TY - JOUR
AU - Razmjou,, Elham
AB - Abstract Background Our goal was to use molecular techniques to verify and characterise clinical diagnoses of ocular toxoplasmosis. Clinical cases were evaluated against IgM and IgG Toxoplasma antibodies, and IgG avidity was tested. B1 gene was assessed for molecular detection, and multi-locus genotyping were conducted to type Toxoplasma infections. Methods A cross-sectional study was conducted in 33 patients with suspected active ocular toxoplasmosis. Patients were examined by an ophthalmologist and clinical manifestations were recorded. Toxoplasma gondii IgG and IgM from serum samples were analysed by chemiluminescence immunoassay and ELISA. Acute vs chronic infection was evaluated by IgG avidity testing. Molecular diagnosis of T. gondii infection targeted the B1 gene, and the T. gondii genotype was determined by amplification of the GRA6, SAG2, SAG3, BTUB and APICO loci. The correlation of age, gender, occupation, education, contact with cats or soil, and the consumption of undercooked meat with the incidence of ocular toxoplasmosis was evaluated. Results Twenty-eight patients (84.8%) were seropositive, two (6%) were both IgG and IgM positive, while one (3%) showed IgG avidity <40%. Molecular testing confirmed toxoplasmosis in 27 patients (81.8%). Chorioretinal scarring (p=0.014) and posterior uveitis (p=0.004) was significantly associated with ocular toxoplasmosis patients. Multi-locus genotyping showed genotype I. Ocular toxoplasmosis showed no significant correlation with gender, age, behaviours, occupation or education. Conclusion Clinical manifestations, serological and molecular detection of Toxoplasma were highly correlated in the diagnosis of ocular toxoplasmosis. Genotype I was predominant in ocular toxoplasmosis in northwest Iran. A larger comparative study should be conducted to provide a broader view of the molecular epidemiology of T. gondii genotypes and its role in toxoplasmosis. chorioretinal scarring, genotype, northwest Iran, ocular toxoplasmosis Introduction Toxoplasmosis, caused by Toxoplasma gondii, affects humans who become infected through consuming raw or undercooked meat or food and water contaminated with sporulated oocysts in the environment, which shed from feline faeces as unsporulated oocysts 1–5 d before, and also by vertical transmission.1Toxoplasma affects several organs, including the eyes. Ocular toxoplasmosis may be congenital or acquired after birth.2,3 It is among the most common causes of uveitis worldwide and of posterior uveitis in immunocompetent people.2–5 Ocular toxoplasmosis prevalence varies geographically, but is estimated to range from 0.3–1% in Europeans and North Americans,6–8 and from 2% in north-eastern to 25% in southern Brazil.9–11 Based on retrospective descriptive studies of referrals to eyecare centres, the prevalence of ocular toxoplasmosis was reported to be 10.1 and 19.4% in adults of northern and central Iran, respectively, and 5.4% in children in northwest Iran.12–14 Ocular toxoplasmosis shows diverse symptoms and clinical signs with varied levels of ocular inflammation2 depending on patient age and the location, extent and severity of retinochoroiditis. The most common feature is focal retinitis or chorioretinitis, manifesting as either localised necrotising retinitis with inflammation of the choroid or as an active creamy white focal retinal lesion.2,15 Ocular symptoms include floaters and blurred vision. Reduced visual acuity may occur as a result of macular involvement or severe vitreous inflammation. Toxoplasmic retinochoroiditis is a recurrent disease in two-thirds of patients.2,3,15 The diagnosis of ocular toxoplasmosis is made primarily by observation of focal necrotising retinochoroiditis. Serological tests such as anti-Toxoplasma IgG and IgM serum may confirm diagnosis in atypical cases.16,17 In cases in which the diagnosis is uncertain, demonstration of anti-Toxoplasma antibody titres in the aqueous or vitreous humour can be helpful. PCR of aqueous and vitreous humour and buffy coat samples shows high sensitivity and specificity.18,19 The outcome of toxoplasmosis is related primarily to host and parasite genetics. Genetic analysis has subdivided T. gondii into three clonal lineages, genotypes I, II and III. The genotype of the infective agent may have implications for the prognosis of the disease.3,19 Several investigations have suggested that genotype II is the most prevalent genotype,19–22 and most studies in Europe and North America state that genotype II is the most common cause of congenital infections,20,21 ocular toxoplasmosis22,23 and infection associated with HIV-AIDS.20 Vallochi et al.24 demonstrated that genotype I is predominate in Brazilian ocular toxoplasmosis. Grigg et al.25 suggested that genotype I is associated with recurrent ocular toxoplasmosis in immunocompromised patients and may cause severe ocular disease. Genotype I has been implicated in several outbreaks associated with a high frequency of ocular toxoplasmosis.2,9 Serological Toxoplasma studies have been carried out in several areas of Iran,26 with limited reports of the Toxoplasma genotypes in soil,27 processed meat such as sausage,28 and chicken, beef and lamb.29 A previous study for the genotyping of ocular toxoplasmosis samples in Tehran used only one locus, GRA6.30 Retrospective descriptive studies have been performed on clinical patterns of uveitis in children and adults in a tertiary ophthalmology centre, reporting Toxoplasma as the most common infectious aetiology of uveitis.12–14 However, no research providing detailed clinical information, serological and molecular detection, or genotyping of T. gondii in ocular toxoplasmosis in Iran has been conducted. We evaluated clinical manifestations, IgM and IgG T. gondii antibodies, IgG avidity and conducted molecular detection of Toxoplasma using the B1 gene in suspected cases of ocular toxoplasmosis. To identify the relationship of T. gondii genotypes with clinical manifestations of ocular toxoplasmosis, genotypes of T. gondii were determined based on GRA6, SAG2, SAG3, BTUB and APICO genes. The association of age, gender, occupation and education, as well as contact with cats or soil, and consumption of undercooked meat with the incidence of ocular toxoplasmosis, was evaluated. Materials and methods Study area A cross-sectional study was performed in Niko Kari Hospital in Tabriz, East Azerbaijan Province, from June 2016 to June 2017. The study area is in northwest Iran (38°7’N and 46°20’E) and has a humid continental climate and annual precipitation of 280 mm and mean annual temperature of 12.6°C, with the lowest mean temperature in January (−1°C) and the highest in July (27°C). Patients and clinical examination Thirty-three patients identified by an ophthalmologist as probably exhibiting active ocular toxoplasmosis were included in the study. Fourteen (42%) were male and 19 (56%) were female, with ages ranging from 21–59 years (mean 34.8±9.3 years). Enrolled subjects completed a questionnaire and gave written informed consent after receiving information on study objectives and laboratory procedures. The project questionnaire included questions of demographic data and risk factors including gender, age, occupation, education, level of contact with cats and soil, and consumption of uncooked meat. Patient disease history was reviewed, eyes were examined via fundoscopy, and data were recorded by an ophthalmologist. Laboratory procedures Blood samples were collected and aliquoted to tubes with and without EDTA. The serum of clotted blood samples was collected for serological analysis and the blood samples containing EDTA were used to produce the buffy coat for molecular analysis. Serological analysis Detection of IgM and IgG T. gondii antibodies The T. gondii IgG and IgM in serum samples were analysed by chemiluminescence immunoassay (CLIA) using a Diasorin Liaison calibrated analyser (Germany) with a commercial kit (Diasorin, Italy) following the manufacturer’s instructions. To confirm Toxoplasma IgG- and IgM-positive sera, samples were evaluated by ELISA with the Toxoplasma IgG EIA Test Kit (ACON Laboratories, USA). IgG avidity for discrimination of acute and chronic infection was evaluated by ELISA with Biomerieux kit (Biomerieux, France) and VIDAS. IgG avidity assessment of each sample was duplicated. The avidity for positive IgG samples was calculated according to the manufacturer’s instructions. IgG avidity >40% indicates past toxoplasmosis infection and IgG avidity <40% indicates recent infection. Molecular analysis DNA extraction Genomic DNA was extracted from 200 μl of buffy coat using QIAamp DNA Blood Mini Kit (QIAGEN, Hilden, Germany) and stored at −20°C until use. PCR amplification of B1 gene The target of PCR was the 194 bp fragment of the 35-fold repetitive B1 gene (AF179871). The primers and PCR reaction were performed according to Schwab and McDevitt.31 PCR was conducted with a readymade Taq DNA Polymerase Master Mix (Amplicon III, Denmark, cat. no. 180301). The final mixture of the reaction contained 12.5 μl of Taq Master Mix (2×), 0.15 μM of each primer, 3 μl DNA and 8 μl distilled water. The PCR was carried out under the following conditions: an initial denaturation step at 95°C for 3 min, 40 cycles of denaturation at 95°C for 30 s, annealing at 55°C for 50 s, extension at 72°C for 20 s, and a final extension at 72°C for 5 min. Electrophoresis on 2% agarose gel was carried out for observation of the amplified fragment. Multi-locus genotyping The genotype of T. gondii was determined based on the GRA6, SAG2, SAG3, BTUB and APICO genetic markers.25,32 Five loci of T. gondii in the B1-positive samples were amplified by nested PCR with primers described by Su et al.32 Primary PCR was performed in a 25 μl final reaction mixture comprising 12.5 μl of Taq Master Mix (Amplicon III, Denmark, cat. no. 180301), 3 μL DNA, 0.15 μM of each primer and 7 μl distilled water under the following conditions: initial denaturation at 95°C for 5 min, 35 cycles of denaturation at 95°C for 30 s, annealing at 50°C for 30 s, extension at 72°C for 30 s, and a final extension at 72°C for 5 min. The amplified products were diluted 1:2 in sterile DNase-free distilled water and used as DNA template in secondary PCR with internal primers for each marker. The conditions of secondary PCR consisted of initial denaturation at 95°C for 5 min, 40 cycles of denaturation at 95°C for 20 s, annealing at 55°C for SAG2, 50°C for GRA6, 52°C for SAG3, 48°C for BTUB and 50°C for APICO for 15 s, an extension at 72°C for 20 s, and a final extension at 72°C for 5 min. The PCR products were visualised by electrophoresis on 1.5% agarose gel. Sequencing and phylogenetic analyses of GRA6, SAG2, SAG3, BTUB and APICO loci The amplicon of each marker was purified with the MinElute gel extraction kit (QIAGEN) and sequenced bi-directionally using internal amplification primers (Macrogen Inc., Seoul, South Korea). The sequencing results were visually verified with Chromas software and aligned with reference sequences retrieved from NCBI for each genotype using DNASIS MAX software v. 3.0 (Hitachi, Yokohama, Japan). The phylogenetic tree was constructed with MEGA7 software. Neighbour-joining analysis was used to depict the phylogenetic position of the GRA6, SAG2, SAG3, BTUB and APICO sequences based on the Kimura 2-parameter model of nucleotide substitution search by stepwise addition of 100 random replicates. The reliability of branches in trees was assessed by bootstrap analysis using 1000 replicates. Sequences from this study were submitted to GenBank under the accession numbers LC406298–LC406303, LC406327–LC406334, LC406343–LC406344, LC406346–LC406350, and LC406354–LC406358 for GRA6, SAG2, SAG3, BTUB and APICO, respectively. Data analysis SPSS v. 24 (SPSS Inc., Chicago, IL, USA) was used for statistical analysis. A descriptive analysis was performed to evaluate the frequency of the variables relative to T. gondii antibodies and molecular results. Potential associations were identified using Chi-square tests at a significance level of 0.05. Results Serology Chemiluminescence immunoassay and the Toxoplasma IgG EIA Test Kit showed 28 samples to be IgG positive (84.8%) (Table 1), 3 samples to be borderline (9%) and 2 samples to be negative (6%). Two T. gondii IgG antibody positive samples were also positive for IgM (6%). IgG avidity testing indicated one (3%) patient with recent toxoplasmosis infection (IgG avidity <40%). In 27 patients seropositive for toxoplasmosis (IgG avidity >40%), ocular inflammation may have been a result of reactivation of toxoplasmosis infection. We did not analyse the data to identify other causes of ocular inflammation in two patients seronegative for toxoplasmosis. Table 1. Clinical manifestations in patients with suspected ocular toxoplasmosis with respect to serological and molecular analysis Clinical manifestation Serology n (%) Molecular n (%) B1 PCR Positive Negative p-value Positive Negative p-value Symptoms  Blurring of vision 27 (87.1) 4 (12.9) 0.284 26 (83.9) 5 (16.1) 0.335  Floater 4 (80) 1 (20) 1.000 4 (80) 1 (20) 1.000  Redness 16 (80) 4 (20) 0.625 15 (75) 5 (25) 0.364  Eye pain 20 (80) 5 (20) 0.302 20 (80) 5 (20) 1.000  Photophobia 5 (83.3) 1 (16.7) 1.000 5 (83.3) 1 (16.7) 1.000  Headache 6 (75) 2 (25) 0.574 6 (75) 2 (25) 0.616 Clinical signs  Retinal inflammation 5 (71.4) 2 (28.6) 0.282 5 (71.4) 2 (28.6) 0.584  Anterior uveitis 6 (60) 4 (40) 0.021 7 (70) 3 (30) 0.336  Posterior uveitis 26 (86.7) 4 (13.3) 0.400 27 (90) 3 (10) 0.004  Vasculitis 6 (75) 2 (25) 0.574 6 (75) 2 (25) 0.616  Chorioretinitis 25 (86.2) 4 (13.8) 0.500 26 (89.7) 3 (10.3) 0.014  Chorioretinal scarring 22 (88) 3 (12) 0.574 21 (84) 4 (16) 0.616  Macular lesions 16 (88.9) 2 (11.1) 0.639 15 (83.3) 3 (16.7) 1.000  Total 28 (84.8) 5 (15.2) 27 (81.8) 6 (18.2) Clinical manifestation Serology n (%) Molecular n (%) B1 PCR Positive Negative p-value Positive Negative p-value Symptoms  Blurring of vision 27 (87.1) 4 (12.9) 0.284 26 (83.9) 5 (16.1) 0.335  Floater 4 (80) 1 (20) 1.000 4 (80) 1 (20) 1.000  Redness 16 (80) 4 (20) 0.625 15 (75) 5 (25) 0.364  Eye pain 20 (80) 5 (20) 0.302 20 (80) 5 (20) 1.000  Photophobia 5 (83.3) 1 (16.7) 1.000 5 (83.3) 1 (16.7) 1.000  Headache 6 (75) 2 (25) 0.574 6 (75) 2 (25) 0.616 Clinical signs  Retinal inflammation 5 (71.4) 2 (28.6) 0.282 5 (71.4) 2 (28.6) 0.584  Anterior uveitis 6 (60) 4 (40) 0.021 7 (70) 3 (30) 0.336  Posterior uveitis 26 (86.7) 4 (13.3) 0.400 27 (90) 3 (10) 0.004  Vasculitis 6 (75) 2 (25) 0.574 6 (75) 2 (25) 0.616  Chorioretinitis 25 (86.2) 4 (13.8) 0.500 26 (89.7) 3 (10.3) 0.014  Chorioretinal scarring 22 (88) 3 (12) 0.574 21 (84) 4 (16) 0.616  Macular lesions 16 (88.9) 2 (11.1) 0.639 15 (83.3) 3 (16.7) 1.000  Total 28 (84.8) 5 (15.2) 27 (81.8) 6 (18.2) Table 1. Clinical manifestations in patients with suspected ocular toxoplasmosis with respect to serological and molecular analysis Clinical manifestation Serology n (%) Molecular n (%) B1 PCR Positive Negative p-value Positive Negative p-value Symptoms  Blurring of vision 27 (87.1) 4 (12.9) 0.284 26 (83.9) 5 (16.1) 0.335  Floater 4 (80) 1 (20) 1.000 4 (80) 1 (20) 1.000  Redness 16 (80) 4 (20) 0.625 15 (75) 5 (25) 0.364  Eye pain 20 (80) 5 (20) 0.302 20 (80) 5 (20) 1.000  Photophobia 5 (83.3) 1 (16.7) 1.000 5 (83.3) 1 (16.7) 1.000  Headache 6 (75) 2 (25) 0.574 6 (75) 2 (25) 0.616 Clinical signs  Retinal inflammation 5 (71.4) 2 (28.6) 0.282 5 (71.4) 2 (28.6) 0.584  Anterior uveitis 6 (60) 4 (40) 0.021 7 (70) 3 (30) 0.336  Posterior uveitis 26 (86.7) 4 (13.3) 0.400 27 (90) 3 (10) 0.004  Vasculitis 6 (75) 2 (25) 0.574 6 (75) 2 (25) 0.616  Chorioretinitis 25 (86.2) 4 (13.8) 0.500 26 (89.7) 3 (10.3) 0.014  Chorioretinal scarring 22 (88) 3 (12) 0.574 21 (84) 4 (16) 0.616  Macular lesions 16 (88.9) 2 (11.1) 0.639 15 (83.3) 3 (16.7) 1.000  Total 28 (84.8) 5 (15.2) 27 (81.8) 6 (18.2) Clinical manifestation Serology n (%) Molecular n (%) B1 PCR Positive Negative p-value Positive Negative p-value Symptoms  Blurring of vision 27 (87.1) 4 (12.9) 0.284 26 (83.9) 5 (16.1) 0.335  Floater 4 (80) 1 (20) 1.000 4 (80) 1 (20) 1.000  Redness 16 (80) 4 (20) 0.625 15 (75) 5 (25) 0.364  Eye pain 20 (80) 5 (20) 0.302 20 (80) 5 (20) 1.000  Photophobia 5 (83.3) 1 (16.7) 1.000 5 (83.3) 1 (16.7) 1.000  Headache 6 (75) 2 (25) 0.574 6 (75) 2 (25) 0.616 Clinical signs  Retinal inflammation 5 (71.4) 2 (28.6) 0.282 5 (71.4) 2 (28.6) 0.584  Anterior uveitis 6 (60) 4 (40) 0.021 7 (70) 3 (30) 0.336  Posterior uveitis 26 (86.7) 4 (13.3) 0.400 27 (90) 3 (10) 0.004  Vasculitis 6 (75) 2 (25) 0.574 6 (75) 2 (25) 0.616  Chorioretinitis 25 (86.2) 4 (13.8) 0.500 26 (89.7) 3 (10.3) 0.014  Chorioretinal scarring 22 (88) 3 (12) 0.574 21 (84) 4 (16) 0.616  Macular lesions 16 (88.9) 2 (11.1) 0.639 15 (83.3) 3 (16.7) 1.000  Total 28 (84.8) 5 (15.2) 27 (81.8) 6 (18.2) Molecular analysis Molecular analysis of the 33 seropositive samples amplified the B1 gene in 27 samples (81.8%). Eleven (40.7%; 95% CI 24.5–59.3) B1 positive samples were successfully genotyped with at least one locus. Six, nine, six, five and seven samples were amplified and successfully sequenced for the GRA6, SAG2, SAG3, BTUB and APICO locus, respectively (Table 2). Multi-locus analysis of samples in which the five markers were successfully amplified identified them as genotype I. The nucleotide sequence alignment of samples with retrieved genotype I sequences of GenBank showed 99–100% homology. The phylogenetic trees of SAG2, GRA6, SAG3 and BTUB are presented in Figure 1. Table 2. Multi-locus genotype characterisation of Toxoplasma gondii DNA and serology from patients with suspected ocular toxoplasmosis Patient no. Serology Genotype/genetic marker B1 SAG2 GRA6 BTUB SAG3 APICO H_1 positive positive na na na na na H_2 positive positive na na na na na H_3 positive positive I na na na na H_4 borderline negative nd nd nd nd nd H_5 positive positive I I I I I H_6 positive positive na na na na na H_7 positive positive na na na na na H_8 positive positive na na na na na H_9 positive positive I I I I I H_10 borderline negative nd nd nd nd nd H_11 positive positive na na na na na H_12 positive positive na na na na na H_13 positive positive na na na na na H_14 borderline negative nd nd nd nd nd H_15 positive positive na na na na na H_16 positive positive na na na na na H_17 positive positive na I na na na H_18 positive positive I na na na I H_19 positive positive na na na na na H_20 positive positive I I I I I H_21 positive positive na na na na na H_22 positive positive I na na na na H_23 positive positive na na na na na H_24 positive positive na na na na na H_25 positive positive I I I I na H_26 positive positive na na na na I H_27 positive negative nd nd nd nd nd H_28 negative negative nd nd nd nd nd H_29 negative negative nd nd nd nd nd H_30 positive positive I I I I I H_31 positive positive na na na na na H_32 positive positive na na na na na H_33 positive positive I na na I I Patient no. Serology Genotype/genetic marker B1 SAG2 GRA6 BTUB SAG3 APICO H_1 positive positive na na na na na H_2 positive positive na na na na na H_3 positive positive I na na na na H_4 borderline negative nd nd nd nd nd H_5 positive positive I I I I I H_6 positive positive na na na na na H_7 positive positive na na na na na H_8 positive positive na na na na na H_9 positive positive I I I I I H_10 borderline negative nd nd nd nd nd H_11 positive positive na na na na na H_12 positive positive na na na na na H_13 positive positive na na na na na H_14 borderline negative nd nd nd nd nd H_15 positive positive na na na na na H_16 positive positive na na na na na H_17 positive positive na I na na na H_18 positive positive I na na na I H_19 positive positive na na na na na H_20 positive positive I I I I I H_21 positive positive na na na na na H_22 positive positive I na na na na H_23 positive positive na na na na na H_24 positive positive na na na na na H_25 positive positive I I I I na H_26 positive positive na na na na I H_27 positive negative nd nd nd nd nd H_28 negative negative nd nd nd nd nd H_29 negative negative nd nd nd nd nd H_30 positive positive I I I I I H_31 positive positive na na na na na H_32 positive positive na na na na na H_33 positive positive I na na I I na: not successfully amplified. nd: not down. Table 2. Multi-locus genotype characterisation of Toxoplasma gondii DNA and serology from patients with suspected ocular toxoplasmosis Patient no. Serology Genotype/genetic marker B1 SAG2 GRA6 BTUB SAG3 APICO H_1 positive positive na na na na na H_2 positive positive na na na na na H_3 positive positive I na na na na H_4 borderline negative nd nd nd nd nd H_5 positive positive I I I I I H_6 positive positive na na na na na H_7 positive positive na na na na na H_8 positive positive na na na na na H_9 positive positive I I I I I H_10 borderline negative nd nd nd nd nd H_11 positive positive na na na na na H_12 positive positive na na na na na H_13 positive positive na na na na na H_14 borderline negative nd nd nd nd nd H_15 positive positive na na na na na H_16 positive positive na na na na na H_17 positive positive na I na na na H_18 positive positive I na na na I H_19 positive positive na na na na na H_20 positive positive I I I I I H_21 positive positive na na na na na H_22 positive positive I na na na na H_23 positive positive na na na na na H_24 positive positive na na na na na H_25 positive positive I I I I na H_26 positive positive na na na na I H_27 positive negative nd nd nd nd nd H_28 negative negative nd nd nd nd nd H_29 negative negative nd nd nd nd nd H_30 positive positive I I I I I H_31 positive positive na na na na na H_32 positive positive na na na na na H_33 positive positive I na na I I Patient no. Serology Genotype/genetic marker B1 SAG2 GRA6 BTUB SAG3 APICO H_1 positive positive na na na na na H_2 positive positive na na na na na H_3 positive positive I na na na na H_4 borderline negative nd nd nd nd nd H_5 positive positive I I I I I H_6 positive positive na na na na na H_7 positive positive na na na na na H_8 positive positive na na na na na H_9 positive positive I I I I I H_10 borderline negative nd nd nd nd nd H_11 positive positive na na na na na H_12 positive positive na na na na na H_13 positive positive na na na na na H_14 borderline negative nd nd nd nd nd H_15 positive positive na na na na na H_16 positive positive na na na na na H_17 positive positive na I na na na H_18 positive positive I na na na I H_19 positive positive na na na na na H_20 positive positive I I I I I H_21 positive positive na na na na na H_22 positive positive I na na na na H_23 positive positive na na na na na H_24 positive positive na na na na na H_25 positive positive I I I I na H_26 positive positive na na na na I H_27 positive negative nd nd nd nd nd H_28 negative negative nd nd nd nd nd H_29 negative negative nd nd nd nd nd H_30 positive positive I I I I I H_31 positive positive na na na na na H_32 positive positive na na na na na H_33 positive positive I na na I I na: not successfully amplified. nd: not down. Figure 1. View largeDownload slide Phylograms of Toxoplasma gondii genotypes constructed by neighbour-joining analysis, based on the nucleotide sequences of (A) SAG2, (B) GRA6, (C) SAG3 and (D) BTUB retrieved from this study (clinical samples) and sequences retrieved from GenBank (indicated by accession numbers). The scale bar represents substitutions per nucleotide; bootstrapping is given as the percentage of 1000 replicates. Only bootstrap values >50 are indicated. Figure 1. View largeDownload slide Phylograms of Toxoplasma gondii genotypes constructed by neighbour-joining analysis, based on the nucleotide sequences of (A) SAG2, (B) GRA6, (C) SAG3 and (D) BTUB retrieved from this study (clinical samples) and sequences retrieved from GenBank (indicated by accession numbers). The scale bar represents substitutions per nucleotide; bootstrapping is given as the percentage of 1000 replicates. Only bootstrap values >50 are indicated. Clinical manifestations In ophthalmologist-identified cases, as well as in serological and molecularly positive subjects, the most prevalent clinical signs and symptoms were blurred vision, posterior uveitis and chorioretinitis, followed by chorioretinal scarring, eye pain, redness and macular lesions (Table 1, Figure 2). Chorioretinal scarring and posterior uveitis were significantly more prevalent in ocular toxoplasmosis patients with molecular diagnosis. Recurrent disease was observed in two patients. Figure 2. View largeDownload slide A 21-year-old man with blurred vision for two weeks. The two lower images are the right eye and the top two images are the left eye. In the usual cases, active lesions are seen as yellowish white foci of retinochoroidal mass adjacent to an atrophic retinal scar. Here, arrows indicate yellowing white choroidal lesions with ill-defined borders involving the macular area, and bleaching centres, often in the vicinity of a coloured and/or atrophic area. An active retinochoroidal lesion causes atrophic retinal scar, which resolves from the periphery to the centre of the lesion. Figure 2. View largeDownload slide A 21-year-old man with blurred vision for two weeks. The two lower images are the right eye and the top two images are the left eye. In the usual cases, active lesions are seen as yellowish white foci of retinochoroidal mass adjacent to an atrophic retinal scar. Here, arrows indicate yellowing white choroidal lesions with ill-defined borders involving the macular area, and bleaching centres, often in the vicinity of a coloured and/or atrophic area. An active retinochoroidal lesion causes atrophic retinal scar, which resolves from the periphery to the centre of the lesion. Ocular toxoplasmosis and possible risk factors Serological and molecular detection of ocular toxoplasmosis relative to sociodemographic variables is shown in Table 3. Molecular analyses showed 84.2% (16/19; 95% CI 62.4–94.5) and 78.6% (11/14; 95% CI 52.4–92.2) of females and males were positive for ocular toxoplasmosis, respectively. No correlation of gender and infection with ocular toxoplasmosis was observed (p=1.000). The highest number of positive cases of 48.1% (13/27; 95% CI 30.7–66.0) was reported in patients aged 30–39 years. With respect to the evaluated risk factors, soil contact demonstrated the highest (18/27; 66.7%; 95% CI 47.8–81.4) association with ocular toxoplasmosis. Homemaker (14/27; 51.9%; 95% CI 34.0–69.3) and high school (15/27; 55.6%; 95% CI 37.3–72.4) were the occupation and education level most frequently associated with ocular toxoplasmosis, respectively. However, the disease showed no significant correlation with occupation and education or with gender, age or behaviours. Table 3. Demographic data of the patients suspected to have ocular toxoplasmosis and the results of serological and molecular detection of Toxoplasma gondii Risk factor No. (%) IgG IgG avidity IgM B1 p-value Gender 1.000  Male 14 (42.4) 11 0 0 11  Female 19 (57.6) 17 1 2 16  Total 33 (100.0) 28 1 2 27 Age groups 0.18  20–29 9 (27.3) 8 0 0 7  30–39 16 (48.5) 13 1 1 13  >40 8 (24.2) 7 0 1 7  Total 33 (100.0) 28 1 2 27 Behaviour 0.301  Contact with cat 7 (21.2) 6 1 1 5  Contact with soil 20 (60.6) 18 1 1 18  Undercooked meat 6 (18.2) 7 1 2 4  Total 33 (100.0) Occupation 0.79  Homemaker 18 (54.5) 15 1 2 14  Carpet weaver 3 (9.1) 3 0 0 3  Employee 2 (6.1) 1 0 0 1  Clerk 6 (18.2) 5 0 0 5  Farmer 1 (3.0) 1 0 0 1  Driver 1 (3.0) 1 0 0 1  Student 2 (6.1) 2 0 0 2  Total 33 (100.0) 28 1 2 27 Education level 0.51  Primary school 5 (15.2) 5 0 1 5  High school 19 (57.6) 15 1 1 15  University 9 (27.3) 8 0 0 7  Total 33 (100.0) 28 1 2 27 Risk factor No. (%) IgG IgG avidity IgM B1 p-value Gender 1.000  Male 14 (42.4) 11 0 0 11  Female 19 (57.6) 17 1 2 16  Total 33 (100.0) 28 1 2 27 Age groups 0.18  20–29 9 (27.3) 8 0 0 7  30–39 16 (48.5) 13 1 1 13  >40 8 (24.2) 7 0 1 7  Total 33 (100.0) 28 1 2 27 Behaviour 0.301  Contact with cat 7 (21.2) 6 1 1 5  Contact with soil 20 (60.6) 18 1 1 18  Undercooked meat 6 (18.2) 7 1 2 4  Total 33 (100.0) Occupation 0.79  Homemaker 18 (54.5) 15 1 2 14  Carpet weaver 3 (9.1) 3 0 0 3  Employee 2 (6.1) 1 0 0 1  Clerk 6 (18.2) 5 0 0 5  Farmer 1 (3.0) 1 0 0 1  Driver 1 (3.0) 1 0 0 1  Student 2 (6.1) 2 0 0 2  Total 33 (100.0) 28 1 2 27 Education level 0.51  Primary school 5 (15.2) 5 0 1 5  High school 19 (57.6) 15 1 1 15  University 9 (27.3) 8 0 0 7  Total 33 (100.0) 28 1 2 27 Table 3. Demographic data of the patients suspected to have ocular toxoplasmosis and the results of serological and molecular detection of Toxoplasma gondii Risk factor No. (%) IgG IgG avidity IgM B1 p-value Gender 1.000  Male 14 (42.4) 11 0 0 11  Female 19 (57.6) 17 1 2 16  Total 33 (100.0) 28 1 2 27 Age groups 0.18  20–29 9 (27.3) 8 0 0 7  30–39 16 (48.5) 13 1 1 13  >40 8 (24.2) 7 0 1 7  Total 33 (100.0) 28 1 2 27 Behaviour 0.301  Contact with cat 7 (21.2) 6 1 1 5  Contact with soil 20 (60.6) 18 1 1 18  Undercooked meat 6 (18.2) 7 1 2 4  Total 33 (100.0) Occupation 0.79  Homemaker 18 (54.5) 15 1 2 14  Carpet weaver 3 (9.1) 3 0 0 3  Employee 2 (6.1) 1 0 0 1  Clerk 6 (18.2) 5 0 0 5  Farmer 1 (3.0) 1 0 0 1  Driver 1 (3.0) 1 0 0 1  Student 2 (6.1) 2 0 0 2  Total 33 (100.0) 28 1 2 27 Education level 0.51  Primary school 5 (15.2) 5 0 1 5  High school 19 (57.6) 15 1 1 15  University 9 (27.3) 8 0 0 7  Total 33 (100.0) 28 1 2 27 Risk factor No. (%) IgG IgG avidity IgM B1 p-value Gender 1.000  Male 14 (42.4) 11 0 0 11  Female 19 (57.6) 17 1 2 16  Total 33 (100.0) 28 1 2 27 Age groups 0.18  20–29 9 (27.3) 8 0 0 7  30–39 16 (48.5) 13 1 1 13  >40 8 (24.2) 7 0 1 7  Total 33 (100.0) 28 1 2 27 Behaviour 0.301  Contact with cat 7 (21.2) 6 1 1 5  Contact with soil 20 (60.6) 18 1 1 18  Undercooked meat 6 (18.2) 7 1 2 4  Total 33 (100.0) Occupation 0.79  Homemaker 18 (54.5) 15 1 2 14  Carpet weaver 3 (9.1) 3 0 0 3  Employee 2 (6.1) 1 0 0 1  Clerk 6 (18.2) 5 0 0 5  Farmer 1 (3.0) 1 0 0 1  Driver 1 (3.0) 1 0 0 1  Student 2 (6.1) 2 0 0 2  Total 33 (100.0) 28 1 2 27 Education level 0.51  Primary school 5 (15.2) 5 0 1 5  High school 19 (57.6) 15 1 1 15  University 9 (27.3) 8 0 0 7  Total 33 (100.0) 28 1 2 27 Discussion Serological analysis in patients suspected to have ocular toxoplasmosis in Tabriz, Iran showed ocular inflammation in 3% and 81.8% of patients as a result of recent and reactivated toxoplasmosis infection, respectively. This confirmed the suggestion that most ocular toxoplasmosis is a result of reactivation of a previous infection,17,19 and is in contrast to the findings of Previato et al.33 and Delair et al.,34 who found it more frequent in recent infections. This difference may be due to the geographical distribution of parasite strain genotype. The molecular analysis detected DNA of Toxoplasma in 81.8% (27/33; 95% CI 65.6–91.4) of patients and confirmed 96.4% (27/28; 95% CI 82.3–99.4) of seropositive patients. Detection of T. gondii DNA has been suggested as a useful tool for diagnosis of ocular toxoplasmosis.19,30,35,36 PCR can detect Toxoplasma DNA in aqueous humour, vitreous fluid19,36 and blood18,30,35. As a non-invasive and simpler sampling method, it is preferred to detect DNA circulating in blood. The parasite DNA in blood likely originates from tissue cysts in the eye or other infected tissue or the parasitaemia of T. gondii tachyzoites in the peripheral blood during reactivation of earlier Toxoplasma infection,17 which may be related to the level of virulence of the parasite genotypes.19 Multi-locus genotyping of GRA6, SAG2, SAG3, BTUB and APICO loci provided evidence of genotype I of T. gondii as a causative agent of ocular toxoplasmosis in Tabriz. Genotype I was the only genotype previously reported in processed meat,28 chicken, beef and lamb29 consumed in Tabriz; hence, ocular toxoplasmosis was presumed to be associated with this genotype. Genotype I is also the predominant genotype reported in ocular toxoplasmosis patients in Poland,18 Brazil,24 Indonesia37 and USA.25 However, genotype III was reported in the only previous molecular study of ocular toxoplasmosis patients in Tehran.30 Ocular toxoplasmosis in immunocompetent, as well as immunocompromised individuals, is one of the most common causative agents of chorioretinitis.9,15 Reported clinical manifestations and the outcome of disease range from blurred vision, floaters and chorioretinitis to blindness, according to immune status and host genetics as well as parasite genotype.2,15,38 The most prevalent clinical manifestations revealed in this study were blurred vision, posterior uveitis and chorioretinitis, followed by chorioretinal scarring, eye pain, redness and macular lesions. Genotype II is reported as predominant in acquired20 and congenital toxoplasmosis;39 but compared to genotype II, genotype I18,24,37 and recombinant I and III are more commonly associated with human ocular toxoplasmosis,25,40 except for in Europe where genotype I is very rare.22 Consequently, we suggest that infection with genotype I may be most likely to result in ocular toxoplasmosis due to its higher virulence.10,18,41 We investigated several factors potentially associated with ocular toxoplasmosis, but no significant correlation was found between gender and the development of ocular toxoplasmosis. This finding is in agreement with Ferreira et al.42 in Brazil. The first symptoms of ocular toxoplasmosis are usually reported in the second decade of life, a finding that is remarkably similar among studies.2,7 All patients participating in this study were aged >20 years, consistent with our suggestion, based on serological findings, that reactivation of Toxoplasma infection was the cause of ocular disease in the majority of the patients. We found no correlation of age with ocular toxoplasmosis, in agreement with previous studies.1,42 The incidence of toxoplasmosis is primarily related to infection from cysts, oocysts or tachyzoites of Toxoplasma through consumption of contaminated meat, water, vegetables and fruits, contact with soil, organ transplantation, or congenital infection associated with behaviours, lifestyle and culture, rather than to gender or age.1,19,42 The current study found no relationship between development of ocular toxoplasmosis and behaviours, occupation or education. Among investigated behaviours, contact with soil was the most common risk factor (90.0%; 95% CI 69.9–97.2). Incidence of ocular toxoplasmosis might be associated with parasite genotype as we found genotype I in all patients, which is reported to show higher pathogenicity than other genotypes.10,18,41 However, about half of positive T. gondii DNA in patients were confirmed with just one or two genetic markers, such that it precludes a definite conclusion. A limitation of the study is that it was conducted in patients with suspected active ocular toxoplasmosis referred to a single centre; thus, our findings may not be representative of the general population, although this centre receives referrals from neighbouring provinces. In addition, sample-size limitations may have prevented detection of valid associations of risk factors with ocular toxoplasmosis. Further studies with a larger number of patients could confirm the results reported here. Conclusions This study showed that ophthalmologist diagnosis according to clinical presentation was well correlated to serological and molecular detection of Toxoplasma as an agent of posterior uveitis in patients. It also confirmed that blood is a reliable and non-invasive source for detection of Toxoplasma DNA in ocular toxoplasmosis. Multi-locus genotyping demonstrated genotype I T. gondii as predominant in ocular toxoplasmosis in Tabriz, but we cannot generalise to other areas of Iran. A broader comparative study should be conducted to provide more information concerning molecular epidemiology of T. gondii genotypes and their role in human toxoplasmosis. The high prevalence of ocular toxoplasmosis in Iran demands an educational campaign regarding the dangers of eating raw or undercooked meat and contact with cats, as well as soil-related hygiene, with respect to preventing infection. Author contributions: MA, ER, LA and ARM conceived and designed the study. MA performed the laboratory experiments. LAG, KN and FM performed the ocular examinations. MA and ER provided analysis and interpretation of the data. MA, ER, LA, ARM, MM, ZR and MK contributed reagents/materials/analysis tools. MA and ZR provided statistical analysis. MA and ER wrote the paper. All authors read and approved the final manuscript. ER is the guarantor of the paper. Acknowledgements: None. Funding: This work was supported by Iran University of Medical Sciences [95-01-30-27820]. Competing interests: None declared. Ethics approval: This study was approved by the research council of Iran University of Medical Sciences [IR. IUMS.REC1395.9221577202]. Patient consent was obtained. References 1 Jones JL , Dargelas V , Roberts J , et al. Risk factors for Toxoplasma gondii infection in the United States . Clin Infect Dis . 2009 ; 49 : 878 – 84 . Google Scholar Crossref Search ADS PubMed 2 Pleyer U , Schlüter D , Mänz M . Ocular Toxoplasmosis: Recent Aspects of Pathophysiology and Clinical Implications . Ophthalmic Res . 2014 ; 52 : 116 – 23 . Google Scholar Crossref Search ADS PubMed 3 Kijlstra A , Petersen E . Epidemiology, Pathophysiology, and the Future of Ocular Toxoplasmosis . Ocul Immunol Inflamm . 2014 ; 22 : 138 – 47 . Google Scholar Crossref Search ADS PubMed 4 Maenz M , Schlüter D , Liesenfeld O , et al. Ocular toxoplasmosis past, present and new aspects of an old disease . Prog Retin Eye Res . 2014 ; 39 : 77 – 106 . Google Scholar Crossref Search ADS PubMed 5 Subauste CS , Ajzenberg D , Kijlstra A . Review of the series ‘Disease of the year 2011: toxoplasmosis’ pathophysiology of toxoplasmosis . Ocul Immunol Inflamm . 2011 ; 19 : 297 – 306 . Google Scholar Crossref Search ADS PubMed 6 Burnett AJ , Shortt SG , Isaac-Renton J , et al. Multiple cases of acquired toxoplasmosis retinitis presenting in an outbreak . Ophthalmology . 1998 ; 105 : 1032 – 7 . Google Scholar Crossref Search ADS PubMed 7 Gilbert RE , Stanford MR . Is ocular toxoplasmosis caused by prenatal or postnatal infection? Br J Ophthalmol . 2000 ; 84 : 224 – 6 . Google Scholar Crossref Search ADS PubMed 8 Perkins E . Ocular toxoplasmosis . Br J Ophthalmol . 1973 ; 57 : 1 – 17 . Google Scholar Crossref Search ADS PubMed 9 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 . Google Scholar Crossref Search ADS PubMed 10 Gilbert RE , Freeman K , Lago EG , et al. Ocular sequelae of congenital toxoplasmosis in Brazil compared with Europe . PLoS Negl Trop Dis . 2008 ; 2 : e277 . Google Scholar Crossref Search ADS PubMed 11 de Amorim Garcia CA , Orefice F , de Oliveira Lyra C , et al. Socioeconomic conditions as determining factors in the prevalence of systemic and ocular toxoplasmosis in Northeastern Brazil . Ophthalmic Epidemiol . 2004 ; 11 : 301 – 17 . Google Scholar Crossref Search ADS PubMed 12 Ghavidel LA , Mousavi F , Bagheri M , et al. Clinical Course of Uveitis in Children in a Tertiary Ophthalmology Center in Northwest Iran . Crescent J Med Biol Sci . 2017 ; 4 : 200 – 4 . 13 Kianersi F , Mohammadi Z , Ghanbari H , et al. Clinical patterns of uveitis in an Iranian tertiary eye-care center . Ocul Immunol Inflamm . 2015 ; 23 : 278 – 82 . Google Scholar Crossref Search ADS PubMed 14 Soheilian M , Heidari K , Yazdani S , et al. Patterns of uveitis in a tertiary eye care center in Iran . Ocul Immunol Inflamm . 2004 ; 12 : 297 – 310 . Google Scholar Crossref Search ADS PubMed 15 Butler NJ , Furtado JM , Winthrop KL , et al. Ocular toxoplasmosis II: clinical features, pathology and management . Clin Exp Ophthalmol . 2013 ; 41 : 95 – 108 . Google Scholar Crossref Search ADS PubMed 16 Chan AS , Mudhar H , Shen SY , et al. Serum IgG2 and tissue IgG2 plasma cell elevation in orbital IgG4-related disease (IgG4-RD): Potential use in IgG4-RD assessment . Br J Ophthalmol . 2017 ; 101 : 1576 – 82 . Google Scholar Crossref Search ADS PubMed 17 Silveira C , Vallochi AL , da Silva UR , et al. Toxoplasma gondii in the peripheral blood of patients with acute and chronic toxoplasmosis . Br J Ophthalmol . 2011 ; 95 : 396 – 400 . Google Scholar Crossref Search ADS PubMed 18 Switaj K , Master A , Borkowski PK , et al. Association of ocular toxoplasmosis with type I Toxoplasma gondii strains: direct genotyping from peripheral blood samples . J Clin Microbiol . 2006 ; 44 : 4262 – 4 . Google Scholar Crossref Search ADS PubMed 19 Robert-Gangneux F , Dardé M-L . Epidemiology of and Diagnostic Strategies for Toxoplasmosis . Clin Microbiol Rev . 2012 ; 25 : 264 – 96 . Google Scholar Crossref Search ADS PubMed 20 Howe DK , Sibley LD . Toxoplasma gondii comprises three clonal lineages: correlation of parasite genotype with human disease . J Infect Dis . 1995 ; 172 : 1561 – 6 . Google Scholar Crossref Search ADS PubMed 21 Ajzenberg D , Cogne´ N , Paris L , et al. Genotype of 86 Toxoplasma gondii isolates associated with human congenital toxoplasmosis, and correlation with clinical findings . J Infect Dis . 2002 ; 186 : 684 – 9 . Google Scholar Crossref Search ADS PubMed 22 Herrmann DC , Maksimov P , Hotop A , et al. Genotyping of samples from German patients with ocular, cerebral and systemic toxoplasmosis reveals a predominance of Toxoplasma gondii type II . Int J Med Microbiol . 2014 ; 304 : 911 – 6 . Google Scholar Crossref Search ADS PubMed 23 Fekkar A , Ajzenberg D , Bodaghi B , et al. Direct genotyping of Toxoplasma gondii in ocular fluid samples from 20 patients with ocular toxoplasmosis: predominance of type II in France . J Clin Microbiol . 2011 ; 49 : 1513 – 7 . Google Scholar Crossref Search ADS PubMed 24 Vallochi AL , Muccioli C , Martins MC , et al. The genotype of Toxoplasma gondii strains causing ocular toxoplasmosis in humans in Brazil . Am J Ophthalmol . 2005 ; 139 : 350 – 1 . Google Scholar Crossref Search ADS PubMed 25 Grigg ME , Ganatra J , Boothroyd JC , et al. Unusual abundance of atypical strains associated with human ocular toxoplasmosis . J Infect Dis . 2001 ; 184 : 633 – 9 . Google Scholar Crossref Search ADS PubMed 26 Daryani A , Sarvi S , Aarabi M , et al. Seroprevalence of Toxoplasma gondii in the Iranian general population: a systematic review and meta-analysis . Acta Trop . 2014 ; 137 : 185 – 94 . Google Scholar Crossref Search ADS PubMed 27 Tavalla M , Oormazdi H , Akhlaghi L , et al. Genotyping of Toxoplasma gondii isolates from soil samples in Tehran, Iran . Iran J Parasitol . 2013 ; 8 : 227 – 33 . Google Scholar PubMed 28 Fallah E , Hajizadeh M , Farajnia S , et al. SAG2 locus genotyping of Toxoplasma gondii in meat products of East Azerbaijan Province, North West of Iran During 2010-2011 . Afr J Biotechnol . 2011 ; 10 : 13631 – 5 . Google Scholar Crossref Search ADS 29 Mahami-Oskouei M , Moradi M , Fallah E , et al. Molecular Detection and Genotyping of Toxoplasma gondii in Chicken, Beef, and Lamb Meat Consumed in Northwestern Iran . Iran J Parasitol . 2017 ; 12 : 38 – 45 . Google Scholar PubMed 30 Norouzi M , Tabaei SJS , Niyyati M , et al. Genotyping of Toxoplasma gondii Strains Isolated from Patients with Ocular Toxoplasmosis in Iran . Iran J Parasitol . 2016 ; 11 : 316 – 24 . Google Scholar PubMed 31 Schwab KJ , McDevitt JJ . Development of a PCR-enzyme immunoassay oligoprobe detection method for Toxoplasma gondii oocysts, incorporating PCR controls . Appl Environ Microbiol . 2003 ; 69 : 5819 – 25 . Google Scholar Crossref Search ADS PubMed 32 Su C , Shwab E , Zhou P , et al. Moving towards an integrated approach to molecular detection and identification of Toxoplasma gondii . Parasitology . 2010 ; 137 : 1 – 11 . Google Scholar Crossref Search ADS PubMed 33 Previato M , Frederico FB , Henrique Antunes Murata F , et al. A Brazilian report using serological and molecular diagnosis to monitoring acute ocular toxoplasmosis . BMC Res Notes . 2015 ; 8 : 746 . Google Scholar Crossref Search ADS PubMed 34 Delair E , Monnet D , Grabar S , et al. Respective Roles of Acquired and Congenital Infections in Presumed Ocular Toxoplasmosis . Am J Ophthalmol . 2008 ; 146 : 851 – 5 . Google Scholar Crossref Search ADS PubMed 35 Cavattoni I , Ayuk F , Zander AR , et al. Diagnosis of Toxoplasma gondii infection after allogeneic stem cell transplant can be difficult and requires intensive scrutiny . Leuk Lymphoma . 2010 ; 51 : 1530 – 5 . Google Scholar Crossref Search ADS PubMed 36 Farhadi A , Haniloo A , Fazaeli A , et al. PCR-based Diagnosis of Toxoplasma Parasite in Ocular Infections Having Clinical Indications of Toxoplasmosis . Iran J Parasitol . 2017 ; 12 : 56 – 62 . Google Scholar PubMed 37 Mayashinta DK , Halleyantoro R , Sari IP , et al. Genotyping of Toxoplasma gondii in Cerebral and Ocular Toxoplasmosis . J Trop Life Sci . 2018 ; 8 : 211 – 6 . Google Scholar Crossref Search ADS 38 Boothroyd JC , Grigg ME . Population biology of Toxoplasma gondii and its relevance to human infection: do different strains cause different disease? Curr Opin Microbiol . 2002 ; 5 : 438 – 42 . Google Scholar Crossref Search ADS PubMed 39 Nowakowska D , Colón I , Remington JS , et al. Genotyping of Toxoplasma gondii by multiplex PCR and peptide-based serological testing of samples from infants in Poland diagnosed with congenital toxoplasmosis . J Clin Microbiol . 2006 ; 44 : 1382 – 9 . Google Scholar Crossref Search ADS PubMed 40 Ferreira IMR , Vidal JE , de Mattos Cde C , et al. Toxoplasma gondii isolates: Multilocus RFLP–PCR genotyping from human patients in Sao Paulo State, Brazil identified distinct genotypes . Exp Parasitol . 2011 ; 129 : 190 – 5 . Google Scholar Crossref Search ADS PubMed 41 Yang N , Farrell A , Niedelman W , et al. Genetic basis for phenotypic differences between different Toxoplasma gondii type I strains . BMC Genomics . 2013 ; 14 : 467 . Google Scholar Crossref Search ADS PubMed 42 Ferreira A , De Mattos CB , Frederico F , et al. Risk factors for ocular toxoplasmosis in Brazil . Epidemiol Infect . 2014 ; 142 : 142 – 8 . Google Scholar PubMed © The Author(s) 2019. Published by Oxford University Press on behalf of Royal Society of Tropical Medicine and Hygiene. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)
TI - Sero-molecular detection, multi-locus genotyping, and clinical manifestations of ocular toxoplasmosis in patients in northwest Iran
JO - Transactions of The Royal Society of Tropical Medicine and Hygiene
DO - 10.1093/trstmh/try137
DA - 2019-04-01
UR - https://www.deepdyve.com/lp/oxford-university-press/sero-molecular-detection-multi-locus-genotyping-and-clinical-jKsmRY0TU3
SP - 195
VL - 113
IS - 4
DP - DeepDyve
ER -