A cross-sectional study was conducted on fresh vegetables collected from Al Forda Central Fruits and Vegetables Market in Sulaibiya Kuwait, the country’s largest wholesale market for fresh produce.

From February 2023 to February 2024, a total of 180 locally grown and imported fresh leafy vegetable samples i.e., equal numbers of lettuce (Lactuca sativa), iceberg lettuce (Lactuca sativa var. capitata), arugula (Eruca vesicaria), parsley (Petroselinum crispum), cilantro (Coriandrum sativum), and dill (Anethum graveolens) were randomly selected and purchased from the open market vendors. These vegetables were selected as they are commonly used as “ready to eat” salads without washing or cooking, thus posing a higher risk of infection. The vegetable type, country of origin, and date of collection were recorded, and the vegetables were transported in sealed sterile plastic bags to the Research Core Facility, College of Medicine, Kuwait University for processing.

There are several published methods to analyze vegetable material for the presence of parasitic eggs. A series of methods were performed to standardize our sample processing method. We determined that the most effective method to detect parasites eggs on leafy vegetables was to peel off the vegetables into small pieces before rinsing rather than mincing or homogenizing them to small pieces as that led to many plant cells which interfered with the microscopy of the slides. An equal amount (300 grams) of each vegetable sample was peeled off, broken into small pieces, and thoroughly rinsed in a bowl containing 500 ml of filtered water followed by vigorous shaking. Modified from Healy et al. [10] , the rinsate was then collected and dispensed in 50 ml conical tubes, centrifuged at 2000 × g for 10 min, and the supernatant was discarded. The pellet was resuspended in 5 ml normal saline solution and 200 µl of Lugol’s iodine solution was added to 2 ml of sample solution, from which 50 µl were transferred to a microscope slide and the whole slide was scanned for parasite eggs/cysts/larvae using the Zeiss Axio Imager M1 light microscope with AxioCam camera under 20x objective lens. The parasites were counted, examined for internal structures, and photographed under 40x objective lens.

Safranin-methylene blue staining was used as described previously [19] to facilitate the detection of coccidian parasites that are less than 20 µm in size. Briefly, 30 µl of sample in normal saline were smeared onto a microscope slide, left to air dry, and passed transiently over a flame. Then, the smear was fixed in 3% HCI in absolute methanol for 5 min and then washed with water. The smear was then stained with 1% aqueous safranin for 60 sec, heated thoroughly until steaming, and washed with water. Finally, the smear was counterstained with 1% methylene blue for 30 sec, washed with water, and blotted dry. The stained smear was then screened for parasites using 20x objective lens and the detected parasites which appeared deep pink to red against a blue background were photographed under 63x oil immersion objective lens. The images were then enhanced, and the parasite size was measured using netScope^® Viewer software version 1.11.8543.20354. Parasite identification was confirmed by referring to the Centers for Disease Control and Prevention (CDC) DPDx image gallery and Google Lens image recognition technology.

The parasitic load on the contaminated vegetables was reported as the number of eggs/cysts/larvae per vegetable unit (300 grams). Descriptive statistical analysis was performed, and the chi-square test was used to test the association between the frequency of parasitic contamination and the country of origin, and the association between the frequency of parasitic contamination and the vegetable type using Microsoft Excel^® Analysis ToolPak software. A p-value < 0.05 was considered as significant.

By vegetable type, Jordanian lettuce showed both the highest parasitic prevalence rate (3 out of 20, 15%) and the highest parasitic diversity with 4 species detected (i.e., Hymenolepis diminuta, Entamoeba coli, Iodamoeba spp., and Anoplocephala spp.) ( Figures 1(a)-(d) ).

This finding is comparable to an earlier study from Jordan which reported a high parasitic contamination rate of 29% in 133 salad vegetables screened, from which the highest contamination rate was found on lettuce samples (63%), which was significantly higher in comparison with tomato, parsley and cucumber (p = 0.006, Odds ratio = 4.6; p = 0.001, Odds ratio = 5.5 and p < 0.0001, Odds ratio = 46.6, respectively [22] . Interestingly, several studies from regional countries also reported that lettuce had the highest parasitic contamination rate in comparison with other vegetables, including Iran (91.1%; [14] , Egypt (45.5%; [8] , Syria (29.5%; [15] , Lebanon (23.3%; [16] , and Iraq (15.0%; [17] . While only 1 Jordanian iceberg lettuce head out of 20 (5%) was contaminated with Strongyloides spp. egg containing larva ( Figure 1(e) ), no parasitic contamination was found on any iceberg lettuce samples imported from Oman. A study from India reported a parasitic contamination rate of 19.72% on fruits and vegetables collected from local markets in Tiruchirapalli, Tamilnadu and tomatoes, carrot and beetroot were the most frequently contaminated fruits and vegetables with diverse types of parasites [11] .

Kuwaiti celery leaves had the same prevalence of parasitic contamination as Jordanian lettuce (3 out of 20, 15%), with 3 species detected (i.e., Heterophyes heterophyes, Strongyloides stercoralis larva, and Sarcocystis spp.) ( Figures 2(a)-(c) ).

Although Kuwait is considered non-endemic for strongyloidiasis, several cases have been reported previously; two studies reported 6 cases of fatal strongyloidiasis hyperinfection following kidney transplantation from South Asian donors in conjunction with immunosuppressive therapy [23] [24] , while the third study reported 4 cases (2 Kuwaiti, 1 Iraqi, and 1 Bangladeshi) of disseminated strongyloidiasis following the prescription of prednisolone and other steroids for pre-existing health conditions [25] . Kuwaiti farmers commonly employ workers from Strongyloides-endemic South Asian countries such as India and Bangladesh. Hence, detecting Strongyloides spp. on Kuwaiti celery may be attributed to open-field defecation and/or expectoration by infected workers, or may indicate the use of untreated soil or animal manure fertilizer contaminated with Strongyloides spp. eggs.

To our knowledge, no human cases of sarcocystosis or heterophyiasis have been reported previously in Kuwait. However, an earlier study screened 240 stray cats for enteric parasites and detected Sarcocystis felis oocysts in 1 out of 240 (0.4%) fecal specimens [26] . Another study from Kuwait examined 240 stray cats for trematode infections and reported an overall prevalence rate of 24.6%, from which Heterophyes heterophyes was the most frequently reported (15.8%) trematode among 13 other species identified [27] . Thus, the detection of both Sarcocystis spp. and Heterophyes heterophyes on Kuwaiti celery samples in this study warrants an investigation into the prevalence of these parasites in stray cats located inside and around Kuwaiti farms.

This study detected parasites on 2 out of 20 (10%) Kuwaiti arugula bunches; 1 had Cryptosporidium spp. while the other had Giardia intestinalis cyst, and both were seen in safranin-methylene blue smears ( Figure 2(d) , Figure 2(e) ). In Kuwait, Cryptosporidium spp. and Giardia duodenalis infections have been reported previously among Kuwaiti children suffering from diarrheal illness with prevalence rates of 3.4% and 0.2%, respectively [28] , while a more recent retrospective study reported Giardia duodenalis infection in 0.8% of hospital patients of all ages [29] . A veterinary study in Kuwait reported a high prevalence of Cryptosporidium spp. infection among pre-weaned calves in dairy cattle farms with a rate of 33.1% [30] , a markedly higher rate than that in humans. Accordingly, it is more likely that the Cryptosporidium spp. detected on Kuwaiti arugula were transmitted by infected animals to the soil which was used to cultivate these vegetables. It is important to note that the safranin-methylene blue stain was of limited use in this study since the safranin dye commonly stained non-parasitic structures such as pollen grains and other artifacts of comparable size with coccidian parasites. Therefore, we do not recommend the use of safranin-methylene blue staining on samples of high biological diversity collected from outdoor environments (e.g., soil, water, vegetables and fruits), as these samples often harbor pollen grains, fungal spores and conidia, dinoflagellates, and microalgae.

Only 1 out of 20 (5%) Kuwaiti parsley bunches were contaminated with Eimeria spp. oocyst ( Figure 2(d) ). While there are no reports on human cases of Eimeria spp. infection in Kuwait, a previous study screened fecal specimens of farm animals for parasites and reported Eimeria spp. as the most common parasite in cattle, sheep, and goats [31] . Therefore, the parasitic contamination of locally grown arugula and parsley with Cryptosporidium spp. and Eimeria spp., respectively rather than transmission from an infected human. It was noted that some farmers used animal manure as organic fertilizer for vegetables, especially those who could not afford or had the facility of greenhouse plantation or contamination may have been transmitted by the unhygienic practices of the workers at the forms or vendors at the market, who had come from the regional developing Arab and Asian countries. All the locally grown lettuce, cilantro, and dill samples tested negative for parasitic contamination, which may be attributed to a variation in parasitic burden between individual farms. Accordingly, we expect that future surveillance studies which incorporate a larger sample size of fresh vegetables including fruits collected from different farms in Kuwait will reveal a higher prevalence of parasitic contamination. According to a recent report from the Ministry of Public Health >46% of the population, especially older children and young adults are obese in Kuwait recommending public health awareness on weight control strategies including healthy diet containing fresh salads and fruits. Detecting parasitic contamination on locally grown vegetables in Kuwait paired with an ever-rising trend in RTE salad marketing and consumption necessitates routine surveillance of parasitic contamination on RTE raw foods. This study highlights the importance of effective public health measures, including personal hygiene and vegetable washing, to reduce the risk of transmission of infection by consumption of vegetables.