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Determination of trace elements, biochemical tests and epidemiological aspects of women infected with Toxoplasma gondii.

Introduction

Toxoplasmosis is a disease caused by the protozoan parasite Toxoplasma gondii, affecting a wide range of hosts--animals, and mammals including humans. (1) The more serious outcome of toxoplasmosis is congenital abnormalities (such as microcephaly, hydrocephaly, blindness and mental retardation) and abortion) in the early gestational period. (2)

Pregnancy is associated with increased demand of all the micronutrients such as iron, copper, zinc, vitamin B12, folic acid and ascorbic acid [3]. The deficiency of these nutrients could affect pregnancy, delivery and outcome of pregnancy. The four trace elements, namely Zinc, Manganese, Copper and Chromium, are necessary during pregnancy and these elements should be supplemented as a daily requirement in pregnant women. (4,5)

Zinc is essential for the growth and development of human life and has an active role in body function; its deficiency leads to dwarfism, hypogonadisim, and ataxia in neonate, in addition to skin lesions. (6) During pregnancy, zinc is needed for brain development in the fetus.

Copper, another important trace element, joins to many Cu-dependent enzymes such as lysoyl oxidase (extracellular matrix protein cross-linking), cytochrome c oxidase (energy production), tyrosinase, dopamine-[beta]-hydroxylase, peptidoglycine alpha-amidating mono-oxygenase, and monoamine oxidase (pigment and neurotransmitter production and metabolism); copper-zinc superoxide dismutase and cerulo-plasmin is an [alpha]-2, copper transporting globulin synthesized in liver microsomes and possesses ferroxidase activity. (7) It acts as an antioxidant in serum by oxidizing ferrous iron which could otherwise act as a catalyst in generating toxic free radicals. (8) Hypo-cupremia was recorded among women with toxoplasmosis recorded, (9) while hypercupremia was recorded during some bacterial and viral infections. (10)

Magnesium is a mineral that helps to form bone and cartilage. It is also a component of enzymes that play a role in the formation of carbohydrates, amino acids, and cholesterol. As part of a powerful antioxidant called manganese superoxide dismutase, magnesium helps to protect cells from damage. (11)

Chromium is a mineral that helps the body break down and store fats, carbohydrates, and protein. It also works with the hormone insulin to maintain normal levels. (12)

Iron is needed for additional erythrocyte production during pregnancy. It is estimated approximately 750-800 mg of elemental that iron is required for entire pregnancy. (13) This study was conducted to assess changes in some trace elements in sera of women with toxoplasmosis in Kirkuk province, Iraq.

Material and methods

The study was conducted on 991 women with abortions, attended to in private gynecological clinics with the cooperation of Ibn-Nafies private medical lab; they ranged in age from 15 to 45+ years. The study was done in Kirkuk province Iraq from January to December, 2009. Before blood sampling, information was taken from each patient; 5ml of venous blood was drawn , sera extracted and processed for serological diagnosis using direct agglutination test (DAT), semi-quantitative standard tube method, ELISA technique using IgM and IgG kits purchased from Bio kit company-Spain and Bio-tech company-USA. These tests were performed per kit instructions. (14)

According to the results of ELISA tests, positive samples for toxoplasma antibodies were chosen from the study group of about 122 samples and 68 negative samples for toxoplasma antibodies were taken as control group for biochemical assessments that involved: serum(S) iron, S. total iron binding capacity (TIBC), S. zinc, S.copper, S. magnesium, S. total protein, S. albumin and S. globulin. All biochemical kits were purchased from companies in Germany and the UK. The procedures were performed per instructions in the kits. None of the women in the toxoplasmosis group and control group was taking any medication or trace elements supplements. Statistical analysis using Student t-test was applied to assess the difference between the means, P<0.05.

Results

From the total of 991 sera examinations using ELISA IgM and IgG toxoplasma kits, only 166 (16.75%) were positive for toxoplasma anti-bodies, which included a high rate of toxoplasma IgG antibodies 11.21% followed by 4.33% and 1.21% for mixed antibodies of toxoplasma IgM and IgG.

Table 2 indicates that 166 positive sera for toxoplasma antibodies were distributed into the following rates 19,38[degrees]%, 17.58[degrees]%, 14.62% and 6.66% in sera of women ages15 to 45+, P<0.05

The rate of toxoplasma antibodies 22.56% was higher than that in rural areas where the rate was14.82%, P<0.05. Also toxoplasmas among non-pregnant and homemakers were high, 26.95% and 17.27% respectively, while they were 14.82% and 14.20% in pregnant women and working women sera respectively. The relationship between toxoplasma antibodies distribution and patient residency and women gestation was significant P<0.05 while it was non-significant by occupation, P.>0.05. Table 3. The same table reveals a high rate of toxoplasma antibodies 22.78% in autumn compared to 13.25% in spring <0.05%.

Table 4 shows the relationship between toxoplasma antibodies and abortion via which the toxoplasma rate in sera of women without abortion (21.64%) was higher in sera of women who had abortions (11.67%), P<0.05. Also, sera of women with a single abortion shows 26.60% of toxoplasma antibodies compared to 1.95% in women with twice or double abortions, P<0.05.

Table 5 summarizes trace elements concentration in blood of women with toxoplasmosis and abortion compared to women with abortion negative for toxoplasmosis. No statistically significant difference was found between the mean iron, magnesium, total protein and serum albumin. S. total iron binding capacity was significantly lower in blood of the test group, 243.77 [micro]g/dl as compared to 257.37 [micro]g/dl in the control group. Copper means in both group were low, but copper mean in the study group (67.97 [micro]g/dl) was significantly higher than in the control group (65.03 [micro]g/dl), P<0.05. Zinc levels in both test and control groups were low; significant statistical difference P<0.05 in the zinc level was observed in sera of the study group, 35.91 [micro]g/dl vs. the lower 38.02 [micro]g/dl in the control group. Serum globulin level was also low in the test group, 2.28 mg/dl compared to the slightly elevated 3.51 mg/dl in sera of the control group, but statistically, the relationship was significant P<0.05.

Discussion

The 16.75% rate of toxoplasma antibodies in the present study is high and reflects the degree of contamination of food and water with the infective stage of oocysts by cat feces. This rate is low when it was compared with those recorded in Baghdad, (15) in Mosul, (15,16) in Tikrit, (16) and in Kirkuk (17,18) where the following rates were recorded: 44%, 24% and 40.6, 33.4% and 52.3% 55.3% respectively. Variances in the results may be attributed to differences in the number of the specimens, type of method; source and type of toxoplasma kits or due to large numbers of specimens included in the present study. Classification of the positive cases by detecting IgM and IgG in the present study, the high rate of IgG antibodies means protection against previous toxoplasmosis. Chance of chronic toxoplasmosis can be excluded by increasing the level of IgG while IgM antibodies mean recent, primary or acute toxoplasmosis. Both antibodies IgM plus IgG in the same serum can be solved by rechecking for toxoplasmosis for both antibodies to exclude residual IgM if the result is negative for IgG antibodies. Distribution of cats in nature had a strong role in spreading the infective stage (oocyst) to the environment, but keeping cats in houses as domestic animals, especially in urban areas, can lead to more exposure to toxoplasmosis. This can explain why the rate of toxoplasmosis in the present study is higher in sera of people from urban areas than in rural areas. (20) The humoral response against toxoplasmosis can evoke strong IgG antibodies which had a role in the production of sterile immunity. (21) This conclusion explains the rate of toxoplasma antibodies in non-pregnant women; they definitely acquired the toxoplasma infection previously, so they were negative for toxoplasmosis, while their abortions might be due to other causes.

High incidence of toxoplasma antibodies during autumn can be attributed to the climate. Normally, the weather in Kirkuk province during autumn is such that oocysts shed later because of winds and consequently be transported from one region to another and leads to increasing the rate of toxoplasmosis.

High frequency of toxoplasma antibodies in sera of women with a single abortion can be interpreted by the fact that there were more women who participated in the study at a young age, so their immunity against infectious agents was not as strong as the older ages to fight against toxoplasma. Or the abortions of women in general may be due to other infections, hormonal changes or anatomical defects or deformities. (22)

Regarding zinc deficiency in the present study, a reduction in serum zinc in humans and animals was noted. (23, 24) This means zinc is necessary for immune function and plays a role in immune response against parasites. Because zinc is important for normal placental development, deficiency may result in impairment in utero of the acquisition of maternal antibodies by the fetus. Therefore, one of the potential beneficial approaches to improving the immune defense against T. gondii is zinc supplementation; due to its catalytic and regulatory functions, it can enhance resistance to infections. (25,26)

Hypo-cupremia in the present study was recorded in Kirkuk province, (9,24) while hypercupremia was recorded in Mosul. (23) Normally, copper is important for hemoglobin synthesis and the depletion may lead to improper iron bounding to globulins to form hemoglobin that resulted in iron store followed by increasing TIBC of transferrin, then after evoke, iron deficiency. This can explain why TIBC in this study was low. In addition, both groups in the present study lost much blood from abortion were suffered from abortion. Other explanations to low copper might be that most of the copper will convert to ceruloplasmin. (27) Hypo-gamma-globulinemia in the study group refers to a diminished immune system to produce sufficient antibodies, especially toxoplasma IgG, to prevent reactivation sequels that are obviously seen among women with toxoplasmosis by spontaneous or recurrent abortion. (28)

Main causes of sero-conversion of toxoplasma antibodies are mostly due to parasite challenges against a host immune system such as antigenic coat changing. Also, most of the bradyzoites are hidden inside the cysts that are enclosed by protective fibrotic tissue derived from the tissue of the host, so when the immune system is diminished, the bradyzoites will liberate and continue propagation again.

Conclusion

From the results, it can be concluded that the prevalence of toxoplasma antibodies among young age women in Kirkuk province is high especially in urban areas, in non-pregnant women, and during autumn. Serum copper, zinc, TIBC and globulin should be watched during toxoplasmosis and gestational periods.

References

(1.) Heybas SM, and Al-Mutawa SA. Immuno pathogenesis of Toxoplasmosis. Clinical Exp. Med. 2003; 3(2):89-105.

(2.) Rathore S, Gupta A, Singh Batra H, and Rathore R. Comparative study of trace elements and serum ceruloplasmin level in normal and pre-eclamptic pregnancies with their cord blood. Biomed. Research 2011; 22(2): 207-210

(3.) Black RE. Micronutrients in Pregnancy. Br J Nutr 2001; 85:193-197.

(4.) Kantola M, Purkunen R, Kroger, et al. Accumulation of calcium, zinc and copper in maternal blood and developmental placental tissue: differences between Finland Estonia and St. Petersburg. Envnono-Res 2000; 83:54-66.

(5.) Osendarp SJM, Raaij JMAV, Darmatadt GL, et al. reeZinc supplementation during pregnancy and effects on growth and mobidity in low birth weight infants: a randomized placebo controlled trial. Lancet 2001; 357:1080-5.11

(6.) Devlin TM. Text book of biochemistry with clinical correlations. 6th. edit. Wiley-Liss. 2006 USA:1143-1146.

(7.) Jain S, Sharma P, Kulsheshtha S, Mohan G, Singh S. The Role of Calcium, Magnesium, and Zinc in Pre-Eclampsia. Biol Trace Elem Res 2009; 22: 104-109.

(8.) Batra HS, Singh P, Somani BL, Gupta A, Sampath S, Ambade V. Serum ferroxidase albumin ratio as a marker in pulmonary tuberculosis. Ind. J Clinical Bio-chem 2007; 22: 106-108.

(9.) Mussttafa W GH .The changes in biochemical tests of women during toxoplasmosis in Kirkuk city. 2000. Professional Diploma in clinical lab. College of Medicine .Tikrit Univ.

(10.) KadiMA ;Amin ST and Majeed GK. Comparison between excretion of some metallic elements in normal and parasitized human. Tikrit Med. J. 1995;1:9-16.

(11.) Lou GS, Amirabi A, Yazdian M and Pashapour N. Evaluation of Serum calcium, magnesium, copper and zinc levels in women with pre-eclampsia. Iran. J. Med. Sci. 2008;33:231-234

(12.) Akinloye O; Oyewale O J and OguntibejuO. Evaluation of trace elements in pregnant women with pre-eclampsia. African J Biotechnolo. 2010; Vol. 9(32): 5196-5202.

(13.) National Research Council. Recommended Dietary Allowances. 10th ed. Washington, DC, National Academy Press 1989.

(14.) Ismaiel M S. (2012) Epidemiological and Immunological aspects falloja city Al-Anbar province-Iraq. MSc. thesis .College Sci. Tikrit Univ.

(15.) Niazi, A. D. Prevalence of toxoplasmosis in Iraq. Bull. Endemic. Disease. 1976;17 (1-4) :3-35.

(16.) Abdul-Ridah RR.bBiochemical changes in aborted toxoplasmosis serpositive women. M.Sc. thesis Al-Mustanseryia Univ. 2000.

(17.) Al-Timimi RL. Detecting of Toxoplasmosis among different groups of aborted women during gestational & age of pregnancy. Diploma thesis, college of medical & health technology, Baghdad Iraq. 2004.

(18.) Kadir MA, Abdulatief BM. and Jaftan A. Prevalence of toxoplasmosis among Women in kirkuk. Iraq. J. Fac. Med. Baghdad. 1992;34(3):279_285 .

(19.) Al-Attar Sh A(2000). Epidemiological study of toxoplasmosis in Kirkuk city. M.Sc. thesis, College of Education, Tikrit Univ. 2000.

(20.) Al-Jubori AM. Serological study of toxoplasmosis in Kirkuk province. M.Sc. thesis. College of Technology. Baghdad. Iraq. 2005.

(21.) Markell EK., John DR. , Krotosky W A. Markell & Voges. Medical parasitology. 8thed .W.B. Saunders. Company's USA 2006: 160-170.

(22.) Rock JA and Jones HW. The lindes operative gynecology. 10th edit. Lippincott-Williams and Wilkins USA.2008:776-780.

(23.) Al-Khashab EM and Albakry HS. Zinc and Copper Levels in Seropositive Toxoplasmosis Women in Mosul-Iraq J. Raf. Sci. 2009, Vol. 20(2): 37-41.

(24.) Ageel, NF. 2003. Serological and Biochemistry Study of Toxoplasmosis in TikritTeaching Hospital. M.Sc thesis College of Medicine, University of Tikrit, Iraq.

(25.) Mocchegiani E, Giacconi R. and Muzzioli R. Zinc, Infections and Immuno-senescence. Mech. Aging. Dev. 2001; 121: 21-35.

(26.) Avunduk A M; Avunduk MC; Baltaci A K and Mustafa C. A. Effect of Melatonin and Zinc on the Immune Response in Experimental Toxoplasma Retin-ochoroiditis. Ophthalmologica. 2007; 221: 421-425.

(27.) Denniston KJ, Topping JJ and Caret RL. General organic and biochemistry. 3rd.edit. McGraw-Hill. USA.2006 :57-60.

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Yahya Jirjees Salman, Ph.D., Assistant Professor of Microbiology, Dept. of Medical Microbiology and Immunology, College of Medicine-Kirkuk University, Iraq
Table 1--Incidence rate of toxoplasma antibodies
among patients attending antenatal clinics in 2009

                       No.         %         No.         %
Total No. exam       positive   positive   Negative   Negative

991                    166       16.75       825       83.25
Type of toxoplasma
Toxo-Igm                43        4.33
Toxo-IgG               111      11.21 *
ToxoIgM + ToxoIgG       12        1.21

* P < 0.05.

Table 2--Frequency of toxoplasma antibodies among
women ages 15-45+
                                 No.
Age groups/years   No. Exam.   Positive   % Positive

15 to 25              423         82        8.27 *
26 to 35              383         56         5.65
36 to 45              140         25         2.52
45 up to above        45          3          0.3
Total                 991        166        16.75

* P < 0.05

Table 3--Distribution of toxoplasma antibodies among women
by residency, gestation, occupation and seasons

                               Residency              Gestation

                                                  Non
Total Number Examined    Urban *     Rural    pregnant **   Pregnant

Number Positive             57        109         38          128
% positive                22.26      14.82       26.95       15.04
Total Number Examined      256        735         141         850
Seasons                   Autumn     Winter     Spring       Summer
Number Positive             54         44         33           35

% positive              22 78 ****   16.18       13.25       15.10

                              Occupations

                        Homemakers
Total Number Examined       ***       Officer

Number Positive             142         24
% positive                 17.27       14.20
Total Number Examined       822         169
Seasons                  * P<0.05
Number Positive          ** P<0.05
                        *** P>0.005
% positive              **** P<0.05

Table 4--Frequency of sero-positive toxoplasma
by number of abortions due to toxoplasmosis

                    Number
Abortion number    examined   Percentages

Single abortion      203         34.46
Double abortions     205         38.80
Triple abortions      96         16.29
Four abortions        46          7.80
Five abortions        39          6.62
Total abortions      589         59.43
Non abortion         402         40.73
All total            991        100.00

                      Number       Percentage
Abortion number    positive +ve   Positive +ve

Single abortion         54          26.60 *
Double abortions         4           1.95
Triple abortions        15          15.62
Four abortions           3           6.52
Five abortions           3           7.69
Total abortions         79          11.67
Non abortion            87          21.64 **
All total              166          16.75

*** P<0.05.

Table 5--Effect of Toxoplasma gondii infection on trace elements
and some biochemical tests in sera of women positive and negative
for toxoplasmosis.

                     Test group

Tests          No.             Values
              tested       M [+ or -] S.D

S. Iron        115        68.28 [+ or -] 0.82
S.TIBC         115     * 234.77 [+ or -] 1.33
Copper         120      * 67.97 [+ or -] 0.75
Magnesium      130         2.29 [+ or -] 123
Zinc           120      * 35.91 [+ or -] 0.54
T.S protein    102          7.0 [+ or -] 0.26
S.Albumin      110         4.12 [+ or -] 0.19
S.globulin     110       * 2.28 [+ or -] 0.16

                     Control group

Tests          No.            Values
              Tested      M [+ or -] S.D

S. Iron         68      79.04 [+ or -] 1.12
S.TIBC          68     257.37 [+ or -] 1.95
Copper          68      65.03 [+ or -] 0.98
Magnesium       68       2.23 [+ or -] 0.18
Zinc            68      38.02 [+ or -] 0.75
T.S protein     68        7.7 [+ or -] 1.08
S.Albumin       68       4.19 [+ or -] 0.69
S.globulin      68       3.51 [+ or -] 0.22

Tests             Normal values        Variances

S. Iron       65 to 176 [micro]g/dl      0.23
S.TIBC        262 to 450 [micro]g/dl     2.69
Copper        70 to 150 [micro]g/dl     12.63
Magnesium       1.5 to 2.3 mg/dl.        0.6
Zinc          60 to 110 [micro]g/dl     10.04
T.S protein      6.0 to 7.8 mg/dl        0.85
S.Albumin        3.5 to 5.2 mg/dl        0.04
S.globulin       2.3 to 3.5 mg/dl        10.5

TIBC: Total serum iron binding
capacity S=serum T=total * P<0.05
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Title Annotation:Article 412: 1 Clock Hour
Author:Salman, Yahya Jirjees
Publication:Journal of Continuing Education Topics & Issues
Article Type:Report
Date:Apr 1, 2014
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