quality further improved at the follow-up.

Pathology revealed necrotizing granulomatous inflammation. However, we could not document tuberculosis dactylitis. Mycobacterial culture and polymerase chain reaction (PCR) were negative. There was no bacterial or fungal growth in the cultures. A thorough diagnostic laboratory evaluation (Table 2) showed that the nitroblue tetrazolium (NBT) test was low and the dihydrorhodamine (DHR) test showed decreased oxidative activity compared with the control. Genetic workup revealed the homozygous p47-phox variant. We administered interferon-gamma treatment for about a year, discontinued the tuberculosis treatment, and discharged her from the hospital with itraconazole and trimethoprim–sulfamethoxazole prophylactic treatments. At 15 months, the lytic lesion in the fourth metacarpal bone healed partially, and the fracture in the third metacarpal bone healed (Figure 1C). She used prophylactic treatments (itraconazole and trimethoprim–sulfamethoxazole) for about 5 years. At the fifth-year follow-up, further improvement was recorded (Figure 1D). However, she has not used her medications since 2016 and has not visit the outpatient clinic. The patient had no history of infection or hospitalization during this period. We evaluated her 33-year-old sister for the etiology of chronic cough and mediastinal and hilar lymphadenopathies (Patient 2).

Table 2.

Special Radiological, Microbiological and Genetic Evaluations and Biopsy Results of the Cases

Patient	Chest X-ray	Specific Radiological Evaluations for the Case	Biopsy	Ziehl–Nielsen/PAS Stains	Tuberculosis PCR	Culture Results	DNA Analysis
1	Normal	Radiograph and MRI of the right hand, consistent with tuberculosis dactylitis and tuberculosis osteomyelitis.	Necrotizing granulomatous inflammation (dorsum of radial border of the right hand)	No organism	Negative	Negative	NCF1 Homozygous defect: c.75_76del (p.Tyr26HisfsTer26) *Schematic representation of mutation:
2	Bilateral hilar and mediastinal lymphadenopathy and multiple nodular infiltrations	Tomography of lung, mediastinal and hilar lymphadenopathies and multiple nodular infiltrations that unite at the superior and inferior lobes of both lungs Tomography of neck: normal Tomography of abdomen: hepatomegaly and right nephrolithiasis Cranial MRI: normal	Reactive lymph nodes (from the hilar lymphadenopathy by mediastinoscopy) bone marrow aspiration biopsy: normocellular bone marrow, no malignant cells, no hemophagocytosis	No organism	Negative (both serum and bone marrow aspiration sample)	Negative
3	Normal	Abdominal tomography and MRI: multiple hypodense nodular lesions, multiple abscesses	Many eosinophils in the content of abscess formation	No organism	Negative	Negative
4	–	–	–	–	–	–

*(NM_000265.6):c.75_76del (p.Tyr26HisfsTer26).

PX domain, phosphoinositide-binding structural domain.

SH3 domain, SRC Homology 3 Domain.

Patient 2

A 33-year-old female with a 4-year history of chronic productive cough, recurrent fever, and night sweats, along with a history of weight loss, was diagnosed with CGD after her sister (Patient 1) was diagnosed. She had no complaints of dyspnea or hemoptysis, and a physical examination revealed hepatomegaly. Chest X-ray and computed tomography (CT) showed bilateral hilar and mediastinal lymphadenopathy (LAP) with multiple nodular infiltrations. The patient had chronic hepatitis C, with the liver palpated 4 cm below the costal margin. Microbiological culture and PCR for M. tuberculosis in the sputum sample were negative. To understand the cause of LAP and nodular infiltration on CT, further investigation was not conducted because C-reactive protein (CRP) and sedimentation rates decreased with empirical treatment without antituberculosis therapy, and no growth was observed in the sputum culture. The patient was diagnosed with CGD (Table 2), like her sister, and used prophylactic treatments of trimethoprim–sulfamethoxazole and itraconazole for 4 years without issues. She did not attend follow-up visits or take medication for the past 10 years but had no hospitalizations for infections. She was diagnosed with asthma and treated with salbutamol. Additionally, she had a total thyroidectomy for a non-toxic multinodular goiter (nodular hyperplasia) and a cholecystectomy for cholelithiasis. Her last check-up in June 2021 showed no complaints or abnormalities in the laboratory workup.

Patient 3

A 2-year-old boy, the third child of nonconsanguineous parents, was admitted with a 3-month history of unexplained fever and hepatomegaly. Physical examination revealed firm hepatomegaly 6 cm below the costal margin. His blood eosinophil count was 1000/mm³, and abdominal CT and MRI showed multiple hepatic abscesses. The liver biopsy revealed eosinophilic and neutrophilic infiltration in sinusoids and multiple eosinophilic abscesses (Figure 2A and B). The cyst hydatid serology was 1/320. The NBT test was low, the DHR showed decreased oxidative activity, and the genetic analysis revealed a homozygous p47-phox variant (Table 2). We gave albendazole for the positive cyst hydatid serology. Microbiological analysis did not detect any bacterial and fungal microorganisms. Thus, we administered antimicrobials, including teicoplanin, amikacin, imipenem, ciprofloxacin, voriconazole, isoniazid, and rifampicin. Mycobacterial culture and PCR were negative in the sputum sample. In the follow-up, the liver size increased, and fever, persistent splenomegaly, and progressive decline in hemoglobin and platelet levels developed. Hemophagocytosis was present in the bone marrow smear. We started the HLH-2004 protocol, including 400 mg/kg intravenous immunoglobulin (IVIG) and 5 mg/kg methylprednisolone. We gave granulocyte infusions and added interferon-gamma therapy. Due to multiple draining liver abscesses, persistent transaminase elevation, and an unstable clinical condition, open surgical debridement was not possible. Percutaneous abscess drainage (Figure 3A and B) provided partial relief, but the patient ultimately died from respiratory distress and multi-organ failure due to sepsis.

Figure 2.

(A) Multiple necrotizing granulomas within the liver parenchyma (H&E). (B) Necrotizing granulomas composed of central necrosis and palisaded multinuclear giant cells, epithelioid histiocytes, and a few lymphocytes (H&E). No acid-resistant bacilli were detected by Ziehl–Neelsen stain, and suspicious fungal hyphae-like structures were observed on GMS stain.

Figure 3.

(A, B) Percutaneous drainage of liver abscesses.

Patient 4

A 4-year-old girl, the sister of Patient 3 and the second child of nonconsanguineous parents, was admitted with lymphadenitis in infancy. She was treated for BCG lymphadenitis in infancy and diagnosed with CGD in our department during family screening after her younger sibling's diagnosis. She had mild clinical findings and did not come for follow-up.

Tuberculin skin test (TST) was negative in all 4 patients, and lymphocyte subset values were within normal ranges. Tables 1 and 2 show the detailed laboratory workup.

Table 1.

Laboratory Findings and Immunological Evaluations of the Cases

Patient	Date	Hb (g/dL)	WBC (cells/mL)	Creatinine and Liver Function Tests	IgA (mg/dL)	IgG (mg/dL)	IgM (mg/dL)	IgE (IU/mL)	Nitroblue Tetrazolium (NBT) Test (%)	Dihydrorhodamine Reduction Flow Cytometry Test (DHR)
1	On admission/Follow-up	9.8/12.3	8100/9200	Normal/normal	359	3210	416	172	30	Defective dihydrorhodamine
2	On admission/Follow-up	9.1/12.1	10.000/7000	Normal/normal	347	4200	652	135	–	Defective dihydrorhodamine
3	On admission/follow-up	8.6/9.1	39.600/17.200	Basal levels: ALT: 9 U/L (<39) AST: 22 U/L (<56) ALP: 397 U/L (<281) GGT: 155 U/L (<18) Elevation of transaminases: ALT: 37 U/L (<39) AST:79 U/L (<56)	165	1697	238	108	10	Defective dihydrorhodamine
4	On admission	11.9	10.600	–	125	2430	108	10.4	10	–

Chronic granulomatous disease is a rare primary immunodeficiency disorder with an incidence of 1/250 000 live births.⁷ It results from a genetic defect in 1 of the 5 subunits of the NADPH oxidase complex. Defects in NADPH oxidase impair the production of superoxide and other reactive molecules, preventing the respiratory burst needed to kill pathogens. This leads to recurrent infections by catalase-positive bacteria and fungi, along with granuloma formation. Chronic granulomatous disease can be inherited in an X-linked or autosomal recessive manner, with X-linked defects accounting for 70% of cases in countries with low consanguinity and having a worse prognosis.⁸

Typical CGD presentations are fungal pneumonia (mainly Aspergillus spp.), suppurative adenitis, and subcutaneous or liver abscesses, in which Staphylococcus aureus is the most common microorganism.^3,9 Osteomyelitis due to S. marcescens, BCG lymphadenitis, Salmonella sepsis, pneumonia, Aspergillus spp., or Pseudomonas spp. sepsis may develop during the disease course. Recently, we analyzed the clinical features of 63 CGD patients from a single center in which the most common presentation was lung infection (42.6% n: 20) followed by lymphadenopathy (19.2% n: 9).¹⁰

X-linked CGD patients usually present during the first years of life. However, the disease may manifest later with milder signs and symptoms.³

We report 4 patients with the same homozygous variant in NCF1 (NM_000265.6) (c.75-76del) (p.Tyr26HisfsTer26), who exhibited varying clinical severities and presentations at different ages. Patients 1, 2, and 4 had a relatively better prognosis and survived, whereas Patient 3 had a poor prognosis and died. Patients 1, 2, and 4 presented with infections indicative of mycobacterial disease (Tables 1 and 2). The differences in presentation among the patients may be attributed to pseudogenes of the NCF1 gene, other genetic polymorphisms, or epigenetic factors potentially influencing the degree of oxidative activity impairment. As the patients’ flow cytometric analysis records were unavailable, we could not directly demonstrate differences in oxidative activity. However, given that they share the same mutation, we do not anticipate significant variation in residual oxidative activity.

The first manifestation of CGD may be a BCG-related disease. BCG-related infections can occur in CGD as well as in other primary immunodeficiencies, such as SCID and MSMD. CGD should be considered in any child presenting with regional (BCGitis) or disseminated (BCGosis) BCG infections.¹¹

Mycobacterium tuberculosis infection of metacarpals, metatarsal bones, and phalanges of hands and feet is known as spina ventosa and is uncommon after the age of 5 years. The incidence of tuberculous dactylitis among children is around 0.65%-6.9%. Trauma history is present in one-third of patients, as in the case of our patient.¹² Osteomyelitis may be seen in the small hands and feet and affects multiple sites in CGD. Commonly seen organisms are S. marcescens, Aspergillus spp., and S. aureus.¹³ In a similar case report, familial CGD with recurrent tuberculosis, pulmonary tuberculosis, rib osteomyelitis, and spinal tuberculosis were common infections.¹⁴

Patient 1's older sister, aged 33, was referred with a 4-year history of chronic productive cough, recurrent fever, and night sweats, despite no prior severe infections. This patient also had low NBT and, like her sibling, had CGD. Respiratory symptoms are common in CGD, with recurrent pneumonia being the most frequent. Pneumonia in CGD can present as focal consolidation or a miliary pattern, with complications including tuberculosis, BCG-induced disease, and infections by mycobacterium species and nontuberculous mycobacteria.¹³

The third patient presented with multiple hepatic abscesses at 2 years of age. Previous studies showed that 27% of CGD patients presented with hepatic abscesses.¹⁵ These patients typically have a fever with or without abdominal pain and are often accompanied by constitutional symptoms. Fever is the most common symptom in patients with hepatic abscesses. Aggressive surgical management is needed for abscess treatment and reduces the mortality from 27% to 6%.¹⁵ Staphylococcus aureus was the most frequent organism identified in cultures, and 88% of patients had positive cultures.¹⁶ We could not identify any microorganism in any of the cultures in the present patient, probably due to multiple antimicrobial therapy. In addition to the antibiotics and other treatment options, granulocyte transfusions, interferon-gamma, percutaneous drainage, open surgical debridement, or resection may be necessary to treat the patients.^15,16 However, open surgical debridement and resection could not be performed due to the multiple liver abscesses and persistent deterioration. The patient developed hemophagocytic lymphohistiocytosis (HLH) and died. Hemophagocytic lymphohistiocytosis is a rare disease and was previously described in CGD patients.¹⁷ The high incidence of infection, accompanied by an inappropriately increased inflammatory response and excessive cytokine secretion, may predispose patients with CGD to develop HLH. In a study from North India, 5 of 80 patients diagnosed with CGD developed HLH. Although mortality was present in three children who received only IVIG therapy, the other 2 who received IV methylprednisolone pulse therapy survived.¹⁸ Intravenous immunoglobulin and methylprednisolone treatments were applied together in Patient 3 in this report. We could not give pulse steroids since the patient had sepsis and multi-organ failure.

Although no microorganism was isolated from the granulomatous lesions in any of the patients, we could not rule out the presence of an undetectable pathogen. Therefore, we administered steroids in addition to antimicrobial therapy.

Chronic granulomatous disease patients may present with diverse features, and the infection severity may vary even in patients with the same mutation. This is true in another study with X-linked CGD.¹⁹ Since Patients 1-4 had the same mutation, the difference in the clinical severity may be explained by the NCF1 pseudogenes, other genetic polymorphisms, and epigenetics. Further studies are needed to understand the variation in the clinical course.

Funding Statement

This study received no funding.

Data Availability Statement:

The data that support the findings of this study are available on request from the corresponding author.