Mycobacterium genavense as a cause of subacute pneumonia in patients with severe cellular immunodeficiency

Background Mycobacterium genavense is a rare nontuberculous mycobacteria (NTM). Human infections are mostly disseminated in the setting of the AIDS epidemic or the use of aggressive immunosuppressive treatments. M. genavense culture is fastidious, requiring supplemented media. Pulmonary involvement rarely occurs as a primary localization. Cases presentation We report here two patients with pneumonia as the predominant manifestation of M. genavense infection: one kidney transplanted patient and one HIV-infected patient. Both patients were initially treated with anti-tuberculous drugs before the identification of M. genavense on sputum or broncho-alveolar lavage fluid culture. A four-drug regimen including clarithromycin and rifabutin was started. Gamma interferon has been helpful in addition to antimycobacterial treatment for one patient. Conclusion Clinicians should be aware that M. genavense could be the etiologic agent of sub-acute pneumonia mimicking tuberculosis in patients with cellular immunodeficiency status.


Background
Host defenses against mycobacteria need an immune cellular response (Th1/Th17) activated by cytokines and chemokines produced by innate immunity cells [1]. Consequently, non-tuberculous mycobacteria (NTM) could cause life threatening infection in immunocompromised patients with profound cellular immune deficiency in particular those who are HIV-infected [2]. M. genavense, is a fastidious growing mycobacteria found in water [3] with birds and pets being occasionally infected [4]. Human infections were initially described in HIV-infected patients [5]. Thereafter, few cases have been observed in non-HIV immunocompromised hosts including only two reports in solid organ transplant recipients [6,7]. M. genavense infection preferentially involves bowel and abdominal lymph nodes, a fact being ascribed to the presumed digestive contamination. Mortality of patients with disseminated M. genavense infection is high, ranging from 44% to 71% in 2 series [8,9]. Herein we report two cases of M. genavense infected patients with pulmonary involvement as the main clinical manifestation, one kidney transplant recipient and one HIVinfected patient.

Case one
A 43-year-old woman, of Moroccan origin, was admitted in August 2005 for seizure, an 8 kg loss of body-weight, slowly increasing dyspnea, productive cough, and fever lasting for 4 months. She had been known to be infected by HIV since 1996, but lost to follow-up. Physical examination revealed left lower limb palsy, pulmonary ronchi, hepatomegaly and oropharyngeal candidiasis. Cerebral computed tomography (CT) imaging demonstrated multiple abscesses. Cerebral toxoplasmosis was suspected and treatment with pyrimethamine and sulfadiazine was initiated. Biological results were: C-Reactive Protein  19 mg/L, leukocyte count 3940/mm 3 , hemoglobin 11,1 g/dL, aspartate aminotransferase 88 U/L, alanine aminotransferase 40 U/L, gamma glutamyl transferase 310 U/L, alkaline phosphatases 210 U/L, lactico deshydrogenase 268 U/L. Her peripheral blood CD4+ cell count was 110/mm 3 and HIV viral load was 6.39 log copies/mL. Serological tests for hepatitis B and C were positive. A chest radiograph demonstrated diffuse bilateral pulmonary infiltrates ( Figure 1A). The thoracic CTscan showed mediastinal lymphadenopathies, numerous diffuse pulmonary nodular infiltrates with a cavitation in the left upper lobe ( Figure 2). Bronchial endoscopy did not show abnormalities. Three sputa were smear positive for acid-fast bacilli (AFB). Broncho-alveolar lavage (BAL) fluid cytological analysis confirmed the presence of AFB but was negative for other microorganisms. Isoniazid, rifampicin and pyrazinamide were therefore started. Despite the anti-tuberculous regimen, fever persisted and respiratory symptoms progressively worsened requiring continuous nasal oxygenotherapy. After 10 weeks, mycobacterial culture from the respiratory specimen grew M. genavense. The initial anti-tuberculosis treatment was changed for a multidrug regimen with moxifloxacin (400 mg/d), clarithromycin (500 mg b.i.d), ethambutol (20 mg/kg/d) and amikacin (15 mg/kg/d, during 3 weeks) and HAART (tenofovir, emtricitabine, efavirenz) were introduced. Clarithromycin was increased to 1000 mg b.i. d after 2 weeks of treatment because the treatment was well-tolerated and optimal treatment recommendations were sparse. HIV viral load decreased to 3 log copies/mL after 1 month without increase in CD4+ count (60 cells/ mm 3 ). Despite 3 months of this antimycobacterial treatment, the patient's clinical course worsened and infiltrates in the upper lobes on chest X-ray increased. A second bronchoscopy was performed and BAL fluid cytologic analysis showed the persistence of 20 to 30 AFB/ field, which were again identified as M. genavense, without other pathogens. Then gamma interferon (IFN-γ) was started (50 μg/m 2 sub-cutaneously 3-times a week). The HAART therapy was continued with undetectable viral load but a persistent CD4 lymphopenia (50/mm 3 ). The patient's general condition gradually improved within 1 month of IFN-γ treatment with oxygen weaning, radiological improvement and negative respiratory specimens mycobacterial culture. The CD4 cell count reached 300/mm 3 after twelve months of antimycobacterial treatment, so IFN-γ was withdrawn. The anti-mycobacterial treatment was withdrawn after 2 years. We decided to continue the treatment more than 6 months after immune reconstitution and 12 months after negative culture of respiratory specimen because chest radiograph demonstrated continuous regression of pulmonary infiltrate until 24 months, with minimal sequelae at the end of treatment ( Figure 1B). At the last medical visit in June 2011, the patient remained in good health, without relapse of the mycobacterial disease.

Case two
A 44 year-old renal transplant Guinean patient was admitted in September 2008 for fever, weight loss and A B dissemination occurred in one case. M. genavense infection has previously been reported in only two solid organ (kidney and heart) transplanted patients [10,11]. Both had intestinal involvement leading to secondary dissemination and death in one case [10]. In HIVinfected patients, disseminated M. genavense infection associates weight loss, fever, anemia, digestive manifestations and hepatosplenomegaly [9,12,13]. Pulmonary symptoms such as cough and dyspnea are rarely at the forefront. M. genavense can be cultured from stools, sputum, urine and blood samples and tissue biopsy [14]. However, culture of M. genavense is fastidious, making microbiological diagnosis frequently difficult, requiring mycobactin J supplementation for optimal recovery on culture. Culture must be prolonged up to 4 months with a median positive culture time of 43 days (min. 10 days-max. 6 months) depending on the culture medium [5,9,11,13,[15][16][17][18]. Our observations are in agreement with these findings, In our first case, diagnosis was delayed because the time to positive culture was 70 days.
Molecular tools such as polymerase chain reaction (PCR) are of interest in two ways before cultures become positive. Firstly, M. genavense can eventually be detected on clinical specimens with PCR [19][20][21]. Secondly, a negative result for M. tuberculosis PCR on a positive AFB smear specimen is useful to exclude tuberculosis earlier.
In case one, NTM treatment would have been considered if M. tuberculosis PCR had been performed on sputum. Furthermore, we think that M. genavense should be envisaged in immunocompromised patients with mycobacterial pulmonary disease and digestive involvement or negative mycobacterial culture lasting for more than one month.
Infection control requires decrease of the immunosuppressive regimen in patients who receive such treatment in combination with appropriate antimycobacterial treatment. Human and mouse models have pointed out the role of CD4+ and CD8+ T lymphocytes in M. genavense infection [22,23]. Explaining the crucial role of T lymphocytes, the bacterial load was significantly higher in organs from athymic mice compared to normal mice [23]. During HIV infection, the risk of M. genavense disease is correlated with CD4 cell count. Indeed M. genavense infection occurs in patients with profound CD4+ lymphopenia (50 cells/mm 3 or less). However, CD4+ cell count is not a reliable marker of immunosuppression in non-HIV patients receiving immunosuppressive treatment. For example, Pneumocystis pneumonia is frequently observed in organ transplanted patients with more than 400 CD4+ cells/mm 3 [24]. In our patient, the CD4+ T cell count was moderately diminished (230/mm 3 ). Thus, M. genavense infection may occur in non-HIV patients under an immunosuppressive regimen with a much higher CD4+ cell count than seen in HIV-infected patients.
The optimal anti-mycobacterial therapy in M. genavense infection is unknown. The high effectiveness of clarithromycin and rifabutin has been demonstrated in a murine model [25]. Multidrug therapies including clarithromycin are recommended [26]. Even with appropriate treatment, clinical evolution is long before resolution of symptoms, and patients have to be treated for at least 12 to 24 months [26]. As recommended in ATS/IDSA guidelines for Mycobacterium avium intracellulare infection, treatment was continued 12 months after immune restoration was achieved for Case 1 [26]. Concerning Case 2, antimycobacterial treatment cannot be withdrawn because of reinforcement of immunosuppressive therapy.
In the case of our first patient, despite HAART introduction and appropriate anti-mycobacterial treatment, the infection was not controlled. The administration of IFN-γ was temporally associated with a significant clinical improvement and bacteriological clearance of infection. IFN-γ is essential for the control of M. genavense. Indeed, mice deficient for IFN-γ are unable to clear M. genavense infection [27]. In HIV-infected individuals the monocytes-macrophages phagocytic function is impaired. IFN-γ can restore deficient functions of HIV-infected macrophages and has already been used in the management of M. avium intracellulare complex infection [28]. The classic pathway of IFN-γ-dependent activation of macrophages by T helper 1-type responses is a wellestablished feature of immune response to infection with intracellular pathogens, such as M. tuberculosis and NTM [22].
Finally clinicians should be aware 1) That the main clinical manifestation of M. genavense infection may be a slowly progressive pneumonia mimicking tuberculosis in patients with cellular immunodeficiency; 2) Thus, this diagnosis should be evoked in an immunosuppressed patient who receives empirical anti-tuberculous therapy without improvement; 3) Extra-thoracic manifestations can be absent; 4) the microbiological diagnosis requires prolonged cultures and may be helped by PCR analysis of bronchial and lung specimens; 5) IFN-γ may be useful to control M. genavense pneumonia in addition to the antimycobacterial regimen; 6) CD4+ T cell count is not a reliable marker of the risk of M. genavense infection in non-HIV imunocompromised patients.

Consent
Written informed consent was obtained from the patient for publication of this case report and accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal