Chronic Granulomatous Disease: a Cohort of 173 Patients—10-Years Single Center Experience from Egypt

Chronic granulomatous disease (CGD) is an inherited primary immunodeficiency disorder (PID) of phagocytes, characterized by a defect of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase components [1, 2]. These are responsible for the production of superoxide anion and hydrogen peroxide through the transfer of electrons from NADPH in the cytosol across the phagosome membrane and the delivery of protons that produce hydrogen peroxide. The produced reactive oxygen intermediates (ROI) damage the phagocytosed microorganisms [3]. The enzyme is formed of five subunits: gp91^phox (CYBB) and p22^phox (CYBA), which are integral membrane proteins that form the flavocytochrome b588 (the electron transport center of the enzyme [4] and p47^phox neutrophil cytosolic factor 1(NCF1), p67^phox neutrophil cytosolic factor 2(NCF2), p40^phox neutrophil cytosolic factor4 (NCF4) which are cytosolic components [5, 6]. A CYBC1 gene mutation leads to reduced expression of NADPH oxidase main subunit (gp91phox) and may result in CGD [7]. The gene encoding gp91^phox is found on the X chromosome, while the genes of the other proteins are located on autosomes [8]. CGD is characterized by recurrent fungal and bacterial infections [9, 10] and hyper-inflammatory and autoimmune symptoms [11, 12].

Laboratory diagnosis of CGD can be made by the nitroblue tetrazolium dye reduction test (NBT) [13] or by flow-cytometry-based dihydrorhodamine (DHR) assay, which is the preferred screening test due to its higher reproducibility, sensitivity, rapidity, and ability to detect X-linked carriers [14, 15]. Few reports were published about CGD in Egypt [15, 16]. For the paucity of data on the disease burden in Egypt, we present a large cohort of patients through our 10 years of experience.

This is a retrospective study conducted at the PID Unit, Pediatric Department of Cairo University Children’s Hospital, Egypt, from 2011 to 2021. The study included cases with a confirmed CGD diagnosis. Detailed history and clinical examination were recorded as well as the microbiological workup and the vaccination history. Glucose-6-phosphate dehydrogenase (G6PD) deficiency was excluded as none of the patients suffered from hemolytic anemia, and all the patients proved to be deficient in one of the NADPH components.

One hundred seventy-three patients from 149 different kindreds were diagnosed with CGD based on DHR test results: 107 males (61.8%) and 66 females (38.2 %). Based on the defective NADPH component, autosomal recessive CGD (AR-CGD) was diagnosed in 132 patients (76.3%) (70 males and 62 females). This included 83 patients (48%) with p47^phox defect, 44 patients (25.4%) with p22^phox defect, and 5 patients (2.9%) with p67^phox defect. X-linked CGD (XL-CGD) was diagnosed in 25 patients (14.4%); all have maternal carrier DHR pattern.

Meanwhile, 16 patients (9.2%) could not be categorized; this included 3 male patients with gp91^phox and p22^phox deficiency, whose mothers were not available to be tested for carrier pattern of DHR which could differentiate between P22^phox and gp91^phox deficiency. Another 13 patients (4 females and 9 males) who had abnormal DHR results could not be categorized due to the transient unavailability of testing either by the intracellular NADPH protein component expression by flow cytometry or the molecular analysis (Fig. 1).

The geographical distribution of CGD patients in Egypt was the following:

While, we had some patients from nearby countries including 9 patients with p22^phox defect from Libya, 4 patients with p47^phox defect from Yemen. One patient with p22^phox defect, and another with XL-CGD from Palestine, one patient with p47^phox defect and one XL-CGD patient from Sudan, and one patient with p22^phox defect from Syria. It was noted that p22^phox deficient patients were prevalent in the North Coast cities as well as in Eastern Libyan cities, while p47^phox deficient patients were prevalent among the Delta, Nile cities, and Sinai. All the Yemeni patients were diagnosed with p47^phox defect (Supplemental Figure 1).

Positive consanguinity was reported in 141 (81.5%) patients with 91.7% consanguinity among AR-CGD. Fifty-seven (32.9%) patients had a history of previous sibling death and 28% with siblings’ affection. The median age at diagnosis was 48 months (range 1–186 months), while the median age at presentation was 7 months (range 0.2–180 months). P47^phox deficient patients were the oldest, while p22^phox/gp91 deficient patients were the youngest at both presentation and diagnosis (Table 6).

CGD is an inherited primary immunodeficiency disorder of phagocytes; few studies have documented CGD patients from Egypt [15, 16]. Herein, we report 173 patients, which is the largest cohort being reported from Egypt. The significant prevalence of AR forms of CGD (76.3%) is related to the high rate of consanguinity (81.5%), consistent with data from Egypt, Israel, Iran, and Turkey that were previously published [15, 25‐29]. The male-to-female ratio in the AR group was 53 to 46% which is similar to the world-wide’s reports [30, 31]. The median age at first manifestation in our cohort was 7 months (ranging from 0.2 to 180 months), which is consistent with studies in India, Mexico, and Israel stating that the first presentations were in the first year of life [10, 28, 32]. While the median age at diagnosis was 48 months, similar to what had been reported in Israel’s and Palestine’s reports [33], it was around 2 years in India and Europe [10, 12] and 30 months in Mexico [32]. Patients with XL-CGD had an earlier age at presentation compared to AR-CGD patients. Patients with p22^phox and gp91^phox deficiency presented and were diagnosed earlier than p47^phox deficient patients. Several reports agreed with our data [10, 33]. However, other countries reported otherwise on comparing between XL and AR groups [30, 34]. The p47^phox group had significantly higher SI as well as overall survival rate, and these findings were reported in several previous studies [30, 33]. The plausible explanation is that in the absence of p47^phox, some electrons are immediately transferred from the FAD prosthetic group in gp91^phox to oxygen, directly producing some H₂O₂. The small quantities of reactive oxygen species (ROS) appear to be adequate to cope with some infections [35]. There was no difference in either the survival or the mortality rates between the XL and AR groups. Nevertheless, a significant statistical difference was noted in the mortality and survival rates between the four groups. The p47^phox deficient patients had the longest overall survival rate, which can be attributed to the same explanation regarding SI. Also, the delay in diagnosis of patients with AR-CGD and lack of awareness among physicians with the mild AR phenotype could explain the missed undiagnosed cases; this contributed to the statistical indifference in the survival and mortality rates between the XL and AR groups. It is important to mention that we diagnosed three male patients with gp91^phox/ p22^phox deficiency, and their mothers were not available to be tested for carrier pattern with the possibility of being CYBC1 deficient patients. It is well known that CYBC1 deficiency can lead to reduced expression of NADPH oxidase’s main subunit (gp91^phox) [7].

The most common isolated organism from pulmonary infections was Aspergillus sp., followed by gram-negative bacteria then Mycobacterial sp., like in many reports [10, 29, 30]. While we reported that the lungs, followed by the skin, and subcutaneous and deep organ abscesses were the most common sites of fungal infections in our cohort, there was only one lymph node fungal isolate. Another study reported that the lung and lymph nodes were the most common sites [36], and others reported that lymph nodes were the leading infection site [29, 37]. The clinical presentations of fungal infection in CGD were highly mutable, ranging from indolent with minimal symptoms to severe life-threatening infections [38]. Fungal CNS infections could be the first presentation of CGD patients, as we had two patients, one with a brain space-occupying lesion, while the second patient had brain and spinal cord involvement. So, the identification of invasive fungal infection in a patient with unknown risk factors should raise attention for further investigation [9].

Although Candida krusei was reported previously as an infectious organism in severe combined immunodeficiency, combined immunodeficiency, and immunocompromised patients [9, 39], to the best of our knowledge, it is the first report of Candida krusei in CGD patients. The peculiar importance of this organism is its intrinsic resistance to fluconazole [39]. Kodamaea ohmeri which inhabits the environment has emerged during the last decades as a human pathogen that can cause life-threatening infections especially in immunocompromised patients [40, 41]. We reported a 3-month-old P22^phox deficient patient who was hospital admitted with pneumonia and liver abscesses. Kodamaea ohmeri, Klebsiella pneumonia, and Acinetobacter were isolated from the bronchoalveolar lavage. During the treatment, the patient developed drug-induced sweet syndrome which was diagnosed according to the diagnostic criteria [42], and the condition resolved completely after treatment. To the best of our knowledge, this is the first report of Kodamaea ohmeri infection in primary immunodeficiency diseases.

The wide spectrum of bacterial species that had been isolated from the patients during the infections highlights the importance of proper microorganisms’ isolation and targeted treatment according to sensitivity. Staphylococcus species were the most commonly isolated bacteria from the skin and deep organ abscesses followed by Klebsiella and Pseudomonas species; these infections were similar to other reports [10, 12, 30, 32]. We are raising the alarm about the emergence of the drug-resistant species among the isolated gram-positive and negative bacteria. Drug-resistant bacterial organisms were isolated from 23/91 (25.2%) patients (17 MRSA, 4 Klebsiella MDR, 1 Pseudomonas MDR, and 1 Acinetobacter MDR). Antimicrobial resistance is a major global health burden and an urgent problem especially among immunocompromised patients all over the world [43‐45].

In addition to infections with the signature microorganisms such as Aspergillus and Staphylococcus species, Mycobacterial species were documented among our patients. In a country with compulsory BCG vaccination in the first month of life, regional BCG-itis was recorded in 13 patients (7.8 %) among the vaccinated patients, which was the first presenting symptom among 4% of them. Similar observation was reported in an Indian report (8%) [10]. While in a Mexican cohort, 58% presented with an adverse reaction, which constituted the first manifestation of CGD in 27 patients (30%), similar to what was reported in Iran [32, 29]. While in Turkey, it was 22% among the vaccinated patients [30], and in Israel, BCG-itis was 2.3% among CGD patients (2 patients from Palestine) as the BCG vaccine is no longer a compulsory vaccine in Israel [28]. The lower percent of BCG complications in our cohort could be attributed to the possibility that minor reaction to BCG vaccine (BCG-itis) induration and ulceration at the site of vaccination, which passed unnoticed or were forgotten by the parents especially in the older age group.

The most common site of mycobacterial infection was the lymph nodes 17/44 (38.6%), followed by the lungs 13/44 (29.5%). Unlike what was reported in the Indian and European experiences as the lung was the most affected site followed by lymph nodes [10, 12]. Mycobacterium tuberculosis complex was the most common causative organism 24/44 (54.5%) among the Mycobacterial sp. infections followed by BCG then atypical Mycobacteria. This was similar to the Indian study [10], while other studies reported that BCG strains were the most common followed by Mycobacterium tuberculosis [12, 32, 46]. This difference could be due to the variability in disease endemicity in the different countries. While it was reported in the European experience that there was no association between the occurrence of BCG-itis and CGD type [12], we noticed a statistically significant higher incidence of regional BCG-itis among P22^phox deficient patients. It is worth noting that mycobacterial infection was the main presentation among many of the P47^phox patients whether with CNS, vertebral, pulmonary, or skin involvement.

In addition to increased susceptibility to infection, patients with CGD have a dysregulated inflammatory response that may require immunosuppressive drugs besides the antimicrobial medications [47, 48]. We reported the hyper-inflammatory clinical manifestations, which included granuloma, IBD, arthritis, secondary HLH, and MISC-post-COVID infection. IBD manifestations were reported in 5.2% of our patients, similar to what was reported in the Indian, Iranian, and Turkish groups [10, 29, 30], but at a lower rate than what was reported in Europe and North America [12, 49]. The difference could be explained by the prevalence of XL-CGD in Western countries, which was associated with a high incidence of colitis [50]. Despite the difficulty in the treatment of IBD in patients with CGD [51], our patients’ responses to treatment were positive.

Genetic diagnosis utilizing Sanger sequencing was done for 44 CGD patients only. It revealed variants in CYBA in 22 patients from 19 different families; almost all of them had the same mutation (p.Val99ProfsTer90), and they originated from the western cities of the Delta and North Coast, while NCF1 mutations were found in 13 patients from 9 different families showing only one variant, p.Tyr26HisfsTer26 in all of them, and they originated from the eastern cities of the Delta. This may point to a founder effect and the importance of the geographic distribution of the patients in correlation with patients’ genotypes.

As Interferon-gamma is not available in Egypt, so it was not used on any of our patients. Despite the antimicrobial prophylaxis, our patients still experienced frequent infections, highlighting the importance of HSCT to CGD patients which was carried out on only 6 patients. The mortality rate in our cohort was 31.4% which is lower than Indian and Mexican reports [10, 32] and higher than those reported in Europe and Israel [12, 28]. Pneumonia was the most common cause of mortality, similar to the European and Indian reports [10, 12]. The overall survival rate in the XL and AR groups of patients is lower than what was reported in Europe, Israel, and Mexico [12, 28, 32]. Nevertheless, the median age of death in both groups was similar to the Mexican report, higher than the Indian report [10], and was lower than what was recorded in Europe and Israel [12, 28].

In conclusion, we documented the different clinical presentations in CGD patients and highlighted the predominance of AR-CGD in our cohort. The delayed diagnosis and the low number of transplanted patients indicate the importance of early diagnosis and initiating proper management for patients to have a better quality of life. The alarm to the emergence of the drug-resistant species is raised, which necessitates antimicrobial stewardship. It is worth mentioning that due to the high incidence of mycobacterial tuberculosis infection among our cohort, any patient with a typical/atypical mycobacterial infection should be screened with a DHR test to role out CGD.