**The Influence of Thermo-Mechanical Activation of Bentonite on the Mechanical and Durability Performance of Concrete**

#### **Safi Ur Rehman 1,\*, Muhammad Yaqub 1, Muhammad Noman 2, Babar Ali 1, Muhammad Nasir Ayaz Khan 3, Muhammad Fahad 4, Malik Muneeb Abid 5 and Akhtar Gul 6**

Received: 27 September 2019; Accepted: 19 November 2019; Published: 17 December 2019

**Abstract:** Despite presenting a very high global warming toll, Portland cement concrete is the most widely used construction material in the world. The eco-efficiency, economy, and the overall mechanical and durability performances of concrete can be improved by incorporating supplementary cementitious materials (SCMs) as partial substitutions to ordinary Portland cement (OPC). Naturally found bentonite possesses pozzolanic properties and has very low carbon footprint compared to OPC. By applying activation techniques, the reactivity of bentonite can be improved, and its incorporation levels can be maximized. In this study, the influence of mechanical and thermo-mechanical activation of bentonite is investigated on properties of concrete. Bentonite was used for 0%, 10%, 15%, 20%, 25%, 30%, and 35% mass replacements of OPC. Mechanical (compressive strength and split tensile strength) and durability (water absorption, sorptivity coefficient, and acid attack resistance) properties were studied. Results of experimental testing revealed that, concrete containing bentonite showed good mechanical performance, while durability was significantly improved relative to control mix. Application of thermo-mechanical activation can enhance the incorporation levels of bentonite in concrete. At 15% and 25%, bentonite produced optimum results for mechanical and thermo-mechanical activation, respectively. Bentonite inclusion is more beneficial to the durability than the mechanical strength of concrete.

**Keywords:** bentonite; compressive strength; sorptivity; acid attack resistance; durability of concrete; eco-efficient binder; low-cost binder

#### **1. Introduction**

In developing countries like Pakistan, the demands for housing and infrastructure development are increasing each day due to rapid urbanization and population growth. Being a major construction material, the demand for cement has increased abruptly in the recent past to fulfill the infrastructure needs. The production of cement exceeded 4.1 billion tons in 2018 alone, which makes it the most used

material after water [1]. Large scale, global, ordinary Portland cement (OPC) production has always been a big threat to the environment, resources, and economy. Nowadays, cement manufacturing accounts for more than 5% of worldwide CO2 emissions [2]. Moreover, the cost of cement has increased almost 150% in the short span of 10 years [3].

The use of supplementary cementitious materials (SCMs) as replacements of OPC is of great interest for limiting the environmental impact of construction industry [4,5]. It is reported that CO2 emissions can be minimized by replacing OPC with SCMs [6–9]. SCMs, which do not require any additional clinker processing, can significantly reduce the CO2 emissions per ton of cementitious materials [4]. The use of natural pozzolans in binder or concrete can result in various beneficial properties, such as low hydration heat, improved later-strength, low permeability, high sulfate-attack resistance, and low alkali-silica reactivity [10,11]. It is estimated that concrete works in a building cost about 31% of the total, and OPC is the main cost-controlling constituent of concrete [12]. SCMs can be utilized for low-cost concrete construction as well [3,13].

Different types of SCMs such as fly ash, bentonite, metakaolin, silica fume, calcined shale, etc., have been used to improve the performance of cement-based composites [14–16]. Till now, SCMs have been extensively used (60%) in ready-mixed concrete owing to their pozzolanic nature [17]. SCMs give comparable results in terms of mechanical performance and show outstanding performances in terms of durability [10,18]. By blending SCMs with OPC, the penetration of harmful chemicals into concrete can be controlled. After the hydration of OPC, a primary by-product, calcium hydroxide (CH), is produced, which is the main target of corrosive media to deteriorate the concrete [19,20]. Supplementary pozzolanic material consumes the CH during the pozzolanic reaction and forms additional calcium silicate hydrate (C-S-H) gel, leading to a durable concrete [21–25]. Pozzolanic cement blends show resistance to thermal cracking due to its low heats of hydration. Moreover, these blended cements have improved ultimate compressive strengths and low permeability because of pore refinement [20,26,27].

In certain locations, natural SCMs or pozzolans are abundant and widely used in countries such as Italy, Germany, Greece, and China as supplements to OPC [28]. Natural SCMs have a wider range of composition and larger variability in physical characteristics compared to traditional or industrial SCMs. The applications of natural SCMs are limited and controlled by their local availability and competition with the industrially produced SCMs (by-products). In future, the use of natural SCMs will expand more because of their extensive deposits all over the world and proven technical advantages [29]. Clay minerals are hydrous aluminum phyllosilicates, composed of repeated two dimensional, tetrahedral (T) and octahedral (O), sheets in different ratios [30]. The two sheets are hold together by an exchangeable interlayer cation coordinated to H2O molecules in the interlayer region.

When T-sheets and O-sheets combine 2:1, the smectite group of clay minerals is formed. Bentonite clay (montmorillonite mineral) belongs to this group [31]. Bentonite is a natural pozzolana found in different regions of the world. The word bentonite was first proposed by Knight in 1898 [32]. Bentonite is also found in different areas of Pakistan, including the province Khyber Pakhtunkhwa [13]. Bentonite is basically an impure clay mainly consisting of montmorillonite mineral. Bentonite clay fulfills all the requirements of ASTMC-618C [33] to be used as a cement replacement material, as shown in Tables 1 and 2. Moreover, by heating this clay, its pozzolanic reactivity can be enhanced further compared to unheated condition [31,34]. By using bentonite clay as a supplement to OPC, an eco-friendly, economical, and durable concrete can be achieved [3,13].

Pozzolana can be activated by different methods; i.e., mechanical, thermal, and chemical activation. Mechanical activation means to ground the SCMs into powder form to increase their fineness and surface areas. Previous studies found that the pozzolanic reactivity of SCMs can be increased by mechanical activation (grinding) [35,36]. As in a pozzolanic reaction, the silica (SiO2) part of pozzolan reacts with CH in the presence of moisture and forms CSH gel. It was reported that the pozzolanic reaction is determined by rate of dissolution of SiO2 in water, and the dissolution rate of SiO2 is proportional to its surface area [37,38]. Despite increasing surface area prolonging the grinding of a

material, it also increases the number of active centers at the edges of the material structure which are more energetic than the normal structure; the pozzolanic reactivity of the material depends on the number of active centers [39,40]. Thermal activation means heat treatment (calcination). It can be heating of the pozzolan or the high temperature curing of the specimens made with pozzolans. It was reported that thermal treat of clays produce a metastable state due to structural disorder and an amorphous reactive structure is formed [41,42]. Different clays have different calcination effects on their pozzolanic reactivities. Mielenz et al. [43] studied thermal treatment effect on the pozzolanic reactivity of 70 different types of pozzolana and reported the optimum activation temperature range of 650–870 ◦C. In previous studies, the optimum activation temperature for Bentonite was reported as the range of 800–830 ◦C [28,31]. In our previous research [34], the optimum activation temperature of the bentonite clay was found to be 800 ◦C.

**Table 1.** Physical properties of ordinary Portland cement (OPC) and bentonite clay.

**Table 2.** Chemical composition of OPC and bentonite clay.

Many studies have been conducted in Pakistan for utilizing industrial wastes, such as bagasse ash [44], rice husk ash [45], fly ash [46,47], etc., as SCMs. Some natural pozzolana have also been studied [3,13]. In the existing literature, very few studies have been conducted to explore the potential of bentonite clay as partial replacement of cement. A study of bentonite on the mechanical and durability properties of concrete needs to be done in more detail. Memon et al., [13] studied sawabi bentonite clay (3%, 6%, 9%, 12%, 15%, 18%, and 21% by mass) as an OPC replacement and reported positive results of bentonite on the fresh and mechanical properties of concrete. Also, further study was recommended [13] to explore the mechanical and thermal activation of bentonite clay and to study its effects on the properties of concrete. Moreover, high replacement levels of bentonite clay need to be explored. This study explored the mechanical and thermo-mechanical activation of bentonite clay to enhance its incorporation level in concrete. The performance of concrete was studied, both for mechanically activated bentonite (MAB) and thermo-mechanically activated bentonite (TMAB) (at

the levels of 10%, 15%, 20%, 25%, 30%, and 35% by mass of OPC), in terms of compressive strength, split tensile strength, water absorption, sorptivity, and acid attack resistance up to 180 days. The results concluded that 15% mechanically-activated and 25% thermo-mechanically activated bentonite can be used (significantly) as substitutes of OPC, with good mechanical and even better durability properties for concrete.

#### **2. Materials and Methods**

#### *2.1. Materials*

#### 2.1.1. Binder

Ordinary Portland cement (OPC) Type-I was used in the research as a binder. Its physical properties and chemical composition are given in the Tables 1 and 2, respectively.

Bentonite clay taken from district Sawabi, KPK, Pakistan, was used in this research. The clay collected from the natural source was in crude form and could not be used directly in concrete, so after collection, the clay was transferred to ball mill machine to obtain the passing size of number 200 sieve with a nominal sieve opening of 0.074 mm and packed in polythene bags protected from light and moisture. Physical properties and the chemical composition of bentonite clay are illustrated in Tables 1 and 2, respectively. The Bentonite clay fulfils the requirements of ASTM C618-12 [33] to be used as natural pozzolana.

#### 2.1.2. Aggregates

Locally available Lawrencepur sand was used as a fine aggregate. Its properties are given in Table 3. Its particle size distribution as per ASTM C136-06 [48] is shown in Figure 1a. Margalla crush was used as coarse aggregate. Its properties are also given in Table 3. Its particle size distribution as per ASTM C136-06 is shown in Figure 1b.

**Table 3.** General properties of aggregates.

**Figure 1.** Particle size distribution of the (**a**) fine aggregate and (**b**) coarse aggregate.

#### *2.2. Activation of Bentonite*

#### 2.2.1. Mechanical Activation (Grinding)

The clay collected from the natural source was first oven-dried for 24 h at 100 ◦C. After drying, the clay was ground in Loss Angeles abrasion machine. A total of 4500 revolutions were performed on each 5 kg batch of the clay to maintain uniform thickness; the powdered clay was then passed through sieve number 200 with a nominal sieve opening of 0.074 mm and packed in polythene bags to protect it from moisture.

#### 2.2.2. Thermal Activation (Heating)

After mechanical grinding, bentonite clay was heated at 800 ◦C for 3 h. For control and uniform heating, the temperature was allowed to reach the target limit before placing the 5 kg clay batch in the furnace. Then the clay was put in the furnace for the required duration. The furnace took 24 h to reach room temperature. In the meantime, the sample remained in the furnace. The clay, after reaching room temperature, was packed into PVC bags in order to protect it from moisture.

#### *2.3. Composition of Concrete Mixes*

A total of 13 concrete mixes were produced in this study. Mechanically activated bentonite (MAB) and thermo-mechanically activated bentonite (TMAB) were used as 0, 10, 15, 20, 25, 30, and 35% by mass replacements of OPC. A constant water-cementitious material ratio of 0.5 was chosen for all mixes. The nomenclature and the composition of each mixture are presented in Table 4.

**Table 4.** Nomenclature and the compositions of mixtures.

Where "B" denotes "bentonite"; "G" denotes "grinding"; "T" denotes "thermal"; "MAB" denotes "mechanically activated bentonite"; and "TMAB" denotes "thermo-mechanically activated bentonite".

Mixing of all concrete mixes was done in a mechanical mixer of 0.15 m3 capacity at the speed of 35 revolutions per minute. First, all solid ingredients were mixed for about 4 min. Subsequently, water was added, and mixing of concrete continued for the next 4 min. The total duration of mixing was kept to 8 min for all mixes.

#### *2.4. Specimen Preparation and Testing*

To assess the performance of concrete modified with bentonite clay, the following methodology was adopted. To evaluate mechanical performance compression and split tensile tests were performed. For compression testing, cylinders of 150 mm diameter and 300 mm height were cast as per ASTM C39 [49] for three different curing durations; i.e., 7, 28, and 180 days. For split tensile testing, cylinders of 150 mm diameter and 300 mm height were cast as per ASTM C496 [50] for curing durations of 7, 28,

and 180 days. For each test 3 specimens were cast and tested. The cylinders were demolded after 24 h of casting. After demolding, all the cylinders were cured in water for the required durations.

For durability assessment, water absorption, sorptivity coefficient, and H2SO4-acid attack resistance were determined. For sorptivity 50 mm thick and 100 mm diameter discs were prepared and tested as per ASTM C1585 [51]. Similarly, for water absorption, 50 mm thick and 100 mm diameter discs were tested as per ASTM C642 [52]. To evaluate acid attack resistance, for each mix a cubic specimen of 100 mm was cast and cured in normal water for 14 days to gain some strength. After 14 days each specimen was kept in 4% sulfuric acid (H2SO4) solution for 7, 28, 56, and 90 days. To maintain 4% concentration, the acid solution was changed every week. The acid attack resistance was measured in terms of mass loss (%).

#### **3. Results and Discussion**

#### *3.1. Mechanical Properties*

#### 3.1.1. Compressive Strength

Compressive strength is an important parameter of cement-based materials. Many durability and strength parameters of concrete can be predicted accurately using its compressive strength [53,54]. The compressive strength of each mix at 7, 28, and 180-days is presented in Figure 2. The general trend in results indicates that an increasing level of mechanically activated bentonite (MAB) reduces the compressive strength. The use of 10% MAB gives optimum results after 28 and 180-days compared to the control mix (CON). Compressive strength at all ages is badly affected by increasing levels of MAB. After 180 days, mixes with 10%–20% MAB gave better compressive strength than the strength of the control mix at day 28. The improvements in the compressive strength of concrete with the inclusion of 10% MAB can be ascribed to (1) its smaller particle size than OPC, which improves the particle size distribution in the binder matrix and (2) the reaction of bentonite particles with free CH (a primary hydration product) to produce secondary CSH-gels [21]. The reduction in compressive strength with an increasing incorporation level of MAB can be ascribed to reduction in overall CH content in the binder owing to lower calcium oxide percentage of bentonite which reduces the chance of MAB consumption in pozzolanic reaction. Naturally, bentonite is found in consolidated form; therefore, without a suitable treatment/activation method, it cannot be used directly in "as is" condition as a supplement for OPC. Memon et al., [13] reported that an MAB replacement of up to 21% does not affect the compressive strength of concrete compared to the control mix.

Concrete mixes with thermo-mechanically activated bentonite (TMAB) showed higher compressive strength than MAB mixes for a given age and replacement level. After 28-days, 10% incorporation of MAB and TMAB showed 1% and 4.3% higher compressive strengths than the control (CON), respectively, whereas, after 180-days, incorporation of 10% MAB and TMAB resulted in 3.5% and 9.8% higher compressive strengths than CON, respectively. Using the thermo-mechanical activation process, the replacement level of bentonite can be enhanced from 10% to 20% to produce a higher compressive strength than CON at 28-days. Clays may consist of some fixed crystalline structures and many of them possess pozzolanic properties. Calcination of a clay mineral alters its crystalline structure into an amorphous one and increases its pozzolanic reactivity considerably [55]. The heating of clay, shale or other mineral changes their structures to alumina or quasi-amorphous silica, which have good pozzolanic reactivities with CH [56].

Strength activity indices (SAIs) of all mixes at days 7 and 28 are displayed in Figure 3. ASTM C618 [33] states that for any material to be considered pozzolanic, the SAI should be at least 75% of the control blend at both day 7 and day 28. From the SAI values, it can be seen that MAB can be used up to 15% with a SAI higher than 75%. Whereas for TMAB up to a 25% level, SAI is well above 75% for both days 7 and 28. It can be concluded that thermo-mechanical activation produces mixes with more compressive strength than mechanical activation, which can help to maximize the level of bentonite in concrete production.

**Figure 2.** Compressive strength of each mix at 7, 28 and 180-days e clay.

**Figure 3.** Strength activity indices of mixes after 7 and 28 days.

A relative analysis of compressive strength is also illustrated in the Figure 4. Compressive strength of the control mix (B0 (CON)) at day 28 was taken as the reference. It can be observed from Figure 4, that the day 28 compressive strength of concrete with 10%–20% MAB almost overlaps with the compressive strength of the reference mix and a notable decrease is noted for 20%–35% MAB. Similarly, for day 28's compressive strength of concrete modified with 10%–25% TMAB, it overlaps or nearly coincides with the compressive strength of the reference mix and a decline was observed for 25%–35% MAB. At day 180, mixes with 10%–20% MAB and 10%–25% MAB show significantly higher compressive strength than the reference.

**Figure 4.** Compressive strength of each mix relative to the day 28 strength of reference mix (CON).

#### 3.1.2. Split Tensile Strength

Results of split tensile testing are shown in Figure 5. At day 7, the tensile strengths of all MAB modified mixes were lower than that of the CON. Similarly to the compressive strength results, MAB incorporation of up to 10% produced maximal compressive strength after 28 and 180 days compared to CON. It is also worth mentioning here, that split tensile strength is more negatively influenced than compressive strength with increasing the incorporation of bentonite. This might be due to different natures of failure for the specimens under compression and split tensile tests. At higher levels of MAB, the filling effect of unreacted bentonite particles can play a good role in resisting compressive stresses, whereas, under splitting tension unreacted particles of bentonite may offer low resistance to failure (two failure surfaces can easily separate due to low cohesion in the binder); therefore, high levels of bentonite are more detrimental to tensile strength than compressive strength.

TMAB showed significant improvement over MAB, due to increased reactivity of clay minerals in pozzolanic reaction. Split tensile strength was reduced by 12% at a 35% level of TMAB after 28 days, whereas after 180 days, all TMAB incorporated mixes exhibited comparable or higher tensile strengths than the CON mix. MAB showed 3% and TMAB 5% higher split tensile strength than CON mix at day 180. But at higher levels, TMAB mixes performed 10% better than MAB. TMAB may have more reactive alumina and silica to react with CH, so have large pozzolanic reactivity [57,58]. Therefore, higher levels of TMAB show more strength development than those of the MAB.

Relative analysis of the split tensile strength results is presented in Figure 6. 28 days' strength of the control mix (B0 (CON)) was taken as the benchmark. At day 28, there is no notable surpassing form the loop of reference mix/benchmark. For mixes modified with MAB (10%–15%), the replacement overlapped the reference loop. For mixes modified with TMAB (at 10%–25% level), they coincided with the benchmark. At day 180, mixes with MAB (10%–15%) outperformed the reference mix, while all the mixes with TMAB, except B35/G/T, beat the reference mix and crossed the loop. Overall no significant difference was evident for the split tensile strength of TMAB incorporated concrete.

**Figure 5.** Split tensile strength of each mix after 7, 28, and 180 days.

**Figure 6.** Split tensile strength of each mix relative to day 28's strength of reference mix (CON).

As, already mentioned, variation in compressive strength and split tensile strength with increasing incorporation level of bentonite follows a similar trend. Therefore, a strong relationship exists between compressive strength and split tensile strength. In Figure 7, the compressive strength of each mix at a given age and replacement level of bentonite is plotted against the corresponding value of split tensile strength. Equation (1) can be used to predict the cylindrical split tensile strength of concrete mix with a coefficient of determination (R2) higher than 0.9.

$$\text{If } \mathbf{f\_{ts}} = 0.40 \times \sqrt{\mathbf{f\_c}} \text{ (for 7, 28, and 180 days, } \mathbf{R}^2 = 0.92\text{)}\text{.}\tag{1}$$

where fts (MPa) is split tensile strength and fc (MPa) is compressive strength of cylindrical specimen.

**Figure 7.** Correlation between experimental values of compressive strength and split tensile strength.

#### *3.2. Durability Properties*

#### 3.2.1. Water Absorption (WA)

WA is the measure of water accessible porosity of a material. WA is the indirect measurement durability of concrete, since water can carry harmful chemicals into concrete which may react with its constituents and result in changes in the properties of the material. WA tests were conducted on each mix after 28 and 180 days, and the results are given in Figure 8.

**Figure 8.** Water absorption test results after 7, 28, and 180 days.

A general trend indicates that the addition of 10%–15% MAB reduces the WA of concrete after 28 days, compared to CON. After 180 days, 10%–20% MAB reduces the WA of concrete compared to CON. This behavior can be ascribed to an improvement in particle size distribution which results in a reduction in effective pore volume of concrete. Reduction in pore volume results in reduction in WA at all ages, whereas chemical reaction between bentonite particles and free CH can decrease the length of interconnected pores. Studies [59,60] have reported that filler effect of pozzolans (fly ash, GGBS, metakaolin, etc.) decreases the permeability of concrete, despite reducing the early age mechanical strength. After 180 days, the mix with 25% MAB shows WA comparable to that of the CON.

After 28 days, mixes with TMAB up to 25%, show higher resistance to WA compared to CON. After 180 days, a mix with any level of TMAB incorporated shows higher WA resistance than CON. This can be ascribed to improved reactivity of bentonite particles and better distribution of particles in the binder matrix. It was noticed that for a given age and replacement level of bentonite, TMAB-added mixes outperformed MAB by a notable margin. For example, at a 10% replacement of bentonite after 28 days, the MAB-added mix showed a 7% lower WA value compared to Con, whereas TMAB-added mix's value was 24% lower.

The experimental relationship between WA and compressive strength values is presented in Figure 9. Generally, compressive strength value increases with decreasing WA. An inverse-linear relationship is formed is between two parameters, as given by Equation (2). The proposed relationship has good predictability, showing an R2 of 0.80.

$$\text{WA} \left( \% \right) = 18.45 \times \text{e}^{-0.017 \times f\_c} \left( \text{for } 7, 28, \text{ and } 180 \text{ days, } \text{R}^2 \right. \\ \tag{2}$$

where WA is water absorption in percentage, and fc is cylindrical compressive strength of concrete in MPa.

#### 3.2.2. Sorptivity Coefficient (SC)

SC can be defined as the absorption time rate of water by hydraulic cement concrete. It is one of the main properties of concrete for assessing its durability [61]. SC values are given in Figure 10. A similar trend like WA was observed for the results of SC with the varying incorporation levels of MAB and TMAB. For the same age and incorporation level of bentonite, TMAB mixes showed more resistance to sorptivity than MAB-added mixes. Since both WA and SC are functions of porosity and microstructural development of concrete, no significant difference in the results of SC and WA was observed with varying dosages of bentonite. After 180 days, almost all bentonite-added mixes showed higher resistance to sorptivity than CON, whereas after 28 days, up to 25% replacement of bentonite (either MAB or TMAB) with OPC lowered the sorptivity compared to the CON mix.

**Figure 10.** Sorptivity coefficient (SC) test results at days 7, 28, and 180.

Similarly to WA, SC has a good relationship with compressive strength; see Figure 11. Both SC and compressive strength are inversely related to each other in an exponential equation; see Equation (3). Since microstructural development and density due to bentonite inclusion leads to reduction in volume and connectivity of pores, these developments can also improve the compressive strength of concrete. Therefore, permeability-related durability and strength parameters are inversely related to each other.

$$\text{SC} \left( \text{mm} / \text{min}^{0.5} \right) = 0.255 \times \text{e}^{-0.019 \times f\_c} \left( \text{for } 7, 28, \text{ and } 180 \text{ days, } \text{R}^2 = 0.85 \right), \tag{3}$$

where SC is sorptivity coefficient and fc (MPa) is cylindrical compressive strength of concrete.

**Figure 11.** Relationship between experimental values of compressive strength (fc) sorptivity coefficient (SC).

#### 3.2.3. Acid Attack Resistance (H2SO4 Attack)

Acids have a strong tendency to react with the CHs of concrete in order to produce a calcium salt and water. There are many aggressive acids, such as acetic acids, nitric acids, hydrochloric acids, and sulfuric acids (H2SO4). Concrete structures handling the acidic wastewaters of different industries are extremely vulnerable to deterioration. Therefore, it is very necessary to decrease the overall CH content of concrete. In this research, H2SO4 was used for acid attacks on concrete mixes with various levels of MAB and TMAB. H2SO4 reacts with CH and forms calcium sulfate which causes deterioration of the microstructure (due to increased internal pressure) and leads to loss in mass and mechanical strength of concrete. The results of acid attack are presented in terms of loss in mass of the specimens after 7, 28, 56, and 90 days for each mix, see Figure 12.

**Figure 12.** Loss in mass of bentonite-added mixes due to H2SO4 attack.

The general trend in results indicates that the incorporation of either MAB or TMAB results in significant improvement in the H2SO4 attack resistance of concrete. Increasing the incorporation level of bentonite not only consumes the CH in a pozzolanic reaction, but results in an overall reduction of the net CH content of binder [21,22]. OPC contains a very high content of CaO, whereas bentonite is rich in SiO2 and Al2O3, therefore, increasing MAB/TMAB level results in the reduction of CH (a primary hydration product of cement). It was also noticed that for a given age and incorporation level, TMAB-added mixes were more resistant to acid attack than MAB-added mixes. Not only does bentonite reduce the CH content of concrete, but it reduces the pore volume. Reduction in pore volume can also control the penetration of SO4 −2 ions from the solution into the microstructure that may cause the deterioration of concrete.

#### **4. Conclusions**

In this research, the influences on mechanical strength and durability of concrete, of various levels (0%, 10%, 15%, 20%, 25%, 30%, and 35% mass replacements of OPC) of mechanically activated (MAB) and thermo-mechanically activated (TMAB) bentonite clays, were investigated. The following conclusions can be drawn from the experimental results:

Compressive strength is badly affected when bentonite incorporation is increased beyond 25%. The maximum incorporation level of MAB is 10% to produce optimum performance after 28 and 180 days compared to the control mix and 20% MAB produces a comparable performance at 180 days. Contrastingly, maximum incorporation level of TMAB is 20%, in order to produce optimum performance after 28 and 180 days, and 25% to produce comparable results after 180 days. Split tensile strength

varies similarly to compressive strength with varying incorporation levels of bentonite. MAB/TMAB added mixes showed more strength development between 28 and 180 days than the control mix.

In general, more positive influence of bentonite incorporation was noticed on durability properties; i.e., WA, SC, and H2SO4 attack resistance. Due to improved reactivity, TMAB mixes show notably improved durability properties over MAB mixes for a given level of bentonite. With an up to 20% level of bentonite, both MAB and TMAB mixes indicate high durability in terms of SC and WA after 28 and 180 days. All mixes incorporating MAB/TMAB, showed very high resistance to acid attack due to reductions in the overall CH contents of the concretes by both pozzolanic reactions and net reductions in CaO contents of binders.

**Author Contributions:** Conceptualization, S.U.R. and M.Y.; methodology, S.U.R. and B.A.; formal analysis, M.N. and M.M.A.; investigation, M.N.A.K.; resources, A.G., M.N.A.K. and M.F.; writing—original draft preparation, S.U.R.; writing—review and editing, M.Y., B.A. and M.N.; supervision, M.Y.

**Funding:** The authors received no specific funding for this work.

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

#### **References**

© 2019 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 (http://creativecommons.org/licenses/by/4.0/).


**1. Introduction**

#### *1.1.* Helicobacter Pylori *Colonization of the Stomach*

*Helicobacter* is a group of Gram-negative, curved or spiral-shaped bacteria, of which *Helicobacter pylori* is the most commonly known species [1]. *H. pylori* is able to colonize the human stomach by moving through the gastric mucin layer, and long-term colonization may increase the risk for development of atrophic gastritis, peptic ulcers and ultimately gastric cancer [2–5]. Approximately 30% of adults in developed countries and 80% of adults in developing countries are colonized with *H. pylori*, and of these, 1–3% further develop gastric cancer [6,7].

*H. pylori* has only been shown as a natural colonizer in humans, but another *Helicobacter* species capable of gastric colonization in humans is *Helicobacter heilmanii* [8,9]. The prevalence of infection with *H. heilmanii* is much lower than of *H. pylori* and it has been described less than 0.5% of patients undergoing upper gastric endoscopy [10].

Due to gastric peristalsis, mucus thickness, low pH and secretion of bile and acid, it was initially believed that no bacteria could survive in the inhospitable stomach environment [2,3,11]. Later, studies detected *H. pylori* as the only bacterium in stomachs with a healthy low pH, while more bacteria were detected at higher pH [12]. The mucus thickness and viscosity are pH-dependent, and a decreased acidity caused by *H. pylori* colonization, atrophic gastritis, treatment with antibiotics or proton pump inhibitors (PPI) may reduce the acidic protection of the stomach, leading to bacterial overgrowth and a higher diversity [13–16]. An alteration of the gastric bacterial composition has also been reported in cases of gastric cancer, with an increase of both oral and intestinal bacterial groups [16]. Recent studies have described the presence of a non-*Helicobacter* gastric microbiota, suggesting that other bacteria are able to live in the strict environment of the stomach [13].

#### *1.2. Previous Studies of the Gastric Microbiota*

Initial studies on gastric bacterial communities were reported in the 1980s [17]. Since then, several studies have investigated the presence of *H. pylori* and non-*Helicobacter* bacteria by cultivation methods and DNA-based methods [16,17]. Zilberstein et al. cultured gastric biopsies in aerobic and anaerobic conditions and found *Veillonella, Lactobacillus* and *Clostridium* to be the predominant bacterial groups [18]. They concluded that species of these bacterial groups may be transiently present [18]. Li et al. performed 16S rRNA gene amplicon sequencing on gastric biopsies from healthy people and patients with gastritis and observed a community dominated by species of *Prevotella, Streptococcus, Veillonella, Rothia* and *Haemophilus* [14]. Similar results were obtained by Bik et al. by 16S rRNA gene amplicon sequencing, where the dominating genera were found to be *Streptococcus, Prevotella, Rothia, Fusobacterium* and *Veillonella* [2]. A study by Delgado et al. on healthy patients identified *Lactobacillus* as one of the most abundant genera in the stomach as well as *Streptococcus* and *Propionibacterium* by 16S rRNA gene amplicon pyrosequencing [19]. A study by Dicksved et al. detected a bacterial community of *Streptococcus*, *Lactobacillus*, *Veillonella* and *Prevotella*, with a low abundance of *Helicobacter* [20]. The study found no significant differences in the gastric bacterial communities of patients with gastric cancer or dyspepsia [8]. Maldonado-Contreras et al. investigated the microbial composition by 16S rRNA gene amplicon hybridization and found non-*Helicobacter* bacteria such as *Proteobacteria, Firmicutes, Actinobacteria* and *Bacteroidetes* to be dominating [21]. Yu et al. identified similar phyla in the gastric stomach area and other body sites [22]. The authors performed a functional profiling of the stomach microbiota and concluded that though similar phyla were present in these areas, the microbiota of the different areas presented different functions [22]. A systematic review by Rajilic-Stojanovic et al. compared the results of papers investigating the gastric microbiota by next-generation sequencing (NGS) [16]. Approximately 2/3 of the described papers detected species of *Prevotella, Streptococcus, Veillonella, Neisseria, Fusobacterium* and *Haemophilus* and have described that the microbiota is subject-specific and differ between individuals [16]. More than 65% of the bacterial groups found in the stomach have also been identified in the human oral cavity, and many bacteria identified in the stomach may originate from the oral cavity or as reflux from the intestine [3]. Previous studies have not been conclusive about whether the detected non-*Helicobacter* bacteria represent a transient contamination of the stomach or if they belong to the persistent gastric microbiota.

#### *1.3.* H. pylori *and the Non-Helicobacter Microbial Community*

The human stomach has been described to contain a core microbiome mainly consisting of *Prevotella, Streptococcus, Veillonella, Rothia and Haemophilus* spp., influenced by diet, inflammation and medication [3]. However, the effect of *H. pylori* colonization on the bacterial diversity has not been established yet. Andersson et al. investigated the bacterial composition with 16S rRNA gene amplicon pyrosequencing and found complex microbial communities with a great diversity in the absence of *H. pylori* [23]. When present, *H. pylori* accounted for 90% of the bacteria, and diversity was decreased [23]. This agrees with other findings of a large variation in the bacterial community depending on the presence or absence of *H. pylori* [4,12,16,21]. The study by Maldonado-Contreras associated *H. pylori* colonization with an increase of *Proteobacteria*, *Spirochetes* and *Acidobacteria* and decrease of *Actinobacteria*, *Bacteroidetes* and *Firmicutes* [21]. Other studies have opposed the suggestion that the diversity of bacteria in the stomach is negatively affected by the presence of *H. pylori* [2,22,24]. As such, the significance of *H. pylori* on the presence of other bacteria in the gastric environment remains unknown. In a study by Sanduleanu et al., the non-*Helicobacter* bacteria in the stomach were found to contaminate both the gastric juices and the mucosa during gastric acid inhibition [15]. A study by Li et al. investigated the effect of washing on the bacterial content of gastric biopsies [14]. The majority of bacteria remained in the biopsies even after several washing steps, and in particular, *Streptococcus* were not removed [14]. Colonization with *H. pylori* has been associated with increased inflammation and development of gastric atrophy, which may cause overgrowth of other bacteria as the environment turns less acidic [25]. The presence of a complex microbial community in an atrophic stomach may further promote inflammation, malignancies and cancer development [25].

#### *1.4. Microbial E*ff*ects on the Development of Gastric Cancer*

*H. pylori* is classified as a class I carcinogen, and colonization with this species is associated with an increased risk for gastric cancer [26]. The precise mechanism for this development is not known in detail, but it may be affected by diet and interactions with the gastric microbiota [27]. It has been hypothesized that the presence of a specific microbiota promotes inflammation and that other bacteria in addition to *H. pylori* may further promote cancer development [26]. The pH is usually increased in the stomachs of gastric cancer patients, resulting in atrophic gastritis and subsequent loss of *H. pylori,* as the environment becomes less acidic [28]. Several studies of the gastric microbiota in the settings of gastric cancer have been performed. A study by Dicksved et al. compared the microbiota of biopsies from patients with gastric cancer and dyspepsia by terminal restriction fragment length polymorphism [20]. They identified complex compositions of bacteria in gastric cancer biopsies but with no significant differences in the microbial compositions compared to dyspepsia patient biopsies [20]. A low abundance of *H. pylori* and a dominance of species from *Streptococcus, Lactobacillus, Veillonella* and *Prevotella* were observed in biopsies from gastric cancer patients [20]. Another study by Ferreira et al. investigated the bacterial composition in biopsies from patients with atrophic gastritis and gastric cancer [29]. They detected a reduced microbial diversity in gastric cancer patients and found an abundance of less than 5% *H. pylori* in most gastric cancer patients [29]. Based on calculations of a microbial dysbiosis index, the presence of a dysbiotic microbiota in the gastric cancer patients was suggested, compared to the microbiota of individuals with atrophic gastritis [29]. In particular, the microbiota of gastric cancer biopsies showed increased abundances of the groups *Actinobacteria* and *Firmicutes*, while the presence of the groups *Bacteroidetes* and *Fusobacteria* were decreased [29]. Yu et al. analyzed the microbiota of gastric cancer biopsies and identified *H. pylori* as the most abundant bacterial species present, followed by bacteria associated with the oral environment [22]. A decrease in *Proteobacteria* and increase in *Bacteroidetes, Firmicutes* and *Spirochetes* were observed in gastric cancer tissue, but comparisons of microbiota in the corpus and antrum areas of the stomach showed no differences [22]. An altered bacterial composition has also been reported in cases of gastric cancer, with an increase of both oral and intestinal bacterial groups [16].

#### *1.5. Definition of Persistent and Transient Microbiota and Microbiome*

In this study, the gastric microbiota is defined as the persistent bacteria that adhere to the gastric mucosa. The gastric microbiome is defined as the collection of microbial genes in the stomach, which also include the community of non-adherent, transient bacteria [30]. Previous studies have not been able to agree on whether the bacteria identified in the stomach represent a true microbiota or are contamination from the oral cavity. It is therefore not clear which bacteria belong to the true gastric microbiota.

The main aim of this study was to investigate the presence of persistent and transient bacterial communities by comparing the changes in bacterial composition of washed and unwashed gastric biopsies. It is the first comprehensive attempt to distinguish between the transient and resident bacteria of the stomach using 16S rRNA gene sequencing.

The second aim of this study was to investigate and compare the gastric microbiota of gastric cancer patients and dyspepsia patients. The results will contribute to the ongoing debate about whether the process of gastric carcinogenesis is mediated through a microbial shift caused by *H. pylori*. Biopsies from both cancerous and non-cancerous tissue were included, and dyspepsia patients were involved as controls of a healthy microbiota.

The hypothesis of our experimental setup is that *H. pylori* is the only true gastric microbiota of the stomach and will be the predominant bacterium that is able to remain in the washed biopsies. Although other studies have shown the presence of non-*Helicobacter* bacteria in the stomach, these will be expected to be present at least in the unwashed biopsies, as transient contamination from other niches [3].

#### **2. Experimental Section**/**Materials and Methods**

#### *2.1. Sampling of Gastric Biopsies*

Twenty-two patients with dyspepsia and twelve patients with gastric cancer were included in the study. The distribution was 68% female (*n* = 23 patients), 32% male (*n* = 11 patients) and an age range of 22–91 years (median 53 years). Clinical information about the patients is listed in Table 1. The exclusion criteria for the patients were age below 18 years, a previous history of *H. pylori* eradication, use of PPI within the last 3 months, use of antibiotics within the last 3 months, and previous treatment of gastric cancer. Three antral biopsies were taken about 4 cm from pylorus from patients with dyspepsia (*n* = 22 patients) by gastroscopy. From patients with gastric cancer (*n* = 12 patients), three antrum biopsies were taken about 4 cm from pylorus, and three biopsies were taken from the cancer area in the corpus. The biopsies were obtained between November 2017 and June 2019. The first biopsy was immediately used for histology to examine the presence of *H. pylori*. Histological slides were stained with haematoxylin/eosin, and a Giemsa stain was used to confirm the presence or absence of *H. pylori*. Figure 1 shows a histological sample positive for *H. pylori* infection.

**Figure 1.** The positive results of *Helicobacter pylori* infection by Giemsa stain. Black circles indicate stained *H. pylori* (blue) that are attached to the gastric epithelial cells.

All patients participating in the study have signed an informed consent form. The study protocol has been approved by Kaunas Regional Bioethics Committee (Protocol No: BE-2-10; P1-BE-2-31).

The second biopsy was immediately transferred to the transport medium Portagerm pylori (bioMérieux, Marcy L'Etoile, France) and stored at −80 ◦C. The third biopsy was placed in a tube with sterile 4 ◦C PBS, collected by sterile forceps and washed for 15 s in 4 ◦C PBS and transferred to a new sterile tube containing 4 ◦C PBS. This step was repeated twice, after which the biopsy was placed in the transport medium Portagerm pylori (bioMérieux, Marcy L'Etoile, France) and stored at −80 ◦C. These biopsies were then transported to the Copenhagen University Hospital (Rigshospitalet) for culture.

#### *2.2. Culture of Gastric Biopsies and Identification of Single Colonies*

Biopsies were cultured on 7% defibrinated horse blood agar plates (SSI Diagnostica A/S, Hillerød, Denmark), placed in serum bouillon with 10% glycerol (SSI Diagnostica, Hillerød, Denmark), and frozen at −80 ◦C. The agar plates were incubated 6 days at 37 ◦C in microaerobic conditions (10% CO2, 5% O2); the observed growth was noted, and single colonies of each morphology were isolated on new 5% horse blood agar plates (SSI Diagnostica A/S, Hillerød, Denmark). The plates with the isolated cultures were incubated at 37 ◦C in microaerobic conditions until visible growth was seen (1–3 days). Each isolate was transferred to a MALDI-TOF target plate, treated with 1μL HCAA matrix (Bruker Daltonics, Billerica, MA, USA), analyzed with MALDI-TOF Mass Spectrometry (Bruker Daltonics, Billerica, MA, USA), and the protein profile was compared to the database Compass (Bruker Daltonics, Billerica, MA, USA) to identify the species. The biopsies were then transported to Statens Serum Institute for microbiome analysis (16S rRNA gene amplicon sequencing).

#### *2.3. Microbiome Analysis (16S rRNA Gene Amplicon Sequencing)*

DNA was extracted from the biopsies using a QIAamp DNA mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's instruction for tissues. For each batch of DNA extraction, a negative

control without sample material was included for downstream analysis. DNA was amplified using a two-step PCR using a modified version of the published universal prokaryotic primers 341F (ACTCCTAYGGGRBGCASCAG) and 806R (ACTCCTAYGGGRBGCASCAG) targeting the V3-V4 regions of the 16S rRNA gene. Amplicons were sequenced on the Illumina MiSeq desktop sequencer (Illumina Inc., San Diego, CA, USA), using the V2 Reagent Kit.


**Table 1.**

*Comparison of treatment methods for bone injuries along with advantages and disadvantages.*

*Recent Advances, Challenges and Future Opportunities for the Use of 3D Bioprinting in Large… DOI: http://dx.doi.org/10.5772/intechopen.111495*

#### *4.1.2 Low-intensity pulsed ultrasonography*

Ultrasound treatment is a safe non-invasive method and it was started in the early 1930s for therapy applications [44]. For bone healing Low-Intensity pulsed ultrasonography, the ultrasound is applied every day at 1.5 MHz, with 1 kHz pulse and 30 mW/cm2 for 20 min time periods [45]. Both bone-forming osteoblasts and mechano-sensitive osteocyte cells can sense the applied ultrasound. Osteoblast accelerates bone formation while regulating inflammatory responses. On the other hand, osteocytes that consisted high percentage of bone cells can sense acoustic radiation force created by ultrasound which leads to biological signals and enhance bone formation [46]. However, some limitation was detected in Low-Intensity pulsed ultrasonography such as low effect on pain reduction, poor functional recovery, and poor capability to reduce recurrent fracture [44].

#### *4.1.3 Extracorporeal shock wave therapy*

Extracorporeal shock wave therapy is a non-invasive therapy technique and can be defined as a sequence of high-energy acoustic impulses with a different course that can produce pressure changes while it propagates [47]. This technique has some advantages such as being safe, non-invasive, easy, and can apply to most patients, however, some surgeons believed that it has no significant difference from a placebo [48]. While it was demonstrated that extracorporeal shock wave therapy can be involved in the activation of various biochemical signals that trigger neoangiogenesis, fibroblast proliferation, collagen synthesis, and finally tissue regeneration. On the other hand, the negative phase of the shocks can enhance cell membrane permeability and inhibit calcification deposits and reduce pain [49]. The involved cellular signaling pathways during extracorporeal shock wave therapy are focal adhesion kinase, Extracellular-signal-regulated kinase, Wnt/β-catenin, Protein kinase R-like endoplasmic reticulum kinase/activated transcription factor, ATP/P2X7, Brain-derived neurotrophic factor [50].

#### **4.2 Surgical techniques**

Ilizarov method is a simple, effective, common, minimally invasive external fixation technique based on applying compression or distraction forces to the bone defects due to osteogenesis induced by tension stress [51]. Although the Ilizarov method is an applicable technique, especially for large bone defects, in some cases it may be associated with dissatisfaction such as pin tract infection, unconnected bone, re-fracture, bone malformation and transform death reported [52, 53]. One of the other popular surgical methods especially in large bone defects is Masquelet induced membrane [54]. This procedure includes two main steps: first, the created cavity is carefully debridement then is filled with polymethyl methacrylate cement to produce a membrane and inhibit soft tissue formation. In the second step, the cement is removed and the iliac crest cancellous bone or RIA (Reamer-Irrigator-Aspirator) cancellous bone is used to fill the cavity [54, 55].


**About the Editor**

**Catherine E. Housecroft** is Professor of Chemistry in the Department of Chemistry at the University of Basel, Switzerland. Her research group, run jointly with Professor Edwin Constable, is engaged in aspects of materials, coordination, structural and supramolecular chemistries. Much of the emphasis is upon the design and development of materials which will contribute to mankind achieving the 17 Sustainable Development Goals designated in 2015 by the United Nations General Assembly. Compounds of interest range from coordination compounds which can absorb or emit light to coordination networks which can act as hosts for small molecules.


*2.2. Experimental Material*

The experimental material was the 1060 aluminum alloy with the chemical composition listed in Table 1.

**Table 1.** Chemical compositions of the 1060 aluminium alloy (wt%).


**7. Future prospects**

abundant bacterial counts, followed by the hindgut and foregut. Interestingly, the fluorescent *E. mundtii* were also detected in the eggs of *S. littoralis* [14], supporting a direct symbiont transmission from one generation to another. Other studies have shown that fluorescent bacteria were transmitted from the gut to the eggs in *Tribolium castaneum* [120]. The symbiotic *E. mundtii* was transmitted to the second-generation progeny, suggesting that the bacteria co-evolve with the insect host (**Figure 4D**). In addition, the fluorescent bacteria were detected in fecal samples of the larvae, indicating they had traveled successfully along the intestinal tract of *S. littoralis* (data not shown). The details of how a bacterial symbiont is transmitted from one generation to the next remain to be clarified. The symbiont that co-evolves with a host has a great chance to secure vertical transmission, for example, a symbiotic relationship exists between the aphid and its endosymbiont *Buchnera aphidicola*. It has been shown that the GFP-tagged *Asaia* strain is vertically transmitted from the mother to the offspring in *Anopheles stephensi* [121]. Bacterial symbionts can be horizontally transferred via "egg smearing," a phenomenon that involves a female stinkbug covering the surface of its eggs with symbiotic bacteria during oviposition.

survive first-generation and colonize second-generation first-instar progeny. Scale bars: 10–20 μm [14].

90 Metagenomics for Gut Microbes

**Figure 4.** Photo showing the localization of fluorescent *E. mundtii* in the intestinal tract of *S. littoralis* at different life stages. (A) Bacterial cells accumulate on the peritrophic matrix separated between gut lumen and epithelium of fourth-instar larvae. (B) Bacteria cluster in the gut of fifth-instar larvae. (C) Fluorescent bacteria are visibly colonizing the tissue of pupae, although no gut tissue has been formed. (D) Vertical transmission of symbiont is evident as fluorescent *E. mundtii*

The newly hatched juveniles acquire the symbionts by ingesting the egg case [122].

Several factors, including the pH, redox potential, oxygen availability, and the nutrient and immune systems, can shape the microbial composition of the gut of insects [123]. Furthermore, constant change in gut contents due to molting and metamorphosis can affect the colonization of microorganisms. Many insects have an intestinal pH in the range of 6–8, and some

The survival strategies of *E. mundtii* in the gut of *S. littoralis* have been unveiled, yet the mechanisms employed by host insect to control the bacterium remain poorly understood. Transcriptomic analyses of the reporter organism indicated already a pattern of relevant enzymes allowing the microbes to adapt to the harsh conditions of the insect gut. The studies can be extended to the very special conditions in the pupae where fluorescent bacteria could be observed. Thus, the concept of using a fluorescent reporter organism that can be recovered at any time from any area of the intestinal tract will allow a holistic analysis of adaptation strategies used by the microbes to adapt to the different developmental stages of the insect, as well as to study the impact of food-ingested plant toxins. In combination with the analysis of transcript patterns from the gut membranes, a first insight into the molecular interaction between the insect host and the microbiome can be expected. In conjunction with CRISPR/ CAS9-created specific knock downs of defined metabolic capacities of the insect, detailed questions concerning the molecular dialog between the insect host and the microbial consortium can be answered.

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#### Rosa Arboretti a , Riccardo Ceccato b, Luigi Salmaso b a Department of Civil, Environmental and Architectural Engineering, University of Padova, Padova, Italy; **Nonparametric methods for stratified C-sample designs: a case study**

Nonparametric methods for stratified C-sample designs: a case study

b Department of Management and Engineering, University of Padova, Vicenza, Italy; Rosa Arboretti, Riccardo Ceccato, Luigi Salmaso

## 1. Introduction

The analysis of C-sample designs in the presence of stratification is a problem frequently faced by practitioners.

In the industrial field a variety of stratified analysis scenarios present themselves. Take, for example, a company that wishes to assess the performance of three different formulas for a new dishwasher detergent. Multiple dishwashers are used and multiple washes are carried out. At the end of each wash, an expert provides an evaluation of the cleaning performance of the formula. When analyzing the resulting data, the effect of using one dishwasher instead of another cannot be ignored, so each dishwasher is considered to be a separate stratum. Likewise, in the healthcare field it is quite common for multiple drugs to be tested on patients of different age groups. Each age group is again considered to be a stratum.

In this paper we focus on a scenario from the field of education. We are interested in assessing how the performance of students from different degree programs at the University of Padova changes, in terms of university credits and grades, when compared with their entrance exam results. In other words, we want to assess whether people who achieved the best results in this exam perform best during their academic career.

The entrance exam can have three possible outcomes (i.e. it is an ordinal variable). This is therefore a typical stochastic ordering problem (Basso et al., 2009; Basso and Salmaso, 2011; Bonnini et al., 2014), that is a problem in which the main interest lies in evaluating the null hypothesis Y1 d = ... d = YC against the alternative hypothesis Y1 d ≤ ... d ≤ YC and E[ψ(Y1)] ≤ ... ≤ E[ψ(YC)], where at least one inequality is strict, and ψ(·) is an increasing function (Pesarin and Salmaso, 2010). Our aim is in fact to assess whether by comparing increasing entrance exam outcomes, the C = 3 corresponding distributions of the student's performance measure Y are stochastically ordered.

A few nonparametric methods have been proposed in the literature to address these problems. Among them, Jonckheere's test (Jonckheere, 1954; Terpstra, 1952) is one of the first nonparametric solutions to test for ordered alternatives and is based on use of the Mann-Whitney test (Mann and Whitney, 1947) to perform all the possible [C × (C − 1)]/2 pairwise comparisons between C groups. Neuhauser et al. (1998) also proposed a modification of this test that ¨ appears to be more powerful than the original test with small sample sizes (Shan et al., 2014). Additionally, permutation-based solutions involving the Non-Parametric Combination (NPC) technique (Pesarin and Salmaso, 2010; Klingenberg et al., 2009; Finos et al., 2007, 2008) were introduced.

We propose a further extension of the NPC technique to address stochastic ordering problems in the presence of stratification. Indeed, the impact of the student's choice of degree program cannot be ignored, therefore stratification must be considered in the testing procedure.

In section 2 we are going to describe the proposed permutation-based approach. In section 3 we apply it to the case study of interest related to university education. Finally, section 4

9 Rosa Arboretti, University of Padua, Italy<EMAIL_ADDRESS>0000-0003-1263-0440 Riccardo Ceccato, University of Padua, Italy<EMAIL_ADDRESS>0000-0002-8629-8439 Luigi Salmaso, University of Padua, Italy<EMAIL_ADDRESS>0000-0001-6501-1585

FUP Best Practice in Scholarly Publishing (DOI 10.36253/fup\_best\_practice)

Rosa Arboretti, Riccardo Ceccato, Luigi Salmaso, *Nonparametric methods for stratified C-sample designs: a case study*, pp. 17-22, © 2021 Author(s), CC BY 4.0 International, DOI 10.36253/978-88-5518-304-8.05, in Bruno Bertaccini, Luigi Fabbris, Alessandra Petrucci, *ASA 2021 Statistics and Information Systems for Policy Evaluation. Book of short papers of the opening conference*, © 2021 Author(s), content CC BY 4.0 International, metadata CC0 1.0 Universal, published by Firenze University Press (www. fupress.com), ISSN 2704-5846 (online), ISBN 978-88-5518-304-8 (PDF), DOI 10.36253/978-88-5518-304-8

provides the results and conclusions.

## 2. Methodology

Firstly, let us further describe the stochastic ordering problem. The main interest lies in evaluating the system of hypotheses:

$$\begin{cases} H\_0: Y\_1 \stackrel{d}{=} \dots \stackrel{d}{=} Y\_C\\ H\_1: Y\_1 \stackrel{d}{\le} \dots \stackrel{d}{\le} Y\_C \text{ and at least one strict inequality} \stackrel{d}{<}, \end{cases}$$

where the symbol d = denotes equality in distribution and d < denotes stochastic dominance, i.e. Y1 d < Y2 if and only if F1(z) ≥ F2(z), ∀z and ∃I : F1(z) > F2(z), z ∈ I with P r(I) > 0, where Fj is the cumulative distribution function. An alternative way to write this is:

$$\begin{cases} H\_0: F\_1 = F\_2 = \dots = F\_{(C-1)} = F\_C \\ H\_1: F\_1 \ge F\_2 \ge \dots \ge F\_{(C-1)} \ge F\_C \text{ and at least one strict inequality.} \end{cases} \tag{1}$$

NPC-based solutions generally consider a particular decomposition. The hypotheses are split in order to recreate the conditions of a set of two-sample problems as follows:

$$\begin{cases} H\_0: \bigcap\_{i=1}^{C-1} H\_{i0} = \bigcap\_{i=1}^{C-1} [(F\_1 = \dots = F\_i) = (F\_{(i+1)} = \dots = F\_C)],\\ H\_1: \bigcup\_{i=1}^{C-1} H\_{i1} = \bigcup\_{i=1}^{C-1} [(F\_1 = \dots = F\_i) > (F\_{(i+1)} = \dots = F\_C)]. \end{cases}$$

where the null hypothesis H0 is the intersection of a number of partial hypotheses and the alternative hypothesis H1 is the union of C − 1 sub-hypotheses.

For each pair of sub-hypotheses Hi0 and Hi1, the first i and the last (C − i) samples are pooled, so that two new samples X1 and X2 are achieved, with sizes N and M. The subproblem can therefore be rewritten as:

$$\begin{cases} H\_{i0} : X\_1 \stackrel{d}{=} X\_2\\ H\_{i1} : X\_1 \stackrel{d}{<} X\_2. \end{cases}$$

Each sub-hypothesis is then tested separately, using appropriate permutation tests. The adopted test statistic can differ according to the nature of the data, but a common and versatile choice is the modified version of the Anderson-Darling test statistic:

$$T = \sum\_{j}^{n} [\hat{F}\_1(X\_j) - \hat{F}\_2(X\_j)] / \{\hat{F}(X\_j)[1 - \hat{F}(X\_j)]\}^{\frac{1}{2}} \tag{2}$$

where X = {X1, X2} is the pooled sample, Fˆ1(t) = N j I(Xj1 ≤ t)/N, Fˆ2(t) = M j I(Xj2 ≤ t)/M, F¯(t) = n j I(Xj ≤ t)/n, n = N + M, t ∈ R1 and I(·) is the indicator function which is 1 if (·) is satisfied and 0 otherwise.

According to the NPC algorithm (Pesarin and Salmaso, 2010), B permuted datasets are independently generated for each sub-problem and the related values of the test statistic T∗ b , b = 1,...,B are calculated to simulate the null distribution of T. Partial p-values (λi) and λ∗ ib, b = 1,...,B estimating their distributions can therefore be achieved. It is worth noting that the same permutation design is adopted for each sub-problem, to implicitly take into account the existing dependency among sub-problems.

A combination step now needs to be performed. The partial p-values λi, i = 1,...,C − 1 related to the C − 1 sub-problems {Hi0 vs Hi1} are combined using an adequate combining function, such as Fisher's combining function T F = −2 · C−1 i=1 log(λi). The same is done for each of the B vectors λ∗ ib, i = 1,...,C − 1. The elements of the new resulting vector represent the second-order test statistics, from which it is finally possible to achieve the global p-value λ to assess the system of hypotheses 1.

Given that stratification needs to be included, we propose firstly applying this procedure to each of the S strata, testing S systems of hypotheses:

$$\begin{cases} H\_{0s}: F\_{1s} = F\_{2s} = \dots = F\_{(C-1)s} = F\_{Cs} \\ H\_{1s}: F\_{1s} \ge F\_{2s} \ge \dots \ge F\_{(C-1)s} \ge F\_{Cs} \text{ and at least one strict inequality.} \end{cases} \tag{3}$$

After applying the aforementioned NPC-based approach to each stratum, the global p-values λs , ∀s = 1,...,S (and the λ∗ sb estimating their distributions) are thus retained. Then we adopt a further combination step, using the Fisher combining function, and retrieve a final p-value λ. In this way, by comparing λ to the desired significance level α, we are able to solve the global stochastic ordering problem H0 vs H1.

Given that multiple systems of hypotheses Hs0 vs Hs1, ∀s = 1,...,S are assessed, we then apply an appropriate multiplicity correction to control the false discovery rate (FDR). Our choice is the Benjamini-Hochberg procedure (Benjamini and Hochberg, 1995).

## 3. A case study

Let us now focus on the real stratified C-sample problem at hand. As mentioned before, we are interested in evaluating the performances of students from different degree programs at the University of Padova. In particular, we want to understand if the university credits gained at the end of the first year (Y a), the credits gained at the end of the third year (Y b ) and the final average grade (Y c ) somehow depend on the results achieved by the student in the entrance exam. In other words, we try to indirectly assess the efficacy of this exam in evaluating and selecting future students. The analysis is performed using R (R Core Team, 2020).

Let us briefly describe the data. The total sample size is 3083 students. Firstly, the degree programs are grouped into 4 classes (identified by their Italian subject titles):

The different classes represent different strata (i.e. S = 4) and have different sample sizes (see Figure 1). The variable reporting the outcome of the entrance exam has three modalities (i.e. C = 3), namely INSUFFICIENTE, SUFFICIENTE and PIU' CHE SUFFICIENTE (Insufficient, Sufficient and More Than Sufficient). For the sake of simplicity, we are going to refer to them as INS, SUF and PIU in our notation. In Figure 1, the possible outcomes are ordered from worst to best.

For each response variable Y j , ∀j ∈ {a, b, c}, we want to assess if Y j INS d ≤ Y j SUF d ≤ Y j PIU, with at least one strict inequality, taking into account the effect of the degree program class.

Looking at credits gained at the end of the first year, a first descriptive analysis (see Figure 2) appears to support the alternative hypothesis. Indeed, in all strata, students achieving INS at the entrance exam appear to perform worse than students achieving SUF, and students achieving PIU at the entrance exam tend to perform better than students achieving SUF.

Similar conclusions can be drawn about both credits gained at the end of the third year (see Figure 3) and the average grade at the end of the academic career (see Figure 4).

Applying our testing procedure, we managed to confirm these hypotheses. We set B = 10000 and used the test statistic in Equation 2 and Fisher's combining function. When looking at Y a (see Table 1), all the partial p-values and the global p-value proved to be substantially smaller than 1%. The only exceptions were ING CIVILE AMBIENTALE L7 (S2) and ING INFORMAZIONE L8 (S3), for which the descriptive analysis shows that the order among entrance exam outcomes is less evident.

Figure 1: Description of the sample.

Figure 2: Credits at the end of the first year.

Figure 3: Credits at the end of the third year.

Figure 4: Average grade at the end of the academic career.

Table 1: Table of p-values for Y a, Y b and Y c .

# 4. Conclusions

In this paper we presented a new solution to C-sample stochastic ordering problems in the presence of stratification, focusing on its application to a case study from the field of education.

Our proposal takes advantage of the Non-Parametric Combination (NPC) procedure (Pesarin and Salmaso, 2010), a versatile permutation-based methodology allowing us to solve several different complex problems, such as stochastic ordering. We apply this technique to evaluate the presence of stochastic ordering in each of the S existing strata and then use an appropriate combining function to assess the stochastic ordering in all the samples.

The application of this procedure allowed us to assess the efficacy of the University of Padova's entrance exams in evaluating and selecting future students. Indeed, it emerged that students with the worst results in the entrance exam tended to perform the worst during their academic career, in terms of both university credits achieved at the end of the first and third years and in terms of the final average grade, independently of the chosen degree program. The only exception was people from ING CIVILE AMBIENTALE L7 and ING INFORMAZIONE L8. For these two strata, when the credits at the end of the third year were considered, it was not possible to find enough evidence in favor of the stochastic ordering hypothesis.

Overall, this approach appears to be significantly promising and a simulation study has been planned to further explore its performances.

# References


**Preface**

In times of global crises such as the COVID-19 pandemic, climate change, or financial crises, the social cohesion of democracies is threatened. The geopolitical battle for global influence and a public communication model that takes place in social networks encourage polarization and disinformation, having consequences for the public sphere. This reprint, "Geopolitics, Public Communication, and Social Cohesion Facing the Crisis of Democracy: Risks and Challenges", contributes to understanding the communication of the main conflicts that put integrity and geopolitics at risk.

Leaders, the media, and citizens, as digital consumers, build stories on digital platforms that can revert to the unity of territories, consolidate extremist ideologies, or foster fragmentation between states. Conversely, reports and opinion polls, from electoral contests or otherwise, highlight audiences' distrust in politics, citizens' disaffection with the media, and institutions' loss of credibility. On this matter, the articles included use innovative approaches based on mixed methods in different countries.

Our reprint aims to provide an academic overview of the role of communication by public institutions in crisis management and the responsibility of journalism and citizens.

> **Concha P ´erez Curiel and Rub ´en Rivas-de-Roca** *Editors*


*2.2. Definitions*

Preeclampsia was defined according to American College of Obstetricians and Gynecologists (ACOG) Task Force on Hypertension in Pregnancy (2013) and the revised statement (2014) of the International Society for the Study of Hypertension in Pregnancy (ISSHP) [9,10]. We considered the diagnosis of preeclampsia in CKD patients in every pregnant woman with pre-existing CKD who developed hypertension (≥140 mmHg systolic pressure or ≥90 mmHg diastolic pressure) after 20 weeks of gestation or a sudden exacerbation of preexisting hypertension or the need to improve antihypertensive therapy, plus the coexistence of one or more of the following new-onset conditions: proteinuria ≥ 300 mg/day or a significant sustained increase of preexisting proteinuria; liver involvement (elevated transaminases and/or severe right upper quadrant or epigastric pain); neurological complications (eclampsia, altered mental status, blindness, stroke, hyperreflexia when accompanied by clonus, severe headaches when accompanied by hyperreflexia, persistent visual scotomata); hematological complications (thrombocytopenia, disseminated intravascular coagulation, hemolysis); and fetal growth restriction. CKD was defined according to Kidney Disease Improving Global Outcomes (KDIGO) guidelines [11]. Referral was defined as the first visit of the patients to our clinic, between the beginning of pregnancy and week 20 of gestation.


**1. Introduction**

The secondary information has extensively recognized the key role of infrastructure growth in fascinating FDI inflow. A sound developed infrastructure strategy boosts markets integration and entices FDI inflow in any nation [1], whereas the deficiency of comprehensive infrastructure interrupts markets relationship and herewith slowdown the foreign direct investment (FDI) inflow in particularly developing countries [2]. The obtainability of advanced infrastructure principally decreases the cost of trade, boosts the ease of doing business and invites the foreign direct investment (FDI) inflow. Infrastructure tool is used in this paper as an engine for economic growth and facilitate a comparative advantage to a developing nation in terms of foreign direct investment inflow [3]. Additionally, the secondary data has shown evidence that the nation with good infrastructure engrossed more foreign direct investment inflow [4], whereas the nation's deficient with infrastructure development are stereotypically unsuccessful to attract the FDI inflow [5] and those nation economies also shown that the poor condition [6]. Moreover, it also determined that the impact of the infrastructure development on FDI is positive and significant in a growing economy, preceding research [7] assessed that a deficiency of Infrastructure castigates FDI inflow. The significance of the infrastructure plays a vital role in the promotion of foreign direct investment inflow. The research data extensively explore the query of how the nonexistence of infrastructure can affect foreign direct investment inflow along with the different results of foreign direct investment in the host nation. Though the literature review has given little concentration to examine the role of FDI in improving the obtainability and quality of infrastructure in developing nations' economies. According to Pradhan et al. [8] illustrated that the foreign organizations carried progressive technology and skills to the host nation herewith encouraged new technological dissemination in the nation along with investment. FDI inflow also facilitates home organizations with an unintended opportunity to learn from the foreign firms by studying and permeating an intelligence of plenteous needed competition [9]: which develops a cumulative output of the ant nation economy. Foreign direct investment plays a significant role in economic growth [10], the positive impact of FDI inflow is not only inadequate to the transfer of better technology, in circumstance, it also needs any nation to develop the quality infrastructure [11]. As a developing nation economy like Indian have enough resources but do not advance technology to effective utilization of the resources, to improve the infrastructure facilities in India, advanced technology is required, it is required the support of overseas capital to improve their infrastructure facilities. Foreign companies cooperate in R&D in enhancing innovative technology and development of any nation, specifically in infrastructure development to bond up with various markets in the different nations. The literature review broadly examined the various determinants factors of FDI like population, political stability and institutional quality etc. [12]. As this critical point lacks in review, this paper aims to examine the causal two functional relationships between total FDI and total infrastructure and enhance the literature review on this vital singularity. Furthermore, the literature discloses that prevailing research articles on the subject matter hurt from numerous data limitations [13]. The original paper on infrastructure focuses on variables representative of infrastructure for a large nation sample during 1990–2018 but it does not formulate an index of cumulative infrastructure. Likewise, extensively the review on infrastructure projects in different sectors depend on a very inadequate description of infrastructure while examining its stimulus on different economic indicators are investment, trade, and growth. Gnangnon (2018) assessed the impact of the telecommunication infrastructure on economic growth in a developing nation like India. Chakraborty and Nunnenkamp [14] uses ITC (international telephone circuits), the inclusive road infrastructure length and the number of airways as a proxy for the infrastructure development to examine the relationship between public infrastructure and foreign capital. Hall et al. [15] investigated broader insight and apprehension of the various infrastructure components to assess the association between transportation cost and infrastructure growth.

As for the given information limitations, this research paper employs a recently established inclusive using global infrastructure index 2020 which comprehends numerous infrastructure extents for India to overcome statistics limitations in secondary information. Predominantly the global infrastructure index 2020 is grounded on an annual wide-ranging of minimum 15 indicators datasheet of the obtainability and quality of infrastructure during 1995–2018 formulated by Khan et al. [16, 17]. In this research paper, the following infrastructure parameters (like power sector, construction, transportation, telecommunication, health, finance and energy) are used. UCM (Unobserved Components Model) is employed expedient infrastructure from the sub-parameters of infrastructure development. Additionally, the paper highlights some important points According to the Reserve Bank of India, infrastructure covers the following sectors also Power, Telecommunications, Railways, Roads including bridges, Seaport and airport, Industrial parks, Urban infrastructure, Mining, exploration and refining, and Cold storage and cold room facility, including for farm level pre-cooling for the preservation or storage of agricultural and allied produce, marine products and meat.


**A. Appendix A**

The drift velocity of positive ion neon gas is: *w*þ ¼ ð Þ 11*:*27*E=n =* ð Þ 1 þ 0*:*01288*E=n* 0*:*5 (m/s) [52] where E/N is in Td. The metastable lifetime of neon gas is equal to 14*:*73 s [53]. The diffusion coefficient of metastable atoms is *Dm* ¼ 150 cm2 s �1 Torr [54]. The energy loss of an excited atom is 16.6 eV. The Ionization energy of neon gas is equal to 21*:*56454 eV. The energy loss (Pec) per electron due to elastic collision of electrons with the background gas is calculated according to [31, 55]. The rate coefficient of chemo-ionization processes is *Kci* ¼ 3*:*6 � 10�10 cm3 s �1 for T = 310 K [56]


**5. Conclusions**

In summary, this research elucidated the complexities of engaging PLWH-SU in treatment. It identified distinct healthcare barrier profiles among PLWH-SU enrolled in CTN-0049, each with different responses to PN interventions. These results help to inform the use of PN programs and provide a more efficient target for PN resources by more narrowly defining the patient population for which PN is effective.

**Supplementary Materials:** The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/jcm12010114/s1, Figure S1: Differences in 3-Class Latent Profile Analysis model by gender. Figure S2: Differences in 3-Class Latent Profile Analysis model by race. Figure S3: Differences in 3-Class Latent Profile Analysis model by southern/non-southern residence.

**Author Contributions:** Conceptualization, S.M.T., L.R.M. and D.J.F.; formal analysis, S.M.T. and D.J.F.; methodology, S.M.T., A.W.C. and D.J.F.; project administration, L.R.M. and D.J.F.; supervision, A.W.C., V.E.H., L.R.M. and D.J.F.; writing—original draft, S.M.T.; writing—review and editing, R.D.S., L.K.G., T.M., L.H., A.R., M.M., P.J., R.M., C.D.R., A.W.C., V.E.H., L.R.M. and D.J.F. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by the National Institutes on Drug Abuse [U10DA013720 and UG1DA013720], the Miami CFAR [P30AI073961], the Emory Center for AIDS Research [P30AI050409] and the Center for HIV and Research in Mental Health [P30MH116867].

**Institutional Review Board Statement:** The parent study received approval from the respective Institutional Review Boards for each study site. However, the current study only used de-identified data and, therefore, was exempt from ethical review.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** CTN 0049 trial data are publicly available on NIDA Data Share: https:// datashare.nida.nih.gov/ (accessed on 7 September 2018).

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

**Disclaimer:** The authors are solely responsible for the content of this article, which does not necessarily represent the official views of the National Institute on Drug Abuse. Dr. Raul Mandler, an employee of the National Institute on Drug Abuse, is an author; he reviewed and approved the manuscript as a part of his authorship role as a Scientific Official.


**Author details**

Said M. Yaiesh1 \*, Abdullatif E. Al-Terki2 and Tariq F. Al-Shaiji2

1 Mubarak Al-Kabeer Hospital, Kuwait City, Kuwait

2 Amiri Hospital, Kuwait City, Kuwait

\*Address all correspondence to<EMAIL_ADDRESS>
© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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**Preface to "Synthesis of Marine Natural Products and Molecules Inspired by Marine Substances II"**

The sea covers more than 70% of Earth's surface and contains more than 300,000 organisms with huge biodiversity. These organisms represent an enormous tank of substances whose chemical structures are the result of the enormous ecological pressure for survival. Given their high chemical diversity, biological activity, biochemical specificity and other crucial molecular properties, marine natural products have always played a crucial role in the search and discovery of novel bioactive molecules that are potentially useful for pharmacological applications. In fact, the development of new 'lead' compounds from the sea has always been and is one of the main purposes of drug discovery.

The study and advanced progress of these products cannot be separated from the development of chemical synthesis and synthetic strategies aimed at the preparation and optimization of these substances and/or analogs, leading the way to new classes of biologically active compounds with pharmacological potential.

> **Emiliano Manzo** *Editor*


**8. Staff training**

There is nothing wrong with using one approach, if this 'one approach' is science [15]. Countless procedures have been developed from the science of behaviour analysis, many specifically for ASD, e.g., Discrete Trail Teaching (DTT); Pivotal Response Training (PRT); Natural Environment Training (NET); Verbal Behaviour Approach (VB); while other proce‐ dures have been developed for more general applications, e.g., Functional Analysis and Functional Assessment; Preference Assessments; shaping, forward chaining, backward chaining; differential reinforcement of low or zero rate and/or incompatible or alternative behaviours; Time-out from Positive Reinforcement (TOR); etc. . Some of these procedures have been combined into comprehensive packages for autism, such as Early Intensive Behavioural Interventions (EIBI) or Early Start Denver Model (ESDM), while others are used more gener‐ ally, e.g., Programmed Instruction, Generative Instruction; Peer Tutoring; Habit Reversal Training; etc. Given that the science of behaviour analysis underpins all of these programmes/ procedures and continuous data-based decision making is part and parcel of ABA, new procedures and progammes are developed continuously to meet the individual or group needs

ABA has been further ridiculed and accused of intending to change the person, while others pride themselves for accepting the person for who they are [69, 80]. In fact, the targets of ABAbased interventions are socially relevant behaviours, linked to cultural and personal norms and preferences [4]. The curricula are agreed with individuals with ASD and/or their caregiv‐ ers. They are generally based on wide-ranging target behaviours, including life skills, such as dressing, toileting, attending; social skills, such as playing or imitation; academic skills, including attending, reading, drawing, writing, and maths, and work/employment based

Basically, the aim of ABA is to enhance all skills necessary to lead a fulfilled life for individuals who would otherwise be limited in the quality of life they experience. These are the same aims that most parents have for all of their children, irrespective of a diagnosis. As such, ABA does not intend to 'change the person', but to enhance skills and help individuals to break down barriers to learning and achieve their full potential. After all, enhancing skills development

Once a new idea can no longer be ridiculed, the second point Schoppenhauer made comes to play: the new idea is opposed. In the case of ABA, this refers to statements such as there is no evidence to support ABA and therefore no recommendation can be made [64]. We have outlined the wealth of evidence in favour of ABA-based interventions earlier in this chapter. Given that behaviour analysts commonly are not included in review bodies, at least in Europe,

When the evidence can no longer be denied, the opposition turns to the behaviour analytic scientists themselves, stating that research conducted by behaviour analysts is biased and therefore not to be taken seriously. The idea, that it is objectionable that scientist conduct scientific research in their own subject area is rather intriguing. Given that it is against ethical guidelines of all social and health care as well as education professionals to work outside their own area of expertise [90], clearly, multidisciplinary practice and interdisciplinary research teams in ASD, should routinely include behaviour analysts, not least because others are not

this mountain of evidence generally is excluded from reviews [68].

qualified to make authoritative statements about behaviour analysis [16].

of service users.

256 Autism Spectrum Disorder - Recent Advances

increases choice.

skills, including interviewing or team work.

A Board Certified Behaviour Analyst (BCBA®) has received fully approved training in the science of behaviour analysis either at Masters or doctoral level, including at least 270 hours of course work and 1500 hours of supervised practice in ABA [3].

While NICE [64] did not make any recommendations regarding ABA or staff qualifications, they recommend a 'social-communication intervention' that includes play-based strategies with parents, carers and teachers to increase joint attention, engagement and reciprocal communication in the child or young person.

Table 1 offers a direct comparison of the basics of ABA-based interventions that were stipulated by the [8] and the NICE [64] recommendations.

**Table 1.** Comparision of ABA-based interventions and NICE [64] recommendation

#### **9. Conclusion**

In a recent review for the Canadian Medical Journal, [1] summed up the evidence for ABAbased intervention in ASD when they stated:

Current best practices for preschool-aged children with ASD include a focus on improving language, cognitive and adaptive skills using applied behaviour analysis (ABA) techniques. Applied behaviour analysis refers to the application of empiri‐ cally derived learning principles (i. e., the antecedent–behaviour–consequence contingency) to produce meaningful changes in behaviour. Such strategies are carefully engineered and implemented through a variety of approaches (e.g., discrete trial teaching to more naturalistic learning contexts) to teach skills and reduce problem behaviour. Applied behaviour analysis interventions can be provided in a variety of settings (e.g., home, specialized treatment centres, special‐ ized or public schools) by a range of front-line therapists, ideally supervised by a psychologist or board-certified behaviour analyst who specializes in ASD. (p. 515)

It is, therefore, not surprising that increasingly reports link ABA-based intervention with optimal outcomes. Individuals previously diagnosed with ASD are now living independent productive happy lives or no longer meeting diagnostic criteria. Mukaddes, Tutkunkardas, Sari, Aydin, and Kozanoglu (2014) suggested that '[i]t could be concluded that a group of children with an autism diagnosis could lose the diagnosis of autism upon early intervention' (p. 1). [65] for example, report a strong statistical significance of early intensive behaviour analytic interventions for children previously reported to have optimal outcomes [28], while [85] report on reductions of restricted and repetitive behaviours and [85] focus on improve‐ ments in academic skills following early intensive behavioural interventions.

Evidently, ABA translates into evidence-based interventions that allow individuals with ASD to overcome barriers by ensuring choice, human rights, equality and true active participation. ABA helps achieve potential by cherishing the person for who they really are and, by accepting difference, it values the difference we can make in people's lives.


Contents

### **Preface XI**

Preface

Endotracheal intubation is the most important component of airway management and is of vital importance. Airway management is a complicated medical practice in which many medical disciplines are involved. These medical disciplines include anesthesiology, emergen‐ cy medicine, critical care, pulmonary medicine, and surgery. Various examinations, maneu‐ vers, and positions may also be required for tracheal intubation. In addition, if the tracheal intubation could not be performed, surgical intervention should be implemented and oxy‐ genation should be provided by tracheotomy or cricothyrotomy. Thus, if intubation difficulty is predicted in medical situations where the airway opening should be provided, or if intuba‐ tion is not possible, a "difficult airway algorithm" should be used for life-saving procedures. In this book, the medical basis of hypoxia and oxygenation, functional airway anatomy and physiology, and difficult airway management are described. I am grateful to the authors for

their contributions to this book and hope that this book will be helpful to the readers.

**Prof.Dr.Riza Hakan Erbay, M.D.**

Faculty of Medicine Pamukkale University Denizli, Turkey

Chairman of Department of Anesthesiology and Reanimation