Improving Diagnosis of Intestinal Parasites Towards a Migrant-Friendly Health System

This study describes the results obtained by the World Health Organization (WHO) collaborating centre (CC) for the diagnosis of intestinal helminths and protozoa (WHO CC ITA-116) during the first 2 years of its activity on (i) the prevalence of intestinal parasites in migrants in southern Italy and (ii) the development and application of new diagnostic tools for intestinal helminths (e.g. FLOTAC, Mini-FLOTAC Kit 200 tests, Kubic FLOTAC microscope (KFM)). Almost 23.3% of migrants examined were positive for at least one intestinal parasite. Moreover, a significant correlation was found between the duration of stay in Italy and positivity for intestinal parasites. The results of the comparison between diagnostic techniques showed a perfect agreement between FLOTAC and Mini-FLOTAC Kit 200 tests whereas no statistically significant differences were found between the count of helminth eggs obtained by Mini-FLOTAC with optical microscope and KFM. The results obtained by WHO CC ITA-116 during the first 2 years of its activity provide important information on innovations in parasitological diagnosis and add data to the parasitological scenario of migrants arriving in southern Italy, highlighting the importance of regular parasitological monitoring.


Introduction
Over the past decade, the number of migrants and refugees in Europe has increased dramatically due to war, violence, persecution, or poor health in their home countries [1,2]. According to the United Nations High Commissioner for Refugees (UNHCR), about 200,000 people irregularly crossed European land borders in 2021 (this number has increased by 60% compared to 2020), more than 114,000 by sea, mainly in Italy, Greece, Spain, Cyprus, and Malta. In 2021, asylum seekers in Europe came from about 140 countries, in particular from Syria, Afghanistan, and Iraq (https:// ec. europa. eu/ info/ strat egy/ prior ities-2019-2024/ promo tingour-europ ean-way-life/ stati stics-migra tion-europe_ en# Refug eesin Europe).
Despite the sharp increase in the number of migrants, refugees, and asylum seekers (hereafter "migrants") worldwide in recent years, insufficient attention has been paid to their health needs as the World Health Organization (WHO) points out [3••]. This entails developing appropriate free health care and comprehensive medical screening, incorporating psychological health care, and breaking down cultural and linguistic barriers [3••, 4]. Knowledge of the health status of migrants is important for a civil reception and a more effective integration process that respects human rights [4]. These people are at very high risk of infection, not only in their countries but also during the travel, due to sub-optimal hygiene standards, malnutrition, and lack of access to health services [5]. Therefore, a full health examination that includes thorough parasitological screening could have far-reaching public 1 3 health implications and be of great importance in revising the policies and practices applied from the arrival to the final destination of migrants [3••].
Intestinal parasites (helminths and protozoa) are likely to be worth diagnosing through screening strategies. Indeed, many migrants come from countries where parasitic diseases are endemic, e.g. those caused by soiltransmitted helminths (STHs), Schistosoma spp., and other helminths and protozoa [6]. Although these parasites can cause substantial morbidity and mortality, they are seldom diagnosed [7]. As a consequence, data on the prevalence and burden of parasitic diseases among newly arriving migrants in Italy are still scant, with only a few papers published that focused on schistosomiasis and helminthiasis, particularly strongyloidiasis [5,[7][8][9][10]. The Italian guidelines on health assessment for migrants recommend parasitological screening (serology) for Strongyloides stercoralis and Schistosoma spp. when the patients come from endemic areas and copromicroscopy for other intestinal parasites only in presence of symptoms or eosinophilia [11].
To date, the recommended technique for the qualitative diagnosis of intestinal parasites is the formol-ether concentration method (FECM) [12] performed on three different samples. As for quantitative diagnosis, the Kato-Katz technique has been the diagnostic method recommended by WHO since the 1990s for quantifying eggs in stool and then classifying the infection intensity based on faecal egg counts (FECs; expressed as eggs per gram (EPG) of stool) [13]. In the last decade, a variety of new diagnostic methods have been introduced, such as McMaster [14], (Mini-) FLOTAC [15,16], and FECPAKG2 [17]. More recently, the use of advanced technologies, such as automated methods for copromicroscopy based on artificial intelligence, has begun to offer solutions to overcome gaps and limitations of FEC techniques (i.e. human errors and time for analysis) for the diagnosis of various parasites of public health relevance (e.g. STHs, Schistosoma spp., protozoa, etc.) [18].
For more than 20 years, the Centre for Monitoring of Parasites (CREMOPAR, at the University of Napoli Federico II) has been involved in supporting the diagnosis of protozoa and helminths in endemic populations, using multivalent and accurate diagnostic tools [8,16,18,19]. Moreover, CREMOPAR was designated as collaborating centre of the World Health Organization (WHO) for the diagnosis of intestinal helminths and protozoa (WHO CC ITA-116; https:// www. whocc. ita116. unina. it/). Beyond supporting endemic countries in monitoring preventive chemotherapy programmes [20], the WHO CC ITA-116 carries out regular parasitological surveillance and health care for migrants in southern Italy. The present study provides results of two main activities of the WHO CC ITA-116: (i) updating data on the prevalence of intestinal parasites among migrants in southern Italy and (ii) improving diagnostic approaches for intestinal parasites.

Materials and Methods
From July 2020 to March 2022, 373 migrants who arrived in the Campania region (southern Italy) were screened for intestinal parasites. The spokes for the stool collection were set in three different migrant assistance centres in the region: (i) extraordinary reception centres (CAS), (ii) Protection System for Beneficiaries of International Protection and for Unaccompanied Foreign Minors (SIPROIMI)/ Reception and Integration System (SAI), and (iii) Medical Centre for the Health Protection of migrants (ASL-NA1). At the time of stool collection, patients were not taking drugs or other substances that could affect the results of the parasitological examination. The samples were collected after the signature of an informed consent document by the participants indicating that they understood the purpose and the procedures required for the study and that they were willing to let their child participate in the study (in the case of parents of children). This study was approved by the ethics committee of the University of Naples Federico II.
All samples were analysed in the laboratories of the WHO CC ITA-116 as described in the study design ( Fig. 1), and all individuals resulted positive for intestinal parasites (helminths and protozoa) received targeted antiparasitic treatment.
Second, a sub-sample of 90 stool samples was used to validate for the first time the Mini-FLOTAC Kit 200 tests ( Fig. 2) for field diagnosis of helminth infections. Each sample was homogenized and analysed by Mini-FLOTAC using FS2 and FS7, according to the protocol described by Cringoli et al. [16] and recommended by the WHO Bench Aids [21].
Finally, the first validation of the Kubic FLOTAC microscope (KFM) [18] was performed on 23 stools resulted positive for STHs (16 for hookworms and 7 for Trichuris trichiura) using the Mini-FLOTAC technique. The performance of the Mini-FLOTAC was evaluated using either a traditional optical microscope (OM) or KFM.
For each positive subject, the number of cysts/oocysts/ eggs and larvae per gram (CPG/OPG/EPG/LPG) of faeces was calculated. Furthermore, a questionnaire was filled out for each patient, reporting age, sex, country of origin, alimentary habits, health status, and duration of stay in Italy. The country of origin of each migrant was georeferenced in a geographical information system (GIS) using ArcGIS Pro 2.7 (Environmental Systems Research Institute, Inc., Redlands, California, USA).

Statistical Analyses
The overall positivity was analysed in correlation with the variables: gender, age class, geographic origin of migrants, and duration of stay in Italy using Pearson's chi-squared test. The association was considered significant at P < 0.05.
The performance comparison (i.e. sensitivity and negative predictive value, NPV) of the diagnostic methods used for the detection of intestinal parasitic infections was calculated considering the combined results as a "Gold standard" [22]. The agreement between the diagnostic techniques (FLO-TAC and Mini-FLOTAC) for detecting intestinal parasites was calculated using Cohen's κ statistic [23]. The κ measure was interpreted as follows: 0, no agreement; 0.01-0.20, poor agreement; 0.21-0.40, fair agreement; 0.41-0.60, moderate agreement; 0.61-0.80, substantial agreement; and 0.81-1.0, nearly perfect agreement.
All statistical analyses were performed using version 23 of SPSS software for Windows (SPSS Inc., Chicago, IL, USA).
A total of 87 migrants resulted positive for at least one intestinal parasite with an overall prevalence of 23.3% (95%CI = 19.2-28.0); of these 19 subjects (21.8%; 95%CI = 14.0-32.2) were co-infected with two or more helminth and/or protozoa species. Positivity to intestinal parasites according to the country of origin is summarized in Table 1. Table 2 reports the number of positives, the prevalence (%), and EPG/LPG/OPG/CPG (min-max or single value in case of one positive sample) for each helminth/protozoa detected. No significant association (P > 0.05) was found between the positivity to parasites and the following independent variables: age, sex, country of origin, alimentary habits, and health status. However, a statistically significant difference was found between the duration of stay in Italy and the presence of intestinal parasites (63.6% of migrants resulted positive for intestinal parasites stayed in Italy for less than 1 year; P = 0.0076). Table 3 shows the results of the comparison between the diagnostic techniques. Out of the 90 subsamples, the number of samples that resulted positive for helminths using each technique was as follows: 52 (57.8%, 95%CI = 46.9-68.0) with  Table 4 shows the performance of each technique. A nearly perfect agreement (κ = 0.93; P < 0.01) was observed between FLOTAC and Mini-FLOTAC Kit 200 tests.
No statistically significant differences (P > 0.05) were found between the total STH eggs (only hookworms and T. trichiura, since any sample was positive for Ascaris lumbricoides), counted by the Mini-FLOTAC with OM and with KFM (320 EPG vs 320 EPG). The time for counting helminth eggs in the Mini-FLOTAC chambers under the OM  was ∼1-5 min, whereas the time for reading the Mini-FLO-TAC with KFM was ∼3-8 min. Finally, 1185 images were acquired for the development of artificial intelligence (AI) and deep learning (DL) software for the automatic detection of hookworms and T. trichiura (Fig. 4) (a paper on validation of KFM and AI and DL software training in the human field is in preparation).

Discussion
The present study showed an overall prevalence of parasites in migrants in southern Italy of 23.3% (13.9% for helminths and 14.7% for protozoa), similar to other studies conducted in Italy [7,8,24,25]. However, it is not always easy to compare data on the prevalence of parasites in different areas/countries, due to a variety of confounding factors affecting the results of parasitological screenings, such as selection bias (i.e. mainly enrolled subjects attending hospitals, outpatient clinics) [7], as well as differences in diagnostic approaches (i.e. coprology, immunology, molecular biology), and laboratory protocols [26•].
We found a higher prevalence of intestinal parasites in migrants from African and Asian countries that are endemic for parasitic infections [27], although the prevalence registered in Italy is different (not only in terms of rate, but also in terms of parasite species) from the geographical regions of birth, as also reported by other authors [7,28]. According to these authors, this may be due to the migration route which is sometimes very long and crosses many countries, and to the mixing of different ethnic groups which could affect the species of intestinal parasites in different migrant populations.
The most common helminths found in this study were hookworms (28.7%) and T. trichiura (19.5%). This is consistent with data from the WHO which indicated that STHs are among the most prevalent parasites worldwide, with approximately 1.5 billion people infected and 2.0 billion people at risk of disease by STH [3••]. Ascaris lumbricoides was not detected in any of the studied subjects, although it was previously found with a low prevalence (0.7-1.4%) in migrants in the Campania region [8,19,29]. Further studies could be interesting to understand how the parasitological scenario in migrants in this Italian region has changed over the years and in relation to the origin of migrants.
A significant association was found between the decreasing positivity to parasites and duration of stay in Italy (63.6% of migrants who tested positive for intestinal parasites had lived in Italy for less than 1 year). This could be due to the improvement in social conditions (i.e. stable accommodation, job opportunities, availability of health care, etc.) as reported by other authors [6,7,19].
Moreover, this paper provided interesting insights into the performance of copromicroscopic techniques. Most intestinal parasites do not give specific clinical signs and therefore can only be detected by laboratory diagnosis [3 ••]. As previously reported, FLOTAC and Mini-FLOTAC are highly sensitive, accurate, and precise techniques for the diagnosis of intestinal parasites in humans [15,16,19,[31][32][33][34][35][36][37][38][39]. The only three stool samples that were negative with Mini-FLOTAC had parasitic levels (4 EPG) below the detection limit of this technique (in this case, the analytic sensitivity is 10 EPG at a dilution factor of 1:20).
A rapid diagnosis is necessary for an appropriate strategy of intervention. A field technique such as the Mini-FLOTAC Kit 200 tests could be very useful to perform efficient, rapid, and reliable diagnosis directly upon on the arrival of migrants in our country, thus preventing chronic diseases and complications and ensuring public health [7]. The main limitation of on-site diagnosis with a portable microscope is that it is not able to achieve high magnification to enable the detection of protozoa.
For this reason, we validated the Mini-FLOTAC Kit 200 test for helminths only.
The use of innovative AI-based tools, such as KFM, could be very important to complete and improve on-site diagnosis Fig. 4 Development of the AI software for the recognition of hookworms in human stool samples of intestinal parasites (i.e. for all helminths and protozoa) and overcome the gaps and limitations of quantitative techniques (i.e. human errors and time for analysis), increasing diagnostic efficiency. This tool has already been successfully used for the detection of gastrointestinal nematodes in cattle [18] and in companion animals [40]. In this study, the KFM was successfully used to count STH eggs, and no differences were observed between the total number of eggs counted using the Mini-FLOTAC at OM and the KFM (320 EPG vs 320 EPG), although only positive samples were used to assess the diagnostic accuracy of KFM. Further studies are needed for the validation of KFM, also using negative samples. In addition, the AI software was trained for the first time to detect STH eggs in stool samples, which yielded promising results. In the future, the KFM will be trained to detect other helminths and protozoa. Thanks to remote control via software using a smartphone, tablet, or PC, or via the Internet, it is possible to transmit the acquired images to other laboratories, which could be very useful to create a network or to support operators directly in the field.
Therefore, the combination of these innovative tools (i.e. Mini-FLOTAC Kit 200 tests and KFM) could be very important to improve the diagnosis of intestinal parasites, as well as to implement the parasitological assays provided in the "Italian guidelines on Health assessment for migrants and asylum seekers upon arrival and while hosted in reception centres" [11], towards a migrant-friendly health system.

Conclusion
The results of this study add data to the parasitological scenario of migrants arriving in southern Italy and provide important information on innovations in parasitological diagnosis, highlighting the importance of regular parasitological screening as soon as possible after their arrival.
These activities are in line with the mission statement of our WHO CC ITA-116 collaborating centre focused to improve public health worldwide and to achieve the main goals of the Neglected Tropical Diseases (NTD) 2021-2030 roadmap to validate and standardize innovative diagnostics for the control and elimination of intestinal parasites.

Data Availability
The data that support the findings of this study are available on request from the corresponding author.

Conflict of Interest
The authors declare no competing interests.

Human and Animal Rights and Informed Consent
This article does not contain any studies with human or animal subjects performed by any of the authors.
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