Toxoplasma gondii Infection and Premenstrual Dysphoric Disorder: A Cross-Sectional Study.

BACKGROUND: Premenstrual dysphoric disorder is a severe form of premenstrual syndrome. The influence of Toxoplasma gondii (T. gondii) infection on clinical features in women with this disorder has not been studied. Therefore, we determined the association of T. gondii infection with symptoms and signs in women suffering from premenstrual dysphoric disorder.
METHODS: We performed a cross-sectional study of 151 women suffering from premenstrual dysphoric disorder. Anti-Toxoplasma IgG and IgM antibodies were detected in sera of the participants using enzyme-linked immunoassays (EIAs). In addition, T. gondii DNA was detected in whole blood of IgG seropositive participants using polymerase chain reaction. We obtained the clinical data of women with the aid of a questionnaire. The association of T. gondii infection with clinical characteristics of women was assessed by bivariate and multivariate analyses.
RESULTS: Anti-T. gondii IgG antibodies were found in 10 (6.6%) of the 151 women studied. Of the 10 IgG seropositive women, four (40.0%) were positive for anti-T. gondii IgM antibodies, and one (10.0%) for T. gondii DNA. Mean number (25.8 ± 7.58) of premenstrual clinical characteristics in seropositive women was similar to that (29.22 ± 9.13) found in seronegative women (P = 0.25). Logistic regression showed that seropositivity to T. gondii was negatively associated with difficulty concentrating (OR: 0.18; 95% CI: 0.03 - 0.91; P = 0.03), and positively associated with out of control feeling or overwhelmed (OR: 9.00; 95% CI: 1.32 - 62.00; P = 0.02).
CONCLUSIONS: Results of this first study on the association of T. gondii infection and clinical characteristics of premenstrual dysphoric disorder suggest that this infection might be linked to some symptoms of this disorder. We report for the first time the association of T. gondii infection and out of control feeling or overwhelmed. Results warrant for further research on the role of T. gondii in premenstrual dysphoric disorder.

Introduction

The coccidian parasite Toxoplasma gondii (T. gondii) causes infections in humans worldwide [1]. Cats are the definitive host of T. gondii, whereas humans and other warm-blooded animals are intermediate hosts [2]. Most infections with T. gondii in humans are acquired by ingestion of food or water contaminated with T. gondii oocysts shed by cats, or by the ingestion of raw or undercooked meat containing tissue cysts [3]. Less frequently, infection with T. gondii may occur by organ transplantation [4], and blood transfusion [5]. In addition, primary infection with T. gondii during pregnancy may lead to vertical transmission and congenital disease [3, 6]. Infections with T. gondii are usually asymptomatic [3]. Subjects with clinical manifestations of infection (toxoplasmosis) may present with disease in eyes, lymph nodes and central nervous system [3, 7, 8]. Toxoplasmosis is particularly severe in immunocompromised individuals [9]. Common symptoms of toxoplasmosis include fatigue, headache, muscle aches, and difficulty concentrating [10]. Furthermore, infection with T. gondii has been associated with a number of psychiatric disorders including depression [11], schizophrenia [11, 12], impulsive aggression [13], generalized anxiety disorder [14], and suicide attempts [15].

Premenstrual dysphoric disorder is a severe form of premenstrual syndrome with serious psychological symptoms [16]. This disorder is characterized by cognitive-affective symptoms that appear in a cyclic manner during the premenstrual period [17]. This illness has been recently designated as a disorder in the Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition (DSM-5) (http://www.dsm5.org/Pages/Default.aspx). Prevalence of premenstrual dysphoric disorder varies from 1.3% to 8% [18, 19]. Symptoms of this disorder significantly impair daily functioning [20, 21], and its etiology is unclear [16, 18, 20]. It raises the question whether T. gondii infection plays a role in this disorder as it does in other psychiatric disorders. To our knowledge, the link of T. gondii infection and premenstrual dysphoric disorder has not been studied. Therefore, we sought to determine the association of T. gondii infection with clinical characteristics of premenstrual dysphoric disorder in a sample of women in Durango City, Mexico.

Materials and Methods

Study design and population studied

Through a cross-sectional study, we examined 151 women with premenstrual dysphoric disorder. Women studied were enrolled in two public primary healthcare centers: Centro de Salud #2 of the Secretary of Health (n = 78), and Clinic of Family Medicine of the Institute of Security and Social Services of State Workers (n = 73) in the northern Mexican city of Durango. Participants were examined from February to April 2016. Inclusion criteria for enrollment were women suffering from premenstrual dysphoric disorder, aged 30 - 40 years old, and who accepted to participate in the study. Diagnosis of premenstrual dysphoric disorder was made according to the DSM-5 criteria [22]. Occupation, civil status, and socioeconomic level of women were not restrictive criteria for enrollment. Pregnant women were not included in the study. Mean age in women examined was 35.52 ± 3.59.

Clinical data of the women studied

Signs and symptoms of women were obtained with the aid of a questionnaire. In total, 59 clinical characteristics of women were recorded including presence of irregular periods, severity of menstruation, vaginal infections, painful periods, fatigue, memory lapses, difficulty concentrating, confusion, judgment problems, suffering from mental illness, mood changes, low self-esteem, depression, guilty feeling, increase of fears, panic attacks, anxiety, tension, nervousness, irritability, aggressiveness, lack of interest in daily activities, lack of interest in social relations, out of control feeling or overwhelmed, reduced tolerance to noises and lights, dizziness, headache, migraine, allergy, breast pain, arterial hypertension, bouts of rapid heartbeat, decrease in muscle power, joint pain, low back pain, muscle tension, clumsiness, tingling extremities, electric shock sensation, bruises, edema in ankles, hands or feet, decreased libido, increased libido, dyspareunia, abdominal bloating, gas, abdominal pain, constipation, diarrhea, nauseas, abdominal inflammation, appetite disturbance, desire to eat certain food or eat a lot, weight gain, respiratory problems, presence of acne, presence of herpes labialis, sleep problems, thyroid disease, and obesity.

Detection of anti-T. gondii IgG and IgM antibodies

A serum sample from each woman was obtained. Serum samples were stored at -20 °C until analyzed. Anti-T. gondii IgG antibodies were detected in serum samples with the commercially available enzyme immunoassay (EIA) kit “Toxoplasma IgG” (Diagnostic Automation/Cortez Diagnostics Inc., Woodland Hills, CA, USA). This kit was also used to quantitate the IgG levels. Anti-T. gondii IgG antibody levels were expressed as International Units (IU)/mL. A cut off of ≥ 8 IU/mL of IgG was used for seropositivity. Sera reactive for anti-T. gondii IgG antibodies were further analyzed for anti-T. gondii IgM antibodies by the commercially available EIA “Toxoplasma IgM” kit (Diagnostic Automation/Cortez Diagnostics Inc.). All EIAs were performed following the instructions of the manufacturer.

Detection of T. gondii DNA

Whole blood of women with anti-T. gondii IgG antibodies was analyzed to detect T. gondii DNA by nested-polymerase chain reaction (PCR). DNA of whole blood was extracted following a protocol describe elsewhere (http://www.protocol-online.org/prot/Protocols/Rapid-Extraction-of-High-Quality-DNA-from-Whole-Blood-Stored-at-4-C-for-Long-Period-4175.html). Primers directed against the B1 gene of T. gondii and a PCR protocol previously described [23] were used. Sensitivity and specificity of this test were previously examined [24]. Amplification products were analyzed by electrophoresis using 2% agarose gels, stained with ethidium bromide, and visualized using ultraviolet illumination.

Statistical analysis

Results were analyzed with the aid of the software SPSS 15.0 (SPSS Inc. Chicago, IL), Microsoft Excel, and Epi Info 7. For calculation of the sample size, we used: 1) a reference seroprevalence of 6.1% [25] as the expected frequency for the factor under study, 2) 100,000 as the population size from which the sample was selected, 3) a 4.0% of confidence limits, and 4) a 95% confidence level. The result of the sample size calculation was 137 subjects. The association of T. gondii infection and the clinical characteristics of women was assessed with the Pearson’s Chi-squared test or the two-tailed Fisher’s exact test (when values were five or less). In the multivariate analysis, we included only variables with a P value ≤ 0.20 obtained in the bivariate analysis. We calculated odds ratio (OR) and 95% confidence interval (CI) using logistic regression with the Enter method. A P value less than 0.05 was considered statistically significant.

Ethical aspects

This study was approved by the Ethics Committees of the General Hospital of the Secretary of Health, and the Institute of Security and Social Services of State Workers in Durango City, Mexico. Participation of women was voluntary. The purpose and procedures of this study were explained to all participants, and a written informed consent was obtained from all of them.

Results

Anti-T. gondii IgG antibodies were found in 10 (6.6%) of the 151 women with premenstrual dysphoric disorder studied. Of the 10 anti-T. gondii IgG positive women, five (50.0%) had IgG levels > 150 IU/mL, two (20.0%) between 100 and 150 IU/mL, and three (30.0%) between 8 and 99 IU/mL. Anti-T. gondii IgM antibodies were found in four (40.0%) of the 10 IgG seropositive women. DNA of T. gondii was detected in one (10.0%) of the 10 women with IgG antibodies against T. gondii. IgG levels in this women were 146 IU/mL.

Women seropositive to anti-T. gondii IgG antibodies showed from 13 to 40 (mean: 25.8 ± 7.58) signs or symptoms, whereas seronegative women (n = 141) had from 7 to 48 (mean: 29.22 ± 9.13) signs or symptoms. Mean number of clinical characteristics in seropositive women was similar to that found in seronegative women (P = 0.25).

Concerning clinical characteristics, bivariate analysis showed seven variables potentially (P ≤ 0.20) associated with IgG seropositivity to T. gondii: irregular periods, difficulty concentrating, low self-esteem, guilty feeling, increase of fears, out of control feeling or overwhelmed, and tingling extremities. Other clinical characteristics studied showed P values higher than 0.20 by bivariate analysis. Results of bivariate analysis of a selection of clinical characteristics of women and IgG seropositivity to T. gondii are shown in Table 1. Further analysis by logistic regression of variables with P ≤ 0.20 obtained by bivariate analysis showed that seropositivity to T. gondii was negatively associated with difficulty concentrating (OR: 0.18; 95% CI: 0.03 - 0.91; P = 0.03), and positively associated with out of control feeling or overwhelmed (OR: 9.00; 95% CI: 1.32 - 62.00; P = 0.02) (Table 2).

Table 1

Results of Bivariate Analysis of a Selection of Clinical Data and Seropositivity to T. gondii

Characteristics	No. of women tested	Prevalence of T. gondii infection		P value
		No.	%
Irregular periods
Yes	100	9	9	0.16
No	50	1	2
Sleep problems
Yes	85	4	4.7	0.33
No	66	6	9.1
Difficulty concentrating
Yes	109	4	3.7	0.02
No	41	6	14.6
Judgment problems
Yes	59	2	3.4	0.31
No	91	8	8.8
Mood changes
Yes	145	9	6.2	0.29
No	5	1	20
Low self-esteem
Yes	114	5	4.4	0.06
No	37	5	13.5
Guilty feeling
Yes	80	3	3.8	0.19
No	71	7	9.9
Increase of fears
Yes	83	3	3.6	0.11
No	68	7	10.3
Anxiety
Yes	103	5	4.9	0.29
No	48	5	10.4
Tension
Yes	111	6	5.4	0.45
No	40	4	10
Nervousness
Yes	119	7	5.9	0.44
No	32	3	9.4
Irritability
Yes	124	7	5.6	0.38
No	27	3	11.1
Lack of interest in social relations
Yes	92	8	8.7	0.31
No	59	2	3.4
Out of control feeling or overwhelmed
Yes	86	8	9.3	0.18
No	65	2	3.1
Reduced tolerance to noises and lights
Yes	89	4	4.5	0.31
No	62	6	9.7
Joint pain
Yes	96	8	8.3	0.32
No	55	2	3.6
Low back pain
Yes	117	9	7.7	0.45
No	34	1	2.9
Tingling extremities
Yes	96	4	4.2	0.17
No	55	6	10.9
Decreased libido
Yes	81	7	8.6	0.34
No	68	3	4.4
Dyspareunia
Yes	42	1	2.4	0.28
No	108	9	8.3
Presence of acne
Yes	85	4	4.7	0.33
No	65	6	9.2

*Sum may not add up to 151 because of missing values.

Table 2

Results of the Multivariate Analysis of Clinical Characteristics and IgG Seropositivity to T. gondii

Characteristics	Odds ratio	95% confidence interval	P value
Irregular periods	7.91	0.75 - 82.5	0.08
Difficulty concentrating	0.18	0.03 - 0.91	0.03
Low self-esteem	0.48	0.08 - 2.57	0.39
Guilty feeling	0.45	0.08 - 2.46	0.36
Increase of fears	0.58	0.11 - 3.10	0.53
Out of control feeling or overwhelmed	9.00	1.32 - 62.0	0.02
Tingling extremities	0.40	0.08 - 1.86	0.24

Bivariate analysis showed that the prevalence of high (> 150 IU/mL) IgG levels to T. gondii was significantly (P = 0.02) lower in women with difficulty concentrating (1/109: 0.9%) than in those without this clinical feature (4/41: 9.8%). Whereas prevalence of high IgG levels was higher, but not statistically significant (P = 0.07), in women suffering from out of control feeling or overwhelmed (5/86: 5.8%) than in those without this clinical characteristic (0/65: 0%). Concerning the association of clinical data and seropositivity to both anti-T. gondii IgG and IgM antibodies, bivariate analysis showed no significant associations. DNA of T. gondii was found in only one woman who was seropositive to anti-T. gondii IgG antibodies. Due to a limited number of cases with high IgG levels, seropositivity to IgM, and positivity to T. gondii DNA, no further regression analysis of the association of these laboratory results and the clinical variables was performed.

Discussion

Premenstrual dysphoric disorder is a clinical entity of unclear pathogenesis [18]. This psychiatric disorder is currently affecting up to 8% of women at reproductive age and focuses on psychological symptoms, whereas physical symptoms prevail in premenstrual syndrome [21]. Infection with T. gondii leads to a wide parasite spread in the host from the intestine to many organs in the body including the brain [26]. Infection with T. gondii has been associated with psychiatric disorders in general [27, 28], and it has been linked to changes in behavior [29, 30]. To our knowledge, the association between T. gondii infection and signs and symptoms of premenstrual dysphoric disorder was not assessed previously. Therefore, the present work aimed to determine whether infection with T. gondii was associated with clinical characteristics of premenstrual dysphoric disorder in a sample of women in the northern Mexican city of Durango. We observed that women with T. gondii IgG antibodies had a similar mean number of signs or symptoms to seronegative women. Nevertheless, multivariate analysis showed that seropositivity to T. gondii is associated with specific clinical characteristics of premenstrual dysphoric disorder in particular. Interestingly, IgG seropositivity to T. gondii was associated with the clinical manifestation of out of control feeling or overwhelmed. It is not clear why infection with T. gondii was associated with this psychological symptom in women with premenstrual dysphoric disorder. We did not find any report in the medical literature about the association of T. gondii and the clinical manifestation of out of control feeling or overwhelmed. The subjective sense of being overwhelmed or out of control has been recognized as a diagnostic symptom of premenstrual dysphoric disorder for about 20 years [31]. In a retrospective study of Brazilian college students, researchers found that out of control feeling or overwhelmed were major symptoms of premenstrual dysphoric disorder [32]. Brazil has a high (64.9%) prevalence of T. gondii infection among women of childbearing age [33], and our finding raises the question whether T. gondii might be linked to these symptoms in Brazilian women with premenstrual dysphoric disorder. In a study in Casablanca, Morocco, researchers found that the subjective sense of being overwhelmed or out of control was present in 55.7% of women with premenstrual dysphoric disorder studied [34]. We may hypothesize that T. gondii in brain may lead to psychological symptoms as out of control feeling or overwhelmed in women suffering from premenstrual dysphoric disorder. Infection with T. gondii may cause changes in neurotransmitters, i.e., dopamine and serotonin, that could lead to mood and behavioral changes [11, 35]. On the other hand, in the present study, infection with T. gondii was negatively associated with difficulty concentrating. This finding suggests a protective effect of T. gondii on this clinical characteristic. Nevertheless, this finding may also suggest that T. gondii was not an important factor for this symptom in women with premenstrual dysphoric disorder. Difficulty concentrating has been linked to T. gondii infection in immunocompetent adult patients suffering from acute toxoplasmic lymphadenitis [10]. Difference in results among the studies might be explained by difference in the characteristics of the patients studied including clinical diagnosis of patients, duration of infection (acute vs. chronic), and gender.

This study has limitations including small sample size of women with premenstrual dysphoric disorder and a low frequency of anti-T. gondii IgG, IgM and PCR positivity. In addition, criteria for diagnosis of this disorder have been recently described, and it is unclear whether this disorder requires further characterization. For the above reasons results of the present study should be interpreted with care.

Conclusions

Results of this first study on the association of T. gondii infection and clinical characteristics of premenstrual dysphoric disorder suggest that this infection might be linked to some symptoms of this disorder. We report for the first time the association of T. gondii infection and out of control feeling or overwhelmed. Results warrant for further research on the role of T. gondii in premenstrual dysphoric disorder.