Acute Conjunctivitis with Episcleritis and Anterior Uveitis Linked to Adiaspiromycosis and Freshwater Sponges, Amazon Region, Brazil, 2005

An epidemiologic investigation of an ocular disease outbreak among children was linked to the unusual fungus Emmonsia sp., an agent of adiaspiromycosis.


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Learning Objectives
Upon completion of this activity, participants will be able to: Describe the mechanism of infection for adiaspiromycosis.   A diaspiromycosis, caused by the fungus Emmonsia sp., was fi rst identifi ed in Brazil during pathologic examination of lung tissue in a patient with pneumonia who died unexpectedly during treatment (1). Conidia of Emmonsia sp. are commonly present in the environment, mainly in soil and dust, and some studies have shown that pulmonary infection most often results from inhalation (2)(3)(4). Conidia, which also affect other mammals, including marsupials and rodents, do not cause infection; rather, disease is caused by the robust, multicellular immunologic response in tissue against the growing conidia, which results in noncaseating granulomas (2). Human pulmonary adiaspiromycosis has been reported in the literature from multiple countries, including Russia, the Czech Republic, Guatemala, Brazil, and the United States (1-4); disseminated infection may occur in immunocompromised persons. Diagnosis is most frequently made by experienced pathologists who microscopically exam tissue using various stains, including periodic acid-Shiff, which shows large, round, multiwalled structures with surrounding foreign-body-type mixed cellular reactions (3)(4)(5).
On October 26, 2006, local ophthalmologists notifi ed the State Health Secretariat in Tocantins, located in the northern Amazon region of Brazil, of an unusual outbreak of conjunctivitis with ocular nodules of unknown etiology among children. Illness was identifi ed in 17 children, 16 of whom were <15 years of age; all were residents of Araguatins (population = 29,336), a city located along the slow-moving Araguaia River. The disease had remained underdiagnosed and underreported in Araguatins until an ophthalmologic service was initiated in the neighboring city of Augustinópolis, where the initial case-patients were referred, examined, and subsequently reported to local health authorities. Shortly after the condition was reported to Brazil's Ministry of Health, a team of epidemiologists, laboratorians, and ophthalmologists began an investigation with the following objectives: 1) determine the magnitude of the problem, 2) identify the cause, and 3) implement prevention and control activities.

Screening
Because 16 (94%) of the 17 initial case-patients reported were 5-15 years of age, active searches were conducted in 40 of the 41 primary schools in Araguatins. Health workers were trained by ophthalmologists to identify children with clinical signs that were similar to the initial 17 case reports. Children with blurred vision, or any of the following, were referred for an ophthalmologic examination: conjunctival injection or infl ammation, nodules on conjunctiva or sclera, or cornea with any discoloration or opacifi cation.

History
We defi ned a case of confi rmed ocular disease (COD) in a child with any of the following physical signs: conjunctival injection or infl ammation, nodules on sclera, or conjunctival or corneal opacities with anterior uveitis identifi ed during ophthalmologic examination (including by slit-lamp and microscopy). Patients with COD were interviewed by using a standardized semistructured questionnaire; parents served as proxies for young children. Information was collected about basic demographic characteristics, duration and type(s) of symptoms, source of drinking water, frequency and specifi c locations where children had exposure to the local freshwater river, and similar illness in family members.

Case-Control Study
We hypothesized that exposure to the Araguaia River played a role in the chain of events resulting in ocular disease. This hypothesis was tested by using an unmatched case-control study design, based on an estimated 90% of case-patients having prior ocular exposure to river water; and by using 80% power, an α level of 0.05, and a case:control ratio of 1:3, which yielded a study sample size of 62 case-patients and 186 controls. The 62 case-patients included in this study were randomly selected from a total of 91 children with COD identifi ed and interviewed. Two separate control groups were selected for interviews. The fi rst group (community controls) included 186 asymptomatic persons ranging from 5 to 20 years of age living in households systematically selected from randomly chosen residential blocks in the urban area of Araguatins municipality. A second control group (household controls) comprised all asymptomatic residents of case-patient households.

Statistical Analysis
In the univariate analysis of the case-control study data, categorical variables were tested by using a χ 2 test, and continuous variables were compared by using a Kruskall-Wallis or t test, as appropriate. The odds ratio (OR) was used as the measure of association, 95% confi dence intervals (CIs) were calculated, and p<0.05 was considered signifi cant. Using a stepwise backward elimination strategy to calculate the adjusted OR, an unconditional logistic regression model was used for the multivariate analysis.

Laboratory Methods
Serologic tests from children with COD included ELI-SA tests for onchocercosis (immunoglobulin [Ig] G), toxoplasmosis (IgM), and toxocariasis (IgG). Blood smears and aqueous humor from selected patients were examined microscopically for evidence of microfi laria. Biopsy samples from COD case-patients with scleral nodules or corneal abnormalities were fi xed in formalin, stained with hemotoxylin and eosin, and periodic acid-Schiff, and examined microscopically. Soil samples were examined for helminth eggs and larvae. Water samples were collected from areas of the Araguaia River where case-patients reportedly swam. These samples were examined for 1) freshwater sponges, which were identifi ed to species, and 2) silicious spicules (gemmoscleres) of these sponges; details of the methods and results of this sampling have been published (6,7).

Results
In addition to the initial 17 COD case-patients who were examined by ophthalmologists and reported to the Ministry of Health, a total of 5,084 children 5-15 years of age (corresponding to 83% of this age group in the population) were examined at 40 schools by health workers. During these active searches, of 235 students triaged and referred for evaluation of possible ocular abnormalities, 64 (27%) were categorized by ophthalmologists as having COD and 103 (44%) had sequelae. In addition to the total 81 COD case-patients identifi ed above by November 26, 2005, COD was diagnosed for an additional 18 by January 26, 2006, identifi ed initially by local clinicians or through patient self-referral.
Environmental exposures most strongly associated with increased risk for disease, which was signifi cant when compared with both household and community controls ( Table 2), were swimming or diving in the Araguaia River and frequenting Cais beach on the bank of the Araguaia River. Fishing in the river was associated with disease but only when case-patients were compared with community controls. Factors not signifi cantly associated with disease  (using either control group) were drinking untreated river water, washing clothes in the river, contact with various types of domesticated animals, a history of exposure to ticks, or a history of allergies. Frequency of river contact was also signifi cantly associated with disease (Table 3). According to multivariate analysis, factors most strongly associated with disease were being of male sex, frequenting the Cais beach area, and diving underwater in the Araguaia River (Table 4). Among 32 case-patients treated with corticosteroid (oral and/or topical prednisone) by ophthalmologists, disease was resolved or cured in 25 (78%); 7 (22%) casepatients had more severe symptoms and were referred to the Sao Geraldo Hospital in Belo Horizonte, Minas Gerais State.
Among those with nodules, 14 had biopsy samples taken under sterile surgical conditions for diagnostic purposes. Microscopic examination of nodules identifi ed microulcerations of corneal epithelium (Figure 4), and a mixed acute infl ammatory response mainly consisting of leukocytes, with some eosinophils, and lymphohistocytic and diffuse neutrophilic infi ltrates with edema. Twelve case-patients (13%) had a granuloma of the anterior chamber of the eye unilaterally; 1 case-patient had bilateral anterior chamber granulomas. In addition, in 2 biopsy samples, subconjunctival infl ammation was present surrounding large, 200-600micron, thick-walled, spherical foreign bodies ( Figure 5) consistent with adiaconidia of Emmonsia sp. fungus, a cause of adiaspiromycosis.

Discussion
These results confi rm the existence of an outbreak of conjunctivitis and severe ocular disease probably caused by adiaspiromycosis, mainly among school-aged children. Risk factors for COD identifi ed in this investigation included diving underwater and frequenting a specifi c beach (Cais) on the Araguia River in the Amazon region of Brazil. The precise reasons eye contact with river water increased risk remain unclear. Perhaps exposure to freshwater sponge spicules caused an initial conjunctival irritation, as suggested in previous publications (6)(7)(8). However, the microscopic identifi cation of probable adiaconidia of Emmonsia sp. fungus in the scleral biopsy samples from children with severe disease in this outbreak suggests that conjunctival irritation was most likely followed by conjunctival exposure to conidia of this fungus, perhaps in dust caused in part by dry environmental conditions, similar to the exposure of respiratory mucosa described in case reports of pulmonary adiaspiromycosis (1)(2)(3)(4)(5)9). Adiaspiromycosis causes an infl ammatory and often granulomatous response in tissue because of the presence of nonbudding, thick-walled adiaconidia of Emmonsia sp. fungus (10). Disease is thought to result from exposure to conidia (through inhalation or mucosal contact with dust); these conidia subsequently cause a marked infl ammatory response and enlarge to become adiaconidia ranging in diameter from 300 to 600-micorons (1)(2)(3)(4)(5)9,10).
Boys were at higher risk than girls most likely because boys had more facial and eye contact with the river water while swimming and diving. To minimize bias, we randomly selected asymptomatic controls among persons 5-25 years of age in the community, but some selection bias may have resulted because boys and adolescents were absent at the time of interview (only 42% of communitybased controls were boys). The clinical characteristics of conjunctivitis in this outbreak were unusual for several reasons. First, unlike conjunctivitis caused by common bacterial or viral pathogens, neither purulent conjunctival discharge nor hemorrhage was reported, and family members of case-patient households were not commonly affected. In addition, disease was characterized by unusual, single or multiple, white, opaque scleral nodules, often with hyperemia or local edema, and in some cases with opacifi cation (changes in the normally transparent characteristics of the cornea or superfi cially on scleral tissue) extending to the limbus, or angular corneal opacities and anterior uveitis with granulomas in the anterior chamber. We believe that the clinical improvement of nearly all patients treated with corticosteroids also argues strongly against a bacterial cause or fungal species other than Emmonsia because conidia of Emmonsia sp. enlarge and cause a localized infl ammatory response but do not commonly have the potential to disseminate.
Characteristics of this outbreak are similar to those of an outbreak of anterior uveitis and granuloma previously reported in India, where the etiology was traced to trematodes (11). Although the thick-walled foreign body we observed microscopically on slides from 2 case-patients was initially suspected to be trematodes, the round, apparently spherical shape, thick walls, and vacuous central area with lack of organized, internal structures is most consistent with the adiaconidia of the Emmonsia sp. Morphologic appearance differs from that of the fungus Coccidiodes immitis, in which spores contain internal microsporules (10).  The natural history of this disease is unknown. However, we identifi ed COD case-patients in several stages of disease, including patients with sequalae. Moreover, after obtaining school surveys, we identifi ed ≈5% of children with ocular abnormalities; COD was diagnosed in one third of children after an ophthalmologic exam. We educated the population about risks for eye contact with river water; active searches were conducted to identify all ill persons in the population and in neighboring cities, and health offi cials limited recreational access to the Araguaia River. These fi ndings suggest that the extent of this problem may be more widespread in the Amazon region than is currently recognized.