Chronic toxoplasmosis and possible risk factors associated with pregnant women in Khyber Pakhtunkhwa

ABSTRACT

Toxoplasmosis is the major cause of abortion and stillbirths among women of childbearing age. The aim of this study is to determine chronic Toxoplasma gondii infection at various trimesters of pregnancy and to identify various possible routes of transmission and contamination in North-West Pakistan. A total of 733 blood samples were randomly obtained from antenatal/maternity centres. A detail questionnaire was designed to analyse the clinical history of the subjects tested for specific anti-Toxoplasma IgG antibodies by using immunofluorescence assay. Overall toxoplasmosis was detected in 18.41% of the studied population. Anti-Toxoplasma IgG was found 7.17% in first trimester with highest prevalence 31.28% in third trimester. Increase in the prevalence of parasite was seen with rise in age highest found in higher age group (P ˂ 0.05). As compared to district Lower Dir and Swat, Upper Dir had high rate 33.03% of chronic infection. Consumption of undercooked meat was also found 45.22% related with the prevalence of infection. A significant relation was observed with level of education (P ˂ 0.05) and prevalence of toxoplasmosis. An association was observed between prevalence of T. gondii infection and various risk factors, i.e. consumption of raw eggs, vegetables, contact with cats, cattle, soil, water sources and use of unpasteurized milk. This study shows high prevalence of the parasite in the studied area. Pregnant women in contact with cats were found more at risk. Education of childbearing age women, personal hygiene and contact with soil was also found important possible risk factors in the spread of infection.

KEYWORDS:

• Toxoplasma gondii
• pregnancy
• immunofluorescence assay (IFA)

Introduction

Toxoplasmosis is a zoonotic disease caused by a coccidian protozoan obligate parasite Toxoplasma gondii. The parasite resides intracellularly infecting a variety of warm-blooded species including human beings.[1] Sexual life cycle of the parasite is completed inside the intestinal epithelial cells of feline's particularly domestic cats, which act as definitive host. Unsporulated oocysts are then shed to the environment with feline faeces. Human beings acquire toxoplasmosis through contact with feline faeces, consumption of partially cooked meat, contaminated fruits and vegetables and infrequently by drinking contaminated water. Another possible route of acquiring infection includes blood transfusions and organ transplants.[2,3]

Vertical transmission of T. gondii during pregnancy may lead to severe complications such as still births or abortion. Infants with exposure to the parasite in utero may face congenital toxoplasmosis with severe nervous and ocular complications.[3,4] Other congenital disorders include hydrocephaly, epilepsy, internal calcification, encephalitis, blindness and serious damage to the central nervous system.[5,6]

Estimated sero-prevalence of human toxoplasmosis varies throughout the world, among different countries, among different geographical regions and it also varies among various cultural groups in the same population. It is estimated that about one third of the world population is infected. Relatively high prevalence of toxoplasmosis has been reported in pregnant women from Africa, Middle East, Europe and South-East Asia.[1,7]

The sero-prevalence studies against T. gondii in pregnant women performed in Pakistan show diversity. From Punjab toxoplasmosis among pregnant women was reported (63%) followed by Azad Kashmir (48%), (19.25%) from district Sawabi, Khyber Pakhtunkhwa and (14.4%) from district Kohat, Khyber Pakhtunkhwa.[5] The presence of felines and favourable environmental condition for the survival of T. gondii in the study area support the presence of infection, yet there is no data available on toxoplasmosis. Similarly, there are no appropriate serological screening facilities available for pregnant women at women health care centres in Pakistan. Keeping in view the importance of congenital toxoplasmosis, favourable environmental fluctuation and the feline host susceptibility in the zoonotic transmission of the parasite, thisstudy is designed to determine the sero-prevalence of T. gondii and possible risk factors associated with pregnancy in the north-west zone of Pakistan.

Materials and methods

Before blood collection informed consent was obtained from each participant, either directly or from the subjects’ guardian. Through a detailed questionnaire, all personal and clinical informations regarding toxoplasmosis were collected from all the subjects included in the study. The ethical approval of the study was granted by Research Ethical Committee, University of Peshawar.

Blood samples were collected in gel and clot activator tubes from pregnant women attending Department of Gynecology, District Head Quarter Hospital, Upper Dir, Department of Gynecology, District Head Quarter Hospital, Lower Dir and Department of Gynecology, District Head Quarter Hospital, Swat. Gel and clot activator tubes (Kantex, Meerbusch, Germany) were kept for one hour to get clear serum. Serum was then transferred to sterilized Eppendorf tubes with the help of jester pipette, brought to the Molecular Laboratory, Department of Animal Sciences, Quaid-e-Azam University Islamabad, Pakistan and stored in low deep freezer (Kaltis, Downey, USA) at −20 °C till further serological examination.

The extracted serum was screened for the presence of T. gondii using IFA kit (Biocintefica, S.A. Iturri 232 (C1427ADD) Buenos Aires, Argentina) as recommended by the manufacturer.

The data was statistically analysed by using latest version of SPSS (version 20). The (P) value of less than 0.05 (by the application of ONE-ANOVA) was assumed to be statistically significant.

Results and discussion

Exposure to the parasite during pregnancy is pathogenic which can lead to fetal complications, i.e. cerebral calcification, hydrocephaly, chorioretinitis and even miscarriage. Transmission of the parasite to human beings occurs by coming in contact with contaminate water, vegetables and soil with cat faeces or by consumption of partially cooked meat, unpasteurized milk and raw eggs.[1,3] Prevalence of toxoplasmosis ranges from 15% to 77% in various parts of the globe.[1,8,9] Vertical transmission is one of the serious forms of T. gondii infection which leads to a number of fetal complications. In this study, prevalence of IgG-specific antibodies against T. gondii was found 18.41% which is in agreement with the study conducted at Swabi, Pakistan, where sero-prevalence was found 19.25% [10] in Rawalpindi--Islamabad, Pakistan 17%.[5,6] The results of this study are also in alliance with studies conducted at Singapore, Thailand and London where sero-prevalence of T. gondii was reported 17.2%, 21.6% and 17.32%, respectively, in women of childbearing age.[11–13] On the other hand, many studies reported higher rate of prevalence from this study, 63% and 48% from Punjab and Azad Kashmir, Pakistan, respectively,[5] study from Turkey reported 30.1% prevalence of the infection [1] in Nigeria 32.6%,[14] Colombia 45.8%,[15] Brazil 49.2% [14,16] and south-western Ethiopia 81.1%.[7]

Various studies have reported that T. gondii infection increases with age. This increase might be correlated with the risk of exposure of individuals to the infection with the increase in age. This study found that prevalence of toxoplasmosis was higher in age group 36–40 years (35.80%) statistically highly significant difference was found (P ˂ 0.05). Sero-prevalence of toxoplasmosis was reported high in higher age groups 35–45 years (48.8%) from Japan.[17] Rosso et al. from Colombia 30–39 years (55.3%) [15] and Ertug et al. from Turkey 30–40 years (51.1%).[1]

A total of 733 pregnant women participated in this study. Overall chronic infection was observed (18.41%). The age range of the studied population was observed from 15–40 years, of which the highest number of participants 197 (26.87%) were found in between the age of 21–25 years. A significant difference was found (P ˂ 0.05) among various age groups with highest prevalence (35.80%) in age group ranging 36–40 years (Table 1). Gestational age was also reported to have a relation with the prevalence of the infection. Most of the participants in the study were in their first trimester; however, infection was found more prevalent (31.28%) in women in their third trimester. A significant difference (P ˂ 0.05) was observed among various districts studied with highest prevalence (33.03%) of parasite in district Upper Dir. Among 177 women who were in contact with cats, 73 (62.39%) showed positive confirmation to anti-Toxoplasma IgG. Significant association was observed (P ˂ 0.05) between practice of consuming undercooked meat and prevalence of toxoplasmosis. This study showed that 137(18.69%) participants were using municipal/pipe water where 37 (27.01%) cases were reported positive against T. gondii, while among 83 (11.32%) of the participants using filtered water for drinking purposes 2 (2.41%) were found positive for anti-Toxoplasma IgG. About one third of the studied population 283 (38.60%) were uneducated, incapable of reading and writing. Antibodies were found more prevalent 83 (29.32) in uneducated community as compared to educated which was statistically significant (P ˂ 0.05). Association of the infection with other possible risk factors like consumption of raw eggs, type of house, consumption of unwashed vegetables and unpasteurized milk is shown in Table 2.

Table 1. Toxoplasmosis and socio-demographic factors in the study population.

Socio-demographic factors	N	Positive (%)	Negative (%)	P value
Age
15–20	163	7.98	92.02	0.0000
21–25	197	11.68	88.32
26–30	171	18.13	81.87
31–35	121	32.23	67.77
36–40	81	35.80	64.20
Trimesters				0.0001
First trimester	293	7.17	92.83
Second trimester	213	20.19	79.81
Third trimester	227	31.28	68.72
Area				0.0001
Upper Dir	221	33.03	66.97
Lower Dir	263	10.27	89.73
Swat	249	14.06	85.94

Table 2. Toxoplasmosis and various associated risk factors.

	Possible risk factors	Anti-Toxoplasma IgG
No. of sources	Contact with cats and cattle	N	Positive (%)	Negative (%)	P value
1	Contact with cats only	117	73 (62.39)	44 (37.61)	0.0000
	Contact with cattle only	308	33 (10.71)	275 (89.29)
	Contact with both	221	28 (12.67)	193 (87.33)
	No contact	87	1 (1.15)	86 (98.85)
2	Water sources				0.0000
	Spring water	251	53 (21.12)	198 (78.88)
	Municipal/pipe water	137	37 (27.01)	100 (72.99)
	Filter water	83	2 (2.41)	81 (97.59)
	Well water	262	43 (16.41)	219 (83.59)
3	Milk				0.0000
	Unpasteurized milk	320	118 (36.88)	202 (63.13)
	Pasteurized milk	413	17 (4.12)	396 (95.88)
4	Vegetables				0.0018
	Unwashed vegetables	389	103 (26.48)	265 (68.12)
	Washed vegetables	344	32 (9.30)	333 (96.80)
5	Meat				0.0000
	Undercooked	272	123 (45.22)	149 (54.77)
	Well cooked	461	12 (2.06)	449 (97.39)
6	Eggs				0.0001
	Consumption raw eggs	263	81 (30.80)	142 (53.99)
	Consumption cooked eggs	470	54 (11.48)	456 (97.02)
7	Type of house				0.0000
	Mud house	331	114 (34.44)	217 (65.55)
	Concrete house	402	21 (5.22)	381 (94.77)
8	Education level				0.0000
	Primary	198	32 (16.16)	166 (83.83)
	Secondary	163	15 (9.20)	148 (90.79)
	College/university	89	5 (5.61)	84 (94.38)
	No education	283	83 (29.32)	200 (70.67)

The obtained data suggests that risk of congenital toxoplasmosis is related with gestational age, i.e. the sero-prevalence was found higher in the third trimester of pregnancy (31.28%), which is supported by the reports from Nigeria, where high prevalence was detected in third trimester of pregnancy (46.7%) [16] and a study from south-western Ethiopia also reported high prevalence of infection in the third trimester of pregnancy (86.8%).[7]

Among the three districts included in this study prevalence of the parasite was found higher in district Upper Dir (33.03%). Upper Dir is a rural area, contact of people with animals is frequent, and most of the people are living in mud-made houses, poor sanitation system and most importantly with low level of education.

Sero-prevalence of Toxoplasma IgG associated with various risk factors like contact with cats and cattle, water sources, consumption of unpasteurized milk, consumption of undercooked meat, consumption of raw vegetables, consumption of raw eggs and type of house were found statistically significant in this study. Various studies showed that use of raw or undercook meat increases chances of acquiring the parasite.[13,16,17]

This study showed that uneducated community is at high risk of acquiring T. gondii infection. Toxoplasmosis was found high (29.32%) in uneducated community and was statistically significant (P ˂ 0.05). Many researchers around the world also pointed out that low level of education increases the risk of acquiring the parasite.[16,18]

In the studied areas stray cats and dogs were significantly associated with high risk of infection as shown in Table 2. Cats actively participate in the spread of T. gondii infection and increase the prevalence of toxoplasmosis.[7,12,13,16,17] Studies from Nigeria and Turkey indicated that sero-prevalence of toxoplasmosis is associated with the consumption of eating raw vegetables, milk and eggs.[1,16]

Conclusion

From this study it was concluded that pregnant women are at high risk to Toxoplasmosis due to their education level, contact with the definitive feline host and poor sanitation system. Therefore, the awareness of childbearing age women is very necessary regarding the transmission and prevention of the disease. Control of the domestic and particularly stray cats should be very useful in limiting the transmission of infection. Sanitation system should be made hygienic in order to control the infection.

Disclosure statement

No potential conflict of interest was reported by the authors.

Additional information

Funding

This work was a part of a project, financially supported by Higher Education Commission Pakistan under the National Research Program for Universities (NRPUS) [grant number 840].