Sero-molecular survey on Toxoplasma gondii infection among drug addicted and non-addicted individuals: a case–control study

The current case–control study obtained ethical approval from the Ethics Committee of the Jahrom University of Medical Sciences, Jahrom, Iran (No. IR.JUMS.REC.1397.154). All sampling processes was done during April 2019 to December 2019 from drug addicted individuals referred to the laboratories of the medical centers of Lar city in Fars Province, Iran. Regarding the criteria, confirmed addiction test and consent for participating were considered as inclusion criteria, and presence of immunodeficiency diseases were considered as exclusion criteria. G*Power software was applied to calculate the sample size in this case–control study. We used prevalence reference of 7.7% (IgG) [28], at a confidence intervals (CI) of 95%, type I error-α of 0.05, and the power of the test (1−β) of 90% to estimate the sample size. The calculation outcome was 135 individuals in each group, although 137 confirmed drug addicted individuals and 141 non-addicted individuals were employed for more accuracy. The control group were people who had no history of addiction. For a more accurate assessment, the age and sex of the control group were matched with the case group. An informed consent form as well as a standard questionnaire were designed and prepared for all participants. Each participant was interviewed orally by the interviewer. This questionnaire included the sociodemographic variables, and risk factors related to T. gondii infection. Afterwards, approximately 10 cc of blood samples were gathered of each participant with and without EDTA. At first, a rapid HIV test was performed on samples from all participants, and if the result was negative, they entered the next stage. Then, the all samples were transported to the laboratory via the cold chain conditions. Blood samples were permitted to clot and centrifuged at 600×g for 10 min to extract the serum and buffy coats [30]. Finally, the separated serum (without EDTA) for serological assay and buffy coats (with EDTA) for PCR-based molecular assay were stored at − 20 °C until examination.

All serum samples were surveyed for anti-Toxoplasma IgG and IgM antibodies through a commercially available enzyme-linked immunosorbent assay (ELISA) kits (Pishtazteb Co, Iran) according to the manufacturer's protocol. This ELISA kits had the 100% sensitivity and 99% specificity. Each run was accompanied by positive and negative controls. Anti-T. gondii IgG and IgM antibodies were estimated via dividing the sample optical density value by the cut-off calibrator value. Considering the kit protocol, when an IgG index or an IgM index was ≥ 1.1, we considered that sample to be positive for IgG or IgM.

To detection of T. gondii, the PCR reaction was arranged in a 20 μL total reaction volume comprising 10 μL of Master mix, 5 μL of DNA, 3 μL deionized sterile water, and 1 μL of each primer TOXO4 (forward: 5′-CGCTGCAGGGAGGAAGACGAAAGTTG-3′) and TOXO5 (reverse: 5′-CGCTGCAGACACAGTGCATCTGGATT-3′) targeting the RE gene of T. gondii [31]. Thermal cycling profile was 94 °C for 5 min, followed by 35 cycles of 30 s at 94 °C, 30 s at 63 °C and 30 s at 72 °C with a final step at 72 °C for 10 min. The PCR amplification is characterized by the production of a 529 bp band for the RE gene [31].

To identify the genotypes of T. gondii isolates, nested PCR were conducted by a set of specific primers targeting a GRA6 gene of T. gondii. At the first step, PCR was performed using GRA6-F1 (forward: 5′-ATTTGTGTTTCCGAGCAGGT-3′) and GRA6-R1 (reverse: 5′-GCACCTTCGCTTGTGGTT-3′), which together amplify a 546-bp specific fragment [32]. Amplification was accomplished in final volume of 20 μL, comprising 10 μL of Master mix, 5 μL of DNA, 3 μL deionized sterile water, and 1 μL of each primer. The PCR amplification conditions were 94 °C for 5 min followed by 40 cycles of 94 °C for 30 s, 56 °C for 30 s, and 72 °C for 30 s, and final extension of 72 °C for 10 min [32]. In the next step, a second PCR was performed using the forward primer GRA6-F2 (5′-TTTCCGAGCAGGTGACCT-3′) and the reverse primer GRA6-R2 (5′-TCGCCGAAGAGTTGACATAG-3′) in order to amplify a 344-bp internal fragment used for genotype identification [32]. The second PCR reaction used 94 °C for 5 min, followed by 30 cycles of 94 °C for 30 s, 59 °C for 30 s, and 72 °C for 30 s, plus a final extension step at 72 °C for 10 min [32].

At the end, PCR products (both RE and GRA6 genes) were electrophoresed on a1.2% agarose gel with SYBR^® Safe stain and visualized using a UV Transilluminator.

PCR products of positive samples were sequenced using ABI 3730 Analyzers (Bioneer, Korea) and the results were compared with sequences available in the GenBank database by BLAST software.