The

n e w e ng l a n d j o u r na l

of

m e dic i n e

case records of the massachusetts general hospital

Founded by Richard C. Cabot Nancy Lee Harris, m.d., Editor Eric S. Rosenberg, m.d., Associate Editor Jo-Anne O. Shepard, m.d., Associate Editor Alice M. Cort, m.d., Associate Editor Sally H. Ebeling, Assistant Editor Christine C. Peters, Assistant Editor

Case 21-2008: An 11-Month-Old Boy with Fever and Pulmonary Infiltrates

Jason B. Harris, M.D., Ian C. Michelow, M.D., Sjirk J. Westra, M.D., and Richard L. Kradin, M.D.

Pr e sen tat ion of C a se

From the Departments of Pediatrics Pediatric Infectious Disease (J.B.H., I.C.M.), Radiology (S.J.W.), and Pathology (R.L.K.), Massachusetts General Hospital; and the Departments of Pediatrics (J.B.H., I.C.M.), Radiology (S.J.W.), and Pathology (R.L.K.), Harvard Medical School. N Engl J Med 2008;359:178-87.
Copyright 2008 Massachusetts Medical Society.

An 11-month-old boy was admitted to this hospital because of fevers and pulmonary infiltrates. One month before admission, a month after beginning a visit to India with his parents, daily fevers, with temperatures up to 38.9C, developed, without localizing signs or symptoms. Before the family left for India, the boys primary care physician gave him one dose of immunoglobulin intramuscularly for hepatitis A prophylaxis and prescribed mefloquine weekly for malaria prophylaxis. He was evaluated at an Indian health care facility, and amoxicillinclavulanate was administered, but there was no improvement. On the sixth day after the onset of fevers, mefloquine was stopped and chloroquine was administered for 3 days, followed by ciprofloxacin for 3 days. Fever persisted, and a nonproductive cough developed. Nineteen days before his admission to this hospital, the patient was admitted to a hospital in India. His blood pressure was 90/60 mm Hg and his pulse 138 beats per minute; although he had been febrile, his temperature was normal. Hepato splenomegaly was noted, but the results of the remainder of the examination were normal. Laboratory-test results are shown in Table 1. No parasites were seen on examination of a peripheral-blood smear, and a test for serum antibodies to Salmonella typhi was negative. A chest radiograph showed an irregular perihilar parenchymal opacity with air bronchograms and multiple smaller air-space opacities in the left perihilar region. Ampicillinsulbactam and amikacin were administered intravenously. A tuberculin skin test showed no induration after 48 hours, and the temperature, which had been fluctuating, returned to normal on the fifth hospital day. Intravenous fluid being infused through a catheter inserted in the dorsum of the right distal forearm infiltrated into the tissue, and an adhesive bandage was applied over the site. The patient was discharged on the sixth hospital day, receiving oral amoxicillinclavulanate and clarithromycin, and his cough gradually resolved. Nine days after discharge, and 5 days before admission to this hospital, he returned to the United States. Two days after his return to the United States, his temperature rose to 39C, and fever recurred daily thereafter. The amoxicillinclavulanate was stopped after completion of the course of therapy; the day before admission to this hospital, the clarithromycin was completed. On the day of admission, he was evaluated at the office of his primary care physician; his temperature was 38.7C. An erythematous
n engl j med 359;2 www.nejm.org july 10, 2008

178

Downloaded from www.nejm.org at HINARI BAND 2 on January 12, 2010 . Copyright 2008 Massachusetts Medical Society. All rights reserved.

case records of the massachusetts gener al hospital

Table 1. Results of Laboratory Tests. Reference Range (Age-Adjusted) 33.039.0 10.513.5

3)

Variable Hematocrit (%) Hemoglobin (g/dl) White cells (per mm Neutrophils Lymphocytes Monocytes Eosinophils Basophils Band forms Atypical lymphocytes Platelets (per mm
3)

20 Days before Admission in India* 11.0 32,800 42 56 2

Current Admission 33.7 11.5 28,900 37 61 1 0 0 1 396,000 66 3+ microcytes

4th Hospital Day 33.8 11.1 36,400 39 47 7 0 0 7 465,000 67 3+ microcytes, 1+ hypochromia

6th Hospital Day 26.8 8.9 24,700 45 31 8 0 1 12 3 319,000 67 3+ microcytes, 2+ hypochromia, rouleaux formation present

6.0x10317.5x103 1749 6777 411 08 03 010 0 150,000450,000 7086

Differential count (%)

Mean corpuscular volume (m3) Red-cell morphology

Erythrocyte sedimentation rate (mm/hr) C-reactive protein (mg/liter) Immunoglobulin (mg/dl) IgG IgA IgM

017 <15 2831026 1682 39141

75

59 143.8 815 61 60

* Reference ranges for laboratory testing performed in India include hemoglobin 13 to 18 g per deciliter, white cells 4000 to 10,000 per cubic millimeter, neutrophils 40 to 75%, lymphocytes 20 to 45%, and eosinophils 1 to 6%.

nodule, 15 to 18 mm in diameter, was present on the dorsum of the right distal forearm, at the site of the previous venipuncture. There were no other localizing signs or symptoms. A chest radiograph showed bilateral pulmonary infiltrates. A biopsy of the nodule on the forearm was performed, and specimens were sent for culture and pathological examination. The patient was referred to this hospital for admission. The patient was born in the United States after a full-term gestation to a primigravid mother; delivery was by cesarean section because of cephalopelvic disproportion. At birth, the patients weight was 3.075 kg (21st percentile) and his length 53.3 cm (90th percentile). At 6 weeks of age, he had been admitted to another hospital for a self-limited episode of fever and poor feeding. A palpable spleen tip was noted on examination, and ultrasonography of the abdomen revealed

that the spleen was 6.2 cm in length, the upper limit of normal for his age. A culture of the blood was sterile; his temperature returned to normal and he was discharged. Follow-up ultrasonography of the abdomen 1 month after discharge was normal. Routine immunizations were given, and developmental milestones and growth parameters were normal. At 6 months of age, his weight was 7.1 kg (18th percentile) and length 71 cm (92nd percentile). While in India, the patient and his parents had stayed with relatives in an urban area, and the patient had close contact with many people, including a relative with pulmonary tuberculosis who was receiving triple-drug therapy. There were no exposures to animals. The patient had no allergies to medications, and in the United States he lived with his parents and did not attend day care. His parents were professionals who had emigrated from India. There was no
179

n engl j med 359;2 www.nejm.org july 10, 2008

Downloaded from www.nejm.org at HINARI BAND 2 on January 12, 2010 . Copyright 2008 Massachusetts Medical Society. All rights reserved.

The

n e w e ng l a n d j o u r na l

of

m e dic i n e

family history of recurrent febrile illnesses, rheumatologic diseases, or immunodeficiency. On examination, he appeared well he was playful and breathing comfortably. His weight was 8.2 kg (3rd percentile), temperature 39.3C, pulse 140 beats per minute, respiratory rate 42 breaths per minute, and oxygen saturation 99% while breathing ambient air. A well-circumscribed area of erythema and induration, 1 cm by 2 cm, was present on the dorsum of the right forearm, and the incision from the biopsy was clean and dry. The lungs were clear, and the results of the remainder of the physical examination were normal. Levels of serum electrolytes and glucose were normal, as were the test results for renal and liver function; the results of other laboratory tests are shown in Table 1. Tests of nasal secretions for antigens of adenovirus, influenza virus types A and B, parainfluenza virus types 1, 2, and 3, and respiratory syncytial virus were negative. Computed tomography (CT) of the chest showed nodular areas of consolidation throughout both lungs, some with calcification. The larger opacities were in the upper lobes, up to 2.6 cm by 1.7 cm by 1.9 cm. Some were contiguous with the hila and extended to the pleural surface. Softtissue fullness in the mediastinum, hila, and left axilla suggested lymphadenopathy. Acetaminophen and ibuprofen were administered orally and ceftriaxone intravenously. On the second hospital day, the temperature rose to 39.7C. A tuberculin skin test was performed, and an aspirate of gastric secretions obtained in the early morning was sent for acidfast staining and mycobacterial culture. Isoniazid, ethambutol, pyrazinamide, and rifampin were begun. Analysis of a urine specimen was normal, and a culture of the urine specimen, collected in an external bag, grew few mixed flora. Specimens of stool were sent for culture of enteric pathogens, presence of Clostridium difficile toxin, and ova and parasitologic analysis. During the next 3 days, two additional earlymorning specimens of gastric aspirate were obtained; staining did not reveal acid-fast bacilli and cultures were pending. On the fourth hospital day, the temperature rose to 40C and the patient vomited intermittently, occasionally after episodes of coughing productive of white sputum. The site of the tuberculin skin test showed no induration. The next day, the temperature fluctuated between 34.6 and 40.1C. Serum IgG antibodies to cytomegalovirus were present; test
180

results for EpsteinBarr virus and human immunodeficiency virus antibodies in the serum and legionella and histoplasma urine antigens were negative. Other laboratory-test results are shown in Table 1. Additional specimens of blood were sent for culture; results of other tests were pending. A review of the slides from the skin biopsy that had been performed earlier revealed scattered nuclear debris and rare structures suggestive of fungal hyphae; in deeper sections stained for organisms, these structures were no longer visible. Ceftriaxone was stopped and meropenem and liposomal amphotericin were administered intravenously. On the sixth hospital day, emesis was tinged with blood, and a sample of stool was guaiacpositive. The respiratory rate transiently increased to 66 to 75 breaths per minute, with oxygen saturations between 98 and 100%, then returned to 32 to 44 breaths per minute. The temperature ranged from 33.9 to 39.6C. Additional chest radiography continued to show bilateral pulmonary infiltrates. A diagnostic procedure was performed on the seventh hospital day.

Differ en t i a l Di agnosis
Dr. Jason B. Harris: May we review the radiologic studies? Dr. Sjirk J. Westra: CT of the chest, performed on admission without the administration of contrast material, showed several ill-defined nodules in the posterior lung zones, several of which contained calcifications; the nodules blended with the pulmonary hila (Fig. 1A), and some extended to the pleura. One week later, CT of the chest performed with the administration of intravenous contrast material showed fullness of the soft tissues in the mediastinum, which could be explained by the normal thymus, and mild prominence of the hilar lymph nodes. The tracheobronchial tree was normally patent. Large nodules were present, predominantly in the upper lobes of the lungs, with ill-defined and spiculated margins, which was probably indicative of inflammation; the larger nodules were calcified (Fig. 1B). A chest radiograph taken 5 days later (Fig. 1C) showed ill-defined nodular infiltrates, predominantly in the middle- and upper-lung zones, and ill-defined hilar markings. The differential diagnosis of bilateral nodular pulmonary consolidation with calcifications includes granulomatous infection, particularly tu-

n engl j med 359;2 www.nejm.org july 10, 2008

Downloaded from www.nejm.org at HINARI BAND 2 on January 12, 2010 . Copyright 2008 Massachusetts Medical Society. All rights reserved.

case records of the massachusetts gener al hospital

berculosis. However, calcification does not occur in primary tuberculosis, and postprimary tuberculosis would be unusual in this young child. Chronic fungal infections frequently calcify, whereas bacterial and pneumocystic infections are not usually associated with calcification. Given the clinical presentation and the appearance of these nodules, a calcifying neoplastic process, such as inflammatory myofibroblastic tumor (inflammatory pseudotumor) or a metastatic malignant tumor, was believed to be less likely than an infection. Dr. Harris: I participated in the care of this patient and am aware of the diagnosis. Fever and a cough developed in this infant during travel in India, and they recurred despite antibiotic therapy. On admission, his weight gain had slowed. Imaging studies showed a chronic multinodular pneumonia with calcification. Our differential diagnosis included infectious and noninfectious conditions, and we considered the possibility that his condition might be a manifestation of primary immunodeficiency.
Infection with Mycobacterium tuberculosis

This patients young age and close contact with an adult with pulmonary tuberculosis suggested a diagnosis of pulmonary tuberculosis, the most common cause of chronic pneumonia in the world, C with approximately 2 million cases per year in 1 India. Infants younger than 1 year of age who are infected with Mycobacterium tuberculosis are at extremely high risk for the development of active disease, estimated to approach 40% in the first 2 years of life as compared with a 5 to 10% cumulative lifetime risk of disease in immunocompetent adults.2 Does this patients clinical presentation support the diagnosis of pulmonary tuberculosis? First, is the incubation period consistent with tuberculosis? This childs symptoms developed 1 month after arriving in India. Infants, unlike older children or adults, often present with symptomatic primary tuberculosis 3 to 8 weeks after exposure, and disseminated disease, such as milFigure 1. CT of the Chest. iary or meningeal tuberculosis, may develop 2 to An axial CT study of the lungs shows ill-defined nodular pul3 6 months after exposure. Are the other clinical monary consolidations in the posterior zones (Panel A, archaracteristics of the patients illness consistent rows). The right-sided nodule is partially calcified. A coronal RETAKE 1st AUTHOR Kradin ICM with tuberculosis? Although infants are more 2nd reformatted image of the CT scan shows three nodules in REG F FIGURE 1a-c 3rd the CASE upper lung zones, with calcifications on the right (Panel likely than adults to present with extrapulmoTITLE Revised EMail B). A chest radiograph showsLine ill-defined nodular opacities, 4-C nary disease, the majority of infants diagnosed SIZElung Enon predominantly in the right upper right middle ARTIST: mst H/T andH/T with tuberculosis in the United States do in fact 16p6 FILL with ill-defined hilar vascular Combo markings zones, (Panel C). 4 cough and fever, as have isolated pulmonary disease. They typically present with nonproductive AUTHOR, PLEASE NOTE:
Figure has been redrawn and type has been reset. Please check carefully. n engl j med 359;2 www.nejm.org july 10, 2008 JOB: 35902 ISSUE: 7-10-08

181

Downloaded from www.nejm.org at HINARI BAND 2 on January 12, 2010 . Copyright 2008 Massachusetts Medical Society. All rights reserved.

The

n e w e ng l a n d j o u r na l

of

m e dic i n e

present with nonproductive cough and fever, as this patient did. The erythematous cutaneous nodule at the site of an intravenous catheter could be a cutaneous manifestation of tuberculosis, although this would be unusual. However, the nodule might have resulted from an unrelated infection, and it appeared to be resolving at the time of admission. A transient response to amikacin may also be seen in M. tuberculosis infection. Are the radiographic findings consistent with tu berculosis? Parenchymal lung disease and mediastinal lymphadenopathy are characteristic of pul monary tuberculosis in infants. Often there is a primary complex, consisting of a parenchymal lesion with associated lymphadenitis. Although miliary involvement can occur in congenital tuberculosis, the presence of multiple bilateral pulmonary nodules, some with calcification, suggesting multi focal granulomatous inflammation, is atypical. Finally, what can we learn from the diagnostic studies that were obtained in the first few days of the patients hospitalization? Acid-fast bacilli stains of early-morning gastric aspirates are often negative in children with pulmonary tuberculosis and have little negative predictive value; however, cultures will be positive in up to 70% of infants with pulmonary tuberculosis.4 This patient also had a negative response to a purified-protein-derivative (PPD) skin test for tuberculosis. Although the majority of infants with disseminated tuberculosis initially have a negative response to this test, 80 to 90% of immunocompetent infants with isolated pulmonary tuberculosis test positive.4,5 Although not definitive, the negative result on the PPD skin test suggests that this patient either did not have tuberculosis or was immunocompromised. Although we were concerned enough to treat this patient empirically for possible tuberculosis, we considered other infectious and noninfectious causes of his illness.
Other Causes of Chronic Multinodular Pneumonia

Histoplasmosis is endemic in India and in the United States and can mimic tuberculosis. It can manifest as a primary pulmonary infection, though infants are predisposed to progressive reticuloendothelial infection characterized by fever, failure to thrive, and hepatosplenomegaly. In this case, a urine antigen test was negative. Blastomyces and cryptococcus are also found in India and can cause chronic pneumonia and cutaneous mani182

festations, including isolated skin nodules and ulcers. However, these infections rarely result in calcified lung lesions. Aspergillus species are an important cause of nodular pneumonia in immunocompromised hosts, particularly patients with hematologic malignancies, neutropenia, or phagocyte disorders, and chronic infections may calcify. Thus, chronic pulmonary aspergillosis due to an underlying immunodeficiency was a consideration, despite the fact that this child had been healthy for the first 10 months of life. Nontuberculous mycobacteria need to be considered, especially in view of the negative finding on the PPD skin test and the partial clinical response to amikacin, which is active against nontuberculous mycobacteria. However, pulmonary infection of this magnitude with nontuberculous mycobacteria is rare in a healthy host, and symptoms recurred on use of clarithromycin, which is highly active against most nontuberculous mycobacterial infections. Burkholderia pseudomallei, the causative agent of melioidosis, is endemic in India and can manifest as a chronic, multinodular pneumonia. Skin manifestations, including ulcers and abscesses, can occur in isolation or as part of disseminated disease; thus, melioidosis was a possible diagnosis in this case. Rhodococcus equi is an increasingly recognized cause of chronic nodular pneumonia in immunocompromised patients; however, infection with R. equi is typically associated with exposures to animals or soil, neither of which was present in this case. Nocardia species can cause chronic lung infections, including multinodular pneumonia, particularly in immunocompromised hosts. Paragonimus, a lung fluke that is endemic in India, can present as chronic pneumonia with multiple calcified nodules; however, most patients have peripheral blood eosinophilia and a history of shellfish exposure, neither of which applied in this case. Sarcoidosis is a chronic pulmonary granulomatous disease; however, the infantile form, Blau syndrome, does not typically involve the lungs. Inflammatory myofibroblastic tumor (also known as inflammatory pseudotumor or plasma-cell granuloma), the most common primary lung tumor in children, is often associated with calcification.6 However, most of these tumors are solitary lung lesions and are not associated with fever. Metastatic Wilms tumor or neuroblastoma can occur at this age, but there is no evidence of a primary tumor in the abdomen.

n engl j med 359;2 www.nejm.org july 10, 2008

Downloaded from www.nejm.org at HINARI BAND 2 on January 12, 2010 . Copyright 2008 Massachusetts Medical Society. All rights reserved.

case records of the massachusetts gener al hospital

Chronic Multinodular Pneumonia as a Manifestation of Primary Immunodeficiency

An early concern was that the patient had a primary immunodeficiency. Primary immunodeficiency disorders are typically suspected in the presence of repeated infections, opportunistic infections, failure to thrive, a positive family history for such a disorder, hematologic abnormalities, abnormal responses to common infections, or characteristic features of a syndrome associated with immunodeficiency, such as DiGeorges syndrome, which is distinguished by cleft palate, endocrine abnormalities, and congenital heart defects. Because many immunodeficiency syndromes can present after 6 months of age, the fact that this child had thrived in early infancy does not exclude such a diagnosis. Although we had not documented the presence of an opportunistic infection, the finding of multiple calcified pulmonary nodules suggested that a chronic granulomatous process preceded the visit to India and the development of symptoms. This could represent an abnormal response to infection a characteristic feature of chronic granulomatous disease. Chronic granulomatous disease, which is due to a primary defect of the phagocytic NADPH oxidase pathway, is one of the most common serious primary immunodeficiency syndromes, with an incidence of at least 1 in 200,000 births

in the United States.7 The clinical manifestations are listed in Table 2. Pulmonary infection occurs in more than 80% of cases, particularly in a form of calcifying multinodular pneumonia known as encapsulating pneumonia.7,8 Another common clinical manifestation is the formation of relatively painless subcutaneous nodules, known as cold abscesses, which are a classic manifestation of phagocytic dysfunction. This patients lung and skin lesions are thus highly characteristic of chronic granulomatous disease. The diagnosis of chronic granulomatous disease is often suggested by a microbiologic diagnosis, since the majority of infections in this disease are due to five catalase-producing microorganisms: Staphylococcus aureus, B. cepacia, Serratia marcescens, nocardia species, and aspergillus species. Aspergillus is the predominant causative agent of pulmonary infections, accounting for more than 40% of cases in which a causative organism is identified.7 Aspergillus and B. cepacia account for many of the deaths in patients with chronic granulomatous disease.7 During the course of our patients treatment at this hospital, additional information was obtained that increased our concern for chronic granulomatous disease. Although there was no family history of immunodeficiency, we learned that the patients parents were closely related. Consanguinity greatly increases the likelihood of

Table 2. Commonly Reported Clinical Manifestations of Chronic Granulomatous Disease in the 368 Patients in the U.S. National Registry.* Clinical Syndrome Pneumonia Abscesses (any) Subcutaneous Liver Lung Perirectal Lymphadenitis Osteomyelitis Bacteremia/fungemia Colitis and enteritis syndromes Gastric-outlet obstruction Urinary-outlet obstruction Proportion of Patients (%) 79 68 42 27 16 15 53 25 18 18 15 10 Staphylococcus species, serratia species Staphylococcus species Aspergillus species None predominates Staphylococcus species Serratia species, aspergillus species Salmonella species, B. cepacia, candida species Common Microbiologic Causes in Order of Prevalence Aspergillus species, staphylococcus species, Burkholderia cepacia, nocardia species

* Reported in 10% or more of cases. Adapted from Winkelstein et al.7 Organisms listed have been identified in more than 10% of cases for each syndrome listed.

n engl j med 359;2 www.nejm.org july 10, 2008

183

Downloaded from www.nejm.org at HINARI BAND 2 on January 12, 2010 . Copyright 2008 Massachusetts Medical Society. All rights reserved.

The

n e w e ng l a n d j o u r na l

of

m e dic i n e

an autosomal recessive disorder. In addition, the skin biopsy was interpreted as showing a possible fungal hypha, which heightened our suspicion that the patient had chronic granulomatous disease. Other causes of primary immunodeficiency are much less likely. Cystic fibrosis and ciliary defects are associated with chronic sinopulmonary disease, but they are usually associated with bronchiectasis. T-cell and B-cell immunodeficiencies can present with pneumonia, but in this case the normal immunoglobulin levels, the lack of lymphopenia, and the lack of prior recurrent infections or chronic diarrhea argue against these immunodeficiencies. Other phagocytic disorders include deficiencies in the interferon-inter leukin-12 receptor signaling pathway.9 Like chronic granulomatous disease, these disorders are associated with a remarkably specific microbiology typically mycobacterial infections or nontyphoid salmonellosis. However, these disorders are much less common than chronic granulomatous disease.
Diagnostic Testing

To evaluate for chronic granulomatous disease, on the fourth hospital day we sent peripheralblood leukocytes from this patient to another laboratory for an oxidative burst assay. While this result was pending, we obtained a specimen from the involved lung tissue for histopathology and culture, which we considered essential to establishing a diagnosis.

Dr . Ja son B . H a r r iss Di agnosis

Infectious pneumonia, probably due to aspergillus species and probably complicating chronic granulomatous disease.

Pathol o gic a l Discussion

Dr. Richard L. Kradin: Pathological examination of a video-assisted thoracoscopic biopsy specimen of the right upper lobe of the lung revealed necrotizing granulomas, primarily centered on small airways (Fig. 2A), with extensive necrosis and dystrophic calcifications (Fig. 2B). Special stains were negative for mycobacteria, but Gomoris methenamine silver staining (Fig. 2C) revealed fragmented, varicose, septate hyphae with rare orthogonal pyriform conidia in areas of necrosis. The presence of fragmented varicose hyphae with small, lateral, pyriform conidiophores and conid184
Figure 2. Lung-Biopsy Specimen. A view of the biopsy specimen of the right upper lung lobe at low magnification revealed several necrotizing RETAKE 1st AUTHOR Kradin (Panel A). A focus ICM bronchocentric granulomas of ne2nd REGcontains F FIGURE 2a-c crosis dystrophic calcification (Panel B). At 3rd CASE TITLE higher magnification (Panel C), a granuloma with Revised EMail 4-C multinucleated giant cells isLine seen. Staining with GoSIZE Enon ARTIST: mst H/T moris methenamine silver (Panel C,H/T inset, lower left) 16p6 FILL Combo reveals a fragmented septate fungal hypha with a conAUTHOR, PLEASE stricted right-angle branch point.NOTE: The fungal hypha is Figure has been redrawn and type has been reset. stained with antibody specific for aspergillus species Please check carefully. (Panel C, inset, lower right).
JOB: 35902 ISSUE: 7-10-08

n engl j med 359;2 www.nejm.org july 10, 2008

Downloaded from www.nejm.org at HINARI BAND 2 on January 12, 2010 . Copyright 2008 Massachusetts Medical Society. All rights reserved.

case records of the massachusetts gener al hospital

A Unstimulated
Unrelated Healthy Control
250 200 150 100 50 0 100
101 102 103 104

Patient
250 200 150 100 50 0 100

Cell Number

101

102

103

104

Fluorescence Intensity

Fluorescence Intensity

B PMA-Stimulated
Unrelated Healthy Control
250 200 250 200 150 100 50 0 100

Patient

Cell Number

150 100 50 0 100

101

102

103

104

101

102

103

104

Fluorescence Intensity

Fluorescence Intensity

Figure 3. Dihydrorhodamine-123 Fluorescence Assay of Peripheral-Blood Neutrophils from the Patient and an Unrelated Healthy Control. 1st RETAKE AUTHOR: Kradin ICM In this fluorescence assay of peripheral-blood neutrophils from the patient and an 2nd unrelated healthy control, neutro3 of 4 REG F FIGURE: 3rd phils are stimulated with phorbol myristate acetate (PMA) in the presence of dihydrorhodamine-123, and flow cyCASE Revised tometry is used to measure the oxidative burst mediated by NADPH oxidase by quantifying the fluorescent product, 4-C Line EMail SIZE ARTIST: ts healthy rhodamine. Shown are baseline histograms from the control and the patient H/T H/T 33p9 before stimulation with PMA Enon (Panel A). After PMA stimulation (Panel B), the histogram Combo from the control shows a unimodal shift of fluorescence AUTHOR, PLEASE NOTE: far to the right, whereas the patient has a broad-based shift in fluorescence that is only approximately 1/10 the size Figure has been redrawn and type of has been reset. recessive chronic granulomatous of the shift observed in the control. This abnormality is suggestive autosomal Please check carefully. disease.
JOB: 35902 ISSUE: 07-10-08

ia in tissue are typical of aspergillus of the flavipesterreus group.10 A hyphal form was highlighted by immunohistochemical staining for aspergillus species. Cultures of the specimen showed no growth of fungi or bacteria, but a culture of the skin-biopsy specimen that had been obtained before admission grew Aspergillus terreus. Patients with chronic granulomatous disease are most commonly infected with A. fumigatus or A. nidulans.11 A. terreus is a ubiquitous environmental fungus, which is emerging as an important pathogen in pulmonary infections in immunocompromised hosts.12,13 The host response to aspergillus in this case mimics that of a mycobacterial or fungal yeast infection. Whereas invasive infections due to asper-

gillus species are well recognized in chronic granulomatous disease, well-defined necrotizing granulomatous inflammation is infrequent. As there was no evidence of angioinvasion in the sampled lung tissue, the infection is best classified as invasive chronic necrotizing aspergillosis, which is distinguished specifically by the absence of vascular invasion. Dr. Ian C. Michelow: The dihydrorhodamine assay, a quantitative measure of NADPH oxidase function during the respiratory burst of phagocytic cells, is the diagnostic test for chronic granulomatous disease. Chronic granulomatous disease is caused by mutations in genes that encode for one or more of the six components of the NADPH oxidase complex, an enzyme that is
185

n engl j med 359;2 www.nejm.org july 10, 2008

Downloaded from www.nejm.org at HINARI BAND 2 on January 12, 2010 . Copyright 2008 Massachusetts Medical Society. All rights reserved.

The

n e w e ng l a n d j o u r na l

of

m e dic i n e

present in phagocytes such as neutrophils, monocytes, and macrophages and is critical to the Plasma membrane Secretory vesicle production of the reactive oxygen species that or specific granule help eradicate organisms that have been subject to phagocytosis. In our patient, the assay (Fig. 3) gp91phox showed a broad peak of fluorescence that was lower than that of normal neutrophils, a pattern typically associated with autosomal recessive Phagosome p22phox variants of chronic granulomatous disease.14 AsRac2 says from patients with X-linked chronic granulomatous disease typically demonstrate a narrow phox p67 Cytosol peak, with activation in response to stimulation p40phox virtually absent. Test results for the parents were NADPH oxidase complex normal; since a carrier of the X-linked form would p47phox be expected to have a dual population of neutrophils, some showing normal activation and others with decreased activation, this finding sugB gests a diagnosis of autosomal recessive chronic Phagosome granulomatous disease. We then proceeded to genetic testing. The 30 36 ? ? majority of cases of chronic granulomatous disease are due to mutations in the gene gp91phox, which causes the X-linked disorder (accounting 10 for approximately 70% of cases). Autosomal re50 92 110 Cytosol cessive cases are caused by abnormalities in the genes p47phox (approximately 20%), p22phox (apNH2 proximately 5%), and p67phox (approximately 5%). 195 151 160 COOH Rarely, abnormalities in Rac2, a small guano 129 p47phox binding site sine triphosphatase, or GTPase, can cause a chronic granulomatous diseaselike phenotype. phox In this patient, sequencing of the relevant genes Figure 4. The NADPH Oxidase Complex and a Proposed Model for p22 identified a novel missense mutation in the Topology. COLOR FIGURE p22phox gene, in which lysine was substituted for When a phagocyte ingests a pathogen, secretory vesicles or specific granules fuse with the phagosome (Panel A). This activates oxidase Rev4 the NADPH 06/16/08 glutamic acid at amino acid 129 of exon 6. The complex, which has six components, including gp91phox (91-kD glycoprotein, Author Dr. Kradin interactions among the six NADPH oxidase comalso known as cytochrome b- subunit, or heavy chain) and p22phox (cytoFig # 4 ponents are shown in Figure 4A. Figure 4B shows chrome b- subunit, or light chain), which are both membrane-bound. The Title phox a proposed model for p22phox topology.15 An alterother four components of the complex are located in the cytosol: p47 ME phox phox native model exists,17,18 but there is consensus (neutrophil cytosolic factor 1), p67 (neutrophil cytosolic factor 2), p40 Harris DE (neutrophil cytosolic factor 4), and the small GTPase Rac2. The activated about the location of the target for p47phox bindDaniel Muller Artist complex assembles on the phagosome membrane. Once activated, the ing as shown in the figure. The patients mutaPLEASE NADPH oxidase transfers electrons from NADPHAUTHOR to molecular ONOTE: 2 , resultFigure has been redrawn and type has been reset tion at amino acid 129 appears to affect the ing in the formation of superoxide anions in the phagosome. enzyme Please checkThe carefully ability of p22phox and gp91phox to form the flavosuperoxide dismutase catalyzes the formation of H2O2, the fate of which Issue date 07-10-2008 cytochrome b heterodimer (Dinauer M: personal depends on the presence of myeloperoxidase, an enzyme that converts H2O2 to hypochlorous acid and hydroxyl radicals, or catalase, an enzyme communication), which presumably causes lack that converts H2O2 to H2O and O2. The reactive oxygen species are reof expression of flavocytochrome b (gp91phox and sponsible for killing pathogens. Normal oxidative activity of the NADPH p22phox complex) in neutrophils, as in most other complex requires fully functional individual components. A proposed model patients with a documented p22phox mutation. for p22phox topology is shown in Panel B. The proline-rich domain from amino The treatment of documented or suspected acids 151 to 160 (each number refers to the corresponding amino acid) is a well-characterized target for p47phox binding. The patients mutation at amiinvasive infections in a patient such as this with no acid 129 (arrow) presumably causes lack of expression of the flavocytochronic granulomatous disease is the same as for chrome b (the gp91phox and p22phox complex) in neutrophils. NH2 denotes other immunocompromised hosts. Long-term the amino terminal, and COOH the carboxy terminal. Adapted from Dahan 15 16 treatment with interferon- has been shown to et al. and Roos et al. reduce the frequency of opportunistic infections, A
186
n engl j med 359;2 www.nejm.org july 10, 2008

Downloaded from www.nejm.org at HINARI BAND 2 on January 12, 2010 . Copyright 2008 Massachusetts Medical Society. All rights reserved.

case records of the massachusetts gener al hospital

as has prophylaxis with trimethoprimsulfa meth ox azole and itraconazole. Currently, the only widely accepted definitive treatment is bone marrow transplantation.19,20 Dr. Harris: Unfortunately the patients condition continued to deteriorate, and a seeming Pandoras box of microbiology was opened. A blood culture obtained on hospital day 4 grew Candida parapsilosis at 72 hours, the skin biopsy obtained before admission grew A. terreus after 7 days, and blood cultures obtained on hospital days 11 through 17 were persistently positive for B. cepacia, despite the organisms susceptibility to meropenem, which the patient had been taking since hospital day 4. In addition to antimicrobial chemotherapy, treatReferences
1. Global tuberculosis control: surveil-

ment included interferon- and eventually a granulocyte transfusion, which was performed in the setting of sepsis and multiorgan failure. The patient died on hospital day 17, and permission for an autopsy was not obtained. The results of the genetic tests were obtained after the patient died.

A nat omic a l Di agnosis

Necrotizing granulomatous pneumonitis due to A. terreus, complicating chronic granulomatous disease.
No potential conflict of interest relevant to this article was reported. We thank Mary Dinauer, M.D., Ph.D., for her insightful comments.

lance, planning, financing. Geneva: World Health Organization, 2006. (WHO report no. WHO/HTM/TB/2006.362.) 2. Starke JR, Smith KC. Tuberculosis. In: Feigen RD, Cherry JD, Demmler GJ, Kap lan SL, eds. Pediatric infectious diseases. 5th ed. Philadelphia: Saunders, 2004: 1337-79. 3. Wallgren A. The time-table of tuberculosis. Tubercle 1948;29:245-51. 4. Vallejo JG, Ong LT, Starke JR. Clinical features, diagnosis, and treatment of tuberculosis in infants. Pediatrics 1994; 94:1-7. 5. Steiner P, Rao M, Victoria MS, Jabbar H, Steiner M. Persistently negative tuberculin reactions: their presence among children with culture positive for Mycobacterium tuberculosis (tuberculin-negative tuberculosis). Am J Dis Child 1980; 134:747-50. 6. Moran CA, Suster S. Unusual nonneoplastic lesions of the lung. Semin Diagn Pathol 2007;24:199-208. 7. Winkelstein JA, Marino MC, Johnston RB Jr, et al. Chronic granulomatous disease: report on a national registry of 368 patients. Medicine (Baltimore) 2000;79:155-69. 8. Wolfson JJ, Quie PG, Laxdal SD, Good RA. Roentgenologic manifestations in children with a genetic defect of polymor-

phonuclear leukocyte function: chronic granulomatous disease of childhood. Radiology 1968;91:37-48. 9. Rosenzweig SD, Holland SM. Defects in the interferon-gamma and interleukin-12 pathways. Immunol Rev 2005;203: 38-47. 10. Chandler FW, Watts JC. Pathologic diagnosis of fungal infections. Chicago: American Society of Clinical Pathologists, 1987. 11. Segal BH, DeCarlo ES, Kwon-Chung KJ, Malech HL, Gallin JI, Holland SM. Aspergillus nidulans infection in chronic granulomatous disease. Medicine (Baltimore) 1998;77:345-54. 12. Lass-Flrl C, Griff K, Mayr A, et al. Epidemiology and outcome of infections due to Aspergillus terreus: 10-year single centre experience. Br J Haematol 2005; 131:201-7. 13. Baddley JW, Pappas PG, Smith AC, Moser SA. Epidemiology of Aspergillus terreus at a university hospital. J Clin Microbiol 2003;41:5525-9. 14. Segal BH, Leto TL, Gallin JI, Malech HL, Holland SM. Genetic, biochemical, and clinical features of chronic granulomatous disease. Medicine (Baltimore) 2000;79:170-200. 15. Dahan I, Issaeve I, Gorzalczany Y, Sigal N, Hirshberg M, Pick E. Mapping of

functional domains in the p22(phox) subunit of flavocytochrome b(559) participating in the assembly of the NADPH oxidase complex by peptide walking. J Biol Chem 2002;277:8421-32. 16. Roos D, van Bruggen R, Meischl C. Oxidative killing of microbes by neutrophils. Microbes Infect 2003;5:1307-15. 17. Taylor RM, Burritt JB, Baniulis D, et al. Site-specific inhibitors of NADPH oxidase activity and structural probes of flavocytochrome b: characterization of six monoclonal antibodies to the p22phox subunit. J Immunol 2004;173:7349-57. 18. Taylor RM, Lord CI, Riesselman MH, et al. Characterization of surface structure and p47phox SH3 domain-mediated conformational changes for human neutrophil flavocytochrome b. Biochemistry 2007;46:14291-304. 19. Sastry J, Kakakios A, Tugwell H, Shaw PJ. Allogeneic bone marrow transplantation with reduced intensity conditioning for chronic granulomatous disease complicated by invasive Aspergillus infection. Pediatr Blood Cancer 2006;47:327-9. 20. Gngr T, Halter J, Klink A, et al. Successful low toxicity hematopoietic stem cell transplantation for high-risk adult chronic granulomatous disease patients. Transplantation 2005;79:1596-606.
Copyright 2008 Massachusetts Medical Society.

Lantern Slides Updated: Complete PowerPoint Slide Sets from the Clinicopathological Conferences
Any reader of the Journal who uses the Case Records of the Massachusetts General Hospital as a teaching exercise or reference material is now eligible to receive a complete set of PowerPoint slides, including digital images, with identifying legends, shown at the live Clinicopathological Conference (CPC) that is the basis of the Case Record. This slide set contains all of the images from the CPC, not only those published in the Journal. Radiographic, neurologic, and cardiac studies, gross specimens, and photomicrographs, as well as unpublished text slides, tables, and diagrams, are included. Every year 40 sets are produced, averaging 50-60 slides per set. Each set is supplied on a compact disc and is mailed to coincide with the publication of the Case Record. The cost of an annual subscription is $600, or individual sets may be purchased for $50 each. Application forms for the current subscription year, which began in January, may be obtained from the Lantern Slides Service, Department of Pathology, Massachusetts General Hospital, Boston, MA 02114 (telephone 617-726-2974) or e-mail Pathphotoslides@partners.org.

n engl j med 359;2 www.nejm.org july 10, 2008

187

Downloaded from www.nejm.org at HINARI BAND 2 on January 12, 2010 . Copyright 2008 Massachusetts Medical Society. All rights reserved.