Final Case Study

1. Final Case Study Trinidad Cisneros, Ruby Khan, Patricia Nykaza California State Univesity Los Angeles

2. 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

3. 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

4. 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.

5. 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.

6. 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.

7. 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

8. 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

9. 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

10. 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

11. 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.

12. 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)

13. 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.

14. 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

15. 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.

16. 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

17. 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.

18. NORMAL Key Figure from the Article • DHR Flow cytometry assay is a quick 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) CARRIER X-linked CGD

19. 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.