# Seroprevalence and Associated Risk Factors forToxoplasma gondii Infection in Healthy Blood Donors: A Cross-Sectional Study in Sonora, Mexico

Authors: Cosme Alvarado-Esquivel; Antonio Rascón-Careaga; Jesús Hernández-Tinoco; María Alba Guadalupe Corella-Madueño; Luis Francisco Sánchez-Anguiano; María Lourdes Aldana-Madrid; Edgar Velasquez-Vega; Trinidad Quizán-Plata; José Luis Navarro-Henze; Joel Alberto Badell-Luzardo; José María Gastélum-Cano; Oliver Liesenfeld
Journal: BioMed Research International
 (2016)

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## Abstract

Toxoplasma gondii (T.gondii) can be transmitted by blood transfusion. We determined the prevalence ofT.gondii infection in healthy blood donors in Hermosillo city, Mexico, and the association of infection withT.gondii with the sociodemographic, clinical, and behavioral characteristics of blood donors. Four hundred and eight blood donors who attended two public blood banks in Hermosillo city were examined for anti-T.gondii IgG and IgM antibodies by using enzyme-linked immunoassays. Of the 408 blood donors (mean age 31.77
±
9.52; range 18–60 years old) studied, 55 (13.5%) were positive for anti-T.gondii IgG antibodies, and 12 (21.8%) of them were also positive for anti-T.gondii IgM antibodies. Multivariate analysis showed that seropositivity toT.gondii was associated with age (OR = 1.74; 95% CI: 1.03–2.94; P
=
0.03) and tobacco use (OR = 2.09; 95% CI: 1.02–4.29; P
=
0.04). Seropositivity toT.gondii was correlated with the number of pregnancies, deliveries, and cesarean sections. The seroprevalence ofT.gondii infection in blood donors in Sonora is the highest reported in blood donors in northern Mexico so far. This is the first report of an association ofT.gondii exposure and tobacco use. Further research to confirm this association is needed.


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## Body

## 1. Introduction

The protozoan parasiteToxoplasma gondii (T.gondii) causes infections in humans and animals all around the world [1, 2]. Infection withT.gondii is often acquired by ingesting food or water contaminated with oocyst shed by cats or by eating raw or undercooked meat containing tissue cysts [2–4]. However,T.gondii infection can also be acquired by blood transfusion [5] and organ transplantation [6]. Dissemination ofT.gondii occurs via blood flow to a large variety of body organs [7–9]. Primary infection during pregnancy may lead to congenital disease [2, 10]. Reactivation ofT.gondii infection in immunocompromised patients may cause a life-threatening disease of the central nervous system [2, 11]. Signs and symptoms of toxoplasmosis usually include retinochoroiditis, enlargement of cervical lymph nodes, or encephalitis [1, 2]. In addition, infection withT.gondii has been linked to schizophrenia and other mental disorders [12, 13].Very little is known about the seroepidemiology ofT.gondii infection in blood donors in Mexico. We previously reported a 7.4% seroprevalence ofT.gondii infection in blood donors in Durango city, Mexico [14], whereas two other studies of blood donors in central and southern Mexico reported seroprevalences of 29% [15] and 69% [16], respectively. There is no information about the magnitude of infection withT.gondii in blood donors in Hermosillo city, in the northwestern Mexican state of Sonora. Therefore, we sought to determine the seroprevalence ofT.gondii infection in blood donors in Hermosillo city, Mexico, and the seroprevalence association with the sociodemographic, clinical, and behavioral characteristics of blood donors.


## 2. Materials and Methods

### 2.1. Study Design and Study Population

Through a cross-sectional study, we examined 408 blood donors who attended two blood banks (Mexican Institute of Social Insurance and State Center for Blood Transfusion of the Secretary of Health) in Hermosillo city, Mexico. Blood donors were enrolled consecutively. Blood sampling of blood donors was performed from November to December 2015. Inclusion criteria for enrollment of participants were (1) blood donors attending any of the participating blood banks; (2) those aged 18 years and older; and (3) those who voluntarily accepted to participate in the study. Of the 408 blood donors studied, 308 were enrolled in the blood bank of the Mexican Institute of Social Insurance and 100 in the State Center for Blood Transfusion.


### 2.2. Detection of Anti-T.gondii IgG and IgM Antibodies

Sera of blood donors were obtained by centrifugation of fresh whole blood and stored at −20°C until analyzed. Sera were analyzed for anti-T.gondii IgG antibodies by a commercially available enzyme immunoassay “Toxoplasma IgG” kit (Diagnostic Automation Inc., Woodland Hills, CA, USA). This test has a cut-off of 8 IU/mL of anti-T.gondii IgG antibodies for seropositivity. In addition, sera positive for anti-T.gondii IgG antibodies were further analyzed for anti-T.gondii IgM antibodies by a commercially available enzyme immunoassay “Toxoplasma IgM” kit (Diagnostic Automation Inc.). All tests were performed according to manufacturer’s instructions, and positive and negative controls were included in each assay.


### 2.3. Sociodemographic, Clinical, and Behavioral Data

Through a face-to-face interview using a standardized questionnaire, we obtained the sociodemographic, clinical, and behavioral data of blood donors. Sociodemographic data included age, gender, birth place, residence, educational level, occupation, and socioeconomic status. Clinical data included health status, presence or history of lymphadenopathy, frequent abdominal pain or headache, dizziness, impairment of memory, reflexes, hearing, or vision, and history of surgery, blood transfusion, or organ transplantation. In women, obstetric history was obtained. Behavioral data included contact with animals, cleaning cat excrements, traveling, soil contact, type of flooring at home, frequency of eating away from home (in restaurants and fast food outlets), eating raw or undercooked meat, washing hands before eating, history of sexual promiscuity, and tobacco, alcohol, or drug use. In addition, behavioral items included frequency of meat consumption, type of meat consumed (pork, lamb, beef, goat, boar, chicken, turkey, rabbit, deer, squirrel, horse, fish, and others), consumption of dried or cured meat (chorizo, ham, sausages, or salami), beef intestine or stomach, animal brains, unwashed raw vegetables or fruits, and unpasteurized milk or untreated water.


### 2.4. Statistical Analysis

Data was analyzed with the aid of the software Epi-Info version 7 and SPSS version 15 (SPSS Inc., Chicago, IL, USA). For calculation of the sample size we used a reference seroprevalence of 7.4% [14] as expected frequency of the factor under study, 15,000 as the population size from which the sample was selected, confidence limits of 2.6%, and a confidence level of 95%. The result of the calculation was 380 subjects. To avoid bias in the statistical analysis, blood donors with missing data were excluded from the study. For comparison of the frequencies among groups we used Pearson’s chi square test and the Fisher exact test (when cells values were small). The association between the characteristics of blood donors andT.gondii seropositivity was assessed by bivariate and multivariate analyses. Only variables with a P value < 0.05 obtained in the bivariate analysis were included in the multivariate analysis. Odds ratio (OR) and 95% confidence interval (CI) were calculated by multivariate analysis using the Enter method. Statistical significance was set at a P value < 0.05.


### 2.5. Ethical Aspects

This study was approved by the Institutional Ethics Committee of the University of Sonora, Mexico. All participants were informed about the purpose and procedures of the survey, and a written informed consent was obtained from all of them.


## 2.1. Study Design and Study Population

Through a cross-sectional study, we examined 408 blood donors who attended two blood banks (Mexican Institute of Social Insurance and State Center for Blood Transfusion of the Secretary of Health) in Hermosillo city, Mexico. Blood donors were enrolled consecutively. Blood sampling of blood donors was performed from November to December 2015. Inclusion criteria for enrollment of participants were (1) blood donors attending any of the participating blood banks; (2) those aged 18 years and older; and (3) those who voluntarily accepted to participate in the study. Of the 408 blood donors studied, 308 were enrolled in the blood bank of the Mexican Institute of Social Insurance and 100 in the State Center for Blood Transfusion.


## 2.2. Detection of Anti-T.gondii IgG and IgM Antibodies

Sera of blood donors were obtained by centrifugation of fresh whole blood and stored at −20°C until analyzed. Sera were analyzed for anti-T.gondii IgG antibodies by a commercially available enzyme immunoassay “Toxoplasma IgG” kit (Diagnostic Automation Inc., Woodland Hills, CA, USA). This test has a cut-off of 8 IU/mL of anti-T.gondii IgG antibodies for seropositivity. In addition, sera positive for anti-T.gondii IgG antibodies were further analyzed for anti-T.gondii IgM antibodies by a commercially available enzyme immunoassay “Toxoplasma IgM” kit (Diagnostic Automation Inc.). All tests were performed according to manufacturer’s instructions, and positive and negative controls were included in each assay.


## 2.3. Sociodemographic, Clinical, and Behavioral Data

Through a face-to-face interview using a standardized questionnaire, we obtained the sociodemographic, clinical, and behavioral data of blood donors. Sociodemographic data included age, gender, birth place, residence, educational level, occupation, and socioeconomic status. Clinical data included health status, presence or history of lymphadenopathy, frequent abdominal pain or headache, dizziness, impairment of memory, reflexes, hearing, or vision, and history of surgery, blood transfusion, or organ transplantation. In women, obstetric history was obtained. Behavioral data included contact with animals, cleaning cat excrements, traveling, soil contact, type of flooring at home, frequency of eating away from home (in restaurants and fast food outlets), eating raw or undercooked meat, washing hands before eating, history of sexual promiscuity, and tobacco, alcohol, or drug use. In addition, behavioral items included frequency of meat consumption, type of meat consumed (pork, lamb, beef, goat, boar, chicken, turkey, rabbit, deer, squirrel, horse, fish, and others), consumption of dried or cured meat (chorizo, ham, sausages, or salami), beef intestine or stomach, animal brains, unwashed raw vegetables or fruits, and unpasteurized milk or untreated water.


## 2.4. Statistical Analysis

Data was analyzed with the aid of the software Epi-Info version 7 and SPSS version 15 (SPSS Inc., Chicago, IL, USA). For calculation of the sample size we used a reference seroprevalence of 7.4% [14] as expected frequency of the factor under study, 15,000 as the population size from which the sample was selected, confidence limits of 2.6%, and a confidence level of 95%. The result of the calculation was 380 subjects. To avoid bias in the statistical analysis, blood donors with missing data were excluded from the study. For comparison of the frequencies among groups we used Pearson’s chi square test and the Fisher exact test (when cells values were small). The association between the characteristics of blood donors andT.gondii seropositivity was assessed by bivariate and multivariate analyses. Only variables with a P value < 0.05 obtained in the bivariate analysis were included in the multivariate analysis. Odds ratio (OR) and 95% confidence interval (CI) were calculated by multivariate analysis using the Enter method. Statistical significance was set at a P value < 0.05.


## 2.5. Ethical Aspects

This study was approved by the Institutional Ethics Committee of the University of Sonora, Mexico. All participants were informed about the purpose and procedures of the survey, and a written informed consent was obtained from all of them.


## 3. Results

Of the 408 blood donors (mean age31.77
±
9.52; range 18–60 years old) 55 (13.5%) were positive for anti-T.gondii IgG antibodies. Of these 55 seropositive donors, 32 (58.2%) had IgG levels higher than 150 IU/mL, 3 (5.4%) between 100 and 150 IU/mL, and 20 (36.4%) between 12 to 97 IU/mL. Seroprevalence of anti-T.gondii IgG antibodies in blood donors from the Mexican Institute of Social Insurance (43/308: 14.0%) was similar (P
=
0.61) to the one (12/100: 12.0%) in the State Center for Blood Transfusion. Of the 55 IgG seropositive blood donors, 12 (21.8%) were also positive for anti-T.gondii IgM antibodies.Concerning the correlation ofT.gondii IgG seropositivity with the sociodemographic characteristics of blood donors, bivariate analysis showed a number of characteristics with a P value < 0.05 including age, residence, educational level, and occupation. Other sociodemographic characteristics of blood donors, that is, gender, birth place, and socioeconomic status, showed P values > 0.05 by bivariate analysis. A selection of sociodemographic characteristics and their correlation withT.gondii seropositivity is shown in Table 1.Table 1
Sociodemographic characteristics of blood donors and prevalence ofT. gondii infection.
Characteristic
Number
⁢Prevalence ofT. gondii infection
P value
Number
%
Age groups (years)
30 or less
206
15
7.3
0.001
31–50
186
38
20.4
>50
16
2
12.5
Gender
Male
334
47
14.1
0.45
Female
74
8
10.8
Birth place
Sonora state
366
49
13.4
0.59
Other Mexican states
39
5
12.8
Abroad
3
1
33.3
Residence area
Urban
373
45
12.1
0.008
Suburban
27
9
33.3
Rural
8
1
12.5
Educational level
No education
10
4
40.0
0.004
1 to 6 years
48
6
12.5
7–12 years
216
36
16.7
>12 years
134
9
6.7
Occupation
Laborera
328
51
15.5
0.01
Nonlaborerb
80
4
5.0
Socioeconomic level
Low
195
32
16.4
0.24
Medium
212
23
10.8
High
1
0
0.0
aLaborer: construction, professional, business, agriculture, factory worker, and others.
bNonlaborer: housewife, student, or no occupation.Of the clinical characteristics of blood donors, seropositivity toT.gondii did not correlate (P
>
0.05) with health status, presence or history of lymphadenopathy, frequent abdominal pain or headache, dizziness, impairment of memory, reflexes, hearing, or vision, and history of surgery, blood transfusion, or organ transplantation. In women, seropositivity toT.gondii correlated with number of pregnancies, deliveries, and cesarean sections (P
<
0.05). Table 2 shows the correlation ofT.gondii seropositivity and the obstetric data of the female blood donors studied.Table 2
Bivariate analysis of obstetric data and infection withT. gondii in female blood donors.
Characteristic
Subjects tested
⁢Prevalence ofT. gondiiinfection
P value
Number
Number
%
Pregnancies
None
21
1
4.8
0.01
One
6
0
0
Two
17
1
5.9
Three
13
1
7.7
Four
7
2
28.6
Five
1
1
100
Deliveries
None
36
4
11.1
0.03
One
8
1
12.5
Two
13
0
0
Three
6
0
0
Four
1
0
0
Five
1
1
100
Cesarean sections
None
43
2
4.7
0.01
One
10
0
0
Two
4
1
25
Three
8
3
37.5
Stillbirths
None
64
6
9.4
1.00
One
1
0
0
Miscarriages
None
59
6
10.2
1.00
One
6
0
0With respect to the correlation of seropositivity toT.gondii and behavioral characteristics of blood donors, a number of characteristics showed P values < 0.05 by bivariate analysis including cleaning cat excrement, tobacco consumption, and type of flooring at home. Other behavioral characteristics, that is, contact with animals, traveling, soil contact, frequency of eating away from home, eating raw or undercooked meat, washing hands before eating, history of sexual promiscuity, alcohol consumption, and drug use, showed P values > 0.05 by bivariate analysis. Table 3 shows a selection of behavioral characteristics of blood donors and their correlation withT.gondii seropositivity. Further analysis of sociodemographic and behavioral characteristics of blood donors by multivariate analysis showed that seropositivity toT.gondii was associated with age (OR = 1.74; 95% CI: 1.03–2.94; P
=
0.03) and tobacco use (OR = 2.09; 95% CI: 1.02–4.29; P
=
0.04) (Table 4).Table 3
Bivariate analysis of selected putative risk factors for infection withT. gondiiin blood donors.
Characteristic
Subjects tested
⁢Prevalence ofT. gondii infection
P value
Number
Number
%
Cats in the neighborhood
Yes
265
39
14.7
0.31
No
143
16
11.2
Cleaning cat excrement
Yes
42
10
23.8
0.03
No
366
45
12.3
Pork meat consumption
Yes
269
39
14.5
0.40
No
139
16
11.5
Sheep meat consumption
Yes
24
1
4.2
0.22
No
384
54
14.1
Pigeon meat consumption
Yes
2
1
50.0
0.25
No
406
54
13.3
Duck meat consumption
Yes
2
1
50.0
0.25
No
406
54
13.3
Quail meat consumption
Yes
4
1
25.0
0.44
No
404
54
13.4
Venison consumption
Yes
40
3
7.5
0.24
No
368
52
14.1
Iguana meat consumption
Yes
2
1
50.0
0.25
No
406
54
13.3
Fish consumption
Yes
244
39
16.0
0.07
No
164
16
9.8
Alcohol consumption
Yes
171
23
13.5
0.98
No
237
32
13.5
Tobacco consumption
Yes
61
14
23.0
0.01
No
347
41
11.8
Drug use
Yes
45
10
22.2
0.06
No
363
45
12.4
Soil contact
Yes
61
11
18.0
0.25
No
347
44
12.7
Washing hands before eating
Yes
390
50
12.8
0.08
No
18
5
27.8
Floor at home
Ceramic or wood
204
26
12.7
0.03
Concrete
203
28
13.8
Soil
1
1
100.0Table 4
Multivariate analysis of selected characteristics of blood donors and their association withT. gondii infection.
Characteristic
Odds ratio
95% confidence interval
P value
Age
1.74
1.03–2.94
0.03
Suburban residence
1.56
0.78–3.12
0.20
Education
0.76
0.48–1.19
0.23
Laborer
0.38
0.13–1.15
0.08
Cleaning cat excrement
0.47
0.20–1.09
0.08
Flooring at home
0.83
0.43–1.58
0.57
Tobacco use
2.09
1.02–4.29
0.04The correlation of IgM seropositivity with the blood bank and the sociodemographic, clinical, and behavioral characteristics of the 55 blood donors with IgG seropositivity toT.gondii was also analyzed. The variables blood bank, age, no cats in the neighborhood, and turkey meat consumption showed association with IgM seropositivity by bivariate analysis (Table 5). Other variables examined in this survey did not show a correlation with IgM seropositivity (P
>
0.05).Table 5
Correlation of seroprevalence of anti-T. gondii IgM antibodies with a selection of characteristics of 55 IgG seropositive blood donors.
Characteristic
Number
Prevalence of anti-T. gondii IgM antibodies
P value
Number
%
Blood bank
Mexican Institute of Social Insurance
43
6
14.0
0.01
State Center for Blood Transfusion
12
6
50.0
Age groups (years)
30 or less
15
7
46.7
0.01
31–50
38
4
89.5
>50
2
1
50.0
Cats in the neighborhood
Yes
39
4
10.3
0.003
No
16
8
50.0
Turkey meat consumption
Yes
9
5
55.6
0.01
No
46
7
15.2


## 4. Discussion

The seroepidemiology of infection withT.gondii in blood donors in Mexico has been scantly studied. The present study aimed to know the seroprevalence and associated risk factors of infection withT.gondii in a sample of blood donors who attended two blood banks in the northwestern Mexican city of Hermosillo. We found a 13.5% seroprevalence ofT.gondii infection in the blood donors studied. This seroprevalence is higher than the 7.4% seroprevalence ofT.gondii infection reported in blood donors in the northern Mexican city of Durango [14]. In contrast, the seroprevalence found in blood donors of Hermosillo city is lower than the 29% seroprevalence reported in blood donors in Jalisco state [15] and the 69% seroprevalence reported in a control group of blood donors in Yucatán state [16] in central and south Mexico, respectively. The seropositivity rate forT.gondii in blood donors in Hermosillo is lower than seroprevalences reported in blood donors in other countries in South America where seroprevalences between 20 and 79% have been reported [17, 18]. Seroprevalences reported in blood donors in other countries were 40% in Saudi Arabia [19], 59.6% in Egypt [20], 20.3% in India [21], 42.9% in New Zealand [22], and 32.1% in the Czech Republic [23]. On the other hand, the seroprevalence found in blood donors in Hermosillo is comparable with seroprevalences ofT.gondii infection reported in blood donors in Iran (19.3%) [24], Taiwan (9.3%) [25], Turkey (19.5%) [26], and Thailand (9.6%) [27] and even lower than that reported in blood donors in Shijiazhuang area, China (4.83%) [28]. Differences in seroprevalences ofT.gondii infection in blood donors among countries might be due to differences in the behavioral characteristics of blood donors, that is, extent ofT.gondii exposure, or differences in the environment. Hermosillo city has a hot desert climate and seroprevalence ofT.gondii infection is usually lower in dry climates than in humid climates [1]. Seroprevalence in blood donors does not reflect the seroprevalence in general population because blood donors are usually healthy and have a limited range of age whereas healthy and ill individuals with a wide range of age can exist in the general population.We searched for sociodemographic, clinical, and behavioral factors of blood donors associated withT.gondii seropositivity. Multivariate analysis showed that age was associated withT.gondii seroprevalence, with the highest rate of positivity in the age group of 31–50 years. This result is consistent with that in a study of blood donors in Durango, Mexico, where the highest seroprevalence ofT.gondii infection was found in the 45–60-year-old group [14]. Similarly, in a study of blood donors in Thailand, researchers found the highest rate ofT.gondii seropositivity in the age groups of 20–30 and 31–40 years old [27]. Intriguingly, multivariate analysis also showed thatT.gondii seropositivity was associated with tobacco use in blood donors in Hermosillo. To the best of our knowledge, this is the first report of an association of tobacco use and seropositivity toT.gondii. It is not clear why tobacco users had a higher seroprevalence ofT.gondii infection than blood donors without tobacco use. It is possible that tobacco users might acquireT.gondii infection by carrying the parasite from the hands to the mouth while smoking. In principle, washing hands before smoking is not commonly done; therefore, handling the cigarette and touching the lips with unwashed hands while smoking might contribute to acquiringT.gondii infection. Sharing the cigarette among people might potentially increase this risk of infection since cigarette is touched with more hands of more people. The hand-cigarette-mouth route has been linked toSalmonella infection [29]. Further research to determine the role of smoking inT.gondii transmission is needed. On the other hand, infection withT.gondii may lead to changes in behavior [30, 31]. It is unknown whether infection withT.gondii may lead to a desire to smoke. Of note, both infection withT.gondii and smoking are linked to dopamine release. Experimental infections withT.gondii in mice resulted in an increase of dopamine release in brain [32]. Similarly, nicotine elicits dopamine release in the ventral striatum that is a substance underlying tobacco smoking behaviors and dependence [33]. It is also unclear whether behavioral changes induced by nicotine may favor an increase in the exposure toT.gondii. Tobacco smoking has been associated to a number of health outcomes including infections, that is, hepatitis C virus [34], postoperative infections after dental implants [35], or spine surgeries [36]. However, the mechanisms involved in the association between infection and tobacco smoking are poorly understood [37].Seropositivity toT.gondii was correlated with the number of pregnancies, deliveries, and cesarean sections. This correlation might be related to an increasing seroprevalence ofT.gondii infection with age. The older the women, the higher the likelihood to accumulate obstetric events. In addition, it is also possible that a history of blood transfusion, a known risk factor forT.gondii infection [5], could have contributed to infection in some women with obstetric events.Concerning the seropositivity to anti-T.gondii IgM antibody in anti-T.gondii IgG seropositive blood donors, bivariate analysis showed a higher IgM seropositivity rate in blood donors attended in the State Center for Blood Transfusion than in those attended in the Mexican Institute of Social Insurance. It is possible that differences in the characteristics of blood donors among blood banks might explain the difference in the seroprevalences. IgM seropositivity was also associated with age and turkey meat consumption. Turkeys can be infected byT.gondii. In a study in Egypt, a 29.4% seroprevalence ofT.gondii infection in turkeys was found [38]. In addition, in two previous studies in Mexico,T.gondii seropositivity was associated with consumption of turkey meat in pregnant women [39] and in rural general population [40]. In the present study, IgM seropositivity was higher in subject without cats in the neighborhood than those with cats in the neighborhood. These results can be interpreted as a higher risk ofT.gondii infection by eating tissue cysts of turkey meat than by ingestion of oocysts shed by cats in the blood donors studied. On the other hand, the presence of IgM positive blood donors could suggest the existence of more recent infections. However, IgM positivity should be interpreted with care becauseT.gondii commercial IgM diagnostic test kits can yield a number of false positive results [41].DNA ofT.gondii has been found in 1.9% of IgM seropositive blood donors [24]; therefore, the use of blood fromT.gondii IgM positive blood donors might represent a risk forT.gondii infection.The present study has some limitations. The sample size of one blood bank was small. The small number of IgM seropositive cases did not allow us to perform further multivariate analysis with this infection marker. In addition, we were unable to perform molecular detection ofT.gondii in samples of blood donors. Results from DNA detection may further help to determine the risk forT.gondii infection by blood transfusion. Further research with a large sample size of blood donors and with the use of molecular methods for detection ofT.gondii should be conducted.


## 5. Conclusions

The seroprevalence ofT.gondii infection in blood donors in Hermosillo city in the northwestern Mexican state of Sonora is the highest reported in blood donors in northern Mexico so far. Seropositivity toT.gondii was linked to obstetric events and age. This is the first report of an association ofT.gondii exposure and tobacco use. Further research to confirm this association is needed.



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