Prevalence of Toxoplasma gondii Antibodies and DNA in Iranian HIV Patients

Background & Objective: Toxoplasma gondii infection has public health importance and can lead to serious diseases in immunosuppressed patients, such as HIV cases. Appropriate control of T. gondii infection in HIV patients requires information about the prevalence of T. gondii antibodies and DNA in different population. In this study, we aimed to determine the prevalence of Toxoplasma gondii antibodies and DNA in HIV patients in Tehran, Iran. Methods: A total of 149 HIV patients from the Iranian Research Center for HIV/AIDS, Tehran, Iran were enrolled in the study. Anti-Toxoplasma IgG and IgM were detected by ELISA and T. gondii DNA was evaluated by PCR and quantita- tive real-time PCR. IgG positive samples were also assessed for their avidity. Results: Anti-Toxoplasma IgG and IgM were positive in 46.3% and 2.7% of cases respectively. 92.7% of our patients showed past infection and 4.3% revealed recently acquired toxoplasmosis based on their IgG avidity test. T. gondii DNA was not detected by PCR but real-time PCR results showed DNA in 4.7% of total patients and 13.1% of the IgG seropositive cases. Conclusion: Our findings indicated that latent toxoplasmosis was relatively prevalent in our study population, but new T. gondii infection had low prevalence. Almost half of our patients were IgG negative and at risk of acquiring toxoplasma infection. Low copy numbers of DNA were detected in 4.7% of the cases without any clinical manifestation. Therefore, detection and monitoring of anti-Toxoplasma antibodies and DNA in HIV patients is substantial to estimate the risk of reactivation and new infection.


Introduction
Toxoplasmosis is a parasitic disease caused by Toxoplasma gondii (T. gondii), an obligate intracellular parasite. It has a wide distribution and it affects almost 30% of the world's population, with a variable prevalence in different countries (1).
In the majority of immunocompetent individuals, T. gondii infection is a subclinical and asymptomatic disease which may lead to latent infection characterized by the persistence of the organism within host tissues without any symptoms (2). However T. gondii can lead to acute and life-threatening disease in immunocompromised people such as HIV individuals, cases with malignancy and subjects receiving organ transplantation (3,4). T. gondii infection is considered as one of the most important opportunistic infections in patients with HIV, and is also considered as the main cause of morbidity and mortality in these patients (5,6).
The prevalence of T. gondii infection in HIV patients varies throughout the world. The overall seroprevalence rate reported is 35.8% but based on the region it differs from 60.7% in the Middle East and North Africa to reach 49.1% in Latin America, 44.9% in sub-Saharan Africa, 30.1% in western and central Europe and North America while it is 25.1% in Asia and the Pacific (7).
The main cause of toxoplasmosis in an HIV patient is the reactivation of latent infection particularly in the brain, leading to toxoplasmic encephalitis (TE), especially when CD4 count reduces to less than 100 cells/mm 3 (7). Patients with toxoplasmosis and HIV co-infection have 30 to 40% risk of TE (8). This is a fatal infection in HIV patients without receiving antiretroviral therapy (9).
Serological tests are used for the diagnosis of toxoplasmosis, but these tests are not reliable in immunocompromised patients due to suppression of the immune system. Molecular tests are now considered as important method for the diagnosis of toxoplasma infection in immunosuppressed individuals, especially when serological techniques fail (10). Some authors mentioned the association of T. gondii IgG antibodies and DNA with the reactivation of toxoplasmosis and its clinical manifestation in HIV patients (11,12).
However, appropriate diagnosis and control of T. gondii infection in HIV patients require adequate information regarding the prevalence of T. gondii antibodies and DNA in different population. Due to limited data on molecular diagnosis of Toxoplasma infection and its association to T. gondii antibodies in Iranian HIV patients, we aimed to determine the prevalence of Toxoplasma gondii antibodies and DNA in HIV patients in Tehran, Iran.

Study Population
In this cross-sectional study, 149 HIV patients who were referred to the Iranian Research Center for HIV/ AIDS in Tehran, Iran were consecutively enrolled from May to September 2017. The study protocol was approved by the Pasteur Institute of Iran Ethical Committee and informed consent was obtained from subjects prior to the study.
CD4 count was determined by flowcytometry and defined as cells/mm3.

Detection of Anti-Toxoplasma IgG and IgM Antibodies
All plasma samples were screened using the standard enzyme-linked immunosorbent assay (ELISA) commercial kits for the detection of anti-Toxoplasma IgG and IgM antibodies (EUROIMMUN, Lubek, Germany) in accordance with the manufacturer's instructions.

Assessment of IgG Avidity
For differentiation between recently acquired and past infection of T. gondii infection, anti-Toxoplasma IgG positive specimens were subjected for IgG avidity test using ELISA kit (EUROIMMUN, Lubek, Germany). A result of ˂40% was interpreted as low avidity (T. gondii infection was acquired within the last 3 months) between 40-60% as equivocal range and ˃60% as high avidity (Toxoplasma infection was acquired more than 3 months ago).

DNA Extraction and Polymerase Chain Reaction
Toxoplasma-DNA was extracted from 200μl of whole blood using QIAamp® DNA Mini Kit (QIA-GEN, Hilden, Germany) according to manufacturer's instructions.
In order to evaluate the suitability of the extracted Polymerase chain reaction was carried out using primer pair TOXO1 (CGCTGCAGGGAGGAAGAC-GAAAGTTG) and TOXO2 (CGCTGCAGACA-CAGTGCATCTGGATT) which were selected from the 50 and 30 end of the 529 bp fragment (RE) respectively. The PCR reaction was performed in a 25 ml reaction mixture containing 15 mM Tris-HCl (pH 8), 50 mM KCl, 1.5 mM MgCl 2 , 0.2 mM dNTP, 10 pmol of each primer and 1.5 U Taq polymerase (YTA PCR Master Mix, Iran). Amplification was performed as initial 7 minute (min) denaturation at 94°C, followed by 35 cycles of amplification including denaturation for 1 min at 94°C, annealing for 1 min at 55°C and extension for 1 min at 72°C. Strand synthesis was completed at 72°C for 10 min and stored in 4°C for 5 min. The expected size of the PCR product was 529 bp. Each batch included negative control containing water and extracted DNA from T. gondii tachyzoites RH-strain as positive control.
The PCR products were electrophoresed on a 1.5% agarose gel with the 100-bp DNA ladder (Sinaclon, Iran) and stained with DNA stain and visualized by ultraviolet transillumination.

Quantitative Real Time PCR Assays
Quantitative Real time PCR Assays for REP-529 was performed according to the method previously described by Babaie et al. (13). Real time PCR was conducted using a BIO-RAD DNA Engine Thermal Cycler under the following conditions: an initial denaturation at 95 °C for 10 min, followed by 35 cycles of 30 sec at 95 °C, 30 sec at 58 °C and 30 sec at 72 °C. The final extension step was set for 20 min at 72 °C. The analytical detection limit of assay is 10 copies per PCR reaction (25µl).

Statistical Analysis
Statistical analyses were conducted using SPSS statistics software (version 16, Chicago, IL, USA). The Chi square test or Fisher's exact test was used to compare variables. Data is presented as mean ± SD or, when indicated, as an absolute number and percentage. P-values <0.05 were considered statistically significant.
All cases were categorized in four age groups as: 18-28, 29-39, 40-49, and ≥ 50 years. The highest (49.3%) and the lowest (33.3%) IgG antibody rates were found within the age groups of 40-49 and 18-28 years old, respectively (table 1). The IgG seroprevalence was slightly higher in females (50%) than in males (45.5%) (Not significant). T. gondii DNA was not detected by PCR, but quantitative real-time PCR results showed T. gondii DNA in 7 (4.7%) patients. The titers of T. gondii DNA was from 10 to 28.39 copies per reaction (25µl). From seven DNA positive samples, 4 cases had IgG simultaneously with high IgG avidity. One case had IgM, IgG and DNA concurrently with low IgG avidity (probably ongoing infection) and only 2 subjects were DNA positive. Out of seven DNA positive cases, six subjects were injecting drug users with a past history of incarceration.
From 69 IgG positive cases, T. gondii DNA was detected in 5 (13.1%) patients. Furthermore, 2.5% (2/80) of serology negative patients showed positive PCR results. The rate of Toxoplasma gondii antibodies and DNA based on CD4 counts were shown in Table 2. There was no significant difference between anti-Toxoplasma antibodies rate and DNA frequency with mean CD4 count.

Discussion
This study investigated the prevalence of Toxoplasma gondii antibodies and DNA in HIV patients in Tehran, Iran. Anti-Toxoplasma IgG and IgM were positive in 46.3% and 2.7% of the cases respectively and 4.7% of the subjects were positive for T. gondii DNA by real time PCR. 92.7% of our patients showed past infection and 4.3% revealed recently acquired toxoplasmosis based on their IgG avidity test. and by suppressing host immune system (14). Almost half of the HIV individuals have co-infection with T. gondii (15,16).

Co-infection of T. gondii and HIV infection is a major public health issue which can affect the course of both infections by the interaction of two pathogens
Seroprevalence of Toxoplasma infection varies greatly worldwide due to socioeconomic conditions, cultural habits, different lifestyles, climate, geographical areas, age, residing in rural areas, educational status and having raw or undercooked meat (17). Different studies reported the seroprevalence rate of T. gondii in HIV patients from less than 10% to over 90% in different countries and even within one country, this variation is substantial (7). This seroprevalence is also very variant in the different parts of Iran (15,(18)(19)(20)(21). Even in Tehran, two different seroprevalence rates were reported by Rostami (22)  Serologic tests such as IgM and IgG are the main methods for diagnosis of toxoplasmosis but these assays constitute some difficulties to differentiate acute form from chronic and the reactivation of infection (24). The detection of anti-T.gondii IgM as a routine diagnostic test of toxoplasmosis has some limited value in the management of HIV patients (25). The IgG avidity test is a qualitative assay which distinguishes chronic toxoplasmosis from a recently acquired infection and determines the status of toxoplasma infection. Low IgG avidity suggests acute infection, while high IgG avidity indicates chronic or reactivated infection (26,27 (31,32). We also found low rate of T. gondii IgM in comparison to IgG and three from four our IgM positive cases had IgG simultaneously with low IgG avidity. Additionally, an overall 4.3% of our cases had low IgG avidity which indicates recently acquired Toxoplasma infection and such is not common in our cases. This data emphasizes that the reactivation of chronic T. gondii infection is the dominant way of acquiring toxoplasmosis in HIV cases that is not a new infection (32,33,34). However, incidence of reactivation is associated with the prevalence and concentration of T. gondii IgG antibodies (35).
Today, molecular methods are known as important diagnostic tools for the detection of toxoplasmosis in immunosuppressed hosts (10). Some authors mentioned that the detection of T. gondii DNA in the blood indicates active infection and is closely associated with clinical manifestation of HIV patients (11,12) while other molecular studies on HIV patients without cerebral toxoplasmosis did not confirm this association. Gashout et al. detected T.gondii DNA in 60% of asymptomatic seropositive HIV patients with a CD4 count less than 100 cells/μl (10). In another study, Toxoplasma DNA was detected in 25% of HIV patients in stages 3 and 4 with positive T. gondii IgG (36). Ayi et al. detected T. gondii DNA in 54.7% of HIV seropositive subjects and DNA was found frequently in cases with lower CD4 count (11). In an investigation in Brazil on AIDS patients without cerebral toxoplasmosis, only 2.34% of patients had T.gondii DNA (12). Rostami et al. from Iran reported only one positive PCR sample in their HIV cohort (22). In our study, T. gondii DNA was observed in 13.1% of the seropositive cases, which is less than several surveys conducted on the same population but is in agreement with Rostami et al. and Colombo et al. surveys (12,22). However, we enrolled a low number of patients with a CD4 count less than 100 and most of our patients receiving HAART without clinical manifestation of toxoplasmosis. Hence, we should expect a low number of DNA positive cases in this study. Although the presence of Toxoplasma DNA in blood could indicate a recent infection or clinically active disease but reversely, it can be just due to shedding a low amount of parasites from tissue cysts into the blood at a subclinical value, especially in asymptomatic cases which is detectable only by real time PCR (37).
Furthermore, in our study, two patients (2.5%) with negative serology showed positive real time PCR results, but we should consider that negative serology does not exclude the risk of toxoplasmosis reactivation (38, 39, and 40).

Conclusion
We evaluated the total burden of T. gondii infection by measuring T. gondii IgG, IgM, IgG avidity test and DNA in Iranian HIV patients. Our data showed that latent toxoplasmosis was relatively prevalent in our study population, but new T. gondii infection had low prevalence. However, almost half of our HIV popu-lation was IgG negative and at risk of acquiring infection. Low copy numbers of T. gondii DNA were detected in 4.7% of the total patients and 13.1% of seropositive cases were without any clinical manifestation. As appropriate control of T. gondii infection in HIV patients is very important, the detection and monitoring of anti-Toxoplasma antibodies and DNA in HIV patients is substantial to estimate the risk of reactivation and new infection.