Ocular Immunology & Inflammation, 2014; 22(5): 360–366 ! Informa Healthcare USA, Inc. ISSN: 0927-3948 print / 1744-5078 online DOI: 10.3109/09273948.2013.841491
ORIGINAL ARTICLE
Prevalence of Toxoplasma gondii and Toxocara canis among Patients with Uveitis Su Jin Lim, MD, PhD1, Sang Eun Lee, PhD2, Sun Hyun Kim, MD, PhD1, Sung-Hee Hong, MS2, Yong Sung You, MD, PhD1, Oh Woong Kwon, MD, PhD1, and Hyeun Seung Kim, MD, PhD3 1
The retina center of Nune Eye Hospital, Seoul, Korea, 2Division of Malaria & Parasitic Diseases, Korea National Institute of Health, Centers for Disease Control and Prevention, Korea, and 3Department of Ophthalmology and Visual Science, St. Mary’s Hospital, The Catholic University of Korea
ABSTRACT Purpose: To investigate the prevalence of Toxoplasma gondii and Toxocara canis in patients with uveitis. Method: Patients with uveitis were examined. Serum antibodies to T. gondii and T. canis were tested by using enzyme-linked immunosorbent assay (ELISA). Polymerase chain reaction (PCR) was done using blood and aqueous humor (AH). Results: 98 patients were enrolled. Mean age was 43.5 ± 13.2 years. Six patients were seropositive for T. gondii with the following pattern—anterior uveitis: 1; posterior uveitis with retinitis: 2; pan uveitis: 2. One patient had a positive result of PCR for T.gondii in AH, who showed pan uveitis. 23 patients were positive to serum IgG for T. canis with the following clinical manifestation—granuloma: 6; pigmented scar: 3; virtrits: 6—but none were PCR positive. Conclusion: T.gondii and T.canis are still one of the important causes of uveitis. Ocular toxocariasis is not an uncommon cause of uveitis even in adult. Keywords: Ocular toxocariasis, ocular toxoplasmosis, parasite, serologic test, uveitis
INTRODUCTION
Improvement of social economic status has led to better general hygiene. So parasite infections like toxoplasma and toxocara have come to receive less attention and that they are considered a problem only for developing country or children. As betterment of social economic status, life style and dietary habits have also been evolving. Organic food, free-range farming and companion animals have become popular. So parasite infection like toxoplasma and toxocara can be a reemerging problem.6 Also it is reported that uveitis of unknown origin is identified as ocular toxoplasmosis or toxocariasis with molecular diagnosis.7,8 Hence, it is worth investigating the prevalence of uveitis related T. gondii and T. canis. In this study, we investigated the prevalence of T. gondii and T. canis in patients with uveitis by means
Uveitis is a common cause of visual impairment. The clinical features and causes of uveitis are variable and the correct diagnosis is often challenging. The distribution of the types and etiology of uveitis can be affected by genetic, geographic, social, and environmental factors and it can be changed with time even in same region and race.1 The inflammation is known to result from systemic immune-mediated disease, medication, trauma, operation or infection. Virus, fungus, bacteria, and parasite can cause uveitis. Among them, toxoplasmosis was identified as one of the most common causes of posterior uveitis, accounting for 25–50% of cases.2–4 Toxocariasis is known to account for 1% of posterior uveitis.5 Published online 14 May 2014
Correspondence: Hyun Seung Kim, MD., PhD, Department of Ophthalmology and Visual Science, St. Mary’s Hospital, The Catholic University of Korea, #62 Yeouido-dong, Yeongdeungpo-gu, Seoul 150-713, Korea. Tel: 82-2–3779-1243. Fax: 82-2- 761-6869. E-mail: sara514@ catholic.ac.kr
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Prevalence of Toxoplasma gondii and Toxocara canis of detecting serum antibody and polymerase chain reaction (PCR) determination in serum and aqueous humor and examining clinical manifestation.
METHOD In this prospective, non-comparative, case series, 98 patients with uveitis were enrolled from February 2011 to January 2012 in Nune Eye Hospital, Seoul. This hospital is one of several referral centers in a metropolitan area where about 10 million people are living. Informed consent was obtained from all patients. All patients with anterior, intermediate or posterior uveitis or chorioretinitis were included. Patients with history of major ophthalmic surgery like cataract, vitrectomy, glaucoma surgery or intravitreal injection or antiviral, antifungal or antiparasitic therapy within 6 months were excluded. Clinical characteristics including visual acuity, intraocular pressure (IOP), location and degree of inflammation were recorded in all patients. Also fundus findings presenting ocular toxoplasmosis like chorioretinitis, papillitis and pigmented scar along with clinical manifestations suggesting ocular toxocariasis like posterior granuloma, peripheral inflammatory mass, and vitritis were investigated. Patients filled out questionnaire including environment, occupation, pet ownership, exposure to animal and history of eating raw liver of animal, raw beef or any other raw flesh of animal. In all patients peripheral blood samples were collected to be analyzed for complete blood cell count (CBC) including eosinophil count and antibody for T.gondii and T. canis. Polymerase chain reaction (PCR) was done for detection for T. gondii and T. canis with peripheral blood. Aqueous humors (AH) were simultaneously taken and analyzed for DNA detection of T.gondii and T. canis with PCR. Aqueous humors were also examined by PCR for herpes simplex virus (HSV), varicella zoster virus (VZV) and cytomegalovirus (CMV) to identify other infectious causes of uveitis. The test for tuberculosis was not tested initially because of the low incidence of lesser than 0.1 percent in South Korea.9 Immunologic and genetic factors like rheumatoid factor (RF), ant-nulcear antibody (ANA) and HLA-B27 were tested and VDRL test and antibody to human immunodeficiency virus (HIV) were also tested.
Serological assay for toxoplasmosis and toxocariasis We used the commercial Platelia Toxo IgG and IgM TMB kit (BIO-RAD co., Marnes La Coquette, France) for serological assay for Toxoplasma gondii infection. All process are automatically calculated on Evolis !
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instrument (BIO-RAD co., France).10,11 These kits were strictly carried out according to the manufacturer recommendation. All samples were examined in twice basically and the doubtful samples were examined in triple. In addition, assay for Toxocara canis infection, the serum samples were tested using commercial enzyme-linked immunosorbent assay (ELISA) for the qualitative screening of serum IgG antibodies to Toxocara (SCIMEDX co., USA) according to the manufacturer’s recommendation.
Western blotting for toxoplasmosis and toxocariasis Each lysate of T. canis total lysate antigen (TLA) (20mL) and T. gondii TLA (20mL) were heated with sample buffer at 100 C for 5 min, separated on 10% acrylamide separating gels and then transferred electrophoretically to nitrocellulose sheets at a constant voltage of 100 V for 1 hr at 4 C. The nitrocellulose sheets were incubated for 1 hr with 5% skim milk in PBS at room temperature (RT) for blocking. Each sample was diluted 1:200 in 5% skim milk/PBS and incubated with nitrocellulose strips for 1 hr at RT. After 3 times washing with PBST, the strips were incubated for 1 hr in HRP-conjugate antihuman IgG (sigma) diluted 1:2000 in 5% skim milk/PBS at RT. After 3 times washing, the strips were incubated with 4-chloro-1-naphthol solution for 1 hr at RT.
PCR For detection of T. gondii PCR was performed using specific primers for surface antigen (SAG1) gene of species of T. gondii. The primers were Tg [A+] (50 -ATCGGTCGTCAATAATGTCG-30 ) and Tg[A-] (50 -GCTTCCTTCTTGATCGTTAG-30 ) and amplify a region of 276 bp of the SAG1 of T. gondii.12 For T. canis, PCR was performed using specific primers for internal transcribed spacer (ITS-2) of ribosomal DNA of species of T. canis. The primers were Tcan (50 -AGTATGATGGGCGCCAAT-30 ) and NC2(50 0 13 TTAGTTTCTTTTCCTCCGCT-3 ). PCR was done in a 20-mL mixture containing 1.0 mL of aqueous humor or whole blood, 1 AnyDirect solution (Bioquest, Seoul, Korea), 0.5 mM each primer, 0.2 mM each dNTP, and 2 unit of Taq DNA polymerase (Bioquest, Seoul, Korea). PCR was performed in a thermal cycler (ABI 9700 Thermal Cyclers, U.S.A.) preheated to 94 C. The cycling conditions were: initial denaturation for 5 min at 94 C, followed by 40 cycles of 30 s at 94 C, 30 s at 55 C, 30 s at 72 C, and an additional 10 min at 72 C for final elongation. After the reaction, the PCR mixture (5 mL) was electrophoresed on a 2.0% agarose gel and stained with ethidium bromide.
362 S. J. Lim et al. TABLE 1. Demographic and disease characteristics of patient with uveitis. Variable
Findings
Gender (male/female), n (%) Age at presentation, years (range) Residence, n (%) Urban Suburban Rural Pet ownership, n (%) Laterality (unilateral/bilateral)a, n (%) Recurrence history, n (%) Location, n (%) Anterior Posterior Diffuse
64 (65.3)/34 (34.7) 43.5 ± 13.2 (19–80) 55 (56) 25 (26) 18 (18) 37 (38) 92 (94)/8(6) 43 (43.9) 34 (34.7) 39 (39.8) 25 (25.5)
a
Eye with inflammation presented at the time of enrollment.
TABLE 2. Serologic and aqueous humor test characteristics of patients with uveitis. Test
No./total no. reported (%)
Rheumatoid factor HLA-B27 Antinulcear antibody VDRL test HIV Herpes simplex virus PCR with AH Varicella zoster virus PCR with AH Cytomegalovirus PCR with AH T. gondii PCR with AH T. gondii serum ELISA antibody T. canis PCR with AH* T. canis serum ELISA antibody
1/98 (1) 22/98 (22.4) 6 (6.1) 0/98 (0) 0/98 (0) 2/98 (2.0) 1/98 (1.0) 0/98 (0) 1/98 (1.0) 6/98 (6.1) 0/98 (0) 23 (23.5)
PCR with AH, polymerase chain reaction with aqueous humor.
RESULT In this study, 98 patients with uveitis were enrolled from February 2011 to January 2012 in Nune Eye Hospital, Seoul. Table 1 shows demographic and disease characteristics of patient with uveitis. 64(65.3%) patients were male and 34 (34.7%) patients were female. Mean age was 43.5 ± 13.2 years. 34 (34.7%) patients had anterior uveitis, 39 (39.8%) patients had posterior uveitis and 25 (25.5%) patients had diffuse uveitis. 43(43.9%) patients had past history of uveitis. One (1.0%) of 98 patients showed elevated level of Rheumatoid factor (RF) and 22 (22.4%) patients were positive for HLA-B27; six (6.1%) patients were positive for antinuclear antibody (ANA). Two patients had positive PCR result for HSV and one patient showed positive result for VZV (Table 2). Six (6.1%) patients were seropositive for Toxoplasma gondii. Among the six patients, one was positive for T. gondii IgM while the other four patients were
FIGURE 1. Toxoplasma gondii PCR result (AH: Aqueous humor).
positive for T. gondii IgG and the remaining one patient had both antibodies. Among the six patients with seropositive for T.gondii, DNA for T.gondii was not detected in the aqueous humor or peripheral blood. One of 98 patients had a positive PCR for T. gondii only in aqueous humor and was seronegative for T. gondii (Figure 1). Three patients were coinfected with T.gondii and T. canis. Serum enzyme-linked immunosorbent assay (ELSIA) for IgG antibody to T. canis was reported as positive for 23 (23.5%) patients and the positive ELISA results was confirmed by Western blotting in all patients. In 23 patients with seropositive for T. canis, DNA amplification was not detected in aqueous humor and peripheral blood with PCR. In addition, the rest of the patients with seronegative for T. canis turned out to have a negative PCR for T. canis as well. Among 6 patients with seropositive for T. gondii, one patient showed anterior uveitis while three patients had posterior uveitis with retinitis and the remaining two patients had panuveitis. Patient with a positive PCR result for T. gondii had diffuse uveitis. In fundus photography pigmented scar region was noticed in two patients (Table 3). Four (17.4%) of the 23 patients who was seropositive for T. canis had anterior uveitis while 11 (47.8%) patients had posterior uveitis, and eight (34.8%) had diffuse uveitis. In fundus examination, granuloma was noticed in 6 of 23 patients. Among six patients, in five patients, a granuloma located in peripheral retina or pars plana and in one patient, it located in the macular (Figure 2). In three patients (13%), pigmented scar was noticed in macula and in six patients (26.1%), there were vitritis obscuring retina vessels. Clinically suspected cases for toxoplasmosis showing retinitis and pigmented region were 15 patients. Among 15 patients, three patients showed positive serologic result. Meanwhile, Suspected ocular Ocular Immunology & Inflammation
Prevalence of Toxoplasma gondii and Toxocara canis
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TABLE 3. Characteristics of patients seropositive to Toxoplasma gondii. No. 1 2 3 4 5 6 7
Sex
Age
VA
Laterality
Locationa
IgM
IgG
PCR
Ab for T. canis
IF
Fundus finding
M M F F F M F
39 52 52 57 29 80 37
0.8 0.8 0.1 1.0 1.0 0.1 0.5
Unilateral Unilateral Unilateral Unilateral Unilateral Unilateral Unilateral
3 2 2 3 1 2 3
– + – + _ _ –
+ + + –
– – – – – – +
+ – + + – – –
– – – – – – –
Granuloma Pigmented scar Pigmented scar Nonspecific Nonspecific Nonspecific Nonspecific
+
+ –
a
Location: 1 anterior 2 posterior 3 diffuse. PCR polymerase chain reaction, positive with aqueous humor; Ab, antibody; IF, immunologic factor (RF, ANA, HLA-B27).
FIGURE 2. (A)Fundus finding of representing patients who were seropositive for T. canis A. 49 Year-old men visited at our clinic for photophobia in left eye for 2 years. He had past history of treatment for systemic parasite infection 7 years ago. He didn’t mention the exact kind of parasite. At that time he had no subjective ocular symptom and didn’t receive the exam of eye. At time of visiting our hospital visual acuity in left eye was 0.8. Anterior chamber was clear and vitreous cell was 1+ in left eye. In fuduns examination, fibrous pigmented scar with tractional band was noticed at macula and disc in left eye. On fluorescein angiography, blocked fluorescence was observed in scar region and mild leakage and stain at inferior to scar and mild disk leakage were noticed. Serum toxocara specific antibody was positive. Those finding imply chronic and mild inflammation by previous infection of T. canis. (B). 39 years- old man visited our hospital for blurry vision in left eye for one month. Visual acuity in left eye was 0.8. Anterior chamber was clear and vitreous cell was (+++) in left eye. In fundus exam margin of disc and vessels was not clear due to vitreous inflammation and peripheral inflammatory mass was noticed at inferotemporal area with Goldman three-mirror lens. (back arrow).
toxocariasis cases representing macular granuloma or peripheral granuloma region or severe vitritis was 7 cases. Among of 7 cases, 6 patients showed positive serologic result. We compared patient characteristics between seropositive and seronegative group for T. canis. !
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In positive group for Toxocara, male were 19/23 (82.6%), while 45/75 (60.0%) were male in negative group. Male outnumbered in seropositive group with a statistical significance (p = 0.05). Mean age was older in seropositive group (49.2 ± 12.4 years) than in negative group (41.7 ± 13.1 years) (p = 0.017).
364 S. J. Lim et al. TABLE 4. Comparison of patient characteristics between positive and negative group for Toxocara canis antibody. Variable Gender Age (year) First attack Pet ownership Eating uncooked meat Visual acuity (logMAR) WBC count (103cell/mm2) Eosinophil count (cell/mm2)
Toxocara positive
Toxocara negative
p Value
82.6 % (19/23) 49.2 ± 12.4 78.3% (18/23) 5 (22.7%) 20 (90.1%) 0.68 ± 0.34 7,17 ± 1.80 129 ± 94
60 % (45/75) 41.7 ± 13.1 49.3% (37/75) 19 (52.7%) 67 (92%) 0.64 ± 0.33 6.55 ± 2.03 161 ± 131
0.05a 0.017b 0.012a 0.403c 1.0a 0.612b 0.174b 0.2b
Pearson’s 2 test. Student t-test. c Fisher’s exact test. a
b
The proportion of patients without past history of uveitis is significantly higher in seropositive group (18/23, 78.3%) than in negative group (37/75, 49.3%) (p = 0.012). There was no difference in environment, occupation, pet ownership, or habit of eating uncooked meat and WBC and eosinophil differential number between the two groups (Table 4). There was no complication related with anterior chamber paracentesis like hemorrhage, infection or hypotony in all patients.
DISCUSSION Uveitis can result from various causes like systemic autoimmune disease, infection, trauma or tumor. Severe vision-threatening uveitis remains a significant public health problem, often resulting in vision loss without proper therapy.14 Sometimes the correct diagnosis is difficult and it is reported that 50% of uveitis were described as unclassified intraocular inflammation.2,15 In many cases, uveitis is treated empirically with steroid.16 In undiagnosed uveitis, prolonged empiric steroid treatment may be harmful by delaying diagnosis and it may allow the inflammation to progress more rapidly in uveitis due to infection. In addition, steroid implant is recently used for treating uveitis17–19 and the accurate diagnosis is more important. In our study, 7.1% in patients with uveitis showed positive results of serologic test or PCR for T. gondii. The prevalence of ocular toxocariasis varies from 0.6% to 21.3 % throughout the world.2–4,20,21 In previous studies it was reported that seroprevalence of apparently healthy people was 8% in urban area and 11.3% in rural area in Korea.22 Our study result is similar to the previous report. In this study, among 15 patients who presented chorioretinitis and pigmented scar and were clinically suspected for toxoplasmosis, only three patients showed positive serologic result and none were PCR positive for T. gondii. If the analysis of intraocular
antibody like Goldman-Witmer coefficient (GWC) were done simultaneously, it could have given higher positive rate.7 Interestingly three out of six patients who were positive to T.gondii also showed positive results to T. canis. It is assumed that these two parasite infections have several common risk factors like petownership or eating undercooked meat. Therefore, if the patient is diagnosed with T. gondii or T. canis, it is recommended to have an investigation for the undiagnosed parasite as well. Toxocara canis is known to cause uveitis especially in young patients. Clinical manifestations of ocular toxocariasis (OT) are defined like peripheral or posterior retina granuloma and chronic vitreous inflammation mimicking endophthalmitis and OT occurs in 1% of uveitis.5,23 Ocular toxocariasis inflammation is severe and granuloma with fibrous traction band to adjacent retina and often to disc can cause retinal detachmentand OT usually has a poor visual prognosis.24–26 Generally diagnosis of OT was done based on clinical manifestations. Currently ELISA testing and molecular test of serum are employed for assisting in the diagnosis OT.23 In our study, seroprevalence for T. canis in patients with uveitis is 23.5%. Positive result for Toxocaraspecific IgG is, in itself, not of definitive diagnostic value for OT. However, in this study, a value of 23.5 % is considered to be higher than expected since previous study revealed that a seroprevalence was 5.1% even in rural Korean healthy adults.27 Among 14 of 23 patients who were positive for T. canis, macula and periphery retina were clearly visualized, and six of 14 patients showed dense white granulomatous inflammatory mass or granuloma. In six of 23 patients, vitritis was noticed and in three of 23 patients pigmented scar was observed. Granuloma, vitritis and pigmented scar are consistent findings with OT, and therefore we can resume that T. canis is closely related with uveitis at least in 15 of 23 patients who were seropositive to T. canis. The proportion of patients without past uveitis history is statistically significantly higher (78.3%) Ocular Immunology & Inflammation
Prevalence of Toxoplasma gondii and Toxocara canis than serologic negative group for T.canis (49.3%) (p = 0.012). It also supports that the uveitis of seropositive group for T. canis was related to infection rather than other underlying disease. In this study, the clinical feature of patients with uveitis related with T. canis seemed to be milder than described in previous reports. The mean visual acuity (LogMAR) in patients with seropositive T. canis was 0.68 ± 0.34 and granuloma was localized and was not large in size and vitreous inflammation was not severe. We thought that the several reasons patients showed mild clinical manifestation in our study were as followed: First is age. Generally OT has been a disease of children and the known clinical features of OT are manifestations of patients who are children or young adults.5,23,28 In this study, mean age of patients with seropositive for T. canis was 49.2 ± 12.4 years. The clinical response of disease could be different depending on the immune response of the host and the same infection can have some age-dependent variation.29–31 So the manifestation of adult OT can be milder than that of children. Second is infection route and loading dose. The clinical presentations of OT vary depending on the parasite load.32,33 In children the main route of infection is soil contaminated with eggs. In adult, ingestion of uncooked liver or meat from animal infected with the Toxocara is the main infection routes.34 Thus the repeated low dose exposure in adult can result in the different clinical manifestation from children. This study didn’t reveal the relation between T. canis infection and its known risk factors. Pet ownership, environment, occupations and eating habit of raw meat showed no difference between positive and negative groups. To determine the exact infection route, larger study with more detailed questionnaire including what kind of meat and how often they eat them and number of pets owned is needed. In conclusion, in this study, OT is more common than expected even in adult. If diagnosis is done solely based on pre-existing clinical feature, it can be missed. Clinical manifestation and its course can be varied according to age and infectious loading dose. Considering the possibility of OT and doing serologic test will be helpful for diagnosis and management in patients with uveitis.
DECLARATION OF INTEREST This study was performed to the fund (2011-E5401000) supported by Korea National Institute of Health Korea, Korea Centers for Disease Control and Prevention. The authors have no proprietary or financial interest in any of the products used in this study. !
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Ocular Immunology & Inflammation
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ORIGINAL ARTICLE
Prevalence of Toxoplasma gondii and Toxocara canis among Patients with Uveitis Su Jin Lim, MD, PhD1, Sang Eun Lee, PhD2, Sun Hyun Kim, MD, PhD1, Sung-Hee Hong, MS2, Yong Sung You, MD, PhD1, Oh Woong Kwon, MD, PhD1, and Hyeun Seung Kim, MD, PhD3 1
The retina center of Nune Eye Hospital, Seoul, Korea, 2Division of Malaria & Parasitic Diseases, Korea National Institute of Health, Centers for Disease Control and Prevention, Korea, and 3Department of Ophthalmology and Visual Science, St. Mary’s Hospital, The Catholic University of Korea
ABSTRACT Purpose: To investigate the prevalence of Toxoplasma gondii and Toxocara canis in patients with uveitis. Method: Patients with uveitis were examined. Serum antibodies to T. gondii and T. canis were tested by using enzyme-linked immunosorbent assay (ELISA). Polymerase chain reaction (PCR) was done using blood and aqueous humor (AH). Results: 98 patients were enrolled. Mean age was 43.5 ± 13.2 years. Six patients were seropositive for T. gondii with the following pattern—anterior uveitis: 1; posterior uveitis with retinitis: 2; pan uveitis: 2. One patient had a positive result of PCR for T.gondii in AH, who showed pan uveitis. 23 patients were positive to serum IgG for T. canis with the following clinical manifestation—granuloma: 6; pigmented scar: 3; virtrits: 6—but none were PCR positive. Conclusion: T.gondii and T.canis are still one of the important causes of uveitis. Ocular toxocariasis is not an uncommon cause of uveitis even in adult. Keywords: Ocular toxocariasis, ocular toxoplasmosis, parasite, serologic test, uveitis
INTRODUCTION
Improvement of social economic status has led to better general hygiene. So parasite infections like toxoplasma and toxocara have come to receive less attention and that they are considered a problem only for developing country or children. As betterment of social economic status, life style and dietary habits have also been evolving. Organic food, free-range farming and companion animals have become popular. So parasite infection like toxoplasma and toxocara can be a reemerging problem.6 Also it is reported that uveitis of unknown origin is identified as ocular toxoplasmosis or toxocariasis with molecular diagnosis.7,8 Hence, it is worth investigating the prevalence of uveitis related T. gondii and T. canis. In this study, we investigated the prevalence of T. gondii and T. canis in patients with uveitis by means
Uveitis is a common cause of visual impairment. The clinical features and causes of uveitis are variable and the correct diagnosis is often challenging. The distribution of the types and etiology of uveitis can be affected by genetic, geographic, social, and environmental factors and it can be changed with time even in same region and race.1 The inflammation is known to result from systemic immune-mediated disease, medication, trauma, operation or infection. Virus, fungus, bacteria, and parasite can cause uveitis. Among them, toxoplasmosis was identified as one of the most common causes of posterior uveitis, accounting for 25–50% of cases.2–4 Toxocariasis is known to account for 1% of posterior uveitis.5 Published online 14 May 2014
Correspondence: Hyun Seung Kim, MD., PhD, Department of Ophthalmology and Visual Science, St. Mary’s Hospital, The Catholic University of Korea, #62 Yeouido-dong, Yeongdeungpo-gu, Seoul 150-713, Korea. Tel: 82-2–3779-1243. Fax: 82-2- 761-6869. E-mail: sara514@ catholic.ac.kr
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Prevalence of Toxoplasma gondii and Toxocara canis of detecting serum antibody and polymerase chain reaction (PCR) determination in serum and aqueous humor and examining clinical manifestation.
METHOD In this prospective, non-comparative, case series, 98 patients with uveitis were enrolled from February 2011 to January 2012 in Nune Eye Hospital, Seoul. This hospital is one of several referral centers in a metropolitan area where about 10 million people are living. Informed consent was obtained from all patients. All patients with anterior, intermediate or posterior uveitis or chorioretinitis were included. Patients with history of major ophthalmic surgery like cataract, vitrectomy, glaucoma surgery or intravitreal injection or antiviral, antifungal or antiparasitic therapy within 6 months were excluded. Clinical characteristics including visual acuity, intraocular pressure (IOP), location and degree of inflammation were recorded in all patients. Also fundus findings presenting ocular toxoplasmosis like chorioretinitis, papillitis and pigmented scar along with clinical manifestations suggesting ocular toxocariasis like posterior granuloma, peripheral inflammatory mass, and vitritis were investigated. Patients filled out questionnaire including environment, occupation, pet ownership, exposure to animal and history of eating raw liver of animal, raw beef or any other raw flesh of animal. In all patients peripheral blood samples were collected to be analyzed for complete blood cell count (CBC) including eosinophil count and antibody for T.gondii and T. canis. Polymerase chain reaction (PCR) was done for detection for T. gondii and T. canis with peripheral blood. Aqueous humors (AH) were simultaneously taken and analyzed for DNA detection of T.gondii and T. canis with PCR. Aqueous humors were also examined by PCR for herpes simplex virus (HSV), varicella zoster virus (VZV) and cytomegalovirus (CMV) to identify other infectious causes of uveitis. The test for tuberculosis was not tested initially because of the low incidence of lesser than 0.1 percent in South Korea.9 Immunologic and genetic factors like rheumatoid factor (RF), ant-nulcear antibody (ANA) and HLA-B27 were tested and VDRL test and antibody to human immunodeficiency virus (HIV) were also tested.
Serological assay for toxoplasmosis and toxocariasis We used the commercial Platelia Toxo IgG and IgM TMB kit (BIO-RAD co., Marnes La Coquette, France) for serological assay for Toxoplasma gondii infection. All process are automatically calculated on Evolis !
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instrument (BIO-RAD co., France).10,11 These kits were strictly carried out according to the manufacturer recommendation. All samples were examined in twice basically and the doubtful samples were examined in triple. In addition, assay for Toxocara canis infection, the serum samples were tested using commercial enzyme-linked immunosorbent assay (ELISA) for the qualitative screening of serum IgG antibodies to Toxocara (SCIMEDX co., USA) according to the manufacturer’s recommendation.
Western blotting for toxoplasmosis and toxocariasis Each lysate of T. canis total lysate antigen (TLA) (20mL) and T. gondii TLA (20mL) were heated with sample buffer at 100 C for 5 min, separated on 10% acrylamide separating gels and then transferred electrophoretically to nitrocellulose sheets at a constant voltage of 100 V for 1 hr at 4 C. The nitrocellulose sheets were incubated for 1 hr with 5% skim milk in PBS at room temperature (RT) for blocking. Each sample was diluted 1:200 in 5% skim milk/PBS and incubated with nitrocellulose strips for 1 hr at RT. After 3 times washing with PBST, the strips were incubated for 1 hr in HRP-conjugate antihuman IgG (sigma) diluted 1:2000 in 5% skim milk/PBS at RT. After 3 times washing, the strips were incubated with 4-chloro-1-naphthol solution for 1 hr at RT.
PCR For detection of T. gondii PCR was performed using specific primers for surface antigen (SAG1) gene of species of T. gondii. The primers were Tg [A+] (50 -ATCGGTCGTCAATAATGTCG-30 ) and Tg[A-] (50 -GCTTCCTTCTTGATCGTTAG-30 ) and amplify a region of 276 bp of the SAG1 of T. gondii.12 For T. canis, PCR was performed using specific primers for internal transcribed spacer (ITS-2) of ribosomal DNA of species of T. canis. The primers were Tcan (50 -AGTATGATGGGCGCCAAT-30 ) and NC2(50 0 13 TTAGTTTCTTTTCCTCCGCT-3 ). PCR was done in a 20-mL mixture containing 1.0 mL of aqueous humor or whole blood, 1 AnyDirect solution (Bioquest, Seoul, Korea), 0.5 mM each primer, 0.2 mM each dNTP, and 2 unit of Taq DNA polymerase (Bioquest, Seoul, Korea). PCR was performed in a thermal cycler (ABI 9700 Thermal Cyclers, U.S.A.) preheated to 94 C. The cycling conditions were: initial denaturation for 5 min at 94 C, followed by 40 cycles of 30 s at 94 C, 30 s at 55 C, 30 s at 72 C, and an additional 10 min at 72 C for final elongation. After the reaction, the PCR mixture (5 mL) was electrophoresed on a 2.0% agarose gel and stained with ethidium bromide.
362 S. J. Lim et al. TABLE 1. Demographic and disease characteristics of patient with uveitis. Variable
Findings
Gender (male/female), n (%) Age at presentation, years (range) Residence, n (%) Urban Suburban Rural Pet ownership, n (%) Laterality (unilateral/bilateral)a, n (%) Recurrence history, n (%) Location, n (%) Anterior Posterior Diffuse
64 (65.3)/34 (34.7) 43.5 ± 13.2 (19–80) 55 (56) 25 (26) 18 (18) 37 (38) 92 (94)/8(6) 43 (43.9) 34 (34.7) 39 (39.8) 25 (25.5)
a
Eye with inflammation presented at the time of enrollment.
TABLE 2. Serologic and aqueous humor test characteristics of patients with uveitis. Test
No./total no. reported (%)
Rheumatoid factor HLA-B27 Antinulcear antibody VDRL test HIV Herpes simplex virus PCR with AH Varicella zoster virus PCR with AH Cytomegalovirus PCR with AH T. gondii PCR with AH T. gondii serum ELISA antibody T. canis PCR with AH* T. canis serum ELISA antibody
1/98 (1) 22/98 (22.4) 6 (6.1) 0/98 (0) 0/98 (0) 2/98 (2.0) 1/98 (1.0) 0/98 (0) 1/98 (1.0) 6/98 (6.1) 0/98 (0) 23 (23.5)
PCR with AH, polymerase chain reaction with aqueous humor.
RESULT In this study, 98 patients with uveitis were enrolled from February 2011 to January 2012 in Nune Eye Hospital, Seoul. Table 1 shows demographic and disease characteristics of patient with uveitis. 64(65.3%) patients were male and 34 (34.7%) patients were female. Mean age was 43.5 ± 13.2 years. 34 (34.7%) patients had anterior uveitis, 39 (39.8%) patients had posterior uveitis and 25 (25.5%) patients had diffuse uveitis. 43(43.9%) patients had past history of uveitis. One (1.0%) of 98 patients showed elevated level of Rheumatoid factor (RF) and 22 (22.4%) patients were positive for HLA-B27; six (6.1%) patients were positive for antinuclear antibody (ANA). Two patients had positive PCR result for HSV and one patient showed positive result for VZV (Table 2). Six (6.1%) patients were seropositive for Toxoplasma gondii. Among the six patients, one was positive for T. gondii IgM while the other four patients were
FIGURE 1. Toxoplasma gondii PCR result (AH: Aqueous humor).
positive for T. gondii IgG and the remaining one patient had both antibodies. Among the six patients with seropositive for T.gondii, DNA for T.gondii was not detected in the aqueous humor or peripheral blood. One of 98 patients had a positive PCR for T. gondii only in aqueous humor and was seronegative for T. gondii (Figure 1). Three patients were coinfected with T.gondii and T. canis. Serum enzyme-linked immunosorbent assay (ELSIA) for IgG antibody to T. canis was reported as positive for 23 (23.5%) patients and the positive ELISA results was confirmed by Western blotting in all patients. In 23 patients with seropositive for T. canis, DNA amplification was not detected in aqueous humor and peripheral blood with PCR. In addition, the rest of the patients with seronegative for T. canis turned out to have a negative PCR for T. canis as well. Among 6 patients with seropositive for T. gondii, one patient showed anterior uveitis while three patients had posterior uveitis with retinitis and the remaining two patients had panuveitis. Patient with a positive PCR result for T. gondii had diffuse uveitis. In fundus photography pigmented scar region was noticed in two patients (Table 3). Four (17.4%) of the 23 patients who was seropositive for T. canis had anterior uveitis while 11 (47.8%) patients had posterior uveitis, and eight (34.8%) had diffuse uveitis. In fundus examination, granuloma was noticed in 6 of 23 patients. Among six patients, in five patients, a granuloma located in peripheral retina or pars plana and in one patient, it located in the macular (Figure 2). In three patients (13%), pigmented scar was noticed in macula and in six patients (26.1%), there were vitritis obscuring retina vessels. Clinically suspected cases for toxoplasmosis showing retinitis and pigmented region were 15 patients. Among 15 patients, three patients showed positive serologic result. Meanwhile, Suspected ocular Ocular Immunology & Inflammation
Prevalence of Toxoplasma gondii and Toxocara canis
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TABLE 3. Characteristics of patients seropositive to Toxoplasma gondii. No. 1 2 3 4 5 6 7
Sex
Age
VA
Laterality
Locationa
IgM
IgG
PCR
Ab for T. canis
IF
Fundus finding
M M F F F M F
39 52 52 57 29 80 37
0.8 0.8 0.1 1.0 1.0 0.1 0.5
Unilateral Unilateral Unilateral Unilateral Unilateral Unilateral Unilateral
3 2 2 3 1 2 3
– + – + _ _ –
+ + + –
– – – – – – +
+ – + + – – –
– – – – – – –
Granuloma Pigmented scar Pigmented scar Nonspecific Nonspecific Nonspecific Nonspecific
+
+ –
a
Location: 1 anterior 2 posterior 3 diffuse. PCR polymerase chain reaction, positive with aqueous humor; Ab, antibody; IF, immunologic factor (RF, ANA, HLA-B27).
FIGURE 2. (A)Fundus finding of representing patients who were seropositive for T. canis A. 49 Year-old men visited at our clinic for photophobia in left eye for 2 years. He had past history of treatment for systemic parasite infection 7 years ago. He didn’t mention the exact kind of parasite. At that time he had no subjective ocular symptom and didn’t receive the exam of eye. At time of visiting our hospital visual acuity in left eye was 0.8. Anterior chamber was clear and vitreous cell was 1+ in left eye. In fuduns examination, fibrous pigmented scar with tractional band was noticed at macula and disc in left eye. On fluorescein angiography, blocked fluorescence was observed in scar region and mild leakage and stain at inferior to scar and mild disk leakage were noticed. Serum toxocara specific antibody was positive. Those finding imply chronic and mild inflammation by previous infection of T. canis. (B). 39 years- old man visited our hospital for blurry vision in left eye for one month. Visual acuity in left eye was 0.8. Anterior chamber was clear and vitreous cell was (+++) in left eye. In fundus exam margin of disc and vessels was not clear due to vitreous inflammation and peripheral inflammatory mass was noticed at inferotemporal area with Goldman three-mirror lens. (back arrow).
toxocariasis cases representing macular granuloma or peripheral granuloma region or severe vitritis was 7 cases. Among of 7 cases, 6 patients showed positive serologic result. We compared patient characteristics between seropositive and seronegative group for T. canis. !
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In positive group for Toxocara, male were 19/23 (82.6%), while 45/75 (60.0%) were male in negative group. Male outnumbered in seropositive group with a statistical significance (p = 0.05). Mean age was older in seropositive group (49.2 ± 12.4 years) than in negative group (41.7 ± 13.1 years) (p = 0.017).
364 S. J. Lim et al. TABLE 4. Comparison of patient characteristics between positive and negative group for Toxocara canis antibody. Variable Gender Age (year) First attack Pet ownership Eating uncooked meat Visual acuity (logMAR) WBC count (103cell/mm2) Eosinophil count (cell/mm2)
Toxocara positive
Toxocara negative
p Value
82.6 % (19/23) 49.2 ± 12.4 78.3% (18/23) 5 (22.7%) 20 (90.1%) 0.68 ± 0.34 7,17 ± 1.80 129 ± 94
60 % (45/75) 41.7 ± 13.1 49.3% (37/75) 19 (52.7%) 67 (92%) 0.64 ± 0.33 6.55 ± 2.03 161 ± 131
0.05a 0.017b 0.012a 0.403c 1.0a 0.612b 0.174b 0.2b
Pearson’s 2 test. Student t-test. c Fisher’s exact test. a
b
The proportion of patients without past history of uveitis is significantly higher in seropositive group (18/23, 78.3%) than in negative group (37/75, 49.3%) (p = 0.012). There was no difference in environment, occupation, pet ownership, or habit of eating uncooked meat and WBC and eosinophil differential number between the two groups (Table 4). There was no complication related with anterior chamber paracentesis like hemorrhage, infection or hypotony in all patients.
DISCUSSION Uveitis can result from various causes like systemic autoimmune disease, infection, trauma or tumor. Severe vision-threatening uveitis remains a significant public health problem, often resulting in vision loss without proper therapy.14 Sometimes the correct diagnosis is difficult and it is reported that 50% of uveitis were described as unclassified intraocular inflammation.2,15 In many cases, uveitis is treated empirically with steroid.16 In undiagnosed uveitis, prolonged empiric steroid treatment may be harmful by delaying diagnosis and it may allow the inflammation to progress more rapidly in uveitis due to infection. In addition, steroid implant is recently used for treating uveitis17–19 and the accurate diagnosis is more important. In our study, 7.1% in patients with uveitis showed positive results of serologic test or PCR for T. gondii. The prevalence of ocular toxocariasis varies from 0.6% to 21.3 % throughout the world.2–4,20,21 In previous studies it was reported that seroprevalence of apparently healthy people was 8% in urban area and 11.3% in rural area in Korea.22 Our study result is similar to the previous report. In this study, among 15 patients who presented chorioretinitis and pigmented scar and were clinically suspected for toxoplasmosis, only three patients showed positive serologic result and none were PCR positive for T. gondii. If the analysis of intraocular
antibody like Goldman-Witmer coefficient (GWC) were done simultaneously, it could have given higher positive rate.7 Interestingly three out of six patients who were positive to T.gondii also showed positive results to T. canis. It is assumed that these two parasite infections have several common risk factors like petownership or eating undercooked meat. Therefore, if the patient is diagnosed with T. gondii or T. canis, it is recommended to have an investigation for the undiagnosed parasite as well. Toxocara canis is known to cause uveitis especially in young patients. Clinical manifestations of ocular toxocariasis (OT) are defined like peripheral or posterior retina granuloma and chronic vitreous inflammation mimicking endophthalmitis and OT occurs in 1% of uveitis.5,23 Ocular toxocariasis inflammation is severe and granuloma with fibrous traction band to adjacent retina and often to disc can cause retinal detachmentand OT usually has a poor visual prognosis.24–26 Generally diagnosis of OT was done based on clinical manifestations. Currently ELISA testing and molecular test of serum are employed for assisting in the diagnosis OT.23 In our study, seroprevalence for T. canis in patients with uveitis is 23.5%. Positive result for Toxocaraspecific IgG is, in itself, not of definitive diagnostic value for OT. However, in this study, a value of 23.5 % is considered to be higher than expected since previous study revealed that a seroprevalence was 5.1% even in rural Korean healthy adults.27 Among 14 of 23 patients who were positive for T. canis, macula and periphery retina were clearly visualized, and six of 14 patients showed dense white granulomatous inflammatory mass or granuloma. In six of 23 patients, vitritis was noticed and in three of 23 patients pigmented scar was observed. Granuloma, vitritis and pigmented scar are consistent findings with OT, and therefore we can resume that T. canis is closely related with uveitis at least in 15 of 23 patients who were seropositive to T. canis. The proportion of patients without past uveitis history is statistically significantly higher (78.3%) Ocular Immunology & Inflammation
Prevalence of Toxoplasma gondii and Toxocara canis than serologic negative group for T.canis (49.3%) (p = 0.012). It also supports that the uveitis of seropositive group for T. canis was related to infection rather than other underlying disease. In this study, the clinical feature of patients with uveitis related with T. canis seemed to be milder than described in previous reports. The mean visual acuity (LogMAR) in patients with seropositive T. canis was 0.68 ± 0.34 and granuloma was localized and was not large in size and vitreous inflammation was not severe. We thought that the several reasons patients showed mild clinical manifestation in our study were as followed: First is age. Generally OT has been a disease of children and the known clinical features of OT are manifestations of patients who are children or young adults.5,23,28 In this study, mean age of patients with seropositive for T. canis was 49.2 ± 12.4 years. The clinical response of disease could be different depending on the immune response of the host and the same infection can have some age-dependent variation.29–31 So the manifestation of adult OT can be milder than that of children. Second is infection route and loading dose. The clinical presentations of OT vary depending on the parasite load.32,33 In children the main route of infection is soil contaminated with eggs. In adult, ingestion of uncooked liver or meat from animal infected with the Toxocara is the main infection routes.34 Thus the repeated low dose exposure in adult can result in the different clinical manifestation from children. This study didn’t reveal the relation between T. canis infection and its known risk factors. Pet ownership, environment, occupations and eating habit of raw meat showed no difference between positive and negative groups. To determine the exact infection route, larger study with more detailed questionnaire including what kind of meat and how often they eat them and number of pets owned is needed. In conclusion, in this study, OT is more common than expected even in adult. If diagnosis is done solely based on pre-existing clinical feature, it can be missed. Clinical manifestation and its course can be varied according to age and infectious loading dose. Considering the possibility of OT and doing serologic test will be helpful for diagnosis and management in patients with uveitis.
DECLARATION OF INTEREST This study was performed to the fund (2011-E5401000) supported by Korea National Institute of Health Korea, Korea Centers for Disease Control and Prevention. The authors have no proprietary or financial interest in any of the products used in this study. !
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