**4. HRTEM study of van der Waals structure of silicon nitride and aluminum nitride**

The atomic arrangement of (8 × 8) was also investigated here by the HRTEM method. For these studies, samples with the following sequence of layers were grown on the (111) Si substrate: 2–3 monolayers of silicon nitride with a structure (8 × 8) and thin epitaxial layer of AlN. The interplanar spacing in the Si substrate in the silicon nitride and AlN layers was determined, see **Figure 7**. It turned out that the interplanar spacing between the layers of silicon nitride and also between the last silicon layer and the silicon nitride layer is about 3.3 Å, which is noticeably larger than the interplanar distances in silicon (3.13 Å) and the known thickness of the β-Si3N4 monolayer (2.9 Å). In addition, the layers of silicon nitride differ sharply in contrast from the layers of Si and AlN.

The interplanar distances in silicon nitride of 3.3 Å are larger than the interplanar distances Si 3.13 Å and are larger than the thickness of the monolayer β-Si3N4 (2.9 Å). The interplanar distances in the еpitaxial AlN layer are also larger than normal interplanar distances in bulk wurtzite AlN (2.49 Å). Therefore, this epitaxial structure (SiN)2(AlN)4 turned out to be a van der Waals crystal.

**Figure 7.** *HRTEM image of layers of SiN and AlN on the Si (111) surface.*


3.2.2. Overset

A polyhedral mesh was chosen for the overset region, as shown in Figure 8, due to the possibility to change the volume of cells gradually and prevent wave reflection near the boat where the strongest gradients are solved.

**Figure 8.** Overset polyhedral mesh.

To better capture the pressure gradients and wave generation at the bow and stern, volumetric refinement controls were applied in order to reduce mesh size in these areas: A control at the bow with very fine cells was used to avoid numerical ventilation [30], and a control for the stern guaranteed the correct flux under the hull.

When deciding for the overset dimensions, two main aspects should be considered:


**3. Preliminaries and Notation**

All the algorithms studied in this paper work in planar, eight-connected grid maps with regions of obstacles consisting of blocked cells. A wavefront launched by a cell state change can propagate from one cell to its neighbor in any cardinal or diagonal directions, and the length of the cardinal and diagonal moves are 1 and <sup>√</sup> 2, respectively. We follow the definition of octile distance to compute the heuristic distance between any two cells in a grid map, i.e., the distance between cells *s* and *s'* is computed by following Equation (2):

$$dist(\mathbf{s}, \mathbf{s}') = \sqrt{2} \times min(d\mathbf{x}, dy) + \left| d\mathbf{x} - dy \right| \tag{2}$$

In Equation (2), *dx* and *dy* denote the differences of the 2D coordinates of cell *s* and *s'*. For each cell *s*, *obsts* maintains coordinates of the obstacle cell *so* to which *s* is currently closest, and *dists* maintains the distance between *s* and *so*. The notation *dirs* maintains the direction along with which s would propagate the wavefront. The options of *dirs* include four cardinal directions (denoted as left = 1, up = 3, right = 5, and down = 7), four diagonal directions (denoted as up-left = 2, up-right = 4, down-right = 6, and down-left = 8), and full directions (denoted as full-dir = 0, declaring that *s* is the source of the wavefront; thus, all the eight directions need to be considered in the next round propagation). The notation *raises* shows if the wavefront on *s* is a raise wavefront or a lower one (the differences between raise and lower wavefronts are explained in Section 4.1.1). Given a cell *s*, a direction *d*, and an integer *k*, the notation s' = *s* + *kd* denotes a cell *s'* that is reached from *s* by moving *k* steps along *d*. For two perpendicular directions *c1* and *c2*, we have *d*=*c1*+*c2* to denote that the sum of *c1* and *c2* results in their corresponding diagonal direction *d*.


**Acknowledgements**

We would like to thank the support of K.C. Wong Education Foundation (China). This work was financially supported by the National Natural Science Foundation of China (No. 82071974 and No. 82102107), and the Science and Technology Program of Guangzhou, China<PHONE_NUMBER>06).


*3.5. Morphology Analysis*

Figure 5 shows the surface morphology of TPRH and TPS with irregular (Figure 5a) and spherical (Figure 5b) shape, respectively. The TPS granules with spherical shape caused a reduction in the contact area of the polymer matrix when compared with the irregular shape of TPRH [52]. For bare polymers of PBS (Figure 6a) and PBAT (Figure 6b), smooth surface morphology was observed while with filler loading, a rough surface was observed as shown in Figure 6c–f. When 40% of TPRH was added into PBAT/PBS matrix, the TPRH particles dispersed homogeneously in the PBAT/PBS matrix, as shown in Figure 6c,e. Some voids in the interfacial boundary were observed in TPRH48/12 due to the pull out of the rice husk particle from the PBAT/PBS matrix. This is caused by the difference in polarity between the hydrophilic rice husk and the hydrophobic PBAT. Fewer filler pullouts with good fibers-matrix adhesion were observed in TPRH36/24 (Figure 6e) as compared to TPRH48/12 (Figure 6c). For TPS blends, a similar trend was observed in TPS48/12 (Figure 6d), which showed a higher filler pullout than the TPS36/24 (Figure 6f). The SEM morphology of TPRH36/24 and TPS36/24 exhibited the co-continuous phase as PBS and PBAT are at a comparable amount. Hence, this explains the less pull out circumstances and better tensile strength in TPRH36/24 and TPS36/24 in comparison to TPRH48/12 and TPS48/12.

comparison to TPRH48/12 and TPS48/12.

*Polymers* **2021**, *13*, x 13 of 20

the extent of crystallinity [51].

*3.5. Morphology Analysis*

**Figure 5.** Surface morphology of (**a**) irregular TPRH granules and (**b**) spherical TPS granules. **Figure 5.** Surface morphology of (**a**) irregular TPRH granules and (**b**) spherical TPS granules.

The bare PBS with a high degree of crystallinity (64.03%) was less susceptible to water absorption because of smaller gaps present between the polymer chains. It was evident from the test results that the addition of filler material slightly reduced the melting point of PBS/PBAT blends. The composites TPRH48/12, TPS48/12, TPRH36/24, and TPS36/24 showed a lower degree of crystallization than PBS. This was due to fillers in polymer matrix reduces the mobility of polymer chains, thus causing the steric hindrance effect ascribed to the cross-linked aromatic structure, leading to a reduction in

Relatively, the composites TPRH36/24 and TPS36/24 exhibited a higher degree of crystallization than TPRH48/12 and TPS48/12. This was due to the high amount of PBS, which possesses higher crystallinity. This phenomenon was consistent with the results of the tensile test, whereby TPRH36/24 and TPS36/24 yield higher tensile strength, as shown in Table 3. However, there was no significant difference found in crystallinity between PBS/PBAT/TPRH blends and PBS/PBAT/TPS blends, indicating the potential of

Figure 5 shows the surface morphology of TPRH and TPS with irregular (Figure 5a) and spherical (Figure 5b) shape, respectively. The TPS granules with spherical shape caused a reduction in the contact area of the polymer matrix when compared with the irregular shape of TPRH [52]. For bare polymers of PBS (Figure 6a) and PBAT (Figure 6b), smooth surface morphology was observed while with filler loading, a rough surface was observed as shown in Figure 6c–f. When 40% of TPRH was added into PBAT/PBS matrix, the TPRH particles dispersed homogeneously in the PBAT/PBS matrix, as shown in Figure 6c,e. Some voids in the interfacial boundary were observed in TPRH48/12 due to the pull out of the rice husk particle from the PBAT/PBS matrix. This is caused by the difference in polarity between the hydrophilic rice husk and the hydrophobic PBAT. Fewer filler pullouts with good fibers-matrix adhesion were observed in TPRH36/24 (Figure 6e) as compared to TPRH48/12 (Figure 6c). For TPS blends, a similar trend was observed in TPS48/12 (Figure 6d), which showed a higher filler pullout than the TPS36/24 (Figure 6f). The SEM morphology of TPRH36/24 and TPS36/24 exhibited the co-continuous phase as PBS and PBAT are at a comparable amount. Hence, this explains the less pull out circumstances and better tensile strength in TPRH36/24 and TPS36/24 in

using rice husk waste to substitute starch in the polymer matrix.

**Figure 6.** Fractured morphology of (**a**) PBS, (**b**) PBAT, (**c**) TPRH48/12, (**d**) TPS48/12, (**e**) TPRH36/24, and (**f**) TPS36/24. **Figure 6.** Fractured morphology of (**a**) PBS, (**b**) PBAT, (**c**) TPRH48/12, (**d**) TPS48/12, (**e**) TPRH36/24, and (**f**) TPS36/24.

#### *3.6. Water Absorption*  The water absorption of the bare PBS, PBAT, and PBS/PBAT blends are shown in *3.6. Water Absorption*

Figure 7. All polymers undergo three stages during the water absorption which are absorption, saturation, and swelling. The water absorption of PBAT and PBS film increased slightly and achieved saturation of around 0.35% after 10 days of immersion in water. The low water absorption capacity was due to PBAT and PBS being hydrophobic polymers with the presence of acyl groups [31]. However, at the initial stage (day 3), it was obvious that PBS, having higher crystallinity, possessed a water absorption capacity The water absorption of the bare PBS, PBAT, and PBS/PBAT blends are shown in Figure 7. All polymers undergo three stages during the water absorption which are absorption, saturation, and swelling. The water absorption of PBAT and PBS film increased slightly and achieved saturation of around 0.35% after 10 days of immersion in water.

of 0 wt%, while PBAT had a capacity of 0.39%, which indicated that the degree of crystallinity is an important factor for water absorption of the polymer. Besides this, PBS The low water absorption capacity was due to PBAT and PBS being hydrophobic polymers with the presence of acyl groups [31]. However, at the initial stage (day 3), it was obvious that PBS, having higher crystallinity, possessed a water absorption capacity of 0 wt%, while PBAT had a capacity of 0.39%, which indicated that the degree of crystallinity is an important factor for water absorption of the polymer. Besides this, PBS with a high degree of crystallinity was less prone to water absorption as compared to PBAT due to the smaller amorphous region accessible for water intake.

*Polymers* **2021**, *13*, x 14 of 20

**Figure 7.** Water absorption of bare PBS, bare PBAT, and PBS/PBAT blends with immersion times. The PBS/PBAT/TPRH composites showed a larger water absorption capacity than **Figure 7.** Water absorption of bare PBS, bare PBAT, and PBS/PBAT blends with immersion times.

bare PBAT and PBS. The water absorption rose tremendously in the first 24 h, and reached the saturation limit after 24 h of immersion. The TPRH48/12 and TPRH36/24 showed water absorption capacity at 11.78 and 9.24%, respectively after 24 h. The composite TPRH36/24 possessed a lower water absorption capacity than TPRH48/12 due to the intrinsic nature of PBS with high crystallinity. Moreover, the water absorption capacity of PBS/PBAT/TPRH was also found to be more than PBS/PBAT/TPS, which is an attribute to the lumen and cell wall of rice husk that provide more room for the water absorption [18]. Besides, the hydrophilic nature of rice husk that favors the intermolecular hydrogen bonding enhanced the water absorption of the film. The swelling in composites occured due to the presence of internal stresses that prevents polymer matrix from absorbing water [53]. The swelling effect was observed for both TPRH and TPS blends after 3 days of immersion, which in agreement with the work was reported by Muthuraj et al. [54]. Nevertheless, TPRH36/24 and TPRH48/12 The PBS/PBAT/TPRH composites showed a larger water absorption capacity than bare PBAT and PBS. The water absorption rose tremendously in the first 24 h, and reached the saturation limit after 24 h of immersion. The TPRH48/12 and TPRH36/24 showed water absorption capacity at 11.78 and 9.24%, respectively after 24 h. The composite TPRH36/24 possessed a lower water absorption capacity than TPRH48/12 due to the intrinsic nature of PBS with high crystallinity. Moreover, the water absorption capacity of PBS/PBAT/TPRH was also found to be more than PBS/PBAT/TPS, which is an attribute to the lumen and cell wall of rice husk that provide more room for the water absorption [18]. Besides, the hydrophilic nature of rice husk that favors the intermolecular hydrogen bonding enhanced the water absorption of the film.

showed lower water absorption capacity (5.17 and 8.68%) than the reported LDPE by Sabezadeh et al. [47] with 11% absorption capacity at day 15. Water absorption characteristics influence the water vapor barrier properties of a material. Thus, the results indicated that the fabricated polymer in this work has good water vapor barrier property, which would extend the shelf life of the food. *3.7. Soil Burial Test*  The entire composites showed a smoother surface before the degradation process. The mass change in the PBS/PBAT blended composites as a function of degradation time is shown in Table 5. The macroscopic appearance of biodegradation in PBS/PBS blended composites at different burying time is shown in Figure 8. Matting of the sample surface The swelling in composites occured due to the presence of internal stresses that prevents polymer matrix from absorbing water [53]. The swelling effect was observed for both TPRH and TPS blends after 3 days of immersion, which in agreement with the work was reported by Muthuraj et al. [54]. Nevertheless, TPRH36/24 and TPRH48/12 showed lower water absorption capacity (5.17 and 8.68%) than the reported LDPE by Sabezadeh et al. [47] with 11% absorption capacity at day 15. Water absorption characteristics influence the water vapor barrier properties of a material. Thus, the results indicated that the fabricated polymer in this work has good water vapor barrier property, which would extend the shelf life of the food.

> and color change was noticeable after the degradation process. The mass loss percentage increased with increasing burying time for the entire samples, which affirmed the


**1.1 Structure and properties of cyclodextrin**

In terms of structure, CDs have basket or truncated cone-like structure in which diameter of the inner cavity is a function of the glucopyranose units as shown in **Figure 2** [8]. The spherical arrangement of glucose units with secondary OH groups on wider end of the rim and primary OH groups on narrower end of the rim imparts it basket-like shape since the ability of primary OH groups to freely rotate decreases the diameter of the cavity at one end. Moreover, H-atoms bonded to CH group as well as OH groups form the external and hydrophilic exterior surface. In comparison to rims, internal cavity presents the hydrophobic microenvironment as it is surrounded with carbon and ether oxygen [9].

As cited earlier, there are three kinds of cyclodextrin, that is, Alpha α, Beta β, and Gamma γ also known as first or parent generation CDs. Due to the presence of sugar backbone in their framework, they can also be identified as cycloamyloses or dextrins [10, 11]. As far as the physicochemical properties are concerned, all the cyclodextrin molecules are large, hydrophilic, stable in basic media, hydrolyzable in acidic media,

**Figure 2.** *Schematic diagram of shape and dimensions of parent cyclodextrin [6, 7].*

*Cyclodextrins: An Overview of Fundamentals, Types, and Applications DOI: http://dx.doi.org/10.5772/intechopen.108078*

**Table 1.**

*Important characteristics of parent cyclodextrin [7].*

and similar in their ability to modify the physical, chemical, and biological features of drugs by yielding inclusion complexes (**Table 1**) [12].

The aqueous solubility of natural CDs and their complexes are known to be restricted, especially in case of *β*-CD, despite the fact that they are hydrophilic. This is owed to comparatively strong molecular bonding in CDs in their crystal state. Furthermore, significant number of intermolecular hydrogen bonds between secondary OH groups in the *β*-CD structure makes it stiff and inhibits the overall hydration. Interestingly, substituting any OH-group even by, for example, methyoxy group, can dramatically enhance the solubility. Nonetheless being less bulky, parent cyclodextrins exhibit lower molecular weight relative to their derivatives [13].

## **1.2 Derivatives of the CDs**

Given the lower aqueous solubility, numerous scientists tried to prepare and evaluated a variety of derivatives of the CDs of medicinal interest. The CDs derivatives can be produced by polymerizing or substituting the methyl, carboxymethyl, ethyl, hydroxyethyl, sulfabutyl, or even saccharides. Bonding various functional groups causes chemical alterations into the main and secondary OH groups of the parent CD molecules [13, 14]. These derivatizations are carried out to achieve the following goals:

Remarkably, till date, a myriad of CD derivatives have successfully been produced and analyzed, yet only a small number including methylated, hydroxy alkylated, and ether substituted derivatives have been employed in studies involving novel pharmaceutical applications [15].

1.Methylated derivatives can be prepared by randomly methylating any secondary OH group in C2, C3, or C6 locations or by selectively methylating all secondary OH groups of C2 position and primary OH groups of C6. In relation to the natural CDs, methylated ones exhibit altered physical, chemical, and structural characteristics. Solubility of methylated CD is also substantially greater;

however, the solubility is inversely proportional to the temperature as it diminishes when temperature rises.


Meet the editor

Nicolae Tudoroiu holds a BS and MS in Automation and Computers, a BS in Mathematics, and a Ph.D. in Automation and Electrical and Computer Engineering. From 1979 to 1993 he was a professor in the Department of Automation and Control, University of Craiova and the Technical University of Timisoara, Romania. Since 2001 he has held the positions of postdoctoral fellow and research associate at Concordia University,

Canada, and professor at John Abbott College, Canada, and Spiru Haret University, Romania. His research interests are in automation and control, including process identification, modeling, state and parameter estimation, fault detection and isolation, battery management systems for electric and hybrid electric vehicles, simulation and control of HVACs, aircraft systems, intelligent control strategies using artificial intelligence, and control and processes optimization.


**5. Conclusions**

The surface properties of nano-gold colloidal surfaces due to adsorption of amyloidogenic peptides were successfully monitored and characterized by observing the response of spectroscopic features as a function of an external pH change. This surface property change was found to be linearly correlated with the coverage ratio of the peptide onto the colloid, Θ. With the simplification of the space occupied by a peptide into a prolate, the Θ could be extracted through a simplified tessellation logic applied for a sphere. The simulation suggested that a prolate needs to have a spiking-out orientation with representative prolate axial length of (*a*, *b*) = (1.4 nm, 2.2 nm) for Aβ1–40, (*a*, *b*) = (4.6 nm, 7.4 nm) for α-syn, and (*a*, *b*) = (2.5 nm, 4.6 nm) for β2m. Of note, these values were similar to the values estimated by the reported protein structural data. However, the above-mentioned prolate dimensions could not reproduce the cases when Θ is less than ~0.50. A lower Θ is required to have less unit coverage area; this increase of unit area was interpreted as a gyration motion of each peptide, which kept a fixed contact spot but changed the tilting angle. The average tilting angle of the prolate was (θτ) (Aβ1–40) = 35 ± 2◦, (θτ) (α-syn) = 18 ± 2◦, and (θτ) (β2m) = 29 ± 6◦, indicating that when the colloid coverage ratio is below 0.5 a prolate can possess high degree of freedom in mobility while still maintaining a high level of interaction with the gold nano-particle surface. At the same time, it indicated many other possibilities of conformation including multiple contacting points or an ensemble of different adsorption orientations. The resulting Θ was fully explained by a relationship between the distances of each unit monomer under a given colloidal area. However, the degree of affinity for a second layer required us to account for the distribution of partially positive charge (δ+) over a peptide. The segment possesses a δ+ that was considered to be highly used when Aβ1–40 and α-syn each interacted with a nano-gold colloidal surface. This possesses a distribution of centering around the prolate axis. On the other hand, the δ+ of β2m was used to interact with each monomer, and the charge distribution was spread around with a distortion, resulting in a high exposure for the counter acting monomer. Therefore, it guided us to predict that β2m possesses different charge distributions than Aβ1–40 and α-syn, and sequences or sections of peptide corresponding to δ+ or δ– thus postulated. In closing, we demonstrated that nano-scale geometrical simulation with a simplified protein structure (i.e., prolate) successfully represents peptide adsorption orientation, providing insights into interfacial conformation and indicating the presence of electrostatic intermolecular and interfacial interactions for these pathophysiologic peptide cases.

**Supplementary Materials:** Supplementary materials can be found at http://www.mdpi.com/1422-0067/20/21/ 5354/s1.

**Author Contributions:** Conceptualization, K.Y.; methodology, K.Y. and E.D.; software, K.Y. and J.J.; validation, K.Y., and A.I.; formal analysis, K.Y., K.B., P.S., J.J., E.D., N.R., J.B., I.D., and A.I.; investigation, K.Y., K.B., J.J. and A.I.; resources, K.Y., I.D., and A.I.; data curation, K.Y.; writing—original draft preparation, K.Y.; writing—review and editing, K.Y., J.B., N.R., and A.I.; visualization, K.Y., E.D., and N.R.; supervision, K.Y.; project administration, K.Y.; funding acquisition, K.Y.

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

**Acknowledgments:** We are grateful for the support by Geneseo Foundation at the initial stage of this project. Ishan Deshmukh and Akane Ichiki thank for the gracious support by SUNY Geneseo Chemistry Department Alumni Summer Research Scholarship. We also thank Jonathan Bourne for helpful discussion during preparation of this manuscript.

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

#### **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/).


*3.1.1 Colorimetric quantitative ALP activity*

*Isolation and Expansion of Mesenchymal Stem/Stromal Cells, Functional Assays and Long-Term… DOI: http://dx.doi.org/10.5772/intechopen.100286*

	- 1.Aspirate or pipette out all culture medium from each well of the 6-well culture plate that contains induced or control cells to be tested.
	- 2.Rinse the cells twice with PBS. Add 200 μL of PBS to the side of each well, not to dislodge the cells. Aspirate off the PBS and re-rinse.
	- 3.Add 125 μL of 0.5 N HCl to each well.
	- 4.Scrape the cells off of the surface using a cell scraper and transfer the cells and HCl to a 1.5 mL polypropylene microcentrifuge tube with a tight fitting cap.
	- 5.Add an additional 125 μL of 0.5 N HCl to each cell to recover any cells remaining in the well, and transfer this to the appropriate tube.
	- 6.Samples may be capped tightly and stored at −20°C if they are not to be tested immediately.
	- 7.Extract the calcium from the cells by shaking the tubes on an orbital shaker for 4 hours at 4°C. If using frozen samples, allow extra time for samples to thaw.
	- 8.Centrifuge the sample tubes at 500 g for 2 minutes.
	- 9.Carefully collect the supernatant with extracted calcium, without disrupting the cell pellets, and transfer these to a new tube.
	- 10.Prepare a standard curve with the calcium standard and determine the amount of calcium in each control and osteogenesis-induced samples. Follow the instructions provided in the Stanbio Total Calcium LiquiColor® Procedure No. 0150 (Stanbio Laboratory).

PuraMatrix™ hydrogel (BD Biosciences) is a 16-amino acid synthetic peptide hydrogel composed of a repeating sequence of arginine, alanine, aspartate, and alanine (RADA16) [31], which is widely used for 3-D culture.

*Isolation and Expansion of Mesenchymal Stem/Stromal Cells, Functional Assays and Long-Term… DOI: http://dx.doi.org/10.5772/intechopen.100286*


*4.2.2. Electromagnetic torque error fuzzification*

Three trapezoidal membership functions are selected to fuzzify the torque error as shown in figure 14, so the following three fuzzy sets are used, N signify Negative, EZ for Zero and P for Positive.

Improved DTC Algorithms for Reducing Torque and Flux Ripples of PMSM Based on Fuzzy Logic and PWM Techniques 179

**Figure 14.** Membership functions for electromagnetic torque error

## *4.2.3. Stator flux angle fuzzification*

178 MATLAB – A Fundamental Tool for Scientific Computing and Engineering Applications – Volume 1

**Figure 12.** Matlab/Simulink design of the fuzzy logic switching table used in FDTC

**4.2. Inputs fuzzification and outputs defuzzification**

*4.2.1. Stator flux error fuzzification*

these two functions are indicated in figure 13.

**Figure 13.** Membership functions for stator flux error

*4.2.2. Electromagnetic torque error fuzzification*

for Positive.

configuration of its inputs and outputs as membership functions.

Figure 12 shows the design of this fuzzy logic system in Matlab/Simulink and also the

In order to examine the fuzzy logic contribution to DTC, the choice of the membership functions number for the fuzzification of flux and torque errors has been repected in this part, i.e two membership functions for ΔФs, because a two level hysteresis controller was utilized to control the stator flux in basic DTC. Whereas three membership functions for the fuzzification of ∆Г because a three level hysteris controller was used to adjust the torque.

Two trapezoidal membership functions are selected to fuzzify the stator flux error, so the following two fuzzy sets are used, N signify Negative and P for Positive. The parameters of

Three trapezoidal membership functions are selected to fuzzify the torque error as shown in figure 14, so the following three fuzzy sets are used, N signify Negative, EZ for Zero and P The flux angle has a universe of discours equal 2π radians, as shown in figure 15. It is divided into six zones or sectors in order to be equivalent to that of the basic DTC. "thetai" means sector i, i.e "theta1" means sector 1 (θ1) and so on.

**Figure 15.** Membership functions for stator flux position

#### *4.2.4. State switches defuzzification*

The sole output control variable of fuzzy logic system is the inverter switching states S1, S3 and S5 or the selected voltage vector. Figure 16, illustrates the suggested output fuzzy set as singletons. Indeed, the choice of the stator volt dge vector is based on the rules indicated in table 3. Each control rule can be described using the state variables ΔФ, ΔΓ and θs and the control variables. The ith rule Ri can be written as:

Ri : if ΔФ, is Ai, ΔΓ is Bi and θs is Ci then S1 is a, S3 is b and S5 is c

Where a, b and c are a boolean variable. Ai, Bi and Ci denote the fuzzy set of the variables ΔФ, ΔΓ and θs, respectively.whereas Ri is the control rule number i.

**Figure 16.** Membership functions for state switches as singletons

### **4.3. Fuzzy logic switcher rules**

The most important part of designing the fuzzy controller (fuzzy logic system) is to design the rule base, because it gouverns the behiviour of fuzzy controller and stores the expert knowledge on how to control the plant. The fuzzy associative memory of Mamdani rule base model to develop DTFC is as shown in Table 3.

**Table 3.** Fuzzy logic switcher rules

#### **4.4. PI-Fuzzy speed controller synthesis**

The speed closed loop with the PI-Fuzzy controller structure is shown in Figure 17. The inputs of this FLC are the normalized values of the speed error denoted "e" and its rate of change denoted "de" that should remain between ±1. Wherefore, two scaling factors (Kne and KΔne) are used to normalize the actual speed error and its rate of change. The output of the controller is the normalized change of the motor torque command which generates the actual value of the motor torque demand when it's multiplied by a third scaling factor (Knc).

Improved DTC Algorithms for Reducing Torque and Flux Ripples of PMSM Based on Fuzzy Logic and PWM Techniques 181

**Figure 17.** Closed loop PI-Fuzzy speed controller

180 MATLAB – A Fundamental Tool for Scientific Computing and Engineering Applications – Volume 1

The most important part of designing the fuzzy controller (fuzzy logic system) is to design the rule base, because it gouverns the behiviour of fuzzy controller and stores the expert knowledge on how to control the plant. The fuzzy associative memory of Mamdani rule

θ2

θ3

**∆Г\∆Φ P N**

 **P** V1 V2

 **Z** V0 V7

θ6

**∆Г\∆Φ P N**

 **P** V4 V5

 **Z** V7 V0

**∆Г\∆Φ P N**

 **P** V6 V1

 **Z** V7 V0

θ5

**∆Г\∆Φ P N**

 **P** V3 V4

 **Z** V0 V7

The speed closed loop with the PI-Fuzzy controller structure is shown in Figure 17. The inputs of this FLC are the normalized values of the speed error denoted "e" and its rate of change denoted "de" that should remain between ±1. Wherefore, two scaling factors (Kne and KΔne) are used to normalize the actual speed error and its rate of change. The output of the controller is the normalized change of the motor torque command which generates the actual value of the motor torque demand when it's multiplied by a third scaling factor (Knc).

**Figure 16.** Membership functions for state switches as singletons

base model to develop DTFC is as shown in Table 3.

**4.3. Fuzzy logic switcher rules**

θ1**,** θ7

**∆Г\∆Φ P N**

 **P** V5 V6

 **Z** V0 V7

θ4

**∆Г\∆Φ P N**

 **P** V2 V3

 **Z** V7 V0

**Table 3.** Fuzzy logic switcher rules

**4.4. PI-Fuzzy speed controller synthesis**

**Figure 18.** Membership functions for speed error

The membership functions used, in this chapter, for the inputs and the output are the same, as shown in figure 18. Where, the following fuzzy sets used in these membership functions: NG is negative big, NM is negative medium, NP is negative small, EZ is equal zero, PP is positive small, PM is positive medium and PG is positive big.

From the speed behavior analysis, the table 4 has been developed to obtain a good performance in the speed closed loop. Whereas, from the membership functions of inputs and the output, and the rules presented in this table, the FLC elaborates the electromagnetic torque reference to be developed by the PMSM.

**Table 4.** PI-Fuzzy speed controller rules

### **4.5. Simulation results**

The sampling period has been chosen equal to 100 µs (10 KHz) for FDTC; in order to compare this strategy with basic DTC; despite the fact that the sampling time used to simulate DTC is less than that used in case of FDTC.

**Figure 19.** Mechanical speed (on the left) and electromagnetic torque (on the right) tracking performance under load variations in case of FDTC

**Figure 20.** Stator current waveform at 800 rpm with nominal load under FDTC

**Figure 21.** Stator current spectrum at 800 rpm with nominal load (on the left) and Stator flux in (α,β) axes under load variations (on the right) in case of FDTC


**5. Conclusions**

This is the first evidence that topically applied HMWHA eye drops induced a significant neurotrophic effect on the subbasal nerve plexus in humans. When applied after any kind of ocular surgery, HMWHA eye drops may serve to support the recovery of damaged nerves. Moreover, HMWHA eye drops offered a new therapeutic option in preventing and treating ocular surface disease, in particular diseases associated with nerve damage like diabetic keratopathy and all forms of neuropathic keratopathy. Future research will focus on the question if patients with diabetic keratopathy and other forms of neuropathic keratopathy could benefit from HMWHA eye drops. HMWHA eye drops provide a holistic approach while simultaneously addressing different interacting pathomechanisms of DED.

**Author Contributions:** B.D., S.A.T., B.S., and H.R. were the investigators taking IVCM images of their patients within the HYLAN M study; R.F.G. provided advice during the design of the HYLAN M study investigation plan and in the interpretation of the results of IVCM; O.S. provided the precise work instruction for IVCM and served as masked reading center for the IVCM images; K.W. performed the digital assessment of the images; G.-B.v.S. served as the coordinating investigator of the HYLAN M study; W.G.K.M.-L. had an overall responsibility for the HYLAN M study design, organization, regulatory compliance, monitoring, and study report; O.S., K.W., G.-B.v.S., J.H.-W., and W.G.K.M.-L. prepared the manuscript of this publication. All authors have read and agreed to the published version of the manuscript.

**Funding:** The HYLAN M study received unrestricted funding from i.com medical GmbH, Munich, Germany.

**Conflicts of Interest:** The study director Wolfgang G.K. Müller-Lierheim is also the CEO of the company i.com medical GmbH, Munich, Germany. The remaining authors declare no conflict of interest.


**8. Factors influencing clinical course of acute disease and/or long‐term outcome**

## **8.1. Subtype of TBEV**

Subtype of TBEV influences the course of acute TBE as well as its long‐term outcome. The disease caused by the European TBEV subtype usually has a biphasic course, around 10% of adult patients have a severe neurologic deficit, case‐fatality rate is <2% [12, 32]. According to a prospective study the abortive form of TBE is rare—the initial phase most of the time move on to the second phase of the disease [51]. Long‐lasting sequelae are identified in up to 50% of adult patients [103]. The disease is less severe and has a better outcome in children than in adults [34, 50, 104, 105].

Symptomatic infections with Far Eastern TBEV subtype often cause an illness with a gradual onset, more severe course, higher rates of severe neurologic sequelae, and a fatality rate of 20–40%; the severity and outcome in adults and children are similar. Limited information about the clinical course of the disease is available for Siberian TBEV subtype. The case-fatality rate is 2–3%; some reports from Russia suggest an association with a chronic progressive form of TBE [1, 11].

## **8.2. Age of patients**

Published data suggest the relationship between age of patients and the severity of TBE and its outcome — the severity of acute illness and the proportion of patients with unfavorable outcome increase with age [33, 47, 50, 106].

The disease caused by European subtype of TBEV generally has a milder course and better outcome in children than in adults. The predominant form of TBE in children and adolescents is meningitis. A summary of 8 studies on 1169 children with TBE showed that meningitis was present in 802 (69%), meningoencephalitis in 356 (30%), and meningoencephalomyelitis in 11 (1%) patients. A total of 20 out of 945 patients (2.1%) had long‐term neurologic sequelae [34]. In contrary to children, in adults, and especially in elderly patients with TBE caused by European subtype of TBEV, the most frequent presentation is meningoencephalitis [33, 47].

Furthermore, fatality rate, the ratio of patients who develop pareses, and the frequency of postencephalic syndrome is also parallel with the increasing age [33, 47, 106].

#### **8.3. Other factors associated with severe acute disease**

Some clinical studies have shown that TBE with monophasic presentation is associated with a more severe course of the acute disease [42, 107–111].

Concomitant TBE and Lyme neuroborreliosis may occur with a more severe clinical course [59, 112, 113].

#### **8.4. Severity of acute illness and other risk factors for unfavorable outcome**

The outcome of TBE is associated with clinical presentation. The risk of incomplete recovery is higher for patients who have more severe clinical illness during acute phase of TBE [45, 47].

Other identified risk factors found to be associated with unfavorable outcome are CSF cell count > 300 cells/μL, impaired blood‐brain barrier (total protein >600 mg/L) and abnormal findings on MRI [46].


**8. Conclusion**

*Capsicum* is a well-diverse shrub commonly grown in different parts of the world. It is genetically diverse, with numerous cultivars and taxonomic derivatives. It has a widely described chromosome number of 2n = 24; however, some wild *Capsicum* species, such as *C. buforum, C. capylopodiume,* and *C. capylopodiume* have a ploidy level of 2n = 26. *C. frutescens* showed karyotypic formulae for 11M + 1SM + 1A and 11M + 1A. *Capsicum* has a relatively similar genomic content compared to tomato, as evidenced by the presence of Capsicum sequences complementary to many tomato cDNAs. To date, more than a thousand Capsicum genes have been annotated in the GenBank that will aid future research on improving the yield and biotic and abiotic traits of *Capsicum*. This comprehensive review is essential in understanding *Capsicum's* biology, genetics, and genomics toward improving its horticultural traits and nutritional and medicinal values.


**Acknowledgements**

and free-flowing traffic data were used to build up queues of heavy vehicles and cars, with gaps varying from 0.9 to 1.8 m; a simple modelling of lane choice of vehicles approaching a

In North America, the American Society of Civil Engineers (ASCE) recommended a load model for the design of spans up to 1951 m [93]. A UDL is to be applied in conjunction with a point load, whose values depend on span length and truck percentage. The ASCE loading is mainly based on truck data from crossings of the Second Vancouver Narrows bridge [7]. Notably, 800

In this chapter, traditional approaches and recent advances in highway bridge traffic loading are described. These are of great significance for structural safety assessment of bridges, where there is a potential for substantial savings by considering site-specific traffic conditions. An introduction to traffic theory and modelling relevant to bridge-loading applications is given, as well as an overview of extreme value statistics, since a probabilistic approach is now well

In bridge traffic loading, it is convenient to distinguish between short-span bridges, which are governed by free-flowing traffic plus an allowance for dynamic vehicle-bridge interaction, and long-span bridges, which are governed by congested conditions with no allowance for dynamic effects. The bridge length threshold between the two modes is not clear-cut but is

Current technologies allow the collection of a great deal of traffic data during uncongested traffic conditions, mainly from *weigh-in-motion* stations. Such data can be used for the analysis of short-span bridges. Importantly, recent studies have shown that dynamic allowances may be significantly smaller than those considered in the main codes of practice, especially when favourable site-specific conditions are accounted for. This implies that the threshold between short- and long-span bridges may be lower than currently thought and that recent techniques to simulate congested traffic, such as those described in this chapter, may have a wider

On the other hand, a shortage of suitable congested data has led to the fact that traffic loading on long-span bridges is often based on conservative assumptions, traditionally a queue of vehicles at minimum bumper-to-bumper distances. Traffic *microsimulation* is a powerful tool to generate realistic congestion patterns based on the widely available free-flowing traffic measurements. Among microsimulation models, the *Intelligent Driver Model* provides an optimal balance between accuracy and computational speed and can be extended with the lane-changing model MOBIL to simulate the remixing of cars and trucks occurring as traffic gets congested. Calibration of the model parameters can be based on site-specific traffic data

full-stop events per year were considered with vehicles spaced at 1.5 m.

queue was also considered.

48 Structural Bridge Engineering

**6. Summary and conclusions**

established when studying bridge loading.

thought to lie between 30 and 50 m.

application than expected.

or on available data in the literature.

This chapter draws several findings from the research conducted by the author at University College Dublin under the supervision of Prof E. OBrien and Dr C. Caprani within the TEAM (*Training in European Asset Management*) project and funded by the European Commission 7th Framework Programme. The author is most grateful to all contributors to that project and also acknowledges the valuable comments of Dr M. Treiber at Technische Universität Dresden on Section 2.


**1. Introduction**

116 Health Management – Different Approaches and Solutions

Mann, Andrea G.; Tam, Clarence C.; Higgins, Craig D. & Rodrigues, Laura C. (2007). The

Marco, Vinceti.; Guglielmina, Fantuzzi.; Lucia, Monici.; Mariateresa, Cassinadri.; Guerrino,

Nilles, E.; Sayward, H. & D'Onofrio, G. (2009). Vascular endothelial growth factor and acute

Tunga, Salthammer.; Sibel, Mentese. & Rainer, Marutzky. (2010). Formaldehyde in the

Wang, JL. & Yau, SC. (2002). Building Pathology, China Electric Power Press, Beijing, China Watt, David S. (2007). *Building Pathology* (2nd Ed.), Blackwell publishing, ISBN 978-1-4051-

Wu, PC.; Li, YY.; Lee, CC.; Chiang, CM. & Su, HJJ. (2003). Risk assessment of formaldehyde

mountain sickness, Journal of Emergencies Trauma Shock , pp. 6–9

2458

53, ISSN 0048-9697

ISSN 0009-2665

359-363, ISSN 0905-6947

6103-9, UK

association between drinking water turbidity and gastrointestinal illness: a systematic review. Bmcpublic Health, Vol.7, No.256, (September 2007), ISSN 1471-

Predieri. & Gabriella, Aggazzotti. (2004). A retrospective cohort study of trihalomethane exposure through drinking water and cancer mortality in northern Italy. Science of The Total Environment, Vol.330, No.1-3, (September 2004), pp. 47-

Indoor Environment. *Chemical Reviews*, Vol.110, No.4, (April 2010), pp. 2536–2572,

in typical office buildings in Taiwan, *Indoor Air*, Vol.13, No.4, (December 2003), pp.

When fungi grow on a living organism or on stored food material that we consume, they may produce harmful metabolites that diffuse into their food (Garcia et al., 2009; Kabak and Dobson, 2009). It is believed that fungi evolved these metabolites as a means of protecting their food supply by preventing other organisms from eating it. These metabolites are referred to as mycotoxins, which literally mean "fungus poisons". Fungi that produce mycotoxins do not have to be present to do harm. When a fungus grows grains in storage, the environment may become unsuitable for the fungus and it dies. Although the fungus dies, during the growth stage, if it produces mycotoxins, this can poison the grains (Fokunang et al., 2006). The effects of poisoning by mycotoxin are referred to as mycotoxicoses. The knowledge that mycotoxicoses is the result of fungal actions was a relatively, recent discovery (Lackner et al., 2009). This is understandable since illnesses in this case are due to consumption of mycotoxins that has been released by the fungus and is not directly caused by the fungus (Coppock and Jacobsen, 2009).

#### **1.1 The mycotoxin system**

The mycotoxin system as shown in figure 1 may be considered in terms of four interacting subsystems namely; toxicology, metabolism, health, productivity and wealth. After exposure through ingestion, inhalation or skin contact, the toxicity of a mycotoxin is determined by a sequence of events such as metabolism, involving the administration, absorption, transformation, pharmacokinetics, molecular interactions, distribution, and excretion of the toxin and its metabolites (Fokunang et al., 2006). In turn, the toxicity of the mycotoxin will be manifested by its effect on the health and productivity of crops, human efforts and agricultural and livestock products**.**

\* O. Y. Tabi1, V. N. Ndikum1, E. A. Tembe-Fokunang1, F. A. Kechia1, B. Ngameni1, N. Guedje1,

R. B. Jiofack1, J. Ngoupayo1, E. A. Asongalem1, J. N. Torimiro1, H. K. Gonsu1, S. Barkwan2, P. Tomkins2,

B. T. Ngadjui1, J. Y. Ngogang1, T. Asonganyi1 and O. M. T. Abena1

*1 Faculty of Medicine and Biomedical Sciences, University of Yaoundé 1, Republic of Cameroon,*

*2 Centre for Biopolymer and Biomolecular Research, Athlone Institute of Technology, Republic of Ireland.*

Mycotoxins: Quality Management, Prevention, Metabolism, Toxicity and Biomonitoring 119

Fig. 3. Mycotoxins of worldwide importance (Coker, 1999)

Fig. 1. The Mycotoxins system

#### **1.2 The spoilage system**

Biodeterioration is the net result of many interacting spoilage agents which may be widely described as biological, chemical, physical, micro-environmental and macro-environmental (Figure 2). However, the relative impact of these agents will often be mainly determined by the nature and extent of human intervention, occurring within the socio-economic system (Coker 1998). The factors that contribute to bio deterioration within an ecosystem are mainly moisture, temperature and pests status (Christensen, 1975; Hussein and Brasel 2001).

Fig. 2. The mycotoxin spoilage system

#### **1.3 Mycotoxins of economic importance**

The moulds and mycotoxins which are now considered to be of world-wide importance are shown in figure 3. Some mycotoxins are however of regional importance as illustrated in table 1. Regional myxcotoxins are specific to certain regions and the occurrence is link to climatic changes. The important mycotoxins have shown the capacity to have a significant impact upon human health and animal productivity in a wider distribution in a number of countries, most especially in the warm humid countries in sub-saharan Africa and Asia (Fokunang et al., 2006; Khlangwiset and Wu 2010).

Biodeterioration is the net result of many interacting spoilage agents which may be widely described as biological, chemical, physical, micro-environmental and macro-environmental (Figure 2). However, the relative impact of these agents will often be mainly determined by the nature and extent of human intervention, occurring within the socio-economic system (Coker 1998). The factors that contribute to bio deterioration within an ecosystem are mainly

The moulds and mycotoxins which are now considered to be of world-wide importance are shown in figure 3. Some mycotoxins are however of regional importance as illustrated in table 1. Regional myxcotoxins are specific to certain regions and the occurrence is link to climatic changes. The important mycotoxins have shown the capacity to have a significant impact upon human health and animal productivity in a wider distribution in a number of countries, most especially in the warm humid countries in sub-saharan Africa and Asia

moisture, temperature and pests status (Christensen, 1975; Hussein and Brasel 2001).

Fig. 1. The Mycotoxins system

Fig. 2. The mycotoxin spoilage system

**1.3 Mycotoxins of economic importance**

(Fokunang et al., 2006; Khlangwiset and Wu 2010).

**1.2 The spoilage system**

Fig. 3. Mycotoxins of worldwide importance (Coker, 1999)

Mycotoxins: Quality Management, Prevention, Metabolism, Toxicity and Biomonitoring 121

can the grain be invaded (He and Zhou, 2008). Although conditions favourable for growth of the *A. flavus* and production of aflatoxin is narrow, the fungus is common and widespread in nature. Under warm humid condition *A flavus* can invade stored gains such as corn as shown in figure 4. It can be found growing on various decaying vegetation where it may heat up the substrate to as high as 113-122°F as it consumes the material (Hedyati et

Fig. 4. *Aspergillus flavus* infestation on corn *Zea mays* (Hedyati et al., 2007)

growth of A. flavus producing aflatoxin in peanuts is shown in figure 5.

Fig. 5. Stored peanuts infected by *A flavus* producing aflatoxin (Kios et al., 2004)

The term aflatoxins was derived in the early 1960s when the death of thousands of turkeys (Turkey X' disease) ducklings and other domestic animals was attributed to the presence of *A. flavus* toxins in groundnut meals imported from South America (Nageswara et al., 2002). The amount of aflatoxin formed differs as to the substrate on which it is growing. Although the mycelial mass may be the same in each substrate, the aflatoxin produced would be far greater in peanuts than in say soybeans, where relatively very little would be produced. The

al., 2007; Lee, 2009).

Table 1. Fungi species and mycotoxins of regional importance (Fokunang et al., 2006


**NEW PEDAGOGICAL CHALLENGES IN THE 21ST CENTURY - CONTRIBUTIONS OF RESEARCH IN EDUCATION**

Edited by **Olga Bernad Cavero** and **Núria Llevot-Calvet**

#### **New Pedagogical Challenges in the 21st Century - Contributions of Research in Education**

http://dx.doi.org/10.5772/66552 Edited by Olga Bernad Cavero and Núria Llevot-Calvet

#### **Contributors**

Ajitha Nayar, Srikirupa V, Diana Marcela Pedraza Díaz, Eleni Mousena, Trifeni Sidiropoulou, Carlos Renato Carola, Victorița Trif, Peter Burridge, Ewa Ogrodzka-Mazur, Aigerim Mynbayeva, Zukhra Sadvakassova, Bakhytkul Akshalova, Miguel Navarro Rodríguez, Yaneth Soto Ruiz, Rubén Edel Navarro, Lindy-Anne Abawi, Susan Carter, Andrews Dorothy, Joan Conway, Bing Ngu, Christine Edwards-Groves, Catrin Rutland, Aziza Alibhai, Emilia Harding, Mariam Moiz Saigar, Oscar Jerez, Cesar Orsini, Marcos Rojas, Catalina Ortiz, Zdenko Medveš, John Felyp Tredinnick-Rowe

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

The rights of the editor(s) and the author(s) have been asserted in accordance with the Copyright, Designs and Patents Act 1988. All rights to the book as a whole are reserved by INTECHOPEN LIMITED. The book as a whole (compilation) cannot be reproduced, distributed or used for commercial or non-commercial purposes without INTECHOPEN LIMITED's written permission. Enquiries concerning the use of the book should be directed to INTECHOPEN LIMITED rights and permissions department (permissions@intechopen.com). Violations are liable to prosecution under the governing Copyright Law.

Individual chapters of this publication are distributed under the terms of the Creative Commons Attribution 3.0 Unported License which permits commercial use, distribution and reproduction of the individual chapters, provided the original author(s) and source publication are appropriately acknowledged. If so indicated, certain images may not be included under the Creative Commons license. In such cases users will need to obtain permission from the license holder to reproduce the material. More details and guidelines concerning content reuse and adaptation can be foundat http://www.intechopen.com/copyright-policy.html.

#### **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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**5.14 Physical refining**

Operations involved in physical refining are degumming, bleaching, filtration, steam stripping, deodourisation, and polishing. Crude palm oil is mixed with 0.21% phosphoric acid followed by bleaching with 1–2% earth under vacuum at 100–150°C and the spent earth is removed by filtration. The refined and bleached oil was deaerated and steamed under a vacuum at 240–2270°C. Finally, the oil is cooled and passed through a polishing filter. The physical method will yield better-quality oil and avoid contamination. The final product of both refining methods is the same *viz.*, refined and deodorized palm oil (RBDPO).


**Author details**

Rahma Al Kindi\*, Asma Al Salmani, Rahma Al Hadhrami, Sanaa Al Sumri and Hana Al Sumri Department of Family Medicine and Public Health, Sultan Qaboos University Hospital, Muscat, Oman

\*Address all correspondence to<EMAIL_ADDRESS>
© 2022 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.

*Perspective Chapter: Modern Birth Control Methods DOI: http://dx.doi.org/10.5772/intechopen.103858*


*Article* **Gambiense Human African Trypanosomiasis Sequelae after Treatment: A Follow-Up Study 12 Years after Treatment**

**Junior Mudji 1,2,**†**, Anna Blum 3,4,**†**, Leticia Grize 3,4, Rahel Wampfler 3,4, Marie-Thérèse Ruf 3,4, Lieselotte Cnops 5, Beatrice Nickel 3,4, Christian Burri 3,4 and Johannes Blum 3,4,\***

Received: 8 November 2019; Accepted: 8 January 2020; Published: 11 January 2020

**Abstract:** The clinical presentation of Human African Trypanosomiasis (HAT) due to *Trypanosoma brucei gambiense* is well known, but knowledge on long-term sequelae is limited. In the frame of studies conducted between 2004 and 2005 in the Democratic Republic of the Congo (DRC), the prevalence of HAT related signs and symptoms were evaluated before the start of treatment and at the end of treatment. To explore possible long-term sequelae, the same clinical parameters were assessed in 2017 in 51 first stage and 18 second stage HAT patients. Signs and symptoms 12–13 years after treatment were compared to before and immediately after treatment and to controls matched for sex and age (±5 years). In first stage HAT patients, the prevalence of all signs and symptoms decreased compared to before treatment but were still higher after 12–13 years than immediately at the end of treatment and in the control group. In second stage HAT patients, all HAT-specific findings had continuously decreased to the point where they were in the range of the healthy control group. In a selection of oligosymptomatic first stage HAT patients, no trypanosomes were detected in the blood by microscopic examination or PCR. An oligosymptomatic presentation of HAT due to the persistence of parasites in compartments, where first stage HAT medications do not penetrate, could not be ruled out.

**Keywords:** human African trypanosomiasis; sequelae; serology; treatment; oligosymptomatic HAT

#### **1. Introduction**

Human African trypanosomiasis (HAT) is caused by the protozoan parasites *Trypanosoma brucei gambiense (T.b. gambiense)* and *Trypanosoma brucei rhodesiense (T.b. rhodesiense*), which are transmitted by tsetse flies. Whereas the *T.b. gambiense* form is characterized by a progressive course typically lasting three years [1], the *T.b. rhodesiense* form is usually acute, and death occurs within weeks or months of infection.

*T.b. gambiense* is endemic in foci in Western and Central Africa and today causes more than 98% of reported cases of HAT. The disease occurs in two stages, the first, or hemolymphatic, stage without invasion of the central nervous system (CNS) and the second, or neurological, stage with invasion of the CNS by the trypanosomes.

According to the last WHO report (WHO interim guidelines for treatment of gambiense human African trypanosomiasis, August 2019) [2], the worldwide number of *T.b. gambiense* HAT cases dropped from over 25,000 in the year 2000 to below 1000 reported cases worldwide in 2018 [2].

Fever, headache, pruritus, lymphadenopathy, and, to a lesser extent, hepato-splenomegaly are the leading signs and symptoms of the first stage but may also be present, to a lesser degree, in the second stage.  The neurological symptoms include tremor, general motor weakness, paralysis of an extremity, epilepsy, akinesia, and abnormal movements (dyskinesia, unspecific movement disorders, Parkinson-like movements, speech disorders) [3–8]. Sleep disorder with somnolence and short interposed sleeping episodes during the day and at night are imposing clinical symptoms from which "sleeping sickness" derives its name. Total sleep duration, however, remains normal [9].

HAT had always been perceived and described as inevitably fatal if untreated. However, oligosymptomatic forms of HAT with few symptoms, non-detectible parasites, and persistent serological titers were recently described along with their potential role for transmission of HAT [10,11]. The clinical presentation of *T.b. gambiense* HAT has been well documented, but studies on long-term sequelae of HAT have not been performed. The present observational case control study describes the clinical signs and symptoms of HAT patients before treatment and 12–13 years after.

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

#### *2.1. Study Design and Setting (See also Flowcharts below)*

The present study assessed the prevalence of HAT related long-term clinical sequelae (signs and symptoms 12–13 years after treatment) and compared signs and symptoms of the HAT patients before, immediately after, and 12–13 years after treatment. Patients at follow-up time were also compared with controls matched by sex and age (±5 years).

This follow-up study was conducted in two phases from 19 July to 14 September 2017 and from 3 May to 30 May 2019 at the Hôpital Evangélique de Vanga, located in the Kwilu province of the Democratic Republic of the Congo (DRC). The area is rural; villages are very remote and only accessible with major efforts by very difficult roads.

#### *2.2. Participants*

In a clinical study carried out in 2004 on endocrinological changes and the involvement of the heart in second stage HAT (detection of parasite, pathological cerebrospinal fluid), clinical parameters from 29 patients were assessed before treatment, at the end of treatment, and after a follow-up of three months [12–14]. Additionally, in the framework of clinical trials carried out between 2004 and 2005, clinical parameters were documented before and after treatment (but without a follow-up of three months) in 96 first stage HAT patients (parasitology confirmed) in Vanga. In these trials, the safety and efficacy of the newly developed drug pafuramidine was compared to the standard treatment with pentamidine for first stage HAT [15,16].

A list of all formerly recruited patients in the above-mentioned studies was compiled, and their place of residence (village) was traced. The villages for the recruitment of study participants were chosen according to the number of eligible patients living there and the accessibility of the villages (distance to Vanga hospital, navigability of roads, and distance between affected villages). According to these parameters, visits were planned, starting with a visit that allowed the enrollment of the largest possible number of patients. Accessible persons were included in the study after informed consent with the exception of patients with one of the following exclusion criteria: history of severe chronic diseases such as tuberculosis, HIV, cancer, liver cirrhosis, and diabetes mellitus. In patients who died or were not accessible, a third-party history by a family member or friend was taken. These patients were not included in the analysis but were discussed separately.

#### *2.3. Sample Size*

Different clinical parameters of HAT have largely different frequencies. The calculation of a sample size yielding 80% power (α = 0.05) was, e.g., 11 patients for sleeping disorders versus 485 patients for abnormal behavior. It was therefore decided to aim at a minimum 60 patients, which would be sufficient to allow the covering of the major signs and symptoms. Due to the limited number of available patients, the sample size calculation was only performed for the first stage HAT patients. Ninety-six first stage and 29 second stage patients who participated in the former studies lived in the perimeter of the Vanga hospital.

#### *2.4. Study Procedure*/*Clinical Examination*

The study was conducted in two parts—a site visit to the villages as described above, followed by a laboratory assessment at the hospital for the first stage HAT patients with clinical signs and symptoms (see below). The original working hypothesis was that no or only rare sequelae or signs of continued infection would be found in treated HAT patients revisited more than 10 years after treatment. Therefore, the study focused on clinical signs and symptoms of HAT patients 12–13 years after treatment. The second part of the study emerged from the findings, which had revealed a significant number of first stage HAT patients with continued or returning complaints. The resulting follow-up study, comprising blood examinations, could only be done with about two years of delay, due to the necessary protocol amendment, time-consuming ethical clearance, and challenges of accessibility and free movement in the election period in the DRC.

For each former patient (=a case), a control person matched by age (±5 years) and sex was enrolled. Family members were preferred and, if not available, hospital personnel or patients with minor surgical problems (i.e., herniotomy) were enrolled instead.

In all HAT patients and controls, a short history and physical examination was established. Questions regarding the symptoms of HAT were asked in the same manner as in the former original study and trials, and the case report form used to compare signs and symptoms was based on those used in the previous trials. The data collected were compared to those before treatment and after treatment from the former studies.

#### *2.5. Additional Blood Examination on Patients with Symptoms*

For the second part of the study, 15 first stage HAT patients who were still oligosymptomatic and had shown clinical signs or symptoms such as headache, sleep disorders, pruritus, or minor neurological problems during the village visits were invited to report to the hospital for additional tests. The following laboratory tests were performed: Microscopic examination of the blood and the aspiration fluid of enlarged lymphatic glands (if present) for the presence of trypanosomes, serologic testing for trypanosomiasis (CATT/*T.b.gambiense*, Institute of Tropical Medicine, Antwerp, Belgium;IFA, in-house test, Swiss Tropical and Public Health Institute, Basel, Switzerland), and PCR from blood samples. CATT was performed immediately after blood withdrawal in the DRC, and EDTA-blood and serum samples for IFA and PCR were stored in aliquots at −20 ◦C at the study site until transfer on dry ice to Switzerland.

#### *2.6. T.b. gambiense Card Agglutination Test for Trypanosomiasis (CATT)*

The CATT is a direct agglutination test using lyophilized bloodstream forms of *T.b. gambiense* variable antigen type LiTat 1.3. It detects specific antibodies in the blood, serum, or plasma of patients infected with *T.b. gambiense* [17]. The test was performed according to the kit manual. For each participant, one CATT test was performed on undiluted whole blood.

The CATT exhibits a sensitivity of 87%–98% [17,18] and a specificity of 95.9% on undiluted whole blood. Cross reactivity with antibodies generated against *Plasmodium* spp. and other parasites is possible.

#### *2.7. Trypanosoma brucei IFA, Malaria IFA, and Leishmania IFA Serological Tests*

Immunofluorescence staining was performed with bloodstream forms of *T.b. brucei* (strain STIB 345) in addition with *Plasmodium falciparum* (strain NF54) or liver sections of *Leishmania donovani* (strain MHOM-ET-67/L82) infected hamster mounted on glass slides. Slides were stored at −80 ◦C until day of use. For IFA analysis, slides were quickly air-dried at room temperature and fixed with acetone. Sera were diluted in PBS pH 7.2 and applied to the slide-slots. Three control sera were present on each slide; one positive, one equivocal, and one negative control. After 25 min of incubation at 37 ◦C in a wet chamber, slides were washed with PBS pH 7.2 and air-dried. FITC conjugated F(ab)'2 anti-IgG/A/M (BioRad, #30244) diluted in 0.01% Evans blue in PBS was added, slides were incubated for 25 min at 37 ◦C, washed, dried, and a cover glass was mounted with buffered glycerol. Slides were examined immediately with a fluorescence microscope. The serodiagnostic test exhibits a sensitivity of 98% for infections with *T.b. rhodesiense* or *T.b. gambiense* and a specificity of >99% for serum samples from healthy blood donors. The IFA exhibits a sensitivity of >99% for *Plasmodium* spp. and a specificity of 98% for healthy blood donor samples. The test has a sensitivity of >96% for visceral leishmaniasis and a specificity of >99% for heathy blood donor sera. Cross reactivity between the mentioned parasites antigens is possible.

#### *2.8. Plasmodium falciparum spp. ELISA*

Malaria serology was performed with an in-house screening ELISA for detection of specific *Plasmodium* spp. antibodies. *P. falciparum antigen* (strain NF54) was coated in 0.05 M sodium carbonate buffer, pH 9.6, to Immulon 2HB plates (, Thermo Scientific, Wohlen, Switzerland,). After washing, diluted sera were added to the plates and incubated for 15 min at 37 ◦C. After additional washing steps, horseradish peroxidase conjugated goat-anti-human-IgG (KPL, 474-1006, BioConcept Ltd, Allschwil, Switzerland) was added. Plates were incubated for 15 min at 37 ◦C, subsequently washed, and o-Phenylendiamine Dihydrochloride (OPD, Sigma, Buchs, Switzerland) was added. Reaction was stopped with 8 M H2SO4, and absorption was read with a Multiscan FC reader (ThermoScientific, Wohlen, Switzerland) at 492 nm. All sera giving positive or equivocal results were additionally tested with an in-house confirmatory Malaria IFA.

#### *2.9. Real-Time PCR*

For molecular analysis, 5 mL of whole blood samples were lysed and stored at the hospital site by adding an equal volume of guanidine-HCl 6 M with 0.1 M EDTA pH = 8, according to previous publications [19,20]. These samples were kept at 4 ◦C until shipment to Switzerland. Upon arrival, samples were immediately processed. For this, samples were heated at 100 ◦C for 15 min, and 500 μL of the guanidine-HCl-EDTA whole blood was used for extraction using the QIAamp DNA Mini Kit from Qiagen (Hilden, Germany), according to the manufacturer's instructions. DNA was eluted in 100 μL AE buffer (provided by the kit). The PCR targets the 18S rRNA gene of the *Trypanozoon* spp. (including the detection of *T.b. brucei, T.b. gambiense, T.b. rhodesiense, T. evansi*, and *T. equiperdum*) as previously described for a conventional PCR format [21]. Primer and probe sequences were newly designed and adapted to the real-time PCR format (forward primer: 5 -TAGTTTTGTGCCGTGCCAGT-3 , reverse primer: 5 -CGCTCCCGTGTTTCTTGTATC-3 , and TaqMan probe: 5 -FAM-TCGGACGTG-iQ500-TTTTGACCCACGC-BHQ1-3 ), resulting in an amplicon of 97 basepairs. The PCR reaction mixture contained 5 μL DNA, 1x GoTaq Probe Master Mix from Promega (Madison, USA), 800 nmol forward and reverse primer, and 200 nmol probe in a total reaction volume of 25 μL. The PCR program started with a step at 50 ◦C for 2 min and 95 ◦C for 10 min, followed by 45 cycles of 95 ◦C for 15 s and 58 ◦C for 1 min. Plasmid containing the

97 bp amplicon sequence, as well as adjacent base pairs, was used as a positive control. The assay was optimised on *T.b. gambiense* and *T.b. rhodesiense* culture samples and spiked blood samples. Analytical sensitivity of the assay was 1 plasmid copy/μL DNA, and 5–50 parasites/mL spiked whole blood. Specificity was tested against DNA samples from *Babesia divergensis*, *Cryptosporidium* spp., *Cyclospora cayetanensis*, *Leishmania aethiopica*, *L. braziliensis*, *L. donovani*, *L. infantum*, *L. mexicana*, *Neospora caninum*, *Plasmodium falciparum*, *P. vivax*, *P. knowlesi*, *P. malariae*, *P. ovale*, *Sarcoycstis hominis*, *Toxoplasma gondii*, and *Trypanosoma cruzi,* and were found to be 100%.

#### *2.10. Statistical Analysis*

Categorical characteristics were summarized as counts and proportions, continuous characteristics as means, standard deviations, medians, and interquartile ranges. Paired comparisons were tested using McNemars' exact test or the Wilcoxon signed rank test depending on the nature of the compared factors. SAS version 9.4 (2002–2012, SAS Institute, Cary, NC, USA) was used to perform the statistical analysis. The level of significance was set to an alpha level <0.05.

#### *2.11. Ethics Statement*

The study protocol was approved by Ethikkommission Nordwest-und Zentralschweiz (EKNZ) (2017-00471; 12.4.2017) and Comité d'Éthique de l'Université Protestante au Congo (UPC) (CEUPC) (0044; 13.6.2017). Prior to enrolment, all participants gave informed written consent.

#### **3. Results**

In the region of Vanga, 96 first stage and 29 second stage HAT patients were recruited into several studies and trials between 2004 and 2005 [12–16]. The recruitment of participants for the present study took place during three visits to the following villages: (i) Kikongo Tango-Milundu, (ii) Nsalu, and (iii) Mayoko-Nkai.

The flowchart of the study, including the number of participants, is shown in Figure 1.

In 69 out of 92 (75%) of the eligible patients, a history and clinical examination could be performed. Fifty-one were previous first stage and 18 were previous second stage HAT patients. Ten out of 92 (11%) were not accessible but, according to oral communication by relatives or friends, were all in good health without any long-term sequelae of HAT. Five out of 92 (5%) died, two of them in the peripartum period of causes unrelated to HAT, and three without a clear diagnosis. Of these three patients, one died due to malnutrition, probably unrelated to HAT, one due to an undefined disease with unspecified swelling but without fever, and the third one due to unknown causes one month after HAT treatment. No information was available for eight out of 92 (9%) of the former patients. There was no history of a confirmed relapse for any of the patients followed up in this study. During the village visits, only clinical investigations, but no specific HAT diagnostic tests, were performed. At this point, no patient was suspected to have HAT; therefore, no blood tests had been planned for this phase of the study.

Table 1 shows the characteristics for patients with previously first (n = 51) and second (n = 18) stage HAT and their matched controls. Tables 2 and 3 show the prevalence of signs and symptoms before treatment and at the long-term follow-up, for first and second stage HAT, respectively. Figures 2 and 3 show the development of these signs and symptoms at the different examined time points.

**Figure 1.** Flow chart of the Human African Trypanosomiasis (HAT) study.

**Table 1.** Descriptive characteristics for first and second stage HAT patients and their controls.

\* Age of controls matched within ±5 years of age of patients; SD = standard deviation; IQR = interquartile range (first to third quartile).

**Table 2.** First stage HAT: Signs and symptoms for patients before treatment and at 12 year follow-up and for their controls.

McNemar's exact test; † Each patient was matched to a control by sex and age (±5 years); § For patients at 12 year follow-up and their healthy controls; (x) Number of new

#### *Trop. Med. Infect. Dis.* **2020**, *5*, 10

**Figure 2.** First stage HAT patients: Prevalence of signs and symptoms at different time points. Absence of a bar indicates 0% prevalence at a certain time point, except for dyspnea, chest pain, abnormal cardiac rhythm, and oedema, which were determined only at 12 year follow-up.

**Figure 3.** Second stage HAT patients: Prevalence of signs and symptoms at different time points. Absence of a bar indicates 0% prevalence at a certain time point.

#### *3.1. Lymphadenopathy*

Lymphadenopathy was the most frequently observed sign before treatment in first stage (98%) and second stage (94.4%) HAT patients. Twelve years after treatment, it was detected only in 5.9% first and in 5.6% second stage patients, which was comparable to the control groups (3.9% and 0%).

#### *3.2. Headache*

Headache was the most frequent complaint of HAT patients before treatment and was more frequent in the first stage (74%, including 2% with unbearable headache) than in the second stage (55.6%). In first stage HAT, headaches decreased to 6% at the end of treatment but increased thereafter to 30% (none with unbearable headache) 13 years later, which is significantly higher than in the control group (14%). One patient asked for further examination pertaining to the headache, which revealed the cause of the headache to be posttraumatic after a head injury without association to HAT. In second stage HAT, headaches decreased to 11% 12–13 years after treatment, compared to 5.6% in the control group.

#### *3.3. Pruritus*

Pruritus was the second most frequent complaint of HAT patients and was less frequent in the first stage (27.4%, including 6% with scratching marks) than in the second stage (50%) before treatment. It decreased to 4% after treatment and re-increased to 11.8% at the 12 year follow-up (none with scratching marks) in first stage and decreased to none in second stage HAT, comparable to the control group (2%).

#### *3.4. Sleep Disorder*

In first stage HAT, sleep disorders were neither observed before nor after treatment, but 3.9% reported daytime sleep and 7.8% reported sleep disorders during the night 12–13 years later. In second stage HAT, daytime sleep decreased from 33% and night time sleep disorders from 44% before treatment stepwise to none 12 years after treatment, equal to the healthy control group.

#### *3.5. Neurological Disorders*

In first stage HAT, neurological disorders were not described, before or after treatment, but tremor was reported in 6% and speech disorders in 3% at follow-up. In the second stage, neurological disorders such as speech impairment (11%), walking disability (28%), motor weakness (22%), and unusual behavior (22%) were observed before treatment, decreasing continuously post treatment, and had disappeared completely after 12–13 years.

#### *3.6. Blood Analysis4*

Blood analysis was performed only in the second phase of the study for symptomatic follow-up patients from the group with previous first stage HAT. Trypanosomes could not be detected in any of these 15 patients, neither by direct blood examination nor by real time PCR. The CATT was negative for all patients, but IFA using *T. b. brucei* antigen was positive in most patients (see Table 4). None of the 15 patients showed a positive *Leishmania* serology. By contrast, all patients exhibited a positive malaria serology in ELISA and IFA, which can be explained by the region being an endemic area for malaria.

**Table 4.** Real-time PCR, CATT, *T. brucei*, Malaria, and *Leishmania* serology of 15 follow-up patients.

Cutoffs, T. brucei IFA: <1/160 negative, 1/160 equivocal, ≥1/320 positive; Malaria ELISA: <0.15 negative, 0.15–0.29 equivocal, ≥0.30 positive; Malaria IFA: <1/40 negative, 1/40 equivocal, ≥1/80 positive; Leishmania IFA: <1/80 negative, 1/80 equivocal, ≥1/160 positive.

#### **4. Discussion**

To the best of our knowledge, this is the first study assessing the long-term sequelae of first and second stage HAT patients more than 10 years after initial treatment, comparing them to healthy controls. The study evaluated the clinical signs and symptoms before treatment, immediately after treatment, and 12–13 years after treatment in 51 first stage and 18 second stage HAT patients. A surprisingly high percentage (75%) of the eligible and accessible patients could be enrolled in the present study, while information on the health condition (including death) of 15% of the patients could be gathered from relatives and friends. In only 10% of the eligible patients was no information available.

Five cases were lost to follow-up because of death. In two of these, HAT could be ruled out as the cause of death, and in the other three, HAT was unlikely as the cause of death according to information from friends and relatives.

In first stage HAT patients, the prevalence of all signs and symptoms (except lymphadenopathy) had decreased 12–13 years after treatment compared to before treatment, but was still higher than immediately after the end of treatment and in the control group. The high prevalence of headaches (31%), pruritus (12%), and even the presence of neuropsychiatric symptoms such as tremor (6%), speech impairment (2%), and sleeping problems (12%) is worrying. All patients with neuropsychiatric symptoms also suffered from headaches. No relapse was clinically suspected in any of the patients or reported by the patients in the time before the long-term follow-up.

In second stage HAT patients treated with melarsoprol, all HAT specific signs and symptoms decreased steadily at the examined time-points towards a range of the healthy control group 12–13 years post-treatment. Only headache was reported in 11%, but this was not significantly different compared to the control group.

The higher prevalence of symptoms in first stage than in second stage HAT patients at follow-up is surprising and contradictory to former studies. In children treated with melarsoprol, signs and symptoms one month to four years after treatment were more frequently reported in second stage patients (22 symptoms in 28 patients) than in their first stage counterparts (25 symptoms in 92 patients) [22]. In contrast to our results, neither headache nor pruritus were the main symptoms, but were primarily neuropsychiatric symptoms: mental confusion (7.5%), changes in character (11.5%), memory problems (11.5%), language problem (3%), and sleeping disorders (5%). Furthermore, changes in growth in young participants, 6–20 years of age, formerly treated for HAT, were more accentuated

in second stage HAT compared to first stage HAT [23]. The subjects treated for first and second stage HAT had a smaller body weight (−4.25 kg), were 3 cm shorter, and had a smaller mid-upper-arm circumference (−1.15 cm) than the control group matched for age, sex and residence. These changes were more accentuated in second stage than in first stage patients: body weight from −4.9 to −2.6 kg, height from −4.9 to −2 cm, and mid-upper-arm circumference from −2 to −1 cm [23]. Differences to these studies could eventually be explained by a relatively low number of patients and a different study design (i.e., age of patients, different follow-up period, different context).

In first stage HAT patients, the clear improvement of the signs and symptoms after treatment was expected, but the reappearance of such symptoms 12–13 years post-treatment is surprising. In a follow-up examination of the patients, no trypanosomes could be detected in any of them, neither by direct microscopic examination nor by real time PCR of the blood. The CATT was negative for all 15 former first stage HAT patients with persistent symptoms, but 80% of them were positive in the *Trypanosoma* serology by IFA. In addition, 13% of the 15 participants showed an equivocal result in IFA, and only 7% were negative. The positive *Trypanosoma* serology by IFA could be explained by either long term-persisting anti-trypanosomal antibodies, a boost of immunity without an actual measurable infection due to repetitive infected tsetse bites [24], the presence of subclinical infection, or by cross-reactivity of malaria antibodies to a certain extent.

There are several hypotheses to explain these unexpected results:

First, the patients improved after the treatment and subjectively did not notice remaining minor symptoms at the end of treatment. All these symptoms were generally mild, and the patients did not seek medical help. It could be possible that the symptoms did not really disappear but persisted at a level below immediate perception. This would mean that sequelae persist even more than a decade after treatment and include headaches, pruritus, sleeping disorders, and rare neurological deficits in the absence of persisting parasites. The description of the signs and symptoms described in children [22,23] and case series of adults [10] strengthens the hypothesis of persistence of sequelae.

Secondly, after an initial cure, some patients could have had a relapse or a reinfection with HAT with an oligosymptomatic presentation and trypanosomes below the detection level in the blood. Even if HAT infections are rare events, mainly in the last decade [25], the study design does not allow the exclusion of such reinfection or relapse.

The third hypothesis could be that a few parasites have survived (hiding in other compartments than the blood) and may have caused a persisting oligosymptomatic low-level infection. Treatment with melarsoprol, which reaches nearly all compartments of the body, killed the trypanosomes, but neither pentamidine nor pafuramidine, used against first stage HAT, can penetrate the blood brain barrier and may not have reached all body compartments. Tanowitz described replicating trypanosomes in adipose tissue interacting with adipocytes [26]. Since most drugs used for the treatment of HAT are hydrophilic, their efficacy could be limited in a lipophilic environment, and adipose tissue could represent a safe site for trypanosomes to hide [26]. It has been described that the skin or the CSF could also represent an anatomic reservoir of trypanosomes [21,27]. In addition, the persistence of trypanosomes in the blood below the detection level cannot be ruled out.

Of interest are the results of a study where untreated HAT patients were compared to formerly treated HAT patients [10]. Eleven HAT patients refused treatment, survived, and were evaluated 2–9 years later. Parasites were detected in only one out of 11 patients in the blood (by microscopy and PCR), and HAT specific antibody titres had decreased in only seven out of 10 patients. The study suggests that oligo-or asymptomatic HAT infections may show spontaneous cure over time or persist as low level infection without passage to the CNS [10]. The control group included 18 first stage and 21 second stage HAT patients treated 15 years prior. No parasites could be detected in the blood of any patients, neither by PCR nor by microscopy after mini anion exchange centrifugation technique. A combination of a positive immune trypanolysis test [28] with a negative CATT test was found in 61.5% [10]. In 58% of the treated and in 68% of the untreated patients, non-specific symptoms such as fever and headaches were observed, showing that such symptoms may persist for a long time [10].

The findings lead to the hypothesis that a few surviving trypanosomes in certain compartments could partially be controlled by the immune system, leading to minor persisting symptoms without, however, the development of apparent clinical signs of a relapse of the disease. The persistent survival of trypanosomes could be a source of transmission [10,11] and could explain the reactivity of the serum in the *Trypanosoma* IFA in our study. The negative CATT results could possibly be explained by either a lower sensitivity than the IFA, by the occurrence of trypanosome strains that lack or do not express the LiTat 1.3 gene that has been described for some endemic areas in Nigeria [17], or by the rapid decline of CATT reactivity in treated HAT patients within a few years [29] while other serological tests are still positive. On the other hand, the negative CATT results could also mean that anti-trypanosomal antibodies are no longer present and the positive IFA results are caused by cross-reacting malaria antibodies.

A review of the natural progression of HAT concludes that, in rare cases, an oligosymptomatic chronic carrier stage exists, but duration beyond six to seven years is exceptional [30]. It is also worth paying attention to the broad spectrum of clinical presentations of *T.b. gambiense* HAT, ranging from a chronic oligosymptomatic form to the frequently observed, usually fatal chronic disease in endemic populations to the acute clinical presentation in non-endemic populations [31].

The presented results would be compatible with such a hypothesis, and the presence of antibody titres even 14–15 years after the end of treatment supports this hypothesis, although it needs to be confirmed by more robust data. The study demonstrates a surprisingly high percentage of HAT patients still suffering from symptoms even more than 10 years after treatment. The study was not designed to specify if re-infections, relapses, or parasites surviving in not yet well described body compartments cause the signs and symptoms or if the patients have sequelae without any surviving trypanosomes. These questions should be addressed in further studies.

#### **5. Limitations**

The limitations of this study are the subjective character of the symptoms, the difficulties associated with properly assessing third party history, and the relatively low number of patients. Another limitation of the study is that no *Trypanosoma* real time PCR and no *Trypanosoma* and malaria serology was performed for treated asymptomatic patients or control group participants. These data could not be collected due to the study design and the unexpected outcome of the first phase of the study. Finally, cross-reactivity of malaria antibodies against *T. brucei* antigens or vice versa cannot be ruled out.

#### **6. Conclusions**

We demonstrated HAT-related signs and symptoms more than 10 years after treatment for the first time in first stage HAT patients without detectable levels of parasites. In first stage HAT patients, the prevalence of all signs and symptoms decreased compared to before treatment, but were still higher than at the end of treatment and in the control group. In second stage HAT patients, all HAT specific findings had continuously decreased to the point where they were in the range of the healthy control group. Persistence of symptoms without detectable levels of parasites in the blood could suggest either parasitaemia below the detection level or the persistence of parasites in some other body compartments. Further studies, including skin or tissue biopsies for detection of trypanosomes by PCR and serologic testing before treatment as well as ten years later, could indicate if there is a reservoir, which could be relevant for subclinical forms and transmission.

**Author Contributions:** Conceptualization, J.B., C.B., A.B., J.M. and L.G.; methodology, J.B., C.B., A.B., J.M. and L.G.; software, L.G.; validation, J.B. and C.B.; formal analysis, L.G.; investigation, J.B., A.B., J.M., B.N., R.W., M.-T.R. and L.C.; resources, B.N., R.W., M.-T.R., L.C. and J.M.; data curation, L.G., A.B., J.B. and B.N.; writing—original draft preparation, J.B., C.B. and B.N.; writing—review and editing, J.B., C.B., L.G., B.N., M.-T.R., R.W., L.C. and A.B.; visualization, L.G.; supervision, C.B., J.B. and B.N.; project administration, J.B. All authors have read and agreed to the published version of the manuscript.

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

**Acknowledgments:** We thank Monica Cal from the Parasite Chemotherapy Unit, Department of Medical Parasitology and Infection Biology of the Swiss TPH for provision of trypanosomes, Karin Stoll-Rudin from the Diagnostic Center of Swiss TPH, and Patrick Ntumba and Jaques Pombo from the Hôpital Evangélique de Vanga for expert technical assistance. We thank Sven Poppert from the Diagnostic Center of Swiss TPH for his support and critical reading.

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

#### **References**

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