Transactions of the Royal Society of Tropical Medicine and Hygiene Advance Access published August 5, 2014
ORIGINAL ARTICLE
Trans R Soc Trop Med Hyg doi:10.1093/trstmh/tru113
Epidemiological and serological profiles of ocular toxoplasmosis in the municipality of Natal, northeastern Brazil Norma H. D. Mendesa, Claudio B. S. Oliveiraa, Carlos A. Garciab, Cecı´lia M. X. C. Holandac and Valter F. Andrade-Netoa,* a
Laborato´rio de Biologia da Mala´ria e Toxoplasmose - LaBMAT, Departamento de Microbiologia e Parasitologia. Centro de Biocieˆncias. Universidade Federal do Rio Grande do Norte (UFRN), 59078–970, Natal/RN, Brazil; bDepartamento de Oftalmologia, Hospital Universita´rio Onofre Lopes, Complexo Hospitalar e de Sau´de, UFRN, 59.012–300, Natal/RN, Brazil; cLaborato´rio de Ana´lises Clı´nicas Central, Hospital Universita´rio Onofre Lopes, Complexo Hospitalar e de Sau´de, UFRN, 59.012–300, Natal/RN, Brazil
Received 1 April 2014; revised 19 April 2014; accepted 4 June 2014 Background: Toxoplasma gondii is the main culprit in most cases of infectious uveitis, in both acute and recurrent cases of congenital toxoplasmosis and acquired infections. Methods: The ocular toxoplasmosis was evaluated in patients at the the reference unit in ophthalmology, in Rio Grande do Norte State, determining the risk factors, and the epidemic, serological and clinical profiles. The production of IgM and IgG antibodies to T. gondii was evaluated by microparticle enzyme immunoassay (MEIA). The same patients diagnosed with fundoscopic alterations have been subjected to the fundus photography procedure. Results: Of the 116 patients with positive serology, 66 patients had bilateral ocular damage and 38 patients showed a higher frequency of lesions of type I. The epidemiological investigation showed that direct contact with cats, the consumption of raw or undercooked meat and direct contact with soil are factors not related to ocular toxoplasmosis development. The characterization of the sample was significant for patients aged 31–40 years. Conclusions: Ocular toxoplasmosis is widely distributed in Natal and other cities in Rio Grande do Norte state, with special relevance for bilateral lesions in 56.9% of the patients assessed, the most frequent being type I with intraocular disposition in the macula. Keywords: Brazil, Ocular toxoplasmosis, Risk factors, Seroprevalence, Uveitis
Introduction Toxoplasmosis is one of the most seroprevalent protozoonoses worldwide. The infection is caused by an intracellular parasite, the protozoan Toxoplasma gondii. This parasite is found on all continents and in various climates, characterizing it as a cosmopolitan etiological agent.1,2 Its evolutionary success is likely due to its immunomodulatory mechanisms, which allow it to avoid or penetrate host cell defenses.3 The ocular form of toxoplasmosis may be congenital, which exhibits early or late clinical manifestations, or may be acquired after birth.1 This ocular involvement is more common and more severe in patients whose immune system is compromised.4 The importance of ocular research is given by the high incidence of ocular lesions associated with the severity of some infections in visual function, in addition to the high frequency in women of childbearing age who are vulnerable to the parasite, in Natal
and some cities in the metropolitan area of Rio Grande do Norte state.5 Susceptibility to the development of severe eye lesions may be related to the virulence of the parasite, size of infecting inoculum, and changes in the environment, which affect the host–parasite relationship, particularly the immune system of the host. 6 Due to the severity and irreversibility of lesions caused by T. gondii, knowledge about the social and epidemiological factors of this infection is of paramount importance in order to apply the adequate intervention, primarily in cases where the clinical profile and serological test results are inconclusive or late. In a number of countries, the prevalence of ocular infections attributed to this parasite is high.7–9 According to Abreu et al. 38% of the posterior uveitis cases were of toxoplasmic aetiology.10 In a study conducted in Sa˜o Paulo, Abreu et al. found an incidence of 88% of posterior uveitis caused by T. gondii.11
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*Corresponding author: Tel: +55 84 3215-3437; Fax: +55 84 3211-9210; E-mail: [email protected]
N. H. D. Mendes et al.
However, in studies of younger patients (≤12 years old) in Natal, Garcia12 found a prevalence of 1.2%. Thus, further studies should be conducted, considering the lack of information on the epidemiological chain of ocular toxoplasmosis in the state of Rio Grande do Norte. This study aims at contributing to better understanding of the clinical form of this disease, by relating it to factors associated with the lifestyle of patients as well as describing the epidemic, serological and clinical profile of the affected individuals.
Methodology Delimitation of the research and application of the epidemiological questionnaire
Statistical analysis TheYates-corrected x2 test was used to compare proportions. Fisher’s exact test was used to calculate the qualitative variables. Association of risk was determined using the OR. The values were considered significant when the p-value was ≤0.05.
Results Population A total of 190 patients agreed to participate. Among the population diagnosed with uveitis (n¼159), a total of 73.0% (116) were positive for high IgG anti-T. gondii avidity. The remaining 27.0% (n¼43), non-reactive to the immunoglobulins analyzed, were classified as having uveitis resulting from other causes. Most patients with positive serology for ocular toxoplasmosis (61.2%, n¼71) had anti-T. gondii IgG levels between 40 and 99 IU/mL. No patient was positive for IgM anti-T. gondii, thereby ruling out active infections.
Immunoenzymatic assay
Epidemiological characteristics
Blood samples were initially obtained by venipuncture. The serum was separated by centrifugation and preserved in a freezer at 2208C until its use. The production of anti-Toxoplasma gondii IgM and IgG antibodies was evaluated by microparticle enzyme immunoassay (MEIA) (Abbott AxSYM system, Abbott Laboratories, Chicago, IL, USA). The sensitivity and specificity of the test was 96.3%, and 99.8%, respectively. Patients with positive IgG but negative IgM titers were considered chronically infected. Patients with positive titers for both IgG and IgM, were considered suggestive for recent infections. The IgG avidity test was used to discriminate between old and recent infections. A group of patients with negative serology for T. gondii was used as a control group.
No statistical significance was found for the following risk factors analyzed in the present study: regular trash collection/presence in rural areas; contact with insect vectors; contact with soil/gardening, hygiene habits/basic sanitation; contact with soil/gardening; consumption of raw or undercooked foods; intake of unfiltered and/or unboiled water; and raising domestic animals, including dogs and cats. With respect to the geographical distribution of patients with ocular toxoplasmosis, 64.6% (n¼75) were from Natal and 35.4% (n¼41) were from other municipalities in the state of Rio Grande do Norte. Distribution in the districts of Natal was as follows: 20.0% (n¼15) from the north, 8.0% (n¼6) from the south, 37.3% (n¼28) from the east, and 34.7% (n¼26) from the west (Table 1). A higher seropositivity was observed in females, 64.6% (n¼75), compared to males, 35.4% (n¼41) (Table 1). The most significant age range was between 31 and 40 years old (n¼33, p¼0.04). A monthly income of up to one minimum wage (53.5%) was the most commonly observed (n¼62, p¼0.52), with a trend to a positive association for a larger number of cases in individuals with no schooling (36.2%; n¼42, p¼0.06).
Clinical ophthalmological examinations The patients underwent an eye examination, consisting of ocular inspection with a flashlight directing the beam parallel to the outer palpebral junction. This method evaluated anterior chamber depth, near visual acuity of both eyes (using Jaeger’s optotypes placed 33 cm from the eyeball) and investigated extrinsic eye muscle function. Fundoscopy was performed by a retinologist using binocular indirect ophthalmoscopy (Slit Lamp Haag Streit Keeler and external lens of 78 diopters, Keeler Instruments Inc., Broomall, PA, USA). This occurred after one drop of tropicamide (Mydriacyl,
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Clinical characterization of the lesions Among the 116 ocular toxoplasmosis-reactive patients, 56.9% exhibited bilateral lesions (n¼66). The analysis of 49 fundus
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The study was conducted at the Ophthalmology Ambulatory Clinic of the Onofre Lopes University Hospital, Federal University of Rio Grande do Norte, Natal, Brazil. Patients who visited the Clinic were invited to participate in the study after giving their written informed consent. A cross-sectional study was performed between September 2008 and September 2011, with a population of individuals from both sexes ranging from 15–60 years old and who were subjected to any ophthalmological assistance. A questionnaire was given to the patients in order to obtain details that could be associated with toxoplasmosis, as follows: age; place of residence, including the regular trash collection and the presence in rural areas; socioeconomic and cultural status; contact with soil/gardening, hygiene habits/basic sanitation; consumption of raw or undercooked food; intake of unfiltered and/or unboiled water; raising domestic animals, including dogs or cats; and the presence of potential vectors in the residence (cockroaches and flies).
Alcon, Sa˜o Paulo, Brazil) eye drop (1%) was applied to the conjunctival sac (2x every 10 minutes) of each eye in order to promote mydriasis. Patients diagnosed with fundoscopic alterations underwent retinography with a digital retinographer (Zaiss 450 Retinograph; Carl Zeiss Meditec, Vertriebsgesellschaft mbH, Oberkochen, Germany). Images were evaluated by OIS digital imaging system. Ocular differential diagnoses for other diseases were excluded from the study. A patient group with a negative serology for T. gondii and lesions caused by other etiologic agents was used as a control.
Transactions of the Royal Society of Tropical Medicine and Hygiene
Table 1. Epidemiological characteristics of the reactive or non-reactive patients treated at the Hospital Universita´rio Onofre Lopes, Natal/Rio Grande do Norte, towards Toxoplasma gondii IgG antibodies %
Non-reactive n¼43
%
75 41
64.7 35.3
8 35
18.6 81.4
75 15 6 28 26
64.6 20.0 8.0 37.3 34.7
22 8 2 7 5
51.2 36.4 9.1 31.8 22.8
15 20 33 29 19
12.9 17.2 28.4 25.0 16.3
9 15 8 5 6
20.9 34.9 18.6 11.7 13.5
photographs (Table 2), using established parameters,13 obtained from 38 patients showed a higher frequency of type I lesions (80%). Type III lesions always occurred together with Type I or, less frequently, with the Type I and II. In relation to intraocular disposition (Table 3), 49% of the lesions (n¼24) were observed in the macular region while 47% were retinal (n¼23). Macular and retinal lesions were detected in 4% (2) of the subjects (Figure 1).
Discussion Toxoplasmosis is a parasitic disease that is still neglected in many parts of the world and consequently both the infection and its clinical manifestations are poorly understood in these regions. The scarcity of studies on ocular toxoplasmosis, in Brazil, especially in the north and northeast regions, is worrisome, since the parasite is the main agent involved in posterior uveitis.9,14 This becomes even more alarming when we observe that more than half of the world’s population may be infected with the protozoan.15 T. gondii is a ubiquitous parasite with various infectious mechanisms16; the infection can be congenital or acquired and, in both cases, the parasite may invade different cell types, including neuroretina and choroid cells. At these sites, the parasite usually assumes a latent form (bradyzoites) and remains in this stage for many years, with sporadic reactivation episodes, which are likely responsible for immune system activation and resulting eye damage.17 The increase of studies involving ocular toxoplasmosis provides knowledge of variations that can occur within different regions, and socioeconomic and cultural conditions. This fact justifies
Type of lesion
Eyes (n¼49) n (%)
Type I only Type II only Types I and II Types II and III Types I, II and III
39 (80) 4 (8) 2 (4) 3 (6) 1 (2)
The classification of the lesion were considered lesions consistent with toxoplasmosis according to Ore´fice.13 Type I: limits and marked with a halo of hyperpigmentation and central area of chorioretinal atrophy. Type II: lesions with hyperpigmented halo and hypopigmented central area. Type III: hyperpigmented or hypopigmented lesions compatible with hyperplasia or atrophy of the retinal pigment epithelium.
Table 3. Classification of the ocular toxoplasmosis lesions, according to the intraocular disposition, Hospital Universita´rio Onofre Lopes, Natal/Rio Grande do Norte Classification of lesions
Eyes (n¼49) n (%)
Macular only Retinal only Both macular and retinal
24 (49) 23 (47) 2 (4)
further studies to define the problem and present the case series to public health authorities. The prevalence of the toxoplasmic uveitis observed in this study was high in relation to other regions of Brazil, as reported by Abreu et al.10 in Venda Nova do Imigrante, Espirito Santo state and Ore´fice; Fernandes18 in Minas Gerais state. A comparison between the results obtained in this study and those reported in international publications revealed a higher frequency than that found by Ronday et al.19 in Sierra Leone, Africa, and by London9 in California, indicating that this region harbors an atypical form of circulating parasites, which could explain the high prevalence. In regard to the origin of the patients, data show a predominance of individuals from Natal, which was expected, given that the reference hospital in this study is located in this city. A majority of the patients were from the eastern and western districts. This is likely because the university hospital treats patients from the public health system, which serves lower income individuals who are not covered by health insurance plans. This also explains the finding, albeit not statistically significant, that the most common monthly income found was up to one minimum wage, corroborating the results reported by Ore´fice and Fernandes.18
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Gender Female Male District Natal (Total) North South East West Age 15–20 21–30 31–40 41–50 .50
Reactive n¼116
Table 2. Classification of the ocular toxoplasmosis lesions, according to the type of lesion, Hospital Universita´rio Onofre Lopes, Natal/Rio Grande do Norte
N. H. D. Mendes et al.
In terms of age, the range of significance (31–40 years old) was higher than that found by Tabatabaei et al.20 and very close to that observed by Aleixo et al.21 The significant age group of this study is expected considering that the prevalence of toxoplasmosis-reactive patients generally increases with age, due to longer exposure to the risk factors.22 The higher incidence of the disease, which is observed in women, can be explained by their high level of exposure to the cysts found in raw meat during its preparation and no improved hygiene practices in meat production.23 This finding is similar to that observed by Garcia et al.24 and Tabatabaei et al.20 Camargo et al.25 observed increased toxoplasmosis incidence in men. This difference is likely associated with greater consumption of raw meat or exposure to oocysts in soil. Despite the high frequency of ocular toxoplasmosis, the bilateral lesions found in most patients were the most surprising finding, since most national and international studies show that ocular toxoplasmosis affects only one eye, causing unilateral damage.9,18,21,24 It is suggested that this is a common characteristic in Brazil.18,21 Ocular toxoplasmosis, which can cause long-lasting or permanent effects, is found in cataracts, glaucoma, macular edema, scarring, and retinal detachment. Despite causing more damage in immunocompromised patients, and being normally asymptomatic in immunocompetent individuals, additional damage may occur progressively in reactivation episodes, thereby affecting visual acuity.26 These progressive losses become particularly significant when the lesion is bilateral since it will result in greater host morbidity due to the loss of visual acuity. Our
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Figure 1. Fundus photographs of the lesions of the type I (A), type II (B), types I and II (C) and types II and III (D). In (A), there is a clearly marked lesion with a hyperpigmented halo (arrow) and a hypopigmented center. In (B), there is an unmarked lesion with a hypopigmented halo and hyperpigmented core. In (C), there is a clearly marked central lesion with hyperpigmented core and halo. In (D), multiple lesions can be observed. This figure is available in black and white in print and in colour at Transactions online.
group is investigating some of the causes of immunophysiopathological bilaterality (data not shown), and the findings should be published soon (probably 2015). The bilaterality of these lesions, also observed by Soares et al.,27 can be partially explained by the characteristics of the parasite. Recent studies have demonstrated that the serotype of T. gondii appears to be associated with the development of ocular toxoplasmosis.28 Thus, the fact that this protozoan exhibits polymorphisms,29 mainly in South American countries such as Brazil,30 might partially explain this divergence in regional casuistics. In Sa˜o Paulo Ferreira et al.31 showed that patients harbored polymorphic strains, although two of them showed a single genotype at nine genetic loci. These data indicate a high rate of recombination of archetypes I, II and III. These characteristics may be correlated with the disease severity observed in Brazilian patients. Although genotypes I and II of Toxoplasma have predominated in Brazil, exhibiting high and moderate pathogenicity, respectively,30,32 our group has recently identified a number of atypical genotypes of this protozoan, widely circulating in various animals used for human consumption. Some of these isolates were unprecedented in the literature and most were classified as pathogenics.33 The finding of atypical and pathogenic strains circulating in the region could also explain the clinical features observed in this study. Another possible explanation is the difference in patient susceptibility, due to ethnic variability. According to Sibley et al.34 the factors that control the occurrence of ocular diseases are poorly understood, although a variety of components, including genetic susceptibility of the host, nutritional, immunological and parasitic load status may be involved in the development of infection. Type I ocular lesions with hyperpigmented edges are considered the gold standard for clinical diagnosis of ocular toxoplasmosis. Thus, the prevalence of this lesion was consistent with other studies.21,35 The large number of type I lesions is justified given the high prevalence of ocular toxoplasmosis observed here. Type III lesions, although nonspecific, were included in the study because the literature shows that atypical lesions may also recur with a typical clinical picture.36 However, no cases of this lesion isolated were observed, possibly because of its low incidence. Usually, this type of injury does not manifest separately. It was possible to observe a predominance of macular lesions, followed by retinal lesions, corroborating studies conducted with children and the general population.20,27 According to Yang et al.,37 anatomical and microvascular differences between the macula and the peripheral region of the retina may create a microenvironment that influences the central location of the lesion, which may be a predisposing factor for the higher frequency of lesions in the macula lutea. Moreover, a survey carried out on the eyes of postmortem patients showed a higher number of macrophages defending against T. gondii in the retina,37 which once again could explain the higher frequency of macular lesions. Although not statistically significant, risk factors such as direct contact with cats, consumption of raw or undercooked meat and contact with soil were associated with ocular lesions caused by T. gondii, as demonstrated by the OR test. These data differ from those reported by a study in Natal/RN conducted by Barbosa, Holanda and Andrade-Neto.5 The authors concluded that contact with cats was associated with toxoplasmosis. However, this positive association between these risk factors and ocular toxoplasmosis in Rio Grande do Norte should not be
Transactions of the Royal Society of Tropical Medicine and Hygiene
dismissed. In addition, a recent study in Brazil showed that the presence of dogs and cats, as well as the consumption of raw or undercooked meat did not influence the development of ocular toxoplasmosis, despite being associated with increased risk for T. gondii infection.38 Finally, this study is of great importance to public health, since it is the first to show the prevalence of ocular toxoplasmosis in the general population of Rio Grande do Norte state. Moreover, it demonstrated the high frequency of uveitis cases and, notably, a pattern of bilateral damage, suggesting that these lesions may be associated with the atypical histologic patterns of local circulating strains. Further studies should be conducted to characterize the immunopathological pattern related to this type of injury and its association with the field isolates obtained.
Funding: This work was supported by grants from National Council for Scientific and Technological Development-CNPq [575840/2008–0 and 301837/2012–0]. Competing interests: None declared. Ethical approval: The study was approved by the Ethical Committee of Onofre Lopes University Hospital, Federal University of Rio Grande do Norte, Natal, Brazil (CEP/HUOL 013/07).
References 1 Dressen DW. Toxoplasma gondii. J Am Vet Med Assoc 1990;196:274–6. 2 Jones JL, Muccioli C, Belfort R et al. Recently acquired Toxoplasma gondii infection, Brazil. Emerg Infect Dis 2006;172:1561–6. 3 Carruthers VB, Suzuki Y. Effects of Toxoplasma gondii infection on the brain. Schizophr Bull 2007;33:745–51. 4 Hovakimyan A, Cunningham ET. Ocular toxoplasmosis. Ophthalmol Clin North Am 2002;15:32–32. 5 Barbosa IR, Holanda CMCX, Andrade-Neto VF. Toxoplasmosis screening and risk factors amongst pregnant females in Natal, northeastern Brazil. T Roy Soc Trop Med H 2009;103:377–82. 6 Holland GN, Crespi CM, Ten Dam-Vam N et al. Analysis of recurrence patterns associated with toxopla´smica retinochoroiditis. Am J Ophthalmol 2008;145:1007–13.
11 Abreu MT, Hirata OS, Belfort R JR et al. Uveı´tes em Sa˜o Paulo – Estudo epidemiolo´gico, clı´nico e terapeˆutico. Arq Bras Oftal 1980;43:10–6. 12 Garcia CA, Ore´fice F, Oliveira LC et al. Socioeconomic conditions as determining factors in the prevalence of systemic and ocular toxoplasmosis in Northeastern Brazil. Ophthalmic Epidemiol 2004; 11:301–17. 13 Ore´fice F, Bahia-Oliveira LMG. Toxoplasmose. In: Ore´fice F. Uveı´te clı´nica e ciru´rgica. 2. ed. Rio de Janeiro: Cultura Me´dica; 2005; p. 699–703. 14 Hooper C, McCluskey P. Intraocular inflammation: its causes and investigations. Curr Allergy Asthma Rep 2008;8:331–8. 15 Jones JL, Kruszon-Moran D, Wilson M et al. Toxoplasma gondii infection in the United States: seroprevalence and risk factors. Am J Epidemiol 2001;154:357–65. 16 Dubey JP, Lindsay DS, Speer CA. Structures of Toxoplasma gondii tachyzoites, bradizoytes, and sporozoytes and biology and development of tissue cysts. Clin Microbiol Rev 1998;11:267–99. 17 Pineles S, Rikkers SM, Levinson RD. Ocular toxoplasmosis. In: Yanoff M, Duker JS (eds.). Ophthalmology. 3rd ed. Maryland Heights, MO: Mosby; 2009. 18 Ore´fice F, Fernandes LC. Aspectos clı´nicos e epidemiolo´gicos das uveı´tes em Servic¸os de Refereˆ ncia em Belo Horizonte de 1970 a 1993. Rev Bras Oftalmol 1996;55:569–78. 19 Ronday MJ, Stilma JS, Barbe RF et al. Blindness from uveitis in a hospital population in Sierra Leone. Br J Ophthalmol 1994;78:690–3. 20 Tabatabaei S, Soleimani M, Foroutan A et al. Ocular toxoplasmosis in Iran: 40 cases analysis. Int J Ophthalmol 2011;4:199–01. 21 Aleixo AL, Benchimol EI, Neves ES et al. Frequency of lesions suggestive of ocular toxoplasmosis among a rural population in the state of Rio de Janeiro. Rev Soc Bras Med Trop 2009;42:165–9. 22 Labalette P, Delhaes L, Margaran F et al. Ocular toxoplasmosis after the fifth decade. Am J Ophthalmology 2002;133:506–15. 23 Nogareda F, Le Strat Y, Villena I et al. Incidence and prevalence of Toxoplasma gondii infection in women in France, 1980–2020: model-based estimation. Epidemiol Infect 2014;142:1661–70. 24 Garcia JL, Navarro IT, Ogawa L et al. Soroprevaleˆncia, epidemiologia e avaliac¸a˜o ocular da toxoplasmose humana na zona rural de Jaguapita˜ (Parana´), Brasil. Rev Panam Salud Publica 1999;6:157–63. 25 Camargo NE, Anele E, Rubim R et al. High prevalence of congenital toxoplasmosis in Brazil estimated in a 3-year prospective neonatal screening study. Int J Epidemiol 2000;29:941–7. 26 Saffra NA, Seidman CJ, Weiss LM. Ocular toxoplasmosis: controversies in primary and secondary prevention. J Neuroinfect Dis 2013; 4: 235689. 27 Soares JAS, Na´sser LS, Carvalho SFG et al. Achados oculares em crianc¸as com toxoplasmose congeˆnita. Arq Bras Oftalmol 2011;74:255–7.
7 Talabani H, Mergey T, Yera H et al. Factors of occurrence of ocular toxoplasmosis. A review. Parasite 2010;17:177–82.
28 Shobab L, Pleyer U, Johnsen J et al. Toxoplasma serotype is associated with development of ocular toxoplasmosis. J Infect Dis 2013;208: 1520–8.
8 Jakob E, Reuland MS, Mackensen F et al. Uveitis subtypes in a German interdisciplinary uveitis center-analysis of 1916 patients. J Rheumatol 2009;36:127–36.
29 Dutra MS, Bela SR, Peixoto-Rangel AL et al. Association of a NOD2 gene polymorphism and t-helper 17 cells with presumed ocular toxoplasmosis. J Infect Dis 2013;207:152–63.
9 London NJS, Hovakimyan A, Cubillan LDP et al. Prevalence, clinical characteristics, and causes of vision loss in patients with ocular toxoplasmosis. Eur J Ophthalmol 2011;21:811–9.
30 Khan A, Jordan C, Muccioli C et al. Genetic divergence of Toxoplasma gondii strains associated with ocular toxoplasmosis, Brazil. Emerg Infect Dis 2006;12:942–9.
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Authors’ contributions: VFAN participated in the conception and design of the study; NHDM carried out all the tests, CBSO and VFAN supervised data collection and interpreted the data; CMCXH supervised and provided substantial assistance in many of the immunoassays; CAG supervised and significantly helped with much of the clinical assessment; all authors read and approved the final manuscript. VFAN is the guarantor of the paper.
10 Abreu MT, Belfort R Jr, Passos A et al. Toxoplasmose ocular em Venda Nova do Imigrante, ES, Brasil. Arq Bras Oftal 1998;61:540–5.
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31 Ferreira I, Mattos C, Ferreira A et al. Genotipagem de isolados de Toxoplasma gondii provenientes de pacientes com toxoplasmose ocular residentes em Sa˜o Jose do Rio Preto, SP. Rev Sau´de 2010;4:1.
35 Glasner PD, Silveira C, Kruszon-Moran D et al. An unusually high prevalence of ocular toxoplasmosis in southern Brazil. Am J Ophthalmol 1992;114:136–44.
32 Vallochi AL, Nakamura MV, Achlensinger D et al. Ocular toxoplasmosis: more than just what meet the eye. Scand J Immunol 2002;55:324–8.
36 Silveira C, Belfort R JR, Muccioli C et al. A follow-up study of Toxoplasma gondii infection in southern Brazil. Am J Ophthalmol 2001;131:351–4.
33 Andrade MMC, Pinheiro BV, Cunha MM et al. New gentotypes of Toxoplasma gondii obtained from farm animals in Northeast Brazil. Res Vet Sci 2013;94:587–9. 34 Sibley LD, Mordue DG, Su C et al. Genetic approaches to studying virulence and pathogenesis in Toxoplama gondii. Philos Trans R Soc Lond B Bio Sci. 2002;357:81–8.
37 Yang HJ, Jin KN, Park YK et al. Seroprevalence of toxoplasmosis in the residents of Cheju island, Korea. Korean J Parasitol 2000; 38:91–3. 38 Ferreira AIC, Branda˜o de Mattos CC, Frederico FB et al. Risk factors for ocular toxoplasmosis in Brazil. Epidemiol Infect 2014;142:142–8.
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ORIGINAL ARTICLE
Trans R Soc Trop Med Hyg doi:10.1093/trstmh/tru113
Epidemiological and serological profiles of ocular toxoplasmosis in the municipality of Natal, northeastern Brazil Norma H. D. Mendesa, Claudio B. S. Oliveiraa, Carlos A. Garciab, Cecı´lia M. X. C. Holandac and Valter F. Andrade-Netoa,* a
Laborato´rio de Biologia da Mala´ria e Toxoplasmose - LaBMAT, Departamento de Microbiologia e Parasitologia. Centro de Biocieˆncias. Universidade Federal do Rio Grande do Norte (UFRN), 59078–970, Natal/RN, Brazil; bDepartamento de Oftalmologia, Hospital Universita´rio Onofre Lopes, Complexo Hospitalar e de Sau´de, UFRN, 59.012–300, Natal/RN, Brazil; cLaborato´rio de Ana´lises Clı´nicas Central, Hospital Universita´rio Onofre Lopes, Complexo Hospitalar e de Sau´de, UFRN, 59.012–300, Natal/RN, Brazil
Received 1 April 2014; revised 19 April 2014; accepted 4 June 2014 Background: Toxoplasma gondii is the main culprit in most cases of infectious uveitis, in both acute and recurrent cases of congenital toxoplasmosis and acquired infections. Methods: The ocular toxoplasmosis was evaluated in patients at the the reference unit in ophthalmology, in Rio Grande do Norte State, determining the risk factors, and the epidemic, serological and clinical profiles. The production of IgM and IgG antibodies to T. gondii was evaluated by microparticle enzyme immunoassay (MEIA). The same patients diagnosed with fundoscopic alterations have been subjected to the fundus photography procedure. Results: Of the 116 patients with positive serology, 66 patients had bilateral ocular damage and 38 patients showed a higher frequency of lesions of type I. The epidemiological investigation showed that direct contact with cats, the consumption of raw or undercooked meat and direct contact with soil are factors not related to ocular toxoplasmosis development. The characterization of the sample was significant for patients aged 31–40 years. Conclusions: Ocular toxoplasmosis is widely distributed in Natal and other cities in Rio Grande do Norte state, with special relevance for bilateral lesions in 56.9% of the patients assessed, the most frequent being type I with intraocular disposition in the macula. Keywords: Brazil, Ocular toxoplasmosis, Risk factors, Seroprevalence, Uveitis
Introduction Toxoplasmosis is one of the most seroprevalent protozoonoses worldwide. The infection is caused by an intracellular parasite, the protozoan Toxoplasma gondii. This parasite is found on all continents and in various climates, characterizing it as a cosmopolitan etiological agent.1,2 Its evolutionary success is likely due to its immunomodulatory mechanisms, which allow it to avoid or penetrate host cell defenses.3 The ocular form of toxoplasmosis may be congenital, which exhibits early or late clinical manifestations, or may be acquired after birth.1 This ocular involvement is more common and more severe in patients whose immune system is compromised.4 The importance of ocular research is given by the high incidence of ocular lesions associated with the severity of some infections in visual function, in addition to the high frequency in women of childbearing age who are vulnerable to the parasite, in Natal
and some cities in the metropolitan area of Rio Grande do Norte state.5 Susceptibility to the development of severe eye lesions may be related to the virulence of the parasite, size of infecting inoculum, and changes in the environment, which affect the host–parasite relationship, particularly the immune system of the host. 6 Due to the severity and irreversibility of lesions caused by T. gondii, knowledge about the social and epidemiological factors of this infection is of paramount importance in order to apply the adequate intervention, primarily in cases where the clinical profile and serological test results are inconclusive or late. In a number of countries, the prevalence of ocular infections attributed to this parasite is high.7–9 According to Abreu et al. 38% of the posterior uveitis cases were of toxoplasmic aetiology.10 In a study conducted in Sa˜o Paulo, Abreu et al. found an incidence of 88% of posterior uveitis caused by T. gondii.11
# The Author 2014. Published by Oxford University Press on behalf of Royal Society of Tropical Medicine and Hygiene. All rights reserved. For permissions, please e-mail: [email protected].
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*Corresponding author: Tel: +55 84 3215-3437; Fax: +55 84 3211-9210; E-mail: [email protected]
N. H. D. Mendes et al.
However, in studies of younger patients (≤12 years old) in Natal, Garcia12 found a prevalence of 1.2%. Thus, further studies should be conducted, considering the lack of information on the epidemiological chain of ocular toxoplasmosis in the state of Rio Grande do Norte. This study aims at contributing to better understanding of the clinical form of this disease, by relating it to factors associated with the lifestyle of patients as well as describing the epidemic, serological and clinical profile of the affected individuals.
Methodology Delimitation of the research and application of the epidemiological questionnaire
Statistical analysis TheYates-corrected x2 test was used to compare proportions. Fisher’s exact test was used to calculate the qualitative variables. Association of risk was determined using the OR. The values were considered significant when the p-value was ≤0.05.
Results Population A total of 190 patients agreed to participate. Among the population diagnosed with uveitis (n¼159), a total of 73.0% (116) were positive for high IgG anti-T. gondii avidity. The remaining 27.0% (n¼43), non-reactive to the immunoglobulins analyzed, were classified as having uveitis resulting from other causes. Most patients with positive serology for ocular toxoplasmosis (61.2%, n¼71) had anti-T. gondii IgG levels between 40 and 99 IU/mL. No patient was positive for IgM anti-T. gondii, thereby ruling out active infections.
Immunoenzymatic assay
Epidemiological characteristics
Blood samples were initially obtained by venipuncture. The serum was separated by centrifugation and preserved in a freezer at 2208C until its use. The production of anti-Toxoplasma gondii IgM and IgG antibodies was evaluated by microparticle enzyme immunoassay (MEIA) (Abbott AxSYM system, Abbott Laboratories, Chicago, IL, USA). The sensitivity and specificity of the test was 96.3%, and 99.8%, respectively. Patients with positive IgG but negative IgM titers were considered chronically infected. Patients with positive titers for both IgG and IgM, were considered suggestive for recent infections. The IgG avidity test was used to discriminate between old and recent infections. A group of patients with negative serology for T. gondii was used as a control group.
No statistical significance was found for the following risk factors analyzed in the present study: regular trash collection/presence in rural areas; contact with insect vectors; contact with soil/gardening, hygiene habits/basic sanitation; contact with soil/gardening; consumption of raw or undercooked foods; intake of unfiltered and/or unboiled water; and raising domestic animals, including dogs and cats. With respect to the geographical distribution of patients with ocular toxoplasmosis, 64.6% (n¼75) were from Natal and 35.4% (n¼41) were from other municipalities in the state of Rio Grande do Norte. Distribution in the districts of Natal was as follows: 20.0% (n¼15) from the north, 8.0% (n¼6) from the south, 37.3% (n¼28) from the east, and 34.7% (n¼26) from the west (Table 1). A higher seropositivity was observed in females, 64.6% (n¼75), compared to males, 35.4% (n¼41) (Table 1). The most significant age range was between 31 and 40 years old (n¼33, p¼0.04). A monthly income of up to one minimum wage (53.5%) was the most commonly observed (n¼62, p¼0.52), with a trend to a positive association for a larger number of cases in individuals with no schooling (36.2%; n¼42, p¼0.06).
Clinical ophthalmological examinations The patients underwent an eye examination, consisting of ocular inspection with a flashlight directing the beam parallel to the outer palpebral junction. This method evaluated anterior chamber depth, near visual acuity of both eyes (using Jaeger’s optotypes placed 33 cm from the eyeball) and investigated extrinsic eye muscle function. Fundoscopy was performed by a retinologist using binocular indirect ophthalmoscopy (Slit Lamp Haag Streit Keeler and external lens of 78 diopters, Keeler Instruments Inc., Broomall, PA, USA). This occurred after one drop of tropicamide (Mydriacyl,
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Clinical characterization of the lesions Among the 116 ocular toxoplasmosis-reactive patients, 56.9% exhibited bilateral lesions (n¼66). The analysis of 49 fundus
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The study was conducted at the Ophthalmology Ambulatory Clinic of the Onofre Lopes University Hospital, Federal University of Rio Grande do Norte, Natal, Brazil. Patients who visited the Clinic were invited to participate in the study after giving their written informed consent. A cross-sectional study was performed between September 2008 and September 2011, with a population of individuals from both sexes ranging from 15–60 years old and who were subjected to any ophthalmological assistance. A questionnaire was given to the patients in order to obtain details that could be associated with toxoplasmosis, as follows: age; place of residence, including the regular trash collection and the presence in rural areas; socioeconomic and cultural status; contact with soil/gardening, hygiene habits/basic sanitation; consumption of raw or undercooked food; intake of unfiltered and/or unboiled water; raising domestic animals, including dogs or cats; and the presence of potential vectors in the residence (cockroaches and flies).
Alcon, Sa˜o Paulo, Brazil) eye drop (1%) was applied to the conjunctival sac (2x every 10 minutes) of each eye in order to promote mydriasis. Patients diagnosed with fundoscopic alterations underwent retinography with a digital retinographer (Zaiss 450 Retinograph; Carl Zeiss Meditec, Vertriebsgesellschaft mbH, Oberkochen, Germany). Images were evaluated by OIS digital imaging system. Ocular differential diagnoses for other diseases were excluded from the study. A patient group with a negative serology for T. gondii and lesions caused by other etiologic agents was used as a control.
Transactions of the Royal Society of Tropical Medicine and Hygiene
Table 1. Epidemiological characteristics of the reactive or non-reactive patients treated at the Hospital Universita´rio Onofre Lopes, Natal/Rio Grande do Norte, towards Toxoplasma gondii IgG antibodies %
Non-reactive n¼43
%
75 41
64.7 35.3
8 35
18.6 81.4
75 15 6 28 26
64.6 20.0 8.0 37.3 34.7
22 8 2 7 5
51.2 36.4 9.1 31.8 22.8
15 20 33 29 19
12.9 17.2 28.4 25.0 16.3
9 15 8 5 6
20.9 34.9 18.6 11.7 13.5
photographs (Table 2), using established parameters,13 obtained from 38 patients showed a higher frequency of type I lesions (80%). Type III lesions always occurred together with Type I or, less frequently, with the Type I and II. In relation to intraocular disposition (Table 3), 49% of the lesions (n¼24) were observed in the macular region while 47% were retinal (n¼23). Macular and retinal lesions were detected in 4% (2) of the subjects (Figure 1).
Discussion Toxoplasmosis is a parasitic disease that is still neglected in many parts of the world and consequently both the infection and its clinical manifestations are poorly understood in these regions. The scarcity of studies on ocular toxoplasmosis, in Brazil, especially in the north and northeast regions, is worrisome, since the parasite is the main agent involved in posterior uveitis.9,14 This becomes even more alarming when we observe that more than half of the world’s population may be infected with the protozoan.15 T. gondii is a ubiquitous parasite with various infectious mechanisms16; the infection can be congenital or acquired and, in both cases, the parasite may invade different cell types, including neuroretina and choroid cells. At these sites, the parasite usually assumes a latent form (bradyzoites) and remains in this stage for many years, with sporadic reactivation episodes, which are likely responsible for immune system activation and resulting eye damage.17 The increase of studies involving ocular toxoplasmosis provides knowledge of variations that can occur within different regions, and socioeconomic and cultural conditions. This fact justifies
Type of lesion
Eyes (n¼49) n (%)
Type I only Type II only Types I and II Types II and III Types I, II and III
39 (80) 4 (8) 2 (4) 3 (6) 1 (2)
The classification of the lesion were considered lesions consistent with toxoplasmosis according to Ore´fice.13 Type I: limits and marked with a halo of hyperpigmentation and central area of chorioretinal atrophy. Type II: lesions with hyperpigmented halo and hypopigmented central area. Type III: hyperpigmented or hypopigmented lesions compatible with hyperplasia or atrophy of the retinal pigment epithelium.
Table 3. Classification of the ocular toxoplasmosis lesions, according to the intraocular disposition, Hospital Universita´rio Onofre Lopes, Natal/Rio Grande do Norte Classification of lesions
Eyes (n¼49) n (%)
Macular only Retinal only Both macular and retinal
24 (49) 23 (47) 2 (4)
further studies to define the problem and present the case series to public health authorities. The prevalence of the toxoplasmic uveitis observed in this study was high in relation to other regions of Brazil, as reported by Abreu et al.10 in Venda Nova do Imigrante, Espirito Santo state and Ore´fice; Fernandes18 in Minas Gerais state. A comparison between the results obtained in this study and those reported in international publications revealed a higher frequency than that found by Ronday et al.19 in Sierra Leone, Africa, and by London9 in California, indicating that this region harbors an atypical form of circulating parasites, which could explain the high prevalence. In regard to the origin of the patients, data show a predominance of individuals from Natal, which was expected, given that the reference hospital in this study is located in this city. A majority of the patients were from the eastern and western districts. This is likely because the university hospital treats patients from the public health system, which serves lower income individuals who are not covered by health insurance plans. This also explains the finding, albeit not statistically significant, that the most common monthly income found was up to one minimum wage, corroborating the results reported by Ore´fice and Fernandes.18
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Gender Female Male District Natal (Total) North South East West Age 15–20 21–30 31–40 41–50 .50
Reactive n¼116
Table 2. Classification of the ocular toxoplasmosis lesions, according to the type of lesion, Hospital Universita´rio Onofre Lopes, Natal/Rio Grande do Norte
N. H. D. Mendes et al.
In terms of age, the range of significance (31–40 years old) was higher than that found by Tabatabaei et al.20 and very close to that observed by Aleixo et al.21 The significant age group of this study is expected considering that the prevalence of toxoplasmosis-reactive patients generally increases with age, due to longer exposure to the risk factors.22 The higher incidence of the disease, which is observed in women, can be explained by their high level of exposure to the cysts found in raw meat during its preparation and no improved hygiene practices in meat production.23 This finding is similar to that observed by Garcia et al.24 and Tabatabaei et al.20 Camargo et al.25 observed increased toxoplasmosis incidence in men. This difference is likely associated with greater consumption of raw meat or exposure to oocysts in soil. Despite the high frequency of ocular toxoplasmosis, the bilateral lesions found in most patients were the most surprising finding, since most national and international studies show that ocular toxoplasmosis affects only one eye, causing unilateral damage.9,18,21,24 It is suggested that this is a common characteristic in Brazil.18,21 Ocular toxoplasmosis, which can cause long-lasting or permanent effects, is found in cataracts, glaucoma, macular edema, scarring, and retinal detachment. Despite causing more damage in immunocompromised patients, and being normally asymptomatic in immunocompetent individuals, additional damage may occur progressively in reactivation episodes, thereby affecting visual acuity.26 These progressive losses become particularly significant when the lesion is bilateral since it will result in greater host morbidity due to the loss of visual acuity. Our
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Figure 1. Fundus photographs of the lesions of the type I (A), type II (B), types I and II (C) and types II and III (D). In (A), there is a clearly marked lesion with a hyperpigmented halo (arrow) and a hypopigmented center. In (B), there is an unmarked lesion with a hypopigmented halo and hyperpigmented core. In (C), there is a clearly marked central lesion with hyperpigmented core and halo. In (D), multiple lesions can be observed. This figure is available in black and white in print and in colour at Transactions online.
group is investigating some of the causes of immunophysiopathological bilaterality (data not shown), and the findings should be published soon (probably 2015). The bilaterality of these lesions, also observed by Soares et al.,27 can be partially explained by the characteristics of the parasite. Recent studies have demonstrated that the serotype of T. gondii appears to be associated with the development of ocular toxoplasmosis.28 Thus, the fact that this protozoan exhibits polymorphisms,29 mainly in South American countries such as Brazil,30 might partially explain this divergence in regional casuistics. In Sa˜o Paulo Ferreira et al.31 showed that patients harbored polymorphic strains, although two of them showed a single genotype at nine genetic loci. These data indicate a high rate of recombination of archetypes I, II and III. These characteristics may be correlated with the disease severity observed in Brazilian patients. Although genotypes I and II of Toxoplasma have predominated in Brazil, exhibiting high and moderate pathogenicity, respectively,30,32 our group has recently identified a number of atypical genotypes of this protozoan, widely circulating in various animals used for human consumption. Some of these isolates were unprecedented in the literature and most were classified as pathogenics.33 The finding of atypical and pathogenic strains circulating in the region could also explain the clinical features observed in this study. Another possible explanation is the difference in patient susceptibility, due to ethnic variability. According to Sibley et al.34 the factors that control the occurrence of ocular diseases are poorly understood, although a variety of components, including genetic susceptibility of the host, nutritional, immunological and parasitic load status may be involved in the development of infection. Type I ocular lesions with hyperpigmented edges are considered the gold standard for clinical diagnosis of ocular toxoplasmosis. Thus, the prevalence of this lesion was consistent with other studies.21,35 The large number of type I lesions is justified given the high prevalence of ocular toxoplasmosis observed here. Type III lesions, although nonspecific, were included in the study because the literature shows that atypical lesions may also recur with a typical clinical picture.36 However, no cases of this lesion isolated were observed, possibly because of its low incidence. Usually, this type of injury does not manifest separately. It was possible to observe a predominance of macular lesions, followed by retinal lesions, corroborating studies conducted with children and the general population.20,27 According to Yang et al.,37 anatomical and microvascular differences between the macula and the peripheral region of the retina may create a microenvironment that influences the central location of the lesion, which may be a predisposing factor for the higher frequency of lesions in the macula lutea. Moreover, a survey carried out on the eyes of postmortem patients showed a higher number of macrophages defending against T. gondii in the retina,37 which once again could explain the higher frequency of macular lesions. Although not statistically significant, risk factors such as direct contact with cats, consumption of raw or undercooked meat and contact with soil were associated with ocular lesions caused by T. gondii, as demonstrated by the OR test. These data differ from those reported by a study in Natal/RN conducted by Barbosa, Holanda and Andrade-Neto.5 The authors concluded that contact with cats was associated with toxoplasmosis. However, this positive association between these risk factors and ocular toxoplasmosis in Rio Grande do Norte should not be
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dismissed. In addition, a recent study in Brazil showed that the presence of dogs and cats, as well as the consumption of raw or undercooked meat did not influence the development of ocular toxoplasmosis, despite being associated with increased risk for T. gondii infection.38 Finally, this study is of great importance to public health, since it is the first to show the prevalence of ocular toxoplasmosis in the general population of Rio Grande do Norte state. Moreover, it demonstrated the high frequency of uveitis cases and, notably, a pattern of bilateral damage, suggesting that these lesions may be associated with the atypical histologic patterns of local circulating strains. Further studies should be conducted to characterize the immunopathological pattern related to this type of injury and its association with the field isolates obtained.
Funding: This work was supported by grants from National Council for Scientific and Technological Development-CNPq [575840/2008–0 and 301837/2012–0]. Competing interests: None declared. Ethical approval: The study was approved by the Ethical Committee of Onofre Lopes University Hospital, Federal University of Rio Grande do Norte, Natal, Brazil (CEP/HUOL 013/07).
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Authors’ contributions: VFAN participated in the conception and design of the study; NHDM carried out all the tests, CBSO and VFAN supervised data collection and interpreted the data; CMCXH supervised and provided substantial assistance in many of the immunoassays; CAG supervised and significantly helped with much of the clinical assessment; all authors read and approved the final manuscript. VFAN is the guarantor of the paper.
10 Abreu MT, Belfort R Jr, Passos A et al. Toxoplasmose ocular em Venda Nova do Imigrante, ES, Brasil. Arq Bras Oftal 1998;61:540–5.
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