Testing For Chronic Granulomatous Disease Research Articles

UNEXPECTED DIAGNOSIS FOLLOWING IMMUNODEFICIENCY WORK-UP

<h3>Introduction</h3> A 15-month-old infant with severe recurrent sinopulmonary infections was evaluated for a primary immunodeficiency. Investigation resulted in a diagnosis that initially wasn't suspected. <h3>Case Description</h3> Our patient was born at term and had no medical concerns until 10-months-old when she developed recurrent otitis accompanied by fevers, diarrhea, and failure to gain weight appropriately. Within five months she was diagnosed with four separate ear infections, each requiring multiple rounds of antibiotics to treat. She was also diagnosed with anemia, splenomegaly, and two episodes of pneumonia. She was hospitalized for one episode of pneumonia and staphylococcus aureus was isolated from a bronchopulmonary lavage. An extensive immunodeficiency laboratory investigation revealed a borderline abnormal oxidative burst test and selective IgA deficiency. Genetic testing for chronic granulomatous disease was negative. Celiac testing was inconclusive based on her IgA deficiency, so an upper GI endoscopy was completed to rule out this condition. She was ultimately diagnosed with eosinophilic esophagitis based on esophageal biopsies. <h3>Discussion</h3> Eosinophilic esophagitis was not considered in the initial differential diagnosis as this typically does not result in recurrent infections. This case demonstrates the important link between malnutrition and immune dysregulation; poor nutrition can lead to secondary immune dysfunction. Recent studies have postulated a correlation between IgA deficiency and eosinophilic esophagitis, this case adds data to that observation. After appropriate treatment, this patient's symptoms dramatically improved with no continued infections, appropriate weight gain, and resolution of diarrhea and splenomegaly.

Clinical and laboratory predictors of monogenic very early onset inflammatory bowel disease

BackgroundInflammatory bowel disease (IBD) is a chronic inflammatory disease of the gastrointestinal tract. Treatment for patients who have a monogenic cause of their IBD, often the youngest children, known as very early onset IBD (VEO-IBD), can be different from standard treatment for polygenic cases. Yet, ascertainment of these patients is difficult. MethodsWe analyzed cases of VEO-IBD to understand the breadth of monogenic etiology and to identify clinical, laboratory, and flow cytometric correlates of this subpopulation. ResultsGenetic causes of very early onset inflammatory bowel disease are highly diverse ranging from pure epithelial defects to classic T cell defects. Flow cytometry, other than testing for chronic granulomatous disease, has a low sensitivity for monogenic etiologies. Poor growth was a clinical feature associated with monogenic causality. ConclusionsGenetic testing is, at this moment, the most robust method for the identification of monogenic cases of very early onset IBD.

Acetaminophen Inhibits the Neutrophil Oxidative Burst: Implications for Diagnostic Testing

Chronic granulomatous disease is a primary immunodeficiency characterized by recurrent bacterial and fungal infections, granuloma formation, and inflammatory disease. Impaired neutrophil oxidative function is an essential diagnostic criterion. Invitro exposure of neutrophils to acetaminophen, a commonly used over-the-counter medication, has been associated with reduced neutrophil oxidative function. The clinical implications of acetaminophen intake for dihydrorhodamine (DHR) testing remain unknown. To evaluate the effect of invivo administration of therapeutic doses of acetaminophen on DHR diagnostic testing. We performed DHR testing in 15 healthy adults before and after administering a single dose of acetaminophen. We retrospectively reviewed 195 DHR test results from hospitalized patients who had received acetaminophen, nonsteroidal anti-inflammatory drug, or corticosteroid before testing. DHR testing result was abnormal in 100% (n= 15) of healthy adults 2 hours after acetaminophen intake. We identified 195 instances of DHR testing less than or equal to 72 hours after acetaminophen ingestion in hospitalized patients who did not have chronic granulomatous disease. DHR results were abnormal in 43 of 195 cases (22.1%). Frequency of false-positive testing was increased in patients who received acetaminophen within 24 hours of testing, and in patients who received more than 1 dose of acetaminophen. Nonsteroidal anti-inflammatory drug and corticosteroid intakes were not associated with abnormal DHR result. Patients treated with acetaminophen have decreased neutrophil oxidative burst as measured by DHR testing. To avoid falsely abnormal testing for chronic granulomatous disease, patients should be advised to avoid acetaminophen for at least 24 hours before DHR testing.

“Clinical Aspects of Chronic Granulomatous Disease in Upper Egypt”

ABSTRACT Chronic granulomatous disease (CGD) is a rare inherited primary immunodeficiency disorder that affects phagocytes and is characterized by a marked increased susceptibility to severe bacterial and fungal infections. We aimed to describe the clinical presentations of pediatric patients with CGD in Upper Egypt and to identify the defective component of NADPH oxidase. Pediatric patients diagnosed with CGD within one year from January 2018 to January 2019 were enrolled in the study. Patient history, clinical and laboratory investigations were carried out, including nitroblue tetrazolium test and flow cytometry DHR analysis. Infectious microorganisms were isolated from infected sites to identify the causative agents and their resistance profile. A total of 15 patients were diagnosed with CGD. Failure to thrive and lymphadenopathy were the most common presentations. The median age of clinical onset was 1.17 years of age. The most common gene mutations were observed in the CYBA gene. All cases showed pulmonary infections followed by abscesses. Staphylococcus aureus and Klebsiella pneumoniae were the most frequently isolated bacterial pathogens, Aspergillus spp and Candida spp were isolated from fungal infections. 4/15 (26.7%) children died due to severe serious infections. We concluded that CGD is common in Upper Egypt, and we recommend raising the awareness and testing for CGD in pediatric patients with recurrent or persistent infections, especially those with a familiar history of similar manifestations to avoid delays in proper diagnosis and deterioration of cases. Abbreviations: CGD: chronic granulomatous disease; XL: X-linked; AR: autosomal recessive

Inherited p40phox deficiency differs from classic chronic granulomatous disease.

Biallelic loss-of-function (LOF) mutations of the NCF4 gene, encoding the p40phox subunit of the phagocyte NADPH oxidase, have been described in only 1 patient. We report on 24 p40phox-deficient patients from 12 additional families in 8 countries. These patients display 8 different in-frame or out-of-frame mutations of NCF4 that are homozygous in 11 of the families and compound heterozygous in another. When overexpressed in NB4 neutrophil-like cells and EBV-transformed B cells in vitro, the mutant alleles were found to be LOF, with the exception of the p.R58C and c.120_134del alleles, which were hypomorphic. Particle-induced NADPH oxidase activity was severely impaired in the patients' neutrophils, whereas PMA-induced dihydrorhodamine-1,2,3 (DHR) oxidation, which is widely used as a diagnostic test for chronic granulomatous disease (CGD), was normal or mildly impaired in the patients. Moreover, the NADPH oxidase activity of EBV-transformed B cells was also severely impaired, whereas that of mononuclear phagocytes was normal. Finally, the killing of Candida albicans and Aspergillus fumigatus hyphae by neutrophils was conserved in these patients, unlike in patients with CGD. The patients suffer from hyperinflammation and peripheral infections, but they do not have any of the invasive bacterial or fungal infections seen in CGD. Inherited p40phox deficiency underlies a distinctive condition, resembling a mild, atypical form of CGD.

Purulent Pericardial Effusion in a 14-Year-Old Girl

Purulent Pericardial Effusion in a 14-Year-Old Girl

Chronic Granulomatous Disease (CGD) and Complete Myeloperoxidase Deficiency Both Yield Strongly Reduced Dihydrorhodamine 123 Test Signals but Can Be Easily Discerned in Routine Testing for CGD

The flow cytometric dihydrorhodamine 123 (DHR) assay is used as a screening test for chronic granulomatous disease (CGD), but complete myeloperoxidase (MPO) deficiency can also lead to a strongly decreased DHR signal. Our aim was to devise simple laboratory methods to differentiate MPO deficiency (false positive for CGD) and NADPH oxidase abnormalities (true CGD). We measured NADPH-oxidase and MPO activity in neutrophils from MPO-deficient patients, CGD patients, NADPH-oxidase-transfected K562 cells and cells with inhibited and substituted MPO. Eosinophils from MPO-deficient individuals retain eosinophilic peroxidase and therefore generate a normal DHR signal. The addition of recombinant human MPO enhances the DHR signal when simply added to a suspension of MPO-deficient cells but not when added to NADPH-oxidase-deficient (CGD) cells. Lucigenin-enhanced chemiluminescence (LCL) is increased in neutrophils from MPO-deficient patients, whereas neutrophils from patients with CGD show a decreased response. A false-positive result caused by MPO deficiency can be easily ascertained because, unlike cells from a CGD patient, cells from MPO-deficient patients (a) contain functionally normal eosinophils, (b) show a significant enhancement of the DHR signal following addition of rhMPO, and (c) generate a strong LCL signal.

DISSEMINATED DEEP TISSUE INFECTION WITH DRAINING SINUS TRACTS IN A HEALTHY SEVEN-YEAR-OLD

An apparently healthy 7-year-old Haitian boy with purulent draining sinuses of the left thoracic paravertebral area was admitted to the hospital for evaluation. Three days before admission a small nodule was noticed on the left side of his back which progressed to a larger tender red mass which drained pus on the day of admission. There was no fever, cough, arthralgia, weight loss or back pain. There was no history of insect bite, trauma or travel. The patient had had a minor fall on his back 3 months earlier, but no serious injury was reported. The child had dental work done 4 to 5 months before presentation, details of which could not be obtained. Physical examination revealed an active thin, pale appearing 7-year-old with no fever. Three distinct erythematous fluctuant tender masses each measuring approximately 3 by 4, 4 by 3 and 2 by 3 cm were present along the left paravertebral area at T7 to T8 which extended to intercostal spaces. Moderate tenderness was elicited on palpation of this area. The thoracic and lumbar spine did not have any bony deformity. Thoracic bony structures were normal. There were several palpable axillary lymph nodes about 1 to 2 cm in size bilaterally. The remainder of the physical examination was normal. Laboratory studies revealed a white blood cell count of 13 600/mm3 with 84% neutrophils, 10% lymphocytes, 5% monocytes and 1% eosinophils. Hemoglobin was 7 g/dl with a hematocrit of 22% and mean corpuscular volume of 65 fl. Platelets were 554 000/mm3. Serum iron was less than 5 μg/ml with total iron-binding capacity of 155 μg/ml and ferritin concentration of 135 mg/ml. The erythrocyte sedimentation rate was 136 mm/h. The serum electrolytes were as follows: sodium, 133 mmol/l; potassium, 5.2 mmol/l; chlorine, 95 mmol/l; CO2, 3 mmol/l; blood urea nitrogen, 11 mg/dl; creatinine, 0.4 mg/dl. Serum albumin was 2.7 g/dl with total protein of 10.2 g/dl. Serum glucose was 71 mg/dl with aspartate aminotransferase of 19 units/l. Urinalysis was normal. The immunologic workup included a nonreactive HIV antibody test by enzyme-linked immunosorbent assay. Absolute CD4 cell count was 632/mm3 with CD4% of 29 and absolute CD8 of 670/mm3 with CD8% of 30. IgG was 5379 mg/dl, IgA was 514 mg/dl, IgM was 173 mg/dl and IgE was 196 mg/dl. Nitroblue tetrazolium test for chronic granulomatous disease was normal. Leukocyte adhesion deficiency was ruled out because monoclonal antibodies against leukocyte function antigen of the CD18 integrin family showed presence of these adhesion markers. A purified protein derivative skin test was negative. Echocardiogram revealed no abnormalities. A chest roentgenogram was normal. Roentgenogram examination of the spine and ribs showed no bony involvement. Computerized tomographic scan of the chest and abdomen with and without contrast revealed prominence of the left paraspinal soft tissue, extending from the lower thoracic spine to the level of L2 vertebra. A discrete fluid collection with associated air bubble was seen in the left paraspinal region, suggesting a paraspinal abscess (Fig. 1). Multiple defects were noted within the spleen; five to six were ring-enhanced measuring 0.5 to 1 cm, suggesting the presence of multiple microabscesses. In addition the patient had a wedge-shaped low attenuation area within the left kidney and nonspecific thickening of the left psoas muscle and posterior pararenal fascia. Multiple tiny nodules were noted within the right upper lobe of the lung, with two prominent nodules measuring 0.4 to 0.75 cm, respectively. Computerized tomography of the head was normal. Dental evaluation during hospitalization showed normal gums.Fig. 1: Computerized tomography scan of the patient.Incision and drainage of the abscesses was performed, and the specimens were sent for Gram stain, bacterial, fungal and acid-fast bacillus cultures. Histopathology of the abscess cavities revealed necrotic inflamed granulating tissue. The results of the culture revealed the etiologic agent.

A rapid, specific assay for superoxide release from phagocytes in small volumes of whole blood.

A rapid, specific assay was developed to measure the release of superoxide anion from stimulated phagocytic cells in small volumes of whole blood. The assay is based on the chemiluminescence that results when lucigenin (bis-N-methyl acridinium nitrate) is reduced by superoxide. Heparinized whole blood, at volumes from 0.2 mL to a single drop from a 20-gauge needle, was mixed with lucigenin and either a soluble or particulate stimulus (phorbol myristate acetate or opsonized zymosan particles, respectively) in a standard volume of 1 mL. The chemiluminescence was measured at 3-minute intervals for a 30-minute period in a luminometer capable of automated operation. Characteristic plots of chemiluminescence versus time were obtained. This assay is rapid and simple, obviates the need to isolate leukocytes from whole blood, is specific for superoxide, can be performed with both soluble and particulate stimuli, and is sensitive enough to detect characteristic responses with as little as a single drop of blood. Patients with chronic granulomatous disease were readily identified using this assay. The ability to use very small blood volumes makes the assay suitable for use in prenatal diagnosis. The main disadvantage of this assay compared to the often-employed slide nitroblue tetrazolium test, with respect to testing for chronic granulomatous disease, is that carriers of x-linked chronic granulomatous disease are not readily detected with this chemiluminescence assay, as they are with the nitroblue tetrazolium test.

Staining of Eosinophils with Nitroblue Tetrazolium in Patients with Chronic Granulomatous Disease

The nitroblue tetrazolium (NBT) test is the primary screening test for chronic granulomatous disease (CGD). Neutrophils and other phagocytic cells readily reduce NBT to blue formazan after oxidative stimulation whereas CGD cells remain colorless. In the present study purified eosinophil populations were obtained from CGD patients and normals by exposing peripheral blood to the peptide N-formyl-methionyl-leucyl-phenylalanine and then centrifuged over a discontinuous Percoll gradient. The eosinophils were then incubated in 0.1% NBT (37 degrees C, 15 min) with either phorbol myristate acetate or buffer alone (HEPES with calcium and magnesium). Blue staining characteristic of NBT reduction occurred in the phorbol myristate acetate-stimulated eosinophils of half (4/8) of the CGD patients tested. The staining was most intense between the nuclear lobes of the eosinophil with little staining near the cell periphery. The staining pattern was present in 75.5 +/- 5.3% of the purified eosinophils in those patients in which the phenomenon occurred and was reproducible in the same patients over a 6-month period. Eosinophils from normal individuals tested with NBT and phorbol myristate acetate showed intense staining over the entire cell cytoplasm as did normal neutrophils. Purified CGD eosinophils that did show the staining pattern were not able to produce superoxide (as measured by cytochrome C reduction) when stimulated by phorbol myristate acetate indicating the staining was probably not related to superoxide production. Patients with CGD have a mild eosinophilia (4.6 +/- 0.7% in 11 patients at the National Institutes of Health) and eosinophil staining may account for the small number of positive NBT cells reported in some patients.

A radiometric whole blood test for chronic granulomatous disease

Chronic granulomatous disease (CGD), caused by an inherited deficiency in the oxygen metabolism of granulocytes and monocytes, is often a diagnostic problem in patients with severe infections. CGD can be diagnosed by several methods, all requiring some knowledge of cell handling and specialized equipment. We describe a simple screening test for CGD, based on the production of 14CO2 from glucose-1-14C following stimulation of the granulocytes in whole blood with zymosan, CV: 14.8%, intraassay. The test can be carried out with equipment available in most routine laboratories and without expertise in granulocyte function tests.

A simple screening test for chronic granulomatous disease.

Leukocytes from 42 normal subjects were consistently stimulated to reduce nitroblue-tetrazolium (NBT) when incubated with heparin in a concentration of 100 units per ml. of whole blood. The same concentration of heparin did not cause NBT reduction by leukocytes from three patients with chronic granulomatous disease. Heparin-stimulated NBT reduction may prove useful as a simple screening procedure for chronic granulomatous disease.

14 C-glucose oxidation in whole blood: a clinical assay for phagocyte dysfunction.

A screening test for chronic granulomatous disease is described; it is based on abnormal oxidation of glucose-1-(14)C to (14)CO(2) during phagocytosis by leukocytes in whole blood.

Oxygen consumption during the initial stage of human leucocyte phagocytosis as a test for chronic granulomatous disease.

It has been demonstrated that an oxygraph technique can be used as a test for chronic granulomatous disease.