Seasonal influence on TORCH infection and analysis of multi‐positive samples with indirect immunofluorescence assay

Abstract Background TORCH including the pathogens of Toxoplasma gondii (TOX), rubella virus (RV), cytomegalovirus (CMV), and herpes simplex virus (HSV) causes intrauterine infections and poses a worldwide threat to women especially in pregnancy. In this study, we described the seasonal difference in TORCH infection and analyzed the anti‐TORCH IgM multipositive serum samples by the indirect immunofluorescence assays (IFA). Methods To observe the seasonal influence of the anti‐TORCH IgG and IgM antibodies, a retrospective study was conducted with 10 669 women (20–40 y old) before pregnancy from August 2016 to July 2017. Totally 199 ELISA anti‐TORCH IgM multipositive serum samples were further tested by IFAs for false‐positive analysis. Results The prevalence of positive HSV1‐IgM, RV‐IgM, HSV2‐IgM, CMV‐IgM, and TOX‐IgM in the present population was 6.30%, 2.55%, 1.94%, 1.24%, and 0.67%, respectively. Additionally, the prevalence of positive RV‐IgM, CMV‐IgM, and HSV1‐IgM was statistically different among four seasons, with the highest positive rates of RV‐IgM (4.12%) in autumn, CMV‐IgM (1.75%) in summer, and HSV1‐IgM (7.53%) in winter. The confirmatory IFAs showed that the positive rates of RUV‐IgM, CMV‐IgM, and HSV2‐IgM were significantly different from those in ELISA screening experiments. Interestingly, only 32.7% (65/199) of the TORCH IgM multipositive results were consistent with those by the IFA, indicating that cross‐reaction caused false positives were common in ELISA IgM antibody screening. Conclusion The TORCH infection displayed different prevalence among four seasons in our 12‐month retrospective study. The IgM multipositives by ELISA screening may need further confirmation analysis due to its relatively high cross‐reaction rate.


| INTRODUC TI ON
TORCH is an acronym that stands for a group of pathogens including Toxoplasma gondii (TOX), rubella virus (RV), cytomegalovirus (CMV), and herpes simplex virus (HSV) causing perinatal infections with similar symptoms. 1 TOX is a common protozoan parasite in the phylum Apicomplexa, which is usually asymptomatic or in the form of a mild, self-limiting illness characterized by fever, malaise, and lymphadenopathy. Nevertheless, in immunodeficiency or when congenitally acquired, it can result in severe disease even death if not treated properly. 2 RV can lead to a devastating congenital rubella syndrome (CRS) of which manifestations are variable with deafness being most common. 3 CMV infections may cause widespread dissemination throughout the body, with diverse cell types such as epithelial, endothelial, fibroblast, and smooth muscle cells supporting productive viral infection. 4 The three major forms of neonatal HSV infection are disseminated disease, central nervous system (CNS) disease, and skin, eye, and mouth diseases. 5 The serum TORCH antibody screening is crucial for early infection diagnosis, 6  In this study, a twelve-month retrospective evaluation with 10 669 women at childbearing age for the TORCH screening assays was conducted, to find out any seasonal influence on the infection rates. In addition, two serum samples that were tested TORCH IgM multipositive by enzyme-linked immunosorbent assay (ELISA) were confirmed and analyzed by the "gold-standard" indirect immunofluorescence assay (IFA).

| Subjects
The TORCH screening results of 10 669 women before pregnancy in Beijing Obstetrics and Gynecology Hospital from August 2016 to July 2017 were used in seasonal analysis. In the period of January 2016 to July 2017 during which 19 652 women were screened for before-pregnancy TORCH infection screening, a total of 199 serum samples that were tested IgM multipositive from the TORCH assays were collected and saved at −80°C.

| Reagents and methods
The TORCH ELISA screening assays were performed on the TECAN  Cover each well with fluorescein-conjugated goat anti-human IgM.
After incubation and washing steps as described above, place a small drop of buffered glycerol in each well and cover with a coverslip, then the slides were read immediately at a magnification of 500-fold, and a positive reaction for TORCH antibodies was indicated by the presence of diffuse nuclear inclusion bodies that emit green fluorescence ( Figure 1). SPSS 21.0 was used for data analysis (IBM Corporation. Chicago, United States). TORCH IgM infection differences were calculated by chi-square test; two-sided P < .05 was considered significant.

| TORCH screening results in childbearing age women before pregnancy
With the ELISA screening assays described above, the positive rates of serum IgM antibodies to TORCH are listed in the order of decreasing prevalence: HSV1 (6.30%), RV (2.55%), HSV2 (1.94%), CMV (1.24%), and TOX (0.67%). As the IgM positivity is a strong indicator for primary infection, the TORCH IgG prevalence reflecting past exposure or vaccination was much higher than IgM, with CMV (92.05%), RV (89.74%), and HSV1 (81.56%) as the top prevalence IgG antibodies in the study population. See Table 1 for more details.

| Seasonal influence on TORCH IgM positive rates
To find out whether the TORCH primary infection in our patients was different among four seasons, statistical analysis (chi-square test, P < .05) was applied to the following study (Table 2) (Table 2).  (Table S1). To make data more compara-  (Table 1), the most common co-infection pattern was HSV1+HSV2, followed by TOX + HSV1 + HSV2 and CMV + HSV1 + HSV2. The cross-reaction between anti-HSV1 and anti-HSV2-IgM antibodies in ELISA was common when compared with IFA results (Table S1).

| D ISCUSS I ON
As seen in The CMV-IgG had the highest prevalence in this study with 92.05%, which was consistent with the data reported previously, 60% in developed countries and almost 100% in developing countries. 10 In other areas of China, CMV infection was also reported to have the highest prevalence in Baotou, Yan'an, and Zhengzhou. [11][12][13] With regard to the TORCH seasonal difference, Feng's article showed that CMV-IgM infection was higher in summer and autumn, whereas it was found higher in spring and winter according to another study with women from Shandong province of China, suggesting the impact of geographic locations in CMV infection. 14 Interestingly, we also observed a higher CMV-IgM prevalence in spring and summer. The HSV1-IgM prevalence was the highest in TORCH in this study In spite of Su's study in which the highest HSV-IgM seroprevalence was found in summer, our data presented an opposite result that HSV1-IgM showed lowest prevalence in summer. There are also reports about the HSV infection which showed no seasonal variation. 19 As seen in In our study, the most common TORCH co-infection patterns were HSV1 + HSV2, TOX + HSV1 + HSV2 and CMV + HSV1 + HSV2.
The anti-HSV1 and anti-HSV2-IgM antibodies tended to cross-react with each other relatively easily due to their similar antigenic determinants. 23 Marawan's article pointed that TOX-IgM-positive tests were associated with seropositivity to HSV1-IgG and HSV2-IgM antibodies and that TOX-seropositive patients were 1.94 and 1.35 times more likely than seronegative patients to have previous exposure to CMV and HSV-1, respectively, which could be caused by that these infections are all linked to the poor socioeconomic conditions. 24 In the study by Rasti et al, 25 TOX-IgG + CMV-IgM and TOX-IgG + HSV-IgG + CMV-IgM were the most frequent TORCH co-infection patterns in the patients with abortion experience. Interestingly, the apparent TORCH co-infection rates (201/19 000) by ELISA in Rasti's report were pretty much comparable to that in our data, suggesting the similarity of the ELISA TORCH IgM screening power.
As an essential part of decreasing the congenital diseases, TORCH-specific antibody screening results should be evaluated with extra care due to the existence of high rates of false-positive IgM reactions, to decrease the unnecessary psychological and economic burdens of patients.