n e w e ng l a n d j o u r na l
of
m e dic i n e
178
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
Variable
Hematocrit (%)
33.039.0
Hemoglobin (g/dl)
10.513.5
3)
6.0x10317.5x103
20 Days before
Admission
in India*
Current
Admission
11.0
33.7
33.8
26.8
11.5
11.1
8.9
32,800
28,900
36,400
24,700
1749
42
37
39
45
Lymphocytes
6777
56
61
47
31
Monocytes
411
Eosinophils
08
Basophils
03
Band forms
010
12
Atypical lymphocytes
Platelets (per mm
3)
150,000450,000
396,000
465,000
319,000
66
67
67
7086
Red-cell morphology
3+ microcytes
017
<15
75
3+ microcytes, 1+
hypochromia
3+ microcytes, 2+
hypochromia,
rouleaux formation present
59
143.8
Immunoglobulin (mg/dl)
IgG
2831026
815
IgA
1682
61
IgM
39141
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%.
179
The
n e w e ng l a n d j o u r na l
of
m e dic i n e
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-
181
The
n e w e ng l a n d j o u r na l
of
m e dic i n e
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
Table 2. Commonly Reported Clinical Manifestations of Chronic Granulomatous Disease in the 368 Patients in the U.S.
National Registry.*
Clinical Syndrome
Proportion of
Patients (%)
Pneumonia
79
Abscesses (any)
68
Subcutaneous
42
Liver
27
Staphylococcus species
Lung
16
Aspergillus species
Perirectal
15
None predominates
Lymphadenitis
53
Staphylococcus species
Osteomyelitis
25
Bacteremia/fungemia
18
18
Gastric-outlet obstruction
15
Urinary-outlet obstruction
10
183
The
n e w e ng l a n d j o u r na l
of
m e dic i n e
Diagnostic Testing
35902
ISSUE:
7-10-08
A Unstimulated
Cell Number
Patient
250
250
200
200
150
150
100
100
50
50
0
100
101
102
103
104
0
100
101
Fluorescence Intensity
102
103
104
Fluorescence Intensity
B PMA-Stimulated
Cell Number
Patient
250
250
200
200
150
150
100
100
50
50
0
100
101
102
103
104
0
100
Fluorescence Intensity
101
102
103
104
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
quantifying the fluorescent product,
4-Coxidase by
Line
EMail
SIZE
ARTIST:
ts healthy
rhodamine. Shown are baseline histograms
from the
and the patient
H/T
H/Tcontrol
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
hasautosomal
been reset. recessive chronic granulomatous
of the shift observed in the control. This
abnormality
is suggestive
Please check carefully.
disease.
JOB: 35902
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-
ISSUE: 07-10-08
185
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 NADPH06/16/08
glutamic acid at amino acid 129 of exon 6. The
phox
complex, which has six components, including
(91-kD
glycoprotein,
Authorgp91 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
Mullerthe
Artist
complex assembles on the phagosome membrane.
Once
activated,
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
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.
References
1. Global tuberculosis control: surveil-
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.
187