The loop-mediated isothermal amplification technique to determine T. gondii infection in women with spontaneous abortion

Toxoplasmosis caused by Toxoplasma gondii is well-known as one of the most widespread parasitic diseases, which has now infected nearly a third of the world's population. Among the infectious agents, T. gondii is considered as one of the main causes of spontaneous abortion (SA). The present study aimed to use the loop-mediated isothermal amplification (LAMP) technique in comparison with serological tests to determine the rate of T. gondii infection in women suffering from SA in Lorestan Province, Western Iran.

a significant difference between age and positivity in the LAMP test (P = 0.017), no significant difference was observed between positivity in the LAMP test and residence, education, job, and contact with cats.

Conclusion
The findings of the present investigation suggest that LAMP is a preferred method for determining Toxoplasma infection in pregnant women suffering from spontaneous abortion compared with other routine serological tests.

Background
Toxoplasmosis caused by Toxoplasma gondii is well-known as one of the most widespread parasitic diseases, which has now infected nearly a third of the world's population. [1][2][3] The main ways to contract this disease are consumption of undercooked meat with T. gondii tissue cysts, drinking of water and food contaminated with excreted oocysts of cat faeces, as well as congenitally. [4][5][6] Although toxoplasmosis is almost asymptomatic in healthy and immunocompetent people, it results in severe complications in immunocompromised individuals. 4,7 Among the serious complications of toxoplasmosis, congenital infection is considered the most important complication in pregnant women, with a worldwide prevalence rate of nearly 200,000 cases every year. 8 During pregnancy, especially in the first trimester, if a mother gets infected with the T. gondii, the parasite is capable of passing through the placental barriers and causing stillbirth or spontaneous abortion (SA) Mycoplasma hominis, cytomegalovirus, human papillomaviruses, and T. gondii are the main causes of SA. 12,13 Previous studies have shown the association between toxoplasmosis and SA. [11][12][13][14][15] However, there are few studies regarding the connection between toxoplasmosis and SA in Iran. The majority of studies regarding the prevalence of toxoplasmosis in SA have been carried out based on serological and some molecular tests, such as conventional polymerase chain reaction (PCR). The loop-mediated isothermal amplification (LAMP) technique has some unique properties, such as high sensitivity and specificity, field usability, and isothermal reaction conditions, without the need for expensive equipment, such as thermal cycling. It is well-known as one of the important techniques for determining microbial pathogenesis. [16][17][18][19] The present study aimed to use the LAMP technique together with serological tests to determine the rate of T. gondii infection in women suffering from SA in Lorestan Province, Western Iran.

Participants
A total of 140 women suffering from their first SA , and who had been referred to the Obstetrics and Gynecology Department, Asalian Hospital, Khorramabad, Iran, were investigated. The fact of SA was diagnosed by a gynecologist, based on a case history and clinical tests, and also from the results of a sonography, to rule out other possible causes of SA for example Rh-incompatibility, threatened abortion, incompetent cervix, as well as some uterine disorders. All the enrolled participants were informed about study and written informed consent was obtained . For women under 15 years of age, written consent was given to their spouse .

Sample collection
Aborted fetal remains and blood samples (5 ml) were collected from each patient in sterilized conditions and stored at a temperature of −20°C until testing. Moreover, a questionnaire including some demographic and risk factors such as the mother's age, gestational age, contact with cats, etc was completed by each patient.

Serological test
The serum samples of all patients were examined to determine the specific IgM and IgG anti-Toxoplasma antibodies using ELISA commercial kits (de EIA de Toxoplasma IgG Foresight® ACON) according to the manufacturer's instructions.

Molecular test by LAMP technique
In this study, the genomic DNA was extracted from the remains of aborted fetuses using a DNA Extraction Kit (Yekta Tajhiz Azma, Iran). The nucleotide sequences of four Descriptive statistics were shown in terms of percent (for categorical) and mean (SD) (for continuous) variables. The Chi-square test was applied to determine the univariate association between independent variables and the outcome. Multifactorial logistic regression models were also used to assess the association between T. gondii positivity and the present risk factors. P<0.05 was considered to be statistically significant.

Participants
In this study, a total of 140 pregnant women suffering from their first SA were evaluated for toxoplasmosis infection by molecular (LAMP) tests. The mean age of the participants was 26.7 ± 6.6 years, ranging from 15 to 44 years old. In term of education, the majority of participants had secondary education and diplomas (39.3%). The majority of participants were also housewives (70.7%), resident in urban areas (75.7%). Table 1 shows the demographic characteristics of the participants in the study.
The epidemiologic and clinical characteristics of pregnant women suffering from their first SA are presented in Table 1 In term of age, there was a significant difference between age and positivity in the LAMP test (P = 0.017), so that all positive cases for Toxoplasma parasite infection based on LAMP test were reported from the age group of lower than 25 years. The majority of positive cases (8.5%) were observed in women with lower education (≤diploma). However, there was no significant difference between positivity to toxoplasmosis by LAMP and education (P = 0.073).
Based on the LAMP test, the highest number of positive cases was observed in housewives (4%). However, no statistically significant difference was observed between positivity to toxoplasmosis by LAMP and profession (P = 0.813). The highest number of positive cases of T. gondii infection was observed in pregnant women who lived in rural areas (5.9%), but by Fisher's exact test, the difference between positivity to toxoplasmosis by LAMP and residence was not statistically significant (P = 0.595). Moreover, the results demonstrate that no statistically significant difference was observed between positivity to toxoplasmosis by LAMP and contact with cats (P = 0.588) and pregnancy grade (P= 0.193) (

Discussion
Congenital toxoplasmosis, an infectious disease that is found in fetuses infected with Toxoplasma gondii, may result in severe consequences, such as ocular, hearing, cognitive, and mental complications, and may even cause SA , miscarriage or stillbirth. 1 Since the accurate mechanism of transmission of the Toxoplasma across the human placenta is not completely understood, the rate of congenital toxoplasmosis in pregnant women varies, depending on the trimester during which the congenital infection occurred. 1,4 So far, it has been proven that in the first trimester, the rate of transmission of toxoplasmosis is nearly 25%, while the transmission rates in the second and third trimester are 50 and 65%, respectively. 8,9 SA occurring before 20 weeks of pregnancy has been reported in about 1 to 2% of pregnant women. 10  Usually, identification of acute primary infection, especially in pregnant women, is very challenging, but the IgM antibody can be used to identify early acute toxoplasmosis. 20,21 In line with our results, Elamin Elhag et al (2015) showed that among 99 women who had suffered SA in Khartoum State, T. gondii IgM was found in 5 women (5.3%), whereas 27 women (28.4%) were found to be positive for T. gondii IgG. 14 Zargar et al. (1998) have demonstrated the prevalence of the IgM anti-Toxoplasma antibody in 49.47% of women with recurrent SA (49.47%) in Kashmir, Pakistan. 22 In Iran, in a study conducted by Matin et al (2017) in Ardebil Province, Iran, the prevalence of anti-Toxoplasma IgG and IgM in women with a history of SA or stillbirth in Sari, Iran was 4.0% and 43.0%, respectively. 23 Saki et al (2015) reported that IgG and IgM anti-Toxoplasma antibodies were found in 32 (24.6%) and 1 (0.77%) of women suffering SA in Ahvaz, Southwest of Iran. 24  Iran by semi-nested PCR. 29 In recent years, the loop-mediated isothermal amplification (LAMP) technique has become well-known as one of the important techniques to determine microbial pathogens because of its unique properties, such as high sensitivity and specificity, field usability, and isothermal reaction condition that don't need expensive equipment, such as thermal cycling. 30,5,17 However, the difference between the serological tests and LAMP for the detection of acute infection may be affected by some factors, including the short duration of parasitemia and the low numbers of trophozoites circulating in peripheral blood, which can cause a sampling error that will create falsenegative results in such cases. [31][32][33][34][35] Among the demographic and risk factors studied in the present investigation, such as age, residence, education, contact with cats, etc, a significant correlation was only observed between age (<25 yrs) and positivity to T. gondii (p=0.017). However, there is no significant association between other risk factors and positivity to T. gondii. Consistent with our results, Elamin Elhag et al (2015) showed that among 99 women suffering SA in Khartoum State, the highest percentage of a positive result was observed between 20-29 years. 14 Moreover, in the study conducted by Saki et al (2015) in line with our results, there was no significant association between some risk factors such as contact with cats and positivity to T. gondii in women who had spontaneously aborted. 24 However, to reach a more accurate conclusion the sample size needs to be increased.

Conclusion
The results of the present study, in line with previous studies, indicate the importance of this parasite as a cause of SA. The findings of the present investigation suggested that LAMP is a preferred method to determine Toxoplasma infection in SA by pregnant women, compared with other routine serological tests.

Declarations
Ethics approval and consent to participate A written consent was taken from the participants before sampling; moreover, the study was also approved by the Ethical Committee of Lorestan University of Medical Sciences (Khorramabad, Iran). The present cross-sectional investigation was carried out from February 2016 to March 2017.

Consent for publication
Not applicable Availability of data and material All data generated or analyzed during this study are included in this published article.

Competing interests
The authors declare that they have no competing interests