Dihydrorhodamine Flow Research Articles

An Infant with Cervical Lymphadenitis Secondary to Nontuberculous Mycobacteria Refractory to Conventional Treatment, Responsive to Interferon-Gamma.

To the Editors: The interferon gamma/interleukin-12 positive feedback loop is critical for immunity against nontuberculous mycobacterium and mycobacterium tuberculosis.1 The use of IFN-γ as an effective treatment for these infections has been poorly described, particularly in the pediatric population. Diniz et al2 and Alangari et al3 describe cases of individuals with an underlying impairment in the interferon gamma/interleukin-12 receptor pathway with nontuberculous mycobacterial infections that were responsive to IFN- γ as an adjunctive treatment to their regimen. Our case presents an infant with lymphadenitis secondary to a nontuberculous mycobacterium that was refractory to conventional treatment and responsive to adjunctive IFN-γ. It is suspected that our infant’s response was due to an impairment in the IL-12/IFN-γ receptor signaling pathway. A previously healthy 9-month-old male presented to his pediatrician with an inflamed cervical lymph node. Ultrasonography revealed a complex cystic lesion. He completed a 10-day cefdinir course. Despite antibiotic treatment, the patient showed progression of the lesions and presented to the emergency department. Workup yielded elevated levels of inflammatory markers, with a white blood cell of 25 (ref. 5.5–17.0 k/µL) and a lymphocytic predominance of 61%. Computed tomography imaging revealed 2 possible retropharyngeal abscesses. The patient was started on clindamycin. Incision and drainage of the lesions were completed, and no growth was observed in tissue cultures. Aspirate culture of the retropharyngeal abscess showed 2+ Haemophilus parainfluenzae. Purified protein derivative was weakly positive at 7 mm of induration. He was discharged with amoxicillin, clarithromycin, and rifampicin. Ten days later, the patient’s physical examination was significant for 3 distinct areas of fistulation on the left side of the neck with no active drainage or tenderness (Fig. 1). Immunologic evaluation showed serum immunoglobulins within the age-appropriate reference range, adequate percentage stimulation and median fluorescence intensity of granulocytes on dihydrorhodamine flow with phorbol myristate acetate stimulation and inappropriate titers to Streptococcus pneumoniae with 1/23 ≥1.3, however, he had pending repeat doses as per the U.S. Centers for Disease Control and Prevention schedule. Immunoglobulin and lymphocyte subsets were within the reference range for age. Pathogenic variants impairing IFN-γ/IL-2 receptor signaling include AR IRF8 LOF, AD STAT1 LOF, AR STAT1 LOF, AR IFN-γR1 LOF, AR IFN-γR2 LOF, AR IKBK2 LOF, AR IL-12Rβ1 LOF, AR IRF8 LOF or AR TYK2 LOF. An X-linked pathology affecting IKBKG could not be excluded but was less likely given the absence of typical associated physical features. This does not rule out the presence of pseudogenes that can lead to a range of symptoms associated with NFkB essential modulator operon deficiency.FIGURE 1.: Demonstrating 3 distinct areas of fistulation on the left side of the neck with no active drainage.Given the progression of his lesions while on guideline-directed therapy, off-label use of subcutaneous IFN-γ treatment (Actimmune) was initiated every Mon, Wed, and Fri. (15 mcg/kg). The patient tolerated the medication without adverse effects. One month after initiating treatment, the patient showed significant improvement in both swelling and appetite, with his weight percentile returning to the previous baseline. Our case, in combination with other studies on the topic, shows great promise in utilizing IFN-γ as an adjunctive treatment for patients with mycobacterial infections that are refractory to conventional antimycobacterial treatments, with few adverse effects.

NAPDH Oxidase-Specific Flow Cytometry Allows for Rapid Genetic Triage and Classification of Novel Variants in Chronic Granulomatous Disease.

Chronic granulomatous disease (CGD) is an innate immune deficiency, primarily affecting the phagocytic compartment, and presenting with a diverse phenotypic spectrum ranging from severe childhood infections to monogenic inflammatory bowel disease. Dihydrorhodamine (DHR) flow cytometry is the standard diagnostic test for CGD, and correlates with NADPH oxidase activity. While there may be genotype correlation with the DHR flow pattern in some patients, in several others, there is no correlation. In such patients, assessment by flow cytometric evaluation of NADPH oxidase-specific (NOX) proteins provides a convenient and rapid means of genetic triage, though immunoblotting has long been used for this purpose. We describe the clinical utility of the NOX flow cytometry assay through assessment of X-linked and autosomal recessive CGD patients and their first-degree relatives. The assessment of specific NOX proteins was correlated with overall NADPH oxidase function (DHR flow), clinical phenotype and genotype. NOX-specific protein assessment is a valuable adjunct to DHR assessment and genotyping to classify and characterize CGD patients. The atypical clinical presentation of some CGD patients can make genotype-phenotype correlation with DHR flow data challenging. Genetic testing, while useful for confirmation of diagnosis, can take several weeks, and in some patients does not provide a conclusive answer. However, NADPH-oxidase-specific protein flow assessment offers a rapid alternative to identification of the underlying genetic defect in cellular subsets, and can be utilized as a reflex test to an abnormal DHR flow. Further, it can provide insight into correlation between oxidative burst relative to protein expression in granulocytes and monocytes.

Newly diagnosed chronic granulomatous disease in a 44 year old male presenting with recurrent groin cellulitis and colitis

Background Chronic Granulomatous Disease (CGD) is an inherited deficiency which results from the absence or dysfunction of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunits in phagocytic cells. This condition is usually diagnosed in early childhood and presents with recurrent infections at epithelial surfaces such as the skin, lungs, and gut. We present a case of CGD which was diagnosed late in adulthood. Case presentation A forty-four year old male was admitted to hospital with fever, testicular pain and an acute onset of swelling of his penis and scrotum. His past history was significant for Crohn’s disease diagnosed at age sixteen; stable on treatment with mesalamine (Asacol W ). He also had recurrent soft tissue infections of his penis and/or scrotum since childhood, approximately 2 or 3 in total, to his recollection. There was no history of abscesses or sexually transmitted infections. However, the patient did complain of frequent upper respiratory tract infections each winter. He had no family history of primary immunodeficiency disease. During admission, he was assessed by urology and general surgery. Both confirmed the absence of any anatomic abnormalities. An immunologist suggested work up for immunodeficiency. Basic laboratory investigations were normal, with a white blood cell count of 7.8 × 10 9 L. C-reactive protein was elevated at 69.7 mg/L. Both his rheumatoid factor and HIV testing were negative. Serum levels of IgA, IgM and IgG levels were all within normal limits (Table 1 reports full details of his immunology investigations) Blood cultures were negative. A CT scan of the chest was normal. CT scan of the abdomen and pelvis revealed no subcutaneous air to suggest a necrotizing infection. Quantitative dihydrorhodamine (DHR)123 flow cytometry assay revealed the complete absence of an oxidative burst response; this was confirmed three times. This was suggestive of a neutrophil NADPH oxidase deficiency associated with CGD. The patient was treated with intravenous cefazolin and metronidazole and discharged home on clindamycin. His cellulitis resolved within weeks, but he experienced a recurrence approximately 6 months later. He was started on trimethoprim-sulfamethoxazole (TMP-SMX), amoxicillin, and itraconazole as prophylactic therapy to prevent future infections. He was assessed by the Infectious Disease service, who simplified his prophylactic regimen to TMP-SMX 160/800 q Mon/Wed/Fri and Penicillin V K 300mg OD based on risk assessment and previous infective patterns. Interferon-gamma was considered, but based on restricted availability at the time and his relatively stable clinical course to date, we elected to pursue antibiotic prophylaxis and active surveillance alone. Genetic testing, performed by the Hospital for Sick Children in Toronto, confirmed a diagnosis of CGD and revealed a homozygous mutation in the NCFI gene (P47). It was recommended that directly family members receive DHR flow cytometry screening but this was declined.