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Year : 2018  |  Volume : 61  |  Issue : 4  |  Page : 557-560
Chronic granulomatous disease presenting with small bone osteomyelitis in a young child: A case report


1 Hematopathology and Immunohematology, Anand Diagnostic Laboratory, Bengaluru, Karnataka, India
2 Department of Pediatrics, Motherhood Hospital, Bengaluru, Karnataka, India
3 Department of Pediatric Immunology, Aster CMI Hospital, Bengaluru, Karnataka, India

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Date of Web Publication10-Oct-2018
 

   Abstract 


Chronic granulomatous disease (CGD) is a life threatening inherited disorder with varied clinical presentations often characterized by recurrent bacterial and fungal infections along with widespread granulomatous tissue response. The disease results from phagocytic defects characterized by deficiencies in oxidative burst of neutrophils. Nitroblue tetrazolium reduction test (NBT) and Dihydrorhodamine (DHR) with PMA stimulation by flow cytometry are quick, simple, sensitive and specific laboratory tests that help establish early and reliable diagnosis of CGD with an overall improvement in survival and disease prognosis. We report a case of 2-year old child who presented with small bone osteomyelitis involving bilateral feet and was later diagnosed to have autosomal recessive CGD due to mutation in NCF1 gene.

Keywords: Chronic granulomatous disease, dihydrorhodamine, nitroblue tetrazolium

How to cite this article:
Chari PS, Chandra P, Prasad S, Bhattad S. Chronic granulomatous disease presenting with small bone osteomyelitis in a young child: A case report. Indian J Pathol Microbiol 2018;61:557-60

How to cite this URL:
Chari PS, Chandra P, Prasad S, Bhattad S. Chronic granulomatous disease presenting with small bone osteomyelitis in a young child: A case report. Indian J Pathol Microbiol [serial online] 2018 [cited 2018 Dec 15];61:557-60. Available from: http://www.ijpmonline.org/text.asp?2018/61/4/557/242982





   Introduction Top


Chronic granulomatous disease (CGD) is a life-threatening inherited disorder with varied clinical presentations often characterized by recurrent bacterial and fungal infections along with widespread granulomatous tissue response. The disease results from phagocytic defects characterized by deficiencies in oxidative burst of neutrophils.[1] It is associated with high fatality, especially where there are delays in diagnosis and treatment. Nitroblue tetrazolium (NBT) reduction test and dihydrorhodamine (DHR) with phorbol 12-myristate 13-acetate (PMA) stimulation by flow cytometry are quick, simple, sensitive, and specific laboratory tests that help establish early and reliable diagnosis of CGD with an overall improvement in survival and disease prognosis.


   Case Report Top


A 2-year-old male child who was born to a third-degree consanguineously married couple presented with fever and right foot swelling at 9 months of age. A week later, he was noted to have swelling of the left foot. He was diagnosed to have osteomyelitis affecting metatarsal bones of both the feet [Figure 1]. He received intravenous antimicrobials for 1 month but continued to remain febrile and symptomatic with persistence of swelling of the feet. Antibiotic-impregnated pellets were inserted surgically into metatarsal bones. The symptoms resolved a month later.
Figure 1: Radiograph of the feet showing osteomyelitis involving the left first and right four metatarsal bones

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Between 12 and 15 months of age, he had repeated episodes of bloody diarrhea and received several courses of oral antimicrobials. At 22 months of age, he developed bilateral cervical suppurative lymphadenitis. Despite being treated with oral antimicrobials for 1 month, he remained symptomatic. Incision and drainage was performed and the pus was drained. Culture of the pus yielded Staphylococcus aureus. The swelling eventually subsided after 8 weeks of antibiotics.

Laboratory workup showed persistent neutrophilic leukocytosis with total white cell counts ranging from 13 to 20 × 109/L and absolute neutrophil count of 11 × 109/L. There was persistent thrombocytosis with platelet counts of approximately 800 × 109/L. Erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) were also raised throughout the course of disease. Human Immunodeficiency virus ELISA test was negative.

In view of recurrent infections, incomplete and slow response to antibiotics, immunological tests were carried out. Serum immunoglobulins were as follows – IgG: 3231 mg/dl, IgM: 545 mg/dl, IgA: <40 mg/dl, and IgE: 386.80 IU/ml. The first three assays were done by turbidimetry and IgE by electrochemiluminescence.

NBT dye and DHR tests were performed and both the results were abnormal.

Nitroblue tetrazolium test

NBT dye (Sigma-Aldrich) was added to the patient's leukocyte rich-heparinized plasma as well as a healthy control sample, each without and with yeast. The test showed no formazan pigment formation with yeast stimulation in comparison to pigment formation noted in 80% of neutrophils in the control sample [Figure 2].
Figure 2: Nitroblue tetrazolium test with yeast stimulation. Control (left) – 80% of neutrophils in the sample show formation of blue-black formazan pigment. Patient (right) – None of the neutrophils in the sample show pigment formation

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DHR123 (Sigma-Aldrich) test was carried out. PMA (Sigma-Aldrich) was used as a stimulant. The hydrogen peroxide that is produced by the neutrophils oxidizes the dye, leading to the emission of fluorescence. Mean fluorescence intensity of the activated neutrophils correlates directly with superoxide production.[2] Quantifying the amount of fluorescence using mean fluorescence intensity after PMA stimulation is relevant and essential, and it can be correlated to the NADPH oxidase activity. Three tubes each for patient's sample and control heparinized whole venous blood were used as follows:

  • Blood
  • Blood + DHR (unstimulated)
  • Blood + DHR + PMA (stimulated).


The samples were acquired on flow cytometer, FC500 (Beckman Coulter), and at least 100,000 events were acquired for each tube. The results were analyzed using Beckman Coulter software (CXP version 2.2). Oxidative index (OI) of neutrophils for control as well as patient was calculated using the formula:



In the index case, the Mean Fluorescence Intensity MFI of unstimulated control sample was 3.78 and stimulated control sample was 499 with an OI of 132. The MFI of unstimulated patient's sample was 2.83 and stimulated patient's sample was 9.7 with an OI of 3.42. This clearly indicated an abnormality in neutrophil function in the case. The first tube (only blood) and lack of oxidative burst by lymphocytes served as internal negative control [Figure 3]. DHR test was also performed on the child's mother, and the result was found to be normal ruling out an X-linked pattern of inheritance.
Figure 3: Dihydrorhodamine test. (Left) Control: MFI of simulated sample – 499; oxidative index: 132. (Right) Patient: MFI of simulated sample: 9.7; oxidative index: 3.42

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Enumeration of T and B lymphocyte subsets and NK cells was done using monoclonal antibodies tagged with fluorochrome. CD3, CD4, CD8, CD19, CD 3−, and CD56 + were evaluated by flow cytometry, and the results obtained were within normal limits for age.

A homozygous nonsense variation in exon 7 of the NCF1 gene (chr7:74197874; G > A; depth: 33x) that results in a stop codon and premature truncation of the protein at codon 194 (p. Trp194Ter; ENST00000289473) was detected on next-generation sequencing. The child was thus diagnosed to have autosomal recessive CGD due to NCF1 gene mutation.

He was initiated on treatment with co-trimoxazole and itraconazole prophylaxis. At 4 months of follow-up, he continues to remain well, awaiting a stem cell transplant.


   Discussion Top


CGD is a genetically heterogeneous disease that was first described by Janeway et al. in 1954[3] and later became a well-characterized entity by Bridges et al. in 1959.[4] There is a paucity of data on the incidence of this disease in our country while the estimated prevalence in the western countries is 1 in 250,000.[5] Although the overall incidence of the disease is low, part of this may be attributed to lack of diagnosis of new cases. Lack of resourceful laboratories is one of the main reasons for this.

The disease results from mutation in one of five members of genes (gp91phox, p47phox, p22phox, p67phox, and p40phox) responsible for nicotinamide dinucleotide phosphate (NADPH) oxidase complex. The mutations result in complete to nearly absent NADPH oxidase activity.[6],[7] The neutrophils in these individuals are unable to assemble the NADPH complex which leads to lack of formation of superoxide anions (O2) and reactive oxygen species such as hydrogen peroxide (H2O2). This, in turn, leads to inability of the phagocytes to kill pathogens, thereby leading to recurrent episodes of infection.[1],[8],[9]

The disease most often presents in toddlers and children. The frequent sites of infection are lung, skin, lymph nodes, and liver. Majority of the infections are due to S. aureus followed by Pseudomonas, Klebsiella, and Salmonella. The disease often takes a fatal course following fungal infections.[1],[10]

Small bone osteomyelitis with constellation of persistently elevated total white blood cell count, platelet count, ESR, and CRP raised a strong clinical suspicion of CGD and urged us to investigate further in those lines. Although low IgG levels are expected in primary immunodeficiency, CGD often presents with a very high IgG as was noted in the index case.[11],[12]

A child with suspected CGD can be screened with tests such as NBT and DHR while genetic studies are confirmatory.

NBT reduction test is a simple test that measures the neutrophil function. The test involves in vitro stimulation of the phagocytic system using yeast and NBT. When plasma from a healthy individual is incubated with yeast and NBT, the normal neutrophils show intracytoplasmic deposits of formazan pigment when examined microscopically. The number of NBT-positive cells is markedly decreased to absent in cases of CGD. This is attributed to the deficient oxidative burst in the neutrophils of CGD patients. The test is useful in screening cases with suspected CGD. It is inexpensive and is very useful in clinical setups with poor resources. It is, however, laborious with high interobserver variability and lacks sensitivity and specificity.[9] Our case showed a complete absence of formazan pigment with yeast stimulation, clearly indicating abnormal neutrophil function. Confirmatory test by DHR was carried out.

DHR with PMA stimulation is a flow-based assay which is analytically more sensitive than NBT. It is the test of choice for diagnosis of CGD. When the test is properly standardized, it is less laborious and provides objectivity in test analysis. It has an added advantage of being able to distinguish X-linked and autosomal forms of CGD. Furthermore, mosaic forms consisting of two distinct populations of NADPH oxidase-positive and NADPH oxidase-negative cells that is seen in female carriers of X-linked CGD.[13],[14],[15] The child's result showed an expectedly abnormal neutrophil OI, confirming the clinical diagnosis of CGD.

Morbidity and mortality of the CGD largely depends on the time of initial diagnosis. Detailed clinical examination along with specific laboratory investigations becomes crucial in establishing a timely diagnosis and makes early treatment interventions.


   Conclusion Top


We present a 2-year-old boy with CGD who presented small bone osteomyelitis and suppurative lymphadenitis. NBT and DHR are screening tools of choice, and these tests can easily be established at most of the standard laboratories. Better awareness of primary immune deficiencies among clinicians and judicious application of these tests would lead to timely diagnosis of this disease.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
   References Top

1.
Rezaei N. Primary immunodeficiency diseases. In: Phagocytic Defects. 2nd ed. Springer, 2017. p. 245-94.  Back to cited text no. 1
    
2.
Kuhns DB, Alvord WG, Heller T, Feld JJ, Pike KM, Marciano BE, et al. Residual NADPH oxidase and survival in chronic granulomatous disease. N Engl J Med 2010;363:2600-10.  Back to cited text no. 2
    
3.
Janeway CA, Craig J, Davidson M, Downey W, Gitlin D, Sullivan JC. Hypergammaglobulinemia associated with severe, recurrent and chronic non-specific infection. Am J Dis Child 1954; 88: 388-92  Back to cited text no. 3
    
4.
Bridges RA, Berendes H, Good RA. A fatal granulomatous disease of childhood; the clinical, pathological, and laboratory features of a new syndrome. AMA J Dis Child 1959;97:387-408.  Back to cited text no. 4
    
5.
Winkelstein JA, Marino MC, Johnston RB Jr., Boyle J, Curnutte J, Gallin JI, et al. Chronic granulomatous disease. Report on a national registry of 368 patients. Medicine (Baltimore) 2000;79:155-69.  Back to cited text no. 5
    
6.
Roos D, de Boer M. Molecular diagnosis of chronic granulomatous disease. Clin Exp Immunol 2014;175:139-49.  Back to cited text no. 6
    
7.
Vowells SJ, Fleisher TA, Sekhsaria S, Alling DW, Maguire TE, Malech HL, et al. Genotype-dependent variability in flow cytometric evaluation of reduced nicotinamide adenine dinucleotide phosphate oxidase function in patients with chronic granulomatous disease. J Pediatr 1996;128:104-7.  Back to cited text no. 7
    
8.
Afzal MM, Jeshtadi A, Mohmmed AK. Study of neutrophilic function by nitroblue tetrazolium test in septicemias and immunodeficiency diseases. Int J Res Health Sci 2014;2:581-90.  Back to cited text no. 8
    
9.
Golightly C, Candace Golightly, Melis McHenry, Peter Racanelli. Neutrophil Oxidative Burst Assay: A Dihydrorhodamine (DHR) based testing of Chronic Granulomatous Disease (CGD) with CytoFlex Flow Cytometer; 2011.  Back to cited text no. 9
    
10.
Madkaikar M, Mishra A, Ghosh K. Diagnostic approach to primary immunodeficiency disorders. Indian Pediatr 2013;50:579-86.  Back to cited text no. 10
    
11.
Al-Herz W, Bousfiha A, Casanova JL, Chatila T, Conley ME, Cunningham-Rundles C, et al. Primary immunodeficiency diseases: An update on the classification from the international union of immunological societies expert committee for primary immunodeficiency. Front Immunol 2014;5:162.  Back to cited text no. 11
    
12.
Leiding JW, Holland SM. Chronic Granulomatous Disease; 9 August 2012. In: Pagon RA, Adam MP, Ardinger H, Pagon RA, Wallace SE, Bean LJH, Stephens K, Amemiya A, et al., editors. GeneReviews®. Seattle (WA): University of Washington, Seattle; 1993-2017. Available from: https://www.ncbi.nlm.nih.gov/pubmed/22876374. [Last updated on 2016 Feb 11].  Back to cited text no. 12
    
13.
Emmendörffer A, Nakamura M, Rothe G, Spiekermann K, Lohmann-Matthes ML, Roesler J, et al. Evaluation of flow cytometric methods for diagnosis of chronic granulomatous disease variants under routine laboratory conditions. Cytometry 1994;18:147-55.  Back to cited text no. 13
    
14.
Abraham RS, Aubert G. Flow cytometry, a versatile tool for diagnosis and monitoring of primary immunodeficiencies. Clin Vaccine Immunol 2016;23:254-71.  Back to cited text no. 14
    
15.
Rawat A, Singh S, Suri D, Gupta A, Saikia B, Minz RW, et al. Chronic granulomatous disease: Two decades of experience from a tertiary care centre in North West India. J Clin Immunol 2014;34:58-67.  Back to cited text no. 15
    

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Correspondence Address:
Preethi S Chari
54, Bowring Tower, Bowring Hospital Road, Shivajinagar, Bengaluru - 560 001, Karnataka
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/IJPM.IJPM_458_17

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