Role of IgG avidity in the eyes with active Toxoplasma retinochoroiditis

doi:10.21203/rs.3.rs-2735047/v2

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Role of IgG avidity in the eyes with active Toxoplasma retinochoroiditis

https://doi.org/10.21203/rs.3.rs-2735047/v2

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Purpose: To study toxoplasma IgG avidity to evaluate the stage of infection and clinical implications in cases of toxoplasma retinochoroiditis in eastern India.

Methods: Retrospective review of toxoplasma retinochoroiditis cases with toxoplasma serology for IgG, IgM, and IgG avidity.

Results: Included in this study were 17 eyes of 17 patients which had active retinitis located in the macula (14), mid-periphery (2), or periphery (1). They were either primary lesions (12) or reactivations (5). All the cases had toxoplasma IgG positive; one case had IgM positivity while all the cases had high IgG avidity values. IgG avidity had a positive correlation with the duration of symptoms.

Conclusions: We observed high IgG avidity values in active retinochoroiditis in both primary ocular toxoplasmosis and reactivation subgroups. These results indicate a late ocular manifestation after initial systemic infection.

Toxoplasma retinochoroiditis

Toxoplasma IgG avidity

IgM

IgG

Ocular toxoplasmosis (OT) is often a clinical diagnosis with a typical clinical presentation of active focal round/oval retinitis accompanied by a pigmented retinochoroidal atrophic scar [[i]]. Toxoplasma serology plays a crucial role in suspected OT cases, primarily by excluding Toxoplasma gondii infection when IgG is negative. A positive toxoplasma IgM in a setting of focal necrotizing retinitis is an additional classification criterion for acute Toxoplasma retinitis.

In immunocompetent cases, the systemic infection caused by Toxoplasma gondii is often asymptomatic and results in a self-limiting febrile illness. In systemic infections, the mainstay of diagnosis is often serology with the detection of anti-toxoplasma IgG, IgM, and IgA antibodies [[ii]]. Specific IgM usually appears approximately a week after exposure, and IgG appears 1 to 3 weeks after IgM. However, distinguishing between past and recent infections can be challenging since Toxoplasma IgG may persist for years following the primary infection. IgG avidity testing has proven valuable in confirming acute infections and distinguishing between reactivations and primary infections using a single serum sample. Avidity refers to the aggregate strength of the reaction between a mixture of polyclonal IgG molecules and multiple protein epitopes [[iii]]. A high Toxoplasma IgG avidity index excludes a recent infection (within the past four to five months), whereas a low index indicates a likely current infection without ruling out older infections [[iv]]. Intermediate and moderate Toxoplasma IgG avidity results pose difficulties in interpretation for risk assessment [[v]].

Recently, toxoplasma IgG avidity testing has gained significance in obstetrics, particularly in pregnant women, to determine the timing of infection and guide management strategies. IgG avidity, combined with IgM testing, aids in differentiating acute infection from chronic and is often deployed as a first-line investigation strategy for toxoplasmosis [2,[vi]].

IgG avidity testing in OT has demonstrated its ability to differentiate between recently acquired infections and the reactivation of past infections [[vii]]. The patterns of infection and the presentation of retinitis following the initial infection may vary across different populations and regions. In this study, we aim to investigate the relevance of IgG avidity testing and the distribution of IgM positivity in cases of active retinochoroiditis presenting with toxoplasma IgG positivity in eastern India.

We retrospectively analyzed electronic medical records of cases diagnosed with Toxoplasma retinochoroiditis from a tertiary eye care centre in eastern India. The study was approved by the institutional review board (2021-107-BHR-41; dated 03/August/2022) and adhered to the guidelines of the Declaration of Helsinki.

All cases diagnosed as Toxoplasma retinochoroiditis with adequate documentation of comprehensive ocular examination including best‑corrected visual acuity (BCVA), slit‑lamp examination, intraocular pressures (IOPs), fundus photography and toxoplasma serology (IgG, IgM, and IgG avidity) were included in the study during the study period of January 2020 to June 2022. The cases were examined, and the records were reviewed by a uveitis consultant. Cases with inadequate documentation, inadequate investigations or absence of serology reports were excluded from the study.

The site and size of retinochoroiditis lesions were noted from the fundus photograph and the location of the lesions was divided into macular, mid-peripheral and periphery. The presence/absence of old healed toxoplasma retinochoroiditis lesions, retinal vasculitis and or anterior segments signs of inflammation was noted and analyzed. Cases were divided into two subgroups ‘Primary ocular toxoplasmosis and ‘Reactivation’.

Study definition:

‘Primary ocular toxoplasmosis’ was defined as cases which satisfied all of the following criteria (Figure 1a,1b):

active focal full-thickness retinochoroiditis
absence of adjacent or isolated old healed retinochoroiditis scars in either of the eyes
IgG positivity
Full-thickness retinitis on OCT along with underlying choroidal thickening.

‘reactivation subgroup’ was defined as cases with the typical or classical pattern of focal active full-thickness retinochoroiditis along with adjacent/isolated retinochoroiditis toxoplasma scar along with toxoplasma IgG positivity.

Toxoplasma IgG, IgM and IgG avidity values were noted and analyzed in all cases. Toxoplasma serology was done in all cases by the ELISA method by the same commercially available kit and manufacturer instructions were followed. The blood samples with Toxoplasma IgG > 8.8 IU/ml were considered positive while <7.2 IU/ml were considered negative; Toxoplasma IgG upper limit for detection was 400 IU/ml. Toxoplasma IgM <6 AU/ml were considered negative while >8 AU/ml were considered positive; the lower limit of detection for Toxoplasma IgM was 3 AU/ml. IgG Avidity was calculated in percentage. IgG avidity was labelled high (>35%), doubtful (30-35%) or low (<30%). We used ‘cut off’ values as standardized by the manufacturer for this kit.

Statistical analysis: Visual acuity records with Snellen’s visual acuity charts were converted to log MAR. Categorical variables were presented in number and percentage (%) and continuous variables were presented as mean ± SD and median. Linear regression analysis was used to correlate IgG avidity values with the duration of symptoms, size of retinitis and improvement in visual acuity. The data was entered and analysed in an MS EXCEL spreadsheet, and analysis was done using Statistical Package for Social Sciences (SPSS) version 21.0.

We included 17 eyes from 17 cases with the mean age of presentation 34.7 ± 15.81 years (range 8–79 years). Male preponderance with 70.6% of males (n=12) and 29.4% of females (n=5) was observed [Table 1].

All the cases were unilateral in presentation (right eye 9 eyes: left eye 8 eyes). The mean best corrected Log MAR visual acuity (BCVA) at presentation was 0.71 (±0.63) at presentation. There was an improvement of vision at the last visit by two Snellen’s lines with the mean log MAR BCVA at the final visit 0.51 (±0.43). Visual acuity improvement was not statistically significant (p-value- 0.3). Two eyes had a vision that was worse than baseline, with one case developing Choroidal neovascular membrane (CNVM) subsequently and the other case having a new macular recurrence of retinochoroiditis.

Clinical characteristics

The mean size of retinitis was 1.53 ±0.89-disc diameter (DD). The location of the lesion was either macular (n=14), mid-peripheral (n=2) or peripheral (n=1). Non-macular lesions (mid-peripheral and peripheral) were significantly (p=0.0096) larger (2.66 DD) as compared to macular lesions (1.28 DD). Associated features observed were retinal vasculitis (n=3), and vitritis (grade ≤ 2, n=16; grade > 2, n=1). Anterior chamber (AC) inflammation was seen in seven eyes of which grade one was in three eyes, grade two an d grade four AC cells in two eyes each.

The mean intraocular pressure at baseline was 16.05 (±8.5). Three eyes had elevated baseline IOP (>20 mm Hg). The mean follow-up period was 7.58 weeks. Three eyes developed complications during the follow-up duration (CNVM, epiretinal membrane, new lesions).

The primary toxoplasma retinochoroiditis sub-group had 12 cases while the recurrence sub-group had 5 cases.

Toxoplasma serology – IgG, IgM, IgG avidity

All eyes included in the study had positive Toxoplasma IgG. Three cases had values of IgG above the detectable limit of 400 IU/ml; excluding these three cases rest of the cases had mean IgG of 77.92 (range 5.64-111) which was 10.82 times higher than normal values.

Toxoplasma IgM was seen positive in a single case which also had a macular lesion with no evidence of old healed lesions in either eye. All but two eyes had IgM values below the detectable limit of <3 AU/ml.

The mean IgG avidity in our study was ‘high’ at 80.76 % (±15.53). Interestingly, none of the cases including the one with positive IgM had low avidity.

Serology of primary ocular toxoplasmosis subgroup

Of the 17 cases recruited, 12 cases had no evidence of prior healed retinochoroiditis in either eye. This subgroup had one earlier-mentioned case with IgM positivity while all other cases were IgM negative. The IgG avidity in this subgroup was high in all cases. The mean IgG avidity of the primary OT group (80.74%) was lower than the recurrence subgroup (84.53%), however, the difference was statistically insignificant (p=0.6).

Correlation of IgG avidity with the duration of symptoms size of retinitis and, improvement in visual acuity

The mean duration of symptoms before the presentation of OT was 2.94 weeks (±1.43). The linear regression analysis showed a positive correlation between the duration of symptoms with IgG avidity values (p-value 0.017). (Figure 2) Linear regression analysis predicted mean 5 weeks duration before the presentation when the IgG avidity could have been ‘low’. (Figure 1) There was no statistically significant correlation between IgG avidity values and size or retinitis or improvement in visual acuity.

We describe 17 eyes of 17 cases of Toxoplasma retinochoroiditis from eastern India serologically showing high IgG avidity at baseline. The subgroup of ‘primary ocular toxoplasmosis’ cases also showed higher IgG avidity.

In pregnant women, Toxoplasma IgG avidity testing has been one of the primary strategies for determining infection acquisition time to predict the possibility of transmission of infection to the fetus [3]. Accurate diagnosis of possible time of infection is important for pregnant women to make timely treatment decisions and avoid vertical transmission of the disease [1,5]

Specific IgM after Toxoplasma infection is seen 1 week after the infection; while the absence of IgM denotes old (>3 months) infection [[i],[ii]]. In primary OT, serum IgM positivity is known to be a specific marker for acute infection [[iii]]. However, IgM testing to determine the time of infection has two unique challenges in toxoplasmosis: 1. It has low specificity which can result in false positivity and; 2. Toxoplasma IgM in a subset of cases can persist from months to years [6,[iv],[v]].

In our study we observed active OT presenting during the chronic phase of infection with ‘high IgG avidity’ (meaning the possibility of acquisition of systemic infection 4-5 months prior) [3,[vi]]. Moreover, the linear regression analysis predicted 5 weeks before having symptoms as a possible time during which IgG avidity could have been ‘low’. (Figure 2) Hence, the minimum interval between acquiring infection to ocular symptoms in our series could be around 5 weeks - 5 months before the development of ocular symptoms.

A study conducted by Subramaniam et al in the Malaysian population studied 71 cases of toxoplasma seropositivity [[vii]]. They observed IgM positivity in 5 cases while low IgG avidity was observed in a single case. They concluded that while toxoplasma retinochoroiditis is a clinical diagnosis; serology testing with IgM and IgG avidity aids in differentiating recent and recurrent infection. In the Iranian population, Bahman Rahimi-Esboei et al observed a ‘low’ IgG avidity in 9.4% of cases while IgM positivity was seen in 6.8% of cases [7]. However, this study also included 34.2 % of cases which were IgG negative. Both these studies showed low rates of recently acquired infection. Another study in the Iranian population by Moghaddas et al showed 100% sensitivity for ‘low’ IgG avidity in cases with primary OT [[viii]]. This was in sharp contrast to our study wherein none of the cases with primary OT showed ‘low’ avidity. This difference between our study and the previous studies demonstrate a possibility of late manifestation of OT after systemic infection in our study population (eastern India) as compared with other Asian populations in Malaysia and Iran. We hypothesise that host immunity and regional variation in a predominant infective strain of Toxoplasma might have led to these differences.

Although a low IgG avidity index cannot be used to diagnose recently acquired ocular toxoplasmosis, a high IgG avidity index excludes primary toxoplasmosis developed within the last 5 months [3,12]. While aforementioned studies agree upon the fact that OT is a clinical diagnosis; IgM along with IgG avidity have a role in differentiating recent infection from reactivation. IgG avidity testing costs an additional 1000-1200 Indian rupees (12-14 US Dollars) expense and might not help in the clinical care of both primary OT and reactivation groups. The IgG avidity values did not have any correlation with the final visual outcome or the size of the retinitis. Testing of IgG avidity in cases of primary ‘typical’ OT can be avoided in resource-limited countries like India given the low positive rates, the diagnosis being mostly clinical in OT, additional expenses and non-contribution to clinical care or outcomes. The limitations of our study include a retrospective nature, small sample size, uneven sample size in two subgroups and the regional study population in eastern India. The resolution time of the retinitis and its co-relation with IgG avidity, which were not studied in this study are future research questions. Role of IgG avidity testing in cases of atypical OT and toxoplasma retinitis in pregnant women also needs to be studied further.

Primary ocular toxoplasmosis has been argued to occur late in a few cases due to initial encystation of the parasite within the retina and later local reactivation to cause a full-thickness retinochoroiditis [4,[ix],[x]]. Infection with a successful immune response during first retinal infection may encyst in the outer retina and cause punctate outer retinal toxoplasmosis (PORT) or cause a limited infective lesion like punctate inner retinal toxoplasmosis (PIRT) [[xi],[xii],[xiii]]. Our study also has the limitation of not including any atypical presentation of OT including PORT/PIRT cases. Our series predominantly has cases presenting as primary OT (n=12) with high avidity meaning presenting during the chronic phase of systemic infection. Whether or not these eyes with a late ocular manifestation of retinitis after initial systemic infection have retinal encysted lesions like outer/inner retinal punctate toxoplasmosis during the long incubation period needs to be studied further.

In summary, our study showed higher IgG avidity values denoting a chronic infection in both primary ocular toxoplasmosis and reactivation subgroups. The IgG values had a positive correlation with the duration of symptoms while no correlation with the visual outcomes or size of retinitis lesions was observed.

Funding: Hyderabad Eye Research Foundation.

Declaration of interest: The authors have no relevant financial or non-financial interests to disclose.

Author contributions:

All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Pratik Lakhmawar, Anup Kelgaonkar, Ashish Khalsa and Anamika Patel. The first draft of the manuscript was written by Pratik Lakhmawar and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

Consent to publish:

Authors Affirm that necessary consent to publish images used in figure 1 have been sought.

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Table 1: Demographic data, clinical characteristics, and serology of cases with toxoplasma retinochoroiditis.

	Study cohort (17 cases, 17 eyes)
Mean age ±SD (years)	34.7 ± 15.81
Gender (M: F)	12:5
Mean baseline BCVA (LogMAR)	0.71 (±0.63)
Mean final visit BCVA (LogMAR)	0.51 (±0.43)
Mean duration of symptoms before presentation as retinitis (weeks)	2.94 (±1.43)
Retinitis location	Macular (14), mid-periphery (2), periphery (1)
Primary ocular toxoplasmosis subgroup	12 (70.59%)
Ocular toxoplasmosis – reactivation subgroup	5 (29.41%)
Associated baseline clinical features	Retinal vasculitis (3), Vitritis ≥ 2 (3), AC reaction (7), Elevated IOP (3)
Toxoplasma IgG positivity	17 cases (100%)
Toxoplasma IgM positivity	1 case
Toxoplasma IgG avidity	‘High avidity’ in all the cases Mean 80.76 % (±15.53)

BCVA: best corrected visual acuity, IgG: Immunoglobulin G, IgM: Immunoglobulin M, AC: anterior chamber, IOP: intraocular pressure

No competing interests reported.

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Role of IgG avidity in the eyes with active Toxoplasma retinochoroiditis

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