Disseminated Mycobacterium genavense Infection Mimicking Sarcoidosis: A Case Report and Review of Literature on Japanese Patients

Sarcoidosis is a systemic inflammatory disease characterized by noncaseating epithelioid cell granulomas. However, certain infections can exhibit similar histological findings. We present a case of a 69-year-old man who was initially diagnosed with sarcoidosis and later was confirmed, through 16S rRNA sequencing, to have disseminated Mycobacterium genavense infection. Acid-fast bacteria were detected in the bone marrow biopsy using Ziehl–Neelsen staining, but routine clinical tests did not provide a definitive diagnosis. The patient tested negative for HIV, anti-interferon-gamma antibodies, and genetic immunodeficiency disorders. He was treated with multiple drugs, including aminoglycosides and macrolides, but showed no improvement in fever and pancytopenia. However, these clinical signs responded favorably to steroid therapy. We reviewed 17 Japanese cases of M. genavense infection. All cases were in males; 7/17 (41%) were HIV-negative; and 12/17 (71%) had a decreased CD4 count. Genetic analysis confirmed M. genavense isolation, and macrolides were used universally. Mycobacterium genavense infection is challenging to identify and mimics other systemic inflammatory diseases such as sarcoidosis. There are no standard treatment protocols. Our case report and Japanese case review contribute to understanding this rare disease.


Introduction
Sarcoidosis is a systemic inflammatory disease of unknown etiology, characterized by noncaseating epithelioid cell granulomas in the involved organs. In addition to the presence of histopathological noncaseating epithelioid cell granulomas, an increased lymphocyte count and CD4/8 ratio in bronchoalveolar lavage fluid (BALF), and elevated serum angiotensin-converting enzyme (ACE) levels, can provide valuable clues to the diagnosis. However, sarcoidosis must be differentiated from sarcoid-like lesions secondary to infections, malignancy, or other causes [1]. Differentiation from infection can be challenging, especially when pathogens are rare and difficult to detect. As a therapeutic approach, prednisolone (PSL) is often initiated at approximately 0.5 mg/kg/day. The dose is gradually reduced to 5-10 mg/ day for maintenance treatment in order to prevent relapses and reduce the risk of complications.
Mycobacterium genavense is a nontuberculous mycobacterium that was first reported in 1992 to cause disseminated infection in human immunodeficiency virus (HIV) patients [2]. It also causes disseminated infections in organ transplant recipients and in patients receiving immunosuppressive therapy for autoimmune diseases [3]. Owing to the difficulty of the detection of organisms, identification of the pathogen is often challenging. Thus, cases of M. genavense infection that were initially diagnosed as sarcoidosis or other systemic inflammatory diseases have been reported [1,4]. Here, we present a patient with M. genavense infection who was initially diagnosed with sarcoidosis. We also reviewed Japanese cases of M. genavense infection described in Japanese and English to gather evidence and explore the characteristics of this rare disease.
High-resolution computed tomography (CT) revealed a reticular shadow and thickening of the intralobular septa in the lower zones of both lungs ( Figure 1). No mediastinal lymphadenopathy was observed. A bronchoscopy was also performed, and BALF obtained from the right B 5 yielded 3.0 × 10 5 cells/mL (19% macrophages, 78% lymphocytes, 3% neutrophils, and 0% eosinophils) with a high CD4/CD8 ratio of 5.3. Ziehl-Neelsen staining, polymerase chain reaction (PCR) for M. tuberculosis and MAC, and BALF culture for Mycobacterium species were negative. Pathological examination of specimens obtained using transbronchial lung biopsy through the right B 2 b, B 4 a, and B 8 a bronchi and skin biopsies showed noncaseating granulomas. Bone marrow biopsy also revealed noncaseating granulomas with hypocellular marrow. Ziehl-Neelsen staining and all specimens were negative for Mycobacterium on PCR testing. The patient was accordingly diagnosed with sarcoidosis based on the increased levels of serum ACE, proportion of lymphocytes, CD4/8 ratio in the BALF, and noncaseating granulomas with negative Ziehl-Neelsen staining. PSL 30 mg/day (0.5 mg/kg/day) was initiated. As the patient's fever improved, his WBC count recovered to a level above 5000/µL, with lymphocytes comprising approximately 25%. His Hb level also increased to >14 g/dL, and the PLT count reached approximately 150,000/µL. The PSL dose was gradually reduced to a maintenance dose of 12.5 mg per day.
staining. PSL 30 mg/day (0.5 mg/kg/day) was initiated. As the patient's fever improved, his WBC count recovered to a level above 5000/µL, with lymphocytes comprising approximately 25%. His Hb level also increased to >14 g/dL, and the PLT count reached approximately 150,000/µL. The PSL dose was gradually reduced to a maintenance dose of 12.5 mg per day. Four years later, at the age of 69 years, the patient developed a persistent fever. Hematology revealed a decreased WBC count at 2600 /µL, with lymphocytes accounting for 8.9%. His hemoglobin count was 10.4 g/dL, and his PLT count was 76,000 /µL. Ziehl-Neelsen staining of the pathological specimens obtained by bone marrow aspiration revealed a noncaseating granuloma (Figure 2A,B), although PCR for tuberculosis and MAC was negative. The results of acid-fast bacilli culture were also negative. We performed 16S ribosomal RNA gene sequencing of Mycobacterium species, as described previously [5]. Although a previous study identified Mycobacterium species using an average 550 bp nucleotide sequence, in this study, we improved the identification accuracy further by using a longer nucleotide sequence of 973 bp. Finally, the patient was diagnosed with M. genavense infection. However, M. genavense was not isolated from the sputum, BALF, blood, urine, or stools. Thus, in order to examine drug sensitivity, we attempted to culture acid-resistant bacteria from a re-biopsied bone marrow sample using mycobactin J-containing medium [6]. The culture attempt was unsuccessful, possibly because of the effects of the treatment. Fluorodeoxyglucose positron emission tomography showed abnormal uptake in the bone marrow, particularly in the femur and pelvis. Four years later, at the age of 69 years, the patient developed a persistent fever. Hematology revealed a decreased WBC count at 2600 /µL, with lymphocytes accounting for 8.9%. His hemoglobin count was 10.4 g/dL, and his PLT count was 76,000 /µL. Ziehl-Neelsen staining of the pathological specimens obtained by bone marrow aspiration revealed a noncaseating granuloma (Figure 2A,B), although PCR for tuberculosis and MAC was negative. The results of acid-fast bacilli culture were also negative. We performed 16S ribosomal RNA gene sequencing of Mycobacterium species, as described previously [5]. Although a previous study identified Mycobacterium species using an average 550 bp nucleotide sequence, in this study, we improved the identification accuracy further by using a longer nucleotide sequence of 973 bp. Finally, the patient was diagnosed with M. genavense infection. However, M. genavense was not isolated from the sputum, BALF, blood, urine, or stools. Thus, in order to examine drug sensitivity, we attempted to culture acid-resistant bacteria from a re-biopsied bone marrow sample using mycobactin J-containing medium [6]. The culture attempt was unsuccessful, possibly because of the effects of the treatment. Fluorodeoxyglucose positron emission tomography showed abnormal uptake in the bone marrow, particularly in the femur and pelvis.
staining. PSL 30 mg/day (0.5 mg/kg/day) was initiated. As the patient's fever improved, his WBC count recovered to a level above 5000/µL, with lymphocytes comprising approximately 25%. His Hb level also increased to >14 g/dL, and the PLT count reached approximately 150,000/µL. The PSL dose was gradually reduced to a maintenance dose of 12.5 mg per day. Four years later, at the age of 69 years, the patient developed a persistent fever. Hematology revealed a decreased WBC count at 2600 /µL, with lymphocytes accounting for 8.9%. His hemoglobin count was 10.4 g/dL, and his PLT count was 76,000 /µL. Ziehl-Neelsen staining of the pathological specimens obtained by bone marrow aspiration revealed a noncaseating granuloma (Figure 2A,B), although PCR for tuberculosis and MAC was negative. The results of acid-fast bacilli culture were also negative. We performed 16S ribosomal RNA gene sequencing of Mycobacterium species, as described previously [5]. Although a previous study identified Mycobacterium species using an average 550 bp nucleotide sequence, in this study, we improved the identification accuracy further by using a longer nucleotide sequence of 973 bp. Finally, the patient was diagnosed with M. genavense infection. However, M. genavense was not isolated from the sputum, BALF, blood, urine, or stools. Thus, in order to examine drug sensitivity, we attempted to culture acid-resistant bacteria from a re-biopsied bone marrow sample using mycobactin J-containing medium [6]. The culture attempt was unsuccessful, possibly because of the effects of the treatment. Fluorodeoxyglucose positron emission tomography showed abnormal uptake in the bone marrow, particularly in the femur and pelvis. Although the patient had no history of recurrent opportunistic infections since childhood, his immunological status was re-evaluated. The WBC and lymphocyte counts were 1200/µL and 370/µL, respectively. The proportion of CD4+ T lymphocytes was 64/µL (17.2%), and that of CD3+ and CD19+ T lymphocytes was 74.2% and 0.2%, respectively. Immunoglobulin levels were as follows: IgG, 858 mg/dL; IgA, 112 mg/dL; and IgM 36.4 mg/dL. The patient tested negative on IGRA, and he also tested negative for HIV antigens and antibodies, human T-cell leukemia virus type 1 antibodies, and anti-interferon-gamma autoantibodies. Gene panel tests for congenital immunodeficiency syndrome (IL2RG, JAK3, IL7R, RAG1, RAG2, DCLRE1C, ADA, PNP, ZAP70, LIG4, NHEJ1, and TBX1) and Mendelian susceptibility to mycobacterial diseases (IL12RB1, IL12B, IL12RB2, IL23R, IFNGR1, IFNGR2, STAT1, CYBB, IRF8, TYK2, RORC, JAK1, IKBKG, GATA2) were also negative.
Treatment for M. genavense was initiated with clarithromycin (CAM), rifampicin (RFP), ethambutol (EB), and amikacin (AMK). The patient's fever temporarily improved, and the PSL dose was gradually tapered from 12.5 mg/day. One month after switching to oral therapy with CAM, RFP, EB, and levofloxacin, and 2 weeks after starting AMK treatment, the patient's fever and pancytopenia recurred. The fever resolved immediately after the re-administration of AMK, and the pancytopenia improved. After 6 months of treatment, a treatment-resistant fever over 40 • C recurred, which persisted even with treatment with AMK combined with azithromycin, rifabutin, sitafloxacin, and EB. The patient also had pancytopenia, with a WBC count of 1100 /µL (lymphocytes, 27.0%; CD4 count, 53/µL). The Hb level was 7.8 g/dL, and the PLT count was 79,000/µL. PSL was increased from 7.5 mg/day to 30 mg/day (0.5 mg/kg/day), and the fever rapidly resolved. The pancytopenia improved, although the low CD4 count persisted. Five months later, hematology showed a WBC count of 3100/µL, with a lymphocyte count of 31.4% and a CD4 count of 212/µL. The hemoglobin level was 10.4 g/dL, and the PLT count was 124,000/µL.
We also reviewed Japanese cases of M. genavense infection. We used the Ichushi-Web, a search system specifically designed for Japanese literature, and Google, Google Scholar, PubMed, and reference lists to search for both Japanese and English language reports. The selection criteria for our literature search encompassed keywords "Mycobacterium genavense" or "M. genavense" in conjunction with "Japanese" or "Japan" for both Japanese and English language reports. We did not establish a specific time frame for the search period. A total of 17 Japanese cases of M. genavense infection, including the present case were identified (Tables 1 and 2). All 17 patients were male, 10 (59%) were HIV-positive, and 7 (41%) were HIV-negative. Furthermore, 12 (71%) patients had a decreased CD4 count. The most common clinical manifestations were fever (13/17, 76%) and lymphadenopathy (13/17, 76%). In all cases, the diagnosis was confirmed by genetic analysis. The primary antibiotics used were macrolides (100%), ethambutol (100%), and rifamycin (16/17, 94%).

Discussion
We present here a case of a Japanese man who developed M. genavense infection during sarcoidosis treatment. Acid-fast bacteria detected by Ziehl-Neelsen staining were observed in the histopathological examination of a bone marrow biopsy sample; however, a definitive diagnosis could not be obtained through routine clinical testing. The diagnosis was confirmed using 16S rRNA sequencing. The patient tested negative for HIV and anti-interferon-gamma antibodies and underwent genetic testing for congenital immunodeficiency disorders. Treatment was initiated with a combination of multiple antibiotics, including aminoglycosides and macrolides; however, the patient developed resistance and experienced a persistently high fever and pancytopenia. Ultimately, a favorable response was achieved with steroid therapy. To gather evidence regarding epidemiology and response to treatment, and explore the characteristics of this rare disease, we also conducted a review of 17 Japanese cases of M. genavense infection.
Although most patients with M. genavense infection have HIV infection, M. genavense infection can occur in the absence of HIV infection. In the meta-analysis by Wetzstein et al. [19], 52 of the 223 (23.3%) individuals were HIV-negative, but most of them were also immunocompromised with conditions such as organ transplantation (15 cases, 6.7%), systemic lupus erythematosus (4 cases, 1.7%), and sarcoidosis (4 cases, 1.7%).
Notably, all 17 Japanese patients with M. genavense infection were male. In the metaanalysis by Wetzstein et al. [19], 171 of the 223 (76.7%) patients were HIV-positive, and 79.8% were male. Data from the National Institute of Infectious Diseases in Japan showed that 20,640 of 23,231 cases of HIV infection (88.7%) reported in 2021 were in males (https: //www.niid.go.jp/niid/ja/aids-m/aids-iasrtpc/11555-512t.html). The higher proportion of males among patients with M. genavense infection in Japan can be attributed to a higher proportion of males among individuals with HIV infection. However, seven (41%) of the patients in our review were HIV-negative. It is unknown why all seven HIV-negative individuals were male. In an observational study from the Netherlands, 7 out of 9 (78%) HIV-negative patients with M. genavense infection were male [20]. Collectively, these results suggest that men have a higher risk of M. genavense infection than women, regardless of their HIV status, although further studies are required to confirm this finding.
A decrease in CD4 count is an important mechanism underlying infection, and cases of M. genavense infection have also been reported in individuals with idiopathic CD4 lymphocytopenia [21]. Among the 223 patients, 171 (76.7%) patients had HIV, and the median CD4 count was only 16/µL [19]. In addition, HIV-negative patients had low CD4 counts (median: 150/µL). In this literature review, 10 of the 17 patients had HIV (58.8%), and all these patients had CD4 counts less than 30/µL. Another two non-HIV patients (11.7%) including the present patient also showed lower CD4 counts (88 and 298/µL) [9]. Aside from HIV, immunosuppressant use in organ transplantation and various autoimmune diseases are also associated with lower CD4 counts. Thus, exploring the possibility of diseases associated with IL-12 receptor deficiencies and impairments of the interferongamma pathway might be important [13,20,22]. Among the 17 Japanese patients in this review, 3 (17.6%), 2 (11.8%), and 1 (5.9%) patient had collagen disease, was positive for anti-IFN-γ antibodies and had hypogammaglobulinemia. The possibility of idiopathic CD4 lymphocytopenia is also possible in the present case owing to the sustained low CD4 counts [23]. However, the possibility of bone marrow dysfunction resulting from the bone marrow infection itself should also be considered. The exact cause of the infection remains unknown.
M. genavense is difficult to culture using common mycobacterial culture methods. Diagnosis is challenging in nearly all cases and is confirmed only using molecular techniques such as 16S rRNA, rpoB, or hsp65 gene analysis [6,19,21]. In this literature review, all 17 Japanese patients had a positive smear, which led to the diagnosis. However, successful culture was achieved in only 7 patients (41%), and identification was subsequently confirmed through genetic analysis in all patients.
Available in vitro susceptibility data are limited [6]. However, existing data suggest that most isolates of M. genavense are susceptible to aminoglycosides, macrolides, quinolones, and rifamycins. A regimen containing macrolides has been demonstrated to be more effective than a regimen without macrolides. In the meta-analysis of 223 patients, logistic regression analysis revealed a significant association between macrolide administration and reduced occurrence of lethal events (OR: 0.162) [19]. In the present review, macrolides were used in all patients, and rifamycin was used in 94%. Quinolones and aminoglycosides were used only in 24% and 18% of cases, respectively. According to the American Thoracic Society guidelines, quinolones and aminoglycosides might be used more frequently in Japan than in other countries [6]. In the current case, aminoglycosides, macrolides, and rifamycins were administered as the initial treatment for M. genavense, and quinolones were added to achieve a five-drug regimen. However, the patient remained treatment resistant with prolonged fever and pancytopenia. The initial diagnosis was sarcoidosis 4 years previously, and steroid therapy was gradually tapered following the diagnosis of M. genavense. However, when the steroid dose was increased owing to treatment resistance observed with the five-drug regimen, the patient rapidly improved. Although steroid use should not be taken lightly owing to its side effects, apart from our report, there have been a few reports of the successful use of steroid combination therapy in cases of M. genavense infection in which antibiotic treatment alone failed to control the condition [7,24]. Further investigation is needed to validate this approach.
The initial diagnosis of sarcoidosis was based on the detection of granulomas in the lungs, skin, and bone marrow; increased lymphocyte count; the CD4/8 ratio in the BALF; and serum ACE levels. Multiple acid-fast bacilli smear examinations and culture tests were performed, and PCR tests for M. tuberculosis and MAC were also negative. However, anti-GPL-core IgA antibodies were also present at the initial diagnosis of sarcoidosis. The cause of sarcoidosis remains largely unknown, although mycobacteria have been reported as a contributing factor [25]. A case-control study of sarcoidosis that included 736 patients with sarcoidosis showed that organ involvement in sarcoidosis primarily affects the lungs (95%), skin (15.9%), lymph nodes (15.2%), eyes (11.8%), and liver (11.5%) [26]. Bone marrow involvement in sarcoidosis is relatively rare, occurring in only 3.9% of cases. In contrast, among the 223 patients with M. genavense infections, bone marrow was the most common organ affected, seen in 76 individuals (34%) [19]. Considering the rarity of bone marrow involvement in sarcoidosis, and the possibility of cross-immunity to M. genavense and MAC, it is possible that the initial diagnosis of sarcoidosis was a sarcoidosis-like condition caused by M. genavense infection. Although detecting M. genavense is quite challenging, the presence of granulomas in the bone marrow and anti-GPL-core IgA antibody positivity could signal the need to consider genetic testing for M. genavense.

Conclusions
We reviewed 17 Japanese cases of M. genavense infection, including the present case. We found that all 17 cases were male, and seven (41.2%) cases were HIV-negative, suggesting that males have a higher risk of M. genavense infection than female patients, regardless of their HIV status. Although the present case was HIV-negative, the CD4 count was low. In the search for the causes of M. genavense infection, the possibility of CD4 lymphocytopenia should be considered, not limited to HIV. The presence of granulomas in the bone marrow and the positive anti-GPL-core IgA antibodies may signal to consider genetic testing for M. genavense. Given that drug treatment with macrolides did not resolve the fever and pancytopenia, the steroid dose was increased, which resulted in a rapid improvement. Cases have been reported where despite administering adequate antimicrobial regimens for M. genavense infection, improvement was not achieved and steroids were ultimately required. Although further studies are needed, it is worth considering steroids in patients who do not respond adequately to antibiotic therapy. Our case report and Japanese case review contribute to a deeper understanding of this rare disease.