- Research article
- Open Access
- Open Peer Review
Seroepidemiology of Toxoplasma gondii infection in pregnant women in a public hospital in northern Mexico
BMC Infectious Diseasesvolume 6, Article number: 113 (2006)
Toxoplasma gondii (T. gondii) infection in pregnant women represents a risk for congenital disease. There is scarce information about the epidemiology of T. gondii infection in pregnant women in Mexico. Therefore, we sought to determine the prevalence of T. gondii infection and associated socio-demographic, clinical and behavioural characteristics in a population of pregnant women of Durango City, Mexico.
Three hundred and forty three women seeking prenatal care in a public hospital of Durango City in Mexico were examined for T. gondii infection. All women were tested for anti-T. gondii IgM and IgG antibodies by using IMx Toxo IgM and IMx Toxo IgG 2.0 kits (Abbott Laboratories, Abbott Park, IL, USA), respectively. Socio-demographic, clinical and behavioural characteristics from each participant were also obtained.
Twenty one out of the 343 (6.1%) women had IgG anti-T. gondii antibodies. None of the 343 women had IgM anti-T. gondii antibodies. Multivariate analysis using logic regression showed that T. gondii infection was associated with living in a house with soil floor (adjusted OR = 7.16; 95% CI: 1.39–36.84), residing outside of Durango State (adjusted OR = 4.25; 95% CI: 1.72–10.49), and turkey meat consumption (adjusted OR = 3.85; 95% CI: 1.30–11.44). Other characteristics as cat contact, gardening, and food preferences did not show any association with T. gondii infection.
The prevalence of T. gondii infection in pregnant women of Durango City is low as compared with those reported in other regions of Mexico and the majority of other countries. Poor housing conditions as soil floors, residing in other Mexican States, and turkey meat consumption might contribute to acquire T. gondii infection.
Toxoplasma gondii (T. gondii) is a protozoan parasite widely distributed around the world [1, 2]. It has been estimated that up to one third of the world's population is infected by T. gondii . This parasite is transmitted to humans mainly by ingesting food or water that is contaminated with oocysts shed by cats or by eating undercooked or raw meat containing tissue cysts [3–5]. Infections in humans are usually asymptomatic but in some infected persons cervical lymphadenopathy or ocular disease may occur [1, 3]. However, primary infection acquired during pregnancy may result in severe damage to the foetus [3, 6]. Manifestations of congenital toxoplasmosis include mental retardation, seizures, blindness, and death . Congenital disease may become apparent at birth or not until the second or third decade of life [7–9]. Acute and latent T. gondii infections during pregnancy are mostly diagnosed by serological tests including detection of anti-T. gondii-specific IgM and IgG antibodies [3, 7–9], and avidity of T. gondii-specific IgG antibodies [3, 10]. Reports of epidemiological studies indicate that prevalence of T. gondii infection in pregnant women varies substantially among countries. For instance, in European countries, prevalences of T. gondii infections in pregnant women vary from 9% to 67% [11–20]. In contrast, in Asian countries, low prevalences of T. gondii infection were found in a Korean study , and a Vietnamese study  (0.8% and 11.2%, respectively). While prevalences as high as 41.8% to 55.4% in pregnant women have been reported in Indian [23, 24], Malaysian  and Nepalese  populations. For its part, a Sudanese study showed that 34.1% of the pregnant women studied had anti-T. gondii antibodies . Similarly, a study performed in New Zealand revealed a 33% prevalence of anti-T. gondii antibodies . In the American continent, a study performed in south Brazil revealed that 74.5% of the pregnant women studied had anti-T. gondii IgG antibodies . In a Cuban study, 70.9% of women had anti-T. gondii antibodies 12 weeks before pregnancy . There is scarce information about the epidemiology of T. gondii infection in pregnant women living in northern Mexico. Therefore, we performed a cross-sectional study in order to determine the prevalence of T. gondii infection in pregnant women of Durango City, Mexico and to know whether any characteristic of the women is associated with the infection.
All pregnant women seeking prenatal care from July 2005 to March 2006 at the General Hospital of Durango City, Mexico were invited to participate in the study. During the study period, 408 pregnant women were attended. Out of the 408 pregnant women, 343 were included in the study, and 65 were not included because either they did not accept to participate, or did not provide blood for analysis or did not submit the questionnaire. Inclusion criteria for the study subjects were: 1) pregnant women in any of the three trimesters of pregnancy; 2) aged 13 years and older; and 3) who accepted to participate in the study. Participants were enrolled consecutively.
Socio-demographic, clinical and behavioural data
Socio-demographic data including age, birth place, residence place, marital status, occupation, educational level, socio-economic level and housing conditions index were obtained from all participants. Housing conditions index was obtained by using the Bronfman's criteria . Briefly, five variables were evaluated: number of persons in the house, number of rooms in the house, material of the floor of the house, availability of drinkable water, and form of elimination of excretes. Clinical data including obstetric history, gestational age, blood transfusion or transplant history; and behavioural data including animal contacts, cleaning up cat excrement, foreign travel, kind of meat consumption (pork, lamb, beef, goat, boar, chicken, turkey, rabbit, deer, squirrel, horse, fish and iguana), raw or undercooked meat consumption, unpasteurized milk or milk products consumption, untreated water consumption, eating dried or cured meat (chorizo, ham, sausages or salami), unwashed raw vegetables or fruits consumption, contact with soil (gardening or agriculture), and eating outside of the home from all women studied were obtained.
Sera of the pregnant women were analysed for anti-T. gondii IgM and IgG antibodies by IMx Toxo IgM and IMx Toxo IgG 2.0 kits (both from Abbott Laboratories, Abbott Park, IL, USA), respectively. These tests are based on Microparticle Enzyme Immunoassays technology and performed on the IMx immunoassay automated analyser (Abbott Diagnostics, North Chicago, Il, USA).
This study was approved by the ethical committee of the General Hospital of Durango City. The purpose and procedures of the study were explained to all participants, and a written informed consent was obtained from all of them.
Results were analyzed with the aid of the software Epi Info 6 and SPSS 8.0. For comparison of the frequencies among the groups, the Mantel-Haenszel test, and when indicated the Fisher exact test, were used. Bivariate and multivariate analyses were used to assess the association between the characteristics of the subjects and the T. gondii infection. Variables were included in the multivariate analysis if they had a p value of less than 0.2 in the bivariate analysis. Adjusted odd ratio (OR) and 95% confidence interval (CI) were calculated by multivariate analysis using a multiple, unconditional logic regression model.
Sociodemographic description of the study population
General socio-demographic characteristics of the 343 pregnant women studied are shown in Table 1. The mean age of the pregnant women was 25 years (range: 13 to 44 years), and all studied women belonged to a low socio-economic level. Most women were born in Mexico and resided in urban areas of Durango State. The majority of them were housewives, have studied up to 12 years, and used to live in regular housing conditions. In addition, most women had had two or more pregnancies at the moment of the study.
Serology and prevalence
Out of the 343 pregnant women studied, twenty one were positive for anti-T. gondii IgG antibodies and none were positive for anti-T. gondii IgM antibodies. Therefore, we found a 6.1% prevalence of latent T. gondii infection, and 0% prevalence of acute T. gondii infection.
Factors associated with seropositivity
In the bivariate analysis, seven variables were identified as possible risk factors associated with T. gondii infection: 1) living in a house with soil floors (p = 0.004); 2) a residence outside Durango State (p = 0.009); 3) turkey meat consumption (p = 0.06); 4) deer meat consumption (p = 0.08); 5) squirrel meat consumption (p = 0.1); 6) wild animals meat consumption (p = 0.06); and 7) blood transfusion (p = 0.1). The rest of the sociodemographic, clinical and behavioural characteristics of the studied women did not show any likely association with T. gondii infection. Table 2 shows the results of the bivariate analysis of selected variables and the results of T. gondii seropositivity. As seen in Table 3, by using multivariate analysis, only three variables were associated with T. gondii seropositivity: 1) living in a house with soil floor (adjusted OR = 7.16; 95% CI: 1.39–36.84); 2) residing outside Durango State (adjusted OR = 4.25; 95% CI: 1.72–10.49); and 3) turkey meat consumption (adjusted OR = 3.85; 95% CI: 1.30–11.44).
In spite of about 2 cases of congenital toxoplasmosis per 1000 newborns occur in Mexico City , the epidemiology of T. gondii infection in general, and in pregnant women in particular, have been poorly studied in Mexico. A national survey in the general population showed that T. gondii infections exist in the whole country , and prevalences clearly vary among the regions [33, 34]. With respect to research on T. gondii infection in pregnant women of Mexico, only three studies have been reported. A study performed in a humid tropical State located in the south-east of Mexico showed that 60% of the pregnant women studied were positive for anti-T. gondii antibodies . Other study performed with high risk pregnant women from central Mexico showed that 34.9% had anti-T. gondii IgG antibodies , and a study of women with spontaneous abortions from the south of Mexico showed a 47% prevalence of anti-T. gondii antibodies . In this study, we found a 6.1% prevalence of latent T. gondii infection in pregnant women of Durango City, Mexico. This prevalence is much lower than those reported in the previous studies of pregnant women in other regions of Mexico [35–37]. Similarly, our prevalence found in Durango city is much lower than those reported in pregnant women from European countries [11–20], Vietnam , India [23, 24], Malaysia , Nepal , Sudan , New Zealand , Brazil , and Cuba , where prevalences vary from 9% to 74.5%. In contrast, our prevalence is higher than the one reported in a Korean study, where researchers found a prevalence of about 0.8% in the pregnant women studied . The lower prevalence found in our study compared with those reported elsewhere might be explained by differences in the characteristics of the pregnant women studied and the environment of Durango City. Concerning the characteristics of the pregnant women that may contribute to the low prevalence found could be: 1) the pregnant women studied belonged to a low socio-economic level and although they do eat meat, they certainly do it at a lower frequency and quantity than those with a better socioeconomic level because they can not afford to buy meat in a regular basis. Since eating contaminated meat is a well known route of T. gondii infection, the lower the frequency of meat consumption the lower the risk of infection; and 2) consumption of undercooked or raw meat is a rare practice both in the studied women and in people of the general population of Durango City. The presence of these two characteristics in our infected population points towards transmission that might have occurred in some cases, not by ingesting tissue cysts of the parasite in infected meat but by ingesting parasite oocysts in contaminated food or water. The poorest housing conditions found among the infected population support this statement. With respect to environmental characteristics, we may speculate that the low prevalence found could be also explained by a number of environment characteristics: 1) Durango City has a dry climate, and prevalence of T. gondii infection in dry climates has been reported lower than other climates [3, 19, 33]; 2) Most of the year Durango City usually has high temperatures during the day, and these high temperatures may contribute to reduce infectivity of T. gondii oocysts. This likely explanation could be supported by the observation in an experimental model that the higher the storing temperature of T. gondii oocysts the shorter the survival and infectivity time of the parasites ; and 3) Durango City also has a high altitude (1880 meters above sea level), and prevalences of T. gondii infection have been found lower in high altitudes than in low altitudes in some studies in humans [39, 40] and a study in animals .
In this study, living in a house with soil floors was associated with T. gondii infection (adjusted OR = 7.16; 95% CI: 1.39–36.84). Contaminated soil with parasite oocysts is a source of infection  and might contribute to explain the higher frequency of infection in women living in a house with floors made of soil than those living in a house with floors made of concrete or other materials. Theoretically, infected cats play a major role in contaminating soil; therefore persons living in a house with soil floors need to have a cat or have contact with it to become infected by this route. The fact that in this study the variable "contact with cats" was not associated with infection might be explained by a number of possible reasons: 1) contact with cats was a very common practice among the studied population, but parasite transmission could be more efficient in those houses with soil floors than in those with other kind of floors; 2) the frequency of infection in cats may be higher in those living in the poorest environment than those living in better housing conditions. The food consumed by the cats is certainly different in those living in the poorest housing condition than those living in a regular housing condition. More hunting of possibly infected animals may occur in the poorest environment than in a better one; 3) soil floors reflect extreme poverty and there are always further bad sanitary conditions in women with this status that may all facilitate transmission of T. gondii, and; 4) animals other than domestic cats may also contaminate soil floors. In this study we also found an association of latent T. gondii infection with a residence outside Durango State (adjusted OR = 4.25; 95% CI: 1.72–10.49). This finding suggests that those women could have been infected outside Durango State. In addition, logistic regression showed that turkey meat consumption was also associated with T. gondii infection (adjusted OR = 3.85; 95% CI: 1.30–11.44). Turkey meat consumption could be thus responsible for at least some cases of T. gondii infection among our infected population. This finding was unexpected since meats other than turkey meat have been implicated more frequently as a source of T. gondii infection than turkey meat. This finding deserves further investigation. The rest of the sociodemographic, clinical, and behavioural characteristics of the studied women did not show any association with T. gondii infection. Known factors associated with T. gondii infection in pregnant women such as drinking untreated water [13, 42], contact with garden soil , increasing parity , consumption of cured meat , and contact with cats , were not associated with T. gondii infection in our study.
We performed serum analysis by Microparticle Enzyme Immunoassays for anti-T. gondii IgG and IgM antibodies because these assays have been shown a high overall agreement as compared with the dye test . In addition, routine quality control measure were applied when analysing samples, therefore, we considered results of our assays were reliable.
We concluded that the prevalence of T. gondii infection in pregnant women of Durango City is low as compared with those reported in other regions of Mexico and the majority of other countries. Poor housing conditions as soil floors, residing in other Mexican States, and turkey meat consumption might contribute to acquire T. gondii infection.
Liesenfeld O, Janitschke K: Toxoplasma. Medizinische Mikrobiologie und Infektiologie. Auflage. Edited by: Hahn H, Falke D, Kaufmann SHE, Ullmann U. 2005, Springer Medizin-Verlag. Berlin, 5: 750-753.
Hill DE, Chirukandoth S, Dubey JP: Biology and epidemiology of Toxoplasma gondii in man and animals. Anim Health Res Rev. 2005, 6: 41-61. 10.1079/AHR2005100.
Montoya JG, Liesenfeld O: Toxoplasmosis. Lancet. 2004, 363: 1965-1976. 10.1016/S0140-6736(04)16412-X.
Dubey JP: Toxoplasmosis – a waterborne zoonosis. Vet Parasitol. 2004, 126: 57-72. 10.1016/j.vetpar.2004.09.005.
Dawson D: Foodborne protozoan parasites. Int J Food Microbiol. 2005, 103: 207-227. 10.1016/j.ijfoodmicro.2004.12.032.
Kravetz JD, Federman DG: Toxoplasmosis in pregnancy. Am J Med. 2005, 118: 212-216. 10.1016/j.amjmed.2004.08.023.
Jones JL, Lopez A, Wilson M, Schulkin J, Gibbs R: Congenital toxoplasmosis: a review. Obstet Gynecol Surv. 2001, 56: 296-305. 10.1097/00006254-200105000-00025.
Remington JS, McLeod R, Desmonts G: Toxoplasmosis. Infectious diseases of the foetus and newborn infant. Edited by: Remington JS, Klein JO. 2000, Saunders, Philadelphia, 205-346. 5
Liesenfeld O, Wong SY, Remington JS: Toxoplasmosis. Cecil textbook of medicine. Edited by: Goldmann L, Bennett JC. 2000, Philadelphia, PA, USA: W.B. Saunders, 1963-21
Alvarado Esquivel C, Sethi Shneh, Janitschke K, Hahn H, Liesenfeld O: Comparison of two commercially available avidity tests for Toxoplasma-specific IgG antibodies. Arch Med Re. 2002, 33: 520-523. 10.1016/S0188-4409(02)00411-3.
Nash JQ, Chissel S, Jones J, Warburton F, Verlander NQ: Risk factors for toxoplasmosis in pregnant women in Kent, United Kingdom. Epidemiol Infect. 2005, 133: 475-483. 10.1017/S0950268804003620.
Gutierrez-Zufiaurre N, Sanchez-Hernandez J, Munoz S, Marin R, Delgado N, Saenz MC, Munoz-Bellido JL, Garcia-Rodriguez JA: Seroprevalence of antibodies against Treponema pallidum, Toxoplasma gondii, rubella virus, hepatitis B and C virus, and HIV in pregnant women. Enferm Infecc Microbiol Clin. 2004, 22: 512-516. 10.1157/13067618.
Ertug S, Okyay P, Turkmen M, Yuksel H: Seroprevalence and risk factors for toxoplasma infection among pregnant women in Aydin province, Turkey. BMC Public Health. 2005, 5: 66-10.1186/1471-2458-5-66.
Petersson K, Stray-Pedersen B, Malm G, Forsgren M, Evengard B: Seroprevalence of Toxoplasma gondii among pregnant women in Sweden. Acta Obstet Gynecol Scand. 2000, 79: 824-829. 10.1034/j.1600-0412.2000.079010824.x.
Punda-Polic V, Tonkic M, Capkun V: Prevalence of antibodies to Toxoplasma gondii in the female population of the County of Split Dalmatia, Croatia. Eur J Epidemiol. 2000, 16: 875-877. 10.1023/A:1007606501923.
Cook AJ, Gilbert RE, Buffolano W, Zufferey J, Petersen E, Jenum PA, Foulon W, Semprini AE, Dunn DT: Sources of toxoplasma infection in pregnant women: European multicentre case-control study. European Research Network on Congenital Toxoplasmosis. BMJ. 2000, 321: 142-147. 10.1136/bmj.321.7254.142.
Harma M, Harma M, Gungen N, Demir N: Toxoplasmosis in pregnant women in Sanliurfa, Southeastern Anatolia City, Turkey. J Egypt Soc Parasitol. 2004, 34: 519-525.
Munoz Batet C, Guardia Llobet C, Juncosa Morros T, Vinas Domenech L, Sierra Soler M, Sanfeliu Sala I, Bosch Mestres J, Dopico Ponte E, Lite Lite J, Matas Andreu L, Juste Sanchez C, Barranco Romeu M: Toxoplasmosis and pregnancy. Multicenter study of 16,362 pregnant women in Barcelona. Med Clin. 2004, 123: 12-16.
Jenum PA, Kapperud G, Stray-Pedersen B, Melby KK, Eskild A, Eng J: Prevalence of Toxoplasma gondii specific immunoglobulin G antibodies among pregnant women in Norway. Epidemiol Infect. 1998, 120: 87-92. 10.1017/S0950268897008480.
Jeannel D, Niel G, Costagliola D, Danis M, Traore BM, Gentilini M: Epidemiology of toxoplasmosis among pregnant women in the Paris area. Int J Epidemiol. 1988, 17: 595-602.
Song KJ, Shin JC, Shin HJ, Nam HW: Seroprevalence of toxoplasmosis in Korean pregnant women. Korean J Parasitol. 2005, 43: 69-71.
Buchy P, Follezou JY, Lien TX, An TT, Tram LT, Tri DV, Cuong NM, Glaziou P, Chien BT: Serological study of toxoplasmosis in Vietnam in a population of drug users (Ho Chi Minh city) and pregnant women (Nha Trang). Bull Soc Pathol Exot. 2003, 96: 46-47.
Singh S, Pandit AJ: Incidence and prevalence of toxoplasmosis in Indian pregnant women: a prospective study. Am J Reprod Immunol. 2004, 52: 276-283. 10.1111/j.1600-0897.2004.00222.x.
Akoijam BS, Shashikant , Singh S, Kapoor SK: Seroprevalence of toxoplasma infection among primigravid women attending antenatal clinic at a secondary level hospital in North India. J Indian Med Assoc. 2002, 100: 591-592.
Nissapatorn V, Noor Azmi MA, Cho SM, Fong MY, Init I, Rohela M, Khairul Anuar A, Quek KF, Latt HM: Toxoplasmosis: prevalence and risk factors. J Obstet Gynaecol. 2003, 23: 618-624. 10.1080/01443610310001604376.
Rai SK, Shibata H, Sumi K, Rai G, Rai N, Manandhar R, Gurung G, Ono K, Uga S, Matsuoka A, Shrestha HG, Matsumura T: Toxoplasma antibody prevalence in Nepalese pregnant women and women with bad obstetric history. Southeast Asian J Trop Med Public Health. 1998, 29: 739-743.
Elnahas A, Gerais AS, Elbashir MI, Eldien ES, Adam I: Toxoplasmosis in pregnant Sudanese women. Saudi Med J. 2003, 24: 868-870.
Morris A, Croxson M: Serological evidence of Toxoplasma gondii infection among pregnant women in Auckland. N Z Med J. 2004, 117: U770-
Spalding SM, Amendoeira MR, Klein CH, Ribeiro LC: Serological screening and toxoplasmosis exposure factors among pregnant women in South of Brazil. Rev Soc Bras Med Trop. 2005, 38: 173-177. 10.1590/S0037-86822005000200009.
Gonzalez-Morales T, Bacallo-Gallestey J, Garcia-Santana CA, Molina-Garcia JR: Prevalence of Toxoplasma gondii antibodies in a population of pregnant women in Cuba. Gac Med Mex. 1995, 131: 499-503.
Bronfman M, Guiscafré H, Castro V, Castro R, Gutiérrez G: La medición de la desigualdad: una estrategia metodológica, análisis de las características socioeconómicas de la muestra. Arch Invest Med. 1988, 19: 351-360.
Vela-Amieva M, Canedo-Solares I, Gutierrez-Castrellon P, Perez-Andrade M, Gonzalez-Contreras C, Ortiz-Cortes J, Ortega-Velazquez V, Galvan-Ramirez Mde L, Ruiz-Garcia M, Saltigeral-Simentel P, Ordaz-Favila JC, Sanchez C, Correa D: Short report: neonatal screening pilot study of Toxoplasma gondii congenital infection in Mexico. Am J Trop Med Hyg. 2005, 72: 142-144.
Velasco-Castrejon O, Salvatierra-Izaba B, Valdespino JL, Sedano-Lara AM, Galindo-Virgen S, Magos C, Llausas A, Tapia-Conyer R, Gutierrez G, Sepulveda J: Seroepidemiology of toxoplasmosis in Mexico. Salud Publica Mex. 1992, 34: 222-229.
Goldsmith RS, Kagan IG, Zarate R, Reyes-Gonzalez MA, Cedeno-Ferreira J: Low Toxoplasma antibody prevalence in serologic surveys of humans in southern Mexico. Arch Invest Med. 1991, 22: 63-73.
Fernandez Torrano M: Epidemiology of Toxoplasmosis in pregnant women in Tabasco, Mexico. J Trop Pediatr. 1988, 34: 143-146.
Galvan Ramirez M, de la L, Soto Mancilla JL, Velasco Castrejon O, Perez Medina R: Incidence of anti-Toxoplasma antibodies in women with high-risk pregnancy and habitual abortions. Rev Soc Bras Med Trop. 1995, 28: 333-337.
Zavala-Velazquez J, Guzman-Marin E, Barrera-Perez M, Rodriguez-Felix ME: Toxoplasmosis and abortion in patients at the O'Horan Hospital of Merida, Yucatan. Salud Publica Mex. 1989, 31: 664-668.
Dubey JP: Toxoplasma gondii oocyst survival under defined temperatures. J Parasitol. 1998, 84: 862-865. 10.2307/3284606.
Hershey DW, McGregor JA: Low prevalence of Toxoplasma infection in a Rocky Mountain prenatal population. Obstet Gynecol. 1987, 70: 900-902.
Rai SK, Shibata H, Sumi K, Kubota K, Hirai K, Matsuoka A, Kubo T, Tamura T, Basnet SR, Shrestha HG, et al: Seroepidemiological study of toxoplasmosis in two different geographical areas in Nepal. Southeast Asian J Trop Med Public Health. 1994, 25: 479-484.
Rajkhowa S, Sarma DK, Rajkhowa C: Seroprevalence of Toxoplasma gondii antibodies in captive mithuns (Bos frontalis) from India. Vet Parasitol. 2006, 135: 369-374. 10.1016/j.vetpar.2005.10.007.
Lopez-Castillo CA, Diaz-Ramirez J, Gomez-Marin JE: Risk factors for Toxoplasma gondii infection in pregnant women in Armenia, Colombia. Rev Salud Publica. 2005, 7: 180-190.
Baril L, Ancelle T, Goulet V, Thulliez P, Tirard-Fleury V, Carme B: Risk factors for Toxoplasma infection in pregnancy: a case-control study in France. Scand J Infect Dis. 1999, 31: 305-309. 10.1080/00365549950163626.
Liesenfeld O, Press C, Flanders R, Ramirez R, Remington JS: Study of Abbott Toxo IMx system for detection of immunoglobulin G and immunoglobulin M Toxoplasma antibodies: value of confirmatory testing for diagnosis of acute toxoplasmosis. J Clin Microbiol. 1996, 34: 2526-2530.
The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/6/113/prepub
The author(s) declare that they have no competing interests.
CAE conceived and designed the study protocol, participated in the coordination and management of the study, performed the data analysis and wrote the manuscript. ASA designed the study protocol, applied the questionnaires and performed the data analysis. SGND performed the analysis of the serum samples. SEM performed the statistical analysis. JHDG applied the questionnaires and performed the data analysis. OL designed the study protocol, performed the data analysis, and wrote the manuscript. SAMG performed the data analysis. ACM applied the questionnaires and monitored the study.