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Chronic granulomatous disease
Clinical Microbiology Newsletter August 1,1995
Vol. 17, No. 15
Chronic Granulomatous Joseph B. Domachowske, M.D. Pediatric Infectious Disease SUNY HeaJth Science Center at Syracuse Syracuse, NY 13210
when activated, normal neutrophils pro-
duce large amounts of superoxide, hydrogen peroxide, and other microbicidal oxidants. Activation of the NADPH oxidase requires assembly of an electron transport chain at the plasma membrane ChtNEU 17(15)113-120.1995
Disease or the phagolysosomal membrane. The identification and characterization of the minimal protein components required for the production of superoxide has recently been described (1). This involves the interaction of three cytosolic proteins: p47phox (“ph’*agocyte “ox”idase), p67phox, and rat-2 along with the membrane-bound flavocytochrome b558, which is made up of two peptides, gp9lphox, and p22phox. In the presence of an appropriate stimulus, assembly of the electron transport chain occurs. One of the early events in the activation of NADPH oxidase is the phosphorylation of p47phox (2,3). Once phosphorylated, p47phox. cytoplasmic p67phox, and cytoplasmic rac2 hanslocate to the plasma membrane where they associate with flavocytochrome b558. Rac2 is a low molecular weight guanosine triphosphate (GTP) binding protein that is essential for oxidase activation (4,5). Its function may be to participate in the regulation of the assembly of the cytosolic components and/or their translocation to the cell membrane. At the cell membrane, the cytosolic factors interact with flavocytochrome b558 to form the active oxidase multiplex (6). Flavocytochrome b558 is a heterodimeric iron-containing heme protein consisting of a large transmembrane subunit, gp9 lphox and a small 22 kDa protein, p22phox (7). Recent work suggests that the gp9lphox component of the oxidase molecule binds FAD and NADPH (8). NADPH is the critical substrate for the oxidase and is produced in acEbcvier
tivated cells via the glycolytic pathway and hexose monophosphate shunt. NADPH serves as the electron donor to the electron transport chain, and the flavocytochrome serves as the terminal electron donor to molecular oxygen, which results in the formation of superoxide anion. At slightly acidic pH, superoxide rapidly reacts with water to form hydrogen peroxide, an event that is accelerated by the presence of superoxide dismutase, a ubiquitous cytoplasmic enzyme. Hydrogen peroxide by itself is a potent microbic&u oxidant, but in the presence of neutrophil myeloperoxidase and halide ion (chloride, bromide, or iodide), the hydrogen peroxide is converted to hypohalous acid. These acids, hypochlorous acid being the most abundant, are directly damaging to invading microbes and render the offending pathogens more susceptible to the myriad of nonoxidative antimicrobial agents released from the
In This Issue Chronic Granulomatous Disease.. . . . . . . . . . . . . . . . . . . . . .113 An update and overview of the clinical and laboratory aspects of this disease
Tubo-Ovarian Abscess and Peritoneal Effusion Caused by Arcanobacterium haemolyticutn.. . . . . . . . . , . . . . . . . 118 A case report
Erratum . . . . . . . . . . . . . . . . . . . ...120 01%4399/95/$0.00
+ 09so
Table 1. Types of CGD” and Associated Defects
Membrane associated abnormalities
Protein abnormality
Chromosomal localization
Percent of CGD cases
~91 phox p22 phox
xp21.1 16q24
57%
p47 phox
7q11.23 lq25
33% 5%
Cytosol associated abnormalities
p67 phox
5%
*CGD = chronic granulomatous disease.
neufrophilic
granules.
Hypochlorous
acid is also metabolized to highly cytotoxic chlorine, chloramines, and hydroxyl radicals (9). As would be expected, a defect in the ability to generate reactive oxygen intermediates places the host at a significant disadvantage when encountering microbes.
Chronic Granulomatous Disease Chronic granulomatous disease is a group of four inherited disorders that share a common phenotype. At the cellular level, the common link between these four entities is their inability to produce superoxide. The resultant effect on the patient is the development of recurrent serious infections and the formation of granulomas that have potential to enlarge sufficiently to obstruct the gastrointestinal or genitourinary tract (10). Each of the four genetic forms of the disease involves a defect in one of four “phox” gene products involved in the activation of the NADPH oxidase. The types of CGD and the proportion of patients represented by each defect is shown in Table 1 (11,12). There is some evidence from studies in Europe and Japan that the exact ratios of the different forms of CGD may vary in different ethnic groups. The disease is said to affect approximately one in a million individuals, but may be a bit more common than that. Nevertheless, CGD is the most common qualitative defect in neutrophils, resulting in severe immunodeficiency. The X-linked form of the disease is
the most common subtype and results from abnormalities of the gp9lphox component of cytochrome b558. The specific genetic defects identified have included missense mutations, point mutations, deletions, and splice site mutations (13-16). Plavocytochrome b558 is undetectable in cells from patients with nearly all the different gp9lphox genotypes; however, some of the point mutations and small balanced deletions can lead to production of normal amounts of protein that is functionally deficient. The second most common form of CGD results from abnormalities in the p47phox gene, which are inherited in an autosomal recessive manner. None of the patients with this subtype described to date has detectable p47phox protein in phagocytic cells. Similarly, but much less common, is the rare subtype of CGD caused by defective p67phox. Patients suffering from this entity completely lack detectable p67phox protein in their neutrophils. Point mutations and other abnormal gene arrangements have been identified in the fourth subtype of CGD, p22phox defects (17). Patients with this disease, as well as patients afflicted with abnormalities in the gp9 lphox component, fail to express flavocytochrome b558 peptide on their surface, even though the gene defect involves the gene encoding only one of the two subunits (18,19). This suggests that the production and/or membrane expression of either of the flavocytochrome peptides
relies on the normal production of its partner. Over 90% of patients with CGD have levels of superoxide production that are less than 1% of normal, whereas the remainder have levels ranging from 1 to 10% of normal. The few patients with detectable superoxide production, albeit far below normal, tend to suffer from less severe disease. It should be noted that a diminished respiratory burst has been reported from patients with severe glucose-6phosphate dehydrogenase (G6PD) deficiency (20). Neutrophils from such patients may fail to reduce nitroblue tetrazolium dye similar to what is classically demonstrated by cells from patients with CGD. NADPH is generated by the first two reactions of the hexose monophosphate shunt: glucose-dphosphate dehydrogenase (G6PD) and 6phosphogluconic dehydrogenase. Since G6PD is the fist enzyme in this pathway, its absence results in greatly diminished shunt activity and thus a severe decrease in the amount of available NADPH substrate. As would be expected, severe G6PD deficiency in neutrophils can result in a defective respiratory burst, with a clinical picture similar to that of CGD. Clinical Manifestations of CGD The clinical presentation of CGD can be quite variable. Typically, these patients develop serious infections early in life, often in their first year, though some may present with their index infection in adolescence or even young adulthood. Attesting to the heterogeneity of this group is the highly unusual case of a 69-yr-old man who was diagnosed with X-linked CGD after he developed Burkholderia (Pseudomonas) cepacia septicemia (21). Because of a family history significant for CGD, the diagnosis was entertained and confirmed. His neutrophils contained normal, albeit dysfunctional cytochrome b, and did generate small amounts of su-
NOTE: No responsibility is assumed by the Publisher for any injury and/or damage to persons or propaty BS a matter of products liability. negligence or otherwise, or from any use or operation of any methods, pmduds, instructions or ideas contained in the material herein. No suggested test or procedure should b-z carried out unless, in the reader’s judgment, its risk is justified. Because of rapid advances in medical sciences. we recommend that the independent verification of diagnoses and dmg dosages should be made. Discussions, views, and recommendations as to medical procedures, choice of drugs, and drug dosages are the responsibility of the authors. ClinicalMicrobiology News/e~fer (lSSN 1069417X) is issued twice monthly in one indexed volume per year by Elswier Science Inc., 655 Avenue of the Americas, New York, NY 10010. Subscription price per year: $175.00 including postage and handling in the United States, Canada. and Mexico. Add $59.00 for postage III the rest of the world. Second-class postage paid at New York. NY and at additional mailing offIces. Postmaster: Send addrss changes to C/in&/Microbiology Newslerter, Elsevin Science Inc.. 655 Avenue of the Americas, New York, NY 10010.
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Clinical Microbiology
Newsletter 17:15,1995
peroxide. Perhaps it was this ability to generate some superoxide that delayed the onset of the index infection. Typical infectious syndromes in patients with CGD include pneumonia, pulmonary abscesses, skin and soft tissue infections, suppumtive lymphadenitis, osteomyelitis, and hepatic abscesses. Such infectious complications are frequently associated with early mortality. Cellulitis and lymphadenitis sometimes respond to local care and antibiotic administration, but early intervention with parenteral antibiotics and/or surgical drainage is prudent if improvement is not demonstrated in a day or two. Deep tissue infections, including hepatic abscesses, pneumonia, and osteomyelitis, can be associated with metastatic infection at other remote sites, including the spleen and the brain. A low threshold for embarking on an extensive workup for areas of possible metastatic seeding even in the face of an obvious focus of infection must be maintained. Occasionally, signs and symptoms of infection may be limited to malaise, pain, or weight loss. Fever may be absent or intermittent. Neutrophilia is not necessarily present. An increase in the erythrocyte sedimentation rate from a previously known baseline value can be a valuable indicator of infection, and certainly a useful parameter to monitor during treatment. Patients with CGD arc particularly prone to infections by organisms that produce catalase. The catalase produced by the offending organism deprives the phagocyte of exogenous peroxide as an alternative microbicidal mechanism. There is also in vitro evidence that many of these organisms are less susceptible to oxygen-independent killing mechanisms, thus increasing their virulence in this disease (22). Staphylococcus aureus and members of the Enterobacteriaceae family are the most common bacterial pathogens. Salmonella spp., Serralia spp., and Escherichia co/i, in particular, have been recognized as being responsible for frequent and severe infections (23,24). Burkholderia cepacia, Nocardia spp., and Mycobacterium tuberculosis are also encountered with increased frequency in these patients. In contrast. organisms that produce hydrogen Clinical Microbiology
Newsletter 17 15, I W‘
peroxide but that are catalase negative, such as the streptococci and lactobacilli, are not major pathogens in CGD-afflitted individuals. Fungal infections also play a major role in the morbidity and mortality of these patients. Cohen et al. (25) reviewed the records of 245 individuals with CGD and found that 20% had a history of a fungal infection. Aspergillus spp. were responsible for 78% of all fungal infections including all cases of fungal osteomyelitis in this cohort. Mouy et al. (24) reviewed the infections of 48 individuals with CGD and reported Aspergillus spp. as second only to Staphylococcus aureus as a cause of serious infection. Clinically evident infection with Aspergillus spp. usually begins in the lung. Contiguous spread or dissemination may occur. Vertebral osteomyelitis in individuals with CGD is usually caused by aspergilli (26). Unfortunately, despite medical and surgical therapy of vertebral osteomyelitis secondary to Aspergillus spp. infection, reported cases have been associated with treatment failure. recurrence, and severe disabling orthopedic or neurologic complications or death. identification of a pathogen may require biopsy for histologic tests and culture. This is usually best combined with surgical drainage, debridement, or extirpation of nonviable tissue. Infection with more than one organism is not unusual, especially
in patients with pneumonia, in whom mixed infection with Aspergillus and Nocardia spp. and M. tuberculosis can occur. Histopathologic examination characteristically demonstrates mixed suppura-
tive and necrotizing granulomatous inflammation in skin, lymph nodes, lung, liver. gastrointestinal tract, and many other sites (27). Eventually, these lesions can enlarge, and by virtue of their size and number, can result in organ dysfunction due to mass effect and/or obstruction. Patients may present with symptoms of gastric outlet, esophageal, or urinary bladder obstruction. In
a subset of patients, the complications arising from the granulomas are more problematic than episodes of infection. The obstructive granulomata can result from infection but also occur in the absence of an apparent infectious process. 0 I‘li)’ I.liC\ IL?‘science I”‘
If no pathogen is identified, an empiric course of antibiotics is prudent. If the obstruction is unresponsive to antibiotics and is of recent onset, judicious use of low dose steroids may be beneficial in shrinking them and alleviating the obstructive symptoms (28).
More Reasons to Suspect CGD In addition to the classic presentations discussed above, there are a number of clinical situations in which CGD should be considered. A family history of unexplained deaths of infant or young males may hint at X-linked CGD. CGD should be part of the differential diagnosis in an patient with unexplained pneumonia caused by S. aureus, Aspergillus spp., B. cepacia, or Serraria spp. Recurrent deep tissue infection caused by staphylococci or any deep tissue infection caused by Serratia spp. is highly suspect. A single unexplained hepatic abscess in a child or osteomyelitis caused by any of these organisms should prompt testing for the possibility of CGD. Since CGD patients also have increased susceptibility to M. tuberculosis infection, a child from a population in which this infection is uncommon should be evaluated for possible CGD. Patients have been known to carry a diagnosis of inflammatory bowel disease with radiologic and/or histologic evidence that confirms the diagnosis by demonstrating granulomatous disease in the small or large bowel (29). A low threshold should be maintained for evaluating such patients for possible CGD, especially in atypical circumstances or in the clinical setting of recurrent infections that are otherwise unexplained.
Establishing the Diagnosis of CGD The laboratory diagnosis of CGD is based on the determination that patients’ neutrophils are unable to undergo an oxidative burst, a function that can be measured in a variety of ways. The nitroblue tetrazolium (NBT) dye reduction test is the method employed by most clinical laboratories. It is simple to perform in experienced hands, and provides both a qualitative and semiquantitative result. When neutrophils produce superoxide, the soluble yellow nitroblue tetrazolium is reduced to the in01%4399/95/$0.00
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soluble blue-black formazin that precipitates on and within activated cells. Simply visualizing the cells using light microscopy and counting the percentage that demonstrate reduction of NBT provides the results. Although this assay is generally easy to perform and interpret, problems arise from a lack of understanding about the test. Properly done, an NBT test must be performed in a timed fashion, the assay being completed in 15 to 20 min. Some CGD patients with trace to 5% production of superoxide may weakly reduce the NBT. This is not a problem in a timed assay, but the test may be read as normal if the assay is allowed to proceed for a long time. More comprehensive analysis of the respiratory burst and molecular genetic subtyping are currently available only in research laboratories and use a variety of specialized techniques. Some of these methods include direct measurement of superoxide production, detection of fluorescent substrate oxidation, cytochrome b spectroscopy, immunoblot analysis for ox&se components, and oxidase gene sequencing. The most useful secondary test is Westem immunoblotting of patient neutrophi1 cytosol and membrane fractions followed by detection with antibodies specific for each of the oxidase “phox” components. This, together with NBT testing of the patient and the patient’s mother, will reveal the genetic subtype in the great majority of cases. Knowledge of the genetic subtype can be helpful for genetic counseling of the family but does not change the clinical approach to the patients’ care. Management of Patients with CGD The mainstay for the management of these patients is aggressive antibiotic treatment of infectious complications in concert with early and aggressive surgical drainage of abscesses. White blood cell transfusions have also been used, particularly for serious infectious complications. Because infection can be very difficult to treat in the face of abnormal neutrophil function, prevention of infection should be the primary goal. The administration of prophylactic antibiotics, especially trimethoprim/sul116
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famethoxazole, significantly reduces the incidence of serious infections in patients with CGD (30). Dicloxacillin or another oral antibiotic with anti-staphylococcal activity is a suitable alternative if trimethoprim/sulfamethoxazole is not tolerated. Particular attention to oral hygiene can control the gingivitis and recurrent aphthous ulcers that often become problematic. Patients should be aware that barns, hay, freshly mown grass, and wood shavings all harbor Aspergillus and they should attempt to avoid such contact. The combined use of interferon-y and antibiotics probably provides the optimal prophylaxis against infection. Supporting evidence for this regimen comes from the results of a multicenter placebocontrolled trial in which patients received antibiotics and either interferon-y or placebo (3 1). The results of the study demonstrated a 67% reduction in serious infections suffered by patients who received both interferon-y and antibiotics. At the recommended interferon-y dosage (0.05 mg/b@ of body surface, 3 d a week), side effects are infrequent but include fever, malaise, and headache. The mechanism of the protective effect is not completely understood but may relate to partial amelioration of the defect in the phagocyte oxidative burst in a small subset of individuals (32). Based on these results, as well as in vitro supporting evidence, the U.S. Food and Drug Administration has licensed the use of interferon-y for patients with CGD. Additional administration of the antifungal agent itraconazole to this regimen may help curtail the onset of infection with Aspergillus spp., but results of clinical trials are not yet available to support its routine use. Despite efforts to prevent infection in these patients, complications occur. The general approach to the diagnosis and management of infections in these patients follows the same general rules that apply to diagnosis and management of all immunodeficient patients with signs and symptoms of an infection. Diligent pursuit of a microbiologic diagnosis by obtaining proper cultures is a must. It should be remembered that these patients are particularly prone to aspergillosis and other fungal infec-
tions, so appropriate cultures must be set up. Early recognition of infection, the acquisition of appropriate cultures, and early institution of adequate doses of parenteral antibiotics can reduce the morbidity and mortality in these patients. Surgical extirpation of infected tissue should be considered early on, especially if there is no improvement after a brief irial of medical management. Transfused granulocytes may also be considered as an adjunctive therapy. Ideally, the white cells should be ABO matched and HLA similar, obtained by centrifugation leukopheresis, and should not be irradiated, because this has an adverse effect on oxidase activity. There are no published studies clearly demonstrating enhanced efficacy of transfused granulocytes. CGD-The Potential for Gene Therapy Studies using retroviral vectors containing cDNA for p47phox, gp9lphox. or p22phox have demonstrated that it is possible to restore oxidase activity in cell lines or neutrophils derived from hematopoietic progenitor cells harvested from CGD patients (33-36). The cDNA encoding the normal oxidase component is inserted into the genomic DNA of hematopoietic stem cells obtained from a patient with that specific oxidase defect. These genetically engineered cells could then be returned to the patient with subsequent maturation into normal mature phagocytes. Even the correction of a small percentage of the patient’s cells may result in a clinical cure because cells from heterozygous mothers who carry the gene for the X-linked form of CGD but who are clinically normal, have been found to express as little as 3% functional oxidase activity. The in vitro evidence that gene therapy restores functional oxidase activity predicts that the clinical studies will soon follow. The era of gene therapy is on the horizon, not just for CGD but for a variety of genetic and acquire-d diseases. It is likely that the lessons learned in the early clinical trials will pave the way for gene therapy to enhance host defense in a variety of ways. The possibilities are limited only by our technical resources and our scientific curiosity. Clinical
Microbiology
Newsletter 17:15,1995
References 1.
2.
3.
4.
5.
6.
7.
8.
9.
Rotrosen, D., et al. 1992. Cytochrome b.558: the flavin binding component of the phagocyte NADPH oxidase. Science 256:1459-1462. Abo, A., et al. 1992. Reconstitution of neutrophil NADPH oxidase activity in the cell-free system by four cornponents: p67-phox, p47-phox, p2lrac1, and cytochrome b-245. J. Biol. Chem. 267:16767-16770. Rotrosen, D.. and T.L. Leto. 1990. Phosphorylation of a neutrophil47kD cytosol oxidase factor. Translocation to membrane is associated with distinct phosphotylation events. J. Biol. Chem. 265:1991&19915. Abo. A., et al. 1991. Activation of the NADPH ox&se involvea the small GTP binding protein p2lracl. Nature 353:668-670. Knaus, U.G., et al. 1991. Regulation of phagocyte oxygen radical production by the GTP-binding protein rac2. Science. 254:1512-1515. Kleinberg, M.E., H.L. Malech, and D. Rotrosen. 1990. The phagocyte 47 kilodalton cytosolic oxidase protein is an early reactant in the activation of the respiratory burst. J. Biol. Chem. 265:15577-15583. Parkos, C.A., et al. 1987. The purified cytochrome b from human granulocyte plasma membrane is composed of two polypeptides with relative molecular weights of 91,000 and 22,000. J. Clin. Invest. 80:732-742. Segal. A.W., et al. 1992. Cytochrome b245 is a flavocytochrome containing FAD and the NADPH-binding site of the microbicidal oxidase of phagocytes. Biochem. J. 284:781-788. Klebanoff. S.J. 1992. Oxygen metabclites from phagocytes, p. 541-588. In J-1. Gallin,I.M. Goldstein, and R. Snyderman (eds.). Inflammation: basic principles and clinical correlates, second edition. Raven Press, New York.
10. Gallin, J.I.. and H.L. Malech. 1990. Update on chronic granulomatous diseases of childhood. Immunotherapy and pctential for gene therapy. JAMA 263:153%1537. 11 Clark, R.A., et al. 1989. Genetic variants of chronic granulomatous disease: prevalence of deficiencies of two cytosolic components of the NADPH oxidase system. N. Engl. J. Med. 321647-652.
ClinicalMicrobiology Xewsle~rer 17:15.1991
12. Casimir, C., et al. 1992. Identification of the defective NADPH-oxidase component in chronic granulomatous disease: a study of 57 European families. Eur. J. Clin. Invest. 22:403-406. 13. Dinauer, M.C., et al. 1989. A missense mutation in the neutrophil cytochrome b heavy chain in cytochrome-positive X-linked chronic granulomatous disease. J. Clin. Invest. 84:2012-2016. 14. Bolscher, B.G., et al. 1991. Point mutations in the beta-subunit of cytochrome b558 leading to X-linked chronic granulomatous disease. Blood. 77:24822487. 15. Schapiro. B.L.. et al. 1991. Chronic granulomatous disease presenting in a 69 year old man. N. Engl. J. Med. 325:1786-1790. 16. De Boer, M., et al. 1992. Splice site mutations are a common cause of X-linked chronic granulomatous disease. Blood 80:1553-1558. 17. Dinauer, M.C., et al. 1990. Human neutrophil cytochrome b light chain (p22phox). Gene structure, chromosomal location, and mutations in cytochromenegative autosomal recessive chronic granulomatous disease. J. Clin. Invest. 86:1729-1737. 18. Segal, A.W. 1987. Absence of both cytochrome b245 subunits from neutrophils in X-linked chronic granulomatous disease. Nature 32688-91. 19. Parkas, C.A., et al. 1989. Absence of both the 91 kD and 22 kD subunits of human neutrophil cytochrome b in two genetic forms of chronic granulomatous disease. Blood 73:14161420. 20. Hopkins, P.J., L.S. Bemiller, and J.T. Cumutte. 1992. Chronic granulomatous disease: diagnosis and classification at the molecular level. Clin. Lab. Med. 12:277-304.
ity, and prevention of infections in chronic granulomatous disease. J. Pediatr. 114:555-560. 25. Cohen, M.S., et al. 1981. Fungal infection in chronic granulomatous disease. The importance of the phagocyte in defense against fungi. Am. J. Med. 71:5P66. 26. Sponseller, P.D., et al. 1991. Skeletal involvement in children who have chronic granulotnatous disease. J. Bone Joint Surg. 73: 37-5 1. 27. Baehner, R.L. 1990. Chronic granulomatous disease of childhood: clinical, pathological, biochemical, molecular, and genetic aspects of disease. Pediatr. Pathol. 10:143-153. 28. Chin, T.W., et al. 1987. Corticosteroids in treatment of obstructive lesions of chronic granulotnatous disease. J. Pediatr. 111:349-352. 29. Fisher, I.E., et al. 1990. Chronic granulomatous disease of childhood with acute ulcerative colitis: a unique association. Pediatr. Pathol. 7:91-96. 30. Margolis, D.M., et al. 1990. Trimethoprim-sulfamethoxazole prophylaxis in the management of chronic granulomatous disease. J. Infect. Dis. 162~72% 726. 31. The International Chronic Granulomatous Disease Cooperative Study Group. 199 1. A controlled trial of interferon gamma to prevent infection in chronic granulomatous disease. N. Engl. J. Med. 324:509-5 16. 32. Ezekowitz, R.A.B.. et al. 1988. Partial correction of the phagocyte defect in patients with X-lied chronic granulomatous disease by subcutaneous interferon gamma. N. Engl. J. Med. 319:14&151.
21. Schapiro, B., et al. 1991, Chronic granulomatous disease presenting in a 69 year old man. N. Engl. J. Med. 325:1786-1790.
33. Thrasher, A., et al. 1992. Restoration of superoxide generation to a chronic granulomatous disease-derived B-cell line by retrovirus mediated gene transfer. Blood 80:112>1129.
22. Odell, E.W., and A.W. Segal. 1991. Killing of pathogens associated with chronic granulomatous disease by the non-oxidative microbicidal mechanisms of human neutrophils. J. Med. Microbiol. 34:12!&35.
34. Cobbs, C.S., et al. 1992. Retroviral expression of recombinant p47phox protein by Epstein-Barr virus-transformed B lymphocytes from a patient with autosomal chronic granulomatous disease. Blood 79:1829-1835.
23. Lazarus, G.M., and H.C. Neu. 1975. Agents responsible for infections in chronic granulomatous disease of childhood. J. Pediatr. 86:415419.
35. Li, F., et al. 1994. CD34+ peripheral blood progenitors as a target for genetic correction of the flavocytochrome b558 defective forms of chronic granulomatous disease. Blood 84:5>58.
24. Mouy. R., et al. 1989. Incidence,
Q 19%
t-.lcev1erScmlce1nc
sever-
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117
Vol. 17, No. 15
Chronic Granulomatous Joseph B. Domachowske, M.D. Pediatric Infectious Disease SUNY HeaJth Science Center at Syracuse Syracuse, NY 13210
when activated, normal neutrophils pro-
duce large amounts of superoxide, hydrogen peroxide, and other microbicidal oxidants. Activation of the NADPH oxidase requires assembly of an electron transport chain at the plasma membrane ChtNEU 17(15)113-120.1995
Disease or the phagolysosomal membrane. The identification and characterization of the minimal protein components required for the production of superoxide has recently been described (1). This involves the interaction of three cytosolic proteins: p47phox (“ph’*agocyte “ox”idase), p67phox, and rat-2 along with the membrane-bound flavocytochrome b558, which is made up of two peptides, gp9lphox, and p22phox. In the presence of an appropriate stimulus, assembly of the electron transport chain occurs. One of the early events in the activation of NADPH oxidase is the phosphorylation of p47phox (2,3). Once phosphorylated, p47phox. cytoplasmic p67phox, and cytoplasmic rac2 hanslocate to the plasma membrane where they associate with flavocytochrome b558. Rac2 is a low molecular weight guanosine triphosphate (GTP) binding protein that is essential for oxidase activation (4,5). Its function may be to participate in the regulation of the assembly of the cytosolic components and/or their translocation to the cell membrane. At the cell membrane, the cytosolic factors interact with flavocytochrome b558 to form the active oxidase multiplex (6). Flavocytochrome b558 is a heterodimeric iron-containing heme protein consisting of a large transmembrane subunit, gp9 lphox and a small 22 kDa protein, p22phox (7). Recent work suggests that the gp9lphox component of the oxidase molecule binds FAD and NADPH (8). NADPH is the critical substrate for the oxidase and is produced in acEbcvier
tivated cells via the glycolytic pathway and hexose monophosphate shunt. NADPH serves as the electron donor to the electron transport chain, and the flavocytochrome serves as the terminal electron donor to molecular oxygen, which results in the formation of superoxide anion. At slightly acidic pH, superoxide rapidly reacts with water to form hydrogen peroxide, an event that is accelerated by the presence of superoxide dismutase, a ubiquitous cytoplasmic enzyme. Hydrogen peroxide by itself is a potent microbic&u oxidant, but in the presence of neutrophil myeloperoxidase and halide ion (chloride, bromide, or iodide), the hydrogen peroxide is converted to hypohalous acid. These acids, hypochlorous acid being the most abundant, are directly damaging to invading microbes and render the offending pathogens more susceptible to the myriad of nonoxidative antimicrobial agents released from the
In This Issue Chronic Granulomatous Disease.. . . . . . . . . . . . . . . . . . . . . .113 An update and overview of the clinical and laboratory aspects of this disease
Tubo-Ovarian Abscess and Peritoneal Effusion Caused by Arcanobacterium haemolyticutn.. . . . . . . . . , . . . . . . . 118 A case report
Erratum . . . . . . . . . . . . . . . . . . . ...120 01%4399/95/$0.00
+ 09so
Table 1. Types of CGD” and Associated Defects
Membrane associated abnormalities
Protein abnormality
Chromosomal localization
Percent of CGD cases
~91 phox p22 phox
xp21.1 16q24
57%
p47 phox
7q11.23 lq25
33% 5%
Cytosol associated abnormalities
p67 phox
5%
*CGD = chronic granulomatous disease.
neufrophilic
granules.
Hypochlorous
acid is also metabolized to highly cytotoxic chlorine, chloramines, and hydroxyl radicals (9). As would be expected, a defect in the ability to generate reactive oxygen intermediates places the host at a significant disadvantage when encountering microbes.
Chronic Granulomatous Disease Chronic granulomatous disease is a group of four inherited disorders that share a common phenotype. At the cellular level, the common link between these four entities is their inability to produce superoxide. The resultant effect on the patient is the development of recurrent serious infections and the formation of granulomas that have potential to enlarge sufficiently to obstruct the gastrointestinal or genitourinary tract (10). Each of the four genetic forms of the disease involves a defect in one of four “phox” gene products involved in the activation of the NADPH oxidase. The types of CGD and the proportion of patients represented by each defect is shown in Table 1 (11,12). There is some evidence from studies in Europe and Japan that the exact ratios of the different forms of CGD may vary in different ethnic groups. The disease is said to affect approximately one in a million individuals, but may be a bit more common than that. Nevertheless, CGD is the most common qualitative defect in neutrophils, resulting in severe immunodeficiency. The X-linked form of the disease is
the most common subtype and results from abnormalities of the gp9lphox component of cytochrome b558. The specific genetic defects identified have included missense mutations, point mutations, deletions, and splice site mutations (13-16). Plavocytochrome b558 is undetectable in cells from patients with nearly all the different gp9lphox genotypes; however, some of the point mutations and small balanced deletions can lead to production of normal amounts of protein that is functionally deficient. The second most common form of CGD results from abnormalities in the p47phox gene, which are inherited in an autosomal recessive manner. None of the patients with this subtype described to date has detectable p47phox protein in phagocytic cells. Similarly, but much less common, is the rare subtype of CGD caused by defective p67phox. Patients suffering from this entity completely lack detectable p67phox protein in their neutrophils. Point mutations and other abnormal gene arrangements have been identified in the fourth subtype of CGD, p22phox defects (17). Patients with this disease, as well as patients afflicted with abnormalities in the gp9 lphox component, fail to express flavocytochrome b558 peptide on their surface, even though the gene defect involves the gene encoding only one of the two subunits (18,19). This suggests that the production and/or membrane expression of either of the flavocytochrome peptides
relies on the normal production of its partner. Over 90% of patients with CGD have levels of superoxide production that are less than 1% of normal, whereas the remainder have levels ranging from 1 to 10% of normal. The few patients with detectable superoxide production, albeit far below normal, tend to suffer from less severe disease. It should be noted that a diminished respiratory burst has been reported from patients with severe glucose-6phosphate dehydrogenase (G6PD) deficiency (20). Neutrophils from such patients may fail to reduce nitroblue tetrazolium dye similar to what is classically demonstrated by cells from patients with CGD. NADPH is generated by the first two reactions of the hexose monophosphate shunt: glucose-dphosphate dehydrogenase (G6PD) and 6phosphogluconic dehydrogenase. Since G6PD is the fist enzyme in this pathway, its absence results in greatly diminished shunt activity and thus a severe decrease in the amount of available NADPH substrate. As would be expected, severe G6PD deficiency in neutrophils can result in a defective respiratory burst, with a clinical picture similar to that of CGD. Clinical Manifestations of CGD The clinical presentation of CGD can be quite variable. Typically, these patients develop serious infections early in life, often in their first year, though some may present with their index infection in adolescence or even young adulthood. Attesting to the heterogeneity of this group is the highly unusual case of a 69-yr-old man who was diagnosed with X-linked CGD after he developed Burkholderia (Pseudomonas) cepacia septicemia (21). Because of a family history significant for CGD, the diagnosis was entertained and confirmed. His neutrophils contained normal, albeit dysfunctional cytochrome b, and did generate small amounts of su-
NOTE: No responsibility is assumed by the Publisher for any injury and/or damage to persons or propaty BS a matter of products liability. negligence or otherwise, or from any use or operation of any methods, pmduds, instructions or ideas contained in the material herein. No suggested test or procedure should b-z carried out unless, in the reader’s judgment, its risk is justified. Because of rapid advances in medical sciences. we recommend that the independent verification of diagnoses and dmg dosages should be made. Discussions, views, and recommendations as to medical procedures, choice of drugs, and drug dosages are the responsibility of the authors. ClinicalMicrobiology News/e~fer (lSSN 1069417X) is issued twice monthly in one indexed volume per year by Elswier Science Inc., 655 Avenue of the Americas, New York, NY 10010. Subscription price per year: $175.00 including postage and handling in the United States, Canada. and Mexico. Add $59.00 for postage III the rest of the world. Second-class postage paid at New York. NY and at additional mailing offIces. Postmaster: Send addrss changes to C/in&/Microbiology Newslerter, Elsevin Science Inc.. 655 Avenue of the Americas, New York, NY 10010.
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peroxide. Perhaps it was this ability to generate some superoxide that delayed the onset of the index infection. Typical infectious syndromes in patients with CGD include pneumonia, pulmonary abscesses, skin and soft tissue infections, suppumtive lymphadenitis, osteomyelitis, and hepatic abscesses. Such infectious complications are frequently associated with early mortality. Cellulitis and lymphadenitis sometimes respond to local care and antibiotic administration, but early intervention with parenteral antibiotics and/or surgical drainage is prudent if improvement is not demonstrated in a day or two. Deep tissue infections, including hepatic abscesses, pneumonia, and osteomyelitis, can be associated with metastatic infection at other remote sites, including the spleen and the brain. A low threshold for embarking on an extensive workup for areas of possible metastatic seeding even in the face of an obvious focus of infection must be maintained. Occasionally, signs and symptoms of infection may be limited to malaise, pain, or weight loss. Fever may be absent or intermittent. Neutrophilia is not necessarily present. An increase in the erythrocyte sedimentation rate from a previously known baseline value can be a valuable indicator of infection, and certainly a useful parameter to monitor during treatment. Patients with CGD arc particularly prone to infections by organisms that produce catalase. The catalase produced by the offending organism deprives the phagocyte of exogenous peroxide as an alternative microbicidal mechanism. There is also in vitro evidence that many of these organisms are less susceptible to oxygen-independent killing mechanisms, thus increasing their virulence in this disease (22). Staphylococcus aureus and members of the Enterobacteriaceae family are the most common bacterial pathogens. Salmonella spp., Serralia spp., and Escherichia co/i, in particular, have been recognized as being responsible for frequent and severe infections (23,24). Burkholderia cepacia, Nocardia spp., and Mycobacterium tuberculosis are also encountered with increased frequency in these patients. In contrast. organisms that produce hydrogen Clinical Microbiology
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peroxide but that are catalase negative, such as the streptococci and lactobacilli, are not major pathogens in CGD-afflitted individuals. Fungal infections also play a major role in the morbidity and mortality of these patients. Cohen et al. (25) reviewed the records of 245 individuals with CGD and found that 20% had a history of a fungal infection. Aspergillus spp. were responsible for 78% of all fungal infections including all cases of fungal osteomyelitis in this cohort. Mouy et al. (24) reviewed the infections of 48 individuals with CGD and reported Aspergillus spp. as second only to Staphylococcus aureus as a cause of serious infection. Clinically evident infection with Aspergillus spp. usually begins in the lung. Contiguous spread or dissemination may occur. Vertebral osteomyelitis in individuals with CGD is usually caused by aspergilli (26). Unfortunately, despite medical and surgical therapy of vertebral osteomyelitis secondary to Aspergillus spp. infection, reported cases have been associated with treatment failure. recurrence, and severe disabling orthopedic or neurologic complications or death. identification of a pathogen may require biopsy for histologic tests and culture. This is usually best combined with surgical drainage, debridement, or extirpation of nonviable tissue. Infection with more than one organism is not unusual, especially
in patients with pneumonia, in whom mixed infection with Aspergillus and Nocardia spp. and M. tuberculosis can occur. Histopathologic examination characteristically demonstrates mixed suppura-
tive and necrotizing granulomatous inflammation in skin, lymph nodes, lung, liver. gastrointestinal tract, and many other sites (27). Eventually, these lesions can enlarge, and by virtue of their size and number, can result in organ dysfunction due to mass effect and/or obstruction. Patients may present with symptoms of gastric outlet, esophageal, or urinary bladder obstruction. In
a subset of patients, the complications arising from the granulomas are more problematic than episodes of infection. The obstructive granulomata can result from infection but also occur in the absence of an apparent infectious process. 0 I‘li)’ I.liC\ IL?‘science I”‘
If no pathogen is identified, an empiric course of antibiotics is prudent. If the obstruction is unresponsive to antibiotics and is of recent onset, judicious use of low dose steroids may be beneficial in shrinking them and alleviating the obstructive symptoms (28).
More Reasons to Suspect CGD In addition to the classic presentations discussed above, there are a number of clinical situations in which CGD should be considered. A family history of unexplained deaths of infant or young males may hint at X-linked CGD. CGD should be part of the differential diagnosis in an patient with unexplained pneumonia caused by S. aureus, Aspergillus spp., B. cepacia, or Serraria spp. Recurrent deep tissue infection caused by staphylococci or any deep tissue infection caused by Serratia spp. is highly suspect. A single unexplained hepatic abscess in a child or osteomyelitis caused by any of these organisms should prompt testing for the possibility of CGD. Since CGD patients also have increased susceptibility to M. tuberculosis infection, a child from a population in which this infection is uncommon should be evaluated for possible CGD. Patients have been known to carry a diagnosis of inflammatory bowel disease with radiologic and/or histologic evidence that confirms the diagnosis by demonstrating granulomatous disease in the small or large bowel (29). A low threshold should be maintained for evaluating such patients for possible CGD, especially in atypical circumstances or in the clinical setting of recurrent infections that are otherwise unexplained.
Establishing the Diagnosis of CGD The laboratory diagnosis of CGD is based on the determination that patients’ neutrophils are unable to undergo an oxidative burst, a function that can be measured in a variety of ways. The nitroblue tetrazolium (NBT) dye reduction test is the method employed by most clinical laboratories. It is simple to perform in experienced hands, and provides both a qualitative and semiquantitative result. When neutrophils produce superoxide, the soluble yellow nitroblue tetrazolium is reduced to the in01%4399/95/$0.00
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soluble blue-black formazin that precipitates on and within activated cells. Simply visualizing the cells using light microscopy and counting the percentage that demonstrate reduction of NBT provides the results. Although this assay is generally easy to perform and interpret, problems arise from a lack of understanding about the test. Properly done, an NBT test must be performed in a timed fashion, the assay being completed in 15 to 20 min. Some CGD patients with trace to 5% production of superoxide may weakly reduce the NBT. This is not a problem in a timed assay, but the test may be read as normal if the assay is allowed to proceed for a long time. More comprehensive analysis of the respiratory burst and molecular genetic subtyping are currently available only in research laboratories and use a variety of specialized techniques. Some of these methods include direct measurement of superoxide production, detection of fluorescent substrate oxidation, cytochrome b spectroscopy, immunoblot analysis for ox&se components, and oxidase gene sequencing. The most useful secondary test is Westem immunoblotting of patient neutrophi1 cytosol and membrane fractions followed by detection with antibodies specific for each of the oxidase “phox” components. This, together with NBT testing of the patient and the patient’s mother, will reveal the genetic subtype in the great majority of cases. Knowledge of the genetic subtype can be helpful for genetic counseling of the family but does not change the clinical approach to the patients’ care. Management of Patients with CGD The mainstay for the management of these patients is aggressive antibiotic treatment of infectious complications in concert with early and aggressive surgical drainage of abscesses. White blood cell transfusions have also been used, particularly for serious infectious complications. Because infection can be very difficult to treat in the face of abnormal neutrophil function, prevention of infection should be the primary goal. The administration of prophylactic antibiotics, especially trimethoprim/sul116
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famethoxazole, significantly reduces the incidence of serious infections in patients with CGD (30). Dicloxacillin or another oral antibiotic with anti-staphylococcal activity is a suitable alternative if trimethoprim/sulfamethoxazole is not tolerated. Particular attention to oral hygiene can control the gingivitis and recurrent aphthous ulcers that often become problematic. Patients should be aware that barns, hay, freshly mown grass, and wood shavings all harbor Aspergillus and they should attempt to avoid such contact. The combined use of interferon-y and antibiotics probably provides the optimal prophylaxis against infection. Supporting evidence for this regimen comes from the results of a multicenter placebocontrolled trial in which patients received antibiotics and either interferon-y or placebo (3 1). The results of the study demonstrated a 67% reduction in serious infections suffered by patients who received both interferon-y and antibiotics. At the recommended interferon-y dosage (0.05 mg/b@ of body surface, 3 d a week), side effects are infrequent but include fever, malaise, and headache. The mechanism of the protective effect is not completely understood but may relate to partial amelioration of the defect in the phagocyte oxidative burst in a small subset of individuals (32). Based on these results, as well as in vitro supporting evidence, the U.S. Food and Drug Administration has licensed the use of interferon-y for patients with CGD. Additional administration of the antifungal agent itraconazole to this regimen may help curtail the onset of infection with Aspergillus spp., but results of clinical trials are not yet available to support its routine use. Despite efforts to prevent infection in these patients, complications occur. The general approach to the diagnosis and management of infections in these patients follows the same general rules that apply to diagnosis and management of all immunodeficient patients with signs and symptoms of an infection. Diligent pursuit of a microbiologic diagnosis by obtaining proper cultures is a must. It should be remembered that these patients are particularly prone to aspergillosis and other fungal infec-
tions, so appropriate cultures must be set up. Early recognition of infection, the acquisition of appropriate cultures, and early institution of adequate doses of parenteral antibiotics can reduce the morbidity and mortality in these patients. Surgical extirpation of infected tissue should be considered early on, especially if there is no improvement after a brief irial of medical management. Transfused granulocytes may also be considered as an adjunctive therapy. Ideally, the white cells should be ABO matched and HLA similar, obtained by centrifugation leukopheresis, and should not be irradiated, because this has an adverse effect on oxidase activity. There are no published studies clearly demonstrating enhanced efficacy of transfused granulocytes. CGD-The Potential for Gene Therapy Studies using retroviral vectors containing cDNA for p47phox, gp9lphox. or p22phox have demonstrated that it is possible to restore oxidase activity in cell lines or neutrophils derived from hematopoietic progenitor cells harvested from CGD patients (33-36). The cDNA encoding the normal oxidase component is inserted into the genomic DNA of hematopoietic stem cells obtained from a patient with that specific oxidase defect. These genetically engineered cells could then be returned to the patient with subsequent maturation into normal mature phagocytes. Even the correction of a small percentage of the patient’s cells may result in a clinical cure because cells from heterozygous mothers who carry the gene for the X-linked form of CGD but who are clinically normal, have been found to express as little as 3% functional oxidase activity. The in vitro evidence that gene therapy restores functional oxidase activity predicts that the clinical studies will soon follow. The era of gene therapy is on the horizon, not just for CGD but for a variety of genetic and acquire-d diseases. It is likely that the lessons learned in the early clinical trials will pave the way for gene therapy to enhance host defense in a variety of ways. The possibilities are limited only by our technical resources and our scientific curiosity. Clinical
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