Final Case Study
Trinidad Cisneros, Ruby Khan, Patricia Nykaza
California State Univesity Los Angeles

Case Summary
 15 years old student was admitted to
the hospital due to respiratory failure.
He had been spending the summer as
part of a gardening crew preparing
garden beds by spreading bark.
 Radiological examination of chest
showed cotton ball densities in both
lungs, a lesion was aspirated with a fine
needle to perform a stain and culture
test
Case Summary Continued
 Aspergillus fumigatus was
identified in granuloma-like lesion.
 Randy was started on intravenous
amphotericin B and assisted with
mechanical ventilation through a
tracheotomy. He remained in the
hospital for two months, where he
also contracted Pseudomonas
aeruginosa and Enterococcus
faecalis, these infections were
treated with strong antibiotics.
Dr. Porter Lec 16
Case Summary Lab Results
 At initial admission to hospital:
WBC above normal=11,500/μl Normal value (5000/100 μl)
 Immunoglobulin levels were higher than normal for IgG, IgM
and IgA.
 Family History: His sister are unaffected but his brother Ralph
has had a perirectal abscess in infancy.
 Their current cytochrome b content in granulocytes are well
below normal values.
Key Information Pointing to Diagnosis
 Radiological examination that showed cotton ball densities in both
lungs!
 Staining and culture of aspirate identified Aspergillus fumigatus.
seen in a granuloma-like lesions
Fungus commonly found in mulch, which his line of work
exposed him to. Common symptoms of this infection include
persistent coughing, chest pain and shortness of breath
 Cytochrome b content in granulocytes is <0.1pmol/mg protein
(normal 101pmol.mg)
 Brother with the history of perirectal abscess in infancy may indicate
an inherited immune disorder.
 Nosocomial infections by P. aeruginosa and E. faecalis are common
in immune deficient individuals.
The Diagnosis for Case 1
 X-linked Chronic Granulomatous Disease
 Inherited disorder of phagocytic activity common in boys.
 Characterized by serious susceptibility to recurrent infection
since birth.
 Mutations in NADPH oxidase subunits prevents individuals
from effectively clearing bacterial and fungal infections.
 NADPH oxidase is instrumental in mounting an oxidative
burst. CGD patients are unable to cause superoxide
generation and kill intracellular pathogens.
 Randy has a mild phenotype which would explain his later
onset of symptoms.
Diagnosis continued

What is a granuloma and how is it formed?
 Granuloma is an organized collection of macrophages in a tight ball- like formation
Most common cause is fungal infection in the lungs called histoplasmosis
Granuloma formation
 Triggering of T cell by antigen presenting cells
The release of cytokines and chemokines by macrophages, activated lymphocytes,
dendritic cells, and PMN
The formation of organized structure of granuloma
In granulomatous disease, the last step usually ends in fibrosis
Confirming X-linked CGD
Primary test to confirm deficiency in PMN producing ROS
•
•
Functional and phenotypic analysis of neutrophils: measured the
capacity of PMN to produce ROS by nitroblue tetrazolium slide test and
flow cytometric dihydrohodamine-1,2,3 (DHR) assay.
NBT assay: based on the reduction of nitroblue tetrazolium to
formazan by PMA acivated leukocytes.
•
•
•
Positive Test= Nitro blue tetrazolium is reduced by NADPH oxidase and
forms an insoluble blue formazan salt.
Negative Test=no blue formazan salt is observed on slide.
DHR assay: Red blood cells are lysed and leukocytes loaded with DHR
for 5 min at 37˚C in presence of catalase. Cells are stimulated with
phorbol myristrate acetate for 15 min at 37˚C and analyzed by flow
cytometry.
•
•
•
Positive Test= Single unchanged peak after stimulation with PMA
stimulation
Negative Test=Single peak at higher intensity after PMA stimulation.
Bimodal Peaks=Carrier
Confirming X-linked CGD
Primary test to confirm deficiency in PMN producing ROS
•
NBT assay: based on the reduction of nitroblue tetrazolium to
formazan by PMA acivated leukocytes.
•
Positive Test= Nitro blue tetrazolium is reduced by NADPH oxidase
and forms an insoluble blue formazan salt.
Elloumi, Z. Diagnostic assays
for chronic granulomatous
disease and other neutrophil
disorders. 2007 Methods in
molecular bio. 412: 505-23
Confirming X-linked CGD
Primary test to confirm deficiency in PMN producing ROS
•
DHR assay:
Elloumi, Z. Diagnostic assays
for chronic granulomatous
disease and other neutrophil
disorders. 2007 Methods in
molecular bio. 412: 505-23
Diagnostic Tests for Differentiating X-linked
CGD
Secondary tests to differentiate from other disease by genetic
testing
•
•
•
SSCP analysis of CYBB gene: Genomic DNA extracted by
conventional salting-out, promoter region and 13 exons of CYBB
gene amplified by PCR runs. PCR products are then evaluated 2%
agarose gel followed by SSCP analysis on 6% polyacrylamide gel
followed by silver staining.
DNA Sequencing: In case of abnormal SSCP patterns.
Haplotype analysis: Show inheritance of CYBB mutations in
investigated families X-chromosome marker analysis.
Evaluating Patient’s Health Status
Clinical Tests
 Blood test (monitor WBC and complement counts)
 Urine test (monitor baseline values)
 Chest x-ray (Infection)
 Liver and bone scan (Organ Failure)
Therapy for X-linked CGD
 Early intervention on onset of infection with strong
prophylactics
 Acute infections should be treated aggressively with appropriate
antibiotics.
 Lifestyle accommodations to avoid exposure to infections. i.e.
avoid mulch.
Prognosis for X- linked CGD
 Manageable for mild cases with early intervention at onset of
symptoms/infection.
 Without treatment children often die within the first few years.
 Long-term antibiotic treatments may help to reduce infections,
in severe conditions, early death is typical due to repeated lung
infections.
 Possible complications of CGD include: Bone damage, Chronic
pneumonia, Lung damage, Skin damage
How would you communicate
diagnosis and disease to the patient
(or his/her parents/guardians)
 The lab results show that your sons ailments are a mild form
of an inherited disease called X-linked Chronic Granulomatous
disease. This means they were born with this condition, and
can pass it on to their children. This disease impairs some of
their white blood cells from clearing bacterial and fungal
infections, but your sons can continue to lead a normal life.
They must be careful at exposing themselves to potential
illnesses. If they present any symptoms, they should
immediately seek medical treatment and expect
hospitalization until their conditions improve.
The Theoretical Immunological Basis for X-liked
CGD
Chronic Granulomatous Disease
 NADPH oxidase defect
 Many different gene
mutations
 No superoxide radical
production
 Host forms granulomas to
eliminate pathogens
Primary Research Article Contributing to the
Understanding of this Disease
 Teimourian et al. Molecular diagnosis of X-linked chronic
granulomatous disease in Iran. 2008. Int J. Hematol. 87:398-404
 Research objectives: First study aimed at analyzing the clinical
features and molecular diagnosis of X-CGD in Iranian patients.
 Experimental Design: 2 year study with 11 non-consanguienesou
families with members clinically diagnoses for CGD. Neutrophil
NADPH oxidase measurements, NBT slide test and flow cytometric
dihydrorhodamine-1,2,3 (DHR) assay. Immunoblot detection of
cytosolic and membrane NADPH oxidase performed using western
blot analysis. SSCP mutation analysis of CYBB gene conducted using
conventional salting-out methods, PCR and DNA sequencing
followed by haplotype analysis.
 Novel Findings: Sequencing showed nine different mutations in the
CYBB gene encoding gp91phox two of which are novel.
NORMAL
Key Figure from the
Article
 DHR Flow cytometry assay is a quick
CARRIER
X-linked CGD
and sensitive test to detect carriers of
oxidase-positive and oxidase-negative
neutrophils in mothers with a binomial
histogram demonstrating two
populations of neutrophils.
 One first population is the inactive
neutrophils, and the second is the
active neutrophils.
 Notice in disease patient only a single
population exist that does not change
upon stimulation with phorbol
myristate acetate (PMA)
Take Home Messages
 X-linked CGD involves recurrent infections since birth due to
defects in NADPH oxidase, which prevent effective killing of
bacteria and fungi by phagocytes.
 Recurrent infections include bacterial and fungal infections,
especially infections of the lungs other tissues and organs may be
common.
 X-linked CGD includes various phenotypes from mild to severe, but
prognosis is manageable with early diagnosis, prophylaxis and
strong antibiotics upon infection.
 Functional and phenotypic analysis of neutrophils are primarily
conducted to verify NADPH oxidase disfunction in phagocytosis:
Nitroblue tetrazolium slide test and flow cytometric
dihydrohodamine-1,2,3 (DHR) assay. Genetic testing can confirm
disease.