*2.2. Technology Appplications*

Traditional investments in water resources in the developing world have seldom been conceived, implemented, or operated from a holistic multi-sectoral basin perspective. They often are based on old technologies and have high operational and maintenance costs that are seldom met, leading to poor service delivery exacerbated by deferred maintenance as they age. Even basic monitoring data are usually not accessible in real-time and require different 'ringfenced' legacy software that are not inter-operable.

**Figure 3.** Typology of 'disruptive' technologies.

One of the main ways in which modern technology is reshaping water resources planning and management is through 'disrupting' the data value chain (Figure 4). This is manifested through new inexpensive sensors for in-situ monitoring (tending towards an expansive 'internet of things'), increasingly powerful Earth observations from satellites and drones/unmanned aerial vehicles (UAVs) to provide synoptic views of topography (including high-resolution digital elevation models to identify flood-prone areas and support hydrodynamic modeling), climate, water levels, flows, snow cover, inundated areas, landcover, watershed status, and even some aspects of water quality and groundwater. Earth observations [22], with near global consistent coverage, is rapidly becoming a game-changer for synoptic observations in large basins, where the resolution of even the free resources from NASA and ESA are often adequate for useful water resources analytics. New unmanned on-water and under-water vehicles show promise; they can be outfitted with sensors and autonomous (single or swarm) capability for surveying large water bodies (e.g., for bathymetry, hydraulic safety, water quality, or fish stock assessments).

New analytical tools, increasingly cloud-based—including at the global level, assimilate available data and generate estimates of a range of critical parameters related to snowmelt estimation, water balance, water accounting (e.g., WA+, WAPOR) [38,39], scenario analysis, and forecasts to create 'digital twins' of basins to facilitate analyses. These enable access to curated archives and real-time estimates of the water status for any basin anywhere in the world to support both strategic planning and tactical operations through data visualization, early alerts/warnings, and the development of interactive packaging for data, analytics, and knowledge. Examples include interactive portals, mobile phone applications, and dynamic e-books. These support decisions at all levels, from simple scoping of water resources development, to detailed planning with stakeholder involvement/outreach, as well as real-time operations. Additional systems related to data/text mining, social media integration, advanced cloud-based modeling, machine learning/AI, or 'bots' can help bring in an additional automation and integrated perspectives to support decision-making.

**Figure 4.** Modernizing the data value chain (Data→Information→Knowledge→Decision Support).

These technologies are helping water managers reimagine the way information-based decisions can be made for smart water resources planning and management and are allowing development of integrated basin/aquifer plans based on both analytical and stakeholder approaches. Technologies have made possible new approaches to use and conserve water and administer usage caps (e.g., using satellite-derived actual evapotranspiration estimates), adopting a systems perspective to improve agricultural water productivity, benchmark systems, and incentivize sustainability. New in-situ and Earth observation monitoring and analytics allow for development and customization of tools for water planning, allocation, and coordinated water infrastructure operations in an integrated multi-sectoral systems perspective. Water infrastructure can now be operated in a more coordinated systems context for multiple objectives ranging from service delivery to climate resilience. Continuous innovation, piloting, and learning from global good practices enables quick scaling-up of new technologies and enables more adaptive management.

3D printing, robotics, automated transport, advanced materials, nanotech, biotech, and cleantech are supporting new operational systems that represent a paradigm shifts away from traditional approaches. Examples include irrigation systems that improve water productivity and field-level water use efficiency (especially when combined with policies such as limits on water abstraction); 3D printed monitoring stations (e.g., 3D-PAWS [40]) that reduce costs for monitoring weather and water levels; and ultrasonic control systems (successfully piloted in Lake Quaroun in Lebanon) that can mitigate algae-related water quality problems.

Platforms are emerging to enable people to work together in new ways in the sharing economy, including fintech, crowdsourcing, crowdfunding, block-chain enabled supply chains, asset sharing systems, Digital ID enabled e-governance, and open learning platforms. Many of these platforms have application in water management in large basins, including online/mobile platforms to support learning or interactions among remote or disperse water user associations, and platforms to help farmers access global marketplaces online, with feedbacks into irrigation water requirements.

#### *2.3. Implications for Large Basins*

The implications of these new technologies, with a focus on digital technologies, for water resources planning and management in large basins, are summarized in Table 1. Major changes in water management around the world are likely in very short timeframes. Ref [41] Many of the initial impacts will come from the plummeting costs of sensors, mobile devices and connectivity, cloud services (including to process increasingly powerful earth observation and other big data), interoperability due to online data standards and protocols, and increasing digital literacy.

Water data could be used primarily for water assessments, evaluations, operations, foresight, design, accountability, and education [42]. Many of these are useful at different scales—from monitoring progress towards the SDG-6 global indicators to helping design a culvert.

Many countries are modernizing their water resources institutions and developing national water resource information systems and analytics to support basin planning and disaster management. Some are also strengthening ties among government, academic, civil society organizations (CSO), and private sector institutions to develop a broad stakeholder base for this transformation. Modern water information systems require integration of data (from global, regional, national, to local sources), data quality management, conversion to interoperable data services, and development of interactive dashboards to help access and visualize data services and associated analytics in appropriate formats to support decisions.

Large basins will especially benefit from these changes given both the challenges (e.g., the need to integrate data across large areas; multiple stakeholders wishing to inform coordinated decisions; large water infrastructure investments) and the opportunities of large basins (e.g., application of free Earth observations in the 10–250 m resolution range; the ability to deliver reach large numbers of remote beneficiaries with valuable data services).

Institutions such as the Mekong River Commission and the various Nile Basin Initiative centers have demonstrated the utility of modern data and analytics in basin planning and hydro-meteorological data integration. Other large basins (e.g., the Congo, Ganges-Brahmaputra-Meghna) are in the nascent stages of this journey given capacity constraints and transboundary cooperation challenges.

Many countries are modernizing their water information systems taking advantage of new technologies. The United States and Australia are improving their already well-established systems facilitated by strong national institutions. Europe is increasingly building on its regional institutions (e.g., European Centre for Medium-Range Weather Forecasts, European Organisation for the Exploitation of Meteorological Satellites, Joint Research Centre of the European Commission) to help countries access better data and analytics. China is utilizing evapotranspiration estimates derived from Earth observations to improve irrigation management [43,44] and India is enhancing its national water resources information system [45].

Estimates based on satellite products or global models are increasingly found to be comparable with those based on in-situ observations [46,47]. These techniques, especially when enhanced by a new generation of artificial intelligence (AI)/machine-learning (ML) algorithms and global models, can revolutionize water resources management even in data-poor environments. When accessed through customized interactive dashboards, this information can be especially useful for estimating parts of the water balance, estimating flooding areas, making customized weather/hydrologic/inundation forecasts, managing large water demands (e.g., agriculture) and system losses, while enhancing and benchmarking water productivity [48,49].

**1.**Summaryofdisruptivetechnologiesrelevanttowatermanagementin


**1. Introduction**

Aflatoxins are fungal secondary metabolites toxic to humans and animals, causing carcinogenic, mutagenic, teratogenic, and immunosuppressive effects [1]. Aflatoxins are produced by toxigenic *Aspergillus flavus*, *A. parasiticus*, and *A. nomius* isolates growing in a variety of food and feed commodities [2]. These metabolites are very stable to autoclaving, pasteurization, and other food processing procedures [3].

Aflatoxin M1 (AFM1) is a 4-hydroxy derivative of aflatoxin B1 (AFB1), which, although approximately ten-fold less toxigenic than aflatoxin B1, exerts cytotoxic, genotoxic, and carcinogenic effects in a variety of species [2], being classified as belonging to group 1 (i.e., carcinogenic to humans) by the International Agency for Cancer Research [4]. AFM1 is

**Citation:** Cruz, P.O.d.; Matos, C.J.D.; Nascimento, Y.M.; Tavares, J.F.; Souza, E.L.d.; Magalhães, H.I.F. Efficacy of Potentially Probiotic Fruit-Derived *Lactobacillus fermentum*, *L. paracasei* and *L. plantarum* to Remove Aflatoxin M1 In Vitro. *Toxins* **2020**, *13*, 4. https:// dx.doi.org/10.3390/toxins13010004

Received: 23 September 2020 Accepted: 18 November 2020 Published: 23 December 2020

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

**Copyright:** © 2020 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/).

formed in the liver and excreted through the milk of lactating animals that have consumed feed contaminated with AFB1. Approximately 0.3–6.2% of AFB1 ingested by livestock is converted to AFM1 in milk [5]. In Brazil and the USA, the maximum allowable limit of AFM1 in raw milk is 0.5 μg/L [6,7]. The European Union has set a maximum limit of AFM1 of 0.05 μg/L for raw milk, heat-treated milk, and milk used in dairy products formulation [8].

Control of aflatoxin in food and feed can be primarily achieved by a prevention of mold contamination and growth with the adoption of improved agricultural practices and control of storage conditions, as well as by the detoxification of contaminated products through chemical (e.g., ammonia, hydrogen peroxide, alkalis, and acids) or physical methods (e.g., heat, radiations, ultraviolet, and microwave) [9]. Some methods used for aflatoxins decontamination, although they have been shown to be effective to a certain extent, may have some drawbacks, such as negative impacts on nutritional and sensory characteristics of foods, production of potentially toxic by-products, or non-suitability for use in solid foods [2,9].

Use of lactic acid bacteria (LAB) has been considered a safe and environmentally friendly biological method for the detoxification of aflatoxins in foods and feeds [10,11]. Studies have found a variable capability among probiotic *Lactobacillus* species or isolates to bind aflatoxins [12–14]. These studies have mostly used commercial *Lactobacillus* cultures or isolates from dairy origin. Although a number of *Lactobacillus* isolates recovered from fruit, vegetables, or their processing by-products have shown good performance in in vitro tests for the selection of probiotics [15–17], none of these isolates have been examined for their capacity to remove aflatoxins. The use of select probiotic *Lactobacillus* isolates has been considered a promising biological tool for removing aflatoxins from foods through adsorption when compared to chemical and physical treatments. Furthermore, although still the fastest method for retaining high detoxification efficacy [18,19], many chemical agents are nonedible materials and need to be eliminated after aflatoxin decontamination [20,21], while *Lactobacillus* species have been usually considered safe for use in foods [16,17].

Considering the available evidence, it was expected that fruit-derived *L. fermentum*, *L. paracasei*, and *L. plantarum* isolates with aptitudes to be used as probiotics would be able to remove AFM1 in a prospective view for application in food and feed detoxification. To test this hypothesis, this study evaluated the efficacy of these isolates as viable and non-viable (heat-killed) cells, in the removal of AFM1 in vitro, as well as the recovery of the AFM1 bound to bacterial cells.

#### **2. Results and Discussion**

Chromatograms for the quantification of AFM1 in positive control, negative control, as well as in samples with viable cells of *L. paracasei* 108, *L. plantarum* 49, and *L. fermentum* 111 are shown in Figure 1. Chromatograms for the quantification of AFM1 in assays evaluating the recovery of AFM1 from cells after 1 h of incubation are shown in Figure 2.

Results of the capability of viable and heat-killed (non-viable) cells of *L. paracasei* 108, *L. plantarum* 49, and *L. fermentum* 111 for removing AFM1 in PBS are presented in Table 1. Viable and heat-killed cells of all examined *Lactobacillus* isolates were able to remove AFM1 in PBS, with removal percentage values in the range of 73.0 ± 1.2–80.0 ± 1.7% and 72.9 ± 1.1–78.7 ± 1.2%, respectively. Viable and heat-killed cells of the three examined isolates had similar values (*p* > 0.05) of AFM1 removal. Only *L. paracasei* 108 had higher values (*p* ≤ 0.05) of AFM1 removal after 24 h for both viable and heat-killed cells compared to 1 h. Higher values of AFM1 removal (*p* ≤ 0.05) after 1 h were found for *L. plantarum* 49 and *L. fermentum* 111, but the three examined isolates had similar values of AFM1 removal (*p* > 0.05) after 24 h.

**Figure 1.** Chromatograms of aflatoxin M1 (AFM1) quantification in positive and negative control. (**I**) Positive control: phosphate buffer solution (PBS) with AFM1. Rt = Retention time of AFM1 in phosphate buffer solution; chromatographic peak area corresponding to AFM1; (**II**) Negative control after 1 h of incubation: PBS + *L. paracasei* 108; (**III**) Negative control after 1 h of incubation: PBS + *L. plantarum* 49; (**IV**) Negative control after 1 h of incubation: PBS + *L. fermentum* 111.

**Figure 2.** Chromatograms of aflatoxin M1 (AFM1) quantification in PBS. (**I**) Chromatogram of assays after 1 h of incubation: PBS + AFM1 + *L. paracasei* 108; (**II**) Chromatogram of assays after 1 h of incubation: PBS + AFM1 + *L. plantarum* 49; (**III**) Chromatogram of assays after 1 h of incubation: PBS + AFM1 + *L. fermentum* 111; (**IV**) AFM1 recovery chromatogram of *L. paracasei* 108 and AFM1 complex after 1 h of incubation; (**V**) AFM1 recovery chromatogram of *L. plantarum* 49 and AFM1 complex after 1 h of incubation; (**VI**) AFM1 recovery chromatogram of *L. fermentum* 111 and AFM1 complex after 1 h of incubation. (**A**) Retention time (min) of aflatoxin M1 in phosphate buffer solution; (**B**) chromatographic peak area corresponding to aflatoxin M1.

**Table 1.** Percentage (average values ± standard deviation) of aflatoxin M1 (AFM1) removal in phosphate buffer solution by *L. paracasei* 108, *L. plantarum* 49, and *L. fermentum* 111.

Different small letters in the same row (a,b) denote a significant difference (*p* ≤ 0.05) among values, based on Tukey's test; different capital letters in the same column (A,B) denote a significant difference among values (*p* ≤ 0.05), based on Tukey's test.

Previous studies have also verified that the capacity of LAB—either as viable or non-viable cells, of binding aflatoxins (e.g., aflatoxin B1, ochratoxin, trichothecene, and AFM1) in PBS, laboratory media, or dairy matrices (e.g., milk and yoghurt)—varies in an isolate-dependent manner [2,11,22,23]. Aflatoxins bind to the surface components of LAB

cells and variations in aflatoxin's binding capacities among LAB species or isolates could be associated with differences in the bacterial cell wall and cell envelope structures [7]. Early investigations have found lower capacity of AFM1 removal by viable and/or heatkilled cells of different LAB (e.g., *L. plantarum*, *L. acidophilus*, *L. reuteri*, *L. johnsonii*, *L. rhamnosus*, *L. bulgaricus*, and *Streptococcus thermophilus*) [2,22,23], including probiotic *L. casei* [10], compared to *L. paracasei* 108, *L. plantarum* 49, and *L. fermentum* 111. The efficacy of AFM1 removal from PBS as high (>60%) as those found for *Lactobacillus* isolates examined in this study was reported to *L. plantarum* MON03 and *L. rhamnosus* GAF01 after 6 or 24 h of incubation [24].

Results of the AFM1 retention capacity of the viable and heat-killed cells of *L. paracasei* 108, *L. plantarum* 49, and *L. fermentum* 111 after washing with PBS are presented in Table 2. Percentage values of recovered AFM1 from viable and heat-killed cells were in the range of 13.4 ± 1.5–60.6 ± 1.6% and 10.9 ± 1.2%–47.9 ± 1.5%, respectively. The highest values of recovered AFM1 after 1 and 24 h were found for *L. fermentum* 111 and *L. paracasei* 108, respectively, for both viable and heat-killed cells. Only for *L. fermentum* 111 did the values of recovered AFM1 decrease after 24 h for viable and heat-killed cells; for *L. paracasei* 108 and *L. plantarum* 49, these values varied with the viability/non-viability of cells and incubation time period. Overall, *L. plantarum* 49 had the higher AFM1 retention capacity after washing. Variations in aflatoxin release have been linked to the differences in binding sites in different LAB isolates, or even in these binding sites being very similar. They could have minimal differences depending on each isolate [13,25,26].

**Table 2.** Percentage (average values ± standard deviation) of recovered aflatoxin M1 (AFM1) in solution after washing with phosphate buffer solution.

Different small letters in the same row (a–c) denote a significant difference (*p* ≤ 0.05) among values, based on Tukey's test; different capital letters in the same column (A,B) denote a significant difference among values (*p* ≤ 0.05), based on Tukey's test.

For all examined isolates, the values of recovered AFM1 decreased after 24 h of incubation, indicating that AFM1 retention capacity increased when the length of the contact time increased. There was no clear association between the capability of removing AFM1, initially, and of retaining AFM1 after washing among examined isolates. Interestingly, a study with different *Lactobacillus* species found lower AFM1 removal values than those found in this study, although the recovery of AFM1 from bacterial cells was lower in the former [11].

Heat treatment positively affected the capability of retaining AFM1 in *L. paracasei* 108 after 1 h of incubation, as well as of *L. plantarum* 49 and *L. fermentum* 111 after 24 h of incubation. Heating could increase the interaction capacity of bacterial cells/aflatoxin complexes by causing an increased exposure of the cell wall components, primarily polysaccharides and peptidoglycans, which act as binding sites to aflatoxin [14]. However, the destruction of specific components of the bacterial cell wall by heating, causing the denaturation of proteins and increased cell surface hydrophobicity, has been cited to result in a decreased capability of LAB cells of binding AFM1 [7]. An increased capability of removing aflatoxin B1 was also found in *L. rhamnosus* after heating [27].

The recovery of the AFM1 bound to the cells of examined *Lactobacillus* isolates after washing indicates that the binding was not strong and could not involve a non-covalent weak bond, but probably a physical association of AFM1 with hydrophobic sites in the

bacterial cell wall [13,20,25]. The lower AFM1 recovery values found for the examined isolates could be linked to the interaction of AFM1 molecules retained in the bacterial cell wall with other AFM1 molecules retained in adjacent cells, forming a type of cross-linked matrix that avoids aflatoxin release during washing [10]. Probably, the efficacy of this type of cross-linked matrix decreased over time for *L. paracasei* 108 and *L. plantarum* 49. Although some authors have reported that a part of non-recovered AFM1 might be degraded or biotransformed by a *Lactobacillus* metabolism [2,7], most of the available literature has indicated that aflatoxins are not removed by the metabolism of LAB, but because of a physical bound to the molecular components of bacterial cells, primarily peptidoglycans from the cell wall [19,21,25].

In agreement with available literature, the results of this study showed that the cell viability of the examined isolates is not a prerequisite for the removal and retaining of AFM1 [13,28]. Cell concentration as high as 108–109 CFU/mL of viable or non-viable LAB is typically needed to reach a level of aflatoxins removal of ≥ 50% [22,28].


**2. Hepatocarcinogenesis**

Hepatocarcinogenesis is the development of liver cancer due to the exposure of carcinogen (a chemical that produces cancer). Many hepatocarcinogens such as aflatoxins, acetylaminofluorene, diethylnitrosamine have been successfully used to develop hepatocarcinogenesis in animals. Experimentally, hepatocarcinogenesis is developed using different carcinogens and also in different animal species. Several genetic and epigenetic changes such as chromosomal deletions, rearrangements, aneuploidy, gene amplification, and mutations, formation of DNA adducts, DNA strand-break, modulation of DNA methylation, and modulation of cell signaling pathways, due to direct or indirect effect of carcinogen exposure lead to neoplastic transformation of hepatocytes in experimental animals. Hepatocarcinogenesis is a multistage complex process, which is preceded by early appearance of morphologically and genetically altered hepatic focal lesions, also known as preneoplastic lesions. Initially monoclonal populations of hepatocytes evolve primarily due to carcinogenic insult. These aberrant monoclonal populations of regenerative hepatocytes (focal lesions) develop hyperplastic nodules to dysplastic nodules, leading to hepatocellular carcinoma.


**1. Introduction**

192 Fungicides for Plant and Animal Diseases

Ueno, K. Tamura, Y. & Chibana, H. (2010). Target validation and ligand development for a

Van De Wouw, A. & Howlett, B. (2011). Fungal pathogenicity genes in the age of 'omics'.

Witke, W. (2004). The role of profilin complexes in cell motility and other cellular processes.

Xu, H., Xu, H., Lin, M., Wang, W., Li, Z., Huang, J., Chen, Y., Chen, X. (2007). Learning the

*glabrata* and structure-based ligand design. *Yeast*. 27: 369–378.

drug target-likeness of a protein. *Proteomics. 7: 4255–4263*.

*Molecular Plant Pathology.* 12: 507-514.

*Trends Cell Biology.* 14: 461–469.

pathogenic fungal profilin, using a knock-down strain of pathogenic yeast *Candida*

This chapter aims at describing an integrated approach to control the development of Black Leaf Streak Disease(BLSD) of bananas and plantains plants with a minimum of fungicide applications resulting in lower cost of control, lower risk of fungicide resistance and lower environmental negative impact.

Bananas are one of the main agricultural productions in the world, and the main fruit crop, with an annual production of about 117 Mt (Lescot, 2011). International trade of bananas represents 14Mtyear–1 with a value of more than 4 billion US\$ (Loeillet, 2005). The production of bananas for this international trade is grown in a small number of tropical countries mainly located in Latin America (80%), in African and Asian countries, and in the European community (French West Indies, Canary Islands). In all these countries, this industry is economically important and a source of direct and indirect employment. But one of the main problems is that it relies on a very narrow genetic base, the Cavendish subgroup, which is exposing the industry to important pest and disease threats.

Photo credit: CARBAP (J. Ngando) Photo 1. Banana plants with necrosis of BLSD .

An Integrated Approach to Control the Black Leaf Streak

on plantains (Stover, 1983; Mobambo et al., 1996).

banana industry is regularly increasing.

(altitude zones) (Fouré & Lescot, 1988).

diseases in the world (Pennisi, 2010).

very recent arrival in Martinique (2010).

cycle that are useful for BLSD control.

gradually evolve from stage 1 to stage 6 (Fouré, 1982)

considered to be dispersed at longer distances than conidia.

susceptible to this disease.

control BLSD to a level that does not alter fruit exportation.

Disease (BLSD) of Bananas, while Reducing Fungicide Use and Environmental Impact 195

The first one is a reduction of yield due to a negative effect on the photosynthetic assimilation (up to 50 to 100%) and thus on dry matter production. Bunch weight is strongly affected (Ramsey & Daniells, 1990), and significant yield losses (20% to 50%) were observed

A second one is a reduction of the conservation potential of the fruit, expressed by its "greenlife", which is the time between harvest and ripening, arguing for a direct effect of these foliar diseases on fruit physiology by mechanisms that remain to be discovered (Abadie et al., 2008; Chillet et al., 2009). The impact on export quality is thus very critical as there are high risks of advanced maturation (Stover, 1972). Therefore banana growers must

The economic incidence of BLSD is important since the cost of control can reach more than 25% of the production cost (Stover & Simmonds, 1987) and the BLSD incidence in the

Two species are responsible for the current epidemics, *Mycosphaerella musicola* Leach, causal agent of the Sigatoka Leaf Spot Disease (SLSD) and *Mycosphaerella fijiensis*, causal agent of the Black Leaf Streak Disease (BLSD). The latter is much more virulent than the former resulting in more intense damage and more difficult control. In addition the spectrum of susceptible varieties is much wider. The group of plantains is particularly susceptible to BLSD when it is considered resistant to SLSD, except in special geographical conditions

BLSD is regarded as the most economically important disease (Jones, 2000) for the banana industry and it is a major constraint for small farmers growing banana and plantains for domestic markets (Marin et al., 2003). It is considered as one of the most important crop

Such a situation is mainly related to the fact that the banana trade relies on a unique group of banana cultivars (*Musa acuminata*, AAA, Cavendish subgroup) which are highly

BLSD is present in Latin America since 1972, in Cameroon since 1980 and has appeared in Ivory Coast in 1985. Its expansion in the Caribbean is more recent having started from two independent introductions in the north (Cuba in 1990, and then Jamaica, the Dominican Republic, Haiti and Puerto Rico) and in the Lesser Antilles (through Trinidad in 2003) with a

The life cycle of BLSD has been widely described and starts with leaf infection by either ascospores or conidia (Fig. 1) It is important to highlight the main characteristics of this life

After a period of epiphyllic growth of generally 2-3 days, germ tubes penetrate stomata. In good conditions, the first symptoms appear generally 10-14 days after. The symptoms then

Conidia are produced on young stages of the disease (stage2-4) and have been considered as water dispersed at short distances, even if they are also present in the "airspora". Ascospores are produced at later stage and wind-dispersed after perithecia burst. They are

The Banana leaf spot diseases are a major constraint in most large production areas, resulting in very significant losses in the absence of suitable control system (Stover, 1986; Stover & Simmonds, 1987). There are two major impacts of the disease on the production.

Photos credit: CARBAP (J. Ngando)

Photo 2. Very severe attack of BLSD on a banana plant with a bunch

Photos credit: CARBAP (J. Ngando) Photo 3. Early ripening of the fruits due to severe attack of BLSD

The Banana leaf spot diseases are a major constraint in most large production areas, resulting in very significant losses in the absence of suitable control system (Stover, 1986; Stover & Simmonds, 1987). There are two major impacts of the disease on the production.

Photos credit: CARBAP (J. Ngando)

Photos credit: CARBAP (J. Ngando)

Photo 2. Very severe attack of BLSD on a banana plant with a bunch

Photo 3. Early ripening of the fruits due to severe attack of BLSD

The first one is a reduction of yield due to a negative effect on the photosynthetic assimilation (up to 50 to 100%) and thus on dry matter production. Bunch weight is strongly affected (Ramsey & Daniells, 1990), and significant yield losses (20% to 50%) were observed on plantains (Stover, 1983; Mobambo et al., 1996).

A second one is a reduction of the conservation potential of the fruit, expressed by its "greenlife", which is the time between harvest and ripening, arguing for a direct effect of these foliar diseases on fruit physiology by mechanisms that remain to be discovered (Abadie et al., 2008; Chillet et al., 2009). The impact on export quality is thus very critical as there are high risks of advanced maturation (Stover, 1972). Therefore banana growers must control BLSD to a level that does not alter fruit exportation.

The economic incidence of BLSD is important since the cost of control can reach more than 25% of the production cost (Stover & Simmonds, 1987) and the BLSD incidence in the banana industry is regularly increasing.

Two species are responsible for the current epidemics, *Mycosphaerella musicola* Leach, causal agent of the Sigatoka Leaf Spot Disease (SLSD) and *Mycosphaerella fijiensis*, causal agent of the Black Leaf Streak Disease (BLSD). The latter is much more virulent than the former resulting in more intense damage and more difficult control. In addition the spectrum of susceptible varieties is much wider. The group of plantains is particularly susceptible to BLSD when it is considered resistant to SLSD, except in special geographical conditions (altitude zones) (Fouré & Lescot, 1988).

BLSD is regarded as the most economically important disease (Jones, 2000) for the banana industry and it is a major constraint for small farmers growing banana and plantains for domestic markets (Marin et al., 2003). It is considered as one of the most important crop diseases in the world (Pennisi, 2010).

Such a situation is mainly related to the fact that the banana trade relies on a unique group of banana cultivars (*Musa acuminata*, AAA, Cavendish subgroup) which are highly susceptible to this disease.

BLSD is present in Latin America since 1972, in Cameroon since 1980 and has appeared in Ivory Coast in 1985. Its expansion in the Caribbean is more recent having started from two independent introductions in the north (Cuba in 1990, and then Jamaica, the Dominican Republic, Haiti and Puerto Rico) and in the Lesser Antilles (through Trinidad in 2003) with a very recent arrival in Martinique (2010).

The life cycle of BLSD has been widely described and starts with leaf infection by either ascospores or conidia (Fig. 1) It is important to highlight the main characteristics of this life cycle that are useful for BLSD control.

After a period of epiphyllic growth of generally 2-3 days, germ tubes penetrate stomata. In good conditions, the first symptoms appear generally 10-14 days after. The symptoms then gradually evolve from stage 1 to stage 6 (Fouré, 1982)

Conidia are produced on young stages of the disease (stage2-4) and have been considered as water dispersed at short distances, even if they are also present in the "airspora". Ascospores are produced at later stage and wind-dispersed after perithecia burst. They are considered to be dispersed at longer distances than conidia.

An Integrated Approach to Control the Black Leaf Streak

Disease (BLSD) of Bananas, while Reducing Fungicide Use and Environmental Impact 197

 Photo 4. Stages 1 and 2 of the disease Photo 5. Stages 2 and 3 of the disease

 Photo 6. Stages 4 (adaxial surface) Photo 7. Stages 5 of the disease

Photo 8. Stage 6 of the disease with Photo 9. The density of infection is perithecia (abaxial surface). often very important resulting in necrotic patches by coalescence.

Plate 1. **Illustration of BLSD symptoms** (Photo credit: Cirad /E.Fouré & L. de Lapeyre de

Bellaire)

(adaxial surface) (adaxial surface)

(abaxial surface)

*( in yellow: data for the Sigatoka Disease and in black, data for the BLSD)*

Fig. 1. The biological cycle of the banana leaf spot diseases

#### **2. Principle and various components of an integrated approach to control BLSD of bananas**

The principle and various components of this integrated approach are presented. They are based on a holistic approach including technical aspects and logistical, economic and political aspects as well.

#### **2.1 Historical features**

It is useful to look at some historical features to better understand the genesis of this integrated control of BLSD.

An effective chemical control of SLSD, based on the use of mineral oils, was developed by a French team of IFAC in Guadeloupe in the fifties (Guyot &Cuillé, 1954; Guyot &Cuillé, 1955).

The work done at that time quickly showed that the foliar emission rate of the banana plant would impose a very large number of sprays to ensure that each new leaf is protected in time. This should lead to a minimum of forty sprays per year with an additional processing constraint to apply about 2000 liters of aqueous formulation per hectare, which was quite intolerable.

(adaxial surface) (adaxial surface)

*( in yellow: data for the Sigatoka Disease and in black, data for the BLSD)*  Fig. 1. The biological cycle of the banana leaf spot diseases

**BLSD of bananas**

political aspects as well.

**2.1 Historical features**

intolerable.

integrated control of BLSD.

**2. Principle and various components of an integrated approach to control**

The principle and various components of this integrated approach are presented. They are based on a holistic approach including technical aspects and logistical, economic and

It is useful to look at some historical features to better understand the genesis of this

An effective chemical control of SLSD, based on the use of mineral oils, was developed by a French team of IFAC in Guadeloupe in the fifties (Guyot &Cuillé, 1954; Guyot &Cuillé, 1955). The work done at that time quickly showed that the foliar emission rate of the banana plant would impose a very large number of sprays to ensure that each new leaf is protected in time. This should lead to a minimum of forty sprays per year with an additional processing constraint to apply about 2000 liters of aqueous formulation per hectare, which was quite

Photo 4. Stages 1 and 2 of the disease Photo 5. Stages 2 and 3 of the disease

Photo 6. Stages 4 (adaxial surface) Photo 7. Stages 5 of the disease

(abaxial surface)

Photo 8. Stage 6 of the disease with Photo 9. The density of infection is perithecia (abaxial surface). often very important resulting in

necrotic patches by coalescence.

Plate 1. **Illustration of BLSD symptoms** (Photo credit: Cirad /E.Fouré & L. de Lapeyre de Bellaire)

An Integrated Approach to Control the Black Leaf Streak

crop practices)

(from de Lapeyre et al., 2009)

emulsions or water.

fungicides with mineral oil (oil compatible formulations)

Disease (BLSD) of Bananas, while Reducing Fungicide Use and Environmental Impact 199

Fig. 2. Fungicide use for SLSD control in commercial banana farms of Guadeloupe (1969-2006).

This warning strategy developed by IRFA was very different from the systematic strategy used by U.S. companies using contact fungicides in water or systemic fungicides in oil

The research therefore looked for reducing these constraints i) by studying more closely the biology of the pathogen to intervene only when necessary ii) limiting the volume of mixture applied per hectare and the number of fungicide applications, iii ) focusing on the choice of more effective and persistent fungicides.

The first goal resulted in the development of disease observation and warning methods, with the early work conducted by Cuillé & Guyot in 1954.

To reach the second goal, the work achieved made it possible to replace the copper formulations applied through ground sprays at a rate of 2000 liters per hectare by a low volume of mixtures, initially applied with ground sprayers and later by plane or helicopter, to go down to 15 liters per hectare. This development was made possible by the discovery of the qualities of mineral oils which have proven to be excellent vehicles for fungicides, either alone or mixed with water. These mineral oils (i) showed a fungistatic activity on the pathogen, (ii) ensured a good repartition of droplets at leaf surface and a good penetration of fungicides in the leaf tissues, (iii)avoided the rainfall leaching occurring after treatment.

As a matter of fact, before 1972, aerial and ground sprays were currently done only with oil. With the appearance of systemic fungicides in the 1970', new formulations were introduced coupling the fungistatic action of the oil with the strong curative action of currently marketed systemic fungicides (Ganry & Laville, 1983). Fungicides used belong to different chemical families with different mode of actions such as benzimidazoles, triazoles, morpholins and more recently strobilurins (see Box 4), which have been used practically in this chronological order, taking into account the emergence of resistance of the fungus to benzimidazoles and triazoles..

Very soon appeared the need to abandon a strategy of systematic control and move towards less fungicide applications in order to (i) reduce production costs, (ii) reduce environmental impact and (iii) limit the occurrence of fungicide resistant strains. With such an objective of better management of the control strategy, CIRAD (formerly IRFA) has, since the seventies, focused its work on the development of a warning method in order to apply fungicides only when necessary.

The warning system used is based both on climatic data (Box 1) and on field observations of symptoms evolution in early stages (Box 2).

In Guadeloupe and Martinique, since the 70's, the information has been processed on an aggregate basis for each island and managed by a specialized team attached to the banana growers associations. Aerial sprays are made on a similar base and are generalized for the whole of each island.

As a result of the use of such a disease management strategy the control of the SLSD has been very satisfactory in the French Antilles since the seventies (Fig. 1), with a number of applications ranging from five to eight per year, while applications carried out systematically in similar situations would have required at least 10 to 15 treatments per year (Ganry, 2001).

This success story relies on some basic requirements:

The research therefore looked for reducing these constraints i) by studying more closely the biology of the pathogen to intervene only when necessary ii) limiting the volume of mixture applied per hectare and the number of fungicide applications, iii ) focusing on the choice of

The first goal resulted in the development of disease observation and warning methods,

To reach the second goal, the work achieved made it possible to replace the copper formulations applied through ground sprays at a rate of 2000 liters per hectare by a low volume of mixtures, initially applied with ground sprayers and later by plane or helicopter, to go down to 15 liters per hectare. This development was made possible by the discovery of the qualities of mineral oils which have proven to be excellent vehicles for fungicides, either alone or mixed with water. These mineral oils (i) showed a fungistatic activity on the pathogen, (ii) ensured a good repartition of droplets at leaf surface and a good penetration of fungicides in the leaf tissues, (iii)avoided the rainfall leaching occurring after treatment. As a matter of fact, before 1972, aerial and ground sprays were currently done only with oil. With the appearance of systemic fungicides in the 1970', new formulations were introduced coupling the fungistatic action of the oil with the strong curative action of currently marketed systemic fungicides (Ganry & Laville, 1983). Fungicides used belong to different chemical families with different mode of actions such as benzimidazoles, triazoles, morpholins and more recently strobilurins (see Box 4), which have been used practically in this chronological order, taking into account the emergence of resistance of the fungus to

Very soon appeared the need to abandon a strategy of systematic control and move towards less fungicide applications in order to (i) reduce production costs, (ii) reduce environmental impact and (iii) limit the occurrence of fungicide resistant strains. With such an objective of better management of the control strategy, CIRAD (formerly IRFA) has, since the seventies, focused its work on the development of a warning method in order to apply fungicides only

The warning system used is based both on climatic data (Box 1) and on field observations of

In Guadeloupe and Martinique, since the 70's, the information has been processed on an aggregate basis for each island and managed by a specialized team attached to the banana growers associations. Aerial sprays are made on a similar base and are generalized for the

As a result of the use of such a disease management strategy the control of the SLSD has been very satisfactory in the French Antilles since the seventies (Fig. 1), with a number of applications ranging from five to eight per year, while applications carried out systematically in similar situations would have required at least 10 to 15 treatments per year (Ganry, 2001).

more effective and persistent fungicides.

benzimidazoles and triazoles..

symptoms evolution in early stages (Box 2).

This success story relies on some basic requirements:

the completion of the spray.

when necessary.

whole of each island.

with the early work conducted by Cuillé & Guyot in 1954.

Fig. 2. Fungicide use for SLSD control in commercial banana farms of Guadeloupe (1969-2006). (from de Lapeyre et al., 2009)

This warning strategy developed by IRFA was very different from the systematic strategy used by U.S. companies using contact fungicides in water or systemic fungicides in oil emulsions or water.

An Integrated Approach to Control the Black Leaf Streak

crop density, water management.

curative, fungicides are used (Table 1.).

10 to 15 sprays a year (depending on

curative (systemic) fungicide

location and inoculums)

Oil used with a rate of 12-15 liters/ha with

a vicious circle.

selection pressure.

Disease (BLSD) of Bananas, while Reducing Fungicide Use and Environmental Impact 201

eliminate new sources of inoculum. Other crop practices must be introduced to synergize with fungicide sprays and leaf removal dealing with crop management: plant nutrition,

Depending on the type of fungicide used and on the selection pressure1, there is always a risk of emergence of fungicide resistance in pathogen populations. It is one of the major reasons of the decrease of the chemical control efficiency which is often the starting point of

Therefore it is highly important to monitor the pathogen sensitivity to the fungicides used. This monitoring must be considered as a key decision tool for the choice of appropriate fungicides (when it is possible) and their sequence in alternation, in order to minimize the

Such an approach was adopted for BLSD when it appeared in Gabon on plantains (see §2.2. BLSD control in Gabon in small scale plantain production for domestic markets) and in Cameroon (see §2.2. BLSD control in Cameroon in the banana industry for export), Ivory Coast and Ecuador on banana and plantain. Thanks to a strong experience on SLSD control and to the implementation of such disease management strategies, BLSD in these countries

In comparison, in some Latin American countries, the number of annual sprays can reach 30 to 60 (more than one per week) due to systematic fungicide applications and to the emergence of resistant strains to curative (systemic) fungicides that were used too frequently (Carlier et al., 2000; Romero & Sutton, 1997). As a consequence more contact, non

Water or oil-water emulsions (2 – 8 liters of

oil + 10-20 liters of water) with

3. Contact and curative fungicide 30 to 60 systematic sprays depending on

1. Contact fungicide 2. Curative fungicide

locations

Table 1. Compared characteristics of the IRFA and american methods for BLSD control

The timing of decisions is based on a standardized set of criteria, taking into account the development of the fungus and the growth of the plant, both climate-driven. The first level

2 Cameroon: from 10 to 15 treatments / year have succeeded in the disease control for more than 10 years. However, the emergence of strains resistant to systemic fungicides, due to some failures in the

**2.2.1 Component 1: Timing of decisions through a biological forecasting**

1 Related to the duration of the pathogen exposure to the fungicide

implementation, is currently preventing the use of this disease management.

management strategy was experimented in this country in the late 80's.

has been properly controlled with an average of 10 to 15 treatments per year2.

IRFA method american method

The characteristics of these two control methods are summarized in the table 1.

#### **2.2 The basic principle of an integrated approach to control the banana BLSD**

Inherited from this strong experience with the control of the SLSD, the integrated approach to control BLSD is based on the rational use of curative (systemic) fungicides applied in oil mixtures.

The sequence of fungicide sprays is driven by a forecasting system supported by biological indicators (Fouré & Ganry, 2008; Ganry et al., 2008). Since *M. fijiensis* is more aggressive than *M. musicola*, the system works efficiently for this pathogen when the disease pressure is not too high. It is the reason why it must be supplemented by sanitation practices consisting in necrotic and pre-necrotic leaf removal in order to

The characteristics of these two control methods are summarized in the table 1.

**2.2 The basic principle of an integrated approach to control the banana BLSD**

mixtures.

Inherited from this strong experience with the control of the SLSD, the integrated approach to control BLSD is based on the rational use of curative (systemic) fungicides applied in oil

The sequence of fungicide sprays is driven by a forecasting system supported by biological indicators (Fouré & Ganry, 2008; Ganry et al., 2008). Since *M. fijiensis* is more aggressive than *M. musicola*, the system works efficiently for this pathogen when the disease pressure is not too high. It is the reason why it must be supplemented by sanitation practices consisting in necrotic and pre-necrotic leaf removal in order to eliminate new sources of inoculum. Other crop practices must be introduced to synergize with fungicide sprays and leaf removal dealing with crop management: plant nutrition, crop density, water management.

Depending on the type of fungicide used and on the selection pressure1, there is always a risk of emergence of fungicide resistance in pathogen populations. It is one of the major reasons of the decrease of the chemical control efficiency which is often the starting point of a vicious circle.

Therefore it is highly important to monitor the pathogen sensitivity to the fungicides used. This monitoring must be considered as a key decision tool for the choice of appropriate fungicides (when it is possible) and their sequence in alternation, in order to minimize the selection pressure.

Such an approach was adopted for BLSD when it appeared in Gabon on plantains (see §2.2. BLSD control in Gabon in small scale plantain production for domestic markets) and in Cameroon (see §2.2. BLSD control in Cameroon in the banana industry for export), Ivory Coast and Ecuador on banana and plantain. Thanks to a strong experience on SLSD control and to the implementation of such disease management strategies, BLSD in these countries has been properly controlled with an average of 10 to 15 treatments per year2.

In comparison, in some Latin American countries, the number of annual sprays can reach 30 to 60 (more than one per week) due to systematic fungicide applications and to the emergence of resistant strains to curative (systemic) fungicides that were used too frequently (Carlier et al., 2000; Romero & Sutton, 1997). As a consequence more contact, non curative, fungicides are used (Table 1.).

Table 1. Compared characteristics of the IRFA and american methods for BLSD control

### **2.2.1 Component 1: Timing of decisions through a biological forecasting**

The timing of decisions is based on a standardized set of criteria, taking into account the development of the fungus and the growth of the plant, both climate-driven. The first level

<sup>1</sup> Related to the duration of the pathogen exposure to the fungicide

<sup>2</sup> Cameroon: from 10 to 15 treatments / year have succeeded in the disease control for more than 10 years. However, the emergence of strains resistant to systemic fungicides, due to some failures in the implementation, is currently preventing the use of this disease management.

<sup>-</sup> Ivory Coast: control is achieved with 12-14 treatments / year in most production areas.

<sup>-</sup> Ecuador: from 10 to 12 treatments / year succeeded in disease control when this disease management strategy was experimented in this country in the late 80's.

An Integrated Approach to Control the Black Leaf Streak

efficiency of the chemical strategy at the harvest stage.

Disease (BLSD) of Bananas, while Reducing Fungicide Use and Environmental Impact 203

of elaboration of reliable tools for strategic decisions is to implement a continuous monitoring of disease evolution in the field. It is also informative to fit these weekly data with weekly rainfall, in order to get a better appreciation of epidemiological conditions **(**de Lapeyre de Bellaire et al., 2010a).

SEDb represents the speed of evolution of the disease, which reflects the climatic conditions and the intensity of the infection (Ganry & Meyer, 1972b). The early detection of new attacks on the youngest leaves will allow blocking their evolution towards necroses thanks to the application of a curative fungicide. In order to maintain the field inoculum as low as possible, it is then essential to stop the disease evolution before necrotic formation because sexual sporulation starts in necrotic stages of the disease.

A larger set of disease parameters should also be used and evaluated continuously every week on the same banana plot used for the assessment of SEDb in order to provide complementary information:

of elaboration of reliable tools for strategic decisions is to implement a continuous monitoring of disease evolution in the field. It is also informative to fit these weekly data with weekly rainfall, in order to get a better appreciation of epidemiological conditions **(**de

SEDb represents the speed of evolution of the disease, which reflects the climatic conditions and the intensity of the infection (Ganry & Meyer, 1972b). The early detection of new attacks on the youngest leaves will allow blocking their evolution towards necroses thanks to the application of a curative fungicide. In order to maintain the field inoculum as low as possible, it is then essential to stop the disease evolution before necrotic formation because

A larger set of disease parameters should also be used and evaluated continuously every week on the same banana plot used for the assessment of SEDb in order to provide

Lapeyre de Bellaire et al., 2010a).

leaves (Fig. 3).

complementary information:

the older leaves.

sexual sporulation starts in necrotic stages of the disease.

to slow down the necrotic formation (Fig. 4).

An Integrated Approach to Control the Black Leaf Streak

Disease (BLSD) of Bananas, while Reducing Fungicide Use and Environmental Impact 205

Fig. 4. Illustration of interpretation of the Youngest Leaf Spotted (YLS) from its weekly evolution in banana plantations**.** *Arrows indicate the date at which the whole 10 banana plants where changed At that date, the variation should not be considered as the value of YLS is artificially different. YLS is scored according to Stover's method* (from de Lapeyre de Bellaire et al., 2010a)

The timing of the fungicide applications is driven by SEDb and the analysis of other

The time between decision and execution of one application should not exceed 2 days. Sprays are carried out by airplane, helicopter, or by backpack sprayer on small surfaces. However, care must be taken with the climatic conditions of aerial spraying: only a small window, early in the morning and late in the afternoon, is suitable; otherwise, thermal inversion and air turbulence do not allow correct spray deposition. Aerial application is not possible on rainy and windy days. Logistics available is therefore essential to optimize

**The efficiency of sprays** is dependent on the quality of the foliar application, and a good

The weather conditions the day of application are critical: temperature must be rather cool (less than 28°C) and wind as low as possible (<1.3 m.s-1) and regular. Sprayers and nozzles must be cleaned every day and calibrated frequently (calibration depends on the use of water or oil and on the rate of mixture used per ha). Significant technical improvement has

**2.2.2 Component 2: Timing and quality of the fungicide applications**

spraying during this small window.

been achieved when aircraft are guided precisely by GPS.

coverage is essential.

parameters. It is the reason why it must be implemented by highly skilled teams.

Table 2. Example of a data sheet used for the calculation of the weekly SEDb in banana plants (FER = Foliar Emission Rate) *( from Fouré &Ganry, 2008)*

Fig. 3. Illustration of the Stage of Evolution of Disease's (SEDb) potential to forecast fungicide applications and to monitor the efficiency of the chemical control strategy in banana plantations. *All fungicide applications are indicated by an arrow above the SEDb curve (arrows with different colors correspond to different fungicides applied)* (from de Lapeyre de Bellaire et al., 2010a)

Table 2. Example of a data sheet used for the calculation of the weekly SEDb in banana

Fig. 3. Illustration of the Stage of Evolution of Disease's (SEDb) potential to forecast fungicide

plantations. *All fungicide applications are indicated by an arrow above the SEDb curve (arrows with different colors correspond to different fungicides applied)* (from de Lapeyre de Bellaire et al., 2010a)

applications and to monitor the efficiency of the chemical control strategy in banana

plants (FER = Foliar Emission Rate) *( from Fouré &Ganry, 2008)*

Fig. 4. Illustration of interpretation of the Youngest Leaf Spotted (YLS) from its weekly evolution in banana plantations**.** *Arrows indicate the date at which the whole 10 banana plants where changed At that date, the variation should not be considered as the value of YLS is artificially different. YLS is scored according to Stover's method* (from de Lapeyre de Bellaire et al., 2010a)

#### **2.2.2 Component 2: Timing and quality of the fungicide applications**

The timing of the fungicide applications is driven by SEDb and the analysis of other parameters. It is the reason why it must be implemented by highly skilled teams.

The time between decision and execution of one application should not exceed 2 days. Sprays are carried out by airplane, helicopter, or by backpack sprayer on small surfaces. However, care must be taken with the climatic conditions of aerial spraying: only a small window, early in the morning and late in the afternoon, is suitable; otherwise, thermal inversion and air turbulence do not allow correct spray deposition. Aerial application is not possible on rainy and windy days. Logistics available is therefore essential to optimize spraying during this small window.

**The efficiency of sprays** is dependent on the quality of the foliar application, and a good coverage is essential.

The weather conditions the day of application are critical: temperature must be rather cool (less than 28°C) and wind as low as possible (<1.3 m.s-1) and regular. Sprayers and nozzles must be cleaned every day and calibrated frequently (calibration depends on the use of water or oil and on the rate of mixture used per ha). Significant technical improvement has been achieved when aircraft are guided precisely by GPS.

An Integrated Approach to Control the Black Leaf Streak

preliminary field experiments.

Disease (BLSD) of Bananas, while Reducing Fungicide Use and Environmental Impact 207

The use of these fungicides in pure oil strengthens their curative effect, because mineral oils are fungistatic. Attention should be paid to the formulation of the fungicide used. Only formulations compatible with pure oil should be used. Phytotoxic effects of the oil have to be preliminarily determined. Fungicide concentration must also be determined according to

It is achieved through prophylactic leaf removal coupled with relevant crop management,

The experience of BLSD control in Gabon (see §2.2) has clearly shown that when plantain fields were isolated into a forest environment and far from infected plots, preventing them

Thus, keeping the sources of inoculum at a very low level is very important to ensure the success of the chemical control strategy. Where extensive spotting is present, new infections will develop quickly because chemical sprays do not remove the disease from spotted leaves and the only solution is to remove them mechanically from the banana tree. Therefore, such necrotic leaf removal must be a regular practice in banana growing countries where BLSD is present. It should be based on a risk management approach, taking into account that it could

Experimental leaf removal has shown that bunch weight, the main yield component, is poorly affected as long as 5 to 7 leaves remain on the plant from flowering to harvest (Ramsey et al., 1990; Daniells et al., 1994; Vargas et al., 2009). A stronger leaf removal could affect the final yield. In addition a lack of resources during this phase, due to severe spotting could compromise the yield of the next ratoon (Eckstein et al., 1995; Dens et al., 2008).

Leaf removal is particularly needed in critical areas where regular sprays are not well performed due to natural obstacles or to regulatory measures such as in buffer areas where aerial spraying is prohibited (e.g. 50 m from houses, gardens, rivers and roads in the FWI). A strong communication effort should be done towards the farm managers in order to improve the situation, because the chemical control strategy will not be successful if the leaf removal is not done properly. Such leaf removal might be mandatory on BLSD susceptible banana plants that cannot be part of the spraying agenda: in home gardens, near houses, along roads or rivers. In such situations the best solution would be to replace the susceptible bananas by resistant varieties. It requires to take into account social and political components, such as the acceptability of these varieties by the consumers in order to encourage voluntary behavior. In some cases like in Australia, more coercive measures were used with an objective of complete

In addition it is essential to destroy systematically all abandoned plantations and isolated

In areas where fungicide sprays are done, it is essential to realize necrotic leaf removal before any fungicide spray in order to lower the inoculum pressure and thus enhance the efficiency of the fungicide and to minimize the exposure of the pathogen to the fungicide

**2.2.3 Component 3: The control of the inoculum pressure in the field**

from external contamination, the disease control was strongly facilitated.

eradication which was successfully achieved (Henderson et al., 2006).

and thus the risk of appearance of resistant strains to the fungicide.

roadside banana plants that are potential reservoir of disease inoculums.

such as plant density and water and fertilizer management.

affect the production beyond certain level.

An irregular topography of the zone or the presence of obstacles might alter the uniformity of the treatment. The banana plots must be shaped to facilitate aerial sprays, taking into account the presence of obstacles and wind drift, and the crop density should not be too high to allow good penetration of the fungicide spray. A strong synergy between the aerial spraying companies and the banana growers is also important to ensure the quality of the aerial applications.

The use of mineral oils as a carrier considerably improves the quality of coverage through aerial spraying with low volumes (12–15 Lha–1). The quality of oil is essential and must fit with the norms defined by Cuillé and Blanchet in 1958 and always used. The quality of the mix with the fungicide is important as well. Fungicides must be used at full rate in order to limit the risk of fungicide resistance.

Table 3. Characteristics of oil used for sprays in the control of SLSD (Cuillé & Blanchet, 1958)

The efficiency of treatments relies on a strong curative effect. Thus, systemic fungicides (antimitotics, sterol inhibitors of group 1 and 2, strobilurines, pyrimidins) are preferred to contact fungicides (see Box 4).

An irregular topography of the zone or the presence of obstacles might alter the uniformity of the treatment. The banana plots must be shaped to facilitate aerial sprays, taking into account the presence of obstacles and wind drift, and the crop density should not be too high to allow good penetration of the fungicide spray. A strong synergy between the aerial spraying companies and the banana growers is also important to ensure the quality of the

The use of mineral oils as a carrier considerably improves the quality of coverage through aerial spraying with low volumes (12–15 Lha–1). The quality of oil is essential and must fit with the norms defined by Cuillé and Blanchet in 1958 and always used. The quality of the mix with the fungicide is important as well. Fungicides must be used at full rate in order to

**Characteristics norms**

Viscosity 4 à 7° Engler at 20°C Density 0.83 to 0.90 Sulfonation index 85-90% maximum

Acidity less than 0.16 Addition ingredients no sulfur compound

Table 3. Characteristics of oil used for sprays in the control of SLSD (Cuillé & Blanchet, 1958)

The efficiency of treatments relies on a strong curative effect. Thus, systemic fungicides (antimitotics, sterol inhibitors of group 1 and 2, strobilurines, pyrimidins) are preferred to

> Contact fungicides (mancozeb, chlorothalonil) have only a preventive effect and do not penetrate in the banana leaf. They are generally applied every week in systematic frameworks because all new unfurling banana leaf must be protected. They have a multi-site effect on fungal biology and strains resistant to these fungicides have never been

> Systemic fungicides penetrate in the banana leaf. The systemic properties of these fungicides are variable: some have only a translaminar transport and others can be distributed in the whole banana plant, and the biokinetics of these fungicides confer a more or less pronounced curative effect. The curative effect is more pronounced on young streaks (stages 1, 2), but lower on older lesions (stages 3, 4) and without any effect on necrotic stages (stages 5, 6), even if

> - Inhibitors of ergosterol biosynthesis (IBS) in group 1 that inhibit eburicol demethylation (DMI fungicides mostly

sporulation might be temporarily decreased. Different chemical families have been used:

Type indifferently paraffinic or naphtenic

aerial applications.

limit the risk of fungicide resistance.

contact fungicides (see Box 4).

*a. Protectants*

reported. *b. Systemic fungicides*

**Box 4. Various categories of fungicides used for BLSD control**

Three groups have a strong curative effect:

Two groups have a moderate curative effect:

very high risk of resistance

moderate risk of resistance

resistance

belonging to the triazole family): high risk of resistance

The use of these fungicides in pure oil strengthens their curative effect, because mineral oils are fungistatic. Attention should be paid to the formulation of the fungicide used. Only formulations compatible with pure oil should be used. Phytotoxic effects of the oil have to be preliminarily determined. Fungicide concentration must also be determined according to preliminary field experiments.


**2. Object recognition systems for automated image analysis**

Aerial or satellite survey consists of several steps that need to be followed. Starting from the data acquisition procedures, they include visual observations, the capturing of imagery and the use of metric measurements on the acquired images. Then, this raw information is typically used to produce a set of documents which consists of text and related images. However, the resulting documents are often hard to use since they have been created for a specific application and require expert knowledge to comprehend. In addition, image quality can vary depending on the remote sensing site conditions. Other challenges on image analysis from aerial surveys can include poor lighting conditions, occlusions, and varying depths. Also, limitations on the geospatial information provided with the acquired images can limit their spatial analysis. Restrictions on the field-of-view (FOV) and the image sensor footprint can affect the number of images collected per aerial survey. Then, consequently, a higher number of images require a higher processing time to be completed. Where time of completion is of essence for cost reasons, then someone can expect that automated image analysis tools are necessary for minimising those processing and completion times.

Integral part of such automated image analysis tools plays the object recognition system used which need to be designed in novel architectures, if they are to be applied for solving more complex problems [8]. Recent advances have led to the development of biologically-inspired also known as cognitive architectures [9] of object recognition systems with separate design blocks of a recognition unit and a separate knowledge learning unit [10, 11]. Thus, cognitive architectures need to exploit the non-linearity, the learning and adaptation to the input data, and provide an attentional mechanism for the hybrid system to be able to select certain input information to be included in its learning against other input. Therefore, knowledge representation and learning becomes a central issue in the design and implementation of such hybrid biologically-inspired pattern recognition [12]. Knowledge representation can have altering effects on problem knowledge learning and problem solving [13]. A problem solving system consists of a domain theory which specifies the task to be solved, the initial problem states and the targeted problem goals, and a control knowledge which guides the decision-making process. Thus, knowledge representation can have a direct effect to the efficiency of the problem solving process [14, 15].

## **2.1 Defining the problem**

In this chapter, we describe a new object recognition architecture for improving the speciation of an endangered bird species from aerial surveys. For reasons of confidentiality, we use here the term endangered bird sub-species 1 or, simply, endangered species 1, and the term endangered sub-species 2 or, simply, endangered species 2. In particular, we are looking to improve the accuracy and precision of the recognition of endangered bird sub-species 1and 2 in winter plumage which the task of correctly speciating them becomes harder than in their summer plumage. Our proposed cognitive object recognition architecture incorporates the features of shape, size and colour (3-bands R, G and B) of the endangered sub-species 1 and 2 in the architecture's knowledge representation, and then apply an unsupervised knowledge learning unit for improving the accuracy and precision in recognising them.


**Table 3.**

*Number of confined heads, semi-confined heads, total heads and slaughtered heads, and energy demand (MWh) for the baseline scenario and scenario 1, in Brazil, in 2000, 2005, 2010, 2015, 2020, 2025 and 2030.*

## *Biogas Generation from Bovine Confinement: An Energy Policy Option for Brazil DOI: http://dx.doi.org/10.5772/intechopen.99828*

### **Figure 4.**

*Rate of number of confined cattle on total cattle with some type of confinement and number of semi-confined cattle on total cattle with some confinement in Brazil, % (2000–2015). Source: Prepared by the authors.*

For semi-confined cattle, this average rate was 40.6%. Therefore, the number of confined cattle will rise from a little over four million in 2015 to around 15.9 million in 2030. Semi-confined cattle will increase from almost 2.7 million (2015) to almost 10.9 million. Total cattle with some type of confinement in 2030 will be around 26.8 million, representing an increase of 74.2% in relation to 2015.

## **4.4 Scenario 3: increases in the rate of cattle with some form of confinement and the time of confinement**

Joining scenarios 1 and 2 creates scenario 3, thus, as has been seen in the previous scenarios, only the basic values for the calculation of the creation of biogas and emissions are altered. Permitted in this scenario is the analysis of all the proposed possibilities for the intensification of livestock raising through the confinement of cattle, and through the increase in both confinement time and the total rate of cattle in some form of confinement.

After the analysis of each of the proposed scenarios and how the basic variables are modified in the three scenarios, the biogas generation potential and to what extent this can meet the demand for energy of slaughterhouses and abattoirs, and the agricultural sector, is examined, as well as the impact on emissions of the insertion of biogas in the energy matrix. In short, it shows how cattle confinement can contribute to energy policy as a complementary renewable source capable of guaranteeing energy security from the point of view of supply without harming the environment.

### **5. Results**

There is a high potential for the use of biogas. Even without any intensification policy it will be possible to meet almost 80% of the energy demands of abattoirs and slaughterhouses in 2015. In 2030, not only will it be possible to meet all energy demands necessary for animal slaughterhouses, but also energy can be offered to the Brazilian electricity system with an addition of around 371.1 GW/h year. In the case of the agricultural sector, first, it is estimated that the demand for electricity of slaughterhouses and abattoirs will represent almost 6% of the total electricity consumed by the sector in 2015. By 2030 it is estimated that this value will be approximately 4%. The insertion of biogas as a source of electricity can be noted that renewable resources

### *Biogas Generation from Bovine Confinement: An Energy Policy Option for Brazil DOI: http://dx.doi.org/10.5772/intechopen.99828*

can meet approximately 14.9% of the electricity demands of the agricultural sector in 2030, in a scenario with both an increase in the confinement rate and an expansion of time spent in confinement (Scenario 3). The importance of the use of biogas as an alternative energy source is so relevant that without any alteration in the country's beef productive system in 2015 biogas could generate sufficient electricity to meet about 4.8% of the demands of the agricultural sector, and in 2030 this value would be practically the same, a little more than 4.7% (**Figure 5**). Changes in confinement time or in the confinement rate can increase the offer of biogas and can increase even more the additional offer to the grid with an addition of almost 1402.4 GW/h (scenario 1), 2867.1 GW/h (scenario 2), or 5146.3 GW/h (scenario 3). As **Figure 6** shows, it is possible to create more than three times the energy demand necessary for animal slaughter (offer of biogas/energy demand = 378.6%) in 2030.

It can be noted that increased confinement time (scenario 1) creates a lower increase in the offer of biogas for electric energy when compared to the increase

### **Figure 5.**

*Relation between the supply of biogas and electricity demand for agricultural sector, for each scenario, % (2015, 2010, 2025 and 2030). Source: Prepared by the authors.*

### **Figure 6.**

*Relation between the supply of biogas and the electricity demand for slaughterhouses, % (2015, 2020, 2025 and 2030). Source: Prepared by the authors.*

**Figure 7.**

*Emission reduction with biogas use, for each scenario, in GgCO2e (2015, 2020, 2025 and 2030). Source: Prepared by the authors.*

in the confinement rate and, above all, that the sum of the different intensification polices results in a significant increase. Thus, this result shows the strategic importance of biogas in Brazil as an energy source capable not only of meeting the demand of slaughterhouses and abattoirs, but also part of the energy demands of the agricultural sector.

An additional positive effect of the use of biogas, in addition to the expansion of energy demands, is on the reduction of GHG emissions. Despite having a low grid emission factor due to the high share of hydroelectricity in the Brazilian energetic matrix, the insertion of a renewable source with the low GHG emissions helps keep the emissions of the Brazilian electricity sector low. In the scenario with the implementation of all intensification policies in the agricultural sector (Scenario 3), it is possible to reduce in the long term (2030) a little more than 1201.56 GgCO2e. Without any policy, this reduction will reach 381.12 GgCO2e, as **Figure 7** shows.

### **6. Conclusion**

Despite being one of the largest beef producers in the world, Brazil still has low productivity indices in the livestock sector, in which the predominance of extensive system means that the country has a stocking rate of between 1 and 2 heads of cattle per hectare. Given this fact, the country has great potential to intensify the sector with the possibility of the reduction of the demand for electricity in abattoirs and slaughterhouses using biogas and even the agricultural sector as a whole and to reduce somewhat greenhouse gas emissions generated by the sector through the introduction of a new renewable and cleaner energy source.

In the scenarios analyzed, it was perceived that the use of biogas in beef cattle confinement is an interesting complementary energy policy, which fits into the need to diversify renewable energy sources to reduce potential problems in electricity supply, in relation to an energy matrix that is dependent on hydroelectricity to

### *Biogas Generation from Bovine Confinement: An Energy Policy Option for Brazil DOI: http://dx.doi.org/10.5772/intechopen.99828*

meet the growing demand of the agricultural sector for electricity. In scenario 3, it can be observed that the application of policies to intensify the livestock sector allows a considerable reduction in GHG emissions and to meet the needs of both the agricultural sector and the totality of the energy demand of slaughterhouses and abattoirs, which are close to the herd, facilitating the use of biogas to supply them.

More profound studies, assessing the economic question, such as the costs of expanded confinement and if, from the economic point of view, intensification is attractive, need to carry out to confirm whether, from the technical point of view, increasing beef confinement is an interesting energy policy to increase the offer of electricity and to reinforce the energy security of Brazil. The scenarios show the need to modernize livestock raising through the better use of the waste and the effluents created during the slaughter process and to propose as a short and midterm energy policy.

Therefore, the conclusion is reached that the use of biogas is important to reduce greenhouse gas emissions, contribute to cleaner energy generation and reduce the energy dependence of the agricultural sector in Brazil. In the face of climate change, this energy resource can help the country to help meet the goals proposed under the Paris Agreement and attract foreign investment in view of the enormous potential to generate energy and reduce emissions.


**5. Conclusions**

The chapter outlined the state of the art of Inte.Ri.M. project. The on-site activity will provide answers to the question marks determined by the specific operational conditions established by the mountain environment.

The project will be able to generate benefits that potentially will have an impact in terms of creating value for the area. Beneficial effects are identified on two levels:

#### *Perspectives on Risk, Assessment and Management Paradigms*

The project development is planned at two levels (national and international). Each stage foresees a growing extension (qualitative and quantitative) of Inte.Ri.M. system through applications that imply an increasing complexity, in terms of technical, economic, and management issues to cope with and in number and variety of partners to involve. The needs of time and funding grow, therefore, consistently. The output of our project and actions planned to increase interest and promote cooperation, jointly with the research team's propensity to act internationally, could make international level directly accessible.


6.7.2. Magnesium

Magnesium (Mg2+) is another mineral that inhibits the formation of apatite. In healthy individuals, serum magnesium concentrations are carefully balanced in a narrow range [190]. The kidneys are the main organs controlling systemic Mg2+ homeostasis, where its transport is highly regulated by hormonal and other intrarenal factors [191]. Bones are the main reservoir of Mg2+, holding ~60% of the total body content. Mg2+ in bones is primarily embedded at the surface of the apatite crystals [192]. Because of its inhibitory properties towards calcification and oral bioavailability, Mg2+ supplementation appeared to be a good option to decrease soft tissue calcification in pathologies with calcification such as chronic kidney disease (CKD) [193], but also in PXE, as both share some phenotypical characteristics [194]. Preliminary testing in *Abcc6*−/<sup>−</sup> mice with increased dietary Mg2+ showed results [138,139] encouraging enough that a prospective human clinical trial was initiated (NCT01525875). However, the translation to humans proved to be inconclusive [129], probably because the dosages used in animal models exceeded what could actually be tolerated in humans and/or as a result of the method for phenotype evaluation methodology (*Cf.* Section 6.1). Therefore, Mg2+ supplementation is probably not a viable treatment strategy, at least on its own.


*4.1.1. At the arteriolar level*

The principal manifestation of I/R in arterioles is a loss of the vasodilatation-dependent en‐ dothelium and the appearance of spasms. [78] Widespread endothelial lesions decrease the production of nitric oxide and do not counterbalance the arterioles' tendency toward vaso‐ constriction. This tendency is highlighted in several tissues, such as skeletal muscle, heart, lung and brain. [79]-[82] The combined effects of IR and inflammation on arteriolar vasomo‐ tricity are well documented. The increase in the contractile response of the pulmonary and mesenteric microcirculation after cardiac surgery predisposes the patient to the develop‐ ment of pulmonary shunt or mesenteric ischemia, particularly during the administration of vasopressive drugs in the postextracorporeal circulation. [83 ],[84]


**BREAST CANCER AND SURGERY**

#### **Breast Cancer and Surgery**

http://dx.doi.org/10.5772/intechopen.71739 Edited by Nilufer Bulut

#### **Contributors**

Josie Todd, Anna Badowska-Kozakiewicz, Michał Piotr Budzik, Joanne Chiu, Shadia Al Bahlani, Samiya Salim Al-Jaaidi, Ibrahim Alanazi, Zahid Khan, Fatma Sen, Heidi Abrahamse, Ivan Mfouo-Tynga, Anggorowati Anggorowati, Malgorzata Banys, Tanja Fehm, Florian Reinhardt, Shahnorbanun Sahran, Ashwaq Qasem, Dheeb Albashish, Siti Norul Huda Sheikh Abdullah, Azizi Abdullah, Rizuana Iqbal Hussain, Fuad Ismail, Suria Pauzi, Nordashima Abd Shukor, Khairuddin Omar, Afzan Adam, Norlia Abdullah, Nilufer Bulut

#### **© The Editor(s) and the Author(s) 2018**

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#### **Notice**

Statements and opinions expressed in the chapters are these of the individual contributors and not necessarily those of the editors or publisher. No responsibility is accepted for the accuracy of information contained in the published chapters. The publisher assumes no responsibility for any damage or injury to persons or property arising out of the use of any materials, instructions, methods or ideas contained in the book.

First published in London, United Kingdom, 2018 by IntechOpen eBook (PDF) Published by IntechOpen, 2019 IntechOpen is the global imprint of INTECHOPEN LIMITED, registered in England and Wales, registration number: 11086078, The Shard, 25th floor, 32 London Bridge Street London, SE19SG – United Kingdom Printed in Croatia

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**6. Clinical outcomes of ADC in breast cancer treatment**

### **6.1 T-DM1**

T-DM1 was evaluated in the phase III, randomized clinical trial EMILIA, which enrolled 991 patients with metastatic, HER2+ breast cancer with disease progression after first line trastuzumab plus taxane based chemotherapy for metastatic disease or with disease recurrence during or within six months of completing adjuvant therapy [51]. Patients were randomized 1:1 to T-DM1 or lapatinib and capecitabine. The co-primary endpoints were progression free survival (PFS) and overall survival (OS). Most patients (88%) received prior chemotherapy for metastatic disease with a median of 3 prior lines of treatment. PFS was significantly improved with median PFS of 9.6 months in the T-DM1 arm versus 6.4 months in the control arm [hazard ratio (HR), 0.65; 95% CI, 0.55–0.77; p < 0.0001]. Overall survival was also significantly improved with median OS of 30.9 months in the T-DM1 arm vs. 25.1 months in the control arm (HR, 0.68; 95% CI, 0.55–0.85; P, 0.0006). Based on the results of this study, T-DM1 was FDA approved for the treatment of HER2+ metastatic breast cancer after progression on first line therapy, and T-DM1 became the standard second line treatment in this patient population until recent findings from DESTINY-Breast03 that demonstrated superiority of T-Dxd over T-DM1 as second line treatment for metastatic, HER2+ breast cancer [41].

Adjuvant T-DM1 for early stage, HER2+ breast cancer and residual disease post taxane and trastuzumab based neoadjuvant chemotherapy was evaluated in the KATHERINE trial, a randomized, phase III study [52]. The study enrolled 1486 patients who were randomized 1:1 to adjuvant T-DM1 or trastuzumab for 14 cycles. The primary outcome was invasive disease free survival (IDFS), which was defined as the time from randomization to first local or regional breast cancer recurrence, distant recurrence, or death from any cause. Key secondary outcomes were PFS and OS. Most patients (77%) received anthracycline based neoadjuvant chemotherapy, and 20% of patients received additional anti-HER2 therapy, 94% of which was pertuzumab. At a median follow up of 40 months, IDFS was significantly improved with T-DM1 versus adjuvant trastuzumab (HR, 0.50; 95% CI, 0.39–0.64; P < 0.001). The 3-year IDFS rate was 88.3% in the T-DM1 group and 77.0% in the trastuzumab group. The results of this study led to the FDA approval of T-DM1 as an adjuvant therapy for patients with early stage, HER2+ breast cancer with residual disease after neoadjuvant chemotherapy and surgery and has established adjuvant T-DM1 as a standard therapy in this patient population [2].

#### **6.2 T-Dxd**

T-Dxd was first FDA approved based on results from the single arm, multicenter, phase II DESTINY-Breast 01 trial that enrolled patients with HER2+ metastatic breast cancer who progressed on prior chemotherapy including T-DM1 [53]. The median number of prior lines of treatment was 6 (range, 2–27). T-Dxd was associated with an objective response rate of 60.9% (95% CI, 53.4–68.0) in a heavily pre-treated population. The benefit of T-Dxd for the treatment of HER2+ metastatic breast cancer after progression on T-DM1 was confirmed in the phase III, randomized clinical trial, DESTINY-Breast02 that demonstrated significant improvement in PFS and OS when compared to chemotherapy [54].

The clinical trial that changed practice when choosing a second line treatment for patients with HER2+, metastatic breast cancer whose disease progressed after first line anti-HER2 therapy plus taxane chemotherapy was the DESTINY-Breast03 trial, which was a randomized, phase III study that compared T-Dxd to T-DM1 in the second line setting [41, 55]. A total of 524 patients were randomized in 1:1 to T-Dxd or T-DM1. The primary endpoint of PFS was significantly improved with median PFS of 28.8 months with T-Dxd vs. 6.8 months with T-DM1 (HR, 0.33; P < 0.000001). Overall survival was significantly improved with T-Dxd with the median not reached in either treatment arm although the risk of death was reduced by 36% with T-Dxd (HR, 0.64; P, 0.0037) demonstrating superiority of T-Dxd and establishing its role as a preferred second line treatment in this patient population [2].

DESINY-Breast 04 (DB-04), a phase III randomized clinical trial, has transformed the way breast cancer is categorized and treated [42]. Through demonstrating superior efficacy of T-Dxd in breast cancer cells that has reduced HER2 expression (previously categorized as HER2 negative), DB-04 provided clinical evidence that the bystander effect is an important characteristic of ADCs. Up to 60% of HER2 negative breast cancer cells express low levels of HER2, and more than 50% of HR+ breast cancer is HER2 low making the findings from DB-04 clinically relevant [56, 57]. In DB-04, patients with metastatic, recurrent HER2-low breast cancer defined as IHC 1+ or IHC 2+/ISH- were randomized 2:1 to T-Dxd or chemotherapy (capecitabine, eribulin, gemcitabine, paclitaxel, or nab-paclitaxel). More than 70% of patients with HR+ disease received prior CDK4/6 inhibitors and more than 99% of patients progressed on 1 line of prior chemotherapy. T-Dxd was associated with significant improvement in PFS and OS in the HR+ cohort and all patients. The median PFS in the HR+ cohort was 10.1 vs. 5.4 months (HR, 0.51; 95% CI, 0.40–0.64 P < 0.001) and in HR-negative was 8.5 vs. 2.9 months (HR, 0.46; 95% CI, 0.24–0.89). Median OS in the HR+ cohort was 23.9 vs. 17.5 months (HR, 0.64; P, 0.003) and in HR-negative was 18.2 vs. 8.3 months (HR, 0.48; 95% CI, 0.24–0.95). Benefit was observed across all subgroups including HER2 IHC 1+ and IHC 2+/ISH negative [42]. For patients with HR+ metastatic breast cancer and visceral crisis or with endocrine resistant disease, the National Comprehensive Cancer Network (NCCN) guidelines list T-Dxd as a preferred, category 1 treatment in the second line setting [2]. The NCCN guidelines list SG as a preferred, category 1 option in the second line setting for this patient population if not candidate for T-Dxd. For TNBC with HER2-low, T-Dxd is listed as preferred, category 1 treatment option in the second line setting [2].

#### **6.3 SG**

SG is the first approved ADC targeting Trop-2 [58]. SG was evaluated in the ASCENT, a phase III, randomized clinical trial in patients with metastatic TNBC who

#### *Antibody Drug Conjugates DOI: http://dx.doi.org/10.5772/intechopen.110804*

progressed after at least two lines of chemotherapy, one of which had to be for metastatic disease [59]. Patients (N = 529) were randomized 1:1 to SG or chemotherapy (eribulin, capecitabine, gemcitabine, or vinorelbine). The primary efficacy outcome was PFS in patients without brain metastases. Key secondary outcomes were PFS in all patients and OS. SG was associated with significant improvement in PFS and OS. In patients without brain metastases, median PFS in SG was 5.6 months vs. 1.7 months with chemotherapy (HR, 0.41; 95% CI, 0.32–0.52; P < 0.001). The median OS was 12.1 with SG vs. 6.7 months with chemotherapy (HR, 0.48; 95% CI, 0.38–0.59; P < 0.001). A total of 61 patients had stable, treated brain metastases at baseline, 32 of which were treated with SG. In a subgroup analysis, patients with stable baseline brain metastases treated with SG had median PFS of 2.8 months compared to1.6 months in patients treated with chemotherapy (HR, 0.65; 95% CI, 0.35–1.22). This analysis is exploratory and limited by small sample size [60]. The NCCN lists SG as a preferred, category 1 treatment option in the second line setting for patients with metastatic TNBC who progressed on at least 2 prior chemotherapy lines, at least one of which for metastatic disease [2].

SG was also evaluated in the TROPiCS-02, a phase III, randomized clinical trial evaluating SG in 543 patients with HR+, HER2 negative or low who have progressed on the following: a CDK 4/6 inhibitor, endocrine therapy, and a taxane and at least two prior chemotherapies in the metastatic setting [61]. The primary outcome of PFS was statistically significant with median PFS of 5.5 in the SG arm vs. 4 months in the chemotherapy arm (HR, 0.661; 95% CI, 0.529–0.826; P, 0.0003). The median OS was also significantly improved in these heavily pretreated patients with endocrine resistant disease with median OS of 14.4 in the SG arm vs. 11.2 months in the chemotherapy arm (HR, 0.789; 95% CI, 0.646–0.964; P, 0.020) [62].


**7.2 Assistive devices**

The use of assistive technology eases discomfort and makes daily chores easier (ADL). Nationwide, the availability is uneven (**Figure 1**) [27, 28].

**Figure 1.** *Assistive device, ACL brace with range of motion [29].*

**Figure 2.** *Hand splinting, Cocup Splint [32].*


Meet the editor

Dr. Xuehui He received her PhD at Tuebingen University, Germany, in the field of molecular biology. Thereafter, she moved to Nijmegen, The Netherlands, and started her adventure in the field of immunology. Over the years, the regulation of the immune system in relation to autoimmune diseases has been her main topic of research including the basics of T cell activation, the induction of immune tolerance, and the modulatory effect of

various immune suppressants. Identification of biomarkers for personalized therapy and using the biomarker profile as a co-diagnostic tool is her recent research focus. Her final aim is to capture disease severity in validated outcome measures and assess side effects of treatments in real clinical practice.

Contents

to Immunoactivation

*and Hiroko Shike*

*by Xining Yang and Mark D. Scott*

Rejections in Heart Transplantation

*by Takuya Watanabe and Norihide Fukushima*

Immunosuppression and Viral Infections

**Preface III**

**Chapter 1 1**

**Chapter 2 15**

**Chapter 3 33**

**Chapter 4 51**

**Chapter 5 65**

**Chapter 6 77**

*by Evgeny Grigoryev, Vera Matveeva, Artem Ivkin and Maryam Khanova*

Biological Modulation of the Treg: Teff Ratio: From Immunosuppression

Novel Diagnostic and Therapeutic Approach to Antibody-Mediated

*by Seema Naik, Kevin Rakszawski, Joseph Cioccio, Hong Zheng*

Role of Hybrid Operating Room: Present and Future

*by Evan Qize Yuan and Calvin Sze Hang Ng*

The New Era of Immunotherapy in Bile Duct Cancer Management *by Cosmas Rinaldi Adithya Lesmana and Baiq Kirana D. Mandasari*

Induced Immunosuppression in Critical Care

## Contents

Preface

To safeguard ourselves, our immune system is equipped with a series of defence mechanisms to recognise and respond to non-self molecules. Although essential for fighting off infections and preventing cancers, destructive immune responses pose a considerable challenge in autoinflammation and transplantation. Currently available immunosuppressants help to control destructive immune responses. However, management of side-effects of lifelong immunosuppression, including cancer development and reduced survival, remain major problems. For this reason, an increasing amount of interest is directed towards the natural specific regulatory mechanism of the immune system. A better understanding of these mechanisms

One approach that the immune system employs to induce self-tolerance is via regulatory T cells (Treg). Treg are well known for their immune regulatory potential and are essential for maintaining immune homeostasis. Impaired Treg-mediated immune regulation has been observed in various autoimmune diseases as well as in cancers. Therefore, Treg might provide an ideal therapeutic target for diseases where the immune balance is impaired and could benefit from the regulation of Treg properties. The rationale of Treg-based immunotherapy for treating autoimmunity and transplant rejection is to tip the immune balance of effector T cell-mediated immune activation and Treg-mediated immune inhibition in favour of Treg cells, either through endogenous Treg expansion strategies or adoptive transfer of ex vivo expanded Treg. Human Treg are currently intensively studied for the induction of immunotolerance both in transplantation and autoimmunity. The tumour microenvironment preferably recruits immune cells, which possess a highly immunosuppressive capacity, thus inducing peripheral immune tolerance and facilitating tumour immune escape. Immune checkpoint inhibitors take the brakes off an immune response that has begun thus reactivating the anti-cancer effect.

In this book on immunosuppression, researchers in this area discuss their recent findings in the context of autoimmune diseases as well as cancers. Topics include biological modulation of Treg, monitoring of the complement pathway in the occurrence of antibody-mediated organ rejection, immunotherapy for bile duct cancer, immunosuppression in viral infections, and the management of biliary diseases using endoscopic ultrasound. These reviews are representative of a current active research field that continues to grow and enlighten. We hope the reviews

**Dr. Xuehui He**

Radboud University, Nijmegen, Netherlands

Department of Laboratory Medicine, Medical Immunology Section,

will be of use to experts in the field and new entrants alike.

holds the key.