The province of Thi-Qar is located in the southern region of Iraq at coordinates 31°14'N 46°19'E, with Nasiriyah serving as its administrative center. Covering an area of around 13,000 square kilometers, Thi-Qar is inhabited by a population of nearly 2 million people. The climate of this area is defined by arid and desertic conditions, featuring limited annual rainfall.

This case-control study involved 150 patients suffering from breast cancer (BC) who were referred to general hospitals in Thi-Qar province between July to September 2023 (Figure 1).

Based on the prevalence of IgG positivity related to toxoplasmosis in both BC and healthy groups, as reported in the study by Al-Muskakeh et al. (2022)19, a sample size of 150 individuals was determined for each group. The case and control groups were matched according to age and residence. The diagnosis of breast cancer was based on clinical examinations confirmed by laboratory tests conducted by relevant specialist doctors. The control group involved 150 healthy individuals with no cancer, who were referred to the studied hospitals to do the routine laboratory experiments during the study time. Participants who had taken systemic antibiotics within the past three months, immunocompromised patients, as well as participants who had received intravenous immunoglobulin therapy or immunotherapy before blood collection, were excluded.

Prior to data collection, all the participants or their authorized representatives provided informed consent through both verbal and written means. Participants completed a questionnaire pertaining to demographic variables such a age and place of residence, as well as some T gondii related risk factors such as agricultural activity and consumption of raw or uncooked meat.

Blood samples were acquired from the participants by drawing five milliliters of blood via sterile vein puncture with disposable plastic syringes. The blood was then placed in a gel tube and left to coagulate at ambient temperature for one hour. Following coagulation, the blood underwent centrifugation at a speed of 4000 rpm for a duration of 10 minutes. The resulting serum was split into two equal parts and transferred to Eppendorf tubes for subsequent immunological assessments before being stored at −20 °C.

To assess anti-T gondii antibodies, ELISA kits for Toxoplasma and IgG (cAMP, Romania) were used to test all the collected sera according to the manufacturer's instructions.

The serum levels of PD-1 and PD-L1 were measured using a specific enzyme-linked immunosorbent assay (ELISA) kit sourced from Sunlong Company, China. The assays were performed in accordance with the manufacturer's instructions.

Total RNA was extracted from the peripheral blood by the kit procedure from Qiagen, Germany, and the quality of the extracted RNA was evaluated using a Nanodrop from Biotek Epoch. The sample was then converted to cDNA using the RevertAid First Strand cDNA Synthesis Kit (Fermentas, Sankt Leon-Rot, Germany) according to the manufacturer’s instructions. Quantitative Real-Time PCR was performed by the SYBR Green master mix 2X (Thermo Fisher Scientific, Heiligen, Germany) using primer designed 11 for the PDL-1 gene forward 5′-TGTGAAAGTCAATGCCCCAT-3′ and reverse: 5′-TGTCAGTTCATGTTCAGAGGT-3′, the PD-1 gene forward 5′-CCAAGGCGCAGATCAAAGAGA-3′ and reverse: 5′-TGGGCTGTGGGCACTT-3′ by denaturation at 95 ℃ for 10 min, followed by 40 extension cycles and a final cycle at 95 ℃ for 15 s and annealing and extension at 60 °C for 30 s. The 2-∆∆Ct value was calculated using Bio-Rad iQ5 Optical System Software (USA) to determine the gene expression levels. The quantity of mRNA was assessed using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a reference for quantifying gene expression (Forward: 5′-AACTTTGGCATTGTGGAAGG-3′ and Reverse: 5′-ACACATTGGGGGTAGGAACA-3′). ¹¹

The statistical analysis was done using the SPSS 24.0 software (SPSS Inc., Chicago, IL, USA). Chi-square tests were used to determine the difference in distribution of participants among case and control participants and the associations between infection and the studied factors. Since breast cancer is more common in people over 30 years old, we classified the participants’ age groups into <34, 34–55, and <55 years. Variables significantly related to Toxoplasma prevalence were analyzed as potential risk factors by means of univariate logistic regression. Variables showing a P-value of less than or equal to 0.20 were included as inputs in the final multivariate logistic regression model. In order to determine the chance of exposure to the Toxoplasma parasite in patients with breast cancer compared to healthy subjects, the odds ratio with a confidence level of 95% was used. P-values of <0.05 for associations were considered to indicate statistical significance. Furthermore, to compare the levels of PD1 and PDL-1 among all the groups studied and pairwise (2 by 2 between groups), the One-Way ANOVA and Tukey's post hoc test were used, respectively.

In the present case-control investigation, a total of 300 participants, including 150 patients with breast cancer and 150 healthy individuals referred to the general hospitals of Thi-Qar province, Iraq, were studied. The mean age of the participants in the BC and non-BC groups was 42.6 and 45.3 years, respectively. In terms of residence, 79 (52.60%) and 86 (57.3%) participants in the BC and non-BC groups lived in urban areas, respectively, and the rest lived in rural parts.

Among 150 BC patients, 71 patients (47.3%) exhibited seropositivity for anti-T gondii IgG antibodies, whereas 25 (16.6%) of the samples collected from the non-BC group of healthy subjects demonstrated the presence of anti-T gondii IgG antibodies. Univariate logistic regression indicated a statistically significant difference in the prevalence of anti-T gondii IgG antibodies between BC and non-BC groups (P<0.001); this indicated that the likelihood of Toxoplasma infection is significantly higher in breast cancer patients compared to those without breast cancer (P<0.001, OR=4.49, 95% CI: 2.63-7.68).

In the examination of age-related subcategories, there was no statistically significant correlation discovered between the prevalence of T gondii antibodies and the age of individuals in both the BC (P=0.581) and non-BC (P=0.696) groups. Additionally, a significant association was found between the participants' residential location and the prevalence of T gondii antibodies in the BC (P=0.003) and non-BC participants (P=0.006). In relation to the agricultural activities of the participants, a significant correlation was observed between the agricultural activities and the prevalence of T gondii antibodies in the BC (P=0.047), but no significant correlation was observed between the agricultural activities and the prevalence of T gondii antibodies in non-BC participants (P=0.653). In terms of consumption of raw or uncooked meat, a significant association was found between this risk factor and the prevalence of T gondii antibodies in the non-BC group (P=0.002), but no significant connection was observed between the consumption of raw or uncooked meat and the prevalence of T gondii antibodies and BC participants (P=0.236) (Table. 1).

In the context of multiple logistic regression analysis, it was found that residing in urban areas (P<0.001, OR=3.86, 95% CI: 2.032–4.342) and the consumption of raw or undercooked meat (P=0.014, OR=2.361, 95% CI: 1.164–4.790) were identified as independent risk factors associated with Toxoplasma seropositivity.

Multiple logistic regression analysis, after being adjusted for age, residence, agricultural activity, and consumption of raw/ uncooked meat, showed that the probability of T gondii seropositivity was significantly higher in breast cancer patients (P<0.001, OR=4.95, 95% CI: 2.78–8.82).

Figure 2 exhibits the mean serum level of PD-1 among BC and non-BC people with or without seropositivity to T gondii antibodies. The results showed that BC patients who had seropositive for T gondii antibodies exhibited a high level of PD-1 (1181.5±55.24 pg/mL), compared with T gondii seronegative BC patients (1096.3±27.98 pg/mL). The statistical analysis revealed a significant difference (P<0.001) in the PD-1 levels of breast cancer patients with and without seropositivity to T gondii infection.

As shown in Figure 2, the results showed that BC patients who were seropositive for T gondii antibodies exhibited a high level of PDL-1 (391.8±12.33 pg/mL), compared with T gondii seronegative BC patients (278.5±13.55 pg/ml). The statistical analysis revealed a significant difference (P<0.001) in the PD-1 levels of breast cancer patients with and without seropositivity to T gondii infection.

Figure 3 indicates the gene expression levels of PD-1 and PDL-1 among BC and non-BC people with or without seropositivity to T gondii antibodies. The results showed that the gene expression level of PD-1 among BC patients with or without seropositivity to T gondii antibodies was 3.12±0.21 and 2.39±0.18-fold, respectively. In addition, the gene expression level of PDL-1 among BC patients with or without seropositivity to T gondii antibodies was 2.96±0.24 and 2.16±0.19-fold, respectively. The statistical analysis showed that the gene expression level of PDL-1 was significantly higher among BC patients seropositive for T gondii compared with BC patients who were seronegative for T gondii antibodies (P<0.05).