Cutaneous balamuthiasis: A clinicopathological study.

INTRODUCTION: Balamuthia mandrillaris, a free-living amoeba, causes an uncommon infection that is characterized by cutaneous and neurological involvement, which carries a poor prognosis.
METHODS: This is a retrospective observational study including patients with clinical suspicion of cutaneous balamuthiasis, their skin biopsies, and/or a positive direct immunofluorescence test. The data were collected from the Dermatology and Pathology service of the Hospital Cayetano Heredia and the Instituto Tropical Alexander von Humboldt, Lima, Peru, from January 1985 to June 2007. We identified 60 biopsies from 35 patients, from which clinical data were available in 30.
RESULTS: Twenty-two (73%) patients had centrofacial lesions, mostly located on the nose. The classical lesion was an asymptomatic, erythematous, or violaceous infiltrated plaque. Twenty-two (73%) patients had neurologic involvement. Fifty (83%) biopsies showed granulomatous dermatitis and 75% showed ill-defined tuberculoid granulomas without caseous necrosis. Multinucleated giant cells were observed in 52 (87%) biopsies. Trophozoite forms were identified in the biopsies of 25 (71%) patients. Direct immunofluorescence was positive in 25 (71%) patients.
CONCLUSION: B. mandrillaris is a pathogen that is capable of inducing a characteristic skin lesion with a reaction pattern of ill-defined tuberculoid granulomas and many giant cells.

The present report describes the cutaneous histological findings of Balamuthia mandrillaris infection, along with a precise clinicopathological correlation.

This may allow physicians to lead to an early diagnosis and successful therapy for an otherwise fatal disease.

Introduction

Balamuthia mandrillaris, 1 of the 4 species of free-living amoeba that is pathogenic to humans, is associated with subacute granulomatous encephalitis, a nearly always fatal condition.1, 2, 3, 4, 5, 6, 7

The onset of balamuthiasis is subtle, the course of the disease is insidious, its outcome is poor, and diagnosis is usually made at autopsy, which is when nervous system involvement is identified. The mortality rate of this disease is >98%, although in a single case reported from our institution, a therapeutic regimen including miltefosine resulted in the regression of central nervous system (CNS) and cutaneous involvement and survival of the patient up to 14 years. An early diagnosis can be made by the histologic examination of clinically suspicious skin lesions, dramatically improving the prognosis of these patients. However, the diagnosis is rarely made on skin histology, mainly due to a lack of knowledge about the disease and the difficulty in identifying the amoeba on skin tissue.8, 9, 10

Skin involvement may appear and remain undiagnosed several weeks or months before the onset of neurologic manifestations.11, 12, 13 Thus, an early histopathologic diagnosis followed by treatment may improve the ominous prognosis of the disease. Moreover, in a review of 30 Peruvian cases at our institution, all patients presented at a health care setting or reported a history of a cutaneous lesion, a finding that differs from other published series., Based on this, balamuthiasis should be included in the differential diagnosis of granulomatous diseases primarily of the facial area and else in both immunocompetent and immunocompromised patients.16, 17, 18

Herein, we describe a series of 35 cases of balamuthiasis and the histopathologic characteristics of the cutaneous infection by this free-living amoeba.

Materials and methods

We collected clinical and pathological data of patients with balamuthiasis who were seen at the Dermatology Service of the Hospital Cayetano Heredia and the Instituto de Medicina Tropical Alexander von Humboldt, Universidad Peruana Cayetano Heredia and whose biopsy specimens were evaluated at the Pathology Department of the Hospital Cayetano Heredia from January 1985 to June 2007. Our search criteria included the following: (1) clinical suspicion of free-living amoeba infection of the skin or (2) identification of the parasite in the skin biopsy by routine histology and/or a positive direct immunofluorescence test. We identified 60 skin biopsies from 35 patients, for whom clinical data were available in 30. Institutional review board approval was not required.

Clinical information such as age, sex, place of origin, clinical appearance and location of lesions, disease duration, exposure to stagnant water, coexistent medical conditions, and neurologic involvement were retrieved from patients' records.

All the skin specimens were fixed in 10% buffered formalin, processed, and embedded in paraffin; 4-mm sections were obtained and stained with hematoxylin-eosin. Special stains were performed in most cases and included Ziehl-Neelsen stain for acid-fast bacilli and periodic acid–Schiff for fungus as work-up for granulomatous infiltrates.

Skin biopsies were reviewed by 3 dermatopathologists and were analyzed for the following histopathologic features: the presence of epidermal, dermal, or subcutaneous fat involvement; the composition of the cellular infiltrate (lymphocytes, plasma cells, eosinophils, neutrophils, histiocytes, and multinucleated giant cells); the presence of well-formed or ill-defined tuberculoid granulomas, vasculitis, perineural inflammatory infiltrate; and the presence of the parasite. Direct immunofluorescence studies were performed at the Parasitology Branch of the Center for Disease Control and Prevention, Atlanta, Georgia.

Statistical analysis was performed using Stata version 7.0 (StataCorp).

Results

Clinical data were available for 30 of 35 patients with a suspected diagnosis of B. mandrillaris infection (Table I). The mean age was 21 years (range, 7-57 years). Fifteen (50%) patients were children (≤15 years). Twenty patients were men and 10 were women.

Table I

Clinical characteristics of cutaneous balamuthiasis

1. Age, years
≤15	15 (50%)
>15	15 (50%)
2. Sex
Male	20 (67%)
Female	10 (33%)
3. Place of origin
Piura	13 (43%)
Lima	10 (33%)
Ica	4 (13%)
Trujillo	2 (7%)
Chimbote	1 (4%)
4. Mean time of cutaneous lesion (months)	15
5. No. of lesions
Single	22 (73%)
Multiple	8 (27%)
6. Location of the lesions
Centrofacial	22 (73%)
Lower limb	8 (27%)
Back (dorsolumbar)	4 (13%)
Abdomen	1 (3%)
7. Exposure to stagnant water	17 (57%)
8. Neurologic involvement	22 (73%)
9. Mortality rate
Only skin involvement	0 (0%)
Skin and central nervous system involvement	20 (91%)

Sixteen patients were from cities located on the North coast of Peru (13 from Piura, 2 from Trujillo, 1 from Chimbote); 10 from the capital Lima, situated on the central Pacific coast, and 4 from Ica, 265-km south of Lima. Seventeen patients had a history of recent exposure to stagnant water.

Clinical features

The duration of the skin lesions at the time of diagnosis ranged from 2 months to 5 years (mean, 15 months). Twenty-two (73%) patients had centrofacial lesions, mostly located on the nose. Thirteen patients had extrafacial lesions. This group included lesions on the knees, thighs, lumbar zone, and abdomen (Fig 1, A and B). Most lesions were asymptomatic, erythematous, or violaceous infiltrated plaques. None of the patients complained of cutaneous pain or tenderness. In 2 cases, the lesions were ulcerated. Eight (27%) patients had multiple lesions. Twenty-two (73%) patients had CNS involvement, with a mortality rate of 91%. The most common neurologic manifestations were headache, altered state of consciousness, and focal neurologic deficits with meningeal signs. No additional clinical data were available.

Fig 1

Cutaneous balamuthiasis. A, The classical clinical appearance is a painless plaque on the central face, especially on the nose. B, The knee is the second most common location.

Histopathologic features

Sixty biopsies from 35 patients were evaluated in this study. The histopathologic characteristics of these biopsies are summarized in Table II.

Table II

Histopathologic features of cutaneous balamuthiasis

1. No. of biopsies	60 (100%)
2. Skin layer involvement
Psoriasiform hyperplasia	16 (27%)
Dermal involvement	58 (97%)
Subcutaneous adipose tissue involvement	17 (28%)
3. Type of pattern
a. Granulomatous pattern	50 (83%)
Ill-defined tuberculoid granulomas	45 (75%)
Well-defined tuberculoid granulomas	5 (8%)
b. Lymphoplasmocytic infiltrate	10 (17%)
4. Type of cell
Lymphocytes	60 (100%)
Plasma cells	53 (88%)
Eosinophils	40 (67%)
Neutrophils	29 (48%)
Multinucleated giant cells	52 (87%)
5. Vasculitis	5 (8%)
6. Perineural infiltrate	10 (17%)
7. Amoeba identified in tissue (in 35 patients)	25 (71%)

Most biopsies showed granulomatous dermatitis (50 biopsies, 83%). Psoriasiform hyperplasia was present in 16 (27%) biopsies. Findings were restricted to the dermis in 58 (97%) biopsies, whereas in 17 (28%) biopsies, the subcutaneous fat was also involved. The granulomas were predominantly tuberculoid without caseous necrosis; 45 (75%) cases showed loose and ill-defined granulomas (Fig 2, A and B), whereas only 5 (8%) cases had well-formed, compact epithelioid cell granulomas. Ten biopsies had a lymphoplasmacytic infiltrate without any evidence of granulomas, and only scattered giant cells were present. Fourteen patients did require >2 biopsies for identification of the microorganism on histology.

Fig 2

Cutaneous balamuthiasis. A and B, Histopathologic examination shows a superficial and deep granulomatous infiltrate composed of ill-defined tuberculoid granulomas. Numerous multinucleated giant cells are present within the granulomas and in the interstitial space (A and B, Hematoxylin-eosin stain; original magnifications: A, ×40; B, ×40.)

The accompanying inflammatory infiltrate was predominantly lymphocytic, admixed with other cells, such as plasma cells (88%), eosinophils (67%), and neutrophils (48%). Multinucleated giant cells (Langhans type) were observed in 52 (87%) biopsies, both inside and outside the granulomas (Fig 3). Perineural inflammatory infiltrate was observed in 10 (17%) biopsies. Vasculitis was seen in 5 (8%) biopsies.

Fig 3

Cutaneous balamuthiasis. Granulomatous infiltration with multinucleated giant cells and a Balamuthia mandrillaris trophozoite (Hematoxylin-eosin stain; original magnification: ×400.)

The trophozoite forms of the amoeba, some of them seen within a lacunar space, were identified in biopsies of 25 of 35 (71%) patients. They showed round or irregular morphology (Figs 3 and 4). No cyst forms were detected. Direct immunofluorescence tests for B. mandrillaris were performed in 25 (71%) patients, all of them confirming the diagnosis.

Fig 4

Cutaneous balamuthiasis. A,Balamuthia mandrillaris trophozoite displays the typical oval shape; small, round nucleus with a large nucleolus; and bubbly cytoplasm. An artifactual retraction around the organism gives the appearance of a lacunar space. B, The trophozoites may adopt a more irregular shape and mimic the histiocytes (A and B, Hematoxylin-eosin stain; original magnifications: A, ×1000; B, ×1000.)

Discussion

Four decades ago, the term amoebiasis was restricted to Entamoeba histolytica infection. Since then, free-living amoebas (FLAs) have emerged as important pathogens.,, Among the many FLAs that are found in the environment, 4 genera of FLAs have been associated with human disease. These include 1 species of Naegleria (N. fowleri); 1 species of Balamuthia (B. mandrillaris); several species of Acanthamoeba causing eye, skin, spinal cord, and CNS infections; and Sappinia pedata (previously identified as Sappinia diploidea), for which a single case of amoebic encephalitis has been described.,,

Following the new classification of the eukaryotes by Visvesvara into 6 clusters or “supergroups” (Amoebozoa, Opisthokonta, Rhizaria, Archaeplastida, Chromalveolata, and Excavata), Acanthamoeba and Balamuthia are included under supergroup Amoebozoa: Acanthamoebidae; Naegleria fowleri under supergroup Excavata: Heterolobosia: Vahlkampfiidae, and Sappinia under supergroup Amoebozoa: Flabellinea: Thecamoebidae. From all these, only Acanthamoeba and B. mandrillaris cause cutaneous lesions.

B. mandrillaris was first identified in 1986 in the brain of a pregnant mandrill baboon that died of encephalitis in the San Diego Zoo. The organism was initially classified as leptomyxid amoeba, and in an early publication of human cases, 2 of the 16 patients were from Peru. Subsequent microscopic studies showed that the parasite was morphologically and antigenically different from other amoebas of the order Leptomyxida; thus, a new species-genus and family were established to include this microorganism, which was later renamed in the honor of the 20th century protozoologist, Dr William Balamuth.1, 2, 3,

Epidemiologically, B. mandrillaris infection is reported mainly in the Americas, with few cases reported in other countries such as Japan, Thailand, Australia, Czech Republic, Portugal, and China.,23, 24, 25, 26, 27, 28, 29, 30, 31

To date, approximately 200 cases of granulomatous meningitis by B. mandrillaris have been reported. This may underestimate the real incidence of the disease, considering the lack of pathognomonic findings, the difficulty in recognizing amoebas in skin biopsy tissues, and the fact that the initial cases were regarded as Acanthamoeba sp.,,

The most recent collection of cases of infection by B. mandrillaris from the United States include 109 cases, with 99% seen with a clinical picture of encephalitis. A high proportion of these cases are Hispanic Americans from the southern and southwest states who are more likely to reside in rural settings and therefore may have exposure to soil and a higher chance of contamination of cuts and other injuries.33, 34, 35

Peru has a high prevalence, and 55 cases have been reported from 1975 to 2012, including some of our cases. In addition, cases have been reported in Chile, Argentina, Venezuela, Brazil, and Bolivia during the last 15 years.,,36, 37, 38 This epidemiologic distribution suggests that certain environmental and genetic factors are associated with the development of the disease.,,

Half of the patients in our series were children, an age group that appears to be more susceptible to the infection, in agreement with previous observations from other series.,,,

Our patients came from valleys on the desertic Pacific coast of Peru. This suggests that environmental factors like the ocean-atmosphere system (including the El Nino phenomenon and UV-B radiation) may play a role in this infection. No seasonal variations have been recorded in the epidemiology of B. mandrillaris infection.,,

More than half of our patients had a contact history with stagnant water. Although B. mandrillaris has never been isolated from water, some studies suggest that exposure to stagnant water may be associated with the disease. The most likely source of infection is soil and dust. B. mandrillaris has been isolated from the soil and dust in the United States, Mexico, Iran, Costa Rica, and Jamaica and from the soil in Piura and Lima, Peru.41, 42, 43, 44, 45 A serologic survey of landscapers and blood donors in southern Arizona concluded that exposure to B. mandrillaris (3%) appears to be more common than granulomatous amebic encephalitis.

The route of infection appears to be the respiratory mucosa and skin trauma, a history of which was recorded in 3 of our patients.,, Solid organ transplantation appears to be a concerning and emerging way of transmission.,

None of our patients were immune deficient due to chronic illness, alcoholism, corticosteroid therapy, HIV, or other diseases, in contrast to those affected by Acanthamoeba spp., where immunosuppression is the rule.

A distinct finding in our series is the presence of a cutaneous lesion preceding the CNS involvement in almost all patients. This finding contrasts to what is seen in the United States, where cutaneous lesions were recognized in only 5% of the cases. A recent series of 28 patients from China also demonstrated a high prevalence of cutaneous involvement (100%), and 57% of such cases developed encephalitis. Genetic factors may account for the observed high occurrence of skin involvement in the Peruvian and Chinese patients in contrast to the scarcity of skin lesions reported in the North American cases. Subtle differences in the mechanisms of innate immunity should be investigated to study the role of macrophages at the early phases of the infection.

Cutaneous lesions in our patients were most commonly located in the centrofacial area over the nose, followed by the lower limbs, in agreement with previous reports.,,, Lesions were usually asymptomatic, solitary erythematous to purplish infiltrated plaques with irregular borders and occasional satellites; when multiple, they were fewer than 4. Ulcers were not common.

In some cases, the skin lesion was detected several months or years before the CNS involvement, as in one of our patients, a 21-year-old woman with a history of cutaneous lesions on her right knee for almost 5 years. In other patients, cutaneous lesions and CNS involvement were identified at the same time.

Along with Acanthamoeba spp, B. mandrillaris is the only FLA that has been reported to elicit cutaneous lesions. In contrast to Acanthamoeba skin infections, which commonly present with ulcers and abscesses and histopathologically only rarely show suppurative or necrotizing granulomas with a few multinucleated giant cells (findings that have been attributed to longstanding immunosuppression and inability to mount a granulomatous reaction around the amoebas), our cases of B. mandrillaris infection commonly display a granulomatous pattern on histopathology.51, 52, 53

The most common histopathologic pattern seen in our series was that of ill-defined tuberculoid granulomas with multinucleated giant cells inside and outside the granulomas, associated with a diffuse superficial and deep inflammatory infiltrate composed of lymphocytes, plasma cells, and histiocytes. Neutrophils and eosinophils were rarely present. A novel finding was the presence of perineural inflammation, which was observed in 67% of our biopsies. This suggests that the route of spread from the skin to the nervous system may be ascending along with the adventitial layer of perineural vessels.

We identified trophozoites in 71% of our biopsies, a rate comparable with that of previous studies.,, They can be seen on routine hematoxylin-eosin–staining and are characterized by a spherical nucleus with a large nucleolus and a dense round or irregular cytoplasm containing empty vacuoles, giving a “bubbly” appearance.,, In some cases, the nucleus could have >1 nucleolus and may show a clear cut central space in the nucleoli of the amoebas that was called “nucleolar perforation,” a feature that could distinguish B. mandrillaris from Acanthamoeba.

In our experience, B. mandrillaris trophozoites are difficult to visualize because of their scarcity and histiocyte-like appearance. Sometimes a retraction artifact around the trophozoites was present, giving a lacunar appearance to the organism. Stains such as periodic acid–Schiff or Ziehl-Nielsen did not result in better identification of trophozoites. In contrast to brain lesions of B. mandrillaris, the cutaneous lesions lacked thrombosis, hemorrhage, necrosis, and the cyst stage.,,, Occasional cyst forms have been reported in the skin.,

The clinical and histopathologic differential diagnosis of cutaneous balamuthiasis includes leishmaniasis, sporotrichosis, lupus vulgaris, sarcoidosis, Wegener granulomatosis, necrobiosis lipoidica, and natural killer and T-cell lymphomas.

Ancillary tests such as immunofluorescence, molecular biology techniques, and serology are useful to confirm the infection but are not available in most clinical settings. Thus, routine histopathologic examination remains the most important way to diagnose cutaneous balamuthiasis, with a dramatic impact on the therapy and prognosis of these patients.

Conclusion

Cutaneous balamuthiasis is rare and has a high fatality rate when complicated by CNS involvement. In our experience, the best method for early diagnosis is the integration of clinical and histopathologic findings of skin lesions. In view of that, most of our patients had cutaneous lesions before CNS dissemination. Awareness of the histopathologic findings of B. mandrillaris cutaneous infection, ill-defined granulomas with many multinucleated giant cells and without necrosis, will help dermatopathologists reach the correct diagnosis. With the discovery of the therapeutic effects of new antiparasitic drugs such as miltefosine in treating Balamuthia infections, early diagnosis with subsequent and prompt therapeutic intervention may radically change the prognosis of this otherwise fatal infection.

Conflicts of interest

None disclosed.