**A Fuzzy Comprehensive Assessment and Hierarchical Management System for Urban Lake Health: A Case Study on the Lakes in Wuhan City, Hubei Province, China**

**Teng Wang 1,†, Jingjing Yan 2,†, Jinlong Ma 2, Fei Li 2,\*, Chaoyang Liu 2, Ying Cai 2, Si Chen 3, Jingjing Zeng 2 and Yu Qi 2**

1 Hubei Research Center of Water Affair, Hubei University of Economics, Wuhan 430073, China<EMAIL_ADDRESS>

Received: 13 October 2018; Accepted: 18 November 2018; Published: 22 November 2018

**Abstract:** Environmental assessment of eutrophication or heavy metals in urban lakes is an important reference for identifying the pollution degree and formulating pollution prevention strategies. At present, the most research on lake health states is often evaluated from a single angle for toxic metals pollution or eutrophication using the standard comparison method for both, the comprehensive trophic level index (TLI), and the health risk assessment for toxic metals. Moreover, the above deterministic methods probably lead to biased or unreliable assessment due to the randomness and fuzziness in environment system caused by natural change and human activities. In this paper, a fuzzy comprehensive lake health assessment method (FCLHAM) was established to evaluate comprehensive lake health states more comprehensively and accurately, which integrates quantitative eutrophication and health risk considerations. To test and verify FCLHAM, 21 lakes, scientifically selected from the total 143 lakes in the Chinese Wuhan city as study case, were investigated and analyzed for their state of eutrophication and the health risk posed by heavy metals. According to the FCLHAM, the average comprehensive lake health state decreased in the sequence of L20 (considerate risk level) > L1–L17, L19, L21 (moderate risk level) > L18 (low risk level). Based on the result, lakes were classified into three categories: general management (L18), enhanced management (L1–L17, L19, L21), and priority management (L20). If the 143 lakes in Wuhan were classified by the "area-region-function" classification, they would be assigned to the same category as the representative lakes of the same type. At this point, we will attribute all of Wuhan's lakes to the three types. Depending on the characteristics of each type, a targeted approach to different types of management for each type of lake is a more efficient way to manage many of Wuhan's lakes. This management mode also serves as an effective reference for the environmental management of urban lakes both at home and abroad. In other words, according to the FCLHAM, a hierarchical management system based on lake characteristics classification was obtained.

**Keywords:** urban lake; comprehensive nutrition status Index; heavy metals; health risk; fuzzy comprehensive method


*Editorial* **Editorial for the Special Issue: Diagnosis, Epidemiology and Transmission Dynamics of** *Cryptosporidium* **spp. and** *Giardia duodenalis*

**Pamela C. Köster, David González-Barrio \* and David Carmena**

Parasitology Reference and Research Laboratory, Spanish National Centre for Microbiology, Majadahonda, 28220 Madrid, Spain<EMAIL_ADDRESS>(P.C.K<EMAIL_ADDRESS>(D.C.)

**\*** Correspondence<EMAIL_ADDRESS>Tel.: +34-91-822-364

*Cryptosporidium* spp. and *Giardia duodenalis* are major contributors to the global burden of diarrhoeal disease, primarily affecting young children in poor-resource settings. Cryptosporidiosis ranks second to rotavirus infection as a cause of life-threatening diarrhoea in children younger than two years of age, in sub-Saharan Africa and south Asia [1]. In contrast, giardiasis is rarely a cause of childhood mortality, but the disease has been consistently associated with growth faltering and cognitive impairment in malnourished children [2]. Intriguingly, large epidemiological studies, based on case-control data, have evidenced that *G. duodenalis* was more frequent among controls (individuals without diarrhoea) than in cases (individuals with diarrhoea) [1,3,4]. This finding has been interpreted by some authors as evidence in favour of a potential "protective" effect of the parasite against diarrhoea, and explains why *G. duodenalis* infections are systematically absent in global burden estimations of diarrhoeal diseases [5].

At least 44 *Cryptosporidium* species and more than 120 genotypes, as well as nine *Giardia* species are currently recognised [6]. Only three *Cryptosporidium* species (mainly anthroponotic *C. hominis* and zoonotic *C. parvum* and *C. meleagridis*) are responsible for most (circa 95%) human cases of cryptosporidiosis reported globally. *Giardia duodenalis* is the only *Giardia* species infective to humans, comprising eight (A–H) distinct genetic groups (the so-called assemblages) of which zoonotic assemblages A and B are commonly reported to infect humans [6]. Because of the large number of morphologically identical (but genetically different) species/genotypes, within both groups of protozoa, molecular-based tools, including Sanger sequencing, are needed for detection, differentiation, and subtyping purposes [7]. Molecular data are also essential to characterize the transmission dynamics of *Cryptosporidium* spp. and *G. duodenalis*, including for the identification of sources of infection and spread pathways, the tracking of virulent genetic variants or the assessment of zoonotic potential. The most common genetic markers used in subtyping analyses include the *Cryptosporidium* 60-kDa glycoprotein (*gp60*) gene, and the *G. duodenalis* β-giardin (*bg*), glutamate dehydrogenase (*gdh*), and triosephosphate isomerase (*tpi*) genes [7].

This special issue includes 14 papers that made important contributions in expanding our current knowledge, on aspects relevant to the diagnosis and epidemiology of *Cryptosporidium* spp. and *G. duodenalis*. These include the usefulness of PCR-based methods, for the first-line routine diagnosis of *Cryptosporidium* spp. in clinical settings [8], or for enlarging the available arsenal of subtyping tools, to assess *Cryptosporidium* intra-species genetic diversity in canine-adapted *C. felis* [9] and ovine-adapted *C. xiaoi* [10]. We were lucky enough to receive relevant contributions dealing with the human molecular epidemiology of *Cryptosporidium* spp. and *G. duodenalis* in a low-income country, such as Mozambique [11–13], in medium-income countries, such as Brazil and China [14,15], and in a high-income country, such as Sweden [16]. Finally, this special issue also included molecular epidemiological studies directed to assess the occurrence, genetic diversity and

**Citation:** Köster, P.C.; González -Barrio, D.; Carmena, D. Editorial for the Special Issue: Diagnosis, Epidemiology and Transmission Dynamics of *Cryptosporidium* spp. and *Giardia duodenalis*. *Pathogens* **2022**, *11*, 141. https://doi.org/10.3390/ pathogens11020141

Received: 19 January 2022 Accepted: 21 January 2022 Published: 24 January 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

zoonotic potential of *Cryptosporidium* spp. and *G. duodenalis* in pet dogs and cats [17], livestock [18], and farmed rabbits and rats [19–21], mostly in China.

In their study, Costa et al. [8] evaluated the diagnostic performance of four "in-house" and four commercial PCR methods for the detection of *Cryptosporidium* spp. against a panel of selected stool samples, belonging to the collection of a French reference laboratory. The authors identified significant differences in the performance of the compared assays, highlighting the need for proper validation and standardization before routine clinical use. An asset of this study was the inclusion of less frequent or rare *Cryptosporidium* species and genotypes (*C. cuniculus*, *C. felis*, *C. meleagridis*, *C. ubiquitum*, *C.* sp. chipmunk genotype), in addition to *C. hominis* and *C. parvum*. All four commercial assays were able to identify all *Cryptosporidium* species and genotypes evaluated, but this was not always the case for the "in-house" protocols.

Li et al. [9] confirmed and expanded the usefulness of the 60-kDa glycoprotein (*gp60*) gene subtyping tool for assessing the genetic diversity within *C. felis*, initially developed by Rojas-Lopez et al. [22]. To do so, the authors identified the subtypes of 20 *C. felis* isolates, obtained from stray, sheltered, and pet cats, in Guandong province and Shanghai city in China. A high intra-species genetic diversity was observed, resulting in the identification of 13 novel and two known subtypes of the parasite. The main contribution of this survey was the demonstration that many of these genetic variants were shared between cats and humans, strongly suggesting that there could be cross-species transmission of *C. felis*. Furthermore, Fan et al. [10] developed a new *gp60* subtyping tool for *C. xiaoi*, a *Cryptosporidium* species adapted to infect sheep and goats, for which this tool was previously lacking. The authors tested 355 *C. xiaoi*-positive samples from Chinese sheep and goats and found an extremely large intra-species genetic diversity, resulting in the identification of 12 (XXIIIa to XXIIIl) subtype families. This study complements and expands the available *gp60* subtyping toolbox, including protocols adapted to *Cryptosporidium* species, such as *C. hominis* and *C. parvum* [23], *C. fayeri* [24], *C. meleagridis* [25], *C. ubiquitum* [26], *C. viatorum* [27], *C. felis* [22], *C. ryanae* [28], *C. canis* [29], and *C. bovis* [30].

This special issue brings together new epidemiological data that contribute to improving our understanding on the current situation of human cryptosporidiosis in Mozambique, one of the least developed countries in sub-Saharan Africa. In a seminal molecular-based study conducted in Zambézia province, Muadica et al. [11] investigated the presence and genetic diversity of *Cryptosporidium* spp., and other intestinal micro-eukaryotes, in a large population (*n =* 1093) of asymptomatic and symptomatic schoolchildren. The authors found a low prevalence of *Cryptosporidium* infections (1.6%) and managed to genotype 13 isolates of the parasite. Three species (*C. hominis*, *C. parvum*, and *C. felis*) were identified at equal rates (31% each), with *C. viatorum* being detected in 7% of cases. These preliminary results were further confirmed and expanded in a subsequent retrospective study, conducted by Messa et al. [12], taking advantage of a large panel of *Cryptosporidium*-confirmed DNA samples (*n* = 190), obtained during the Global Enteric Multicenter Study (GEMS) at the Manhiça district in the Maputo province [1]. The GEMS project was specifically designed as a case-control study to determine the etiology and population-based burden of paediatric diarrheal disease in sub-Saharan Africa and South Asia [31]. In their study, Messa et al. [12] identified three *Cryptosporidium* species including *C. hominis* (73%), *C. parvum* (23%), and *C. meleagridis* (4%). Both *C. hominis* and *C. parvum* were more prevalent among children with diarrhoea than in children without it (48% vs. 33%). A large intra-species genetic variability was observed within *C. hominis* (*gp60* subtype families Ia, Ib, Id, Ie, and If) and *C. parvum* (*gp60* subtype families IIb, IIc, IIe, and IIi) but not within *C. meleagridis* (*gp60* subtype family IIIb). Molecular genotyping data, provided by Cossa-Moiane et al. [13], in young children (*n* = 319) presenting with diarrhoea at hospital settings in the Maputo province, pointed in the same direction. In this study, a microscopy-based *Cryptosporidium* prevalence of 11% was obtained. In addition, typing results were available from a subset (*n* = 29) of these *Cryptosporidium*-positive samples, confirming the predominance of *C. hominis* (93%) over *C. parvum* (3%). A mixed infection of *C. hominis* and *C. parvum*

was also detected (3%). Taken together, these three studies strongly suggest that human cryptosporidiosis in Mozambique is mainly of anthropic nature, although domestic dogs, cattle, and avian species can act as source of human infection in certain areas. This situation reflects the coexistence of different transmission pathways of cryptosporidiosis in the country.

Understanding the public health significance of emerging diarrhoea-causing microeukaryotes, including *Cryptosporidium* spp. and *G. duodenalis*, is increasingly attracting research interest in rapidly developing countries, such as Brazil and China [32,33]. In the former country, Köster et al. [14] investigated the occurrence and genetic diversity of *G. duodenalis* in a community survey of indigenous people (*n* = 574) from the Brazilian Amazon. During the four consecutive sampling campaigns of the study, *G. duodenalis* prevalence rates varied from 13–22% and primarily affected individuals younger than 15 years of age. Near 75% of the infections were attributed to the assemblage B of the parasite. Remarkably, no association between the *G. duodenalis* genotype and the occurrence of diarrhoea could be demonstrated. This finding is in agreement with the results obtained in a recent study conducted in Mozambican children younger than five years of age [34]. Considered together, these findings indicate that the parasite genotype does not suffice to explain, per se, the progression from infection to disease. Little information on the molecular variability of *Cryptosporidium* spp. and *G. duodenalis* is still available from Chinese human populations. Zhang et al. [15] attempted to overcome this gap in knowledge by analysing stool samples (*n* = 507) from randomly selected individuals, with and without gastrointestinal manifestations, seeking medical attention at hospital settings in the Yunnan Province. Interestingly, no *Cryptosporidium* infections were identified in the surveyed clinical population, whereas a low *G. duodenalis* occurrence rate (2%) was found. Sequence analyses of the *G. duodenalis*-positive isolates confirmed that assemblage A was far more prevalent than assemblage B (90% vs. 10%, respectively). The large, geographicaldependent difference in assemblage frequencies, documented in the studies mentioned above, may be indicative of different sources of infection and transmission routes.

*Cryptosporidium* spp. and *G. duodenalis* are a public health concern, not only in low- and medium-income countries, but also in developed nations [35]. In their study, Lebbad et al. [16] retrospectively genotyped *Cryptosporidium*-positive stool samples (*n* = 398) collected in 12 of the 21 regional laboratories that carry out routine parasitological diagnosis in Sweden. The cohort of stool samples analysed included patients that acquired the infection in the country and abroad. The authors identified 12 distinct *Cryptosporidium* species/genotypes, with *C. parvum* (75%) and *C. hominis* (12%) accounting for the majority of the cases identified. A very large intra-species genetic diversity was detected, allowing the identification of 29 *gp60* subtype families including four novel ones (*C. parvum* IIr, IIs, IIt, and *Cryptosporidium* horse genotype VIc). The authors also reported a human infection by rodent-adapted *C. ditrichi*, a *Cryptosporidium* species very rarely found in humans [36]. Another major contribution to this survey was the demonstration that almost 8% of human cryptosporidiosis cases in Sweden had a zoonotic origin. However, it is still unclear whether some of these findings (e.g., *C. erinacei*, *C. ditrichi*, *C.* horse genotype) correspond to true or spurious infections.

From the human molecular data presented above, it is clear now that zoonotic transmission is a significant route of *Cryptosporidium* spp. and *G. duodenalis*, spreading in several settings, either in developing or developed nations. Human infections can arise through direct contact with infected animals, or through accidental ingestion of contaminated water or food with the faecal material of these animals [37–39]. This special issue included five articles dealing with the potential role of companion, livestock, and farmed animals as potential sources of human cryptosporidiosis and giardiasis. Most of these studies were conducted in China. In the first one, Wang et al. [17] investigated the presence and genetic diversity of *Cryptosporidium* spp. and *G. duodenalis* in faecal samples from pet dogs (*n* = 262) and cats (*n* = 171), collected in veterinary clinics, markets and shelters, from the Yunnan province. Reported infection rates for *G. duodenalis* and *Cryptosporidium* spp. were 14% and 5% in

dogs, and 1% each in cats, respectively. Sequence analyses revealed that dogs were infected only by canine-adapted *Cryptosporidium* (*C. canis*) and *G. duodenalis* (assemblages C and D) species/genotypes. Similarly, cats were infected by feline-adapted *C. felis* and *G. duodenalis* assemblage F. These data indicate that pet dogs and cats play a marginal role as sources of human cryptosporidiosis and giardiasis. It should be noted that most molecular-based surveys conducted so far, failed to demonstrate zoonotic transmission events between pet dogs/cats and humans [40,41]. In another survey, Cao et al. [18] assessed the occurrence and molecular diversity of *Cryptosporidium* spp. in faecal samples (*n* = 476) from Bactrian camels in the Xinjiang Uygur Autonomous Region. In this Chinese area, the Bactrian camel is one of the few large animal species suitable for livestock production, providing clothing, milk, meat, and transport for many people. The authors found a PCR-based prevalence of 8% and identified six different *Cryptosporidium* species circulating in the surveyed camel population. Of them, *C. andersoni* (67%) and *C. parvum* (17%) accounted for eight out of ten infections. Other, less frequent species, included *C. occultus*, *C. ubiquitum*, *C. hominis*, and *C. bovis*. The study represents the first report of *C. hominis* (*gp60* subtype family Ik) in Bactrian camels, expanding the known range of suitable hosts for this pathogen in China that already included cattle [42], donkeys [43], horses [43], and non-human primates [44], beside humans. Some authors have interpreted all this information as evidence in support of considering *C. hominis* as a zoonotic species [45]. Regarding intensive animal farming, Cui et al. [19] investigated the presence, molecular variability and zoonotic potential of *G. duodenalis* in coypus (*n* =308), reared in fur farms in six Chinese provinces/autonomous regions. The parasite was detected in all farms investigated at variable infection rates, ranging from 1–29%. Subtyping data was available for 38 isolates, of which 95% were assigned to assemblage B and the remaining 5% to assemblage A. These data indicate that giardiasis could be an occupational risk for those individuals working in close contact with the farmed coypus or their excreta. In a similar study conducted in farmed bamboo rats (*n* = 724), in Guangdong province, Li et al. [20] identified *Cryptosporidium* infections in 12% of the investigated animals. Sequence analyses of the isolates positive to the parasite allowed for the identification of five distinct *Cryptosporidium* species/genotypes, with *C.* bamboo rat genotype I (56%) and *C. parvum* (35%) being the most prevalent genetic variants found. The remaining positive samples belonged to *C.* bamboo rat genotype III (6%), *C. occultus* (2), and *C. muris* (1%). All *C. parvum* isolates were assigned to the rare *gp60* subtype families IIo and IIp (not previously identified in human cryptosporidiosis cases). Taken together, these data indicate that farmed bamboo rats were infected by rodent-adapted *Cryptosporidium* species with little, or no zoonotic potential. Finally, Naguib et al. [21] provided novel data on the presence and genetic variability of *Cryptosporidium* spp. in farmed rabbits in Egypt, a country where the molecular epidemiology of this protozoan parasite is poorly understood. The authors collected and analysed faecal samples (*n* = 235) from nine rabbit farms, located in three Egyptian provinces. *Cryptosporidium* infections were confirmed in eight out of the nine farms sampled at variable rates, ranging from 4% to 24%. All *Cryptosporidium*-positive isolates generated (*n* = 28) were identified as *C. cuniculus* and belonged to the *gp60* subtype family Vb. Although *C. cuniculus* is known to be particularly adapted to infect domestic and wild leporids, it has also been identified in clinical human cases [46] and in waterborne outbreaks of cryptosporidiosis [47] and is regarded as an emerging pathogen to humans.

**Author Contributions:** P.C.K., D.G.-B. and D.C. wrote the Editorial. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Acknowledgments:** We are grateful to all the authors for their exciting contributions to this Special Issue. We also thank the reviewers for their helpful recommendations. We would also like to thank the *Pathogens* editorial office staff for their assistance and support and for having given us the opportunity to propose and organize this Special Issue.

**Conflicts of Interest:** The authors declare no conflict of interest.


**4.1 Facility-wide approach**

As demonstrated above, higher dialysate sodium provides questionable and inconsistent benefit for intradialytic hemodynamic stability at the cost of proven exacerbation of hypertension and interdialytic weight gain. "Lower" dialysate sodium should therefore be preferred, however, the exact definition of "lower" concentration is variable between studies. In the prospective studies, "lower" was defined from below 132 mEq/L to 145mEq/L while "higher" was defined from 137 to 155 or higher. Of the 165 patients in these studies, we could identify 131 patients where the exact high and low settings could be identified. The weighted average for the lower sodium was 137mEq/L and 143mEq/L for the higher sodium settings.

Given the number of potential barriers to crafting an individualized approach the sodium prescription for each patient, implementing a facility-wide change to 137mEq/L may be safely recommended. Typically, each dialysis unit sets a 'usual' dialysate sodium concentration based on the decision of the medical director. The 'standard' sodium can serve as the default with each provider making individualized changes based on individual patient's needs. Therefore, the initial step is encouraging dialysis directors to choose a default dialysate sodium concentration at, or close to, 137mEq/L.


**9. Follow-up**

Further analysis of data from EU-AUS and S0033 trials reported that tumor genotype has a significant prognostic impact on PFS and OS, with tumors with mutation of KIT exon 9 having a worse prognosis when compared to tumors with mutation of KIT exon 11 [96, 97]. A subsequent meta-analysis combining S0033 and EU-AUS trials [98] reported a minor albeit significant PFS advantage for a higher imatinib dose of 400 mg twice daily for patients with advanced GIST. The PFS benefit was only evident in patients with KIT exon 9 mutations treated with a high-dose imatinib without difference in OS between the two groups, while patients with KIT exon 11 had a more favorable prognosis. Thus, genotype is required for the

The findings of the results of the previous studies [94, 95, 98] designated imatinib dosage of 400mg/ day to be the standard treatment for patients with advanced GISTs, and patients with advanced GISTs with KIT exon 9 mutations to be started on the higher imatinib dose (400 mg twice daily), keeping in mind that the toxicity of imatinib is dose-dependent [99]. Imatinib treatment should be life long as interruption of treatment has a higher rate of disease progression as proven by the phase III randomized trial [100–102]. Indications of surgery in advanced GISTs are still debatable. Multiple retrospective studies have shown that debulking surgery may be beneficial in patients with a stable disease without generalized progression as surgery may improve the prognosis. However, patients should be treated with imatinib first before attempting surgery [103–108].

**8.3. Alternatives of imatinib in case of resistance or progression of disease with**

in KIT exons 13, 14, and 17 and a D842V mutation in PDGFRA exon 18 [111–113].

second-line therapy like sunitinib may be recommended.

Most patients with advanced GISTs will show improvement with imatinib therapy, although a subgroup of patients will fail to show a response. Resistance to imatinib may be primary or secondary. Primary resistance is defined as continuous growth or growth within 6 months of therapy, and occurs in 15–20% of patients with advanced GIST [109] and occurs frequently in patients with wild-type (WT) GIST or KIT exon 9 mutations or D842V mutation in PDGFRA exon 18 [110]. Unfortunately, most patients develop secondary resistance, which is defined as patients who received treatment with imatinib for longer than 6 months and had an initial response and then developed progressive disease. Secondary KIT mutations occur frequently

If a patient develops resistance, escalating imatinib dosage to 800 mg daily or shifting to a

Sunitinib is considered a second-line therapy for patients with advanced GIST refractory to imatinib therapy. Outcomes of a randomized phase III trial versus placebo reported a prolongation of the time to progression from 1.5 to 6.3 months in patients with GIST who progressed on imatinib treatment [114]. It is approved by the EMA and the FDA for the treatment of patients with GIST resistant to imatinib therapy and for patients who are not tolerant to ima-

In case of progression on imatinib and sunitinib, regorafenib is considered a third-line therapy [4]. It was recently approved by EMA and FDA for the treatment of patients with unresectable or metastatic GISTs who are resistant or intolerant to imatinib and sunitinib, and

treatment of advanced or metastatic GISTs.

**imatinib therapy**

80 Gastric Cancer - An Update

tinib therapy.

There are no studies assessing the efficacy of different follow-up modules. Nevertheless, follow-up strategies were created based on the fact that most recurrences occur within the first 5 years after surgery.

A follow-up schedule frequency is based on the risk of aggressiveness and recurrence of GISTs [4]. CT is favored over other imagings, such as MRI and FDG-PET scan, because it is more readily obtainable, although other modalities can be used in case CT is inconclusive.

## **9.1. Follow-up for localized resectable GISTs**

Very low-risk patients with surgically removed tumor do not require a follow-up. Low-risk patients require an annual CT. Intermediate- and high-risk patients require CT every 4 months for the first 1–2 years, every 6 months for 3–5 years, and then CT every year thereafter [4].

#### **9.2. Follow-up for unresectable/metastatic GISTs**

Follow-up should be done at the start of every 3 months and can be delayed to every 6 months if there is response to the treatment.


**2. Past and present of camel milk**

During the last 50 years, a fundamental shift has occurred in the way of housing and managing the camels. Today, in some countries, the extensive or nomadic production system for camels has become semi-intensive or intensive modern well-organized farms and industries [8]. Camels were known as "ships of the desert" commonly used as a mean of transport for thousands of years, carrying up to 600 lbs. on their backs. Currently, they are mainly considered for their milk and meat production, in particular, their milk. Camel milk contributed to the nonbovine milk production with a total amount of 2.7 million tons in 2016 [1]. Since the quantity and quality of milk production are highly dependent on the housing and management of animals, intensive production systems are currently being set up. All the world over, camel milk has proved to be suitable for producing various derived products with significant nutritional value [9].


*4.1. Applying Citizen Science to the FEEDS Project*

In applying the Bridge Framework, FEEDS incorporates the Smart Framework's principles of integrating citizen science, community-based participatory research, and systems science for population health research [11]. Citizen science, in particular, has played an important role in the ecological sciences to collect data on a range of issues including wildlife movement patterns and climate change-related environmental hazards [13,14]. Citizen science, when combined with digital tools, has great potential to generate big data to address complex public health crises if citizens' data can be anonymized and applied by communities [11,40]. In addition to capturing environmental and health-related data (e.g., weather, permafrost degradation, fire hazards, human movement, etc.), the FEEDS Project uses a custom-built app to engage and enable citizens to report on environmental hazards, changes in biodiversity or wildlife, and related food and mental health issues in their communities [40]. Big data is relayed in real time to a digital dashboard, where citizens and decision-makers have access to valuable information which can be used to mitigate health-related risks of climate change [40].


**4. Timeline of selenium treatment in Chagas Cardiopathy (STCC) clinical trial, the strength of a scientific network and the social arm of the project**

The next step, in 1999, was to enhance the number of patients studied to confirm whether Se levels would be lower in patients with more severe cardiac disease. The first partnership was made with Dr. Alejandro M. Hasslocher-Moreno, who was the physician responsible for the clinic management and follow-up of CD patients at the Evandro Chagas National Institute of Infectious Diseases (INI), Fiocruz, Rio de Janeiro city. Se levels were measured and a very large variation was observed. We then decided that patients with different nutritional habits from another state in Brazil should also be analyzed. It implicated a new partnership with colleagues from Belo Horizonte city and CD patients from Dr. Manoel Otávio Rocha, enrolling a total of 273 patients in the study [4]. We observed that Se levels in patients with moderate and severe cardiopathy were lower than in patients with mild cardiopathy and indeterminate form, as well as in uninfected individuals [4].

In parallel, we performed some proof-of-concept studies, showing in experimentally *T. cruzi*-infected mice—(i) that Se diet deficiency turn mice much more susceptible to acute-phase mortality [1]; (ii) the parasite survival and replication was not affected by Se [2]; (ii) that mice parasitemia did not vary in situations of nutritional deficiency or Se supplementation [2]; (iii) that in the acute [2] and in the chronic [3] phases in mice, treatment with Se was able to reduce cardiac inflammation, regulate arrhythmias, and prevent cardiomegaly; (iv) digestive mega disease was prevented by Se supplementation [22]. A second idea was planted—could Se treatment or supplementation prevent the progression of chronic forms of CCC?

In 2004, we started to move on to the clinical trials stage. We designed a placebo-controlled, double-blind trial, recruiting patients with mild heart disease (stages B1 and B2 defined by the Brazilian Consensus on CD) followed at the Chagas clinic at INI-Fiocruz, for treatment of 100 mcg/day Se for 1 year. Two outcomes were defined—50% reduction in progression of heart disease and a significant reduction in left ventricular ejection fraction (LVEF) value in 5 years of follow-up. However, we did not previously contact a Se supplier. Our first impediment was to think that it should be easy to find a Se source in the Brazilian pharmaceutical market. The saga was in the early beginning. **Figure 2** represent the

historical saga of the Se clinical trial, including the first step (1996–2004 period) of developing ideas, hypothesis, and pre-clinical studies.

*The Saga of Selenium Treatment Investigation in Chagas Disease Cardiopathy: Translational… DOI: http://dx.doi.org/10.5772/intechopen.103772*

(x) Immunological analyzes (humoral and cellular); (xi) Nutritional assessment; (xii) Follow-up ECG and ECO; (xiii) Follow-up echocardiogram. A challenge was the complexity of dealing with so many fields, multiplied by 135 expected participating patients multiplied by 14 visits each, with 10 only in the first year. It resulted in 1890 visits, with all the variables and biomarkers collected in most of them. In 2014, we had overcome the main obstacles to crossing the "valley of death," as shown in Declan Butler's publication in *Nature* in 2008 [23], and we the first patient was enrolled, enabling the publication of the STCC trial protocol in 2014 [24]. But then…

At this point, we decided to develop what we call the social arm of the clinical project.

April 14, 2015, we organized a meeting to make a public launch of the project, and to propose workshops of playful activities to "Talk about Chagas with Science and Art," inspired by the work of the Argentine group "Hablamos de Chagas" [29]. These initiatives quickly produced results—(i) we organized five successive editions of the course "We talk about Chagas with Science and Art" from 2015 to 2019, for people with CD, their family members, and health professionals; (ii) we created a

#### **Figure 3.**

*Time sequence of inclusion of participants on the STCC clinical trial. Black solid line shows the putative participants tracked, dashed line shows the participants included after signing the project voluntary consent form, and solid line with white dots shows the number of patients that completed the one-year treatment. Note that after 2015 the slope angle of the inclusion curve rise, denoting a success in the recruiting phase.*

"Rio Chagas Collective" on social media, which gave rise to the (iii) "Rio Chagas Association," founded on April 8, 2016, in which we participate in the Scientific Council; and (iv) the Rio Chagas Association participants inspired us to conceive the "Chagas Express XXI" social technology [30].

13. In 2018, we created a *YouTube* channel called "We talk about Chagas" (https:// www.youtube.com/c/FalamosdeChagas) to provide videos about Chagas disease, and in 2019, we created the social technology for those affected by the disease named "Expresso Chagas XXI" to talk about Chagas in endemic areas, bringing science, culture, and art to those most in need of information for health promotion, prevention of Chagas disease and access to diagnosis and treatment in the Unified Health System (SUS – Sistema Único de Saúde). In 2019, we carried out a pilot expedition in four cities in an endemic area for CD in northern Minas Gerais. In addition, in 2020, we organized solidarity strategies for coping with the COVID-19 pandemic together with Rio Chagas Association (virtual WhatsApp meetings called "Coffee with Affection").

Chagas Express XXI was created as an "imaginary train" with around 40 ArtScience workshops, games, laboratory activities, and conversation circles. It was structured with an entry and exit point, followed by six more modules of activities that combined a focus on associations of affected people, on opportunities for the public to rediscover Carlos Chagas' discoveries, on microscopic observations and play, on health education in approach. A One Health approach was adopted, with a focus on home care, environment and reservoirs, and wellness activities. Chagas Express XXI was conceived as a social technology since all processes were co-created by scientists and patients with Chagas disease and worked with local cross-sector partnerships. We observed that 81% of the more than 2000 participants were unaware of the possibility of treating Chagas disease and 52% requested a blood test to diagnose CD. From the 1100 adults tested, 20% were diagnosed as positive for *T. cruzi* infection [30].

The fourth idea would then be: if the clinical trial of Se becomes unfeasible, we will have a social legacy to be able to continue later.

14. However, the problem was not only in the communication and mobilization of patients to participate in STCC, since in INI/Fiocruz cohort the number of patients *The Saga of Selenium Treatment Investigation in Chagas Disease Cardiopathy: Translational… DOI: http://dx.doi.org/10.5772/intechopen.103772*

in the B1 and B2 stages of CCC was small as compared to patients in other clinical stages (indeterminate, A and C/D). Besides, a new risk for the project was arising: a long time had passed and the patients in the INI/Fiocruz cohort aged, acquiring comorbidities that were predicted as exclusion criteria and entering other studies that were ongoing, almost making the trial unfeasible due to a very slow rate of inclusion, as shown in **Figure 3**, during the years 2016 and 2017. To overcome this problem, the clinicians proposed to increase the age of recruitment from 65 to 75 years and to consider diabetes mellitus as a non-exclusion criterion.

Besides, the statisticians carried out other scenario studies for the feasibility of the STCC. They concluded that by extending the age range to up to 75 years, reducing the time of follow-up to one year, and focusing as a primary outcome only on the reduction of LVEF values (and not on the rate of CCC progression), we would conclude the inclusion of participants and generate some valid results. Then, in 2018, we published an update of the protocol [31] and started working to include 62 patients, divided into the two groups (placebo and Se treatment) for a follow-up of one year. We were then able to include all the expected patients and on August 8th, 2018, we were able to complete the last 12 months' visit of the last participant included.

15. The next step was to include all the laboratory and the nutritional data in the database, to run quality control for it, and to start the statistical analysis, both for descriptions of the clinical findings of participants and for comparisons between the placebo and Se-treated groups, as well as sub-group analyses, whenever possible. The study results were finally published in September 2021 [5]. When comparing the mean values of LVEF recorded in Se treated with the placebo group at baseline and after 1 year of follow-up, we did not find significant differences in the B1/B2 stages patients (overall), nor in the B1 stage patients, but found a significant effect (*p* = 0.02) in the B2 stage patients (**Figure 4**).

After one year (**Figure 4**, gray bars), all the groups showed a lower mean of LVEF when compared to baseline values (**Figure 4**, black bars). However only those already on the B2 subgroup, with LVEF <45%, showed a high decrease in LVEF in 1-year, which

#### **Figure 4.**

*Mean values for left ventricular ejection fraction recorded for all the patients participating in the STCC clinical trial, at baseline (baseline, black bars) and after 12 months (gray bars). In the X-axis it is shown the comparison of the groups treated with placebo or selenium, considering all the participants in each group and in the subgroups B1 and B2 stages. For details, see reference #5. Figure 4 Was prepared with data published in the STCC results [5].*

was reversed by Se treatment. The differences in LVEF longitudinal changes between groups were evaluated using linear mixed effect models in intention-to-treat analyses. This type of analysis assesses the rate of change of the outcomes by the time X intervention group interaction term, considering the correlations between repeated measures over time and missing data. In the B2 subgroup, the worsening of cardiac dysfunction was significantly reduced by Se treatment, and while the placebo group had a drop in LVEF, the Se group remained stable. In addition, only in the Se group, we observed cases of LVEF increase of more than 10 absolute points in 5 patients (n = 3) and recovery from stage B2 to B1 (n = 2), as reported [5]. In this short-term follow-up (1 year), the secondary outcomes observed were all related to LVEF as the underlying cause and could not be weighted in the analysis. We also observed that treatment with Se was safe for patients with CCC and that the low percentage of adverse effects detected were similar in the two groups. However, there was no complete shift of patients treated with Se to a safe range of serum Se (>100mcg/L), indicating that the dose of 100 mcg per day may have been insufficient. Further studies are needed to explore higher doses and/or associations of Se at different stages of CCC (B2 and C) at a short follow-up (one year) and at early diseases stages (A and B1), with longer follow-up.

16. The paper with STCC results [5] discussed some possibilities and pointed that "new clinical trials with a longer follow-up are needed to investigate the effects of Se in the mild (stages A and B) or severe (stages C and D) CCC, and in the asymptomatic indeterminate clinical form". The limitations of STCC were reported and the future outspread of the project is depicted in **Figure 2** from 2022 to, at least, 2026. The design of new projects (STCC#2 and STCC#3) is underway, and when the adequate industry partnership(s) will be signed, the proposal is that the new clinical trials will have a following of 3 to 10 years to generate the necessary evidence to support public policies recommendations.


**1. Introduction**

In this paper, we are interested in solving the linear response eigenvalue problem (LREP):

$$\mathbf{H}z := \left[ \begin{array}{cc} 0 & M \\ K & 0 \end{array} \right] \left[ \begin{array}{c} u \\ v \end{array} \right] = \lambda \left[ \begin{array}{c} u \\ v \end{array} \right] = \lambda z\_{\text{-}1}$$

where *K* and *M* are *N* × *N* real symmetric positive definite matrices. Such a problem arises from studying the excitation energy of many particle systems in computational quantum chemistry and physics [1–3]. It also known as the Bethe-Salpeter (BS) eigenvalue-problem [4] or the random phase approximation (RPA) eigenvalue problem [5]. There has immense past and recent work in developing efficient numerical algorithms and attractive theories for LREP [6–15].

Since all the eigenvalues of **H** are real nonzero and appear in pairs {*λ*, −*λ*} [6], thus we order the eigenvalues in ascending order, i.e.,

$$-\lambda\_1 \le \cdots \le -\lambda\_N < \lambda\_N \le \cdots \le \lambda\_1.$$

In this paper, we focus on a small portion of the positive eigenvalues for LREP, i.e., *λi*, *i* = *k*, *k* + 1, ··· , - with 1 ≤ *k* ≤ - ≤ *N* and - − *k* + 1 *N*, and their corresponding eigenvectors. We only consider the real case, all the results can be easily applied to the complex case.

The weighted Golub-Kahan-Lanczos method (**wGKL**) for LREP was introduced in [16]. It produces recursively a much small projection **B***j* = ( 0 *Bj BTj* 0 ) of **H** at *j*-th iteration, where *Bj* ∈ R*j*×*j* is upper bidiagonal. Afterwards, the eigenpairs of **H** can be constructed by the singular value

decomposition of *Bj*. The convergence analysis performs that running *k* iterations of **wGKL** is equivalently running 2*k* iterations of a weighted Lanczos algorithm for **H** [16]. Actually, *Bj* can be also a lower bidiagonal matrix, and the same discussion can be taken place as in the case of *Bj* is upper bidiagonal. In the following, we only consider the upper bidiagonal case.

It is well known that the single-vector Lanczos method is widely used for searching a small number of extreme eigenvalues, and it may encounter very slow convergence when the wanted eigenvalues stay in a cluster [17]. Instead, a block Lanczos method with a suitable block size is capable of computing a cluster of eigenvalues including multiple eigenvalues very quickly. Motivated by this idea, we are going to develop a block version of **wGKL** in [16] in order to find efficiently all or some positive eigenvalues within a cluster for LREP. Based on the standard block Lanczos convergence theory in [17], the error bounds of approximation to an eigenvalue cluster, as well as their corresponding eigenspace are established to illustrate the advantage of our weighted block Golub-Kahan-Lanczos algorithm (**wbGKL**).

As the increasing size of the Krylov subspace, the storage demands, computational costs, and numerical stability of a simple version of a block Lanczos method may be affected [18]. Several kinds of efficiently restarting strategies to eliminate these effects are developed for the classic Lanczos method, such as, implicitly restart method [19], thick restart method [20]. In order to make our block method more practical, and using the special structure of LREP, we consider the thick restart strategy to our block method.

The rest of this paper is organized as follows. Section 2 gives some necessary preliminaries for our later use. In Section 3, the weighted block Golub-Kahan-Lanczos algorithm (**wbGKL**) for LREP is presented, and its convergence analysis is discussed. Section 4 proposed the thick restart weighted block Golub-Kahan-Lanczos algorithm (**wbGKL-TR**). The numerical examples are tested in Section 5 to illustrate the efficiency of our new algorithms. Finally, some conclusions are given in Section 6.

Throughout this paper, R*m*×*n* is the set of all *m* × *n* real matrices, R*n* = R*n*×1, and R = R1. *In* (or simply *I* if its dimension is clear from the context) is the *n* × *n* identity matrix, and 0*m*×*n* is an *m* × *n* matrix of zero. The superscript "*T*" denotes transpose. ·*F* denotes the Frobenius norm of a matrix, and ·2 denotes the 2-norm of a matrix or a vector. For a matrix *X* ∈ R*m*×*n*, *rank*(*X*) denotes the rank of *X*, and R(*X*) = *span*(*X*) denotes the column space of *X*; the submatrices *Xi*:*j*,: and *X*:,*k*:- of *X* composed by the intersections of row *i* to row *j* and column *k* to column -, respectively. For matrices or scalars *Xi*, *diag*(*X*1, ··· , *Xk*) denotes the block diagonal matrix with the *i*-th diagonal block *Xi*.