Disseminated Balamuthia mandrillaris infection 1

Katherine R. Schafer1, Neil Shah2, MI Almira-Suarez3, Jennifer M. Reese4, George M. Hoke5, 2

James W. Mandell5, Sharon L. Roy6, Govinda Visvesvara6 3

1 Wake Forest University Health Sciences, 2 University of North Carolina at Chapel Hill, 3 4

George Washington University Hospital, 4 Mid-Atlantic Pathology Services, Inc., 5 University of 5

Virginia Health Sciences, 6 Centers for Disease Control and Prevention 6

Balamuthia mandrillaris is a rare cause of human infection but carries a high morbidity 7

and mortality when infections do occur. A case of disseminated Balamuthia infection is 8

presented. Early diagnosis and initiation of recommended therapy are essential for 9

increased chance of successful outcomes. 10

________________________________________________________ 11

Case Report. The patient was an 82-year-old white male with a history of necrobiosis lipoidica 12

versus granuloma annulare of the right hand who was admitted in April 2012 for acute onset of 13

fevers, chills, nausea, vomiting, lethargy, and altered mental status (AMS). He initially presented 14

to an outside hospital emergency department with a temperature of 38.5°C, heart rate of 97 15

beats per minute, and oxygen saturation of 88% on room air. His oxygen saturation improved 16

with nasal cannula oxygen. A chest x-ray was performed, which demonstrated findings 17

concerning for granulomatous disease. Due to his condition, he was transferred to our tertiary 18

care center for further evaluation and treatment. 19

Per his family, he had been healthy and without complaints prior to his current presentation. For 20

approximately 15 months, he had been treated by a dermatologist for skin lesions located on his 21

right thigh and hand as well as left upper extremity (LUE) and abdomen. He had multiple skin 22

biopsies from the right hand, LUE, and right thigh with negative bacterial, acid-fast bacillus 23

JCM Accepted Manuscript Posted Online 1 July 2015J. Clin. Microbiol. doi:10.1128/JCM.01549-15Copyright © 2015, American Society for Microbiology. All Rights Reserved.

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(AFB), and fungal studies. During this period of time, he took itraconazole for three months 24

without clinical response and was thus started on prednisone. All lesions resolved except those 25

on his right hand. He was treated with a 14-day course of doxycycline and cephalexin for 26

possible cellulitis of the right hand prior to this admission. Previous work-up included a negative 27

syphilis screen and a normal ACE level (drawn for possible sarcoidosis). Chest X-ray (CXR) at 28

that time revealed nonspecific interstitial changes. 29

Review of systems was obtained from family members given the patient’s AMS. Per their report, 30

the patient endorsed a headache, but no neck pain or stiffness. He had no history of mouth 31

sores or genital ulcers and had a remote history of shingles. He had an episode of "bronchitis" 32

one month prior to admission (PTA) that was slow to improve but gradually resolved. 33

In terms of exposures, he had no international travel but did have sea water exposure on the 34

Gulf Coast of Alabama one month prior to admission. He had no pets but had been mowing 35

grass and spreading grass seed PTA. He had no known tick bites but spent time outdoors using 36

a metal detector for treasure hunting and digging in soil. Previously, he taught a bible study 37

group in a prison but had no known tuberculosis (TB) exposure and no previous TB testing. 38

On initial physical exam, he was febrile to 38.2°C and his oxygen saturation was 94% on two 39

liters nasal cannula. He was initially agitated and confused but progressed to somnolence over 40

a period of 12 hours. He had no cervical lymphadenopathy or nuchal rigidity. Cardiac, 41

pulmonary, and abdominal exams were unremarkable. On the right dorsal hand and proximal 42

fingers there was a collection of erythematous punched-out ulcers with overlying crusts that 43

extended onto the palm at the 5th metacarpal-phalangeal surface (Figure 1A). His initial 44

laboratory values were significant for a total white blood cell (WBC) count of 8.60 k/uL with 45

88.3% neutrophils and 4.8% lymphocytes. He also had a hemoglobin of 13.1 g/dL, hematocrit of 46

37.7%, sodium of 129 mmol/L, albumin of 2.9 g/dL, creatinine of 1.3 mg/dL, and glucose of 256 47

g/dL. 48

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Computed Tomography (CT) of the head revealed age-indeterminate infarcts in the anterior limb 49

of the right internal capsule, left parafalcine frontal lobe and inferior left frontal lobe, favored to 50

be chronic infarcts. CXR showed diffuse, bilateral opacities. CT scan of the chest revealed 51

diffuse centrilobular nodules with intralobular septal thickening. Given his presentation and 52

imaging findings, he was empirically started on intravenous (IV) ceftriaxone and azithromycin for 53

coverage of community-acquired pneumonia. 54

On hospital day (HD) 1, infectious diseases was consulted and the patient was empirically 55

started on treatment for meningoencephalitis including IV vancomycin, ceftriaxone, acyclovir 56

and trimethoprim-sulfamethoxazole (an unknown penicillin allergy precluded the use of 57

ampicillin for coverage of Listeria) in addition to the antibiotics for pneumonia. Repeat 58

examination at that time did reveal nuchal rigidity. Cerebrospinal fluid (CSF) analysis via lumbar 59

puncture revealed an opening pressure of 21cm H2O, protein of 54mg/dL and glucose of 60

86mg/dL. Cell count was remarkable for 14 WBCs (57% lymphocytes, 3% neutrophils and 40% 61

monocytes). No organisms were identified on gram stain. Dermatology was also consulted for 62

skin biopsies and cultures of the patient’s right hand. 63

On HD 2, preliminary report of the patient’s skin biopsy revealed a dense mixed acute and 64

chronic dermal inflammatory infiltrate. Scattered organisms, morphologically compatible with 65

ameba, were noted within lacunar spaces (Figure 1B, original magnification x 400). The 66

organisms showed granular to vacuolated cytoplasm with irregular contours, and contained 67

enlarged nuclei with large central karyosomes (Figure 1C, original magnification x 1000). The 68

patient was therefore empirically started on liposomal amphotericin and voriconazole for 69

coverage of disseminated amebiasis and amebic encephalitis (AE). All other antibiotics except 70

for azithromycin were discontinued. Despite treatment with broad spectrum antibiotics, the 71

patient continued to become increasingly obtunded, eventually requiring transfer to the intensive 72

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care unit (ICU) on HD 3 for closer monitoring and worsening hypoxia, intermittently requiring 73

increased oxygen supplementation. 74

75

On HD 4, the Centers for Disease Control and Prevention (CDC) was contacted for further 76

therapeutic options. CDC recommended combination therapy shown to have efficacy in 77

previous cases, including pentamidine, sulfadiazine, flucytosine, fluconazole (or itraconazole), 78

and azithromycin (or clarithromycin) as well as miltefosine, which at the time required approval 79

by the Food and Drug Administration (FDA) and overseas delivery before it could be used (1-4). 80

As there was not yet definitive identification of the ameba species, sulfadiazine 1.5g every 6 81

hours, pyrimethamine 200 mg once then 75 mg daily, and leucovorin 25 mg daily were added, 82

based on case reports of successful treatment of Acanthamoeba encephalitis (5). His 83

azithromycin dose was increased to 1800 mg daily as well. At this time, miltefosine was 84

ordered. 85

Given the persistent AMS and concern for AE, magnetic resonance imaging (MRI) of the brain 86

was obtained, which revealed multiple parenchymal supra- and infratentorial rounded lesions 87

with surrounding white matter edema and faint enhancement. The largest lesions were in the 88

left posterior temporal lobe. Other lesions were seen in the left dorsal pons and left frontal lobe. 89

Radiology favored these lesions to be infectious in nature. Neurosurgery was consulted on HD 4 90

for urgent biopsy of the patient’s largest brain mass. At this time, the patient’s sodium level 91

continued to decrease to a nadir of 121 mmol/L. Because of his worsening hyponatremia and 92

the small size of the lesions, they deferred brain biopsy and recommended repeat MRI for 93

evaluation of progression of his lesions. Also, because of the patient’s worsening respiratory 94

symptoms, he underwent evaluation with bronchoscopy. Fluid analysis of the broncho-alveolar 95

lavage (BAL) was negative for Legionella pneumophila, Pneumocystis jiroveci, acid fast bacilli, 96

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viruses, and fungal etiologies. Gram negative rods were visible from the BAL Gram’s stain so 97

cefepime was added to his antibiotic regimen. 98

On HD 5, the patient’s condition remained unchanged with no signs of improvement. Following 99

CDC recommendations, flucytosine 1750mg IV q 12 hours was added to his treatment regimen. 100

Miltefosine was being held in customs at this time, pending FDA release. 101

On HD 6, a repeat MRI demonstrated increase in the number and size of prior lesions. These 102

lesions also showed peri-lesional edema and faint enhancement. At that time, neurosurgery 103

planned to perform a biopsy with radiologic guidance on HD 10 as his serum sodium 104

normalized. 105

On HD 7, the bacterial culture from BAL grew “few” Pseudomonas aeruginosa which were 106

sensitive to cefepime. The patient’s inability to handle secretions became more pronounced and 107

his clinical status continued to deteriorate, requiring increasing oxygen and low-dose 108

vasopressor support. A family meeting was held to discuss goals of care; the family decided to 109

continue antibiotics but change code status to “Do Not Resuscitate or Intubate.” 110

On HD 8, CDC reported preliminary identification of ameba forms on skin biopsy which favored 111

Balamuthia. Pyrimethamine and leucovorin were discontinued and the patient was started on 112

pentamidine 300mg IV daily. 113

On HD 9, the patient began to have periods of apnea and expired with family at the bedside. 114

The family agreed to proceed with a limited autopsy of brain only. Post mortem gross 115

examination revealed numerous red to brown soft lesions involving bilateral parietal, frontal and 116

temporal lobes, left pons and basal ganglia, and cerebellum, predominantly in subcortical white 117

matter, with many lesions extending to the gray-white junction. Histopathologic examination 118

revealed the granulomatous nature of the lesions, with predominantly cyst forms and occasional 119

trophozoites, consistent with an amebic infection (Figure 2). Additionally, purulent and 120

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granulomatous inflammation was noted in many subpial and perivascular spaces. Real-time 121

PCR assays performed on fresh-frozen autopsy brain material determined the amebic species 122

as Balamuthia mandrillaris. The CDC performed immunofluorescent staining of the skin biopsy, 123

which was positive for Balamuthia mandrillaris (Figures 1D and 1E). The final diagnosis was 124

disseminated Balamuthia mandrillaris infection. 125

_________________________________________________________________________ 126

Microbiology. Balamuthia mandrillaris is a free-living ameba that lives in soil, dust, and water 127

(6). The species name derives from the first site from which it was isolated in 1986—the brain 128

of a mandrill. The first human case of Balamuthia-associated granulomatous encephalitis was 129

described in 1990. In the lab, Balamuthia feeds on small ameba but not bacteria, which makes 130

its growth in lab challenging. Cultivation of this organism in the lab requires axenic culture or 131

growth in mammalian cell cultures (7). The exact pathogenesis of Balamuthia is incompletely 132

defined at this time, although cell-to-cell contact appears necessary for this process and the 133

organism may have an affinity for binding to the extracellular matrix (2, 7-9). However, human 134

infection appears to begin with either inhalation or percutaneous inoculation of Balamuthia cysts 135

or trophozoites. The organisms are then believed to either migrate through tissue or via 136

hematogenous spread to the location or locations where disease becomes manifest, which can 137

include the brain and skin most commonly but other organs as well. A mouse model of 138

Balamuthia encephalitis proposes the following as one possible mode of infection: the organism 139

is inhaled through the nasal passages, adheres to the nasal epithelium, and then travels along 140

the olfactory nerve into the central nervous system (10). 141

Epidemiology. Balamuthia has been identified in infections in both immunocompromised and 142

immunocompetent patients. There is no clear predilection based on age or sex but cases have 143

been reported from individuals at the extremes of age and the earliest reported cases were in 144

patients with AIDS or substance abuse disorders (2, 11). While there is an over-representation 145

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of Hispanic ethnicity among reported cases, the cause of this association is not understood and 146

may be related to surveillance, exposure, biology, or other factors or combinations of factors (9, 147

12). Transmission through organ transplantation has also occurred (13, 14). 148

Clinical Presentation. Balamuthia mandrillaris has two primary clinical presentations: cutaneous 149

and granulomatous amebic encephalitis (GAE), both of which may have a prodrome of weeks to 150

months. There are also case reports of sinus infections and pulmonary involvement, typically 151

abscesses (7). Patients may present with isolated cutaneous findings or typical 152

encephalopathy/encephalitis symptoms. The latter presentation may also include meningeal 153

signs (9). The cutaneous manifestations can vary from ecthyma-like lesions to erythematous 154

plaques. 155

The pulmonary findings in our case represent a different presentation from other cases 156

described in the literature. Although we do not have definitive tissue diagnosis for his 157

pulmonary process, it likely represents disseminated amebic infection given the presence of the 158

organism in two other anatomic locations (skin and brain). 159

Diagnosis. The diagnostic difficulty derives from the non-specific presentation, which may not 160

suggest amebic infection early in the disease course. A thorough exposure history, skin exam, 161

and neurologic assessment may suggest inclusion of this entity on the differential diagnosis. If 162

neurologic symptoms are present, brain MRI may be helpful and may show space-occupying, 163

ring-enhancing lesions. If cutaneous involvement is present, skin biopsy can be diagnostic if 164

trophozoite forms are seen. Balamuthia, although difficult to distinguish from Acanthamoeba 165

species on H &E staining, may have 2–3 nucleoli visible. Immunofluorescent staining, PCR, 166

and an experimental serologic test can be performed at CDC to distinguish between Balamuthia 167

and Acanthamoeba species. Cerebrospinal fluid examination may reveal a lymphocytic 168

pleocytosis, elevated protein, normal or low glucose, and normal or mildly elevated opening 169

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pressure. Brain biopsies may show both trophozoite and cyst forms of Balamuthia that show 170

positive immunofluorescent staining. 171

Consideration of Balamuthia infection early in the disease course is the most important element 172

of diagnostic workup. Once the diagnosis is included in the differential, further diagnostic 173

workup, treatment guidance, and prompt access to miltefosine can be obtained by contacting 174

the CDC Emergency Operations Center at 770-488-7100. Additionally, further information 175

about the clinical features of Balamuthia and its basic workup is available on the CDC website 176

at http://www.cdc.gov/parasites/balamuthia/health_professionals/index.html. 177

Treatment/Prognosis. Prognosis is poor, in part because the diagnosis is often not considered 178

until late in the disease course. There is no clear standard therapy for infections with B. 179

mandrillaris. Pentamidine, flucytosine, fluconazole and sulfadiazine plus either azithromycin or 180

clarithromycin (combined with surgical resection if CNS lesions are present) has been used 181

successfully in a few cases (2). Additionally, miltefosine, an agent used to treat leishmaniasis, 182

has demonstrated some in vitro effect on Balamuthia infections and has been used successfully 183

in combination therapy of Balamuthia GAE and skin infections (4, 15). Miltefosine is not readily 184

available in the United States but can now be obtained directly from CDC under an FDA-185

approved investigational new drug protocol for treatment of free-living ameba infections (16). 186

Amphotericin B, TMP/SMX, and polymixin B have shown little to no in vitro effects on 187

Balamuthia (7). Other work suggests that cyst formation in brain tissue may be hindered by 188

ketoconazole, cytochalasin D, and cycloheximide (17). 189

In summary, Balamuthia mandrillaris infection is a rare, but important, cause of encephalitis and 190

cutaneous infection. It is difficult to diagnose and early tissue biopsy is extremely important. 191

This disease carries a very high mortality. Treatment is poorly defined but early diagnosis, 192

prompt ameba-specific treatment, and increased availability of miltefosine through CDC may 193

have a positive effect on improving outcomes in the future. 194

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195

Disclaimer: The findings and conclusions in this report are those of the authors and do not 196

necessarily represent the official position of the Centers for Disease Control and Prevention. 197

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Figure 1 Dermatologic presentation and pathology. Figure 1A: Erythematous punched out 247

ulcers with overlying crusts on the right dorsal hand. Figure 1B: Histopathology from the right 248

hand showing a dense mixed acute and chronic dermal inflammatory infiltrate and scattered 249

amebic trophozoites within lacunar spaces (arrows). (Original magnification x 400). Figure 1C: 250

High-power view of an amebic organism demonstrating granular to vacuolated cytoplasm with 251

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irregular contours (pseudopods) containing a nucleus with a large central karyosome (arrow). 252

(Original magnification x 1000). Figure 1D: Low power view of immunofluorescence from skin 253

biopsy. (Original magnification x 100). Figure 1E: High power view of immunofluorescence from 254

skin biopsy with visible amoebic organisms. (Original magnification x 1000). 255

256

Figure 2 Central nervous system lesions. Figure 2A: MRI axial FLAIR image demonstrating 257

multiple focal, rounded hyperintense lesions in the pons, cerebellum, and occipital lobes. Figure 258

2B: Gross section of the pons and cerebellum; softened granular lesion in the pons (see arrow). 259

Figure 2C.: Microscopic examination of the pontine lesion; prominent granulomatous 260

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inflammation (yellow asterisk) in abundant necrotic background. A single amebic cyst is seen 261

(black arrow) in the mid-lower field of the picture. 262

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