�������� ����� ��

Contamination of commonly consumed raw vegetables with soil transmittedhelminthes eggs in Mazandaran province, northern Iran

Ali Rostami, Maryam Ebrahimi, Saeed Mehravar, Vahid Fallah Omrani,Shirzad Fallahi, Hamed Behniafar

PII: S0168-1605(16)30118-0DOI: doi: 10.1016/j.ijfoodmicro.2016.03.013Reference: FOOD 7165

To appear in: International Journal of Food Microbiology

Received date: 6 November 2015Revised date: 9 March 2016Accepted date: 13 March 2016

Please cite this article as: Rostami, Ali, Ebrahimi, Maryam, Mehravar, Saeed,Omrani, Vahid Fallah, Fallahi, Shirzad, Behniafar, Hamed, Contamination of com-monly consumed raw vegetables with soil transmitted helminthes eggs in Mazan-daran province, northern Iran, International Journal of Food Microbiology (2016), doi:10.1016/j.ijfoodmicro.2016.03.013

This is a PDF file of an unedited manuscript that has been accepted for publication.As a service to our customers we are providing this early version of the manuscript.The manuscript will undergo copyediting, typesetting, and review of the resulting proofbefore it is published in its final form. Please note that during the production processerrors may be discovered which could affect the content, and all legal disclaimers thatapply to the journal pertain.

ACC

EPTE

D M

ANU

SCR

IPT

ACCEPTED MANUSCRIPT

1

Short communication

Contamination of commonly consumed raw vegetables with soil transmitted

helminthes eggs in Mazandaran province, northern Iran.

Ali Rostamia, Maryam Ebrahimi

a*, Saeed Mehravar

b, Vahid Fallah Omrani

c, Shirzad Fallahi

d,

Hamed Behniafara,

a Department of Medical Parasitology and Mycology, School of Medicine, Shahid Beheshti

University of Medical Sciences, Tehran, Iran

b Department of epidemiology and statistics, School of Public Health, Tehran university of

Medical Science, Tehran, Iran

c Cellular and Molecular Biology Research Center, Shahid Beheshti University of Medical

Sciences, Tehran, Iran

d Department of Parasitology and Mycology, School of Medicine, Lorestan University of

Medical Sciences, Khorramabad, Iran

Corresponding Author: ٭Maryam Ebrahimi, Department of Parasitology and Mycology, School

of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran.

Tel: +982122439962,

E-mail: mebrahimi198463@gmail.com

ACC

EPTE

D M

ANU

SCR

IPT

ACCEPTED MANUSCRIPT

2

Abstract:

Soil-transmitted helminthes (STHs) infections are responsible for significant burden of morbidity

and mortality worldwide. Consumption of raw vegetables without proper washing is one of the

major routes of such infections. We evaluate the prevalence of STH contamination in commonly

used vegetables in Mazandaran province, northern Iran. A total of 772 fresh raw vegetables were

obtained from retail markets. Each sample was divided into two groups. One group was used as

the unwashed sample and the second group was washed with standard washing procedures.

Then, samples were examined for helminth eggs by using standard methods. Data analysis was

performed using SPSS20. The overall prevalence of STHs was 14.89% (115/772). The rate of

STH contamination was significantly higher in warm seasons (20.5%, 79/386) than in cold

seasons (9.32%, 36/386) among the unwashed vegetables (OR= 2.50; CI 95%=1.64-3.8; P <

0.001). No parasites were observed in standard washed samples (OR= 271.40; CI 95%=16.84-

4373.64; P < 0.001). Prevalence of STH contamination was significantly higher in leafy

vegetables than root vegetables (OR= 1.67; CI 95%=1.09-2.55; P < 0.05). The prevalence of STHs

species in all the vegetables were as follows: Ascaris lumbricoides (3.36%), Trichuris trichiura

(2.2%), hookworms (2.9%), Toxocara spp. (1.68%), Trichostrongylus spp. (1.55), Taenia sp.

(0.9%) and Hymenolepis nana (2.2%). The results of the present study emphasized that

vegetables are potential risk factor for transmission of helminthes infection to human in Northern

Iran. It is necessary that health authorities trained the consumers to proper and standard washing

of vegetables before consumption.

Keywords: Vegetables, Contamination, Soil-transmitted helminthes, Northern Iran

ACC

EPTE

D M

ANU

SCR

IPT

ACCEPTED MANUSCRIPT

3

1. Introduction

Soil-transmitted helminthes (STHs), food-borne parasitic infections, are responsible for

significant burden of morbidity and mortality worldwide. It is estimated that 819 million people

are infected with Ascaris lumbricoides, 465 million with Trichuris trichiura, and 439 million

with hookworms globally (Pullan et al., 2014). An estimated 4.98 million or more disability-

adjusted life years have been attributed to STH infections (Pullan et al., 2014). Moreover

morbidity due to STH infections has primarily been associated to a range of complications,

including gastroenteritis, malnutrition, anemia, intestinal obstruction, poor physical and

cognitive development, nutrient absorption and iron loss (Hotez et al., 2008; Nokes and Bundy,

1994).

Vegetables and fruit are an important part of a healthy and balanced meal, providing vitamins,

minerals, and phytonutrient (Losio et al., 2015). The increased global tendency for eating raw

or slightly cooked vegetables and also rapid transport of foods especially soft fruit and

vegetables may increase the risk of foodborne infections (Fallah et al., 2012; Kozan et al., 2005).

Direct ingestion of human-infective eggs due to consumption of raw vegetables and fruits

without proper washing is one of the major routes of infection (Adenusi et al., 2015; Kozan et

al., 2005). In recent years, several studies have shown STH contamination of fresh vegetables

and fruits, suggesting their important role in human infection (Adamu et al., 2012; Adanir and

Tasci, 2013; Daryani et al., 2008; Ezatpour et al., 2013; Fallah et al., 2016; Fallah et al., 2012;

Gupta et al., 2009; Kozan et al., 2005; Maikai et al., 2012).

Northern Iran is a highly endemic area for intestinal parasites (Daryani et al., 2012; Omrani

et al., 2015). Up to our knowledge, there is no previously published study on the STH

ACC

EPTE

D M

ANU

SCR

IPT

ACCEPTED MANUSCRIPT

4

contamination of vegetables in northeastern Iran. Therefore, the main aim of this study was to

determine the prevalence of STH contamination in commonly used vegetables in Mazandaran

province, north of Iran and influence of season and standard washing procedure on burden of the

STH in the vegetables.

2. Materials and methods

2.1. Study area

This cross sectional study was carried out in four cities (Noor, Amol, Babol and Sari) in

Mazandaran Province, Northern Iran, from May 2014 to February 2015. This area (36.5° 25´N

53° 21´E) has a hot-summer Mediterranean climate, a mean annual temperature of 16°C and

about 900 mm of precipitation falls annually. According to Statistical Centre of Iran (SCI), the

number of total population in this area is about 3,100,000 (SCI, 2012).

2.2. Sample collection

A total of 772 samples of commonly consumed raw vegetables (193 samples in each city,

386 samples in warm seasons [193 spring and 193 summer] and 386 samples in cold seasons

[193 autumn and 193 winter]) were obtained (200-300 gr each) from retail markets (Table 1).

The included vegetables for this study were Radish (Raphanus sativus), Scallion (Allium

wakegi), Spinach (Spinacia oleracea), Parsley (Petroselinum crispum), lettuce (Lactuca sativa),

Green onion (Allium ascalonicum) and Mint (Mentha piperita). Vegetables were collected

separately in nylon sterile polythene and transported to the laboratory for parasitological

examination.

ACC

EPTE

D M

ANU

SCR

IPT

ACCEPTED MANUSCRIPT

5

2.3. Sample preparation and examination

Each sample was divided into two groups. One group was used as the unwashed sample

and the second group was washed with standard washing procedures according to Iranian

Ministry of Health and Medical Education protocol. Briefly, in first washing stage, the leaves of

leafy vegetables were separated and immersed in tap water inside a sink. Thus, mud and dust of

these vegetables were removed. In the second stage, separation of helminth eggs was performed

by 3 to 5 droplets of detergent per liter for 5 min. Then, disinfection of vegetables was conducted

by calcium hypochlorite solution (with 200 mg/l free chlorine) for 5 min; and finally the

disinfected vegetables were washed with tap water (Ministry of Health and Medical

Education of Iran, 2010; Yarahmadi et al., 2012). The unwashed vegetable samples were tested

for presence of STH eggs and larvae using the formalin ether concentration technique according

to the methods described previously (Adenusi et al., 2015; Organization, 1991, 1994). Helminth

eggs were identified by microscopic observation (Zeiss, Germany, 100× and 400×

magnification).

2.4. Analysis of data

The data were analyzed using the SPSS software version 20 for windows (SPSS Inc.,

Chicago, IL, USA). The chi-square test was used to compare the differences in rate of

contamination among warm and cold seasons and between unwashed and washed vegetables. At

expected frequencies less than five, the statistical significance was calculated using simulation

by the Monte Carlo method bases on 10,000 replicates. Associations were tested using odds

ratios (OR) and 95% confidence intervals (CI) after adjustments. A P-value <0.05 was

considered statistically significant. The mean number of STH eggs was calculated as described

previously (Choi and Lee, 1972).

ACC

EPTE

D M

ANU

SCR

IPT

ACCEPTED MANUSCRIPT

6

3. Results

Across the 772 vegetable samples, the overall prevalence of STH was 14.89% (115/772). The

distributions of the vegetables sampled according to the each season and percentage of

contaminated samples with STH eggs in different seasons are presented in Table 1. The rate of

parasitic contamination in vegetable samples was the highest in summer (48.3%) and the lowest

in winter (6.7%) (P < 0.001). Table 2 and 3 summarizes the results of parasitic contamination of

vegetables during warm and cold seasons. The rate of STH contamination was significantly

higher in warm seasons (20.5%, 79/386) than in cold seasons (9.3%, 36/386) among the

unwashed vegetables (OR= 2.50; CI 95%=1.64-3.8; P < 0.001). No parasites were observed in

standard washed samples (OR= 271.40; CI 95%=16.84-4373.64; P < 0.001). The prevalences of

STH species in all the vegetables were as follows: Ascaris lumbricoides (3.36%), Trichuris

trichiura (2.2%), hookworms (2.9%), Toxocara spp. (1.68%), Trichostrongylus spp. (1.55),

Taenia spp. (0.9%) and Hymenolepis nana (2.2%). Prevalence of STH contamination was

significantly higher in leafy vegetables (spinach, parsley, lettuce and mint; 17.39% [80/460])

than root vegetables (radish, scallion and green onions; 11.2% [35/312]) (OR= 1.67; CI

95%=1.09-2.55; P < 0.05). There was no significant difference in the incidence of STH

contamination among the four cities (P = 0.72) (fig. 1). Table 4 shows a comparison of the mean

number of STH contamination per 200 grams of vegetables. The mean number of STH

contamination was 0.42 from radish, 0.34 from scallion, 0.46 from spinach, 0.43 from parsley,

0.46 from lettuce, 0.33 from green onion and 0.26 from mint.

ACC

EPTE

D M

ANU

SCR

IPT

ACCEPTED MANUSCRIPT

7

4. Discussion

A downside to the potential health benefits is that raw, leafy, green vegetable products are

responsible for the transmission of foodborne pathogens, including bacteria, protozoa and Soil-

transmitted helminthes (STHs) (Losio et al., 2015). Recent outbreaks of foodborne diseases in

both of Europe and the USA demonstrated that there are a significant relationship between

pathogen contamination and vegetable consumption (Mercanoglu Taban and Halkman, 2011).

Studies on helminthological contamination of vegetables in Iran are rare. This survey is first

study investigating only helminthological contamination of vegetables in Iran. The results

obtained in this study have shown moderate STH contamination (14.89%) of vegetables in

Northern Iran that is lower than the prevalence of parasitic contamination reported in

Shahrekord, southwest Iran (Fallah et al., 2016; Fallah et al., 2012), Khorramabad, western Iran

(Ezatpour et al., 2013), and in Ardebil, northwest Iran (Daryani et al., 2008). The use of rivers

and springs water to irrigate vegetables in this area could be a possible explanation to this lower

prevalence of STH in this region, whereas due to water shortage in other areas of Iran, sewage

effluent or water supplies contaminated with sewage are used to irrigate vegetables. In addition,

previous studies have shown that uncontrolled use of water contaminated with sewage or human

and animal faeces to irrigate of vegetables (at least 20 million hectares in 50 countries) is

responsible for their high rates of contamination with STH eggs, especially in developing

countries (Kozan et al., 2005; Hussain et al., 2001). It is important, because that a proportion of

the vegetables cultivated in these developing countries are exported to the developed world

(Kozan et al., 2005; Robertson & Gjerde, 2001). Also, the overall prevalence of STH in our

study (14.89%) is higher than the 6.3% in Burdur, Turkey (Adanir and Tasci, 2013), 3.5% in

ACC

EPTE

D M

ANU

SCR

IPT

ACCEPTED MANUSCRIPT

8

Maiduguri, northeast Nigeria (Adamu et al., 2012), and 8.44% reported in Ogun State, southwest

Nigeria (Adenusi et al., 2015).

Our finding revealed that the majority of the contaminated vegetable samples were leafy

vegetables (10.36%), especially in spinach, parsley and lettuce. A possible description could be

that vegetables like lettuce have broad leaves and large surface areas, leading to more contact

with the sewage contaminated soil surface (Adamu et al., 2012). The rate of STH contamination

was smaller in root vegetables (4.53%), suggesting that broad leaves and more contact with soil

are important predictors to STH contamination. Similar with our study, lettuce and spinach were

the vegetables contaminated most commonly in Ankara, Turkey (Kozan et al., 2005), Benha,

Egypt (Eraky et al., 2014), Maiduguri, northeast Nigeria (Adamu et al., 2012), Riyadh, Saudi

Arabia (Al-Megrim, 2010), and in Kaduna State, Nigeria (Maikai et al., 2012).

Another important objective of this study was to ascertain whether standard washing procedures

and climate changes have influence in rate of STH contamination. The standard washing

procedures is differing substantially from what is called traditional washing. In traditional

procedure, the vegetables are immersed in tap water inside a sink. After a short period to

sedimentation of mud and dust, they are gently collected, put in a wood or plastic basket, and

rinsed for 1.5-2 min with tap water (Fallah et al, 2012). In fact, unlike standard washing

procedures, no detergent or disinfectant solution is used in traditional washing. No parasites were

observed in standard washed samples. In agreement with our study, Kozan et al. in Ankara,

Turkey, founded STH contamination in 5.9% of unwashed vegetable samples and none in

washed samples (Kozan et al., 2005). Also, Fallah et al. have reported that rate of parasitic

contamination is significantly lower in pre-washed and standard washed samples than unwashed

samples. They founded intestinal parasites from 32.6% of unwashed, 1.3% of traditionally

ACC

EPTE

D M

ANU

SCR

IPT

ACCEPTED MANUSCRIPT

9

washed and not in any standard washed samples (Fallah et al., 2012). Although it should be

noted that the possible unsafety of the water used for the washing of vegetables may be

responsible for some reported contaminations in our investigation and studies conducted by other

researchers. In addition, results of the present study have showed that rate of contamination was

significantly higher in warm seasons. This result is consistent with previous studies in Iran and

the world (Al-Megrin et al., 2010; Eraky et al., 2014; Ezatpour et al., 2013; Fallah et al., 2012).

A possible explanation could be higher shedding of helminthes eggs in warm seasons by humans

and animals. In addition, the majority of days in cold seasons are rainy in northern Iran, resulted

in probable wash of helminthes eggs from the surface of vegetables (Fallah et al., 2016). Climate

change is an important determinant of transmission of STH and moisture and warm temperature

are essential for larval development and eggs survival in the soil. Taking into account that many

of developing countries and countries with not appropriate hygienic condition (such as India and

African, Middle East and Southeast Asian countries) have warm weather in during the year, the

more rate of STH contamination in warm season cannot be overlooked. As reported by Pullan et

al. (2014) the vast majority of STH infections (>75%) and years lived with disability (>70%)

occurred in Asian and African countries. Moreover, de Silva et al. have reported that highest

rates of Ascaris infection occur in China and Southeast Asia, in the coastal regions of West

Africa, and in Central Africa. Trichuris infections reach their highest prevalence in Central

Africa, southern India and Southeast Asia. Hookworm infections, however, are common

throughout much of sub-Saharan Africa, in addition to South China and Southeast Asia (Silva et

al., 2003).

Despite sustained improvement in sanitary conditions over the past three decades, intestinal

parasitic infections are still highly prevalent in Iran. It is estimated that the overall prevalence of

ACC

EPTE

D M

ANU

SCR

IPT

ACCEPTED MANUSCRIPT

10

IPIs in Iran is 38%. The prevalence rate for some of the more common helminthes parasites in

Iran is as follow: A. lumbricoides 10.9%, and H. nana 2.3%, hookworms <1%, T. trichiura <1%,

Strongyloides stercoralis (in endemic areas) 4.9-42%, Taenia spp. <1% (Omrani et al., 2015;

Pullan et al., 2014; Rokni, 2008, 2009). In this study, the most detected STH eggs were A.

lumbricoides, hookworm eggs, T. trichiura and H. nana, respectively. The high fecundity of A.

lumbricoides and T. trichiura, high resistant these helminthes eggs that can persist in the

environment for months to years and high rate of these helminthes infections in the humans

could be significant reasons for high STH contamination of vegetables in this area and other

areas in the world with hot and humid climates.

Northern Iran is highly endemic area for hookworm infections (Necatoriasis) and Taeniasis

(Hydatidosis) (Rokni, 2008, 2009). High prevalence of these infections in human and animals

and poor sanitation in area with agricultural and pastoral culture can be source of vegetables

contamination. In addition, results of this study indicate that vegetables can be an important risk

factor for such infections in human.

The vegetables surveyed in our study are untreated and unwashed and are usually sold

unpacked in variable values as chosen by the customers. Moreover, vegetables from the northern

Iran are exported to neighboring provinces in Iran and neighboring countries. Therefore, this

study is also important with regard to this point of view.

5. Conclusion:

In conclusion, the results of the present study emphasized that if standard washing and

disinfecting procedures of the raw eaten vegetables be neglected, they could be considered as a

potential risk factor for transmission of helminths infection to human. It is necessary an

ACC

EPTE

D M

ANU

SCR

IPT

ACCEPTED MANUSCRIPT

11

integrated approach to improve the sanitary conditions in the areas where the vegetables are

cultivated. In addition, the health authorities should train the consumers to proper and standard

washing of vegetables before consumption.

Acknowledgements

The authors would like to thank Dr. Mazaher Gholipourmalekabadi, for his assistance during the

preparation of this manuscript.

Conflict of interest

The authors declare that there is no conflict of interests regarding the publication of this paper.

References

Adamu, N.B., Adamu, J.Y., Mohammed, D., 2012. Prevalence of helminth parasites found on

vegetables sold in Maiduguri, Northeastern Nigeria. Food. Control. 25, 23–26.

Adanir, R., Tasci, F., 2013. Prevalence of helminth eggs in raw vegetables consumed in Burdur,

Turkey. Food. Control. 31, 482–484.

Adenusi, A.A., Abimbola, W.A., Adewoga, T.O., 2015. Human intestinal helminth

contamination in pre-washed, fresh vegetables for sale in major markets in Ogun State,

southwest Nigeria. Food. Control. 50, 843–849.

Al-Megrm, W.I., 2010. Prevalence of intestinal parasites in leafy vegetables in Riyadh, Saudi

Arabia. Int. J. Trop. Med. 5, 20–23.

Choi, D.W., Lee, S., 1972. Incidence of parasites found on vegetables collected from markets

and vegetable gardens in Taegu area. Korean. J. Parasitol. 10, 44–51.

ACC

EPTE

D M

ANU

SCR

IPT

ACCEPTED MANUSCRIPT

12

Daryani, A., Ettehad, G., Sharif, M., Ghorbani, L., Ziaei, H., 2008. Prevalence of intestinal

parasites in vegetables consumed in Ardabil, Iran. Food. Control. 19, 790–794.

Daryani, A., Sharif, M., Nasrolahei, M., Khalilian, A., Mohammadi, A., Barzegar, G., 2012.

Epidemiological survey of the prevalence of intestinal parasites among schoolchildren in

Sari, northern Iran. Trans. R. Soc. Trop. Med. Hyg. 106, 455–459.

De Silva, N.R., Brooker, S., Hotez, P.J., Montresor, A., Engels, D., Savioli, L., 2003. Soil-

transmitted helminth infections: updating the global picture. Trends. Parasitol. 19, 547–

551.

Eraky, M.A., Rashed, S.M., Nasr, M.E.-S., El-Hamshary, A.M.S., Salah El-Ghannam, A., 2014.

Parasitic contamination of commonly consumed fresh leafy vegetables in Benha, Egypt.

J. Parasitol. Res. 613960.

Ezatpour, B., Chegeni, A.S., Abdollahpour, F., Aazami, M., Alirezaei, M., 2013. Prevalence of

parasitic contamination of raw vegetables in Khorramabad, Iran. Food. Control. 34, 92 –

95.

Fallah, A.A., Makhtumi, Y., Pirali-Kheirabadi, K., 2016. Seasonal study of parasitic

contamination in fresh salad vegetables marketed in Shahrekord, Iran. Food. Control. 60,

538–542.

Fallah, A.A., Pirali-Kheirabadi, K., Shirvani, F., Saei-Dehkordi, S.S., 2012. Prevalence of

parasitic contamination in vegetables used for raw consumption in Shahrekord, Iran:

influence of season and washing procedure. Food. Control. 25, 617–620.

Gupta, N., Khan, D., Santra, S., 2009. Prevalence of intestinal helminth eggs on vegetables

grown in wastewater-irrigated areas of Titagarh, West Bengal, India. Food. Control. 20,

942–945.

ACC

EPTE

D M

ANU

SCR

IPT

ACCEPTED MANUSCRIPT

13

Hotez, P.J., Brindley, P.J., Bethony, J.M., King, C.H., Pearce, E.J., Jacobson, J., 2008. Helminth

infections: the great neglected tropical diseases. J. Clin. Invest. 118, 1311–1321.

Hussain, I., Raschid, L., Hanjra, M., Marikar, F., & Van der Hoek, W., 2001. A

framework for analyzing socioeconomic, health and environmental impacts of

wastewater use in agriculture in developing countries: Working Paper 26. Colombo, Sri

Lanka: International Water Management Institute. IWMI.

Mercanoglu Taban, B., Halkman, A.K., 2011. Do leafy green vegetables and their ready-to-eat

[RTE] salads carry a risk of foodborne pathogens? Anaerobe 17, 286–287.

Ministry of Health and medical education of IRAN (2010). Guidline to disinfection vege-

table: www. behdasht.gov. ir.Ac-cessed .2010.[in persian].

Kozan, E., Gonenc, B., Sarimehmetoglu, O., Aycicek, H., 2005. Prevalence of helminth eggs on

raw vegetables used for salads. Food. Control. 16, 239–242.

Losio, M., Pavoni, E., Bilei, S., Bertasi, B., Bove, D., Capuano, F., Farneti, S., Blasi, G., Comin,

D., Cardamone, C., 2015. Microbiological survey of raw and ready-to-eat leafy green

vegetables marketed in Italy. Int. J. Food. Microbiol. 210, 88–91.

Maikai, B., Elisha, I., Baba-Onoja, E., 2012. Contamination of vegetables sold in markets with

helminth eggs in Zaria metropolis, Kaduna State, Nigeria. Food. Control. 28, 345–348.

Nokes, C., Bundy, D., 1994. Does helminth infection affect mental processing and educational

achievement? Parasitol. Today. 10, 14–18.

Omrani, V.F., Fallahi, S., Rostami, A., Siyadatpanah, A., Barzgarpour, G., Mehravar, S.,

Memari, F., Hajialiani, F., Joneidi, Z., 2015. Prevalence of intestinal parasite infections

and associated clinical symptoms among patients with end-stage renal disease undergoing

hemodialysis. Infection. 43, 537–544.

ACC

EPTE

D M

ANU

SCR

IPT

ACCEPTED MANUSCRIPT

14

Pullan, R.L., Smith, J.L., Jasrasaria, R., Brooker, S.J., 2014. Global numbers of infection and

disease burden of soil transmitted helminth infections in 2010. Parasit. Vectors. 7, 37.

Robertson, L. J., & Gjerde, B., 2001. Occurrence of parasites on fruits and vegetables in Norway.

J. Food. Protect. 64, 1793–1798.

Rokni, M., 2008. The present status of human helminthic diseases in Iran. Ann. Trop. Med.

Parasitol. 102, 283–295.

Rokni, M., 2009. Echinococcosis/hydatidosis in Iran. Iranian. J. Parasitol. 4, 1–16.

WHO., (1991). Basic laboratory methods in medical parasitology. Geneva: World

Health Organization.

WHO., (1994). Bench aids for the diagnosis of intestinal parasites. Geneva: World

Health Organization.

Yarahmadi, M., Yunesian, M., Pourmand, M., Shahsavani, A., Mubedi, I., Nomanpour, B.,

Naddafi, K., 2012. Evaluating the Efficiency of Lettuce Disinfection According to the

Official Protocol in Iran. Iranian. J. Public. Health. 41, 95–103.

ACC

EPTE

D M

ANU

SCR

IPT

ACCEPTED MANUSCRIPT

15

Figure 1: Distribution of contaminated raw vegetables with soil transmitted helminthes (STHs) eggs

according to different cities in Mazandaran province, northern Iran

ACC

EPTE

D M

ANU

SCR

IPT

ACCEPTED MANUSCRIPT

16

Table1: Number of vegetables and percentage of contaminated samples with soil transmitted

helminthes (STH) eggs in different seasons.

.

A= Chi-square Test or Chi-square Test with Monte Carlo simulation

* P ≤ 0.05.

Vegetables Spring

Summer Autumn

Winter P Value A

number Infected

(%) number

Infected

(%) number

Infected

(%) number

Infected

(%)

Radish

29 5 (17.2) 29 7 (24.1) 29 3 (10.3) 29 1 (3.4) 0.623

Scallion

24 2 (8.3) 25 3 (12) 24 4 (16.6) 25 1 (4) 0.477

Spinach 29 7 (24.1) 29 11 (37.9) 29 2 (6.8) 29 3 (10.3) 0.011

*

Parsley

29 8 (27.5) 29 8 (27.5) 29 3 (10.3) 29 1 (3.4) 0.030*

Lettuce

28 6 (21.4) 28 9 (32.1) 28 5 (17.8) 28 2 (7.1) 0.149

Green

onion

25 2 (8) 24 2 (8.3) 25 3 (12) 24 2 (8.3) 1

Mint

29 4 (13.7) 29 5 (17.2) 29 3 (10.3) 29 3 (10.3) 0.929

Total 193 34 (36.5) 193 45 (48.3) 193 23 (11.9) 193 13 (6.7) 0.001

ACC

EPTE

D M

ANU

SCR

IPT

ACCEPTED MANUSCRIPT

17

Table 2: Contamination of vegetables with soil transmitted helminthes (STH) eggs in spring and

summer (warm seasons).

N

o Common STH Uncommon STH

Ascar

is

eggs

no.(

%)

T.

trichiu

ra

eggs

no.(%)

Hookwo

rm

eggs no.

(%)

Toxoca

ra spp.

eggs

no. (%)

Trichostrong

ylus spp. eggs

no. (%)

Taen

ia spp eggs

no.

(%)

H.

nana

eggs

no.(

%)

Total

Radish

(Raphanus

sativus)

58

3

(5.17

)

1

(1.72)

2 (3.44) 0

(0.00)

4 (6.89) 0

(0.00

)

2

(3.44

)

12 (20.6

8)

Scallion

(Allium

wakegi)

49

0

(0.00

)

0

(0.00)

1 (2.04) 3

(6.12)

1 (2.04) 0

(0.00

)

0

(0.00

)

5 (10.2)

Spinach

(Spinacia

oleracea)

58

4

(6.89

)

3

(5.17)

3 (5.17) 2

(3.44)

2 (3.44) 1

(1.72

)

3

(5.17

)

18 (31.0

3)

Parsley

(Petroselin

um

crispum)

58

2

(3.44

)

5

(8.62)

4 (6.89) 0

(0.00)

1 (1.72) 0

(0.00

)

4

(6.89

)

16 (27.5

8)

Lettuce

(Lactuca

sativa)

56

5

(8.92

)

2

(3.57)

3 (5.35) 1

(1.78)

2 (3.57) 2

(3.57

)

0

(0.00

)

15 (26.7

8)

Green

onion

(Allium

ascalonicu

m)

49

0

(0.00

)

1

(2.04)

1 (2.04) 2

(4.08)

0 (0.00) 1

(2.04

)

0

(0.00

)

4 (8.16)

Mint

(Mentha

piperita)

58

1(1.7

2)

2

(3.44)

3 (5.17) 0

(0.00)

0 (0.00) 1

(1.72

)

1

(1.72

)

9 (15.5

1)

Total 38

6

15

(3.88

)

14 (3.62)

17 (4.40) 8 (2.07) 10 (2.59) 5 (1.29)

10 (2.59

)

79

(20.4

6)

ACC

EPTE

D M

ANU

SCR

IPT

ACCEPTED MANUSCRIPT

18

Table 3: Contamination of vegetables with soil transmitted helminthes (STH) eggs in autumn

and winter (cold seasons).

N

o

Common STH Uncommon STH

Ascar

is

eggs

no.(%

)

T.

trichiu

ra

eggs

no.(%)

Hookwo

rm

eggs no.

(%)

Toxoca

ra spp.

eggs

no. (%)

Trichostrong

ylus spp.

eggs

no. (%)

Taen

ia spp.

eggs

no.

(%)

H.

nana

eggs

no.(

%)

Total

Radish

(Raphanus

sativus)

58

2

(3.44)

0

(0.00)

1 (1.72) 0

(0.00)

0 (0.00) 0

(0.00

)

1

(1.72

)

4 (6.89

)

Scallion

(Allium

wakegi)

49

1

(2.04)

0

(0.00)

1 (2.04) 1

(2.04)

0 (0.00) 0

(0.00

)

2

(4.08

)

5 (10.2

)

Spinach

(Spinacia

oleracea)

58

1

(1.72)

1

(1.72)

1 (1.72) 0

(0.00)

0 (0.00) 1

(1.72

)

1

(1.72

)

5 (8.62

)

Parsley

(Petroselin

um

crispum)

58

1

(1.72)

1

(1.72)

0 (0.00) 2

(3.44)

0 (0.00) 0

(0.00

)

0

(0.00

)

4 (6.89

)

Lettuce

(Lactuca

sativa)

56

2

(3.57)

0

(0.00)

1 (1.78) 1

(1.78)

1 (1.78) 1

(1.78

)

1

(1.78

)

7 (12.5

)

Green

onion

(Allium

ascalonicu

m)

49

3

(6.12)

0

(0.00)

0 (0.00) 0

(0.00)

1 (2.04) 0

(0.00

)

1

(2.04

)

5 (10.2

)

Mint

(Mentha

piperita)

58

1

(1.72)

1

(1.72)

2 (3.44) 1

(1.72)

0 (0.00) 0

(0.00

)

1

(1.72

)

6 (10.3

4)

Total

38

6

11

(2.84)

3 (0.77)

6 (1.55) 5 (1.29) 2 (0.51) 2 (0.51)

7 (1.81)

36

(9.32

)

ACC

EPTE

D M

ANU

SCR

IPT

ACCEPTED MANUSCRIPT

19

Table 4: Total and mean number of soil transmitted helminthes (STH) eggs found per 200

gram from each vegetable.

Radish

Scallion

Spinach

Parsley

Lettuce

Green

onion Mint

No of vegetable examined 103 84 110 106 96 82 112

Total vegetable weight

(gram) 27600 20800 29000 25200 29200 21400 30400

Total number (mean number

per 200 gr) of Ascaris eggs

from vegetable

22

(0.15) 8 (0.07)

23

(0.15)

11

(0.08)

21

(0.14)

8

(0.07)

5

(0.03)

Total number (mean number

per 200 gr) of T. Trichiura

eggs of vegetable

7 (0.05) 0 (0.0) 9 (0.06) 14

(0.11)

13

(0.08)

4

(0.03)

8

(0.05)

Total number (mean number

per 200 gr) of Hookworm

eggs of vegetable

11

(0.07) 7 (0.06)

14

(0.09)

15

(0.11)

9

(0.06)

9

(0.08)

13

(0.08)

Total number (mean number

per 200 gr) of other

helminthes eggs of

vegetable

19

(0.13) 21 (0.2)

22

(0.15)

15

(0.11)

25

(0.17)

13

(0.12)

15

(0.09)

Total helminthes eggs 59

(0.42)

36

(0.34)

67

(0.46)

55

(0.43)

68

(0.46)

36

(0.33)

41

(0.26)

ACC

EPTE

D M

ANU

SCR

IPT

ACCEPTED MANUSCRIPT

20

Highlights

We evaluate the prevalence of Soil-transmitted helminthes in vegetables in northern Iran

The overall prevalence of STHs was 14.89% (115/772)

The rate of STHs contamination was associated with climate changes

The rate of STHs contamination was associated with washing procedures

No parasites were observed in standard washed samples