Parasitol Res (2016) 115:18271834

DOI 10.1007/s00436-016-4922-8

ORIGINAL PAPER

Standardization of a method for the detection of helminth eggs

and larvae in lettuce
F. C. Matosinhos 2 & V. C. Valenzuela 2 & J. A. Silveira 1 & E. M. Rabelo 1

Received: 23 December 2015 / Accepted: 11 January 2016 / Published online: 20 January 2016
# Springer-Verlag Berlin Heidelberg 2016

Abstract Despite reports that food-borne parasitic infections

have been increasing worldwide, the methodologies
employed to detect food contamination by helminths are still
largely based on methodologies used to detect these pathogens
in feces and water. This study sought to improve the diagnosis
of parasitic contaminants in lettuce by standardizing a method
for detecting helminth eggs and larvae and estimating their
percentage of recovery. Sanitized lettuces were artificially
contaminated with different amounts of Ascaris suum and
hookworm eggs and larvae. To standardize the method, we
tested liquid extractors, vegetable washing steps, and spontaneous sedimentation times. Higher percentages of egg and
larvae recovery were obtained using 1 M glycine as the liquid
extractor, manual shaking for 3 min and 2 h of sedimentation.
Five different levels of artificial contamination (ten replicates
each; n = 50) were tested using these standardized conditions,
yielding an average recovery of 62.6 % (20.2), 51.9 %
(20.0), and 50.0 % (27.3) for A. suum eggs, hookworm
eggs, and larvae, respectively. Tests were performed with a
different matrix to evaluate the performance of the method.
Furthermore, collaborative analytical studies performed by
different laboratories produced satisfactory results. The method for the identification of helminth eggs and larvae proposed
in this study proved to be simpler and more efficient than
previously published procedures, thereby demonstrating its

* E. M. Rabelo
elidam.rabelo@gmail.com

Departamento de Parasitologia - Instituto de Cincias Biolgicas,

Universidade Federal de Minas Gerais, Avenida Antnio Carlos,
6627, CEP 31270-901 Belo Horizonte, MG, Brazil

Servio de Microscopia de Produtos do Instituto Otvio Magalhes

da Fundao Ezequiel Dias, Belo Horizonte, Brazil

potential contribution to health surveillance and epidemiological studies.

Keywords Food contamination . Helminth eggs . Helminth
larvae . Leafy vegetables . Detection technique

Introduction
Helminth infections are among the most common health problems worldwide, with an estimated two billion people infected
with helminths. The geohelminths are among the most prevalent infections, with a global estimated prevalence of infection
for Ascaris lumbricoides, Trichuris trichiura, and hookworms
of 1.2 billion, 795 million, and 740 million, respectively (de
Silva et al. 2003).
There have been increasing reports of infectious diseases
associated with the consumption of fresh fruits and vegetables. Additionally, there is a growing demand for minimally
processed foods and meals served outside the home. Several
studies have been usually conducted in developing countries
to assess the degree of contamination of vegetables by pathogens. High levels of contamination in vegetables by
enteroparasites were reported, demonstrating the importance
of vegetables (especially those eaten raw) as parasitic infection couriers (Nyarango et al. 2008; Ogbolu et al. 2009;
Al-Megrin 2010; Shahnazi and Jafari-Sabet 2010; Said
2012).
A wide variety of methodologies have been used in the
search for parasites that contaminate vegetables. However,
these methods include well-established techniques for the
analysis of fecal material and water that have been adapted
for the analysis of vegetables. Most of the methods use water,
solutions containing detergents, and physiological solutions as
the extractor solutions (Al-Binali et al. 2006; Damen et al.

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Parasitol Res (2016) 115:18271834

detected in vegetables. Hookworms were chosen due to their

high detection frequency during vegetable parasitological
analyses (Simes et al. 2001; Traviezo-Valles et al. 2004;
Al-Binali et al. 2006). Ascaris suum eggs were obtained from
a female adult worm, cleaned and stored in 10 % formaldehyde solution for conservation in the refrigerator.
Ancylostoma ceylanicum eggs were obtained from the feces
of hamsters (Mesocricetus auratus) experimentally infected.
Fecal material was subjected to a sedimentation technique
involving formalin-ether centrifugation (Blagg et al. 1955).
The larvae were obtained after 7 days of coproculture with
feces of hamsters experimentally infected with A. ceylanicum
and were recovered by the Baermann technique as modified
by Moraes (1948).

2007; Maikai et al. 2013). The extraction of contaminants can

be achieved by simple incubation of the sample in the extraction solution (Al-Megrin 2010), sonication (Shahnazi and
Jafari-Sabet 2010), manual stirring (Chandra et al. 2014), or
using a mechanical shaker (Eraky et al. 2014). Contaminant
recoveries are mainly achieved using different spontaneous
sedimentation and centrifugation conditions (Avcioglu et al.
2011; Kapec and Borecka 2012). The wide variety of techniques used to analyze helminth contamination in vegetables
demonstrates the lack of consensus among the different laboratories that conduct this type of research, thereby hindering
the comparison of surveys performed by different research
groups around the world.
The US FDA (Food and Drug Administration) has published a protocol for the analysis of food contaminated by
helminthes. However, this protocol is an adaptation of protocols designed to isolate protozoans from contaminated water
(Bier 1991) and when the methodology was applied to the
detection of helminthes, it resulted in a very low recovery ratio
(only 10 % for Trichuris sp. and Ascaris sp. eggs).
Parasitological evaluation of vegetables has been neglected
from food regulation committees worldwide. This problem
is aggravated by the absence of reliable protocols to assess
vegetable contamination, mainly regarding helminthes eggs
or larvae. Therefore, the aim of this study was to standardize
a methodology to identify eggs and larvae present on raw
vegetables. This study was able to establish a methodology
for the diagnosis of parasitic contaminants, thus contributing
to health surveillance and epidemiological studies.

Vegetable samples
The vegetable of choice for the standardization of the methodology was lettuce (Lactuca sativa). Because lettuce is consumed raw, it is the most commonly used vegetable in works
studying this subject (Ripabelli et al. 2004). Minimally processed samples were used because they are sanitized and therefore are less likely to contain parasites. Each sample used for
artificial contamination consisted of 30 g of lettuce leaves,
which reflects the average amount consumed in each portion.
After the standardization of the methodology, as described
below, it was applied to another matrix to test its effectiveness.
Arugula (Eruca sativa) was chosen for this testing because it
is also consumed raw most of the time. Our analysis included
five samples minimally processed without artificial contamination and quadruplicate samples artificially contaminated
with each of the five levels of contamination (described in
Table 1).

Materials and methods

All procedures followed the recommendations of the Ethics
Committee on Animal Experimentation of Universidade
Federal de Minas Gerais (CETEA/UFMG), project number
088/09.

Quantification of parasites used in the artificial

contamination
After concentration of the helminth eggs and larvae, ten samples from each suspension were analyzed by optical microscopy. The average of the results was regarded as the estimated
value in the suspensions used for the artificial contamination
of the samples (10.80 1.93, 12.20 2.89, and 5.6 1.5 for

Parasites used for artificial contamination

We used eggs and larvae of Ancylostoma ceylanicum and eggs
of Ascaris suum for the artificial contamination of the samples. These parasites represent the helminth forms that are
Table 1 Estimated number of
eggs and larvae present in each
level of the artificial
contamination

Contamination levels

A. suum eggsa

Hookworm eggsa

Hookworm larvaea

Level 1
Level 2
Level 3
Level 4
Level 5

100
50
20
11
5

100
50
20
12
6

50
25
10
6
3

Estimated number of eggs and larvae added to 30 g of vegetable leaves

Parasitol Res (2016) 115:18271834

10 L samples of A. suum eggs, hookworm eggs, and larvae,

respectively).
Artificial contamination of samples
Leaves from each sample were placed in a container with
minimum overlapping. Ascaris suum eggs and hookworm
eggs and larvae were pipetted onto different areas on the leaf
surfaces, according to the levels described in Table 1. The
number of eggs and larvae are estimated based on triplicates
counting; however, this number may suffer natural deviation
and that is the reason why sometimes the final value found
may even be higher than a 100 %. To ensure that all parasites
had been distributed, the same volume of distilled water was
pipetted at other points of the vegetable surface to wash out
possible eggs and larvae that had been retained in the tip. The
leaves were left to dry at room temperature for approximately
one and a half hours or until their surfaces were dry
(Robertson and Gjerde 2000).

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The stirring method of the liquid with the samples was also
tested. The samples were stirred manually for 1 or 3 min or
using mechanical homogenization with a Stomacher
(Worthing, South Dakota, USA) for 60 s at low speed. The
tests were performed in triplicate using 200 ml of 1 M glycine
solution as the extractor solution. The timing of the sedimentation was also tested in triplicate (1, 2, and 24 h). The conditions of the three parameters that showed a better average of
recovery were chosen for the other tests.
Determination of the recovery percentage of the proposed
method
Following the standardization of the protocol, the samples
were divided into the following groups: (a) control sample:
samples not spiked and supposedly free of parasites and (b)
artificially contaminated samples, divided into five different
levels of contamination (Table 1).
Interlaboratory analyses

Standardization of the contaminant detection method

Each step and its variables were tested in triplicate for the
standardization of the method. We tested different extractor
liquids, types of homogenization, and sedimentation times.
The extractor liquids tested included distilled water, 0.1 %
Tween 80 solution, 1 M glycine solution, and 1 % sodium
lauryl sulfate solution (Chandra et al. 2014; Cook et al.
2006) and it was initially tested only with eggs of nematodes
used in this study. To avoid the presence of foam in the solutions of Tween 80 and sodium lauryl sulfate, one drop of an
antifoam agent (Antifoam AFluka, St. Louis, MO, USA)
was added to these extractor solutions.
For the liquid extractor test, three samples were weighed
(30 g each) and artificially contaminated with 100 eggs of A.
suum and 100 hookworm eggs for an initial test. The samples
were placed in a brand new plastic bag (24 34 0.5 cm), and
200 ml of each liquid to be tested was added to the lettuce
leaves. The bag was manually shaken for 3 min. Then, the
liquid was drained by cutting one of the plastic bag tips and
filtered through a sieve (1 mm mesh) directly into a conical
cup. After settling for 24 h, the supernatant was discarded with
a pipette and the approximately 10 ml pellet was transferred to
a 15 ml centrifuge tube. The cup was washed with 5 ml of
distilled water; the wash water was added to the tube and the
sample was centrifuged at 1120g for 5 min. The supernatant
was discarded, the pellet was homogenized with a Pasteur
pipette, and the slides were prepared for the counting and
identification of recovered eggs. The eggs were visualized
under a light microscope at a magnification of 100 and/or
200. The two extractor solutions that presented the best performances were tested in triplicate with samples contaminated
with approximately 50 hookworm larvae.

Collaborative analyses were performed to test the performance of the method under different conditions than those
tested in the standardization and validation process. The samples were contaminated in triplicate with contaminant levels 1,
2, and 3 (Table 1). Then, the samples were packed in plastic
bag, sealed, stored in polystyrene boxes, and delivered to the
laboratories that had agreed to perform the analyses: Dr.
Adriana Oliveira Costa, Department of Clinical and
Toxicological Analysis of UFMG Faculty of Pharmacy
MG (located in a different building at the same institution as
the original laboratory); Dr. Joziana Muniz de Paiva Barante
from The Veterinary Medicine Department of the Federal
University of Lavras MG (185 Km away from the original
institution); and Dr. Maria Helena Martini, responsible for the
Food Microscopy area of the Regional Laboratory Center of
the Institute Adolfo Lutz Campinas- SP (465 Km away from
the original institution). The established protocol and the required reagents were also shipped to the labs. The samples
were sequentially numbered in a codified manner that did
not reveal the different levels of artificial contamination in
each sample. Therefore, the collaborative testing represented
blind tests.
Statistical analysis
All data were examined for normality using the
Kolmogorov-Smirnov test. For analysis between two groups,
Students t test was used for parametric data and the
Mann-Whitney test for nonparametric data. T he
Kruskal-Wallis test was used followed by Dunns post-test
for comparisons of three or more groups of nonparametric
data. One-way ANOVA followed by Bonferronis post-test

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Parasitol Res (2016) 115:18271834

was used for parametric data. The Grubbs test was used to
detect outliers, which were removed from the sample. All tests
were considered significant when they presented a value of
P < 0.05. All analyses were performed using GraphPad Prism
5.0 for Windows (GraphPad Software, San Diego, CA, USA).

Results
Standardization of the proposed method
Table 2 shows that the 1 M glycine (pH = 5.5, d 1.025) extractor liquid presented the best recoveries of helminth eggs in
lettuce, with an average of 72.3 % (14.5) for hookworm eggs
and 99.3 % (11.5) for the A. suum eggs. Although the distilled water and 0.1 % Tween 80 solution presented also a
good level of recovery, it was slight lower than the 1 M glycine and the 1 % sodium lauryl sulfate solution was the worst
among the four tested extractor liquids. The presence of foam
in the Tween 80 solution was controlled by the addition of an
antifoam agent; however, this reagent had no effect on the
sodium lauryl sulfate solution, and the foam hindered the filtration step and the reading of the slide under the microscope.
Only the sodium lauryl sulfate solution showed significant
differences (P < 0.05) in the recovery of hookworm eggs compared to the other solutions. When considering the recovery of
A. suum eggs, none of the results were significantly different
(P > 0.05).
Further analyses were performed in triplicate using distilled
water and a 1 M glycine solution. These analyses included
contamination of the samples with 50 hookworm larvae in
addition to the helminth eggs. The results are shown in
Fig. 1a. Although a significant difference was not obtained,
we observed that in general, a greater recovery was achieved
using the 1 M glycine solution as the extractor liquid.
Therefore, this solution was chosen as the extractor liquid
for the subsequent standardization tests.
Despite the low speed used during the mechanical agitation, this agitation method produced damage to the lettuce
leaves, generating a greenish sediment and making the
Table 2 Performance evaluation of extractor solutions for egg recovery
after artificial contamination of lettuce samples
N of recovered eggs
Extractor solution

Hookworm eggs

A. suum eggs

Distilled water
0.1 % Tween 80
1 M glycine
1 % sodium lauryl sulfate

70.67 (12,42)
56.00 (2.65)
72.33 (14.57)
16.00 (5.57)

81.67 (24,70)
61.67 (8.08)
99.33 (11.59)
43.33 (21.01)**

**(P < 0.05)

Fig. 1 Standardization of the protocol for helminth recovery from

lettuce. a Test of water and 1 M glycine as extractor liquids. b Test for
the different stirring methods using the extractor liquid (manual stirring
for 1 min, manual stirring for 3 min, and mechanical homogenization
using Stomacher). c Test for different sedimentation times (1, 2, and
24 h)

microscopic analysis of the sediment more difficult. There

was no significant difference (P > 0.05) among any of the
agitation modes tested (Fig. 1b). Therefore, the 3-min manual
stirring was chosen as the sample homogenization procedure,
as it does not require an additional equipment. As shown in
Fig. 1c, the highest level of recovery was obtained with a
sedimentation time of 2 h regardless of the contaminant tested.
However, there was no significant difference among the different sedimentation times. Comparing the results and the
running time of the entire procedure, we chose the spontaneous sedimentation interval of 2 h.
Validation of the protocol for different levels
of contamination
Control samples that were not artificially contaminated and
samples that were artificially contaminated with five different
levels of contamination were analyzed by the standardized

Parasitol Res (2016) 115:18271834

1831

protocol. Only one of the 10 control samples analyzed was

shown to be naturally contaminated, with the presence of two
live nematode larvae (unidentified). All of the other control
samples were found to be free of contaminants.
Table 3 shows the results obtained for the different levels of
artificial contamination (as described in Table 1). No significant difference was found for levels 1, 2, 3, and 4 regardless of
the type of contaminant used. The only difference in recovery
was observed for level 5 contamination, where the average
percentage of hookworm larvae recovered was smaller than
the other samples (30.0 % 36.7; P < 0.05).
The results are shown as percentages and the sum of the
replicates for each level. Based on a total of 50 replicates (10
replicates per level 5 levels), we observed that the best
recovery results were achieved for A. suum eggs. The average
recovery of this contaminant in 50 known positive tests was
62.6 % (20.2) with no false negative results, resulting in a
sensitivity ratio of 100 %.
The average recovery of the 50 artificial contamination
tests for hookworm eggs was 51.9 % (20.0). Only one false
negative result was detected, representing 2 % of the final
results. Therefore, the sensitivity of the method for detecting
hookworm eggs was 98 %. This detection failure occurred at
the lowest level of contamination (level 5), where there were
six eggs per 30 g of lettuce leaves (0.2 egg/g).
The average global recovery of the hookworm larvae was
50.0 % (27.3). We detected five false negative results,
representing 10 % of the total results for that contaminant,
for a sensitivity of 90 %. The misleading results also occurred
at the lowest level of contamination, where only three nematode larvae were added for each lettuce sample of 30 g (0.1
larvae/g).

separated by the type of contaminant. Due to a technical problem, some of the results from laboratory 3 had to be discarded.
Based on the results of the three laboratories and all three
levels of contamination employed in a total of 27 tests, the
average recovery was 52.1 % (37.9) and 27.4 % (20.5) for
the A. suum and hookworm eggs, respectively. Only one false
negative result occurred for each of these contaminants,
resulting in a sensitivity of 96.3 % when executed by other
laboratories. Only 18 results were analyzed for the hookworm
larvae (laboratories 1 and 2), resulting in an average recovery
of 37.4 % (20.9).
Application of the proposed method to another matrix
Table 5 depicts the average percent recovery of helminth eggs
and larvae from arugula. Five minimally processed samples
were analyzed without artificial contamination; one sample
was shown to be naturally contaminated with 27 rotifers
(Rotifera), 1 insect fragment, and 1 whole insect (data not
shown). However, no helminth contamination was detected.
We tested four replicates for each level of contamination
for all five levels for a total of 20 samples. The average recovery for each contaminant was 44.3 % (18.0), 51.2 % (20.4),
and 53.1 % (23.5) for the A. suum eggs, hookworm eggs, and
larvae, respectively. The method had a sensitivity of 100 % for
the helminth eggs, because no false negative results were detected. One of the larvae samples tested showed a false negative result (5 %), producing a sensitivity of 95 % for this
contaminant. This incorrect result was found for the lowest
level of contamination (level 5), in which, three larvae were
added to 30 g of the plant leaves (0.1 larvae/g).

Interlaboratory analyses

Discussion
Table 4 depicts the recovery percentages obtained from the
different laboratories that performed the tests; the results are
Table 3 Average recovery percentages of eggs and larvae using the
proposed detection method. Results of the different levels of artificial
contamination were evaluated
Percentage of eggs and larvae recovereda

Level 1
Level 2
Level 3
Level 4
Level 5
a

A. suum eggsa

Hookworm eggsa

Hookworm larvaea

58.1 (12.7)
61.8 (22.1)
65.0 (15.0)
58.1 (27.6)
70.0 (21.6)

54.2 (8.5)
65.4 (17.4)
51.5 (13.9)
41.6 (18.3)
46.7 (30.2)

56.4 (7.2)
60.8 (16.4)
65.0 (34.4)
38.2 (13.8)
30.0 (36.7)**

Values represent the average of ten replicates. The standard deviation is

shown in parentheses
**P < 0.05

This study aimed to establish a method for the detection of

contaminants in plants using lettuce as a model for the standardization of the method. During separate testing of variations in the most critical steps of the method, we observed that
some parameter interfered with the results in a more forceful
way, while others lead to minor improvements.
The use of 1 % sodium lauryl sulfate solution yielded the
worst recovery percentage due to the presence of large quantities of foam and bubbles in the reading slide. In contrast, Bier
(1991) concluded that the use of a mixture of anionic and
neutral detergents increased recovery compared with the use
of water or saline solution for vegetable washing. Our vegetable washing mode may have interfered with this result,
because the proposed method was performed by manual
shaking, while in the work conducted by Bier, it was performed in an ultrasound bath. Glycine solution has been
also shown to be a good extractor solution for the recovery

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Table 4 Recovery percentage of A. suum eggs, hookworm eggs, and hookworm larvae obtained by the collaborating laboratories after artificial
contamination of lettuce samples
Labs

Rounds

Percentage of A. sum eggs recovery

Percentage of hookworm eggs recovery

Percentage of hookworm larvae recovery

W/Ca

W/Ca

W/Ca

Level 2

Level 3

Level 1

Level 2

Level 3

Level 1

Level 2

Level 3

51

10

30

12

10

38

16

70

52

34

180

17

25

54

20

40

3
1

0
0

43
50

52
102

50
90

0
0

21
31

14
44

15
30

0
0

50
26

32
80

50
10

41

44

140

33

40

85

28

28

50

3
1

0
0

30
79

0
48

45
35

0
0

16
49

8
64

15
55

0
NP

14
NP

8
NP

60
NP

2
3

0
0

25
41

42
28

35
30

0
0

16
13

56
30

30
0

NP
NP

NP
NP

NP
NP

NP
NP

45.8 +/
15.5

40.0 +/
29.0

70.6 +/
54.8

23.1 +/
12.2

29.8 +/
22.4

29.4 +/
26.0

35.0 +/
15.3

30.7 +/
25.6

46.7 +/
20.6

Average +/
SD
a

Level 1

Samples that were not artificially contaminated

NP not performed

of Cryptosporidium parvum oocysts in lettuce (Cook et al.

2006).
In another study, six wash solutions (sterile E-Pure water,
3 % levulinic acid3 % sodium dodecyl sulfate, 1 M glycine,
0.1 M phosphate-buffered saline, 0.1 % Alconox, and 1 %
HClpepsin) were evaluated for their effectiveness in removing Cyclospora cayetanensis, C. parvum, and Toxoplasma
gondii from basil. The lowest variability in the recovering rate
from samples inoculated with 100 oocysts was observed to
1 % HClpepsin wash solution. However, no significant differences between the tested washing solutions were observed
(Chandra et al. 2014).
In the present work, both, manual and mechanical agitation
were tested and observed that the vigorous nature of the latter
caused more damaged to the lettuce leaves, generating
greenish sediment with higher plant debris and making the
microscopic analysis more difficult. Chandra et al. (2014) also

Table 5 Average percentage of recovery of eggs and larvae in arugula

samples after artificial contamination
Percentage of eggs and larvae recovereda

Level 1
Level 2
Level 3
Level 4
Level 5
a

A. suum eggsa

Hookworm eggsa

Hookworm larvaea

53.7 (17.4)
38.0 (18.4)
51.2 (18.0)
33.7 (18.5)
45.0 (19.1)

58.2 (14.8)
33.0 (8.4)
65.0 (28.6)
41.7 (11.8)
58.0 (21.4)

57.0 (7.7)
45.0 (8.9)
72.5 (25.0)
41.5 (9.8)
49.7 (43.0)

Values represent the average of four replicates. The standard deviation is

shown in parentheses

assessed manual and mechanical agitation using the rocker

platform to oocysts recovery in basil samples. They
concluded that the hand shaking method promoted greater
abrasion on the surfaces of the basil leaves, favoring the
removal of oocysts. Therefore, manual shaking was
preferably used in the protocol because the methodology
was simpler and did not require the use of a mechanical
device.
Barnab et al. (2010) compared the sensitivity of the parasitological techniques used by Hoffman, Pons, and Janer
(HPJ) and the Faust technique in lettuce, arugula, and watercress samples. They observed that the first technique, which is
based on spontaneous sedimentation, was more effective for
the detection of helminth eggs and larvae and protozoan cysts
in the plants analyzed. In our work, the timing of spontaneous sedimentation did not affect the detection of the denser
parasite forms, represented here by the A. suum eggs and
hookworm larvae. In contrast, at least 2 h of sedimentation
were required to obtain reasonable recoveries of the hookworm eggs.
When all of the contamination levels (n = 50) were considered together, our proposed method proved to be most effective for the recovery of A. suum eggs (62.6 % 20.2), followed by the recovery of hookworm eggs (51.9 % 20.0) and
larvae (50.0 % 27.3). Bearing in mind that the final results
of parasitological analysis in plants are expressed as the presence or absence of parasites and that the contaminations were
in the range of 3.33 eggs/g to 0.2 egg/g and 1.66 larvae/g to
0.1 larva/g, the average percentage of recovery observed for
the method proposed here is considered satisfactory despite
the standard deviation values. In 1991, Bier presented the
method used by the FDA and evaluated their recovery

Parasitol Res (2016) 115:18271834

percentages. When cabbage leaves were infected with 1 egg/g

and 10 eggs/g, the average recovery for Ascaris sp. or
Trichuris sp. eggs was only 10 %. Robertson and Gjerde
(2000), evaluating the parasite recovery in different vegetables, used a detergent solution in a double washing protocol,
involving mechanical agitation and sonication. In this study,
only four lettuce samples artificially infect (approximately 1.2
eggs/g) were analyzed and the average recovery for Ascaris
eggs was of 68 %.
In this work, we were concerned in establish a methodology that may be easily applied to different laboratories being
reliable and reproducible. These parameters were considered
in the establishment and choice of the methodology developed
in this work. In this sense, the method is simpler and faster
than the method proposed by the FDA. Its robustness was
demonstrated by the application of the protocol to another
matrix (arugula). In this matrix, the average percentage recovery of hookworm eggs and larvae was maintained at the same
level and did not significantly vary from the results found for
the lettuce samples. Although the recovery of A. suum eggs
from arugula was lower compared to the lettuce samples, the
sensitivity of the method was still considered good because a
reasonable percentage of eggs were recovered and no false
negative results were found for this contaminant.
Although the average percentage of recovery obtained by
the collaborative laboratories was lower than the recovery rate
obtained by the original laboratory, these results are considered satisfactory. Of the 27 samples contaminated by parasite
eggs, only two results were mistakenly identified: one false
negative result for A. sum and one for hookworm eggs. The
method was demonstrated to be reliable for the qualitative
detection of A. suum and hookworm eggs and larvae in
lettuce. Cook et al. (2006) standardized a detection method
for C. parvum in lettuce (n = 30) and obtained a mean recovery of 59 12 %. However, the validation of the method by eight other laboratories produced an average recovery of 30.4 %. This result demonstrates that training and
continued practice are required to increase the method
sensitivity.
In addition to the helminth eggs, larval stages that usually
develop in the environment are also often reported as leafy
vegetable contaminants. Barrantes et al. (2011) analyzed 60
samples of lettuce obtained from markets in Lima and Peru; of
these, 38 (63.3 %) were contaminated by filariform and
rhabditoid Strongyloides spp. larvae. In this work, we found
living larvae of nematodes in a lettuce control sample and
other filth materials (i.e., insect fragments and rotifers) in a
control sample of arugula, both of which were minimally
processed. Silva et al. (2007) evaluated the parasitological
quality of 52 minimally processed vegetable samples and detected contamination by Eimeria spp. oocysts in eight samples
(15.3 %), indicating that there was contamination with animal
feces during some stage of vegetable production.

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Conclusion
In conclusion, this study was able to establish a methodology
for detecting eggs and helminth larvae on leaves that could be
used by health surveillance agencies to improve the practice of
supervision of minimally processed foods and those acquired
Bin nature^ and to give support to epidemiological studies.
Acknowledgments We are deeply grateful to Dr. Joziana Barante, Dr.
Adriana Costa, and Maria Helena Martini who performed the interlab
analyses and to Dr. Maringela Carneiro for the critical comments on
the manuscript.
Compliance with ethical standards All procedures followed the recommendations of the Ethics Committee on Animal Experimentation of
Universidade Federal de Minas Gerais (CETEA/UFMG), project number
088/09.

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