Chronic Disseminated Mycobacterium xenopi Infection in a Cat with Idiopathic CD4+ T Lymphocytopenia

Abstract

A 3-year-old castrated male domestic shorthair cat was referred for examination in August 2000 with a 3-week history of poor appetite, vomiting, and weight loss. One week before referral, evaluation by the referring veterinarian identified anemia (PCV 21% [reference range 29–48%]) and moderate renal insufficiency (BUN 55 mg/dL [reference range 14–36 mg/dL], serum creatinine 4.2 mg/dL [reference range 0.6–2.4 mg/dL], urine specific gravity 1.015) with an inflammatory urine sediment (proteinuria, white blood cells, bacteria) on a voided sample. The results of enzyme-linked immunosorbent assays for feline leukemia virus (FeLV) antigen and feline immunodeficiency virus (FIV) antibodies were negative. Radiographs disclosed bilateral renomegaly and microcardia. Abdominal ultrasound examination identified a thin cortex in the left kidney and both kidneys were hyperechoic. The cat was treated with lactated Ringer's solution IV and enrofloxacin 6 mg/kg/d IV for 3 days. Upon examination at the referral hospital, the cat was quiet and responsive. Physical examination abnormalities were limited to bilaterally enlarged kidneys that appeared to be painful on palpation and a body condition score of 3/9. Body weight was 3.6 kg. On CBC there was a severe nonregenerative macrocytic normochromic anemia (PCV 17% [reference range 30–47%], MCV 55 fL [reference range 41–51 fL], MCHC 31 g/dL [reference range 31–35 g/dL], aggregate reticulocytes 20,880/μL) in the presence of normal serum erythropoietin concentration (17 mU/mL [reference range 10–30 mU/mL]). The WBC count (10,200/μL [reference range 5,500–19,500/μL]), differential cell count (segmented neutrophils 7,040/μL [reference range 2,500–12,500/μL], lymphocytes 2,860/μL [reference range 1,500–7,000/μL], monocytes 260/μL [reference range 0–800/μL], eosinophils 50/μL [reference range 0–1,500/μL]), and platelet count (485,000/μL [reference range 300,000–800,000/μL]) were normal. Serum chemistry results indicated moderate azotemia (BUN 36 mg/dL [reference range 16–34 mg/dL], serum creatinine 3.5 mg/dL [reference range 0.7–2.3 mg/dL]), increased AST activity (173 U/L [reference range 5–30 U/L]), and hypoalbuminemia (1.8 g/dL [reference range 2.0–3.1 g/dL]). Results of urinalysis collected by cystocentesis indicated isosthenuria (USG 1.011), proteinuria, and bacteruria. Aerobic urine culture was negative, and plasma prothrombin and partial thromboplastin times were within normal limits. Ultrasonographic examination of the abdomen identified moderate bilateral renomegaly with pyelectasia, mesenteric lymphadenopathy, and an enlarged hypoechoic pancreas. By means of ultrasound guidance, fine-needle tissue aspirates were collected from kidney, mesenteric lymph node, and pancreas, and slides were prepared for cytologic evaluation using Wright-Giemsa stain. Preparations obtained from the kidney were very cellular, consisting primarily of large, epitheliod macrophages arranged in sheets and individually. The cytoplasm of many of the macrophages contained long, thin, negatively staining, rod-shaped inclusions consistent with Mycobacteria spp. (Fig 1). Renal epithelial cells were also observed in the preparation. Macrophages containing similar negatively staining rods were observed in tissue aspirate preparations from the pancreas and a mesenteric lymph node, along with pancreatic epithelium and lymphocytes from each respective organ. Urine sediment was stained for acid-fast organisms and was negative. Photomicrograph of tissue aspirated from the left kidney. A large macrophage (center) contains numerous, negatively staining, rod-shaped bacteria. A cluster of renal epithelium (partially lysed) is seen in the lower right corner (Wright-Giemsa stain). The inset in the upper right corner contains a bone marrow macrophage with several intracytoplasmic, acid-fast positive bacteria indicating organisms of the genus Mycobacterium (Ziehl-Neelsen stain), scale bar = 10 μm. Slide preparations of a bone marrow aspirate stained with Wright-Giemsa contained marrow particles of normal cellularity. Hematopoietic precursor cells from the myeloid, erythroid, and megakaryocytic cell lines were present in normal numbers, and all cell lines had complete and orderly maturation. Several bone marrow macrophages were observed, almost all of which contained numerous, intracytoplasmic, negatively staining rods. Slide preparations of the bone marrow aspirate were fixed in 10% formalin and submitted for Ziehl-Neelsen staining for demonstration of acid-fast bacteria. Rod-shaped inclusions that were negatively stained using Wright-Giemsa were uniformly positive using the acid-fast stain (Fig 1, inset), consistent with the genus Mycobacterium. A sample of the bone marrow aspirate was submitted for mycobacterial culture and antibiotic susceptibility.a A working diagnosis of disseminated mycobacteriosis was made based on the finding of acid-fast bacilli in multiple organs. Initial medical management consisted of IV lactated Ringer's solution, nasogastric (NG) tube feeding, famotidine (0.7 mg/kg IV q12h), metoclopramide (0.4 mg/kg SQ q8h), and attempts to stimulate appetite with diazepam (0.1 mg/kg IV q12h) or oxazepam (0.6 mg/kg q12h). Triple antibiotic therapy was initiated with enrofloxacin (5 mg/kg IV q12h), clarithromycin (8.3 mg/kg via NG tube q12h), and clofazimine (14 mg/kg PO q24h). Major and minor blood cross matches were performed, and 1 unit (60 mL) of fresh whole blood was administered IV. A trial of erythropoietin (100 U/kg SC 3 times/wk) was started during hospitalization because the nonregenerative anemia was thought possibly to have been associated with renal insufficiency and the erythropoietin concentration was in low-normal range. However, it was also considered that the anemia was more likely attributable to the effects of chronic inflammatory disease. Because of persistent anorexia, a low profile gastrostomy tube was surgically placed 8 days after initial presentation. A kidney biopsy performed at that time identified marked interstitial infiltrates of lymphocytes, neutrophils, and macrophages, with acid-fast bacilli within macrophages, consistent with the clinical diagnosis of disseminated mycobacteriosis. Disseminated mycobacteriosis usually is associated with defects in cell-mediated immunity. Peripheral blood lymphocyte subset analysis performed by flow cytometry identified moderately decreased CD4+ T lymphocytes (600/μL [reference range 700–3,000/μL]), normal CD8+ T lymphochytes (1,284/μL [reference range 400–2,000/μL], and a markedly inverted CD4+ : CD8+ ratio (0.53 [reference range 1.5–2.5]). In cats, this pattern most commonly is observed in chronic FIV infection. The serum FeLV antigen and FIV antibody tests were repeated and confirmed to be negative. Peripheral blood was also negative for FeLV and FIV by polymerase chain reaction and by virus cultures performed 3 times over a 1-year period. Bone marrow was negative for FeLV by immunofluorescent antibody assay. Because no other underlying cause for the lymphocyte abnormalities could be identified, a diagnosis of idiopathic CD4+ T lymphopenia was made.1 At discharge 13 days after initial presentation, the cat's clinical condition had improved, but it still would not eat. Clinicopathologic abnormalities were only partially improved at the time of discharge (BUN 24 mg/dL, serum creatinine 2.8 mg/dL, serum albumin 1.4 g/dL, PCV 21%). Treatment at home included enrofloxacin, clarithromycin, clofazimine, famotidine, metoclopramide, and feeding of a nutrient-dense liquified diet through the gastrostomy tube. Based on the experience of persistent infection in immunosuppressed human patients with opportunistic disseminated mycobacteriosis, it was suspected that antibiotic therapy would be required for the life of the cat. The cat was reexamined after 8 weeks of triple-antibiotic therapy. The owner reported that activity was normal, but the cat refused to eat an adequate amount of food voluntarily and received most of its nutrition via the gastrostomy tube. Physical examination was normal, with the exception of persistent renomegaly. Body weight had increased 18% to 4.25 kg. Mild azotemia (BUN 48 mg/dL, serum creatinine 2.5 mg/dL) and normocytic normochromic anemia (PCV 24%) persisted. Fine-needle aspirates were collected from kidney, bone marrow, and mesenteric lymph node and were negative for mycobacterial organisms by cytology and on acid-fast stain. The CD4+ T-lymphocyte count and the CD4+ : CD8+ratio were decreased since the initial visit (Fig 2). Results from the original bone marrow culture submitted 2 months earlier revealed Mycobacterium xenopi susceptible to ciprofloxacin, clarithromycin, and clofazimine (Table 1). Treatment was continued with clarithromycin and ciprofloxacin. Total lymphocyte and CD4+ and CD8+ T-lymphocyte counts over 5 years in a cat diagnosed with disseminated Mycobacterium xenopi infection. Six months later (April 2001), fine-needle aspirates of the bone marrow and kidney were performed. Bone marrow cytology was normal, but macrophages with acid-fast organisms were present in the kidney. Ciprofloxacin and clarithromycin were continued, and rifampin (10 mg/kg via the gastrostomy tube q12h) was added. On 4 more reevaluations (July 2001 to October 2002), stable azotemia was present and anemia had resolved. Acid-fast organisms were identified intermittently on fine-needle aspiration cytology of the kidney. Mycobacterial cultures were negative at all time points except one (July 2001) in which M. xenopi was reisolated from culture of the bone marrow. Kidney size gradually decreased over time to below normal. In October 2002, the cat was referred for evaluation of panleukopenia (WBC 2,940/μL) in the presence of normal PCV and platelet count. Cytologic evaluation of the bone marrow indicated a mild plasmacytosis and an inadequate myeloid response to peripheral neutropenia, but no organisms were seen or cultured. The clarithromycin was discontinued in the event that neutropenia was an adverse reaction to this drug. Five months later, WBC and neutrophil counts were normal, but the lymphocyte count remained decreased. Ciprofloxacin and rifampin were continued. Over the next 3 years, the cat was clinically normal with stable renal insufficiency, recurrence of leukopenia, and continued deterioration of all lymphocyte subset counts (Fig 2). The cat developed malignant melanoma on the maxillary gingiva 6 years after initial presentation, which was surgically excised without complication. In January 2007, a transudative pleural effusion developed, accompanied by severe anemia. An echocardiogram was normal, and no cause for the pleural effusion could be identified. Treatment consisted of blood transfusion and supportive care. Two months later, approximately 7 years after the initial diagnosis of disseminated mycobacteriosis, the cat's condition deteriorated and it died at home. A necropsy was performed and confirmed the persistence of disseminated mycobacteriosis. Macrophages containing acid-fast bacilli were identified in all examined tissues. In the gastrointestinal tract, the infiltrate was sometimes composed of loose nodular aggregates scattered throughout all layers, mostly in the submucosa. In other regions, there were dense, band-like infiltrates of macrophages expanding the lamina propria and submucosa with extension into the muscularis (Fig 3A). Macrophages and multinucleated giant cells containing acid-fast bacilli were observed in the lung (Fig 3B), spleen, and renal interstitium. The splenic white pulp was moderately to markedly depleted of lymphocytes. Occasional scattered renal tubules were surrounded by and contained few to low numbers of neutrophils. These tubules contained luminal extracellular acid-fast bacilli with disruption or loss of the tubular epithelium or contained luminal macrophages with intracytoplasmic acid-fast bacilli. Occasional glomeruli contained focal aggregates of acid-fast bacilli, neutrophils, and cell debris (Fig 3C). Neutrophilic and granulomatous inflammation was observed around the feeding tube; macrophages in this lesion also contained acid-fast bacilli. The adrenal gland contained multifocal necrosis, suppurative inflammation, and acid-fast bacteria (Fig 3D). Centrilobular lipid-type vacuolar degeneration with hepatocellular loss and fibrosis was observed in the liver. Culture of the lung confirmed persistence of M. xenopi infection, which had acquired resistance to ciprofloxacin and rifampin (Table 1). (A) Large intestinal submucosa is expanded by sheets of macrophages and multinucleated giant cells, with focal transmural extension of inflammation (scale bar = 1 mm). Macrophages and giant cells contain large numbers of acid-fast bacilli (inset, scale bar = 100 μm). (B) Pulmonary interstitium is multifocally infiltrated and expanded by large numbers of macrophages (scale bar = 100 μm) and few multinucleated giant cells containing large numbers of acid-fast bacilli (inset, scale bar = 50 μm). (C) Glomerulus containing aggregates of neutrophils (scale bar = 100 μm), cell debris, and acid-fast bacilli (inset, scale bar = 50 μm). Large numbers of macrophages are present in the surrounding interstitium. (D) Focus of necrosis, suppurative inflammation (scale bar = 50 μm), and acid-fast bacilli (inset, scale bar = 50 μm) in the adrenal cortex. Hematoxylin and eosin. Insets: Ziehl-Neelsen. In the case reported here, simultaneous diagnosis of CD4+ T lymphocytopenia and mycobacteriosis made it impossible to know whether the CD4+ T lymphocytopenia predisposed to mycobacteriosis or if the M. xenopi infection caused the depletion of CD4+ T lymphocytes. However, a decrease in CD4+ T lymphocytes associated with infection usually is transient and accompanied by a CD8+ T lymphocytosis.2 In this case, CD4+ T lymphocytopenia preceded progressive depletion of all lymphocyte subpopulations. Therefore, it is more likely that the CD4+ T lymphocytopenia was the primary disease that resulted in susceptibility to the opportunistic mycobacterial infection. To the authors' knowledge, idiopathic CD4+ T lymphocytopenia (ICL) has not been previously reported in veterinary species. In human patients, ICL is defined by the Centers for Disease Control and Prevention as a decreased number of circulating CD4+ T lymphocytes (<300/μL or <20% of total T cells) on more than 1 occasion in the absence of any defined immunodeficiency or immunosuppressive therapy.1, 2 In some cases, ICL is accompanied by panlymphocytopenia, as was observed to develop in this cat. In this cat, the initial CD4+ T lymphocyte count was 600/μL (32% of total T cells), which was below the reference range for cats but did not reach the threshold required for a diagnosis of ICL in humans. The appropriate maximum CD4+ T lymphocyte count for a diagnosis of ICL in cats currently is unknown, but the CD4+ T lymphopenia in this cat subsequently progressed to meet the criteria used for diagnosis in humans. In most cases, ICL is diagnosed after development of opportunistic infections in HIV-negative patients. The pathogenesis is unknown, but several mechanisms have been hypothesized. A case report of 2 siblings who both had marked decreases in CD4+ T lymphocytes suggested that there may be a hereditary component to the disease in some cases.3 The ICL syndrome has also been reported in a variety of immune dysfunctions, including decreased bone marrow clonogenic capability, deficiency of p56Lck kinase activity, decreased progenitor cell recovery, inappropriate thymopoiesis, excessive apoptosis of CD4+ memory cells, ineffective T-cell receptor signal transduction, and TNFα, IL-2, or IL-7 dysregulation, ultimately resulting in a decreased oligoclonal T-cell receptor repertoire.1, 4, 5 Although mechanisms contributing to ICL still are equivocal, it is clear that ICL should be included in the differential diagnosis of unexplained opportunistic infections. M. xenopi is a slow-growing, saprophytic, nontuberculous, acid-fast bacillus that is ubiquitous in soil and water and is also commonly identified in municipal and hospital water supplies.6, 7 In humans, colonization by M. xenopi usually is restricted to individuals with defective cell-mediated immunity, particularly those with HIV infection.7, 8 Pulmonary infections are most common, and disseminated infections, as observed in the cat of this report, are associated with a worse prognosis. M. xenopi infection has been reported in 2 cats previously,9, 10 but the authors are not aware of any other reports of long-term outcomes or successful treatment of M. xenopi in cats. The most common disseminated nontuberculous mycobacterial organism isolated from human patients is Mycobacterium avium complex.8, 11, 12 Prophylactic antibiotic therapy against M. avium complex is recommended for AIDS patients with CD4+ T lymphocytes <50/μL.13 Once infection with disseminated mycobacterial infection is established, treatment is difficult because of resistance to many antibiotics and persistence of the organism during clinical remission with negative cultures. The most successful treatment protocols for human patients with M. avium complex involve the simultaneous use of multiple antibiotics long term.11, 12 Because there are no evidence-based empirical treatment guidelines for M. xenopi at this time, initial treatment should follow guidelines developed for M. avium complex infection pending the results of susceptibility testing and response to therapy.12 A limitation associated with the long-term use of antibiotic therapy is acquired resistance that may develop throughout the course of the disease. In the case reported here, a survival time of approximately 7 years was achieved using combinations of enrofloxacin or ciprofloxacin, rifampin, clofazimine, and clarithromycin. At the time of necropsy, the M. xenopi isolate had developed resistance to two of the antibiotics administered for treatment. Although the long-term prognosis for patients with disseminated mycobacteriosis remains guarded, the use of combination antibiotic therapy can be associated with a long survival time. aNational Jewish Medical and Research Center, Denver, CO