- Email: [email protected]
A serological investigation and genotyping of Toxoplasma gondii among Iranian blood donors indicates threat to health of blood recipients
Journal Pre-proof A serological investigation and genotyping of Toxoplasma gondii among Iranian blood donors indicates threat to health of blood recipients Seyed Abdollah Hosseini, Ehsan Golchin, Mehdi Sharif, Shahabeddin Sarvi, Ehsan Ahmadpour, Alireza Rostamian, Sara Gholami, Afsaneh Amouei, Ahmad Daryani
PII:
S1473-0502(20)30006-9
DOI:
https://doi.org/10.1016/j.transci.2020.102723
Reference:
TRASCI 102723
To appear in:
Transfusion and Apheresis Science
Received Date:
21 October 2019
Revised Date:
11 December 2019
Accepted Date:
23 December 2019
Please cite this article as: Hosseini SA, Golchin E, Sharif M, Sarvi S, Ahmadpour E, Rostamian A, Gholami S, Amouei A, Daryani A, A serological investigation and genotyping of Toxoplasma gondii among Iranian blood donors indicates threat to health of blood recipients, Transfusion and Apheresis Science (2020), doi: https://doi.org/10.1016/j.transci.2020.102723
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier.
A serological investigation and genotyping of Toxoplasma gondii among Iranian blood donors indicates threat to health of blood recipients Seyed Abdollah Hosseini
a, b, 1
, Ehsan Golchin
a,1
, Mehdi Sharif c, Shahabeddin Sarvi
a, b
, Ehsan
Ahmadpour d, Alireza Rostamian e, Sara Gholami b, Afsaneh Amouei a, b, Ahmad Daryani a, b, *
Toxoplasmosis Research Center, Mazandaran University of Medical Science, Sari, Iran
b
Department of Parasitology, School of Medicine, Mazandaran University of Medical Science, Mazandaran, Sari,
of
a
Iran
Department of Parasitology, School of Medicine, Sari Branch, Islamic AZAD University, Sari, Iran
d
Infectious and Tropical Diseases Research Center, Tabriz University of Medical Sciences, Tabriz, Iran
e
Iranian Blood Transfusion Organization, Tehran, Iran
1
Co-first author
Professor of Medical Parasitology
-p re
*Correspondence: Prof. Ahmad Daryani,
ro
c
lP
Department of Medical Parasitology and Mycology 18 Km of Khazar Abad Road, School of Medicine, Mazandaran University of Medical Sciences PO Box 48175-1665, Sari, Iran
ur na
Phone +98 11 33543088 Fax: +98 11 33543249
Email: [email protected]
Jo
ABSTRACT Background: Toxoplasmosis is a zoonotic disease in animals and human caused by the intracellular obligatory protozoan named Toxoplasma gondii. The purpose of this study was to evaluate the sero-molecular prevalence and genotyping T. gondii among healthy blood donors in north of Iran. Methods: In this cross-sectional study, 400 blood donors participated from all Blood Transfusion Organization (BTO) in Mazandaran province during October and November 2014. The blood samples were investigated for seroprevalence, DNA detection and genotyping of T. gondii using ELISA, nested-PCR, and Multilocus nested-PCR-RFLP methods respectively. Results: Among all of blood donors, 294 (73.5%) and 9 (2.2%) cases were seropositive for anti-T. gondii IgG and IgM antibodies. T. gondii DNA was detected in 7 samples. Four genotype of T.
1
gondii were identified in blood donors samples (Genotype ToxoDB#1, #2, #10 and #27), which 50% of T. gondii strains were highly pathogenic. Conclusions: Taking into account survive T. gondii in blood transfusion bag, the high prevalence of T. gondii and existence of pathogenic genotypes in Iranian blood donors, it seems that T. gondii screening should be performed at the BTO to prevent complications of toxoplasmosis in blood recipients.
Jo
ur na
lP
re
-p
ro
of
Key words: Toxoplasma gondii, healthy blood donors, Genotype, Iran
2
1. Introduction Toxoplasmosis is a zoonotic disease and one of the most parasitic infections in animals and human caused by the intracellular protozoan named Toxoplasma gondii (T. gondii) [1, 2]. The infection usually transmitted to humans through ingestion of oocyst-contaminated food and water or eating tissue cysts of T. gondii in undercooked meat. Moreover, during pregnancy transmission from mother to fetus via the placenta has an important role in the infectious disease transmission [3-6].
of
In immunocompetent persons, clinical signs of toxoplasmosis are mild and self-limited including: fever, malaise and cervical lymphadenopathy. But the infection in immunocompromised individuals (such as AIDS patients) and blood recipients (thalassemia, hemophilia, dialysis, organ transplantation persons and neonatal jaundice) is severe and complications include; pneumonitis and encephalitis [7, 8]. Congenital toxoplasmosis, which occurs during pregnancy, can cause miscarriage, splenomegaly, microcephaly, hydrocephalus and chorioretinitis [6].
-p
ro
It is estimated that up to one-third of the world's human are infected with toxoplasmosis [9]. Although the infection occurs worldwide, the seropositivity of T. gondii can vary widely among different regions of the world. In some areas, more than 50% of blood donors are seropositive for toxoplasmosis such as north Brazil (75%) and Egypt (59/6%) [10, 11]. In the other regions, the seroprevalence is low, for example 7.4% in Mexico and 4.1% in North Thailand [12, 13].
ur na
lP
re
The genetic diversity of the pathogen parasite varies based on geographical districts and different hosts. Generally, T. gondii consists of three separate lineages (types I, II and III). Type I is highly virulent to mice. The type II and III are lowly virulent and prevalent throughout all continents. Type II is the most reason of human toxoplasmosis in both congenital infection and HIV positive patients in North America and Europe [14-16]. So far, precise approaches concerning the genetic features of T. gondii isolates from healthy blood donors are not available in Iran and world. Epidemiology and genotyping studies of T. gondii in the individuals could help reevaluate the population genetic structure, population biology, pathogenesis and identification of risk factors affecting the prevalence of this important zoonotic pathogen in Iran. Hence this study was aimed to survey the sero-molecular prevalence and genotyping of T. gondii among blood donors in north of Iran. 2. Material and methods 2.1. Study design
Jo
Mazandaran province is located near the Caspian Sea in north of Iran. This province occupies an area of 23756 km2 and has a population of 3073943. In this cross-sectional study, sampling was carried out from all of the nine blood transfusion organizations in Mazandaran including: Ramsar, Tonekabon, Chalus, Amol, Babol, Qaemshahr, Sari, Jouybar and Behshahr. 2014. The sample size to estimate prevalence was determined by the formula: 𝑛 = z (1-a/2)2*pq)/d2 considering a confidence level of 95% (𝑧 = 1.96), absolute precision of 5% (𝑑), and a prevalence of 75% (𝑝). The estimated prevalence was obtained from a pilot study that included 400 samples. We randomly collected blood samples from the healthy blood donors during October and November 2014. 2.2. Questionnaire
3
In this study, the questionnaire assessed basic demographic data, including; gender, age, blood group, Rh, residence (urban, rural), education level, blood transfusion, and occupation (business, employee, farmer, house wives, un-employee). Moreover, the major risk factors in the distribution of toxoplasmosis, including; raw vegetables and milk consumption, contact with garden soil, existence of cat in the neighborhood and undercooked meat and meat products consumption and sources of drinking water (municipal water, well water, bottle water, filtered water and boiled water) were surveyed. 2.3. Serological test
ro
of
Four ml venous blood was collected in sterile container from each the blood donor. After centrifuging at 3000 rpm for 3 min, the serum and buffy coat were separate and stored at -20°C until assayed. All the serum samples were investigated for T. gondii IgG and IgM antibodies using Enzyme Linked Immune Sorbent Assay (ELISA) kit (Euroimmun Medizinische Labordiagnostika AG, Germany) according to the manufacture's protocol. All the blood samples were routinely examined for Hepatitis B virus (HBV), human immunodeficiency virus (HIV), Hepatitis C virus (HCV) and Terponema palidom (Syphilis) before using for transfusion.
-p
2.4. DNA Extraction and conventional PCR
ur na
lP
re
DNA was extracted from the buffy coat of all blood samples using the blood DNA extraction kit (Dena Zist, Iran) according to the manufacturer’s instructions. Conventional PCR was carried out to amplify a 529 bp fragment of the RE gene(TOX4: 5`-CGCTGCAGGGAGGAAGACGAAAGTTG`,TOX5:5`CGCTGCAGACACAGTGCATCTGGATT-3`).Conventional PCR was performed in a volume of25 µl, containing 100 ng of genomic DNA, and 1X PCR mix (Taq PCR MasterMix, Amplicon), 0.2 mM external primers. PCR conditions were as follows: initial denaturation for 7 min at 94 °C, 30 cycles of denaturing 30 seconds at 94°C, annealing for 30 seconds at 56°C, extension for 45 seconds at 72°C; final extension for 7 min at 72°C (BioRad, USA). PCR products was subjected to electrophoresis on a 1.5% agarose gel in a Tris-boric acid-EDTA (TBE) buffer at 90 V for 20 min and visualized with ultraviolet transilluminator after staining with Syber safe stain (green viewer). Positive (T. gondii DNA) and negative (without DNA template) controls were run with every PCR batch.
Jo
2.5. Genotyping of T. gondii in positive DNA samples DNA samples that were positive for RE gene marker in conventional PCR were genotyped by multiplex-multilocus-nested-PCR-RFLP using the subsequently genetic markers SAG1, SAG2, SAG3, BTUB, GRA6, C22-8, C29-2, L358, PK1,Apico and CS3 [17-22]. Briefly, multiplex PCR was carried out by a set of mixed external primers in a single reaction. Then, the products were diluted 1:1 with nuclease-free water, and used for multilocus nested PCR amplifications with internal primers for each gene marker, respectively. For each gene marker, the PCR mixture consisted of 12.5 μl PCR Premix (Taq PCR MasterMix, Amplicon), 5 μM forward and reverse primers, and 1.5 μl multiplex PCR-generated products in a 25μl reaction volume. The multilocus nested PCR was performed with an annealing temperature at 60°C for 60s for all the genes except BTUB and Apico that was carried out in 58 °C. The final products were digested using restriction endonucleases (New England Biolabs Ipswich, Massachusetts) specific for each genetic marker according to the manufacturer’s guidelines (Table 1). The restriction fragments were imagined by electrophoresis using a 2.5% to 3% agarose gel and photographed by a gel documentation system. 4
2.6. Phylogenetic analysis The Splits Tree software 4 was used for phylogenetic analysis [23]. For phylogenetic reconstruction between genotypes, genotypic data of restriction polymorphism obtained by PCR-RFLP in this study and others previously isolated in the world (which is available at https://toxodb.org/) were transformed into binary data and tabulated then were analyzed. 2.7. Statistical analysis The results were analyzed using the SPSS – 16.0 software package (SPSS Inc, Chicago, IL, USA). P-value less than 0.05 were considered statistically significant. Chi squared and Fisher exact tests were used to compare relation between seroprevalence of T. gondii and the risk factors.
of
3. Results
ro
A total of the 400 healthy blood donors, 381 (95.25%) and 19 (4.75%) participants were male and female, respectively. The mean age were 35.5 years (Rang: 18 to 60 years old). The overall 294 (73.5%) and 9 (2.2%) individuals were seropositive for anti-T. gondii IgG and IgM antibodies, respectively (7 samples of 9 IgM+ were IgG+). 7 blood donors (1.8 %) were seropositive for both IgG and IgM. Titers IgG in positive samples ranged from 31 to 324 IU/ml.
re
-p
The seroprevalence rate of IgG was 72.96 % and 84.21% in men and women, respectively. "O" blood group was the most frequent (37.1%) and "AB" was the least frequent blood group (10.2%) among blood donors (P>0.05). By geographic region, Sari city had the highest seroprevalence rate of T. gondii (82.35%), followed by Amole (81.42%), Tonekabon (80%), Babol (70%), Jouybar (70%), Qaemshahr (68%), Chalus (66.66%), Ramsar (60%) and Behshahr had the lowest seroprevalence rate of T. gondii (56%).
ur na
lP
The seroprevalence of T. gondii based on details of demographic data are summarized in Table 2. Association between risk factors and seropositivity (P value), odds ratio (OR) and 95% CI for univariate analysis are summarized in Table 3. Some risk factors significantly associated with T. gondii seropositivity including; education level (P= 0.01), age (P<0.001), occupation (P=0.02), existence of cat in the neighborhood (P=0.01) and residence (P=0.00). There was no significant relation between the seroprevalence of T. gondii and gender, blood group, Rh, water consumption, blood transfusion, contact with meat and garden soil, animal contact, raw milk consumption, undercooked meat, raw vegetables consumption and washing vegetables (P>0.05).
Jo
All of the samples were initially screened for the four mandatory tests, HIV, HBV, HCV and RPR (rapid plasma regain/syphilis antibodies).In these samples, only two, one and one person showed a positive HBV, HCV and RPR screening test, respectively. In molecular analysis, Toxoplasma DNA was detected in 2 of 7 IgM+/IgG+, 1 of 2 IgM+/IgG-, and 4 of 287 IgM-/IgG+ samples. Of 7 PCR positive samples 4 cases showed complete genotyping results. Four patterns of T. gondii strain were identified in these samples including genotype ToxoDB#1 (Type II clonal), #2 (Type III clonal), #10 (Type I clonal) and #27 (Type I variant). The results of genotyping of T. gondii genomic DNA from humans were illustrated in Table 4. The results of phylogenic analysis revealed that 2 isolates of T. gondii (50%) were classified in phylogeny group 1, one isolate (25%) in phylogeny group 2, and one other isolate (25%) in phylogeny groups 3 (Fig. 1). 4. Discussion 5
Jo
ur na
lP
re
-p
ro
of
This study is important to understand the situation of seroprevalence and molecular epidemiology and determine predominance genotype of T. gondii in the healthy blood donors in north of Iran. Toxoplasmosis can be transmitted to transplant recipients (heart, renal and bone marrow transplant) via blood products [24], therefore it is important to survey T. gondii in healthy blood donors. The present study is the first sero-molecular epidemiologic study of T. gondii infection among blood donors in Mazandaran province, Iran. Prevalence rates of T. gondii among different groups of this province are very high. For example, prevalence rate of T. gondii was 74.6% in women referred to medical health laboratory before marriage [25], 75.6% in AIDS / HIV positive patients [16] and 58.8% in pregnant women [27]. In our study, the prevalence rate of chronic toxoplasmosis (IgG positive) was 73.5 % in the samples. Also, 2.2 % of the blood donors were seropositive for acute toxoplasmosis (IgM positive) and 1.7 % of them were positive for both IgG and IgM. These rates of seroprevalence in healthy blood donors are very high and similar to prevalence that reported from northeast Brazil by Coêlho et al [10]. It is more prevalent than other countries including; Egypt (59.6%), Kenya (54%), Saudi Arabia (52.1%), North India (51.8%), Czech Republic (32%), Iraq (30%), South India (20.3%), Turkish (19.5%), South Iran (12.3%), Taiwan (9.3%), Mexico (7.4%) and North East Thailand (4.1%) [7, 11-13, 28-34]. The level of Toxoplasma infection in different areas depends on different factors such as geographical location and weather conditions, age, host immunity, living location, job, education level, contact with cats and genotype of parasite. The geographical location and weather conditions such as changes in temperature and humidity are effective for survive of oocysts (resistant stage of T. gondii) that excreted by cats. T. gondii is more prevalent in humid areas in comparison to dry areas. The infection risk increases when the weather is both warm and humid, or moderated and less humid [34]. The high prevalence of T. gondii in north of Iran, could be due to many factors, including; the high humidity (up to approximately 90 percent), environmental temperature (18-20°C), existence a lot of cats, and consuming free-range poultry and raw or undercooked meat in this area. Data analysis showed that the prevalence of Toxoplasmasignificantly increased with age. This could be due to increased risk of contact to infection sources with age. These results are in accordance with the results of Sarkari et al and Sunder et al and in contrast to the result of Zghair et al [28, 31, 36]. Our analysis showed that the most rate of toxoplasmosis among blood groups was seen in ‘B’ blood group (77.9%) which is similar to the study done by Mahmoudvand et al and in contrast to the results of Sarkari et al and Chiang et al [31, 33, 34]. Also, the level of infection between Rh positive (73.31%) and Rh negative (74.57%) samples was not different in contrast to result of Sarkari et al ‘study [ 36]. In this present study, the seropositivity of T. gondii increased with decreasing education level. 84.26% of under-diploma individuals were seropositive. We found a significant correlation between education level and seroprevalence of T. gondii. It is agreement with other studies such as Chiang et al and Mahmoudvand et al [34, 37]. This result could be due to a lack of adequate awareness about personal and social hygiene in this group. The rate of infection among housewives (87.5%) was more than other jobs in this study. There is a significant relationship between job and rate of the infection. Our results are accordance with those of studies done by Al-Qurashi et al and Jones et al [38, 39]. The high prevalence of infection in housewives is might be due to spend more time cooking at home, clean vegetables, handle and chop meat without gloves and taste undercooked meat. 6
Jo
ur na
lP
re
-p
ro
of
Among our study groups, the presence of cat in the neighborhood is a significant risk factor for toxoplasmosis. It is similar to reports presented in other countries [34, 40, 41]. Its reason is that cats are a main sources of the infection. They excrete millions of oocysts and sporulated oocysts can survive for months in moist environments. Besides, mechanical transmission by beetles and flies can spread the oocysts from the soil to food. In regard to abundance of oocysts in the environment, prey animals could become infected to toxoplasmosis, which could be lead to increase the infection among the animal. Moreover, during hunting of small mammals, cats may be infected with tissue cysts. Cats can contaminate the water, gardens, farms, and food through excretion of a large amount of oocysts. Accordingly, human can be infected through the contaminated food and water. Consumption of undercooked meat products is a source of Toxoplasma transmission. Transmission of toxoplasmosis through meat depends on nutritional and cultural habits of individuals in special districts or countries [42, 43]. In our study, there was a significant relationship between the prevalence of T. gondii and consumption of raw/undercooked meat products. It is known that tissue cysts of Toxoplasma may survive several weeks between 1 and 4°C and can be inactivated only in the processes over 67°C or below -12°C [44]. Therefore, lack of hygienic preparation of meat products (such as sausages, salami and hamburger) and eating raw/undercooked meat products, serves as a route of disease transmission. Statistical analysis in our study showed that the high seropositivity of T. gondii significantly was showed among persons living in rural areas (83.03%). The reason may be due to abundance of cats, poor environmental sanitation and low standards hygiene. It is similar to the results of Zarkovic et al [41]. In current study, there was no significant difference in Toxoplasma seroprevalence between males and females in the present study. However, seroprevalence of this parasite in females (84.21%) was higher than males (72.96%). These findings are similar to the study conducted by Elhence et al and in contrast to the results of studies done by Zarkovic et al and Abdolghani et al [7, 41, 45]. Similar to other studies, there wasn’t any significant relationship between prevalence rate of T. gondii and consumption of raw/undercooked meat, milk, vegetable, water and contact with garden soil in the present study [33, 35]. Moreover, in this study we investigated the molecular diagnosis of T. gondii using nested PCR. Toxoplasma DNA was detected in 7 cases of 294 seropositive samples and 6/294 (2%) persons with IgM-& IgG+ antibodies. The presence of Toxoplasma DNA in 7 cases of 294 seropositive samples (2.38%) can prove the transmission of the infection through blood transfusion. Unlike the chronic infection, which is characterized by tissue cysts and an absence of parasites in blood circulation, in acute infection, however, small quantities of T. gondii may present in blood stream and hence it detectable. In this infection, the initial spreading of tachyzoites is commonly restricted to less than twenty day’s duration nevertheless this could vary according to the genotype of T. gondii and the host immune reaction [9]. The ability to detect T. gondii DNA in clinical samples made it possible to directly genotype the detected isolates without the need to harvest the T. gondii [16, 46]. In current study, for assessment of T. gondii genotypes, a Multiplex nested PCR–RFLP was used to improve the investigative yields. The sensitivity of this technique is assessed to be 10-genome equivalent each PCR [17], only a little of clinical specimens can be genotyped. Among the 7 clinical DNA samples studied, 4 cases (57.1%) were genotyped. In this investigation, the genotyping of the finding revealed a total of four different genotypes of T. gondii, including ToxoDB genotypes #1, #2, #10, and #27. ToxoDB genotype #1 or type II of 7
ur na
lP
re
-p
ro
of
T. gondii is the most abundant in different host in all of the world. ToxoDB genotype #2 or Type III is almost common in five continents and most countries [19]. ToxoDB genotype #10 or type I of T. gondii is a high pathogenicity strain and the most frequent genotype isolated from Asia, after genotype ToxoDB # 9; however, this genotype has never been reported in Africa, so far [21]. Moreover, ToxoDB genotype #27 or type I variant of T. gondii, at the first time, was reported from cat and bird in South America by Pena et al. (2008) [22]. Overall, the analysis of the multilocus nested PCR-RFLP showed four pattern of genotypes, suggesting relatively high diversity of the T. gondii isolated among the blood donors population in north of Iran. This is the first report of the T. gondii genotypes in blood donors using multilocus PCR-RFLP. The phylogenetic networks presented in Fig. 1 show that the 50% of the samples belonged to group 1, 25% to group 2 and 25% to group 3, respectively. In fact, genotype isolated in the current study is close to clonal genotype type I, which is the most pathogenic type of T. gondii. One of the strengths of this investigation is the use of the Mn-PCR-RFLP technique by 12 gene markers compared to the studies using less than 5 gene markers for the determination of T. gondii genotypes. At the same time, applying bioassay and isolation of Toxoplasma parasite could be helpful for future studies to better understand the virulence status. Our findings provide information about sero-molecular epidemiology and genetic characterization of T. gondii infection among healthy blood donors in north of Iran. Taking into account the high prevalence of T. gondii in this area and survive the parasite in blood transfusion bag, it seems that it is necessary to screen T. gondii in addition to HIV, Hepatitis B, C and Syphilis at the BTO. Our study on genetic characterization of T. gondii in the healthy blood donors showed that there is the most pathogenic genotype in these individuals. Since blood recipients are patients that use immunosuppressive drugs, special measures should be taken to prevent the acute toxoplasmosis and serious consequences. Ethical approval The study protocol was reviewed and approved by the Research Ethics Committee of Mazandaran University of Medical Sciences (No. Ethical approval of MUMS 471). Conflicts of interest The authors declare no conflicts of interest.
Jo
Acknowledgments This study was prepared from E. Golchin 's MSc thesis and supported by grant (No. 471) from the deputy of research, Mazandaran University of medical sciences, Sari, Iran. Moreover, this study was supported by Elite Researcher Grant Committee under award number [963443] from the National Institutes for Medical Research Development (NIMAD), Tehran, Iran which we gratefully acknowledge.
8
References
Jo
ur na
lP
re
-p
ro
of
[1] Hill D, Dubey J. Toxoplasma gondii: transmission, diagnosis and prevention. Clin Microbiol Infect 2002;8:634-640. https://doi.org/10.1046/j.1469-0691.2002.00485.x [2] Daryani A, Sarvi S, Aarabi M, Mizani A, Ahmadpour E, Shokri A, et al. Seroprevalence of Toxoplasma gondii in the Iranian general population: A systematic review and meta-analysis. Acta trop 2014;137:185-194. https://doi.org/10.1016/j.actatropica.2014.05.015 [3] Pappas G, Roussos N, Falagas ME. Toxoplasmosis snapshots: global status of Toxoplasma gondii seroprevalence and implications for pregnancy and congenital toxoplasmosis. Int J Parasitol 2009;39:1385-94. https://doi.org/10.1016/j.ijpara.2009.04.003 [4] Sukthana Y. Toxoplasmosis: beyond animals to humans. Trends Parasitol 2006;22:137142. https://doi.org/10.1016/j.pt.2006.01.007 [5] Arefkhah N, Hosseini SA, Karimzade R, Moshfe A, Hadinia F, Larki RA, Mozaffari MA, Hadinia A. Seroprevalence and risk factors of Toxoplasma gondii infection among Cancer and Hemodialysis Patients in southwest Iran. Clin Epidemiol Glob Health 2019; 30. https://doi.org/10.1016/j.cegh.2019.01.007 [6] Sarvi S, Chegeni TN, Sharif M, Montazeri M, Hosseini SA, Amouei A, et al. Congenital toxoplasmosis among Iranian neonates: a systematic review and meta-analysis. Epidemiol Health 2019;41. https://doi.org/10.4178/epih.e2019021 [7] Elhence P, Agarwal P, Prasad KN, et al. Seroprevalence of Toxoplasma gondii antibodies in North Indian blood donors: implications for transfusion transmissible toxoplasmosis. Transfus Apher Sci 2010;43:37-40. https://doi.org/10.1016/j.transci.2010.05.004 [8] Arefkhah N, Pourabbas B, Asgari Q, Moshfe A, Mikaeili F, Nikbakht G, Sarkari B. Molecular genotyping and serological evaluation of Toxoplasma gondii in mothers and their spontaneous aborted fetuses in Southwest of Iran. Comp Immunol Microbiol Infect Dis 2019;1:66:101342. https://doi.org/10.1016/j.cimid.2019.101342 [9] Robert-Gangneux F, Dardé M-L. Epidemiology of and diagnostic strategies for toxoplasmosis. Clin Microbiol Rev 2012;25:264-96. https://doi.org/10.1128/CMR.05013-11 [10] Coêlho RA, Kobayashi M, Carvalho Jr LB. Prevalence of IgG antibodies specific to Toxoplasma gondii among blood donors in Recife, Northeast Brazil. Rev Inst Med Trop Sao Paulo 2003;45:229-231. http://dx.doi.org/10.1590/S0036-46652003000400011 [11] Elsheikha H, Aboul-Dahab MA, Abdel Maboud AI, et al. Prevalence and risk factors of Toxoplasma gondii antibodies in asymptomatic Egyptian blood donors. J Egypt Soc Parasitol 2009;39:351-361. [12] Alvarado-Esquivel C, Mercado-Suarez MF, Rodríguez-Briones A, et al. Seroepidemiology of infection with Toxoplasma gondii in healthy blood donors of Durango, Mexico. BMC Infect Dis 2007;7:75. https://doi.org/10.1186/1471-2334-7-75 [13] Pinlaor S, Ieamviteevanich K, Pinlaor P, Maleewong W, Pipitgool V. Seroprevalence of specific total immunoglobulin (Ig), IgG and IgM antibodies to Toxoplasma gondii in blood
9
Jo
ur na
lP
re
-p
ro
of
donors from Loei Province, Northeast Thailand. Southeast Asian J Trop Med Public Health 2000;31:123-127. [14] Ajzenberg D, Cogné N, Paris L, Bessières M-H, Thulliez P, Filisetti D, et al. Genotype of 86 Toxoplasma gondii isolates associated with human congenital toxoplasmosis, and correlation with clinical findings. J Infect Dis 2002;186:684-689. https://doi.org/10.1086/342663 [15] Sharif M, Amouei A, Sarvi S, Mizani A, Aarabi M, Hosseini S-A, et al. Genetic diversity of Toxoplasma gondii isolates from ruminants: a systematic review. Int J. Food Microbiol 2017; 258:38-49. https://doi.org/10.1016/j.ijfoodmicro.2017.07.007 [16] Hosseini S, Amouei A, Sharif M, Sarvi S, Galal L, Javidnia J, et al. Human toxoplasmosis: a systematic review for genetic diversity of Toxoplasma gondii in clinical samples. Epidemiol Infect 2019;147. https://doi.org/10.1017/S0950268818002947 [17] Grigg ME, Ganatra J, Boothroyd JC, Margolis TP. Unusual abundance of atypical strains associated with human ocular toxoplasmosis. J Infect Dis 2001;184:633-9. https://doi.org/10.1086/322800 [18] Howe DK, Honoré S, Derouin F, Sibley LD. Determination of genotypes of Toxoplasma gondii strains isolated from patients with toxoplasmosis. J Clin Microbiol 1997;35:1411-4. [19] Khan A, Taylor S, Su C, Mackey AJ, Boyle J, Cole R, Glover D, Tang K, Paulsen IT, Berriman M, Boothroyd JC. Composite genome map and recombination parameters derived from three archetypal lineages of Toxoplasma gondii. Nucleic Acids Res 2005;33:2980-92. https://doi.org/10.1093/nar/gki604 [20] Su C, Zhang X, Dubey JP. Genotyping of Toxoplasma gondii by multilocus PCR-RFLP markers: a high resolution and simple method for identification of parasites. Int J Parasitol 2006;36:841-8. https://doi.org/10.1016/j.ijpara.2006.03.003 [21] Su C, Shwab E, Zhou P, Zhu X, Dubey J. Moving towards an integrated approach to molecular detection and identification of Toxoplasma gondii. Parasitology 2010;137:1-11. https://doi.org/10.1017/S003118200999106 [22] Pena HF, Gennari SM, Dubey JP, Su C. Population structure and mouse-virulence of Toxoplasma gondii in Brazil. Int J Parasitol 2008;38:561-9. https://doi.org/10.1016/j.ijpara.2007.09.004 [23] Huson DH, Bryant D. Estimating phylogenetic trees and networks using SplitsTree 4. Manuscript in preparation, software available from www splitstree org. 2005. [24] Wreghitt T, Hakim M, Gray J, Balfour A, Stovin P, Stewart S, et al. Toxoplasmosis in heart and heart and lung transplant recipients. J Clin Pathol 1989;42:194-9. https://doi.org/10.1136/jcp.42.2.194 [25] Sharif M, Ajami A, Daryani A, Ziaei H, Khalilian A. Serological survey of toxoplasmosis in women referred to medical health laboratory before marriage northern Iran 2003-2004. Int J Mol Med Adv Sci. 2006;2:134-7 [26] Daryani A, Sharif M, Meigouni M. Seroprevalence of IgG and IgM anti—Toxoplasma antibodies in HIV/AIDS patients, northern Iran. Asian Pac J Trop Dis 2011;4:271-4. https://doi.org/10.1016/S1995-7645(11)60084-9 10
ro
of
[27] Hosseini S, A. Dehgani, N. Sharif, M. et al. Serological Survey of Toxoplasmosis in Pregnant Women. J Mazand Univ Med Sci 2014; 24:146-150. [28] Sundar P, Mahadevan A, Jayshree RS, Subbakrishna DK, Shankar SK. Toxoplasma seroprevalence in healthy voluntary blood donors from urban Karnataka. Indian J Med Res 2007;126: 50. [29] Al-Amari O. Prevalence of antibodies to Toxoplasma gondii among blood donors in Abha, Asir Region, south-western Saudi Arabia. The Journal of the Egyptian Public Health Association 1994;69:77-88. [30] Svobodova V, Literak I. Prevalence of IgM and IgG antibodies to Toxoplasma gondii in blood donors in the Czech Republic. Eur J Epidemiol 1998;14:803-805. https://doi.org/10.1023/A:1007589422080 [31] Zghair KH, AL-Qadhi BN, Mahmood SH. The effect of toxoplasmosis on the level of some sex hormones in males blood donors in Baghdad. Journal of Parasitic Diseases 2015; 39:393400.https://doi.org/10.1007/s12639-013-0382-6
Jo
ur na
lP
re
-p
[32] Griffin L, Williams KA. Serological and parasitological survey of blood donors in Kenya for toxoplasmosis. Trans R Soc Trop Med Hyg 1983;77:763-766. https://doi.org/10.1016/00359203(83)90283-3 [33] Yazar S, Eser B, Yay M. Prevalence of anti-Toxoplasma gondii antibodies in Turkish blood donors. Ethiop Med J 2006;44:257-61. [34] Chiang T, Hsieh, HH.,Kuo, MC.,et al. Seroepidemiology of Toxoplasma gondii Infection among healthy blood donors in Taiwan. PLOS one 2012;7:p.e48139. https://doi.org/10.1371/journal.pone.0048139. [35] Meerburg BG, Kijlstra A. Changing climate—changing pathogens: Toxoplasma gondii in North-Western Europe. Parasitol Res 2009;105:17-24. https://doi.org/10.1007/s00436-0091447-4 [36] Sarkari B, Shafiei R, Zare M, Sohrabpour S, Kasraian L. Seroprevalence and molecular diagnosis of Toxoplasma gondii infection among blood donors in southern Iran. J Infect Dev Ctries 2014;8:543-547. https://doi.org/10.3855/jidc.3831 [37] Mahmoudvand H, Saedi Dezaki E, Soleimani S, Baneshi MR, Kheirandish F, Ezatpour B, et al. Seroprevalence and risk factors of Toxoplasma gondii infection among healthy blood donors in southeast of Iran. Parasite Immunol 2015;37:362-367. https://doi.org/10.1111/pim.12198 [38] Al-Qurashi AR, Ghandour AM, Obeid O, Al-Mulhim A, Makki SM.Seroepidemiological study of Toxoplasma gondii infection in the human population in the Eastern Region. Saudi medical journal. 2001; 22:13-18. [39] Jones JL, Kruszon-Moran D, Wilson M, McQuillan G, Navin T, McAuley JB. Toxoplasma gondii infection in the United States: seroprevalence and risk factors. Am J Epidemiol 2001;154:357-365. https://doi.org/10.1093/aje/154.4.357
11
[40] Munsoor MM, Ahmed IM. Sero-prevalence of toxoplasmosis in blood donors in Khartoum State using latex agglutination test. SUST Journal of Science and Technology, 2012;13:3844 [41] Zarkovic A, MacMurray C, Deva N, Ghosh S, Whitley D, Guest S. Seropositivity rates for Bartonella henselae, Toxocara canis and Toxoplasma gondii in New Zealand blood donors. Clin Exp Ophthalmol 2007;35:131-134. https://doi.org/10.1111/j.1442-9071.2006.01406.x
Jo
ur na
lP
re
-p
ro
of
[42] Tenter AM, Heckeroth AR, Weiss LM. Toxoplasma gondii: from animals to humans. Int J Parasitol 2001;31:217-220. https://doi.org/10.1016/S0020-7519(00)00124-7 [43] Dubey J, Jones J. Toxoplasma gondii infection in humans and animals in the United States. Int J Parasitol 2008; 38: 1257-1278. https://doi.org/10.1016/j.ijpara.2008.03.007 [44] Aspinall TV, Marlee D, Hyde JE, Sims PF. Prevalence of Toxoplasma gondii in commercial meat products as monitored by polymerase chain reaction–food for thought? Int J Parasitol 2002;32:1193-1199. https://doi.org/10.1016/S0020-7519(02)00070-X [45] Abdülgani F. Prevalence of IgM and IgG Antibodies to Toxoplasma gondii in Blood Donors in the North Region of Jordan. Journal of Inonu University Medical Faculty 2010;11:14346. [46] Wang L, He L-y, Chen Z-w, Wen H, Fang G-s, Luo Q-l, et al. Seroprevalence and genetic characterization of Toxoplasma gondii in cancer patients in Anhui Province, Eastern China. Parasit Vectors 2015;8:162. https://doi.org/10.1186/s13071-015-0778-5
12
Legend of figures
Jo
ur na
lP
re
-p
ro
of
Fig. 1 NeighborNet phylogenetic network of Toxoplasma gondii isolates in blood donors from northern Iran.
13
Table 1 Summary of external and internal primers and enzymes for multilocus nested PCR-RFLP genotyping of Toxoplasma gondii. Markers
Multiplex PCR primers (external primers)
Nested PCR primers (internal primers)
Nested PCR (bp)
Restriction enzymes, incubation temperature and time
Ref.
SAG1
F: GTTCTAACCACGCACCCTGAG R: AAGAGTGGGAGGCTCTGTGA Not needed. The DNA fragment for 5´-SAG2 is covered by the external primers of alt. SAG2.
F: CAATGTGCACCTGTAGGAAGC R: GTGGTTCTCCGTCGGTGTGAG F: GAAATGTTTCAGGTTGCTGC R: GCAAGAGCGAACTTGAACAC
390
Sau96I+HaeII, .37 0C 1 h, 2.5% gel. MboI, 37 0C 1 h,2.5% gel.
[17]
F: TCTGTTCTCCGAAGTGACTCC R: TCAAAGCGTGCATTATCGC F: GGAACGCGAACAATGAGTTT R: GCACTGTTGTCCAGGGTTTT
F: ATTCTCATGCCTCCGCTTC R: AACGTTTCACGAAGGCACAC F: ACCCATCTGCGAAGAAAACG R: ATTTCGACCAGCGGGAGCAC
222
[18]
F: CAACTCTCACCATTCCACCC R: GCGCGTTGTTAGACAAGACA F: TCCAAAATGAGAGAAATCGT R: AAATTGAAATGACGGAAGAA F: ATTTGTGTTTCCGAGCAGGT R: GCACCTTCGCTTGTGGTT F: TGATGCATCCATGCGTTTAT R: CCTCCACTTCTTCGGTCTCA
F: TCTTGTCGGGTGTTCACTCA R: CACAAGGAGACCGAGAAGGA F: GAGGTCATCTCGGACGAACA R: TTGTAGGAACACCCGGACGC F: TTTCCGAGCAGGTGACCT R: TCGCCGAAGAGTTGACATAG F: TCTCTCTACGTGGACGCC R:AGGTGCTTGGATATTCGC
225
HhaI, 37 0C 1 h, 2.5% gel. HinfI+TaqI, 37 0C 30 min, 65 0C 30 min. 2.5% gel. NciI, 37 0C 1 h, 2.5% gel.
[19]
F: ACCCACTGAGCGAAAAGAAA R: AGGGTCTCTTGCGCATACAT F: TCTCTCGACTTCGCCTCTTC R: GCAATTTCCTCGAAGACAGG F: GAAAGCTGTCCACCCTGAAA R: AGAAAGCTCCGTGCAGTGAT F: TGGTTTTAACCCTAGATTGTGG R: AAACGGAATTAATGAGATTTGAA GTGTATCTCCGAGGGGGTCT TGTGACTTCTTCGCATCGAC
F: AGTTCTGCAGAGTGTCGC R:TGTCTAGGAAAGAGGCGC F: AGGAGGCGTAGCGCAAGT R: CCCTCTGGCTGCAGTGCT F: CGCAAAGGGAGACAATCAGT R: TCATCGCTGAATCTCATTGC F: GCAAATTCTTGAATTCTCAGTT R: GGGATTCGAACCCTTGATA AGCGGATTTCCAACACTGTC CTGCTGCATTCACAAACTCC
BsiEI+TaqI, 60 0C 1 h, 2.5% gel. MseI, 37 0C 1 h, 2.5% gel. BsmAI+MboII, NEB2, BSA, 37 0C 30 min, 55 0C 30min. 2.5% gel HpyCH4IV+RsaI, 37 0C 1 h, 2.5% gel. HaeIII+NlaIII, 37 0C 1 h, 2.5% gel. AvaI+RsaI, 37 0C 1 h, 2.5% gel. AflII+DdeI, 37 0C 1 h, 3% gel. N1aIII and MboI at 37 0C for 60 min
C22-8
C29-2 L358 PK1 Apico CS3
of
546
14
[18]
[19]
ro
GRA6
ur na
BTUB
Jo
SAG3
lP
alt. SAG2
242
[17]
521
[19]
411 344
-p
3´-SAG2
re
5´-SAG2
446 418 903 640 557
[19]
[20] [19, 20] [19, 20] [19, 20] [21, 46]
Table 2 Demographic characteristics of the 400 healthy blood donor in northern Iran. Characteristics
No. blood donors
No. seropositive donors (%)
P-value
Donation Center 10 25 45 70 80 50 85 10
6 20 30 57 56 3 70 7
(60) (80) (66.66) (81.42) (70) (68) (82.35) (70)
25
14
(56)
Male Female
381
278
(72.96)
19
16
≤30 31-39 ≥40
142 122
87 95
136
112
(82.35)
O A B AB
154 119 86
109 88 67
(70.77) (73.94) (77.90)
30
(73.17)
341
250
(73.31)
59
44
(74.57)
108
91
(84.26)
292
203
(69.52)
316 46 8
236 36 7
(74.68) (78.26) (87.50)
30
15
(50)
288 112
20 93
(69.79) (83.03)
Positive Negative
Occupation Business & Employee Farmer & Worker Housewives Student
Urban Rural
Jo
Residence
(84.21) (61.3) (77.9)
-p
ur na
Education Level Under diploma Upper diploma
lP
Rh
re
Blood group
41
ro
Gender
Age
15
0.09
of
Ramsar Tonekabon Chaloos Amol Babol Qaemshahr Sari Joubar Behshahr
0.27
0.000*
0.69
0.83
0.000*
0.01*
0.000*
Table 3
Statistical analysis ofthe seroprevalence ofToxoplasma gondii and the risk factors in blood donors in northern Iran Blood donor No.
No. Seropositive (%) (78.20) (66.86)
0.01*
147 253
108 186
(73.46) (73.51)
0.99
66 374
48 375
(73.07) (73.52)
0.96
378 22
280 14
239 49 73 16 23
184 31 54 11 14
ur na Jo
(74.07) (63.63)
148 52 94
125
0.28
(76.98) (63.26) (73.97) (68.75) (60.86)
0.18
(71.15) (76.47) (75.8)
0.53
ro
-p
re 218
of
183 111
182
16
P-value
234 166
208 68 124
lP
Variable Existence of cat in the neighborhood Yes No Undercooked meat consumption Yes No Raw milk consumption Yes No Raw vegetable consumption Yes No Water consumption Municipal Mineral Filtered Well Boiled Contact with garden soil No Yes with glove Yes without glove Consumption of raw/undercooked meat Products Yes No
(68.68) 0.04*
Table 4 Genotypes of Toxoplasma gondii in blood donors in the Mazandaran province, northern Iran ID
SAG1
5+3SAG2
altSAG2
SAG3
BTUB
GRA6
C228
C292
L358
PK
Apico
CS3
Toxo-DB Genotype
TgBDIr-6
IIorIII
II
II
II
II
II
II
II
II
II
II
II
#1
TgBDIr-19
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
-
TgBDIr-29
I
I
I
I
I
I
I
III
I
I
I
I
#27
TgBDIr-65
IIorIII
III
III
III
III
III
III
III
III
III
III
III
#2
I
I
I
I
I
I
I
I
I
I
I
I
#10
TgBDIr-194
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
-
TgBDIr-227
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
-
Jo
ur na
lP
re
-p
ro
of
TgBDIr-316
17
PII:
S1473-0502(20)30006-9
DOI:
https://doi.org/10.1016/j.transci.2020.102723
Reference:
TRASCI 102723
To appear in:
Transfusion and Apheresis Science
Received Date:
21 October 2019
Revised Date:
11 December 2019
Accepted Date:
23 December 2019
Please cite this article as: Hosseini SA, Golchin E, Sharif M, Sarvi S, Ahmadpour E, Rostamian A, Gholami S, Amouei A, Daryani A, A serological investigation and genotyping of Toxoplasma gondii among Iranian blood donors indicates threat to health of blood recipients, Transfusion and Apheresis Science (2020), doi: https://doi.org/10.1016/j.transci.2020.102723
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier.
A serological investigation and genotyping of Toxoplasma gondii among Iranian blood donors indicates threat to health of blood recipients Seyed Abdollah Hosseini
a, b, 1
, Ehsan Golchin
a,1
, Mehdi Sharif c, Shahabeddin Sarvi
a, b
, Ehsan
Ahmadpour d, Alireza Rostamian e, Sara Gholami b, Afsaneh Amouei a, b, Ahmad Daryani a, b, *
Toxoplasmosis Research Center, Mazandaran University of Medical Science, Sari, Iran
b
Department of Parasitology, School of Medicine, Mazandaran University of Medical Science, Mazandaran, Sari,
of
a
Iran
Department of Parasitology, School of Medicine, Sari Branch, Islamic AZAD University, Sari, Iran
d
Infectious and Tropical Diseases Research Center, Tabriz University of Medical Sciences, Tabriz, Iran
e
Iranian Blood Transfusion Organization, Tehran, Iran
1
Co-first author
Professor of Medical Parasitology
-p re
*Correspondence: Prof. Ahmad Daryani,
ro
c
lP
Department of Medical Parasitology and Mycology 18 Km of Khazar Abad Road, School of Medicine, Mazandaran University of Medical Sciences PO Box 48175-1665, Sari, Iran
ur na
Phone +98 11 33543088 Fax: +98 11 33543249
Email: [email protected]
Jo
ABSTRACT Background: Toxoplasmosis is a zoonotic disease in animals and human caused by the intracellular obligatory protozoan named Toxoplasma gondii. The purpose of this study was to evaluate the sero-molecular prevalence and genotyping T. gondii among healthy blood donors in north of Iran. Methods: In this cross-sectional study, 400 blood donors participated from all Blood Transfusion Organization (BTO) in Mazandaran province during October and November 2014. The blood samples were investigated for seroprevalence, DNA detection and genotyping of T. gondii using ELISA, nested-PCR, and Multilocus nested-PCR-RFLP methods respectively. Results: Among all of blood donors, 294 (73.5%) and 9 (2.2%) cases were seropositive for anti-T. gondii IgG and IgM antibodies. T. gondii DNA was detected in 7 samples. Four genotype of T.
1
gondii were identified in blood donors samples (Genotype ToxoDB#1, #2, #10 and #27), which 50% of T. gondii strains were highly pathogenic. Conclusions: Taking into account survive T. gondii in blood transfusion bag, the high prevalence of T. gondii and existence of pathogenic genotypes in Iranian blood donors, it seems that T. gondii screening should be performed at the BTO to prevent complications of toxoplasmosis in blood recipients.
Jo
ur na
lP
re
-p
ro
of
Key words: Toxoplasma gondii, healthy blood donors, Genotype, Iran
2
1. Introduction Toxoplasmosis is a zoonotic disease and one of the most parasitic infections in animals and human caused by the intracellular protozoan named Toxoplasma gondii (T. gondii) [1, 2]. The infection usually transmitted to humans through ingestion of oocyst-contaminated food and water or eating tissue cysts of T. gondii in undercooked meat. Moreover, during pregnancy transmission from mother to fetus via the placenta has an important role in the infectious disease transmission [3-6].
of
In immunocompetent persons, clinical signs of toxoplasmosis are mild and self-limited including: fever, malaise and cervical lymphadenopathy. But the infection in immunocompromised individuals (such as AIDS patients) and blood recipients (thalassemia, hemophilia, dialysis, organ transplantation persons and neonatal jaundice) is severe and complications include; pneumonitis and encephalitis [7, 8]. Congenital toxoplasmosis, which occurs during pregnancy, can cause miscarriage, splenomegaly, microcephaly, hydrocephalus and chorioretinitis [6].
-p
ro
It is estimated that up to one-third of the world's human are infected with toxoplasmosis [9]. Although the infection occurs worldwide, the seropositivity of T. gondii can vary widely among different regions of the world. In some areas, more than 50% of blood donors are seropositive for toxoplasmosis such as north Brazil (75%) and Egypt (59/6%) [10, 11]. In the other regions, the seroprevalence is low, for example 7.4% in Mexico and 4.1% in North Thailand [12, 13].
ur na
lP
re
The genetic diversity of the pathogen parasite varies based on geographical districts and different hosts. Generally, T. gondii consists of three separate lineages (types I, II and III). Type I is highly virulent to mice. The type II and III are lowly virulent and prevalent throughout all continents. Type II is the most reason of human toxoplasmosis in both congenital infection and HIV positive patients in North America and Europe [14-16]. So far, precise approaches concerning the genetic features of T. gondii isolates from healthy blood donors are not available in Iran and world. Epidemiology and genotyping studies of T. gondii in the individuals could help reevaluate the population genetic structure, population biology, pathogenesis and identification of risk factors affecting the prevalence of this important zoonotic pathogen in Iran. Hence this study was aimed to survey the sero-molecular prevalence and genotyping of T. gondii among blood donors in north of Iran. 2. Material and methods 2.1. Study design
Jo
Mazandaran province is located near the Caspian Sea in north of Iran. This province occupies an area of 23756 km2 and has a population of 3073943. In this cross-sectional study, sampling was carried out from all of the nine blood transfusion organizations in Mazandaran including: Ramsar, Tonekabon, Chalus, Amol, Babol, Qaemshahr, Sari, Jouybar and Behshahr. 2014. The sample size to estimate prevalence was determined by the formula: 𝑛 = z (1-a/2)2*pq)/d2 considering a confidence level of 95% (𝑧 = 1.96), absolute precision of 5% (𝑑), and a prevalence of 75% (𝑝). The estimated prevalence was obtained from a pilot study that included 400 samples. We randomly collected blood samples from the healthy blood donors during October and November 2014. 2.2. Questionnaire
3
In this study, the questionnaire assessed basic demographic data, including; gender, age, blood group, Rh, residence (urban, rural), education level, blood transfusion, and occupation (business, employee, farmer, house wives, un-employee). Moreover, the major risk factors in the distribution of toxoplasmosis, including; raw vegetables and milk consumption, contact with garden soil, existence of cat in the neighborhood and undercooked meat and meat products consumption and sources of drinking water (municipal water, well water, bottle water, filtered water and boiled water) were surveyed. 2.3. Serological test
ro
of
Four ml venous blood was collected in sterile container from each the blood donor. After centrifuging at 3000 rpm for 3 min, the serum and buffy coat were separate and stored at -20°C until assayed. All the serum samples were investigated for T. gondii IgG and IgM antibodies using Enzyme Linked Immune Sorbent Assay (ELISA) kit (Euroimmun Medizinische Labordiagnostika AG, Germany) according to the manufacture's protocol. All the blood samples were routinely examined for Hepatitis B virus (HBV), human immunodeficiency virus (HIV), Hepatitis C virus (HCV) and Terponema palidom (Syphilis) before using for transfusion.
-p
2.4. DNA Extraction and conventional PCR
ur na
lP
re
DNA was extracted from the buffy coat of all blood samples using the blood DNA extraction kit (Dena Zist, Iran) according to the manufacturer’s instructions. Conventional PCR was carried out to amplify a 529 bp fragment of the RE gene(TOX4: 5`-CGCTGCAGGGAGGAAGACGAAAGTTG`,TOX5:5`CGCTGCAGACACAGTGCATCTGGATT-3`).Conventional PCR was performed in a volume of25 µl, containing 100 ng of genomic DNA, and 1X PCR mix (Taq PCR MasterMix, Amplicon), 0.2 mM external primers. PCR conditions were as follows: initial denaturation for 7 min at 94 °C, 30 cycles of denaturing 30 seconds at 94°C, annealing for 30 seconds at 56°C, extension for 45 seconds at 72°C; final extension for 7 min at 72°C (BioRad, USA). PCR products was subjected to electrophoresis on a 1.5% agarose gel in a Tris-boric acid-EDTA (TBE) buffer at 90 V for 20 min and visualized with ultraviolet transilluminator after staining with Syber safe stain (green viewer). Positive (T. gondii DNA) and negative (without DNA template) controls were run with every PCR batch.
Jo
2.5. Genotyping of T. gondii in positive DNA samples DNA samples that were positive for RE gene marker in conventional PCR were genotyped by multiplex-multilocus-nested-PCR-RFLP using the subsequently genetic markers SAG1, SAG2, SAG3, BTUB, GRA6, C22-8, C29-2, L358, PK1,Apico and CS3 [17-22]. Briefly, multiplex PCR was carried out by a set of mixed external primers in a single reaction. Then, the products were diluted 1:1 with nuclease-free water, and used for multilocus nested PCR amplifications with internal primers for each gene marker, respectively. For each gene marker, the PCR mixture consisted of 12.5 μl PCR Premix (Taq PCR MasterMix, Amplicon), 5 μM forward and reverse primers, and 1.5 μl multiplex PCR-generated products in a 25μl reaction volume. The multilocus nested PCR was performed with an annealing temperature at 60°C for 60s for all the genes except BTUB and Apico that was carried out in 58 °C. The final products were digested using restriction endonucleases (New England Biolabs Ipswich, Massachusetts) specific for each genetic marker according to the manufacturer’s guidelines (Table 1). The restriction fragments were imagined by electrophoresis using a 2.5% to 3% agarose gel and photographed by a gel documentation system. 4
2.6. Phylogenetic analysis The Splits Tree software 4 was used for phylogenetic analysis [23]. For phylogenetic reconstruction between genotypes, genotypic data of restriction polymorphism obtained by PCR-RFLP in this study and others previously isolated in the world (which is available at https://toxodb.org/) were transformed into binary data and tabulated then were analyzed. 2.7. Statistical analysis The results were analyzed using the SPSS – 16.0 software package (SPSS Inc, Chicago, IL, USA). P-value less than 0.05 were considered statistically significant. Chi squared and Fisher exact tests were used to compare relation between seroprevalence of T. gondii and the risk factors.
of
3. Results
ro
A total of the 400 healthy blood donors, 381 (95.25%) and 19 (4.75%) participants were male and female, respectively. The mean age were 35.5 years (Rang: 18 to 60 years old). The overall 294 (73.5%) and 9 (2.2%) individuals were seropositive for anti-T. gondii IgG and IgM antibodies, respectively (7 samples of 9 IgM+ were IgG+). 7 blood donors (1.8 %) were seropositive for both IgG and IgM. Titers IgG in positive samples ranged from 31 to 324 IU/ml.
re
-p
The seroprevalence rate of IgG was 72.96 % and 84.21% in men and women, respectively. "O" blood group was the most frequent (37.1%) and "AB" was the least frequent blood group (10.2%) among blood donors (P>0.05). By geographic region, Sari city had the highest seroprevalence rate of T. gondii (82.35%), followed by Amole (81.42%), Tonekabon (80%), Babol (70%), Jouybar (70%), Qaemshahr (68%), Chalus (66.66%), Ramsar (60%) and Behshahr had the lowest seroprevalence rate of T. gondii (56%).
ur na
lP
The seroprevalence of T. gondii based on details of demographic data are summarized in Table 2. Association between risk factors and seropositivity (P value), odds ratio (OR) and 95% CI for univariate analysis are summarized in Table 3. Some risk factors significantly associated with T. gondii seropositivity including; education level (P= 0.01), age (P<0.001), occupation (P=0.02), existence of cat in the neighborhood (P=0.01) and residence (P=0.00). There was no significant relation between the seroprevalence of T. gondii and gender, blood group, Rh, water consumption, blood transfusion, contact with meat and garden soil, animal contact, raw milk consumption, undercooked meat, raw vegetables consumption and washing vegetables (P>0.05).
Jo
All of the samples were initially screened for the four mandatory tests, HIV, HBV, HCV and RPR (rapid plasma regain/syphilis antibodies).In these samples, only two, one and one person showed a positive HBV, HCV and RPR screening test, respectively. In molecular analysis, Toxoplasma DNA was detected in 2 of 7 IgM+/IgG+, 1 of 2 IgM+/IgG-, and 4 of 287 IgM-/IgG+ samples. Of 7 PCR positive samples 4 cases showed complete genotyping results. Four patterns of T. gondii strain were identified in these samples including genotype ToxoDB#1 (Type II clonal), #2 (Type III clonal), #10 (Type I clonal) and #27 (Type I variant). The results of genotyping of T. gondii genomic DNA from humans were illustrated in Table 4. The results of phylogenic analysis revealed that 2 isolates of T. gondii (50%) were classified in phylogeny group 1, one isolate (25%) in phylogeny group 2, and one other isolate (25%) in phylogeny groups 3 (Fig. 1). 4. Discussion 5
Jo
ur na
lP
re
-p
ro
of
This study is important to understand the situation of seroprevalence and molecular epidemiology and determine predominance genotype of T. gondii in the healthy blood donors in north of Iran. Toxoplasmosis can be transmitted to transplant recipients (heart, renal and bone marrow transplant) via blood products [24], therefore it is important to survey T. gondii in healthy blood donors. The present study is the first sero-molecular epidemiologic study of T. gondii infection among blood donors in Mazandaran province, Iran. Prevalence rates of T. gondii among different groups of this province are very high. For example, prevalence rate of T. gondii was 74.6% in women referred to medical health laboratory before marriage [25], 75.6% in AIDS / HIV positive patients [16] and 58.8% in pregnant women [27]. In our study, the prevalence rate of chronic toxoplasmosis (IgG positive) was 73.5 % in the samples. Also, 2.2 % of the blood donors were seropositive for acute toxoplasmosis (IgM positive) and 1.7 % of them were positive for both IgG and IgM. These rates of seroprevalence in healthy blood donors are very high and similar to prevalence that reported from northeast Brazil by Coêlho et al [10]. It is more prevalent than other countries including; Egypt (59.6%), Kenya (54%), Saudi Arabia (52.1%), North India (51.8%), Czech Republic (32%), Iraq (30%), South India (20.3%), Turkish (19.5%), South Iran (12.3%), Taiwan (9.3%), Mexico (7.4%) and North East Thailand (4.1%) [7, 11-13, 28-34]. The level of Toxoplasma infection in different areas depends on different factors such as geographical location and weather conditions, age, host immunity, living location, job, education level, contact with cats and genotype of parasite. The geographical location and weather conditions such as changes in temperature and humidity are effective for survive of oocysts (resistant stage of T. gondii) that excreted by cats. T. gondii is more prevalent in humid areas in comparison to dry areas. The infection risk increases when the weather is both warm and humid, or moderated and less humid [34]. The high prevalence of T. gondii in north of Iran, could be due to many factors, including; the high humidity (up to approximately 90 percent), environmental temperature (18-20°C), existence a lot of cats, and consuming free-range poultry and raw or undercooked meat in this area. Data analysis showed that the prevalence of Toxoplasmasignificantly increased with age. This could be due to increased risk of contact to infection sources with age. These results are in accordance with the results of Sarkari et al and Sunder et al and in contrast to the result of Zghair et al [28, 31, 36]. Our analysis showed that the most rate of toxoplasmosis among blood groups was seen in ‘B’ blood group (77.9%) which is similar to the study done by Mahmoudvand et al and in contrast to the results of Sarkari et al and Chiang et al [31, 33, 34]. Also, the level of infection between Rh positive (73.31%) and Rh negative (74.57%) samples was not different in contrast to result of Sarkari et al ‘study [ 36]. In this present study, the seropositivity of T. gondii increased with decreasing education level. 84.26% of under-diploma individuals were seropositive. We found a significant correlation between education level and seroprevalence of T. gondii. It is agreement with other studies such as Chiang et al and Mahmoudvand et al [34, 37]. This result could be due to a lack of adequate awareness about personal and social hygiene in this group. The rate of infection among housewives (87.5%) was more than other jobs in this study. There is a significant relationship between job and rate of the infection. Our results are accordance with those of studies done by Al-Qurashi et al and Jones et al [38, 39]. The high prevalence of infection in housewives is might be due to spend more time cooking at home, clean vegetables, handle and chop meat without gloves and taste undercooked meat. 6
Jo
ur na
lP
re
-p
ro
of
Among our study groups, the presence of cat in the neighborhood is a significant risk factor for toxoplasmosis. It is similar to reports presented in other countries [34, 40, 41]. Its reason is that cats are a main sources of the infection. They excrete millions of oocysts and sporulated oocysts can survive for months in moist environments. Besides, mechanical transmission by beetles and flies can spread the oocysts from the soil to food. In regard to abundance of oocysts in the environment, prey animals could become infected to toxoplasmosis, which could be lead to increase the infection among the animal. Moreover, during hunting of small mammals, cats may be infected with tissue cysts. Cats can contaminate the water, gardens, farms, and food through excretion of a large amount of oocysts. Accordingly, human can be infected through the contaminated food and water. Consumption of undercooked meat products is a source of Toxoplasma transmission. Transmission of toxoplasmosis through meat depends on nutritional and cultural habits of individuals in special districts or countries [42, 43]. In our study, there was a significant relationship between the prevalence of T. gondii and consumption of raw/undercooked meat products. It is known that tissue cysts of Toxoplasma may survive several weeks between 1 and 4°C and can be inactivated only in the processes over 67°C or below -12°C [44]. Therefore, lack of hygienic preparation of meat products (such as sausages, salami and hamburger) and eating raw/undercooked meat products, serves as a route of disease transmission. Statistical analysis in our study showed that the high seropositivity of T. gondii significantly was showed among persons living in rural areas (83.03%). The reason may be due to abundance of cats, poor environmental sanitation and low standards hygiene. It is similar to the results of Zarkovic et al [41]. In current study, there was no significant difference in Toxoplasma seroprevalence between males and females in the present study. However, seroprevalence of this parasite in females (84.21%) was higher than males (72.96%). These findings are similar to the study conducted by Elhence et al and in contrast to the results of studies done by Zarkovic et al and Abdolghani et al [7, 41, 45]. Similar to other studies, there wasn’t any significant relationship between prevalence rate of T. gondii and consumption of raw/undercooked meat, milk, vegetable, water and contact with garden soil in the present study [33, 35]. Moreover, in this study we investigated the molecular diagnosis of T. gondii using nested PCR. Toxoplasma DNA was detected in 7 cases of 294 seropositive samples and 6/294 (2%) persons with IgM-& IgG+ antibodies. The presence of Toxoplasma DNA in 7 cases of 294 seropositive samples (2.38%) can prove the transmission of the infection through blood transfusion. Unlike the chronic infection, which is characterized by tissue cysts and an absence of parasites in blood circulation, in acute infection, however, small quantities of T. gondii may present in blood stream and hence it detectable. In this infection, the initial spreading of tachyzoites is commonly restricted to less than twenty day’s duration nevertheless this could vary according to the genotype of T. gondii and the host immune reaction [9]. The ability to detect T. gondii DNA in clinical samples made it possible to directly genotype the detected isolates without the need to harvest the T. gondii [16, 46]. In current study, for assessment of T. gondii genotypes, a Multiplex nested PCR–RFLP was used to improve the investigative yields. The sensitivity of this technique is assessed to be 10-genome equivalent each PCR [17], only a little of clinical specimens can be genotyped. Among the 7 clinical DNA samples studied, 4 cases (57.1%) were genotyped. In this investigation, the genotyping of the finding revealed a total of four different genotypes of T. gondii, including ToxoDB genotypes #1, #2, #10, and #27. ToxoDB genotype #1 or type II of 7
ur na
lP
re
-p
ro
of
T. gondii is the most abundant in different host in all of the world. ToxoDB genotype #2 or Type III is almost common in five continents and most countries [19]. ToxoDB genotype #10 or type I of T. gondii is a high pathogenicity strain and the most frequent genotype isolated from Asia, after genotype ToxoDB # 9; however, this genotype has never been reported in Africa, so far [21]. Moreover, ToxoDB genotype #27 or type I variant of T. gondii, at the first time, was reported from cat and bird in South America by Pena et al. (2008) [22]. Overall, the analysis of the multilocus nested PCR-RFLP showed four pattern of genotypes, suggesting relatively high diversity of the T. gondii isolated among the blood donors population in north of Iran. This is the first report of the T. gondii genotypes in blood donors using multilocus PCR-RFLP. The phylogenetic networks presented in Fig. 1 show that the 50% of the samples belonged to group 1, 25% to group 2 and 25% to group 3, respectively. In fact, genotype isolated in the current study is close to clonal genotype type I, which is the most pathogenic type of T. gondii. One of the strengths of this investigation is the use of the Mn-PCR-RFLP technique by 12 gene markers compared to the studies using less than 5 gene markers for the determination of T. gondii genotypes. At the same time, applying bioassay and isolation of Toxoplasma parasite could be helpful for future studies to better understand the virulence status. Our findings provide information about sero-molecular epidemiology and genetic characterization of T. gondii infection among healthy blood donors in north of Iran. Taking into account the high prevalence of T. gondii in this area and survive the parasite in blood transfusion bag, it seems that it is necessary to screen T. gondii in addition to HIV, Hepatitis B, C and Syphilis at the BTO. Our study on genetic characterization of T. gondii in the healthy blood donors showed that there is the most pathogenic genotype in these individuals. Since blood recipients are patients that use immunosuppressive drugs, special measures should be taken to prevent the acute toxoplasmosis and serious consequences. Ethical approval The study protocol was reviewed and approved by the Research Ethics Committee of Mazandaran University of Medical Sciences (No. Ethical approval of MUMS 471). Conflicts of interest The authors declare no conflicts of interest.
Jo
Acknowledgments This study was prepared from E. Golchin 's MSc thesis and supported by grant (No. 471) from the deputy of research, Mazandaran University of medical sciences, Sari, Iran. Moreover, this study was supported by Elite Researcher Grant Committee under award number [963443] from the National Institutes for Medical Research Development (NIMAD), Tehran, Iran which we gratefully acknowledge.
8
References
Jo
ur na
lP
re
-p
ro
of
[1] Hill D, Dubey J. Toxoplasma gondii: transmission, diagnosis and prevention. Clin Microbiol Infect 2002;8:634-640. https://doi.org/10.1046/j.1469-0691.2002.00485.x [2] Daryani A, Sarvi S, Aarabi M, Mizani A, Ahmadpour E, Shokri A, et al. Seroprevalence of Toxoplasma gondii in the Iranian general population: A systematic review and meta-analysis. Acta trop 2014;137:185-194. https://doi.org/10.1016/j.actatropica.2014.05.015 [3] Pappas G, Roussos N, Falagas ME. Toxoplasmosis snapshots: global status of Toxoplasma gondii seroprevalence and implications for pregnancy and congenital toxoplasmosis. Int J Parasitol 2009;39:1385-94. https://doi.org/10.1016/j.ijpara.2009.04.003 [4] Sukthana Y. Toxoplasmosis: beyond animals to humans. Trends Parasitol 2006;22:137142. https://doi.org/10.1016/j.pt.2006.01.007 [5] Arefkhah N, Hosseini SA, Karimzade R, Moshfe A, Hadinia F, Larki RA, Mozaffari MA, Hadinia A. Seroprevalence and risk factors of Toxoplasma gondii infection among Cancer and Hemodialysis Patients in southwest Iran. Clin Epidemiol Glob Health 2019; 30. https://doi.org/10.1016/j.cegh.2019.01.007 [6] Sarvi S, Chegeni TN, Sharif M, Montazeri M, Hosseini SA, Amouei A, et al. Congenital toxoplasmosis among Iranian neonates: a systematic review and meta-analysis. Epidemiol Health 2019;41. https://doi.org/10.4178/epih.e2019021 [7] Elhence P, Agarwal P, Prasad KN, et al. Seroprevalence of Toxoplasma gondii antibodies in North Indian blood donors: implications for transfusion transmissible toxoplasmosis. Transfus Apher Sci 2010;43:37-40. https://doi.org/10.1016/j.transci.2010.05.004 [8] Arefkhah N, Pourabbas B, Asgari Q, Moshfe A, Mikaeili F, Nikbakht G, Sarkari B. Molecular genotyping and serological evaluation of Toxoplasma gondii in mothers and their spontaneous aborted fetuses in Southwest of Iran. Comp Immunol Microbiol Infect Dis 2019;1:66:101342. https://doi.org/10.1016/j.cimid.2019.101342 [9] Robert-Gangneux F, Dardé M-L. Epidemiology of and diagnostic strategies for toxoplasmosis. Clin Microbiol Rev 2012;25:264-96. https://doi.org/10.1128/CMR.05013-11 [10] Coêlho RA, Kobayashi M, Carvalho Jr LB. Prevalence of IgG antibodies specific to Toxoplasma gondii among blood donors in Recife, Northeast Brazil. Rev Inst Med Trop Sao Paulo 2003;45:229-231. http://dx.doi.org/10.1590/S0036-46652003000400011 [11] Elsheikha H, Aboul-Dahab MA, Abdel Maboud AI, et al. Prevalence and risk factors of Toxoplasma gondii antibodies in asymptomatic Egyptian blood donors. J Egypt Soc Parasitol 2009;39:351-361. [12] Alvarado-Esquivel C, Mercado-Suarez MF, Rodríguez-Briones A, et al. Seroepidemiology of infection with Toxoplasma gondii in healthy blood donors of Durango, Mexico. BMC Infect Dis 2007;7:75. https://doi.org/10.1186/1471-2334-7-75 [13] Pinlaor S, Ieamviteevanich K, Pinlaor P, Maleewong W, Pipitgool V. Seroprevalence of specific total immunoglobulin (Ig), IgG and IgM antibodies to Toxoplasma gondii in blood
9
Jo
ur na
lP
re
-p
ro
of
donors from Loei Province, Northeast Thailand. Southeast Asian J Trop Med Public Health 2000;31:123-127. [14] Ajzenberg D, Cogné N, Paris L, Bessières M-H, Thulliez P, Filisetti D, et al. Genotype of 86 Toxoplasma gondii isolates associated with human congenital toxoplasmosis, and correlation with clinical findings. J Infect Dis 2002;186:684-689. https://doi.org/10.1086/342663 [15] Sharif M, Amouei A, Sarvi S, Mizani A, Aarabi M, Hosseini S-A, et al. Genetic diversity of Toxoplasma gondii isolates from ruminants: a systematic review. Int J. Food Microbiol 2017; 258:38-49. https://doi.org/10.1016/j.ijfoodmicro.2017.07.007 [16] Hosseini S, Amouei A, Sharif M, Sarvi S, Galal L, Javidnia J, et al. Human toxoplasmosis: a systematic review for genetic diversity of Toxoplasma gondii in clinical samples. Epidemiol Infect 2019;147. https://doi.org/10.1017/S0950268818002947 [17] Grigg ME, Ganatra J, Boothroyd JC, Margolis TP. Unusual abundance of atypical strains associated with human ocular toxoplasmosis. J Infect Dis 2001;184:633-9. https://doi.org/10.1086/322800 [18] Howe DK, Honoré S, Derouin F, Sibley LD. Determination of genotypes of Toxoplasma gondii strains isolated from patients with toxoplasmosis. J Clin Microbiol 1997;35:1411-4. [19] Khan A, Taylor S, Su C, Mackey AJ, Boyle J, Cole R, Glover D, Tang K, Paulsen IT, Berriman M, Boothroyd JC. Composite genome map and recombination parameters derived from three archetypal lineages of Toxoplasma gondii. Nucleic Acids Res 2005;33:2980-92. https://doi.org/10.1093/nar/gki604 [20] Su C, Zhang X, Dubey JP. Genotyping of Toxoplasma gondii by multilocus PCR-RFLP markers: a high resolution and simple method for identification of parasites. Int J Parasitol 2006;36:841-8. https://doi.org/10.1016/j.ijpara.2006.03.003 [21] Su C, Shwab E, Zhou P, Zhu X, Dubey J. Moving towards an integrated approach to molecular detection and identification of Toxoplasma gondii. Parasitology 2010;137:1-11. https://doi.org/10.1017/S003118200999106 [22] Pena HF, Gennari SM, Dubey JP, Su C. Population structure and mouse-virulence of Toxoplasma gondii in Brazil. Int J Parasitol 2008;38:561-9. https://doi.org/10.1016/j.ijpara.2007.09.004 [23] Huson DH, Bryant D. Estimating phylogenetic trees and networks using SplitsTree 4. Manuscript in preparation, software available from www splitstree org. 2005. [24] Wreghitt T, Hakim M, Gray J, Balfour A, Stovin P, Stewart S, et al. Toxoplasmosis in heart and heart and lung transplant recipients. J Clin Pathol 1989;42:194-9. https://doi.org/10.1136/jcp.42.2.194 [25] Sharif M, Ajami A, Daryani A, Ziaei H, Khalilian A. Serological survey of toxoplasmosis in women referred to medical health laboratory before marriage northern Iran 2003-2004. Int J Mol Med Adv Sci. 2006;2:134-7 [26] Daryani A, Sharif M, Meigouni M. Seroprevalence of IgG and IgM anti—Toxoplasma antibodies in HIV/AIDS patients, northern Iran. Asian Pac J Trop Dis 2011;4:271-4. https://doi.org/10.1016/S1995-7645(11)60084-9 10
ro
of
[27] Hosseini S, A. Dehgani, N. Sharif, M. et al. Serological Survey of Toxoplasmosis in Pregnant Women. J Mazand Univ Med Sci 2014; 24:146-150. [28] Sundar P, Mahadevan A, Jayshree RS, Subbakrishna DK, Shankar SK. Toxoplasma seroprevalence in healthy voluntary blood donors from urban Karnataka. Indian J Med Res 2007;126: 50. [29] Al-Amari O. Prevalence of antibodies to Toxoplasma gondii among blood donors in Abha, Asir Region, south-western Saudi Arabia. The Journal of the Egyptian Public Health Association 1994;69:77-88. [30] Svobodova V, Literak I. Prevalence of IgM and IgG antibodies to Toxoplasma gondii in blood donors in the Czech Republic. Eur J Epidemiol 1998;14:803-805. https://doi.org/10.1023/A:1007589422080 [31] Zghair KH, AL-Qadhi BN, Mahmood SH. The effect of toxoplasmosis on the level of some sex hormones in males blood donors in Baghdad. Journal of Parasitic Diseases 2015; 39:393400.https://doi.org/10.1007/s12639-013-0382-6
Jo
ur na
lP
re
-p
[32] Griffin L, Williams KA. Serological and parasitological survey of blood donors in Kenya for toxoplasmosis. Trans R Soc Trop Med Hyg 1983;77:763-766. https://doi.org/10.1016/00359203(83)90283-3 [33] Yazar S, Eser B, Yay M. Prevalence of anti-Toxoplasma gondii antibodies in Turkish blood donors. Ethiop Med J 2006;44:257-61. [34] Chiang T, Hsieh, HH.,Kuo, MC.,et al. Seroepidemiology of Toxoplasma gondii Infection among healthy blood donors in Taiwan. PLOS one 2012;7:p.e48139. https://doi.org/10.1371/journal.pone.0048139. [35] Meerburg BG, Kijlstra A. Changing climate—changing pathogens: Toxoplasma gondii in North-Western Europe. Parasitol Res 2009;105:17-24. https://doi.org/10.1007/s00436-0091447-4 [36] Sarkari B, Shafiei R, Zare M, Sohrabpour S, Kasraian L. Seroprevalence and molecular diagnosis of Toxoplasma gondii infection among blood donors in southern Iran. J Infect Dev Ctries 2014;8:543-547. https://doi.org/10.3855/jidc.3831 [37] Mahmoudvand H, Saedi Dezaki E, Soleimani S, Baneshi MR, Kheirandish F, Ezatpour B, et al. Seroprevalence and risk factors of Toxoplasma gondii infection among healthy blood donors in southeast of Iran. Parasite Immunol 2015;37:362-367. https://doi.org/10.1111/pim.12198 [38] Al-Qurashi AR, Ghandour AM, Obeid O, Al-Mulhim A, Makki SM.Seroepidemiological study of Toxoplasma gondii infection in the human population in the Eastern Region. Saudi medical journal. 2001; 22:13-18. [39] Jones JL, Kruszon-Moran D, Wilson M, McQuillan G, Navin T, McAuley JB. Toxoplasma gondii infection in the United States: seroprevalence and risk factors. Am J Epidemiol 2001;154:357-365. https://doi.org/10.1093/aje/154.4.357
11
[40] Munsoor MM, Ahmed IM. Sero-prevalence of toxoplasmosis in blood donors in Khartoum State using latex agglutination test. SUST Journal of Science and Technology, 2012;13:3844 [41] Zarkovic A, MacMurray C, Deva N, Ghosh S, Whitley D, Guest S. Seropositivity rates for Bartonella henselae, Toxocara canis and Toxoplasma gondii in New Zealand blood donors. Clin Exp Ophthalmol 2007;35:131-134. https://doi.org/10.1111/j.1442-9071.2006.01406.x
Jo
ur na
lP
re
-p
ro
of
[42] Tenter AM, Heckeroth AR, Weiss LM. Toxoplasma gondii: from animals to humans. Int J Parasitol 2001;31:217-220. https://doi.org/10.1016/S0020-7519(00)00124-7 [43] Dubey J, Jones J. Toxoplasma gondii infection in humans and animals in the United States. Int J Parasitol 2008; 38: 1257-1278. https://doi.org/10.1016/j.ijpara.2008.03.007 [44] Aspinall TV, Marlee D, Hyde JE, Sims PF. Prevalence of Toxoplasma gondii in commercial meat products as monitored by polymerase chain reaction–food for thought? Int J Parasitol 2002;32:1193-1199. https://doi.org/10.1016/S0020-7519(02)00070-X [45] Abdülgani F. Prevalence of IgM and IgG Antibodies to Toxoplasma gondii in Blood Donors in the North Region of Jordan. Journal of Inonu University Medical Faculty 2010;11:14346. [46] Wang L, He L-y, Chen Z-w, Wen H, Fang G-s, Luo Q-l, et al. Seroprevalence and genetic characterization of Toxoplasma gondii in cancer patients in Anhui Province, Eastern China. Parasit Vectors 2015;8:162. https://doi.org/10.1186/s13071-015-0778-5
12
Legend of figures
Jo
ur na
lP
re
-p
ro
of
Fig. 1 NeighborNet phylogenetic network of Toxoplasma gondii isolates in blood donors from northern Iran.
13
Table 1 Summary of external and internal primers and enzymes for multilocus nested PCR-RFLP genotyping of Toxoplasma gondii. Markers
Multiplex PCR primers (external primers)
Nested PCR primers (internal primers)
Nested PCR (bp)
Restriction enzymes, incubation temperature and time
Ref.
SAG1
F: GTTCTAACCACGCACCCTGAG R: AAGAGTGGGAGGCTCTGTGA Not needed. The DNA fragment for 5´-SAG2 is covered by the external primers of alt. SAG2.
F: CAATGTGCACCTGTAGGAAGC R: GTGGTTCTCCGTCGGTGTGAG F: GAAATGTTTCAGGTTGCTGC R: GCAAGAGCGAACTTGAACAC
390
Sau96I+HaeII, .37 0C 1 h, 2.5% gel. MboI, 37 0C 1 h,2.5% gel.
[17]
F: TCTGTTCTCCGAAGTGACTCC R: TCAAAGCGTGCATTATCGC F: GGAACGCGAACAATGAGTTT R: GCACTGTTGTCCAGGGTTTT
F: ATTCTCATGCCTCCGCTTC R: AACGTTTCACGAAGGCACAC F: ACCCATCTGCGAAGAAAACG R: ATTTCGACCAGCGGGAGCAC
222
[18]
F: CAACTCTCACCATTCCACCC R: GCGCGTTGTTAGACAAGACA F: TCCAAAATGAGAGAAATCGT R: AAATTGAAATGACGGAAGAA F: ATTTGTGTTTCCGAGCAGGT R: GCACCTTCGCTTGTGGTT F: TGATGCATCCATGCGTTTAT R: CCTCCACTTCTTCGGTCTCA
F: TCTTGTCGGGTGTTCACTCA R: CACAAGGAGACCGAGAAGGA F: GAGGTCATCTCGGACGAACA R: TTGTAGGAACACCCGGACGC F: TTTCCGAGCAGGTGACCT R: TCGCCGAAGAGTTGACATAG F: TCTCTCTACGTGGACGCC R:AGGTGCTTGGATATTCGC
225
HhaI, 37 0C 1 h, 2.5% gel. HinfI+TaqI, 37 0C 30 min, 65 0C 30 min. 2.5% gel. NciI, 37 0C 1 h, 2.5% gel.
[19]
F: ACCCACTGAGCGAAAAGAAA R: AGGGTCTCTTGCGCATACAT F: TCTCTCGACTTCGCCTCTTC R: GCAATTTCCTCGAAGACAGG F: GAAAGCTGTCCACCCTGAAA R: AGAAAGCTCCGTGCAGTGAT F: TGGTTTTAACCCTAGATTGTGG R: AAACGGAATTAATGAGATTTGAA GTGTATCTCCGAGGGGGTCT TGTGACTTCTTCGCATCGAC
F: AGTTCTGCAGAGTGTCGC R:TGTCTAGGAAAGAGGCGC F: AGGAGGCGTAGCGCAAGT R: CCCTCTGGCTGCAGTGCT F: CGCAAAGGGAGACAATCAGT R: TCATCGCTGAATCTCATTGC F: GCAAATTCTTGAATTCTCAGTT R: GGGATTCGAACCCTTGATA AGCGGATTTCCAACACTGTC CTGCTGCATTCACAAACTCC
BsiEI+TaqI, 60 0C 1 h, 2.5% gel. MseI, 37 0C 1 h, 2.5% gel. BsmAI+MboII, NEB2, BSA, 37 0C 30 min, 55 0C 30min. 2.5% gel HpyCH4IV+RsaI, 37 0C 1 h, 2.5% gel. HaeIII+NlaIII, 37 0C 1 h, 2.5% gel. AvaI+RsaI, 37 0C 1 h, 2.5% gel. AflII+DdeI, 37 0C 1 h, 3% gel. N1aIII and MboI at 37 0C for 60 min
C22-8
C29-2 L358 PK1 Apico CS3
of
546
14
[18]
[19]
ro
GRA6
ur na
BTUB
Jo
SAG3
lP
alt. SAG2
242
[17]
521
[19]
411 344
-p
3´-SAG2
re
5´-SAG2
446 418 903 640 557
[19]
[20] [19, 20] [19, 20] [19, 20] [21, 46]
Table 2 Demographic characteristics of the 400 healthy blood donor in northern Iran. Characteristics
No. blood donors
No. seropositive donors (%)
P-value
Donation Center 10 25 45 70 80 50 85 10
6 20 30 57 56 3 70 7
(60) (80) (66.66) (81.42) (70) (68) (82.35) (70)
25
14
(56)
Male Female
381
278
(72.96)
19
16
≤30 31-39 ≥40
142 122
87 95
136
112
(82.35)
O A B AB
154 119 86
109 88 67
(70.77) (73.94) (77.90)
30
(73.17)
341
250
(73.31)
59
44
(74.57)
108
91
(84.26)
292
203
(69.52)
316 46 8
236 36 7
(74.68) (78.26) (87.50)
30
15
(50)
288 112
20 93
(69.79) (83.03)
Positive Negative
Occupation Business & Employee Farmer & Worker Housewives Student
Urban Rural
Jo
Residence
(84.21) (61.3) (77.9)
-p
ur na
Education Level Under diploma Upper diploma
lP
Rh
re
Blood group
41
ro
Gender
Age
15
0.09
of
Ramsar Tonekabon Chaloos Amol Babol Qaemshahr Sari Joubar Behshahr
0.27
0.000*
0.69
0.83
0.000*
0.01*
0.000*
Table 3
Statistical analysis ofthe seroprevalence ofToxoplasma gondii and the risk factors in blood donors in northern Iran Blood donor No.
No. Seropositive (%) (78.20) (66.86)
0.01*
147 253
108 186
(73.46) (73.51)
0.99
66 374
48 375
(73.07) (73.52)
0.96
378 22
280 14
239 49 73 16 23
184 31 54 11 14
ur na Jo
(74.07) (63.63)
148 52 94
125
0.28
(76.98) (63.26) (73.97) (68.75) (60.86)
0.18
(71.15) (76.47) (75.8)
0.53
ro
-p
re 218
of
183 111
182
16
P-value
234 166
208 68 124
lP
Variable Existence of cat in the neighborhood Yes No Undercooked meat consumption Yes No Raw milk consumption Yes No Raw vegetable consumption Yes No Water consumption Municipal Mineral Filtered Well Boiled Contact with garden soil No Yes with glove Yes without glove Consumption of raw/undercooked meat Products Yes No
(68.68) 0.04*
Table 4 Genotypes of Toxoplasma gondii in blood donors in the Mazandaran province, northern Iran ID
SAG1
5+3SAG2
altSAG2
SAG3
BTUB
GRA6
C228
C292
L358
PK
Apico
CS3
Toxo-DB Genotype
TgBDIr-6
IIorIII
II
II
II
II
II
II
II
II
II
II
II
#1
TgBDIr-19
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
-
TgBDIr-29
I
I
I
I
I
I
I
III
I
I
I
I
#27
TgBDIr-65
IIorIII
III
III
III
III
III
III
III
III
III
III
III
#2
I
I
I
I
I
I
I
I
I
I
I
I
#10
TgBDIr-194
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
-
TgBDIr-227
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
-
Jo
ur na
lP
re
-p
ro
of
TgBDIr-316
17